Novel argyrin derivatives as inhibitors of bacterial infections

Abstract

The present invention relates to novel argyrin derivatives of formula (I) and the use of argyrin derivatives as inhibitors of bacterial infections.

Description

[0001] Novel Argyrin Derivatives as Inhibitors of Bacterial Infections

[0002] The present invention relates to novel argyrin derivatives and the use of argyrin derivatives as inhibitors of bacterial infections.

[0003] Neisseria gonorrhoeae is a bacterial pathogen that infects the eyes, pharynx, reproductive and urinary tracts in both men and women as well as the anal canal in homosexual men. Over the past two decades, there has globally been an increasing trend in both the infection rate and the detection of antibiotic resistant clinical isolates of N. gonorrhoeae. Alarmingly, a growing proportion of detected gonococcal infections demonstrate resistant phenotype to ceftriaxone, an anti-gonococcal antibiotic of last resort. The sluggish rate of new antibiotic development is by no means sufficient to address the challenges posed by the growing rate of infections such as those caused by N. gonorrhoeae. Further, antibiotic resistance has forced health authorities in the past to recommend dual therapy (ceftriaxone and azithromycin), which in turn led to concerns about its effect on human microbiome.

[0004] Argyrins are a family of naturally produced octapeptides that display antimicrobial activity against Pseudomonas aeruginosa (F. Sasse, H. Steinmetz, T. Schupp, F. Petersen, K. Memmert, H. Hofmann, C. Heusser, V. Brinkmann, P. von Matt, G. Hdfle and H. Reichenbach, Journal of Antibiotics 2002, 55, 543-551 ; GB 2 367 553 A). One of the advantages of argyrins is that they show no toxicity against human gut flora. It was revealed in a mouse model study that following treatment with argyrin B, microbiota shows a similar pattern to the control group, making argyrins suitable antibiotics to be administered solo or in combination with other antibiotics. Mechanistically, argyrins bind a unique site in the bacterial elongation factor G, thereby bringing ribosomal protein synthesis complex to a halt. This uniqueness in action mechanism potentially decreases the likelihood of developing cross-resistance among the clinically relevant bacterial pathogens.

[0005] Over the past few years, our lab has developed a heterologous expression system to supply quantitative amounts of naturally occurring argyrins (mainly A-D) and to support a variety of studies including in vivo evaluations (D. Pogorevc, Y. Tang, M. Hoffmann, G. Zipf, H. S. Bernauer, A. Popoff, H. Steinmetz, and S. C. Wenzel, ACS Synthetic Biology 2019, 8, 1121 -1133; D. Pogorevc, and R. Muller, Microbial Biotechnology 2021 , 15, 353- 369). However, there are major hurdles towards developing natural argyrins as viable drug candidates. Those include poor solubility of argyrins A-D, safety issues manifested by the toxicity of argyrins against mammalian cell lines, and sub-optimal potency against important pathogens of clinical relevance. Furthermore, a comprehensive understanding of structure-activity-relationship (SAR) is lacking due to the limited supply of derivatives.

[0006] It has been the object of the present invention to provide novel compounds (especially novel argyrin derivatives) having antibacterial activity, especially against N. gonorrhoeae and C. difficile.

[0007] The present invention provides compounds of formula (I): wherein

[0008] R1is a methyl group or an ethyl group;

[0009] R2is hydrogen, a methyl group or a hydroxymethyl group

[0010] R3is hydrogen or a methyl group; R6is hydrogen or a methyl group; each X is independently selected from N and CR4; each Y is independently selected from N and CR5; each R4is independently selected from hydrogen, halogen, a hydroxy group, an amino group, a cyano group, an azido group, a nitro group, a COOH group, a CONH2 group; or a C1-4 alkyl group, a C2-4 alkenyl group, a C2-4 alkynyl group, or a C1-4 heteroalkyl group, all of which groups may optionally be substituted; and each R5is independently selected from hydrogen, halogen, a hydroxy group, an amino group, a cyano group, an azido group, a nitro group, a COOH group, a CONH2 group; or a C1-4 alkyl group, a C2-4 alkenyl group, a C2-4 alkynyl group, or a C1-4 heteroalkyl group, all of which groups may optionally be substituted; or a salt thereof.

[0011] Preferably, R1is an ethyl group.

[0012] Further preferably, R2is a methyl group.

[0013] Moreover preferably, R3is hydrogen.

[0014] Further preferably, one X is N and the remaining groups X are CR4; wherein each R4is preferably hydrogen.

[0015] Moreover preferably, all groups X are CR4.

[0016] Further preferably, at least one R4is not hydrogen.

[0017] According to a preferred embodiment, if all groups X are CR4, at least one R4is not hydrogen. Moreover preferably, one Y is N and the remaining groups Y are CR5; wherein each R5is preferably hydrogen.

[0018] Further preferably, all Y groups are CR5.

[0019] Moreover preferably, each R4is independently selected from hydrogen, halogen, an amino group, a cyano group, an azido group, a nitro group, a substituted or unsubstituted C1-4 alkyl group, a substituted or unsubstituted C2-4 alkenyl group, a substituted or unsubstituted C2-4 alkynyl group, a C1-4 alkyloxy group, a C1-4 alkylthio group, a C1-4 hydroxyalkyl group, a carboxylic acid, a carboxylic acid ester, a substituted or unsubstituted carboxamide, a substituted or unsubstituted phosphonate, a substituted or unsubstituted sulfonate, or a substituted or an unsubstituted sulfonamide.

[0020] Further preferably, each R5is independently selected from hydrogen, halogen, an amino group, a cyano group, an azido group, a nitro group, a substituted or unsubstituted C1-4 alkyl group, a substituted or unsubstituted C2-4 alkenyl group, a substituted or unsubstituted C2-4 alkynyl group, a C1-4 alkyloxy group, a C1-4 alkylthio group, a C1-4 hydroxyalkyl group, a carboxylic acid, a carboxylic acid ester, a substituted or unsubstituted carboxamide, a substituted or unsubstituted phosphonate, a substituted or unsubstituted sulfonate, or a substituted or an unsubstituted sulfonamide.

[0021] Moreover preferably, each R4is independently selected from hydrogen, halogen, an amino group, a cyano group, a nitro group, a C1-4 alkyl group, a C1-4 alkyloxy group, a C1- 4 alkylthio group, and a C1-4 hydroxyalkyl group.

[0022] Further preferably, each R5is independently selected from hydrogen, halogen, an amino group, a cyano group, a nitro group, a C1-4 alkyl group, a C1-4 alkyloxy group, a C1-4 alkylthio group, and a C1-4 hydroxyalkyl group.

[0023] Moreover preferably, each R4is independently selected from hydrogen, halogen, an amino group, a cyano group, a nitro group, a methyl group, a methoxy group, a thiomethyl group, and a hydroxymethyl group. Further preferably, one R4is selected from hydrogen, halogen, an amino group, a cyano group, a nitro group, a methyl group, a methoxy group, a thiomethyl group, and a hydroxymethyl group, and the remaining R4’s are hydrogen.

[0024] Moreover preferably, one R4is selected from halogen, an amino group, a cyano group, a nitro group, a methyl group, a methoxy group, a thiomethyl group, and a hydroxymethyl group, and the remaining R4’s are hydrogen.

[0025] Further preferably, one R4is a methyl group, and the remaining R4’s are hydrogen.

[0026] Moreover preferably, each R5is independently selected from hydrogen, halogen, an amino group, a cyano group, a nitro group, a methyl group, a methoxy group, a thiomethyl group, and a hydroxymethyl group.

[0027] Further preferably, one or two R5is / are selected from hydrogen, halogen, an amino group, a cyano group, a nitro group, a methyl group, a methoxy group, a thiomethyl group, and a hydroxymethyl group, and the remaining R5’s are hydrogen.

[0028] The term "optionally substituted" refers to a group which is unsubstituted or substituted by one or more (especially by one, two or three; preferably by one or two; especially preferably by one) substituent(s). If a group comprises more than one substituent, these substituents are independently selected, i.e. , they may be the same or different.

[0029] Examples for substituents are fluorine, chlorine, bromine, and iodine and OH, SH, NH2, =0, -SO3H, -SO2NH2, -COOH, -CN, -N3 and -NO2 groups; especially fluorine, chlorine, bromine, and iodine and OH, SH, NH2, =0, -COOH, and -CN groups.

[0030] Further examples of substituents are Ci-Ce alkyl, C2-C6 alkenyl, C2-C6 alkynyl, and Ci-Ce heteroalkyl groups; preferably C1-C4 alkyl and C1-C4 heteroalkyl groups.

[0031] Especially preferably, the compounds of formula (I) are selected from the following compounds, or a salt thereof:

[0032] The most preferred compounds of the present invention are the compounds disclosed in the examples, or a salt thereof.

[0033] It is further preferred to combine the preferred embodiments of the present invention in any desired manner (e.g., any embodiment of X may be combined with any embodiment of Y).

[0034] The present invention further provides the following strains:

[0035] 1. Myxococcus xanthus KT.2.1 = DSM 35027

[0036] 2. Myxococcus xanthus KT.2.32 = DSM 35028

[0037] Depositor: Helmholtz-Zentrum fur Infektionsforschung GmbH, Braunschweig

[0038] Deposition date: May 7th, 2024

[0039] The present invention moreover provides a compound of the following formula:

[0040] for use in the treatment or prophylaxis of bacterial infections caused by / V. gonorrhoeae.

[0041] The expression alkyl refers to a saturated, straight-chain or branched hydrocarbon group. The expression C1-6 alkyl refers to a saturated, straight-chain or branched hydrocarbon group that contains from 1 to 6 carbon atoms. The expression C1-4 alkyl refers to a saturated, straight-chain or branched hydrocarbon group that contains from 1 to 4 carbon atoms. Examples are a methyl (Me, CH3), CF3, CD3, ethyl, propyl, iso-propyl, n-butyl, iso-butyl, sec-butyl, tert-butyl, n-pentyl, iso-pentyl, n-hexyl, or 2,2-dimethylbutyl group.

[0042] The expressions alkenyl and alkynyl refer to at least partially unsaturated, straight-chain or branched hydrocarbon groups. The expression C2-6 alkenyl refers to an unsaturated, straight-chain or branched hydrocarbon group that contains from 2 to 6 carbon atoms and having at least one double bond. The expression C2-4 alkenyl refers to an unsaturated, straight-chain or branched hydrocarbon group that contains from 2 to 4 carbon atoms and having at least one double bond. The expression C2-6 alkynyl refers to an unsaturated, straight-chain or branched hydrocarbon group that contains from 2 to 6 carbon atoms and having at least one triple bond. The expression C2-4 alkynyl refers to an unsaturated, straight-chain or branched hydrocarbon group that contains from 2 to 4 carbon atoms and having at least one triple bond. Examples are an ethenyl (vinyl), propenyl (allyl), isopropenyl, butenyl, ethynyl, propynyl, or butynyl group.

[0043] Furthermore, the terms alkyl, alkenyl and alkynyl refer to groups in which one or more hydrogen atoms have been replaced by a halogen atom (preferably F or Cl) such as, for example, a 2,2,2-trichloroethyl, a -C(CI)=CH2, difluoromethyl, fluoromethyl or a trifluoromethyl group.

[0044] The expression heteroalkyl refers to an alkyl, alkenyl or alkynyl group as defined above in which one or more carbon atoms have been replaced by an oxygen, nitrogen, phosphorus, boron, selenium, silicon or sulfur atom (preferably by an oxygen, sulfur or nitrogen atom) or by a PO, SO or a SO2 group. Accordingly, the expression heteroalkyl also refers to a carboxylic acid or to a group derived from a carboxylic acid, such as, for example, a carboxylic acid ester, a carboxamide, acyl, acylalkyl, alkoxycarbonyl, acyloxy, acyloxyalkyl, carboxyalkylamide or alkoxycarbonyloxy, as well as to a phosphonate, a sulfonate, or a sulfonamide. Furthermore, the term heteroalkyl refers to groups in which one or more hydrogen atoms have been replaced by a halogen atom (preferably F or Cl).

[0045] The term C1-C6 heteroalkyl refers to a heteroalkyl group containing from 1 to 6 carbon atoms. The term C1-C4 heteroalkyl refers to a heteroalkyl group containing from 1 to 4 carbon atoms. Preferably, the term C1-C6 heteroalkyl refers to a heteroalkyl group containing from 1 to 6 carbon atoms and 1 , 2, 3 or 4 heteroatoms selected from 0, S and / or N (especially 0 and / or N). Further preferably, the term C1-C4 heteroalkyl refers to a heteroalkyl group containing from 1 to 4 carbon atoms and 1 , 2 or 3 heteroatoms selected from 0, S and / or N (especially 0 and / or N).

[0046] Specific examples of heteroalkyl groups are methoxy, trifluoromethoxy, -OCD3, ethoxy, n-propyloxy, isopropyloxy, butoxy, te / Y-butyloxy, methoxymethyl, ethoxymethyl, -CH2CH2OH, -CH2OH, -SC Me, -NHAc, -CONH2, methoxyethyl, 1 - methoxyethyl, 1 -ethoxyethyl, 2-m ethoxyethyl or 2-ethoxyethyl, methylamino, ethylamino, propylamino, isopropylamino, dimethylamino, diethylamino, isopropylethylamino, methylamino methyl, ethylamino methyl, diisopropylamino ethyl, methylthio, ethylthio, isopropylthio, enol ether, dimethylamino methyl, dimethylamino ethyl, acetyl, propionyl, butyryloxy, acetyloxy, methoxycarbonyl, ethoxycarbonyl, propionyloxy, acetylamino or propionylamino, carboxym ethyl, carboxyethyl or carboxypropyl, / V-ethyl- / V-methyl- carbamoyl or / V-methylcarbamoyl.

[0047] The term halogen refers to F, Cl, Br, or I. It should be appreciated that compounds of formula (I) may have tautomeric forms from which only one might be specifically mentioned or depicted herein; different geometrical isomers (which are usually denoted as cis / trans isomers or more generally as (E) and (Z) isomers); or different optical isomers as a result of one or more chiral carbon atoms (which are usually nomenclatured under the Cahn-lngold-Prelog or R / S system). All these tautomeric forms, geometrical or optical isomers (as well as racemates and diastereomers) and polymorphous forms are included in the invention. Since the compounds of formula (I) contain asymmetric C-atoms, they may be present either as achiral compounds, mixtures of diastereomers, mixtures of enantiomers or as optically pure compounds. The present invention comprises both all pure enantiomers and all pure diastereomers, and also the mixtures thereof in any mixing ratio.

[0048] According to a further embodiment of the present invention, one or more hydrogen atoms of the compounds of the present invention may be replaced by deuterium. Deuterium modification may improve the metabolic properties of a drug with little or no change in its intrinsic pharmacology. Deuterium substitution at specific molecular positions may improve metabolic stability, reduce formation of toxic metabolites and / or increase the formation of desired active metabolites. Accordingly, the present invention also encompasses the partially and fully deuterated compounds of formula (I). The term hydrogen also encompasses deuterium.

[0049] The therapeutic use of compounds according to formula (I), their salts (especially their pharmacologically acceptable salts), solvates and hydrates, respectively, as well as formulations and pharmaceutical compositions also lie within the scope of the present invention.

[0050] The present invention further provides pharmaceutical compositions comprising one or more compounds described herein or a salt (especially a pharmaceutically acceptable salt), solvate or hydrate thereof, optionally in combination with one or more carrier substances and / or one or more adjuvants. The present invention further provides a compound or a pharmaceutical composition as described herein for use in the treatment or prophylaxis of bacterial infections (especially for use in the treatment or prophylaxis of bacterial infections caused by / V. gonorrhoeae and / or for use in the treatment or prophylaxis of bacterial infections caused by C. difficile).

[0051] The present invention moreover provides the use of a compound or a pharmaceutical composition as described herein for the preparation of a medicament for the treatment or prophylaxis of bacterial infections (especially for the treatment or prophylaxis of bacterial infections caused by N. gonorrhoeae and / or for use in the treatment or prophylaxis of bacterial infections caused by C. difficile).

[0052] According to a further preferred embodiment, the present invention provides a method for the treatment or prevention of a bacterial infection in a subject, which comprises administering to the subject an effective amount of a compound of formula (I), or a salt thereof.

[0053] According to a moreover preferred embodiment, the present invention provides a method for the treatment or prevention of a bacterial infection in a subject, which comprises administering to the subject an effective amount of a pharmaceutical composition comprising a compound of formula (I), or a salt thereof.

[0054] Preferred examples of the bacterial infections are bacterial infections caused by N. gonorrhoeae and bacterial infections caused by C. difficile.

[0055] The compounds of the present invention may be used together with one or more other medicinal agents, especially together with one or more other antibacterial agents. These may e.g. be used as a combined preparation for simultaneous, sequential, or separate use in the treatment of patients with bacterial infections.

[0056] Preferably, the compounds of the present invention may be administered in combination with one or more of the following antibacterial agents or other agents: ceftriaxone, cefixime, gentamicin, azithromycin, spectinomycin, kanamycin, tetracycline, erythromycin, chloramphenicol, ciprofloxacin, moxifloxacin, ertapenem, doxycycline, metronidazole, vancomycin, fidaxomicin, bezlotoxumab, rifaximin, and tigecycline.

[0057] The one or more other medicinal agents and the compounds of the present invention may be administered simultaneously (e.g., in separate compositions or via a single composition) or sequentially in either order. In the latter case, the two or more compounds will be administered within a period and in an amount and manner that is sufficient to ensure that an advantageous or synergistic effect is achieved.

[0058] The present invention also relates to pro-drugs which are composed of a compound of formula (I) and at least one pharmacologically acceptable protective group which will be cleaved off under physiological conditions, such as an alkoxy-, arylalkyloxy-, acyl-, acyloxymethyl group (e.g. pivaloyloxymethyl), an 2-alkyl-, 2-aryl- or 2-arylalkyl- oxycarbonyl-2-alkylidene ethyl group or an acyloxy group as defined herein, e.g. ethoxy, benzyloxy, acetyl or acetyloxy or, especially for a compound of formula (I), (II) or (Illi), carrying a hydroxy group (-OH): a sulfate, a phosphate (-OPO3 or -OCH2OPO3) or an ester of an amino acid.

[0059] Preferably, the present invention also relates to a prodrug, a biohydrolyzable ester, a biohydrolyzable amide, a polymorph, tautomer, stereoisomer, metabolite, N-oxide, biohydrolyzable carbamate, biohydrolyzable ether, physiologically functional derivative, atropisomer, or in vivo-hydrolysable precursor, diastereomer or mixture of diastereomers, chemically protected form, affinity reagent, complex, chelate and a stereoisomer of the compounds of formula (I).

[0060] Examples of salts (especially pharmacologically acceptable salts) of sufficiently basic compounds are salts of physiologically acceptable mineral acids like hydrochloric, hydrobromic, sulfuric and phosphoric acid; or salts of organic acids like methanesulfonic, p-toluenesulfonic, lactic, acetic, trifluoroacetic, citric, succinic, fumaric, maleic and salicylic acid. Further, a sufficiently acidic compound may form alkali or earth alkali metal salts, for example sodium, potassium, lithium, calcium, or magnesium salts; ammonium salts; or organic base salts, for example methylamine, dimethylamine, trimethylamine, triethylamine, ethylenediamine, ethanolamine, choline hydroxide, meglumine, piperidine, morpholine, tris-(2-hydroxyethyl)amine, lysine, or arginine salts; all of which are also further examples of salts of the compounds described herein.

[0061] The compounds described herein may be solvated, especially hydrated. The hydratization / hydration may occur during the process of production or as a consequence of the hygroscopic nature of the initially water-free compounds. The solvates and / or hydrates may e.g. be present in solid or liquid form.

[0062] In general, the compounds and pharmaceutical compositions described herein will be administered by using the known and acceptable modes known in the art.

[0063] For oral administration such therapeutically useful agents can be administered by one of the following routes: oral, e.g. as tablets, dragees, coated tablets, pills, semisolids, soft or hard capsules, for example soft and hard gelatine capsules, aqueous or oily solutions, emulsions, suspensions or syrups, parenteral including intravenous, intramuscular and subcutaneous injection, e.g. as an injectable solution or suspension, rectal as suppositories, by inhalation or insufflation, e.g. as a powder formulation, as microcrystals or as a spray (e.g. liquid aerosol), transdermal, for example via an transdermal delivery system (TDS) such as a plaster containing the active ingredient or intranasal. For the production of such tablets, pills, semisolids, coated tablets, dragees and hard, e.g. gelatine, capsules the therapeutically useful product may be mixed with pharmaceutically inert, inorganic, or organic excipients as are e.g. lactose, sucrose, glucose, gelatine, malt, silica gel, starch, or derivatives thereof, talc, stearic acid or their salts, dried skim milk, and the like. For the production of soft capsules, one may use excipients as are e.g. vegetable, petroleum, animal or synthetic oils, wax, fat, and polyols. For the production of liquid solutions, emulsions or suspensions or syrups one may use as excipients e.g. water, alcohols, aqueous saline, aqueous dextrose, polyols, glycerin, lipids, phospholipids, cyclodextrins, vegetable, petroleum, animal, or synthetic oils. Especially preferred are lipids and more preferred are phospholipids (preferred of natural origin; especially preferred with a particle size between 300 to 350 nm) preferred in phosphate buffered saline (pH = 7 to 8, preferred 7.4). For suppositories one may use excipients as are e.g. vegetable, petroleum, animal or synthetic oils, wax, fat, and polyols. For aerosol formulations one may use compressed gases suitable for this purpose, as are e.g. oxygen, nitrogen, and carbon dioxide. The pharmaceutically useful agents may also contain additives for conservation, stabilization, e.g. UV stabilizers, emulsifiers, sweetener, aromatizers, salts to change the osmotic pressure, buffers, coating additives and antioxidants.

[0064] In general, in the case of oral or parenteral administration to adult humans weighing approximately 80 kg, a daily dosage of about 0.1 mg to about 10,000 mg, preferably from about 1 mg to about 1 ,000 mg, should be appropriate, although the upper limit may be exceeded when indicated. The daily dosage can be administered as a single dose or in divided doses, or for parenteral administration, it may be given as continuous infusion or subcutaneous injection.

[0065] The compounds of the present invention can be prepared by fermentation or by chemical synthesis applying procedures known to a person skilled in the art. The compounds of the present invention may e.g. be synthesised following procedures described in WO 2018 / 167506 A1. Further, the synthetic protocols described in the following publications can be adapted to produce the argyrins of the current invention:

[0066] E. Stempel, R. F.-X. Kami, N. Budisa, and M. Kalesse, Bioorganic & Medicinal Chemistry 2018, 26, 5259-5269; S. V. Ley, and A. Priour, European Journal of Organic Chemistry 2002, 3995-4004; L. Bulow, I. Nickeleit, A.-K. Girbig, T. Brodmann, A. Rentsch, U. Eggert,

[0067] F. Sasse, H. Steinmetz, R. Frank, T. Carlomagno, N. P. Malek, and M. Kalesse, ChemMedChem 2010, 5, 832-836; S. V. Ley, A. Priour, and C. Heusser, Organic Letters 2002, 4, 711 -714.

[0068] EXAMPLES

[0069] A biotechnological approach was employed to produce and purify novel argyrins. An inhouse strain of M. xanthus was chosen as heterologous host in which the mchA gene (part of myxochromide gene cluster) was replaced by tetracycline resistance cassette (figure 1 ). Then a plasmid harboring the argyrins gene cluster (pArg2345) was integrated into the genome of M. xanthus via homologous recombination, as described previously (D. Pogorevc, Y. Tang, M. Hoffmann, G. Zipf, H. S. Bernauer, A. Popoff, H. Steinmetz, and S. C. Wenzel, ACS Synthetic Biology 2019, 8, 1121 -1133; D. Pogorevc, and R. Muller, Microbial Biotechnology 2021 , 15, 353-369). The resulting strain was transformed by a second plasmid harboring arg45 (or arg451), as well tryptophan synthase gene originated from Salmonella enterica (trpAB).

[0070] Figure 1 shows the schematic representation of the molecular cloning approach to generate the heterologous hosts harboring argyrin gene cluster as well as tryptophan synthase genes.

[0071] To generate the second plasmids containing arg45(1)-trpAB the following approach was implemented: primer pairs nptll-trpAB-F / trpAB-Nsil-R (Table 1 ) were used to amplify 2036 bp long Trpfta operon from the pSTB7 expression plasmid ordered from the ATCC (ATCC 37845). The PCR fragment was digested by Nde\ and Nsi\ restriction endonucleases and cloned into pUC18-Zeo-mx8-nptll-corO1 (D. Pogorevc, F. Panter, C. Schillinger, R. Jansen, S. C. Wenzel, and R. Muller, Metabolic Engineering 2019, 55, 201 - 211 ). The sequence of the resulting tryptophan synthase harboring expression construct pUC18-Zeo-mx8-nptll-trpAB was confirmed by Sanger sequencing. The 2222 bp long Trpfta PCR fragment was amplified from pUC18-Zeo-mx8-nptll-trpAB using trpAB- F / trpAB-R primer pair (Table 1 ). The fragment was digested by BglW restriction endonuclease and cloned into similarly digested pUC18-Zeo-mx8-nptll-arg45 and pUC18-Zeo-mx8-nptll-arg451 (D. Pogorevc, and R. Muller, Microbial Biotechnology 2021 , 15, 353-369). The sequences of the resulting plasmids were confirmed by sanger sequencing and subsequently used for transformation into M. xanthus heterologous host. The detailed experimental procedures for PCR amplification, DNA isolation and bacterial transformation has been described in a previous publication (D. Pogorevc, and R. Muller, Microbial Biotechnology 2021 , 15, 353-369).

[0072] Table 1. List of primers used for producing arg45(1)-trpAB constructs.

[0073] The producer strains described above were inoculated from frozen glycerol stocks and grown on CTT agar plates supplemented with kanamycin 50 pg / mL - plus zeocin 50 pg / mL when appropriate - for several days until plates were mostly overgrown with cells. All of the cells were scraped from the plates to inoculate seed culture medium M7 / s4. The composition of M7 / S4 is 0.5% soy flour, 0.5% corn starch, 0.2% glucose, 0.1 % yeast extract, 0.1 % MgSO4.7H2O, 0.1 % CaCl2.2H2O, 1 % HEPES, with final pH 7.4 and supplemented with 0.1 mg / L of vitamin B12 and 5 mg / L of FeCh after autoclaving. For seed cultures, 50 mL of broth was cultivated in 300 mL Erlenmeyer flask at 30 °C, 180 rpm for 48-72 h. The well grown preculture was then diluted 10-fold in the production medium. The production medium was M7 / S4 supplemented with amino acids before autoclaving. The concentration of amino acids was calculated to the final concentration of 10 mM L-serine, 5 mM L-cysteine, 5 mM L-alanine, 10 mM glycine and 10 mM L-Abu. All cultures were supplemented with suitable antibiotics as mentioned above as well as with 1 mg / mL of pyridoxal phosphate and 1 % Amberlite XAD-16 resin. Over the entire cultivation period, the cultures were supplemented twice daily with selected indole derivatives (indole derivatives were ordered from Alfa Aesar, Sigma-Aldrich, or Combi- Blocks). Indole stocks were prepared at 1 M concertation in 100% DMSO and stored at - 20°C. All cultures were cultivated for 6-8 days before harvesting by centrifugation at 8000 rpm for 15 min. Supernatant was discarded and pelleted cells with Amberlite XAD-16 resin were extracted with 50 mL of ethyl acetate, twice. Extracts were filtered and dried in round bottom flasks on rotary evaporator. Crude extracts were dissolved in 1 -2 mL of methanol and stored at -20 prior to analysis and purification. The methanolic solutions were diluted 10x-100x in methanol for LCMS analysis. UPLC-MS measurement were performed on a Dionex (Germering, Germany) Ultimate 3000 RSLC system equipped with Waters (Eschborn, Germany) BEH Cis column (100 x 2.1 mm, 1.7 pm) equipped with a Waters VanGuard BEH Cis 1.7 pm guard column. The flow rate was set to 0.6 mL / min, and the column temperature was 45 °C. An 18 minute gradient was as the following: 0-0.5 min, 95% water with 0.1 % formic acid (A) and 5% acetonitrile with 0.1 % formic acid (B); 0.5-18.5 min, 5-95% B; 18.5-20.5 min, 95% B; 20.5-21 min, 95-5% B; 21 - 22.5 min, 5% B. UV-vis spectra were recorded by a DAD in the range from 200 to 600 nm. The LC flow was split to 75 pL / min before entering the Broker Daltonics (Bremen, Germany) maXis 4G HR-qToF mass spectrometer equipped with an Apollo II ESI source. Mass spectra were acquired in centroid mode ranging from 150-2500 m / z at a 2 Hz full scan rate. Mass spectrometry source parameters were set to 500 V as end plate offset; 4000 V as capillary voltage; nebuliser gas pressure 1 bar; dry gas flow of 5 L / min and a dry temperature of 200 °C. Ion transfer and quadrupole settings were set to funnel RF 350 Vpp.; multipole RF 400 Vpp as transfer settings and ion energy of 5 eV as well as a low mass cut of 300 m / z. Collision cell was set to 5.0 eV and pre-pulse storage time was set to 5 ps. Spectra acquisition rate was set to 2 Hz.

[0074] For MS2experiments, CID (collision-induced dissociation) energy was ramped from 35 eV for 500 m / z to 45 eV for 1 ,000 m / z. MS full scan acquisition rate was set to 2 Hz and MS2spectra acquisition rates were ramped from 1 to 4 Hz for precursor ion intensities of 10 kcts to 1 ,000 kcts.

[0075] Calibration was done automatically before every LC-MS run by injection of a sodium formate solution and calibration on the respective clusters formed in the ESI source. All MS analyses were acquired in the presence of the lock masses (C12H19F12N3O6P3, C18H19O6N3P3F2 and C24H19F36N3O6P3) which generate the [M+H]+ ions of 622.0289; 922.0098 and 1221.9906.

[0076] HPLC purification was performed on an Ultimate 3000 UHPLC system (Thermo Fisher) equipped with a diode array detector and ISQTMEC single quadrupole mass spectrometer. Concentrated methanolic solutions were applied on an XBridge BEH C18 OBD column, with 10 mm x 250 mm dimensions, 130 A pore size, and 5 pm particle size. An optimized gradient of eluent A (0.1 % formic acid in H2O) and eluent B (0.1 % formic acid in acetonitrile) was used to purify the argyrins.

[0077] Table 2. Argyrin derivatives produced by cultivation.

[0078] aOnly substituents other than C-H have been specified in the columns X and Y.bObtained by applying an 18-min gradient of eluent B as described above.cThe m / z values correspond to the [M + H+]+ion, unless otherwise specified.d[M + 2H+]+

[0079] Example 13. White powder, Yield 15 mg / L culture. MS: mlz (+ESI) calculated 869.3399 [M+H]+, found 869.3378.1H NMR (500 MHz, CDC ) <5 (ppm) 10.64 (s, 1 H, NH), 9,29 (s, 1 H, NH), 8.85 (obscure d, 1 H, NH), 8.71 (d, J = 8.01 Hz, 1 H, NH), 8.68 (d, J = 7.27 Hz, 1 H, NH), 8.38 (s, 1 H, NH), 8.05 (s, 1 H, thiazole H), 7.39-7.34 (m, 2H, arom. H), 7.09 (d, J = 2.03 Hz, 1 H, arom. H), 6.97-6.89 (m, 2H, arom. H), 6.80 (d, J = 2.24 Hz, 1 H, arom. H), 6.74 (d, J = 6.30 Hz, 1 H, NH), 6.37 (t, J = 7.53 Hz, arom. H), 5.43 (quint., J = 7.48 Hz, 1 H, alpha CH), 5.35 (d, J = 7.91 Hz, arom. H), 5.13 (s, 1 H, vinylic H), 5.09 (m, 1 H, alpha CH), 4.95 (d, J = 16.78 Hz, 1 H, diastereotopic H), 4.78 (s, 1 H, vinylic H), 4.68 (d, J = 12.61 Hz, 1 H, -CH2-OH), 4.57 (d, J = 12.82 Hz, 1 H, -CH2-OH), 4.49 (m, 1 H, NH), 4.35 (s, 3H, -OCH3), 4.2 (m, 1 H, alpha CH), 3.95 (m, 1 H, alpha CH), 3.56-3.37 (m, 3H, aliphatic H), 3.3 (dd, J = 14.96 and 3.95 Hz, 1 H, diastereotopic H), 3.16 3.14 (s, 3H, -NCH3), 2.85 (dd, J = 15.23 and 3.47 Hz, 1 H, diastereotopic H), 2.09-1 .82 (m, 2H, CH3-CH2), 1 .75 (d, J = 7.05 Hz, 3H, CH-CH3), 0.89 (t, J = 7.37 Hz, 3H, CH2-CH3), 0.85 (overlapped m, 1 H, diastereotopic H).

[0080] Example 44. Yellow powder, Yield 3 mg / L culture. MS: mlz (+ESI) calculated 884.3144 [M+H]+, found 884.3117.1H NMR (700 MHz, CDCI3) 5 (ppm) 11 .83 (s, 1 H, NH), 9.23 (s, 1 H, NH), 8.86 (s, 1 H, NH), 8.72-8.67 (2 overlapping broad singlets, 2H, NH), 8.33 (s, 1 H, NH), 8.05 (s, 1 H, thiazole H), 7.90 (d, J = 7.93 Hz, 1 H, arom. H), 7.43-7.37 (m, 2H, arom. H), 7.33 (obscure s, 1 H, arom. H), 6.97 (d, J = 7.18 Hz, arom. H), 6.79 (m, 2H, arom. H + NH), 6.44 (t, J = 7.85 Hz, arom. H), 5.70 (d, J = 7.63 Hz, 1 H, arom. H), 5.40 (quint., J = 7.37 Hz, 1 H, alpha CH), 5.16 (s, 1 H, vinylic H), 5.14 (m, 1 H, alpha CH), 4.90 (d, J = 17.20 Hz, sarcosine methylene H), 4.87 (s, 1 H, vinylic H), 4.36 (s, 3H, OCH3), 4.24 (m, 1 H, alpha CH), 4.16 (m, 1 H, NH), 4.00 (m, 1 H, alpha CH), 3.57 (m, 2H, diastereotopic H), 3.47 (dd, J = 14.96 and 3.74 Hz, 1 H, diastereotopic H), 3.40 (d, J = 16.16, 1 H, diastereotopic H), 3.32 (dd, J = 14.96 and 3.59 Hz, 1 H, diastereotopic H), 3.19 (s, 3H, NCH3), 2.86 (dd, J = 15.48 and 3.37 Hz, 1 H, diastereotopic H), 2.09-1.84 (m, 2H, CH3- C / - / 2), 1 .78 (d, J = 7.03 Hz, 3H, CH-CH3), 0.93-0.87 (overlapping t, J = 7.30 Hz, CH2-CH3 + diastereotopic H).

[0081] Example 63. White powder, Yield 8 mg / L culture. MS: mlz (+ESI) calculated 857.3199 [M+H]+, found 857.3213.1H NMR (700 MHz, CDCI3) <5 (ppm) 10.69 (s, 1 H, NH), 9.37 (s, 1 H, NH), 8.82 (d, J = 8.48 Hz, 1 H, NH), 8.59 (d, J = 7.06 Hz, 1 H, NH), 8.44 (s, 1 H, NH),

[0082] 8.31 (s, 1 H, NH), 8.06 (s, 1 H, thiazole H), 7.10 (d, J = 8.09 Hz, 1 H arom. H), 7.03 (d, J = 8.61 Hz, 1 H arom. H), 6.99 (obscure d, 1 H, arom. H), 6.93 (t, J = 8.10 Hz, 1 H arom. H), 6.82 (obscure s, 1 H, arom. H), 6.80 (d, J = 6.55 Hz, 1 H, NH), 6.74 (d, J = 10.79 Hz, 1 H arom. H), 6.42 (t, J = 7.45 Hz, 1 H arom. H), 5.66 (d, J = 7.96 Hz, 1 H, arom. H), 5.50 (m, 1 H, alpha CH), 5.06 (m, 1 H, alpha CH), 5.02 (s, 1 H, vinylic H), 4.99 (d, J = 15.67 Hz, diastereotopic H), 4.73 (s, 1 H, vinylic H), 4.56 (m, 1 H, NH), 4.32 (s, 3H, OCH3), 4.21 (m, 1 H, alpha CH), 3.98 (m, 1 H, alpha CH), 3.58 (dd, J = 15.35 and 3.53 Hz, 1 H, diastereotopic H), 3.52 (dd, J = 17.08 and 7.83 Hz, 1 H, diastereotopic H), 3.45 (dd, J = 14.96 and 3.66 Hz, 1 H, diastereotopic H), 3.41 (d, J = 17.08 Hz, 1 H, diastereotopic H),

[0083] 3.31 (dd, J = 14.96 and 4.05 Hz, 1 H, diastereotopic H), 3.11 (s, 3H, NCH3), 2.85 (dd, J = 15.28 and 3.08 Hz, 1 H, diastereotopic H), 2.01 and 1.89 (each m, 1 H, CH3-CH2), 1.73 (d, J = 7.06 Hz, 3H, CH-CH3), 1.12 (dd, J = 17.21 and 5.14 Hz, 1 H, diastereotopic H), 0.89 (t, J = 7.32 Hz, 3H, CH2-CH3).

[0084] Example 78. White powder, Yield 10 mg / L culture. MS: m / z (+ESI) calculated 965.2260 [M+H]+, found 965.2257.1H NMR (700 MHz, CDCI3) 5 (ppm) 10.84 (s, 1 H, NH), 9.37 (s, 1 H, NH), 8.76 (s, 1 H, NH), 8.71 (d, J = 8.12 Hz, 1 H, NH), 8.66 (d, J = 7.27 Hz, 1 H, NH), 8.30 (s, 1 H, NH), 8.05 (s, 1 H, thiazole H), 7.39-7.32 (m, 2H, arom. H), 7.27 (d, J = 6.52 Hz, 1 H, arom. H), 7.14 (m, 1 H, arom. H), 6.94 (d, J = 7.48 Hz, 1 H, NH), 6.79 (m, 1 H, NH), 6.76 (d, J = 6.41 Hz, 1 H, NH), 6.11 (t, J = 7.64 Hz, 1 H, arom. H), 5.42 (m, 1 H, alpha CH), 5.38 (d, J = 7.48 Hz, 1 H, arom. H), 5.08 (m, 1 H, alpha CH), 5.02 (s, 1 H, vinylic H), 4.96 (d, J = 16.88 Hz, 1 H, diastereotopic H), 4.75 (s, 1 H, vinylic H), 4.34 (s, 3H, OCH3), 4.30 (m, 1 H, NH), 4.21 (m, 1 H, alpha CH), 4.05 (m, 1 H, alpha CH), 3.57-3.52 (m, 2H, diastereotopic H), 3.47 (dd, J = 14.91 and 3.79 Hz, 1 H, diastereotopic H), 3.40 (d, J = 16.78 Hz, 1 H, diastereotopic H), 3.31 (dd, J = 15.01 and 3.69 Hz, 1 H, diastereotopic H), 3.18 (s, 3H, sarcosine N-CH3), 2.79 (dd, J = 15.44 and 3.37 Hz, 1 H, diastereotopic H), 2.04 and 1.92 (each m, 1 H, CH3-CH2), 1.77 (d, J = 7.16 Hz, 3H, CH-CH3), 1.10 (dd, J = 17.26 and 5.18 Hz, 1 H, diastereotopic H), 0.91 (t, J = 7.37 Hz, 3H, CH2-CH3).

[0085] Example 82. White powder, Yield 10 mg / L culture. MS: mlz (+ESI) calculated 837.3500 [M+H]+, found 837.3504.1H NMR (700 MHz, CDCI3) 5 (ppm) 9.38 (s, 1 H, NH), 8.77 (m, 2H, NH), 8.28 (s, 1 H, NH), 7.95 (s, 1 H, thiazole H), 7.27 (d, J = 8.10 Hz, 1 H, arom. H), 7.18-7.10 (m, 3H, arom. H), 7.07 (t, J = 7.00 Hz, 1 H, arom. H), 7.03 (m, 1 H, NH), 6.93 (m, 2H, arom. H), 6.85 (d, J = 7.00 Hz, 1 H, arom. H), 6.54 (s, 1 H, NH), 5.49 (quint., J = 7.00 Hz, 1 H, alpha CH), 5.42 (s, 1 H, NH), 5.13 (d, J = 1.5 Hz, 1 H, vinylic H), 5.03-4.94 (m, 2H, alpha CH + sarcosine methylene H), 4.80 (d, J = 1.5 Hz, 1 H, vinylic H), 4.30 (obscure d, 1 H, alpha CH), 4.12 (dt, J = 9.1 , 6.3 Hz, 1 H, Abu alpha CH), 3.75-3.58 (m, 4H, aliphatic H), 3.47 (d, J = 17.00 Hz, 1 H, sarcosine methylene H), 3.18 (s, 3H, sarcosine N-CH3), 3.17-3.11 (m, 1 H, trp methylene H), 2.73 (s, 3H, methylindole CH3), 2.65 (s, 3H, methylindole CH3), 1.98 and 1.86 (2x m, Abu CH2), 1.75 (d, J = 7.1 Hz, 3H, CH3), 0.91 (t, J = 7.4 Hz, 1 H, Abu CH3). Example 93. White powder, Yield 1 mg / L culture. MS: mlz (+ESI) calculated 853.3450 [M+H]+, found 853.3450.1H NMR (500 MHz, CDCb) <5 (ppm) 10.67 (s, 1 H, NH), 9.43 (s, 1 H, NH), 8.79 (d, J = 8.33 Hz, 1 H, NH), 8.75 (obscure d, 1 H, NH), 8.62 (d, J = 7.16 Hz, 1 H, NH), 8.29 (s, 1 H, NH), 8.03 (s, 1 H, thiazole H), 7.35-7.29 (m, 2H, arom. H), 6.98 (d, J = 2.35 Hz, 1 H, arom. H), 6.90 (dd, J = 5.98 and 2.56 Hz, 1 H, arom. H), 6.78 (d, J = 2.24 Hz, 1 H, arom. H), 6.73 (d, J = 6.30 Hz, 1 H, NH), 6.65 (d, J = 7.05 Hz, 1 H, arom. H), 6.25 (t, J = 7.53 Hz, 1 H, arom. H), 5.43 (quint., J = 7.43 Hz, 1 H, alpha CH), 5.23 (d, J = 7.91 Hz, 1 H, arom. H), 5.04 (m, 1 H, alpha CH), 4.93 (m, 2H, sarcosine methylene H + vinylic H), 4.5 (d, J = 1.18 Hz, 1 H, vinylic H), 4.48 (m, 1 H, NH), 4.32 (s, 3H, OCH3), 4.17 (m, 1 H, alpha CH), 3.95 (m, 1 H, alpha CH), 3.55-3.35 (m, 3H, aliphatic H), 3.28 (dd, J = 14.85 and 3.95, diastereotopic H), 3.11 (s, 3H, NCH3), 2.80 (dd, J = 15.28 and 3.31 , diastereotopic H), 2.19 (s, 3H, indole C / - / 3), 2.03-1 .84 (m, 2H, CH3-CH2), 1 .72 (d, J = 7.16 Hz, 3H, CH-CH3), 0.95 (dd, J = 17.36 and 5.29 Hz, 1 H, distereotopic H), 0.87 (t, J = 7.43 Hz, 3H, CH2-CH3).

[0086] Example 94. White powder, Yield 1 mg / L culture. MS: mlz (+ESI) calculated 853.3450 [M+H]+, found 853.3444.1H NMR (700 MHz, CDCI3) <5 (ppm) 10.72 (s, 1 H, NH), 9.25 (s, 1 H, NH), 8.92 (d, J = 7.88 Hz, 1 H, NH), 8.86 (d, J = 1 .85 Hz, 1 H, NH), 8.66 (d, J = 7.01 Hz, 1 H, NH), 8.22 (d, J = 2.09 Hz, 1 H, NH), 8.09 (s, 1 H, thiazole H), 7.15 (d, J = 7.88 Hz, 1 H, arom. H), 7.07 (d, J = 2.09 Hz, 1 H, arom. H), 6.87 (d, J = 7.88 Hz, 1 H, arom. H), 6.83 (d, J = 2.21 Hz, 1 H, arom. H), 6.76 (d, J = 6.40 Hz, 1 H, NH), 6.70 (d, J = 7.01 Hz, 1 H, arom. H), 6.34 (dd, J = 9.04 and 4.25 Hz, 1 H, NH), 6.22 (t, J = 7.30 Hz, 1 H, arom. H), 5.40 (quint., J = 7.35 Hz, 1 H, alpha CH), 5.29 (d, J = 7.88 Hz, 1 H, arom. H), 5.10-5.05 (m, 2H, vinylic H + alpha CH), 5.00 (s, 1 H, vinylic H), 4.57 (m, 1 H, diastereotopic H), 4.48 (m, 1 H, NH), 4.33 (s, 3H, OCH3), 3.97 (m, 1 H, alpha CH), 4.22 (dd, J = 7.32 and 5.48 Hz, 1 H, NH), 3.97 (m, 1 H, alpha CH), 3.66-3.57 (m, 2H, diastereotopic H), 3.52-3.43 (m, 2H, diastereotopic H), 3.31 (dd, J = 11 .00 and 4.90 Hz, 1 H, diastereotopic H), 2.84 (dd, J = 15.38 and 3.32 Hz, 1 H, diastereotopic H), 2.65 (s, 3H, indole C / - / 3), 2.23 (s, 3H, indole C / - / 3), 2.08-1 .89 (m, 2H, CH3-CH2), 1 .79 (d, J = 7.26 Hz, 3H, CH-CH3), 1 .01 (dd, J = 17.23 and 5.29 Hz, 1 H, distereotopic H), 0.92 (t, J = 7.26 Hz, 3H, CH2-CH3).

[0087] Example 96. White powder, Yield 5 mg / L culture. MS: mlz (+ESI) calculated 867.3607 [M+H]+, found 867.3637.1H NMR (500 MHz, CDCI3) <5 (ppm) 10.70 (s, 1 H, NH), 9.46 (s, 1 H, NH), 8.88 (s, 1 H, NH), 8.82 (d, J = 8.33 Hz, 1 H, NH), 8.65 (d, J = 7.05 Hz, 1 H, NH), 8.24 (obscure m, 1 H, NH), 8.06 (s, 1 H, thiazole H), 7.13 (d, J = 8.01 Hz, 1 H, arom. H), 7.01 (d, J = 2.24 Hz, 1 H, arom. H), 6.84 (d, J = 7.91 Hz, 1 H, arom. H), 6.82 (d, J = 2.24 Hz, 1 H, arom. H), 6.78 (d, J = 6.52 Hz, 1 H, NH), 6.68 (d, J = 6.95 Hz, 1 H, arom. H), 6.18 (t, J = 7.53 Hz, 1 H, arom. H), 5.45 (quint, J = 7.45 Hz, 1 H, alpha CH), 5.24 (d, J = 7.80 Hz, 1 H, arom. H), 5.06 (m, 1 H, alpha CH), 4.98 (d, J = 17.2 Hz, 1 H, sarcosine methylene H), 4.95 (d, J = 1.5 Hz, 1 H, vinylic H), 4.68 (obscure d, 1 H, vinylic H), 4.5 (m, 1 H, NH), 4.32 (s, 3H, OCH3), 4.20 (m, 1 H, alpha CH), 3.97 (dt, J = 8.98 and 6.25 Hz, 1 H, alpha CH), 3.57-3.38 (m, 3H, aliphatic H), 3.30 (dd, J = 15.00 and 4.1 Hz, 1 H, distereotopic H), 3.14 (s, 3H, -NCH3), 2.83 (dd, J = 15.33 and 3.26 Hz, 1 H, distereotopic H), 2.63 (s, 3H, indole C / - / 3), 2.22 (s, 3H, indole C / - / 3), 2.07-1.86 (m, 2H, CH3-CH2), 1.75 (d, J = 7.05 Hz, 3H, CH-CH3), 0.96 (dd, J = 17.36 and 5.40 Hz, 1 H, distereotopic H), 0.90 (t, J = 7.37 Hz, 3H, CH2-CH3).

[0088] Example 106. Pale yellow powder, Yield 8 mg / L culture. MS: mlz (+ESI) calculated 855.3065 [M+H]+, found 855.3064.1H NMR (500 MHz, CDCI3) 5 (ppm) 10.69 (s, 1 H, NH), 9.37 (s, 1 H, NH), 8.87 (d, J = 8.53 Hz, 1 H, NH), 8.66 (d, J = 7.15 Hz, 1 H, NH), 8.49 (s, 1 H, NH), 8.02 (s, 1 H, thiazole H), 7.54 (s, 1 H, arom. H), 7.50 (d, J = 8.53 Hz, 1 H, arom. H), 7.37 (dd, J = 8.53 and 1.51 Hz, 1 H, arom. H), 7.34 (s, 1 H, arom. H), 7.15 (d, J = 8.12 Hz, 1 H, arom. H), 7.03 (d, J = 2.2 Hz, arom. H), 6.96 (t, J = 7.70 Hz, 1 H, arom. H), 6.87 (s, 1 H, NH), 6.75 (d, J = 6.46 Hz, 1 H, NH), 6.48-6.40 (m, 2H, arom. H), 5.49 (quint., J = 7.39 Hz, 1 H, alpha CH), 5.20 (m, 1 H, alpha CH), 5.05 (d, J = 1 .38 Hz, 1 H, vinylic H), 5.00 (d, J = 17.19 Hz, 1 H, diastereotopic H), 4.82 (m, 1 H, NH), 4.76 (s, 1 H, vinylic H), 4.27 (m, 1 H, alpha CH), 4.03 (m, 1 H, alpha CH), 3.65-3.54 (m, 2H, aliphatic H), 3.47-3.34 (m, 2H, aliphatic H), 3.27 (dd, J = 15.13 and 4.95 Hz, 1 H, diastereotopic H), 3.14-3.05 (m, 4H, NCH3 + diastereotopic H), 2.52 (s, 3H, SC / - / 3), 1.92 (m, 2H, CH3-CH2), 1.72 (d, J = 7.02 Hz, 3H, CH-CH3), 1.27 (m, 1 H, diastereotopic H), 0.86 (t, J = 7.36 Hz, CH2-CH3).

[0089] Example 107. Pale yellow powder, Yield 4.5 mg / L culture. MS: mlz (+ESI) calculated 901.2942 [M+H]+, found 901.2951.1H NMR (500 MHz, CDCI3) 5 (ppm) 10.80 (s, 1 H, NH), 9.41 (s, 1 H, NH), 8.80 (multiplet, 2H, NH), 8.62 (d, J = 7.29 Hz, 1 H, NH), 7.93 (s, 1 H, thiazole H), 7.70 (s, 1 H, arom. H), 7.52 (d, J = 8.39 Hz, 1 H, arom. H), 7.38 (dd, J = 8.53 and 1.51 Hz, 1 H, arom. H), 7.22 (m, 2H, arom. H), 7.15 (br s, 1 H, arom. H), 7.12 (s, 1 H, arom. H), 7.08 (d, J = 2.06 Hz, 1 H, arom. H), 6.84 (d, J = 6.60 Hz, 1 H, NH), 6.66 (s, 1 H, NH), 5.46 (quint., J = 8.30 Hz, 1 H, alpha CH), 5.08 (s, 1 H, vinylic H), 5.05-4.93 (m, 3H, NH + alpha CH + diastereotopic H), 4.77 (s, 1 H, vinylic H), 4.32 (m, 1 H, alpha CH), 4.06 (m, 1 H, alpha CH), 3.75 (dd, J = 17.40 and 8.05 Hz, diastereotopic H), 3.50-3.33 (m, 3H, aliphatic H), 3.24 (dd, J = 15.27 and 6.46 Hz, diastereotopic H), 3.14 (s, 3H, NCH3), 2.82 (m, 1 H, diastereotopic H), 2.60 and 2.54 (each s, 3H, SCH3), 1 .92 (m, 2H, CH3-CH2), 1 .73 (d, J = 7.15 Hz, 3H, CH-CH3), 1.58 1.27 (m, 1 H, diastereotopic H), 0.90 (t, J = 7.29 Hz, CH2-CH3).

[0090] MIC determination against N. gonorrhoeae

[0091] N. gonorrhoeae strains were purchased from the German Collection of Microorganisms and Cell Cultures (Deutsche Sammlung von Mikroorganismen und Zellkulturen, DSMZ), the American Type Culture Collection (ATCC) and National Collection of Type Cultures (NCTC). All compounds were prepared as DMSO stocks and MIC values were determined in the microbroth dilution assays in 96-well microtiter plates (MTPs). In brief, bacteria were inoculated on Columbia blood agar (BBLTM, BD). Agar plates were incubated 18-24 h at 37 °C in presence of 5% CO2. The following day, colonies from the high viability area were collected with a cotton swab and suspended in supplemented GCP medium. Next, bacterial suspensions were diluted in supplemented GCP medium to the starting ODeoo of 0.1 . Bacteria were grown for 3 h at 37 °C and 180 rpm in normal atmosphere. Next, bacterial suspensions that achieved at least doubling of the ODeoo in 3 h were back diluted to 0.08 in fresh supplemented GCP and added to the 96-well MTPs containing serially diluted test compounds. MTPs were incubated for 24-48 h at 37 °C in the presence of 5% CO2. MIC values were determined as the lowest antibiotic concentration at which no visual growth of bacteria was observed. GCP medium consists of 1.5% proteose peptone #3, 0.4% potassium phosphate dibasic, 0.1 % potassium phosphate monobasic, 0.5% NaCI; 100x dilution of supplement 1 , 1000x dilution of supplement 2, and 10Ox dilution of supplement 3. Supplement 1 consists of 40% glucose, 1 % L-glutamine, and 0.002% co-carboxylase. Supplement 2 consists of 0.125% Fe(NO3)3. Supplement 3 consists of 0.5 M NaHCO3. All the supplement solutions were filter-sterilized after preparation.

[0092] Table 3. Activity of selected argyrins against N. gonorrhoeae strains. an.d.: not determined

[0093] MIC determination against C. difficile

[0094] Following the CLSI protocol (CLSI, 2012), the strains were cultured in brain heart infusion (BHI) broth (COPAN Diagnostics Inc., California, USA) and the MIC values were determined in a Whitley A35 Anaerobic workstation (Don Whitley Scientific Limited, West Yorkshire, UK). The incubation temperature was set at 37°C and under the gas composition of N2 (90.9%), CO2 (9.0%) and O2 (0.1 %). The 100x stocks solutions of the test compound in DMSO were diluted 50x in BHI and serially diluted on 96-well CellStar microtiter plates (Greiner Bio-One, Frickenhausen, Germany). The bacterial suspensions grown overnight in BHI were adjusted in the same medium to the density of 2.0 McFarland using a densitometer (BioMerieux, Marcyl'Etoile, France). The cells were further diluted (100x) after which 50 pL aliquots were added to the microplate containing the same volume of serially diluted argyrins. Positive control consisted of bacteria in the presence of 1 % DMSO, whereas negative control consisted of only growth media in addition to 1% DMSO. MIC values were determined after incubation in the anaerobic chamber for 48 h at 37°C. Table 4. Activity of selected argyrins against C. difficile strains. Table 5. MICso and MIC90 of selected argyrins against C. difficile strains of various ribotypes. aObtained against 55 clinical isolates of C. difficile.

[0095] Metabolic Stability in Liver Microsomes

[0096] For the evaluation of phase I metabolic stability, the test compounds (1 pM) were incubated with 0.5 mg / mL pooled mouse liver microsomes (Xenotech, Kansas City, USA), 2 mM NADPH, and 10 mM MgCL at 37 °C for 120 min on a microplate shaker (Eppendorf, Hamburg, Germany). The metabolic stability of testosterone, verapamil and ketoconazole were determined in parallel to confirm the enzymatic activity of mouse liver microsomes. The incubation was stopped after defined time-points by precipitation of aliquots of enzymes with twice the volumes of cold acetonitrile containing internal standard (15 nM diphenhydramine). Samples were stored on ice until the end of the incubation and precipitated protein was removed by centrifugation (15 min, 4 °C, 4,000 g). The remaining test compound at the different time points quantified by HPLC-MS / MS (Vanquish Flex coupled to a TSQ Altis Plus, Thermo Fisher, Dreieich, Germany) and half-life (ti / 2) values were calculated.

[0097] Table 6. Microsomal stability data of selected examples.

[0098] As shown in table 3, several compounds demonstrate superior activity to that of ceftriaxone against WHO X strain with examples 81 , 82, 84, and 86 proving to be the most potent (MIC = 0.125-0.25 pg / mL). The retained potency of these argyrins against the WHO X strain is remarkable since this strain exhibits a multidrug resistant phenotype against first-line antibiotics such as ceftriaxone and cefixime as well as ciprofloxacin, tetracycline and azithromycin.

[0099] Against C. difficile, selected argyrins showed superior activity to that of argyrin B (table 4 and 5). Once tested against more than 50 strains of C. difficile, examples 44, 63, 93, and 94 were found to be the most promising with the MIC50 / 90 values between 0.002-0.006 pg / mL. Also noteworthy was example 94 which demonstrated superior activity against C. difficile strain in comparison to its N-Me analog (example 96), while demonstrating a higher stability in the presence of liver microsomes (table 6). This suggests that the class of analogs with R6as hydrogen have the potential to yield compounds with further optimized pharmacokinetic properties.

[0100] C. difficile in vivo infection model:

[0101] Example 13 was dissolved in DMSO, gradually diluted with warm 0.5% methylcellulose solution using an ultrasonic bath at 37°C (final DMSO concentration 5%). Male C57BL / 6J mice (14-16 weeks old) were injected intraperitoneally (IP) with 250 pg clindamycin in 100 pl PBS one day before infection. After 24 hours, mice were orally infected with 2x 103C. difficile 630 spores in 100 pl. Example 13 (1 mg / kg or 5 mg / kg body weight) or vancomycin (50 mg / kg in H2O) was administered orally in 100 pL, on day one, two, and three post- infection. The control group received 100 pl of vehicle (0.5% methylcellulose, 5% DMSO). Mouse weight and health were monitored daily. Fecal pellets were collected at various time points in oxygen-reduced PBS, weighed, and homogenized with a TissueLyser II (Qiagen) using a stainless steel bead. Serial dilutions of the homogenate were plated on oxygen-reduced CLO plates (Biomerieux 43431 ) and incubated anaerobically (10% CO2, 10% H2, 80% N2) for 1 -2 days. C. difficile colonies were counted as colony forming units (CFU) per gram of feces, log-transformed, and analyzed by one-way ANOVA with Dunnett’s post-test.

[0102] Figure 2 shows the effect of example 13 and vancomycin on C. difficile colonization in a mouse infection model. Male C57BL / 6J mice were pretreated with clindamycin (250 pg, IP) and orally infected with 2x103C. difficile 630 spores. Post-infection, mice were treated orally with vehicle control, vancomycin at 50 mg / kg), example 13 at 1 mg / kg, or 5 mg / kg once daily on days 1 , 2, and 3 post-infection. Fecal samples were collected across 6 days, and C. difficile burden was quantified as colony forming units (CFU) per gram of feces. Data are shown as logi0CFU / g feces (mean ± SEM, n = 6-10 per group). Statistical significance was determined by one-way ANOVA with Dunnett’s post-test (*p < 0.05, **p < 0.01 , ***p < 0.001 , ****p < 0.0001 vs. control).

[0103] Example 13 significantly reduced C. difficile burden at both doses on multiple days, comparable to vancomycin treatment.

[0104] Cytotoxicity

[0105] HepG2 cells and primary human hepatocytes (pHH) were used to assess cytotoxicity. HepG2 cells were cultured in RPMI1640 supplemented with 10% fetal bovine serum (FBS) and 1 % penicillin / streptomycin under standard conditions (37°C, 5% CO2). pHH cells were cultured using Williams medium supplemented with 25 mM HEPES and 2 mM L-glutamine under standard conditions (37°C, 5% CO2). Test examples, including example 13, 63 and 96, were dissolved in DMSO and diluted in cell culture medium to final concentrations ranging from 3.13 pM to 25 pM. The final DMSO concentration did not exceed 0.5% v / v. HepG2 cells were seeded in 96-well plates and allowed to adhere for 48 hours prior to treatment, pHH cells were treated directly after seeding. Examples were added in triplicate wells and incubated for 24 hours for HepG2 cells and 4h for pHH cells. Cell viability was assessed using a resazurin-based metabolic assay. Fluorescence was measured using a plate reader (Ex / Em: 560 / 590 nm). Fluorescence values were normalized to vehicle controls, and the percentage viability as fractional survival (FS) was calculated. The mean and standard deviation of triplicates were reported for each example concentration. Examples were considered cytotoxic if viability dropped below 80%.

[0106] Table 7. Cytotoxicity assessment of test examples in HepG2 and pHH cells.

[0107] HepG2 exhibited minimal concentration-dependent decrease of cell viability in presence of argyrin B, the rest of the examples showed no cytotoxicity.

[0108] Pharmacokinetic evaluation of example 13 and 82: PK and feces evaluation of example 13:

[0109] Pharmacokinetic evaluation was performed with a CRO (Pharmacelsus, Saarbrucken, Germany). Adult male C57BI / 6 mice (~8 weeks of age) were purchased from Janvier Labs, France. The animals were housed in a temperature-controlled room (20-24°C) and maintained in a 12h light / 12h dark cycle. Food and water were provided ad libitum before and during the study. All experimental procedures were approved by and conducted in accordance with the regulations of the local Animal Welfare authorities (Landesamt fur Gesundheit und Verbraucherschutz, Abteilung Lebensmittel- und Veterinarwesen, Saarbrucken).

[0110] Seven blood sampling time points after oral administration were selected: 15 min, 30 min, 60 min, 120 min, 240 min, 480 min and 1440 min post dose. At each of the designated time points, 20 pL blood was collected from the tail vein into Li-heparin tubes. Whole blood samples were stored on dry ice within 1 -2 minutes of sampling and stored at -20°C until processed for LC-MS analysis. The mice were placed into metabolic cages immediately after the administration for collection of feces after 4h, 8h and 24 h. Feces samples were collected, frozen and stored at -20°C until LC-MS analysis.

[0111] Table 8. PK summary table for example 13.

[0112] Table 9. Amount of example 13 determined in feces 4, 8 and 24h after oral administration.

[0113] Pharmacokinetic analysis of example 13 in male C57BI / 6 mice (10 mg / kg, oral) revealed a moderate systemic exposure (Cmax = 76.9 ng / mL, ti / 2= 1.6 h) and high fecal concentrations, peaking at 8 hours post-dose, indicating significant intestinal exposure.

[0114] PK and reproductive tissue evaluation of example 82:

[0115] Adult female Balb C mice (~8 weeks of age) were purchased from Janvier Labs, France. The animals were housed in a temperature-controlled room (20-24°C) and maintained in a 12h light / 12h dark cycle. Food and water were provided ad libitum before and during the study. All experimental procedures were approved by and conducted in accordance with the regulations of the local Animal Welfare authorities (Landesamt fur Gesundheit und Verbraucherschutz, Abteilung Lebensmittel- und Veterinarwesen, Saarbrucken).

[0116] Seven blood sampling time points after IP administration were selected: 15 min, 30 min, 60 min, 120 min, 240 min, 480 min and 1440 min post dose. At each of the designated time points, 20 pl blood was collected from the tail vein into Li-heparin tubes. Whole blood samples were stored on dry ice within 1 -2 minutes of sampling and stored at -20°C until processed for LC-MS analysis. After the final pharmacokinetic sampling (8 h or 24 h) uterus and vagina were withdrawn. Tissue samples were frozen immediately and stored at -20°C until LC-MS analysis.

[0117] Table 10. PK summary table for example 82.

[0118] Table 11. Amount of example 82 detected in uterus and vagina of Balb C mice 8 and 24h after IP administration.

[0119] Example 82 administered intraperitoneally to female Balb C mice (25 mg / kg) showed high systemic exposure (Cmax = 3071 ng / mL, ti / 2= 8 h) and detectable distribution to reproductive tissues. Example 82 levels in uterus and vagina were 104 ng / g and 128 ng / g at 8 hours, decreasing to 36.2 ng / g and 52.6 ng / g, respectively, by 24 hours.

[0120] (Original in Electronic Form)

[0121] (This sheet is not part of and does not count as a sheet of the international application) (Original in Electronic Form)

[0122] (This sheet is not part of and does not count as a sheet of the international application)

[0123] FOR RECEIVING OFFICE USE ONLY

[0124] FOR INTERNATIONAL BUREAU USE ONLY -5 This form was rec international Bure -5-1 Authorized officer

Claims

Claims1 . A compound of formula (I):whereinR1is a methyl group or an ethyl group;R2is hydrogen, a methyl group or a hydroxymethyl groupR3is hydrogen or a methyl group;R6is hydrogen or a methyl group; each X is independently selected from N and CR4; each Y is independently selected from N and CR5; each R4is independently selected from hydrogen, halogen, a hydroxy group, an amino group, a cyano group, an azido group, a nitro group, a COOH group, a CONH2 group; or a C1-4 alkyl group, a C2-4 alkenyl group, a C2-4 alkynyl group, or a C1-4 heteroalkyl group, all of which groups may be substituted; andeach R5is independently selected from hydrogen, halogen, a hydroxy group, an amino group, a cyano group, an azido group, a nitro group, a COOH group, a CONH2 group; or a C1-4 alkyl group, a C2-4 alkenyl group, a C2-4 alkynyl group, or a C1-4 heteroalkyl group, all of which groups may be substituted; or a salt thereof.

2. A compound according to claim 1 , wherein one X is N and the remaining groups X are CR4; wherein each R4is preferably hydrogen.

3. A compound according to claim 1 , wherein all groups X are CR4.

4. A compound according to any one of the preceding claims, wherein at least one R4is not hydrogen.

5. A compound according to any one of the preceding claims, wherein one Y is N and the remaining groups Y are CR5; wherein each R5is preferably hydrogen.

6. A compound according to any one of the preceding claims 1 to 4, wherein all Y groups are CR5.

7. A compound according to any one of the preceding claims, wherein each R4is independently selected from hydrogen, halogen, an amino group, a cyano group, a nitro group, a methyl group, a methoxy group, a thiomethyl group, and a hydroxymethyl group.

8. A compound according to any one of the preceding claims, wherein one R4is selected from hydrogen, halogen, an amino group, a cyano group, a nitro group, a methyl group, a methoxy group, a thiomethyl group, and a hydroxymethyl group, and the remaining R4’s are hydrogen.

9. A compound according to any one of the preceding claims, wherein one R4is selected from halogen, an amino group, a cyano group, a nitro group, a methyl group, a methoxy group, a thiomethyl group, and a hydroxymethyl group, and the remaining R4’s are hydrogen.

10. A compound according to any one of the preceding claims, wherein one R4is a methyl group, and the remaining R4’s are hydrogen.

11. A compound according to any one of the preceding claims, wherein each R5is independently selected from hydrogen, halogen, an amino group, a cyano group, a nitro group, a methyl group, a methoxy group, a thiomethyl group, and a hydroxymethyl group.

12. A compound according to any one of the preceding claims, wherein one or two R5is / are selected from hydrogen, halogen, an amino group, a cyano group, a nitro group, a methyl group, a methoxy group, a thiomethyl group, and a hydroxymethyl group, and the remaining R5’s are hydrogen.

13. A compound according to any one of the preceding claims, wherein one or two R5is / are selected from halogen, an amino group, a cyano group, a nitro group, a methyl group, a methoxy group, a thiomethyl group, and a hydroxymethyl group, and the remaining R5’s are hydrogen.

14. A compound according to any one of the preceding claims, wherein one R5is a methyl group, and the remaining R5’s are preferably hydrogen.

15. A compound according to any one of the preceding claims, wherein R1is an ethyl group; R2is a methyl group; R3is hydrogen; and R6is a methyl group.

16. A compound according to any one of the preceding claims, wherein R1is an ethyl group; R2is a methyl group; R3is hydrogen; and R6is hydrogen.

17. A compound according to any one of the preceding claims which is selected from the following compounds, or a salt thereof:

18. Pharmaceutical composition comprising a compound according to anyone of the preceding claims and optionally one or more carrier substances and / or one or more adjuvants.

19. Compound according to any one of claims 1 to 17 or pharmaceutical composition according to claim 18 for use in the treatment or prophylaxis of bacterial infections.

20. Compound according to any one of claims 1 to 17 or pharmaceutical composition according to claim 18 for use in the treatment or prophylaxis of bacterial infections caused by / V. gonorrhoeae.21 . Compound according to any one of claims 1 to 17 or pharmaceutical composition according to claim 18 for use in the treatment or prophylaxis of bacterial infections caused by C. difficile.

22. Strains DSM 35027 and DSM 35028.

23. A compound of the following formula:for use in the treatment or prophylaxis of bacterial infections caused by / V. gonorrhoeae.

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