Jagaricin, derivatives, and uses thereof

2022-02-28 来源：客趣旅游网

(19)

TEPZZ 7Z¥4_ZB_T

(11)

EP2 703 410B1

(12)

EUROPEAN PATENT SPECIFICATION

(51)Int Cl.:

C07K7/64(2006.01)A61K38/12(2006.01)A61P35/00(2006.01)

C12P21/00(2006.01)A61P31/10(2006.01)C07K7/56(2006.01)

of the grant of the patent: 14.10.2015Bulletin2015/42

(45)Date of publication and mention

(21)Application number: 12006113.0(22)Date of filing: 28.08.2012

(54)Jagaricin, derivatives, and uses thereof

Jagaricin, Derivate und ihre VerwendungJagaricine, dérivés et leurs utilisations(84)Designated Contracting States:

AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR

•Graupner, Katharina07745 Jena (DE)•Hertweck, Christian04105 Leipzig (DE)•Lackner, Gerald07743 Jena (DE)•Scherlach, Kirstin04105 Leipzig (DE)

(43)Date of publication of application:

05.03.2014Bulletin2014/10

(73)Proprietor: Leibniz-Institut für Naturstoff-Forschung und Infektionsbiologie07745 Jena (DE)

(74)Representative: Forstmeyer, Dietmar et al

BOETERS & LIECK Oberanger 32

80331 München (DE)

(72)Inventors:

•Bretschneider, Tom07747 Jena (DE)

(56)References cited:

EP-A1- 1 698 638

US-A- 4 137 224

EP2 703 410B1Note: Within nine months of the publication of the mention of the grant of the European patent in the European PatentBulletin, any person may give notice to the European Patent Office of opposition to that patent, in accordance with theImplementing Regulations. Notice of opposition shall not be deemed to have been filed until the opposition fee has beenpaid. (Art. 99(1) European Patent Convention).

Printed by Jouve, 75001 PARIS (FR)

EP2 703 410B1

Description

Field of the invention

[0001]The present invention concerns a novel compound termed jagaricin, jagaricin derivatives, pharmaceutical com-positions comprising these compounds, a method for producing jagaricin, and the the novel compound for use asfungicide or antitumor agent.Background of the invention

[0002]Microbial natural products are one of the most promising sources for novel drugs. This is, because naturalproducts own an element of structural complexity which allows for the specific and effective inhibition of many proteintargets. For instance, nonribosomally synthesized peptides (NRPs) or polyketides, are a prosperous source for newbioactive compounds (see, e.g. A. L. Harvey, Drug Discov. Today 2008, 13, 894; D. J. Newman, G. M. Cragg, J. Nat.Prod. 2012, 75, 311). Nonribosomal peptide synthetases (NRPSs) consist of different building blocks, so called modules,that are responsible for the activation and incorporation of one amino acid into the growing peptide chain at a time (R.Finking, M. A. Marahiel, Annu. Rev. Microbiol. 2004, 58, 453; D. Schwarzer, R. Finking, M. A. Marahiel, Nat. Prod. Rep.2003, 20, 275). Thereby, every module can be further dissected into domains which exhibit one enzymatic function each.The adenylation-(A)-domain recognizes and activates the substrate, usually amino acids. These activated amino acidsare transferred to the thiolation-(T)-domain (also peptidyl carrier protein-(PCP)-domain) that is responsible for the trans-port of the substrate between the other catalytic domains. The peptide bond formation is catalyzed by the condensa-tion-(C)-domain. In addition to these core domain several modification domains, like epimerization-(E)-domains, can bea part of NRPSs (C. T. Walsh et al., Curr. Opin. Chem. Biol. 2001, 5, 525). The last module harbors a thioesterase-(Te)-do-main that releases the peptide chain either as a linear or as a cyclic product (Finking and Marahiel, loc. cit.; Schwarzer,Finking, and Marahiel, loc. cit.).

[0003]The research on antifungal medication has been neglected in the past, since fungal diseases were consideredas easily curable (R. Di Santo, Nat. Prod. Rep. 2010, 27, 1084; M. F. Vicente et al., Clin. Microbiol. Infect. 2003, 9, 15).However, an increasing need for antifungal drugs has emerged, as the incidents of severe fungal infections are contin-uously rising. Such fungal infections can be particularly dangerous for immunocompromized patients or persons whoreceived invasive surgeries, especially in view of the fact that resistance against commonly used drugs arises amongfungal human pathogens (R. Di Santo, loc. cit.; N. H. Georgopapadakou, T. J. Walsh, Science 1994, 264, 371).

[0004]Although much progress has been made in the development of antitumor agents, cancer is one of the leadingcauses of death. The most effective chemotherapeutics either interfere with the tumor cell cycle and division or bind toDNA and cause apoptosis through various downstream processes.

[0005]The motile Gram-negative bacterium Janthinobacterium agaricidamnosum causes the soft rot disease of mush-rooms (S. P. Lincoln, T. R. Fermor, B. J. Tindall, Int. J. Syst. Bacteriol. 1999, 49 Pt 4, 1577). For J. lividum, a betterinvestigated bacterium from the genus Janthinobacterium, secondary metabolite production has already been described(J. H. Johnson, A. A. Tymiak, Bolgar, M. S., J. Antibiot. 1990, 43, 920; J. O’Sullivan et al., J. Antibiot. 1990, 43, 913; A.Shirata et al., J. Sericult.Sci. Jpn. 1997, 66, 377). Accordingly, Janthinobacterium agaricidamnosum may also be apromising source for novel bioactive natural products.

[0006]Thus, a need remains to provide novel compounds or compositions that may be used to effectively treat fungalinfections/diseases and/or cancer.

[0007]It is, therefore, an aim of the present invention to provide a novel compound, and derivatives thereof, withantifungal and/or antitumor activity; a pharmaceutical composition comprising the novel compound or derivatives thereof;the novel compound for use as fungicide or antitumor agent, and the novel compound for use in a method of preventingor treating a fungal disease or cancer. Preferably, such treatment is more effective and not as burdensome as currenttreatments and improves the lives of the patients.Summary and Description of the Invention

[0008]The present invention was made in view of the prior art and the needs described above, and, therefore, theobject of the present invention is to provide a novel compound and derivatives thereof. In particular, jagaricin a novelsecondary metabolite from the mushroom pathogen Janthinobacterium agaricidamnosum and derivatives thereof areprovided. Another object of the invention is to provide pharmaceutical compositions comprising the novel compound orderivatives thereof. Other objects of the present invention are to provide a method for producing jagaricin, and the novelcompound for use as fungicide or antitumor agent.

[0009]These objects are solved by the subject matter of the attached claims.

[0010]These and other aspects of the present invention will become apparent upon reference to the following detailed

EP2 703 410B1

description and definitions.

[0011]The inventors established that a gene cluster coding for a nine modular NRPS is responsible for the biosynthesisof the novel secondary metabolite compound - jagaricin - in Janthinobacterium agaricidamnosum.

[0012]The inventors showed that the novel compound has strong antifungal activity against the major human patho-gens Candida albicans, Aspergillus fumigatus and Aspergillus terreus.

[0013]The inventors also showed that the novel compound exhibits antiproliferative and cytotoxic activity.[0014]The inventors further established that the novel compound has little or no antibacterial activity.

[0015]The inventors also established that the novel compound is involved in the soft rot infection process, but is notessential for pathogenicity.

[0016]Taken together, the inventors demonstrate that the novel cyclic lipopeptide jagaricin is produced by the mush-room pathogen Janthinobacterium agaricidamnosum (J. agaricidamnosum) and displays strong antifungal activitiesagainst the major human pathogenic fungi C. albicans and Aspergillus spp as well as antiproliferative activity againsthuman umbilical vein endothelial cells HUVEC, human chronic myeloid leukemia cells K-562 and cytotoxic activityagainst human cervix carcinoma cells HeLa.

[0017]These results for the first time provide the secondary metabolite jagaricin and derivatives thereof, and allow atherapeutic, preventive and/or curative role to be conceived for it or a derivative thereof in the treatment of a fungalinfection/disease and/or cancer. Accordingly, the present invention is directed to a compound of the general formula (I):

or a pharmacologically acceptable salt thereof, wherein

R1 and R2 can each independently represent a hydrogen atom, an optionally substituted alkyl group, an optionallysubstituted alkenyl group, or an optionally substituted alkinyl group, wherein one carbon atom in said alkyl, alkenyl,or alkynyl group may be replaced by an oxygen atom, a sulfur atom, C=O, NR10, CONR11, or NR12CO at anychemically allowable position;

R10, R11, and R12 can each independently represent a hydrogen atom or an alkyl group having 1 to 6 carbon atoms;

R3 can be a hydrogen atom, a halogen atom, a hydroxyl group, a cyano group, a nitro group, an amino group, amercapto group, an optionally substituted alkyl group having 1 to 6 carbon atoms, an alkoxy group having 1 to 6carbon atoms, a SOCH3 group, a SO2CH3 group, or an acyl group having 2 to 6 carbon atoms, or a polyethyleneglycol group of formula -A-[CH2-CH2-O]n-R20, wherein A is -0-, -C(=O)-, -OC(=O)-, or -OC(=O)- (CH2)m-O-; m is aninteger from 1 to 20; n is an integer of from 2 to 100, and R20 is a hydrogen atom, a methyl group, or an ethyl group;R4 and R5 can each independently represent a hydrogen atom, an optionally substituted alkyl group, an optionallysubstituted alkenyl group, or an optionally substituted alkinyl group, wherein one carbon atom in said alkyl, alkenyl,or alkynyl group may be replaced by an oxygen atom, a sulfur atom, C=O, NR13, CONR14, or NR15CO at anychemically allowable position;

R13, R14, and R15 can each independently represent a hydrogen atom or an alkyl group having 1 to 6 carbon atoms;

EP2 703 410B1

R6 can be a hydrogen atom, a halogen atom, a hydroxyl group, a cyano group, a nitro group, an amino group, amercapto group, an optionally substituted alkyl group having 1 to 6 carbon atoms, an alkoxy group having 1 to 6carbon atoms, a SOCH3 group, a SO2CH3 group, an acyl group having 2 to 6 carbon atoms, or a polyethylene glycolgroup of formula -A-[CH2-CH2-O]n-R20, wherein A is -0-, -C(=O)-, -OC(=O)-, or -OC(=O)- (CH2)m-O-; m is an integerfrom 1 to 20; n is an integer of from 2 to 100, and R20 is a hydrogen atom, a methyl group, or an ethyl group; andR7 can be a hydrogen atom, a halogen atom, a hydroxyl group, a cyano group, a nitro group, an amino group, amercapto group, an optionally substituted alkyl group having 1 to 6 carbon atoms, an alkoxy group having 1 to 6carbon atoms, a SOCH3 group, a SO2CH3 group, or an acyl group having 2 to 6 carbon atoms, or a polyethyleneglycol group of formula -A-[CH2-CH2-O]n-R20, wherein A is -0-, -C(=O)-, -OC(=O)-, or -OC(=O)- (CH2)m-O-; m is aninteger from 1 to 20; n is an integer of from 2 to 100, and R20 is a hydrogen atom, a methyl group, or an ethyl group.[0018]Compounds are generally described herein using standard nomenclature. For compounds having asymmetriccenters, it should be understood that, unless otherwise specified, all of the optical isomers and mixtures thereof areencompassed. Compounds with two or more asymmetric elements can also be present as mixtures of diastereomers.In addition, compounds with carbon-carbon double bonds may occur in Z- and E- forms, with all isomeric forms of thecompounds being included in the present invention unless otherwise specified. Where a compound exists in varioustautomeric forms, a recited compound is not limited to any one specific tautomer, but rather is intended to encompassall tautomeric forms. Recited compounds are further intended to encompass compounds in which one or more atomsare replaced with an isotope, i.e., an atom having the same atomic number but a different mass number. By way ofgeneral example, and without limitation, isotopes of hydrogen include tritium and deuterium and isotopes of carboninclude 11C, 13C, and 14C.

[0019]Compounds according to the formulas provided herein, which have one or more stereogenic center(s), havean enantiomeric excess of at least 50%. For example, such compounds may have an enantiomeric excess of at least60%, 70%, 80%, 85%, 90%, 95%, or 98%. Some embodiments of the compounds have an enantiomeric excess of atleast 99%. It will be apparent that single enantiomers (optically active forms) can be obtained by asymmetric synthesis,synthesis from optically pure precursors or by resolution of the racemates. Resolution of the racemates can be accom-plished, for example, by conventional methods such as crystallization in the presence of a resolving agent, or chroma-tography, using, for example, a chiral HPLC column.

[0020]Compounds herein may also be described using a general formula that includes variables such as, e.g., A R1,R2, R3, R4, R5, R6, R7, R10, R11, R12, R13, R14, R15, R20, etc. Unless otherwise specified, each variable within such aformula is defined independently of any other variable, and any variable that occurs more than one time in a formula isdefined independently at each occurrence. Also, combinations of substituents and/or variables are permissible only ifsuch combinations result in stable compounds, i.e., compounds that can be isolated, characterized and tested forbiological activity.

[0021]A \"pharmaceutically acceptable salt\" of a compound disclosed herein preferably is an acid or base salt that isgenerally considered in the art to be suitable for use in contact with the tissues of human beings or animals withoutexcessive toxicity or carcinogenicity, and preferably without irritation, allergic response, or other problem or complication.Such salts include mineral and organic acid salts of basic residues such as amines, as well as alkali or organic salts ofacidic residues such as carboxylic acids. Suitable pharmaceutical salts include, but are not limited to, salts of acids suchas hydrochloric, phosphoric, hydrobromic, malic, glycolic, fumaric, sulfuric, sulfamic, sulfanilic, formic, toluenesulfonic,methanesulfonic, benzene sulfonic, ethane disulfonic, 2-hydroxyethylsulfonic, nitric, benzoic, 2-acetoxybenzoic, citric,tartaric, lactic, stearic, salicylic, glutamic, ascorbic, pamoic, succinic, fumaric, maleic, propionic, hydroxymaleic, hydroiod-ic, phenylacetic, alkanoic such as acetic, HOOC-(CH2)n-COOH where n is any integer from 0 to 4, i.e., 0, 1, 2, 3, or 4,and the like. Similarly, pharmaceutically acceptable cations include, but are not limited to sodium, potassium, calcium,aluminum, lithium and ammonium. Those of ordinary skill in the art will recognize further pharmaceutically acceptablesalts for the compounds provided herein. In general, a pharmaceutically acceptable acid or base salt can be synthesizedfrom a parent compound that contains a basic or acidic moiety by any conventional chemical method. Briefly, such saltscan be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of theappropriate base or acid in water or in an organic solvent, or in a mixture of the two. Generally, the use of nonaqueousmedia, such as ether, ethyl acetate, ethanol, isopropanol or acetonitrile, is preferred.

[0022]It will be apparent that each compound of formula (I) may, but need not, be present as a hydrate, solvate ornon-covalent complex. In addition, the various crystal forms and polymorphs are within the scope of the present invention.[0023]A \"substituent,\" as used herein, refers to a molecular moiety that is covalently bonded to an atom within amolecule of interest, e.g. to a compound of general formula (I) or a prodrug thereof. For example, a \"ring substituent\"may be a moiety such as a halogen, alkyl group, haloalkyl group or other substituent described herein that is covalentlybonded to an atom, preferably a carbon or nitrogen atom, that is a ring member. The term \"substituted,\" as used herein,means that any one or more hydrogens on the designated atom is replaced with a selection from the indicated substituents,

EP2 703 410B1

provided that the designated atom’s normal valence is not exceeded, and that the substitution results in a stable com-pound, i.e., a compound that can be isolated, characterized and tested for biological activity. When a substituent is oxo,i.e., =O, then 2 hydrogens on the atom are replaced.

[0024]The expression alkyl refers to a saturated, straight-chain (or linear) or branched hydrocarbon group that containsfrom 1 to 20 carbon atoms, preferably from 1 to 12 carbon atoms, more preferably from 1 to 6 carbon atoms, for examplea methyl, ethyl, propyl, iso-propyl, n-butyl, iso-butyl, sec-butyl, tert-butyl, n-pentyl, n-hexyl, 2,2-dimethylbutyl or n-octylgroup.

[0025]The expressions alkenyl and alkynyl refer to at least partially unsaturated, straight-chain or branched hydro-carbon groups that contain from 2 to 20 carbon atoms, preferably from 2 to 12 carbon atoms, more preferably from 2 to6 carbon atoms, for example an ethenyl, allyl, acetylenyl, propargyl, isoprenyl or hex-2-enyl group. Preferably, alkenylgroups have one or two, more preferably one, double bond(s) and alkynyl groups have one or two, more preferably one,triple bond(s).

[0026]The expression alkoxy refers to a saturated straight-chain or branched group of the general formula - OR,wherein R represents an alkyl group as defined above. An alkoxy group having 1 to 6 carbon atoms or 1 to 4 carbonatoms is preferred. Preferred examples of an alkoxy group having 1 to 6 carbon atoms include methoxy group, ethoxygroup, n-propoxy group, isopropoxy group, n-butoxy group, isobutoxy group, s-butoxy group, t-butoxy group, n-pentyloxygroup, n-hexyloxy group, and the like.

[0027]The expression \"acyl group\" refers to an alkylcarbonyl group, wherein the alkyl moiety is an alkyl group asdescribed above. An acyl group having 2 to 6 carbon atoms or 2 to 4 carbon atoms is preferred. Examples of an acylgroup include an acetyl group, a propanoyl group, 1-methylpropanoyl group, a butanoyl group, 1-methylpropanoyl group,2-methylpropanoyl group, 1,1-dimethylpropanoyl group, a pentanoyl group, and the like. An acetyl group and a propanoylgroup are mentioned as a preferred example.

[0028]The expression \"halogen\" or \"halogen atom\" as preferably used herein means fluorine, chlorine, bromine, iodine.[0029]The expression \"optionally substituted\" as used in conncetion with any group, preferably refers to a group inwhich one or more hydrogen atoms have been replaced each independently of the others by fluorine, chlorine, bromineor iodine atom; or by OH, =O, SH, =S, NH2, =NH, CN, NO2, or an alkoxy group.

[0030]As for an optionally substituted alkyl group, a group in which one or more hydrogen atoms have been replacedeach independently of the others by a hydroxyl group, a halogen atom, preferably a fluorine or chlorine atom, or amethoxy group can be mentioned as a preferred example. Additionally, an optionally substituted alkyl group may be oneselected from the group consisting of the above described preferred examples of an alkyl group further including atrifluoromethyl group, a difluoromethyl group, a hydroxymethyl group, 2-hydroxyethyl group, and a methoxymethyl group.A methyl group, an ethyl group, a n-propyl group, an isopropyl group, a cyclopropyl group, a trifluoromethyl group, adifluoromethyl group, a hydroxymethyl group, a 2-hydroxyethyl group or a methoxymethyl group are more preferred asan optionally substituted alkyl group. As for a substituent for an optionally substituted alkenyl group, and a substituentfor an optionally substituted alkynyl group, the substituents for an optionally substituted alkyl group as described abovecan be mentioned.

[0031]As used herein a wording defining the limits of a range of length such as, e. g., \"from 1 to 5\" means any integerfrom 1 to 5, i. e. 1, 2, 3, 4 and 5. In other words, any range defined by two integers explicitly mentioned is meant tocomprise and disclose any integer defining said limits and any integer comprised in said range.

[0032]Preferred according to the present invention can be a compound represented by the general formula (I’),

EP2 703 410B1

or a pharmacologically acceptable salt thereof; wherein R1, R2, R3, R4, R5, R6, R7 are defined as in general formula (I)above.

[0033]Preferably, R1 can be a group represented by the general formula (II):

wherein

R8 can be a hydrogen atom, a halogen atom, a hydroxyl group, a cyano group, a nitro group, an amino group, amercapto group, an optionally substituted alkyl group having 1 to 6 carbon atoms, an alkoxy group having 1 to 6carbon atoms, a SOCH3 group, a SO2CH3 group, an acyl group having 2 to 6 carbon atoms, or a polyethylene glycolgroup of formula -A-[CH2-CH2-O]n-R20, wherein A is -0-, -C(=O)-, -OC(=O)-, or -OC(=O)- (CH2)m-O-; m is an integerfrom 1 to 20; n is an integer of from 2 to 100, and R20 is a hydrogen atom, a methyl group, or an ethyl group; morepreferably R8 can be a halogen atom, a hydroxyl group, an optionally substituted alkyl group having 1 to 6 carbonatoms, an alkoxy group having 1 to 6 carbon atoms, a SOCH3 group, a SO2CH3 group, or an acyl group having 2to 6 carbon atoms; especially preferred R8 can be a halogen atom, a hydroxyl group, an amino group, or an optionallysubstituted alkyl group having 1 to 6 carbon atoms; and most preferred R8 can be a hydroxyl group; and

y is an integer from 1 to 20; more preferably an integer from 1 to 15; further preferred an integer from 1 to 10, andmost preferred y is 10.[0034]

Preferably, R1 can be a group represented by the general formula (II’):

wherein R8 and y are defined as in general formula (II) above.

[0035]Also preferred, R2 can be a hydrogen atom or an optionally substituted alkyl group having 1 to 6 carbon atoms;especially preferred R2 represents a hydrogen atom or a methyl group; and most preferred R2 can be a hydrogen atom.[0036]Preferably, R3 can be a halogen atom, a hydroxyl group, an amino group, or an optionally substituted alkylgroup having 1 to 6 carbon atoms; especially preferred R3 represents a halogen atom, a hydroxyl group, or an optionally

EP2 703 410B1

substituted alkyl group having 1 to 6 carbon atoms; more preferably R3 can be a halogen atom, or a hydroxyl group;and most preferred R3 can be a hydroxyl group.

[0037]Preferably, R4 and R5 can each independently represent a hydrogen atom, or an optionally substituted alkylgroup, wherein one carbon atom in said alkyl group may be replaced by an oxygen atom, a sulfur atom, C=O, NR13,CONR14, or NR15CO at any chemically allowable position; and R13, R14, and R15 are as defined above; especiallypreferred R4 and R5 can each independently represent a hydrogen atom, or an optionally substituted alkyl group; andmost preferred R4 and R5 each represents a hydrogen atom.

[0038]Further preferred, R6 can be a halogen atom, a hydroxyl group, an amino group, an optionally substituted alkylgroup having 1 to 6 carbon atoms, an alkoxy group having 1 to 6 carbon atoms, a SOCH3 group, a SO2CH3 group, oran acyl group having 2 to 6 carbon atoms; especially preferred R6 can be a halogen atom, a hydroxyl group, an aminogroup, or an optionally substituted alkyl group having 1 to 6 carbon atoms; and most preferred R6 can be a hydroxyl group..[0039]Preferably, R7 can be a halogen atom, a hydroxyl group, an amino group, an optionally substituted alkyl grouphaving 1 to 6 carbon atoms, an alkoxy group having 1 to 6 carbon atoms, a SOCH3 group, a SO2CH3 group, an acylgroup having 2 to 6 carbon atoms, or a polyethylene glycol group of formula -A-[CH2-CH2-O]n-R20, wherein A -C(=O)-,or -OC(=O)- (CH2)m-O-; m is an integer of from 1 to 6; n is an integer of from 2 to 10, and R20 is a hydrogen atom, or amethyl group; especially preferred R7 can be a halogen atom, a hydroxyl group, an amino group, or an optionallysubstituted alkyl group having 1 to 6 carbon atoms; and most preferred R7 can be a hydroxyl group.

[0040]Especially preferred according to the invention can be a compound represented by formula (III):

or a pharmacologically acceptable salt thereof.

[0041]The compound according to formula (III) may herein also be referred to as jagaricin.

[0042]It is to be noted that the present invention also encompasses all possible combinations of all preferred embod-iments.

[0043]Compounds provided herein exhibit high antifungal activity with an inhibition constant (MIC) at nanomolarconcentrations. Further, the compounds according to the invention may provide antitumor activity on cultured humantumor cell lines, i.e. an antiproliferative activity with an inhibition constant (GI50) and/or a cytotoxic activity with an IC50or CC50 in the micromolar range.

[0044]The activity and more specifically the pharmacological activity of the compounds according to the presentinvention can be assessed using appropriate in vitro assays. For instance, the GI50, CC50, or IC50 values of the compoundsaccording to the present invention may be determined via a cytotoxicity and antiproliferative assay of cell growth. Anti-fungal activities can, for example, be studied qualitatively by agar diffusion tests. Preferred compounds of the inventionhave values in the micromolar range, still more preferably values in the nanomolar range in the assays mentioned above.[0045]Preferably, the compounds of formula (I) according to the present invention each have one or more pharma-cological properties, especially, antiproliferative, antibacterial, antifungal or cytostatic activity, low toxicity, low drug druginteraction, high bioavailability, especially with regard to oral administration, high metabolic stability, and high solubility.[0046]The therapeutic use of one or more compound(s) of formula (I), its/their pharmacologically acceptable salt(s)

EP2 703 410B1

and also formulations and pharmaceutical compositions containing the same are within the scope of the present invention.The present invention also relates to the use of the compound of formula (I) as active ingredient in the preparation ormanufacture of a medicament, especially, a compound of formula (I), its pharmacologically acceptable salt and alsoformulations and pharmaceutical compositions for use in the treatment of fungal infections or cancer as well as its/theiruse for the preparation of a medicament, particularly a medicament for the treatment of fungal infections or cancer.[0047]The pharmaceutical compositions according to the present invention comprise at least one compound of formula(I) and, optionally, one or more carrier substances, excipients and/or adjuvants. Pharmaceutical compositions mayadditionally comprise, for example, one or more of water, buffers such as, e.g., neutral buffered saline or phosphatebuffered saline, ethanol, mineral oil, vegetable oil, dimethylsulfoxide, carbohydrates such as e.g., glucose, mannose,sucrose or dextrans, mannitol, proteins, adjuvants, polypeptides or amino acids such as glycine, antioxidants, chelatingagents such as EDTA or glutathione and/or preservatives. Furthermore, one or more other active ingredients may, butneed not, be included in the pharmaceutical compositions provided herein. For instance, the compounds of the inventionmay advantageously be employed in combination with an antibiotic, another anti-fungal, or anti-viral agent, an-antihistamine, a non-steroidal anti-inflammatory drug, a disease modifying anti-rheumatic drug, another cytostatic drug, adrug with smooth muscle activity modulatory activity, or mixtures of the aforementioned.

[0048]Pharmaceutical compositions may be formulated for any appropriate route of administration, including, forexample, topical such as, e.g., transdermal or ocular, oral, buccal, nasal, vaginal, rectal or parenteral administration.The term parenteral as used herein includes subcutaneous, intradermal, intravascular such as, e.g., intravenous, intra-muscular, spinal, intracranial, intrathecal, intraocular, periocular, intraorbital, intrasynovial and intraperitoneal injection,as well as any similar injection or infusion technique. In certain embodiments, compositions in a form suitable for oraluse are preferred. Such forms include, for example, tablets, troches, lozenges, aqueous or oily suspensions, dispersiblepowders or granules, emulsion, hard or soft capsules, or syrups or elixirs. Within yet other embodiments, compositionsprovided herein may be formulated as a lyophilizate.

[0049]Compositions intended for oral use may further comprise one or more components such as sweetening agents,flavoring agents, coloring agents and/or preserving agents in order to provide appealing and palatable preparations.Tablets contain the active ingredient in admixture with physiologically acceptable excipients that are suitable for themanufacture of tablets. Such excipients include, for example, inert diluents such as, e.g., calcium carbonate, sodiumcarbonate, lactose, calcium phosphate or sodium phosphate, granulating and disintegrating agents such as, e.g., cornstarch or alginic acid, binding agents such as, e.g., starch, gelatin or acacia, and lubricating agents such as, e.g.,magnesium stearate, stearic acid or talc. The tablets may be uncoated or they may be coated by known techniques todelay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longerperiod. For example, a time delay material such as glyceryl monosterate or glyceryl distearate may be employed.

[0050]A composition may further include one or more components adapted to improve the stability or effectivenessof the applied formulation, such as stabilizing agents, suspending agents, emulsifying agents, viscosity adjusters, gellingagents, preservatives, antioxidants, skin penetration enhancers, moisturizers and sustained release materials. Examplesof such components are described in Martindale--The Extra Pharmacopoeia (Pharmaceutical Press, London 1993) andMartin (ed.), Remington’s Pharmaceutical Sciences.

[0051]For the treatment of fungal infections as well as for the treatment of cancer, the dose of the biologically activecompound according to the invention may vary within wide limits and may be adjusted to individual requirements. Therequired dose may be administered as a single dose or in a plurality of doses. The amount of active ingredient that maybe combined with the carrier materials to produce a single dosage form will vary depending upon the host treated andthe particular mode of administration. Dosage unit forms will generally contain a sufficient amount of active ingredient.It will be understood, however, that the specific dose level for any particular patient will depend upon a variety of factorsincluding the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of admin-istration, route of administration, and rate of excretion, drug combination, i.e. other drugs being used to treat the patient,and the severity of the particular disease undergoing therapy.

[0052]Preferred compounds of the invention will have certain pharmacological properties. Such properties include,but are not limited to oral bioavailability, such that the preferred oral dosage forms discussed above can provide thera-peutically effective levels of the compound in vivo.

[0053]Compounds provided herein are preferably administered to a patient such as, e.g., a human, orally or topically,and are present within at least one body fluid or tissue of the patient. Accordingly, further disclosed are methods fortreating patients suffering from a fungal disease or cancer. As used herein, the term \"treatment\" encompasses bothdisease-modifying treatment and symptomatic treatment, either of which may be prophylactic, i.e., before the onset ofsymptoms, in order to prevent, delay or reduce the severity of symptoms, or therapeutic, i.e., after the onset of symptoms,in order to reduce the severity and/or duration of symptoms. Patients may include but are not limited to primates, especiallyhumans, domesticated companion animals such as dogs, cats, horses, and livestock such as cattle, pigs, sheep, withdosages as described herein.

[0054]The compounds of the present invention are useful in the treatment of different cancers, such as, for example,

EP2 703 410B1

breast, colon, lung and prostate tumors, as well as osteosarcoma, acute myeloid leukaemia, sporadic endometrial cancer,melanoma, malignant melanoma, soft tissue Sarcoma, B-cell chronic lymphocytic leukaemia, gastric cancers, cervicalcancer, hepatocellular carcinoma, pancreatic cancer; renal cancer/kidney cancer, or colorectal cancer.

[0055]Also the following methods for producing a compound of formula (I) lie within the scope of the present invention.[0056]The development of natural product based drugs is often hampered by their structural complexity. This factprecludes facile total synthetic access to analogues or the development of natural product libraries. Therefore, semi-synthetic as well as biotechnological approaches are commonly pursued in pharmaceutical research and development(von Nussbaum et al., Angew. Chem. Int. Ed. 2006, 45, 5072-5129). A very interesting strategy combines chemicalsemisynthesis with biosynthesis using genetically engineered microorganisms, a technique which occasionally has beentermed mutational biosynthesis or in short mutasynthesis (Review: S. Weist, R. D. Süssmuth, Appl. Microbiol. Biotechnol.2005, 68, 141-150).

[0057]For instance, a compound of formula (I), e.g. jagaricin, can be produced by culturing Janthinobacterium agar-icidamnosum (DSM 9628). It is understood that the production of compounds of formula (I) is not limited to the use ofthe particular organism described herein, which is given for illustrative purpose only. The invention also includes the useof any mutants which are capable of producing a compound of formula (I) including natural mutants as well as artificialmutants, e.g. genetically manipulated mutants and the expression of the gene cluster responsible for biosynthesis in aproducer strain or by heterologous expression in host strains.

[0058]A compound of formula (I) can be produced in liquid culture, by growing the respective microorganism in mediacontaining one or several different carbon sources, and one or different nitrogen sources. Also salts are essential forgrowth and production. Suitable carbon sources are different mono-, di-, and polysaccharides like maltose, glucose orcarbon from amino acids like peptones. Nitrogen sources are ammonium, nitrate, urea, chitin or nitrogen from aminoacids. The following inorganic ions support the growth or are essential in synthetic media: Mg-ions, Ca-ions, Fe-ions,Mn-ions, Zn-ions, K-ions, sulfate-ions, Cl-ions, phosphate-ions.

[0059]Temperatures for growth and production are between 10°C to 40 °C, preferred temperatures are between 20°C and 30 °C, especially at 22 °C and 28 °C, respectively. The pH of the culture solution is from 5 to 8, preferably 6.5 and 7.5.[0060]A compound of formula (I) can also be obtained by chemical synthesis using usual chemical reactions andsynthesis methods known to a person skilled in the art.Brief Description of the Figures

[0061]

Figure 1. Biosynthesis gene cluster with structure of jagaricin as well as the modular organisation of the jagaricinsynthetase.

Figure 2. HPLC chromatogram of J. agaricidamnosum ΔjagA extract, the wild type extract and medium extract. Thejagaricin peak is framed.

Figure 3. Plasmide map of pKG01. MCS-multi cloning site; AmpR-Ampicillin resistance cassette; KmR-Kanamycinresistance cassette.

[0062]The present invention is now further illustrated by the following examples from which further features, embod-iments and advantages of the present invention may be taken.

EXAMPLES

[0063]All reagents were purchased from commercial suppliers and used without further purification. Additionally,experiments were usually performed according to standard protocols and according to the manufacturer’s protocol,respectively. Specific methods and materials are summarized below.

Bacterial strains and media[0064]Janthinobacterium agaricidamnosum (DSM 9628) was retrieved from the German collection of microorganisms(DSM). The bacteria were cultured in nutrient media (605 DSM without NaCl; 1 g/L beef extract, 2 g/L yeast extract, 5g/L peptone, 15 g/L agar). Plates and liquid cultures (shaken at 150 rpm in baffled flasks) were grown at 22 °C and 28°C, respectively. During screening plates and liquid cultures of modified nutrient agar (additional 4 g/L chitin, 100 g/Lmushroom cubes), M9 (6 g/L Na2HPO4, 3 g/L KH2PO4, 0.5 g/L NaCl, 2 g/L NH4Cl, 4 g/L glucose, 25 g/L FeSO4, 2 mMMgSO4, 15 g/L agar), MS (20 g/L mannite, 20 g/L soy flour, 20 g/L agar) and modified VK (5 g/L glycine, 10 g/L yeast

EP2 703 410B1

extract, 10 g/L glucose, 10 g/L corn steep, 10 g/L CaCO3, 15 g/L agar pH 6.7-7.0) media were used. For the productionof jagaricin J. agaricidamnosum was grown in modified VK medium. The selection of positive J. agaricidamnosummutants was carried out on nutrition agar supplemented with 50 mg/mL kanamycin. For the construction of pKG01,Escherichia coli Top 10 cells and E. coli ER2925 cells were used. E. coli was cultured in LB medium (10 g/L tryptone,5 g/L yeast extract, 5 g/L NaCl, 1 g/L glucose) supplemented with 50 mg/mL kanamycin. Candida albicans, Aspergillusfumigatus and A. terreus were used for the bioactivity tests and they were cultivated on malt agar (C. albicans; 40 g/Lmalt extract, 4 g/L yeast extract, 15 g/L agar, pH 5.7-6.0) and potato glucose agar (A. fumigatus and A. terreus; 4 g/Lpotato starch infusion, 20 g/L dextrose, 15 g/L agar) plates, respectively.Screening for secondary metabolites[0065]Bacterial 20 mL cultures were grown for three days. Then the cultures were extracted twice with 20 mL of ethylacetate. Next, the ethyl acetate was removed under reduced pressure. The residue was dissolved in 0.5 mL methanoland was analyzed via analytical HPLC (Shimadzu LC-10Avp series with autosampler, high pressure pumps, columnoven and DAD detector, C18 column (Eurospher 100-5 250x4.6 mm), 1 mL/min flow rate, gradient elution (MeCN/0.1% TFA 0.5/99.5 to 100/0 within 30 minutes)) and mass spectrometry measurements (direct injection of 10 mL; Exactive,Thermo Scientific).Expression analysis20

[0066]RNA isolation was carried out with TRIsure RNA isolation reagent (Bioline). Thereby, the pellet of a 1 mLovernight culture was resuspended in 1.5 mL reagent. Cell disruption at SpeedMill PLUS (analytikjena) was carried outin lysis tubes (Biospec Products). Subsequently, remaining DNA was removed with Turbo-DNAse (Ambion). A one stepRT-PCR kit (One Step SYBR PrimeScript RT-PCR Kit, TaKaRa) and commercially purchased primers were used forreverse transcription (42 °C 5 min, 95 °C 10 sec) and amplification (95 °C 5 sec, 54 °C 10 sec, 72 °C 15 sec; cycle wasrepeated 40 times).Purification of jagaricin30

[0067]The crude extract of a 50 L fermentation was separated by size exclusion chromatography (Sephadex-LH-20column with methanol as mobile phase). An additional purification step was carried out via preparative HPLC (ShimadzuLC-8a series, DAD detector, C-18 column Grom-Saphir-110C (250x20 mm), 10 mL/min flow rate, 83 % MeCN/0.01 %TFA 10/90 to 100/0 within 25 minutes) and yielded the pure compound.Structural elucidation[0068]The ester bond of the lipopeptide was hydrolyzed by incubation in 1 M NaOH at room temperature for 1 hour.After the neutralization, MS/MS analyses were carried out with the linearized and with the unmodified compound usingthe TSQ Quantum (Thermo Scientific) and the Exactive (Thermo Scientific) MS instrument in order to get informationabout the amino acid sequence. For NMR measurements jagaricin was dissolved in deuterated methanol. NMR spectrawere recorded on Bruker Avance DRX 500 and DRX 600 instruments (Table S2, S11-16). Spectra were referenced tothe residual solvent signals.

Table S2. NMR shifts of β-hydroxymyristic acid and the amino acids starting from the N-terminus.

partial structureβ-hydroxymyristic acid

position12

13C NMR1H NMR (mult., J in Hz)

172.844.769.838.426.6

26.6-30.8 (6C)33.123.7

345

6-111213

-2.39 (1H, *)

2.34 (1H, d, 8.6)3.94 (1H, m)1.45 (2H,*)1.44 (1H,*)1.29 (1H,*)

1.24-1.35 (12H,*)1.27 (2H,*)1.29 (2H,*)

EP2 703 410B1

(continued)

partial structure

position143-OH

13C NMR1H NMR (mult., J in Hz)

14.5-168.3+132.2118.713.8-171.158.872.317.0-172.860.668.7i20.7-173.957.336.9128.9131.3116.4157.4--n.d.131.2i130.8+13.2-174.456.127.932.8178.0--172.344.0-

0.89 (3H, t, 6.9)n. d.

--5.71 (1H, q, 7.3)1.87 (3H, d, 7.3)n. d.

-4.71 (1H,d, 4.1)5.50 (1H, brs)1.34 (3H, d, 6.5)n. d.

-4.34 (1H,*)4.10 (1H,*)

1.18 (3H, t, 5.9*)n. d.

-4.37 (1H,*)

3.24 (1H, dd,**)

2.99 (1H, dd, 14.4,9.2)-7.09 (2H, d, 8.4)6.68 (2H, d, 8.4)-n. d.n. d.

--6.39 (1H, brs)1.60 (3H, d, 6.9)n. d.

-4.29 (1H, dd,**)2.13 (2H, m)2.39 (2H,*)-n. d.n. d.-4.02 (1H, d, 16.6)3.80 (1H, d, 16.6)n. d.

dehydrobutyrine

1234NH

L-threonine

1234NH

D-threonine

1234NH

D-tyrosin

12345,96,877-OHNH

dehydrobutyrine

1234NH

D-glutamine

12345

5-NH2NH12NH

Glycine

EP2 703 410B1

(continued)

partial structureL-threonine

position1234NH

13C NMR1H NMR (mult., J in Hz)

172.461.368.3i20.0-170.354.127.4131.1i135.0119.0--

-4.15 (1H, d, 5.1)4.08 (1H,*)

1.18 (3H, t, 5.9*)n. d.

-4.56 (1H, t, 7.5)3.03 (1H,*)2.91 (1H,*)-8.61 (1H, s)7.35 (1H, s)n. d.n. d.

L-histidine

123

4565-NHNH

* partial overlappingn. d. not detected

+ deduced from 2D couplingsi interchangeable signals

** coupling was observed, but coupling constant not determined

[0069]The amino acid stereochemistry was determined by derivatisation with 1-fluoro-2,4-dinitrophenyl-5-L-alanine-amide (L-FDAA). First, jagaricin was hydrolyzed with 6 M HCl supplemented with 0.05 % phenol overnight at 105 °C.The solvent was removed by reduced pressure and 100 mL 1 M NaHCO3 along with 50 mL L-FDAA (10 mg/mL in acetone)were added to the reaction that was heated at 50 °C for 1 h. Next, 50 mL 2 M HCl were added and the reaction mixturewas diluted with 200 mL 50 % (vol/vol) acetonitrile. The derivatives were analyzed via analytical HPLC (Shimadzu LC-10Avp series, DAD detector, column Grom-Sil 100 ODS-0 AB, 3 mm (250x4.6 mm), 1 mL/min flow rate, MeCN/0.1 %TFA 25/75 to 60/40 within 35 minutes to 100/0 within 8 minutes). The retention times (minutes) of standard amino acidswere as follows: D-His, 5.23; L-His, 6.47; L-Thr, 11.57; L-Gln, 14.4; D-Thr, 15.57; D-Gln, 15.61; L-Tyr, 34.34; D-Tyr,37,36; jagaricin, L-His, 6.47; jagaricin, D-Thr, 11.57; jagaricin, D-Gln, 15.61, jagaricin, D-Tyr, 37,36. D-Thr, D-Gin, L-allo-Thr and D-allo-Thr were additionally analyzed as previously described with the altered gradient (25/75 to 57/43within 35 minutes to 70/30 within 10 minutes to 100/0 within 5 minutes). The retention times (minutes) of standard aminoacids were as follows: L-allo-Thr, 11.66; D-allo-Thr, 12.26; D-Thr, 15.57; D-Gln, 16.25; jagaricin, D-Thr, 15.48; jagaricin,D-Gln, 16.25. Additionally, the free β-hydroxy-myristic acid (HMA) obtained from hydrolysis and the (R)-and (S)-HMAstandards (both TRC) were derivatized after hydrolysis of jagaricin with (R)-(-)-α-methoxy-a-trifluoromethylphenylacetylchloride reagent as previously described (Jenske, R. and W. Vetter, J. Chromatogr. A., 2007. 1146(2): p. 225-31) wherebythe methylation of the fatty acid was carried out in hexan/methanol with trimethylsillyldiazomethan at room temperaturefor 10 minutes. For the subsequent reaction 4-dimethylaminopyridine was used instead of pyridine. For GC-MS meas-urements the samples were dissolved in methanol. The analytics were executed on a Thermo Trace GC Ultra coupledwith a FID and a Thermo Polaris Q electron impact (EI)-ion trap mass spectrometer equipped with Combi PAL autosam-pler. A SGE forte capillary column BPX5 30 m; 0.25 mm inner diameter and 0.25 mm film was used. The column wasoperated with helium carrier gas 1.0 mL/min and splitless injection. Injector temperature was 300°C, splitless time 1.0min, then split flow was set to 15 mL/min. The FID temperature were set to 250°C with 35 mL/min hydrogen, 350 mL/minsynthetic air and 30 mL/min helium make up gas. Method was carried out like previously described (Jenske and Vetter2007, loc.cit.). Total ion current (TIC) were obtained using the mass range of 50-600 amu. 10 mL sample were injectedinto the GC.

Acetylation of jagaricin[0070]9 mg of substance was incubated with 1 mL pyridine (water free) and 1 mL acetic anhydrid over night underlight exclusion. The acetylated product was precipitated with 20 mL ice cold water. Subsequently the substance was

EP2 703 410B1

extracted three times with 20 mL chloroform. The organic phase was washed two times with distilled water and driedwith Na2SO4. After removing the solvents under reduced pressure, the product was analyzed by analytical HPLC andafterwards the acetylated jagaricin was subjected to NMR measurements.

Analysis of C-domain in module 5[0071]The amino acid sequence of core motifes C 1 through C 7 were manually compared with the ones of LCL- andDC-domains that were analyzed by Rausch and co-workers (Rausch, C. et al., BMC Evol. Biol., 2007; 7: p. 78). Addi-L

tionally, a phylogeny of all C-domains of the jagaricin synthetase was constructed (data not shown). Alignment and treeconstruction were performed with Mega 3.1 (Molecular Evolutionary Genetics Analysis, Version 3.1, Kumar, Tamuraand Nei).Bioactivity tests10

[0072]50 mL of different concentrated jagaricin solutions were each pipetted into a pierced hole (9 mm diameter)within an agar plate that was inoculated with the test strains C. albicans, A. fumigatus and A. terreus, respectively. Afterincubation at 24 °C over night the inhibition zone was measured. The antiproliferative and cytotoxic assay were performedas previously described (Abdou, R. et al., Phytochemistry, 2009. 71(1): p. 110-6).Annotation of the jagaricin biosynthesis gene cluster[0073]The J. agaricidamnosum genome was visualized with Artemis (Rutherford, K. et al., Bioinformatics, 2000.16(10): p. 944-5) and manually scanned for long open reading frames which are likely to belong to natural productbiosynthesis gene clusters. The corresponding amino acid sequences were analyzed via NCBI BLAST (Altschul S.F. etal., J. Mol. Biol., 1990. 215(3): p. 403-10; Sayers, E.W. et al., Nucleic. Acids Res., 2011. 39(Database issue): p. D38-51)and the PKS/NRPS analysis web site (Bachmann, B.O. and J. Ravel, Methods Enzymol, 2009. 458: p. 181-217) in orderto search for conserved NRPS and PKS domains. The NRPSpredictor2 (Rottig, M. et al., Nucleic. Acids Res., 2011.39(Web Server issue): p. W362-7) was used to characterize the A-domain specificity.Phylogenetic tree construction of thioesterase domains[0074]Amino acid sequences from thioesterase (Te) domains of cyclic lipopeptides were obtained from the ClustScandata base (Starcevic, A. et al., Nucleic. Acids Res., 2008. 36(21): p. 6882-92). Alignment and tree construction (datanot shown) were performed with Mega 3.1 (Molecular Evolutionary Genetics Analysis, Version 3.1, Kumar, Tamura andNei).

Construction of pKG01[0075]800 bp long homologous regions up- and downstream from the C1-domain coding region were amplified viaPCR using appropriate forward and reverse primer pairs. Additionally, the kanamycin resistance cassette was amplifiedfrom the template pK19 by employing an appropriate primer pair that incorporates a 30 bp overhang which is homologousto the above primer pairs for amplifying the C1-domain coding region. The Taq polymerase (NEB) carried out all ampli-fication reactions. The three PCR products were subjected to an overlapping PCR. For this reaction Phusion Flash PCRmaster mix (Thermo Scientific) and the appropriate pair were used. The product was cut with PstI and subsequentlyligated into the with PstI cut pGem-T Easy (Promega), yielding the plasmide pKG01 (Figure 3). Next, the plasmide wasused to transform E. coli ER2925.Transformation of J. agaricidamnosum20

[0076]20 mL of nutrition media was inoculated with 0.5 mL of a bacterial overnight culture. The flask was shaken untilOD600 reached 0.3. All subsequent steps were carried out on ice. Cells were harvested by centrifuging for 5 min at 5,000rpm at 4 °C. The pellet was washed two times with 20 mL and 10 mL 300mM sucrose, respectively. Cells were dissolvedin 0.5 mL 300 mM sucrose. 1 mL of demethylated plasmide was added to an aliquot of 60 mL competence cells andelectroporation was carried out (2 mm cuvette, 2500 V, 25 mF, 200 Ω). Cells were shaken at 25 °C for 1-2 h for recoveryand plated on nutrition agar supplemented with 50 mg/mL kanamycin. The resulting mutants were checked via PCR. Inaddition, the ability to produce jagaricin was tested as previously described (’Screening for secondary metabolites’).

EP2 703 410B1

Imaging mass spectrometry[0077]All mentioned chemical compounds, soft- and hardware were purchased by Bruker Daltonics. Commercialmushroom fruit bodies (Agaricus bisporus) were cut into approx. 1 mm thin slices that were placed on a conductive glassslide covered with double-sided adhesive coal tape (Plano). The mushroom tissue was inoculated with an overnightculture of J. agaricidamnosum and with jagaricin dissolved in water, respectively. The sample was incubated at roomtemperature for 24 h under moist conditions. Next, the sample was treated with a saturated solution of α-cynano-4-hydroxy cinnamic acid dissolved in a 2:1 mixture of 0.1 % TFA/MeCN. After a final drying step at 37 °C for 2 hours, theglass slide was clamped into a MTP slide adapter II and was subjected to MALDI-MS measurements. For this purposea ultrafleXtreme mass spectrometer was used operating with flexControl 3.0 in positive reflector mode collecting datain the range of m/z 900-2000 Da. The laser intensity was set to 80% with a laser frequency of 1000 Hz. Before the run,the flexControl method was calibrated using peptide calibration standard II. The automatic scanning of the imaging areawas programmed in flexImaging 3.0 with a raster width of 100 mm in XY recording 1000 spectra with a sample rate of2 GS/s at every spot. The resultant sum spectrum was evaluated manually and the mass of interest was visualized inthe logarithmic scale by picking the peak with 1 Da mass range using the brightness optimization as implemented inflexImaging.

Example 1: Assessing the annotated natural product biosynthesis gene cluster in the genome of J. agaricidamnosum20

[0078]One gene cluster coding for a nine modular NRPS was assigned as the potential jagaricin biosynthesis genecluster (Figure 1), as the size of the potential product matched the molecular weight of the isolated compound. Additionally,expression analyses of the biosynthesis gene cluster supported this assumption, as they showed an expression of thegene cluster during growth in producing media but not in non-producing media.

[0079]The jagaricin synthetase shows some interesting features. The first module exhibits a starter C-domain, thatcatalyses the condensation of a CoA-activated fatty acid with the first amino acid, leading to the biosynthesis of alipopeptide. Also it is noteworthy, that the glycine activating module number seven possesses an E-domain, althoughglycine is not a chiral amino acid. However, E-domains do not only have their catalytic function, but they are also importantfor protein-protein interaction between NRPS subunits. Since the described E-domain is located at the C-terminus ofJagC, a structural important role for this domain is very likely. Another noteable feature of the jagaricin biosynthesisgene cluster is the missing A-domain in module number two. A similar domain organization has been described for theyersiniabactin synthetase. During the synthesis of the siderophore yersiniabactin the second A-domain loads, in additionto the T-domain in the same module, two additional T-domains. Therefore, the loading of the second Thr in the jagaricinbiosynthesis is probably carried out by the first A-domain.

[0080]Employing the modular structure of the biosynthesis gene cluster, the assigned A-domain specificities andMS/MS analyses, we were able to predict that jagaricin is a cyclic lipopeptide with the amino acid sequenceC14H26O2-Dhb-Thr-Thr-Tyr-Dhb-Gln-Gly-Thr-His (Figure 1). The ring closure was expected to lie in between His andthe first Thr. NMR studies confirmed the predicted structure of jagaricin and identified the fatty acid chain as β-hydroxy-myristic acid. However, no couplings were observed in 2D NMR experiments that could verify the position of the ringclosure. Yet, a phylogenetic analysis of Te-domains of different cyclic lipopeptides supported the proposed position forthe ring closure. Further NMR experiments with OH-acetylated jagaricin could validate the ring closure position. Theabsolute stereochemistry was elucidated by using derivatisation reactions with Marfey’s and Mosher’s reagent, respec-tively (Figure 1). Thereby, it was at first surprising to identify D-Tyr instead of L-Tyr, as there is no epimerization domainin the fourth module. However, detailed analysis of the C-domain in the downstream module revealed the signaturesequences of a DCL-domain. Hence, the upstream A-domain activates probably D-Tyr that is supplied by an in transacting racemase. This D-Tyr is subsequently build into the growing peptide chain via the DCL-domain. Several exampleswhere D-amino acids are incorporated into NRPs via this mechanism have been studied in the past. Thus, the stereo-chemistry of the amino acids coincided with the modular architecture of the jagaricin synthetase.Example 2: Assessing the bioactivity of jagaricin50

[0081]Bioactivity studies showed strong antifungal activity of jagaricin against the major human pathogens Candidaalbicans, Aspergillus fumigatus and Aspergillus terreus (Table 1), but little or no antibacterial activity (data not shown).In higher concentrations jagaricin exhibits antiproliferative and cytotoxic activity (Table 1).

Table 1. Biological activity of jagaricin [mM].Ate

MIC

0.28

Afu0.41

Cal0.42

HUVEC-K-562-HeLa-

EP2 703 410B1

(continued)

Ate

Afu--

Cal--

HUVEC1-

K-5621-

HeLa-3.8

GI50CC50

Ate: A. terreus; Afu: A. fumigatus; Cal: C. albicans

Example 3: Assessing the involvement of jagaricin in the soft rod infection process10

[0082]Imaging mass spectrometry studies could visualize the production of jagaricin within the damaged tissue (datanot shown). Moreover, appliance of purified jagaricin also caused a superficial lesion on mushroom tissue. In order toevaluate the biological function of jagaricin further and to validate the annotation of the jagaricin biosynthesis genecluster, the jagaricin biosynthesis gene cluster was disrupted by insertion of a kanamycin resistance cassette. The correctinsertion of the kanamycin resistance cassette was checked via PCR. The knock-out mutant ΔjagA showed neitherjagaricin production in production media VK (Figure 2) nor on mushroom tissue. Therefore, the knock-out providesevidence, that jagaricin biosynthesis gene cluster was correctly annotated. Though, the mutant was still able to causelesions on the mushroom fruit bodies.

[0083]These results indicate, that although jagaricin is involved in the infection process, it is not essential for patho-genicity. Thus, enzymes take part in the degradation process of the fruit bodies, as well. Studies of the brown blotchdisease identified tolaasin as the sole virulence factor, while degradation enzymes have been shown to be the onlyvirulence factor in the cavity disease caused by Burkholderia gladioli pv. agaricicola. However, the discovered mode ofaction, where produced toxins are not essential for pathogenicity, but contribute to the disease outcome, has also beendescribed for the plant pathogen Pseudomonas syringae.

Claims1.

A compound of the general formula (I):

or a pharmacologically acceptable salt thereof, wherein

R1 and R2 each independently represents a hydrogen atom, an alkyl group in which one or more hydrogenatoms optionally have been replaced each independently of the others by a fluorine, chlorine, bromine or iodineatom: or by OH, =O, SH. =S, NH2, =NH, CN, NO2, or an alkoxy group, an alkenyl group in which one or morehydrogen atoms optionally have been replaced each independently of the others by a fluorine, chlorine, bromineor iodine atom: or by OH, =O, SH, =S, NH2, =NH, CN, NO2, or an alkoxy group, or an alkinyl group in whichone or more hydrogen atoms optionally have been replaced each independently of the others by a fluorine,chlorine, bromine or iodine atom: or by OH, =O, SH, =S, NH2, =NH, CN, NO2, or an alkoxy group, and whereinone carbon atom in said alkyl, alkenyl, or alkynyl group may be replaced by an oxygen atom, a sulfur atom,15

EP2 703 410B1

C=O, NR10, CONR11, or NR12CO at any chemically allowable position;

R10, R11, and R12 each independently represents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms;R3 is a hydrogen atom, a halogen atom, a hydroxyl group, a cyano group, a nitro group, an amino group, amercapto group, an alkyl group having 1 to 6 carbon atoms in which one or more hydrogen atoms optionallyhave been replaced each independently of the others by a fluorine, chlorine, bromine or iodine atom; or by OH,=O, SH, =S, NH2, =NH, CN, NO2, or an alkoxy group, an alkoxy group having 1 to 6 carbon atoms, a SOCH3group, a SO2CH3 group, or an acyl group having 2 to 6 carbon atoms, or a polyethylene glycol group of formula-A-[CH2-CH2-O]n-R20, wherein A is -O-, -C(=O)-, -OC(=O)-, or -OC(=O)- (CH2)m-O-; m is an integer from 1 to20; n is an integer of from 2 to 100, and R20 is a hydrogen atom, a methyl group, or an ethyl group;

R4 and R5 each independently represents a hydrogen atom, an alkyl group in which one or more hydrogenatoms optionally have been replaced each independently of the others by a fluorine, chlorine, bromine or iodineatom; or by OH, =O, SH, =S, NH2, =NH, CN, NO2, or an alkoxy group, an alkenyl group in which one or morehydrogen atoms optionally have been replaced each independently of the others by a fluorine, chlorine, bromineor iodine atom: or by OH, =O, SH, =S, NH2, =NH, CN, NO2, or an alkoxy group, or an alkinyl group in whichone or more hydrogen atoms optionally have been replaced each independently of the others by a fluorine,chlorine, bromine or iodine atom; or by OH, =O, SH, =S, NH2, =NH, CN, NO2, or an alkoxy group, wherein onecarbon atom in said alkyl, alkenyl, or alkynyl group may be replaced by an oxygen atom, a sulfur atom, C=O,NR13, CONR14, or NR15CO at any chemically allowable position;

R13, R14, and R15 each independently represents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms;R6 is a hydrogen atom, a halogen atom, a hydroxyl group, a cyano group, a nitro group, an amino group, amercapto group, an alkyl group having 1 to 6 carbon atoms in which one or more hydrogen atoms optionallyhave been replaced each independently of the others by a fluorine, chlorine, bromine or iodine atom; or by OH,=O, SH, =S, NH2 =NH, CN, NO2, or an alkoxy group, an alkoxy group having 1 to 6 carbon atoms, a SOCH3group, a SO2CH3 group, an acyl group having 2 to 6 carbon atoms, or a polyethylene glycol group of formula-A-[CH2-CH2-O]n-R20, wherein A is -0-, -C(=O)-, -OC(=O)-, or -OC(=O)- (CH2)m-O-; m is an integer from 1 to20; n is an integer of from 2 to 100, and R20 is a hydrogen atom, a methyl group, or an ethyl group; and

R7 is a hydrogen atom, a halogen atom, a hydroxyl group, a cyano group, a nitro group, an amino group, amercapto group, an alkyl group having 1 to 6 carbon atoms in which one or more hydrogen atoms optionallyhave been replaced each independently of the others by a fluorine, chlorine, bromine or iodine atom; or by OH,=O, SH, =S, NH2, =NH, CN, NO2, or an alkoxy group, an alkoxy group having 1 to 6 carbon atoms, a SOCH3group, a SO2CH3 group, or an acyl group having 2 to 6 carbon atoms, or a polyethylene glycol group of formula-A-[CH2-CH2-O]n-R20, wherein A is -O-, -C(=O)-, -OC(=O)-, or -OC(=O)- (CH2)m-O-; m is an integer from 1 to20; n is an integer of from 2 to 100, and R20 is a hydrogen atom, a methyl group, or an ethyl group.

The compound according to claim 1 or a pharmacologically acceptable salt thereof, wherein the compound isrepresented by the general formula (I’):

EP2 703 410B1

wherein R1, R2, R3, R4, R5, R6, R7 are defined as in claim 1.3.

The compound according to claim 1 or claim 2, or a pharmacologically acceptable salt thereof, wherein R1 is a grouprepresented by the general formula (II):

wherein

R8 can be a hydrogen atom, a halogen atom, a hydroxyl group, a cyano group, a nitro group, an amino, amercapto group, an alkyl group having 1 to 6 carbon atoms in which one or more hydrogen atoms optionallyhave been replaced each independently of the others by a fluorine, chlorine, bromine or iodine atom; or by OH,=O, SH, =S, NH2 =NH, CN, NO2, or an alkoxy group, an alkoxy group having 1 to 6 carbon atoms, a SOCH3group, a SO2CH3 group, an acyl group having 2 to 6 carbon atoms, or a polyethylene glycol group of formula-A-[CH2-CH2-O]n-R20, wherein A is -0-,- C(=O)-, - OC(=O)-, or -OC(=O)- (CH2)m-O-; m is an integer from 1 to20; n is an integer of from 2 to 100; and R20 is a hydrogen atom, a methyl group, or an ethyl group; andy is an integer from 1 to 20.

The compound according to any one of claims 1 to 3, or a pharmacologically acceptable salt thereof, wherein R1 isa group represented by the general formula (II’):

wherein R8 and y are defined as in claim 3.5.

The compound according to any one of claims 1 to 4, or a pharmacologically acceptable salt thereof, wherein R2 ishydrogen atom or an alkyl group having 1 to 6 carbon atoms in which one or more hydrogen atoms optionally havebeen replaced each independently of the others by a fluorine, chlorine, bromine or iodine atom; or by OH, =O, SH,=S, NH2, =NH, CN, NO2, or an alkoxy group.

The compound according to any one of claims 1 to 5, or a pharmacologically acceptable salt thereof, wherein R3 isa halogen atom, a hydroxyl group, an amino group, or an alkyl group having 1 to 6 carbon atoms in which one ormore hydrogen atoms optionally have been replaced each independently of the others by a fluorine, chlorine, bromineor iodine atom; or by OH, =O, SH, =S, NH2, =NH, CN, NO2, or an alkoxy group.

The compound according to any one of claims 1 to 6, or a pharmacologically acceptable salt thereof, wherein R4and R5 each independently represents a hydrogen atom, or an alkyl group in which one or more hydrogen atomsoptionally have been replaced each independently of the others by a fluorine, chlorine, bromine or iodine atom; orby OH, =O, SH, =S, NH2, =NH, CN, NO2, or an alkoxy group, wherein one carbon atom in said alkyl group may bereplaced by an oxygen atom, a sulfur atom, C=O, NR13, CONR14, or NR15CO at any chemically allowable position;and R13, R14, and R15 are defined as in claim 1.

The compound according to any one of claims 1 to 7, or a pharmacologically acceptable salt thereof, wherein R6 isa halogen atom, a hydroxyl group, an amino group, an optionally substituted alkyl group having 1 to 6 carbon atomsin which one or more hydrogen atoms optionally have been replaced each independently of the others by a fluorine,chlorine, bromine or iodine atom; or by OH, =O, SH, =S, NH2, =NH, CN, NO2, or an alkoxy group, an alkoxy grouphaving 1 to 6 carbon atoms, a SOCH3 group, a SO2CH3 group, or an acyl group having 2 to 6 carbon atoms.The compound according to any one of claims 1 to 8, or a pharmacologically acceptable salt thereof, wherein R7 isa halogen atom, a hydroxyl group, an amino group, an optionally substituted alkyl group having 1 to 6 carbon atoms

EP2 703 410B1

in which one or more hydrogen atoms optionally have been replaced each independently of the others by a fluorine,chlorine, bromine or iodine atom; or by OH, =O, SH, =S, NH2, =NH, CN, NO2, or an alkoxy group, an alkoxy grouphaving 1 to 6 carbon atoms, a SOCH3 group, a SO2CH3 group, an acyl group having 2 to 6 carbon atoms, or apolyethylene glycol group of formula -A-[CH2-CH2-O]n-R20, wherein A -C(=O)-, or -OC(=O)- (CH2)m-O-; m is aninteger of from 1 to 6; n is an integer of from 2 to 10, and R20 is a hydrogen atom, or a methyl group.

10.The compound according to any one of claims 1 to 9, or a pharmacologically acceptable salt thereof, wherein the

compound is represented by formula (III):

11.A pharmaceutical composition that comprises at least one compound according to any one of claims 1 to 10, or a

pharmacologically acceptable salt thereof and, optionally, at least one carrier substance and/or at least one adjuvant.

12.A method for the preparation of a compound of formula (I), the method comprising the steps of:

(a) fermenting Janthinobacterium agaricidamnosum (DSM 9628); and

(b) separating and retaining the compound from the culture broth; wherein the compound is a compound ac-cording to claim 10.

13.The compound, or a pharmacologically acceptable salt thereof, or the pharmaceutical composition according to any

one of claims 1 to 11 for use as a medicament.14.The compound, or a pharmacologically acceptable salt thereof, or the pharmaceutical composition according to any

one of claims 1 to 11 for use in the treatment or prevention of a fungal infection or cancer.

Patentansprüche

1.Verbindung der allgemeinen Formel (I):

EP2 703 410B1

oder ein pharmakologisch akzeptables Salz derselben, wobei

R1 und R2 jeweils unabhängig voneinander ein Wasserstoffatom, eine Alkylgruppe, in der gegebenenfalls einoder mehrere Wasserstoffatom(e) jeweils unabhängig von den anderen durch ein Fluor-, Chlor-, Brom- oderJodatom; oder durch OH, =O, SH, =S, NH2, =NH, CN, NO2 oder eine Alkoxygruppe ersetzt worden sein kann/ können; eine Alkenylgruppe, in der gegebenenfalls ein oder mehrere Wasserstoffatom(e) jeweils unabhängigvon den anderen durch ein Fluor-, Chlor-, Brom- oder Jodatom; oder durch OH, =O, SH, =S, NH2, =NH, CN,NO2 oder eine Alkoxygruppe ersetzt worden sein kann / können; oder eine Alkinylgruppe, in der gegebenenfallsein oder mehrere Wasserstoffatom(e) jeweils unabhängig von den anderen durch ein Fluor-, Chlor-, Brom-oderJodatom; oder durch OH, =O, SH, =S, NH2, =NH, CN, NO2 oder eine Alkoxygruppe ersetzt worden sein kann/ können, darstellt; und wobei ein Kohlenstoffatom in der Alkyl-, Alkenyl- oder Alkinylgruppe durch ein Sauer-stoffatom, ein Schwefelatom, C=O, NR10, CONR11 oder NR12CO an jeder chemisch erlaubten Position ersetztsein kann;

R10, R11, und R12 jeweils unabhängig voneinander ein Wasserstoffatom oder eine Alkylgruppe mit 1 bis 6Kohlenstoffatomen darstellt;

R3 ein Wasserstoffatom, ein Halogenatom, eine Hydroxylgruppe, eine Cyanogruppe, eine Nitrogruppe, eineAminogruppe, eine Mercaptogruppe, eine Alkylgruppe mit 1 bis 6 Kohlenstoffatomen, in der gegebenenfallsein oder mehrere Wasserstoffatom(e) jeweils unabhängig von den anderen durch ein Fluor-, Chlor-, Brom- oderJodatom; oder durch OH, =O, SH, =S, NH2, =NH, CN, NO2 oder eine Alkoxygruppe ersetzt worden sein kann/ können; eine Alkoxygruppe mit 1 bis 6 Kohlenstoffatomen, eine SOCH3 -Gruppe, eine SO2CH3 -Gruppe, odereine Acylgruppe mit 2 bis 6 Kohlenstoffatomen, oder eine Polyethylenglycolgruppe der Formel-A-[CH2-CH2-O]n-R20 ist, wobei A -O-, -C(=O)-, -OC(=O)-, oder -OC(=O)- (CH2)m-O- ist; m eine ganze Zahl von1 bis 20 ist; n eine ganze Zahl von 2 bis 100 ist, und R20 ein Wasserstoffatom, eine Methylgruppe, oder eineEthylgruppe ist;

R4 und R5 jeweils unabhängig voneinander ein Wasserstoffatom, eine Alkylgruppe, in der gegebenenfalls einoder mehrere Wasserstoffatom(e) jeweils unabhängig von den anderen durch ein Fluor-, Chlor-, Brom- oderJodatom; oder durch OH, =O, SH, =S, NH2, =NH, CN, NO2 oder eine Alkoxygruppe ersetzt worden sein kann/ können; eine Alkenylgruppe, in der gegebenenfalls ein oder mehrere Wasserstoffatom(e) jeweils unabhängigvon den anderen durch ein Fluor-, Chlor-, Brom- oder Jodatom; oder durch OH, =O, SH, =S, NH2, =NH, CN,NO2 oder eine Alkoxygruppe ersetzt worden sein kann / können; oder eine Alkinylgruppe, in der gegebenenfallsein oder mehrere Wasserstoffatom(e) jeweils unabhängig von den anderen durch ein Fluor-, Chlor-, Brom- oderJodatom; oder durch OH, =O, SH, =S, NH2, =NH, CN, NO2 oder eine Alkoxygruppe ersetzt worden sein kann/ können, darstellt; und wobei ein Kohlenstoffatom in der Alkyl-, Alkenyl- oder Alkinylgruppe durch ein Sauer-stoffatom, ein Schwefelatom, C=O, NR10, CONR11 oder NR12CO an jeder chemisch erlaubten Position ersetztsein kann;

R13, R14, und R15 jeweils unabhängig voneinander ein Wasserstoffatom oder eine Alkylgruppe mit 1 bis 6Kohlenstoffatomen darstellt;

R6 ein Wasserstoffatom, ein Halogenatom, eine Hydroxylgruppe, eine Cyanogruppe, eine Nitrogruppe, eineAminogruppe, eine Mercaptogruppe, eine Alkylgruppe mit 1 bis 6 Kohlenstoffatomen, in der gegebenenfallsein oder mehrere Wasserstoffatom(e) jeweils unabhängig von den anderen durch ein Fluor-, Chlor-, Brom- oder

EP2 703 410B1

Jodatom; oder durch OH, =O, SH, =S, NH2, =NH, CN, NO2 oder eine Alkoxygruppe ersetzt worden sein kann/ können; eine Alkoxygruppe mit 1 bis 6 Kohlenstoffatomen, eine SOCH3 -Gruppe, eine SO2CH3 -Gruppe, odereine Acylgruppe mit 2 bis 6 Kohlenstoffatomen, oder eine Polyethylenglycolgruppe der Formel -A-[CH2-CH2-O]n-R20 ist, wobei A -O-, -C(=O) -, -OC(=O)-, oder -OC(=O)- (CH2)m-O- ist; m eine ganze Zahl von1 bis 20 ist; n eine ganze Zahl von 2 bis 100 ist, und R20 ein Wasserstoffatom, eine Methylgruppe, oder eineEthylgruppe ist; und

R7 ein Wasserstoffatom, ein Halogenatom, eine Hydroxylgruppe, eine Cyanogruppe, eine Nitrogruppe, eineAminogruppe, eine Mercaptogruppe, eine Alkylgruppe mit 1 bis 6 Kohlenstoffatomen, in der gegebenenfallsein oder mehrere Wasserstoffatom(e) jeweils unabhängig von den anderen durch ein Fluor-, Chlor-, Brom- oderJodatom; oder durch OH, =O, SH, =S, NH2, =NH, CN, NO2 oder eine Alkoxygruppe ersetzt worden sein kann/ können; eine Alkoxygruppe mit 1 bis 6 Kohlenstoffatomen, eine SOCH3 -Gruppe, eine SO2CH3 -Gruppe, odereine Acylgruppe mit 2 bis 6 Kohlenstoffatomen, oder eine Polyethylenglycolgruppe der Formel-A-[CH2-CH2-O]n-R20 ist, wobei A -O-, -C(=O)-, -OC(=O)-, oder -OC(=O)- (CH2)m-O- ist; m eine ganze Zahl von1 bis 20 ist; n eine ganze Zahl von 2 bis 100 ist, und R20 ein Wasserstoffatom, eine Methylgruppe, oder eineEthylgruppe ist.

Die Verbindung nach Anspruch 1 oder ein pharmakologisch akzeptables Salz derselben, wobei die Verbindungdurch die allgemeine Formel (I’) dargestellt ist:

wobei R1, R2, R3, R4, R5, R6, R7 wie in Anspruch 1 definiert sind.3.

Die Verbindung nach Anspruch 1 oder Anspruch 2, oder ein pharmakologisch akzeptables Salz derselben, wobeiR1 eine Gruppe ist, die durch die allgemeine Formel (II) dargestellt ist:

wobei

R8 ein Wasserstoffatom, ein Halogenatom, eine Hydroxylgruppe, eine Cyanogruppe, eine Nitrogruppe, eineAminogruppe, eine Mercaptogruppe, eine Alkylgruppe mit 1 bis 6 Kohlenstoffatomen, in der gegebenenfallsein oder mehrere Wasserstoffatom(e) jeweils unabhängig von den anderen durch ein Fluor-, Chlor-, Brom- oderJodatom; oder durch OH, =O, SH, =S, NH2, =NH, CN, NO2 oder eine Alkoxygruppe ersetzt worden sein kann/ können; eine Alkoxygruppe mit 1 bis 6 Kohlenstoffatomen, eine SOCH3 -Gruppe, eine SO2CH3 -Gruppe, oder

EP2 703 410B1

eine Acylgruppe mit 2 bis 6 Kohlenstoffatomen, oder eine Polyethylenglycolgruppe der Formel-A-[CH2-CH2-O]n-R20 sein kann, wobei A -O-, -C(=O)-, -OC(=O)-, oder -OC(=O)- (CH2)m-O-ist; m eine ganzeZahl von 1 bis 20 ist; n eine ganze Zahl von 2 bis 100 ist, und R20 ein Wasserstoffatom, eine Methylgruppe,oder eine Ethylgruppe ist; undy eine ganze Zahl von 1 bis 20 ist.

Die Verbindung nach einem der Ansprüche 1 bis 3, oder ein pharmakologisch akzeptables Salz derselben, wobeiR1 eine Gruppe ist, die durch die allgemeine Formel (II’) dargestellt ist:

wobei R8 und y wie in Anspruch 3 definiert sind.5.

Die Verbindung nach einem der Ansprüche 1 bis 4, oder ein pharmakologisch akzeptables Salz derselben, wobeiR2 ein Wasserstoffatom oder eine Alkylgruppe mit 1 bis 6 Kohlenstoffatomen, in der gegebenenfalls ein oder mehrereWasserstoffatom(e) jeweils unabhängig von den anderen durch ein Fluor-, Chlor-, Brom- oder Jodatom; oder durchOH, =O, SH, =S, NH2, =NH, CN, NO2 oder eine Alkoxygruppe ersetzt worden sein kann / können, ist.

Die Verbindung nach einem der Ansprüche 1 bis 5, oder ein pharmakologisch akzeptables Salz derselben, wobeiR3 ein Halogenatom, eine Hydroxylgruppe, eine Aminogruppe, oder eine Alkylgruppe mit 1 bis 6 Kohlenstoffatomen,in der gegebenenfalls ein oder mehrere Wasserstoffatom(e) jeweils unabhängig von den anderen durch ein Fluor-,Chlor-, Brom-oder Jodatom; oder durch OH, =O, SH, =S, NH2, =NH, CN, NO2 oder eine Alkoxygruppe ersetztworden sein kann / können, ist.

Die Verbindung nach einem der Ansprüche 1 bis 6, oder ein pharmakologisch akzeptables Salz derselben, wobeiR4 und R5 jeweils unabhängig voneinander ein Wasserstoffatom, oder eine Alkylgruppe, in der gegebenenfalls einoder mehrere Wasserstoffatom(e) jeweils unabhängig von den anderen durch ein Fluor-, Chlor-, Brom- oder Jodatom;oder durch OH, =O, SH, =S, NH2, =NH, CN, NO2 oder eine Alkoxygruppe ersetzt worden sein kann / können,darstellt; wobei ein Kohlenstoffatom in der Alkylgruppe durch ein Sauerstoffatom, ein Schwefelatom, C=O, NR13,CONR14 oder NR15CO an jeder chemisch erlaubten Position ersetzt sein kann; und R13, R14, und R15 wie in Anspruch1 definiert sind.

Die Verbindung nach einem der Ansprüche 1 bis 7, oder ein pharmakologisch akzeptables Salz derselben, wobeiR6 ein Halogenatom, eine Hydroxylgruppe, eine Aminogruppe, eine Alkylgruppe mit 1 bis 6 Kohlenstoffatomen, inder gegebenenfalls ein oder mehrere Wasserstoffatom(e) jeweils unabhängig von den anderen durch ein Fluor-,Chlor-, Brom-oder Jodatom; oder durch OH, =O, SH, =S, NH2, =NH, CN, NO2 oder eine Alkoxygruppe ersetztworden sein kann / können; eine Alkoxygruppe mit 1 bis 6 Kohlenstoffatomen, eine SOCH3 -Gruppe, eine SO2CH3-Gruppe, oder eine Acylgruppe mit 2 bis 6 Kohlenstoffatomen ist.

Die Verbindung nach einem der Ansprüche 1 bis 8, oder ein pharmakologisch akzeptables Salz derselben, wobeiR7 ein Halogenatom, eine Hydroxylgruppe, eine Aminogruppe, eine gegebenenfalls substituierte Alkylgruppe mit 1bis 6 Kohlenstoffatomen, in der gegebenenfalls ein oder mehrere Wasserstoffatom(e) jeweils unabhängig von denanderen durch ein Fluor-, Chlor-, Brom- oder Jodatom; oder durch OH, =O, SH, =S, NH2, =NH, CN, NO2 oder eineAlkoxygruppe ersetzt worden sein kann / können; eine Alkoxygruppe mit 1 bis 6 Kohlenstoffatomen, eine SOCH3-Gruppe, eine SOCH3 -Gruppe, eine Acylgruppe mit 2 bis 6 Kohlenstoffatomen, oder eine Polyethylenglycolgruppeder Formel -A-[CH2-CH2-O]n-R20 ist, wobei A -C(=O)-, oder -OC(=O)- (CH2)m-O- ist; m eine ganze Zahl von 1 bis6 ist; n eine ganze Zahl von 2 bis 10 ist, und R20 ein Wasserstoffatom oder eine Methylgruppe ist.

10.Die Verbindung nach einem der Ansprüche 1 bis 9, oder ein pharmakologisch akzeptables Salz derselben, wobei

die Verbindung durch die allgemeine Formel (III) dargestellt ist:

EP2 703 410B1

11.Pharmazeutische Zusammensetzung, die mindestens eine Verbindung gemäß einem der Ansprüche 1 bis 10, oder

ein pharmakologisch akzeptables Salz derselben, und gegebenenfalls mindestens eine Trägersubstanz und / odermindestens ein Adjuvans umfasst.

12.Verfahren zur Herstellung einer Verbindung gemäß der Formel (I), wobei das Verfahren die Schritte umfasst:

(a) Janthinobacterium agaricidamnosum (DSM 9628) fermentieren; und

(b) Abtrennen und Gewinnen der Verbindung aus dem Kulturmedium; wobei die Verbindung eine Verbindunggemäß Anspruch 10 ist.

13.Die Verbindung, oder ein pharmakologisch akzeptables Salz derselben, oder die pharmazeutische Zusammenset-zung nach einem der Ansprüche 1 bis 11 zur Verwendung als Medikament.14.Die Verbindung, oder ein pharmakologisch akzeptables Salz derselben, oder die pharmazeutische Zusammenset-zung nach einem der Ansprüche 1 bis 11 zur Verwendung bei der Behandlung oder Prophylaxe einer Pilzinfektionoder von Krebs.

Revendications

1.Composé de la formule générale (I):

EP2 703 410B1

ou un sel pharmacologiquement acceptable de celui-ci, où

R1 et R2 représentent chacun indépendamment un atome d’hydrogène, un groupe alkyle dans lequel un ouplusieurs atome(s) d’hydrogène a/ont été éventuellement remplacé(s) chacun indépendamment des autres parun atome de fluor, de chlore, de brome ou d’iode; ou par OH, =O, SH, =S, NH2, =NH, CN, NO2, ou un groupealcoxy, un groupe alcényle dans lequel un ou plusieurs atome(s) d’hydrogène a/ont été éventuellement rem-placé(s) chacun indépendamment des autres par un atome de fluor, de chlore, de brome ou d’iode; ou parOH, =O, SH, =S, NH2, =NH, CN, NO2, ou un groupe alcoxy, ou un groupe alcynyle dans lequel un ou plusieursatome(s) d’hydrogène a/ont été éventuellement remplacé(s) chacun indépendamment des autres par un atomede fluor, de chlore, de brome ou d’iode; ou par OH, =O, SH, =S, NH2, =NH, CN, NO2, ou un groupe alcoxy, etoù un atome de carbone dans ledit groupe alkyle, alcényle ou alcynyle peut être remplacé par un atomed’oxygène, un atome de soufre, C=O, NR10, CONR11, ou NR12CO en toute position chimiquement permise;R10, R11, et R12 représentent chacun indépendamment un atome d’hydrogène ou un groupe alkyle ayant 1 à6 atomes de carbone;

R3 est un atome d’hydrogène, un atome d’halogène, un groupe hydroxyle, un groupe cyano, un groupe nitro,un groupe amino, un groupe mercapto, un groupe alkyle ayant 1 à 6 atomes de carbone dans lequel un ouplusieurs atome(s) d’hydrogène a/ont été éventuellement remplacé(s) chacun indépendamment des autres parun atome de fluor, de chlore, de brome ou d’iode; ou par OH, =O, SH, =S, NH2, =NH, CN, NO2, ou un groupealcoxy, un groupe alcoxy ayant 1 à 6 atomes de carbone, un groupe SOCH3, un groupe SO2CH3, ou un groupeacyle ayant 2 à 6 atomes de carbone, ou un groupe polyéthylène glycol de formule -A-[CH2-CH2-O]n-R20, danslequel A est -O-, -C(=O)-, -OC(=O)-, ou - OC(=O)-(CH2)m-O-; m est un nombre entier compris entre 1 et 20;n est un nombre entier compris entre 2 et 100, et R20 est un atome d’hydrogène, un groupe méthyle ou ungroupe éthyle;

R4 et R5 représentent chacun indépendamment un atome d’hydrogène, un groupe alkyle dans lequel un ouplusieurs atome(s) d’hydrogène a/ont été éventuellement remplacé(s) chacun indépendamment des autres parun atome de fluor, de chlore, de brome ou d’iode; ou par OH, =O, SH, =S, NH2, =NH, CN, NO2, ou un groupealcoxy, un groupe alcényle dans lequel un ou plusieurs atome(s) d’hydrogène a/ont été éventuellement rem-placé(s) chacun indépendamment des autres par un atome de fluor, de chlore, de brome ou d’iode; ou parOH, =O, SH, =S, NH2, =NH, CN, NO2, ou un groupe alcoxy, ou un groupe alcynyle dans lequel un ou plusieursatome(s) d’hydrogène a/ont été éventuellement remplacé(s) chacun indépendamment des autres par un atomede fluor, de chlore, de brome ou d’iode; ou par OH, =O, SH, =S, NH2, =NH, CN, NO2, ou un groupe alcoxy,où un atome de carbone dans ledit groupe alkyle, alcényle ou alcynyle peut être remplacé par un atomed’oxygène, un atome de soufre, C=O, NR13, CONR14, ou NR15CO en toute position chimiquement permise;R13, R14, et R15 représentent chacun indépendamment un atome d’hydrogène ou un groupe alkyle ayant 1 à6 atomes de carbone;

R6 est un atome d’hydrogène, un atome d’halogène, un groupe hydroxyle, un groupe cyano, un groupe nitro,un groupe amino, un groupe mercapto, un groupe alkyle ayant 1 à 6 atomes de carbone dans lequel un ouplusieurs atome(s) d’hydrogène a/ont été éventuellement remplacé(s) chacun indépendamment des autres parun atome de fluor, de chlore, de brome ou d’iode; ou par OH, =O, SH, =S, NH2, =NH, CN, NO2, ou un groupe

EP2 703 410B1

alcoxy, un groupe alcoxy ayant 1 à 6 atomes de carbone, un groupe SOCH3, un groupe SO2CH3, un groupeacyle ayant 2 à 6 atomes de carbone, ou un groupe polyéthylène glycol de formule -A-[CH2-CH2-O]n-R20, danslequel A est -O-, -C(=O)-, -OC(=O)-, ou - OC(=O)-(CH2)m-O-; m est un nombre entier compris entre 1 et 20;n est un nombre entier compris entre 2 et 100, et R20 est un atome d’hydrogène, un groupe méthyle ou ungroupe éthyle; et

R7 est un atome d’hydrogène, un atome d’halogène, un groupe hydroxyle, un groupe cyano, un groupe nitro,un groupe amino, un groupe mercapto, un groupe alkyle ayant 1 à 6 atomes de carbone dans lequel un ouplusieurs atome(s) d’hydrogène a/ont été éventuellement remplacé(s) chacun indépendamment des autres parun atome de fluor, de chlore, de brome ou d’iode; ou par OH, =O, SH, =S, NH2, =NH, CN, NO2, ou un groupealcoxy, un groupe alcoxy ayant 1 à 6 atomes de carbone, un groupe SOCH3, un groupe SO2CH3, ou un groupeacyle ayant 2 à 6 atomes de carbone, ou un groupe polyéthylène glycol de formule -A-[CH2-CH2-O]n-R20, oùA est --O-, -C(=O)-, -OC(=O) -, ou -OC(=O) - (CH2)m-O-; m est un nombre entier compris entre 1 et 20; n estun nombre entier compris entre 2 et 100, et R20 est un atome d’hydrogène, un groupe méthyle ou un groupeéthyle.

Composé selon la revendication 1 ou un sel pharmacologiquement acceptable de celui-ci, dans lequel le composéest représenté par la formule générale (I’):

où R1, R2, R3, R4, R5, R6, R7 sont tels que définis dans la revendication 1.3.

Composé selon la revendication 1 ou 2, ou un sel pharmacologiquement acceptable de celui-ci, dans lequel R1 estun groupe représenté par la formule générale (II):

où

R8 peut être un atome d’hydrogène, un atome d’halogène, un groupe hydroxyle, un groupe cyano, un groupenitro, un groupe amino, un groupe mercapto, un groupe alkyle ayant 1 à 6 atomes de carbone dans lequel unou plusieurs atome(s) d’hydrogène a/ont été éventuellement remplacé(s) chacun indépendamment des autrespar un atome de fluor, de chlore, de brome ou d’iode; ou par OH, =O, SH, =S, NH2, =NH, CN, NO2, ou un

EP2 703 410B1

groupe alcoxy, un groupe alcoxy ayant 1 à 6 atomes de carbone, un groupe SOCH3, un groupe SO2CH3, ouun groupe acyle ayant 2 à 6 atomes de carbone, ou un groupe polyéthylène glycol de formule-A-[CH2-CH2-O]n-R20, où A est -O-, -C(=O)-, -OC (=O) -, ou -OC (=O) - (CH2)m-O-; m est un nombre entiercompris entre 1 et 20; n est un nombre entier compris entre 2 et 100, et R20 est un atome d’hydrogène, ungroupe méthyle ou un groupe éthyle; et

y est un nombre entier compris entre 1 et 20.

Composé selon l’une quelconque des revendications 1 à 3, ou un sel pharmacologiquement acceptable de celui-ci, dans lequel R1 est un groupe représenté par la formule générale (II’):

où R8 et Y sont tels que définis dans la revendication 3.

Composé selon l’une quelconque des revendications 1 à 4, ou un sel pharmacologiquement acceptable de celui-ci, dans lequel R2 est un atome d’hydrogène ou un groupe alkyle ayant 1 à 6 atomes de carbone dans lequel unou plusieurs atome(s) d’hydrogène a/ont été éventuellement remplacé(s) chacun indépendamment des autres parun atome de fluor, de chlore, de brome ou d’iode; ou par OH, =O, SH, =S, NH2, =NH, CN, NO2, ou un groupe alcoxy.Composé selon l’une quelconque des revendications 1 à 5, ou un sel pharmacologiquement acceptable de celui-ci, dans lequel R3 est un atome d’halogène, un groupe hydroxyle, un groupe amino, ou un groupe alkyle ayant 1 à6 atomes de carbone dans lequel un ou plusieurs atome(s) d’hydrogène a/ont été éventuellement remplacé(s)chacun indépendamment des autres par un atome de fluor, de chlore, de brome ou d’iode; ou par OH, =O, SH,=S, NH2, =NH, CN, NO2, ou un groupe alcoxy.

Composé selon l’une quelconque des revendications 1 à 6, ou un sel pharmacologiquement acceptable de celui-ci, dans lequel R4 et R5 représentent chacun indépendamment un atome d’hydrogène ou un groupe alkyle danslequel un ou plusieurs atome(s) d’hydrogène a/ont été éventuellement remplacé(s) chacun indépendamment desautres par un atome de fluor, de chlore, de brome ou d’iode; ou par OH, =O, SH, =S, NH2, =NH, CN, NO2, ou ungroupe alcoxy, où un atome de carbone dans ledit groupe alkyle peut être remplacé par un atome d’oxygène, unatome de soufre, C=O, NR13, CONR14, ou NR15CO en toute position chimiquement permise; et R13, R14, et R15sont tels que définis dans la revendication 1.

Composé selon l’une quelconque des revendications 1 à 7, ou un sel pharmacologiquement acceptable de celui-ci, dans lequel R6 est un atome d’halogène, un groupe hydroxyle, un groupe amino, un groupe alkyle ayant 1 à 6atomes de carbone dans lequel un ou plusieurs atome(s) d’hydrogène a/ont été éventuellement remplacé(s) chacunindépendamment des autres par un atome de fluor, de chlore, de brome ou d’iode; ou par OH, =O, SH, =S, NH2,=NH, CN, NO2, ou un groupe alcoxy, un groupe alcoxy ayant 1 à 6 atomes de carbone, un groupe SOCH3, ungroupe SO2CH3, ou un groupe acyle ayant 2 à 6 atomes de carbone.

Composé selon l’une quelconque des revendications 1 à 8, ou un sel pharmacologiquement acceptable de celui-ci, dans lequel R7 est un atome d’halogène, un groupe hydroxyle, un groupe amino, un groupe alkyle ayant 1 à 6atomes de carbone dans lequel un ou plusieurs atome(s) d’hydrogène a/ont été éventuellement remplacé(s) chacunindépendamment des autres par un atome de fluor, de chlore, de brome ou d’iode; ou par OH, =O, SH, =S, NH2,=NH, CN, NO2, ou un groupe alcoxy, un groupe alcoxy ayant 1 à 6 atomes de carbone, un groupe SOCH3, ungroupe SO2CH3, ou un groupe acyle ayant 2 à 6 atomes de carbone, ou un groupe polyéthylène glycol de formule-A- [CH2-CH2-O]n-R20, où A est -C(=O) -, ou -OC(=O)-(CH2)m-O-; m est un nombre entier compris entre 1 et 6; nest un nombre entier compris entre 2 et 10, et R20 est un atome d’hydrogène ou un groupe méthyle.

10.Composé selon l’une quelconque des revendications 1 à 9, ou un sel pharmacologiquement acceptable de celui-ci, dans lequel le composé est représenté par la formule (III):

EP2 703 410B1

11.Composition pharmaceutique qui comprend au moins un composé selon l’une quelconque des revendications 1 à

10, ou un sel pharmacologiquement acceptable de celui-ci et, éventuellement, au moins une substance porteuseet/ou au moins un adjuvant.12.Procédé pour la préparation d’un composé de formule (I), le procédé comprenant les étapes suivantes:

(a) la fermentation de Janthinobacterium agaricidamnosum (DSM 9628); et

(b) la séparation et la rétention du composé provenant du bouillon de culture; où le composé est un composéselon la revendication 10.

13.Composé, ou un sel pharmacologiquement acceptable de celui-ci, ou la composition pharmaceutique selon l’une

quelconque des revendications 1 à 11 pour une utilisation comme médicament.14.Composé, ou un sel pharmacologiquement acceptable de celui-ci, ou la composition pharmaceutique selon l’une

quelconque des revendications 1 à 11 pour une utilisation dans le traitement ou la prévention d’une infection fongiqueou d’un cancer.

EP2 703 410B1

REFERENCES CITED IN THE DESCRIPTION

This list of references cited by the applicant is for the reader’s convenience only. It does not form part of the Europeanpatent document. Even though great care has been taken in compiling the references, errors or omissions cannot beexcluded and the EPO disclaims all liability in this regard.

Non-patent literature cited in the description•A. L. HARVEY. Drug Discov. Today, 2008, vol. 13,•894 [0002]

•D. J. NEWMAN; G. M. CRAGG. J. Nat. Prod., 2012,•vol. 75, 311 [0002]

••R. FINKING; M. A. MARAHIEL. Annu. Rev. Micro-biol., 2004, vol. 58, 453 [0002]

••D. SCHWARZER; R. FINKING; M. A. MARAHIEL.Nat. Prod. Rep., 2003, vol. 20, 275 [0002]

••C. T. WALSH et al. Curr. Opin. Chem. Biol., 2001,vol. 5, 525 [0002]

••R. DI SANTO. Nat. Prod. Rep., 2010, vol. 27, 1084[0003]

••M. F. VICENTE et al. Clin. Microbiol. Infect., 2003,vol. 9, 15 [0003]

••N. H. GEORGOPAPADAKOU; T. J. WALSH. Sci-ence, 1994, vol. 264, 371 [0003]

••S. P. LINCOLN; T. R. FERMOR; B. J. TINDALL.Int. J. Syst. Bacteriol., 1999, vol. 49, 1577 [0005]••J. H. JOHNSON; A. A. TYMIAK; BOLGAR, M. S.J. Antibiot., 1990, vol. 43, 920 [0005]

••J. O’SULLIVAN et al. J. Antibiot., 1990, vol. 43, 913[0005]

••

A. SHIRATA et al. J. Sericult.Sci. Jpn., 1997, vol. 66,377 [0005]

•

Martindale--The Extra Pharmacopoeia. Pharmaceu-tical Press, 1993 [0050]

Remington’s Pharmaceutical Sciences [0050]

VON NUSSBAUM et al. Angew. Chem. Int. Ed.,2006, vol. 45, 5072-5129 [0056]

S. WEIST; R. D. SÜSSMUTH. Appl. Microbiol. Bio-technol., 2005, vol. 68, 141-150 [0056]

JENSKE, R.; W. VETTER. J. Chromatogr. A., 2007,vol. 1146 (2), 225-31 [0069]

RAUSCH, C. et al. BMC Evol. Biol., 2007, vol. 7, 78[0071]

ABDOU, R. et al. Phytochemistry, 2009, vol. 71 (1),110-6 [0072]

RUTHERFORD, K. et al. Bioinformatics, 2000, vol.16 (10), 944-5 [0073]

ALTSCHUL S.F. et al. J. Mol. Biol., 1990, vol. 215(3), 403-10 [0073]

SAYERS, E.W. et al. Nucleic. Acids Res., 2011, vol.39, D38-51 [0073]

BACHMANN, B.O.; J. RAVEL. Methods Enzymol,2009, vol. 458, 181-217 [0073]

ROTTIG, M. et al. Nucleic. Acids Res., 2011, vol. 39,W362-7 [0073]

STARCEVIC, A. et al. Nucleic. Acids Res., 2008, vol.36 (21), 6882-92 [0074]

因篇幅问题不能全部显示，请点此查看更多更全内容

查看全文

全部栏目

Jagaricin, derivatives, and uses thereof