Novel darobactin derivative

Abstract

The present invention relates to darobactin derivatives of formula (I), their methods of use and production. [Formula 1] JPEG2024507521000087.jpg84158

Description

Technical Field

[0001] The present invention relates to novel derivatives of darobactin, methods of using the same, and methods of producing the same.

Background Art

[0002] An increasing number of antibiotic-resistant bacterial pathogens are causing an increase in mortality in humans. Many deaths are caused by particularly troublesome bacteria belonging to the ESKAPE panel (vancomycin-resistant Enterococci, Staphylococcus aureus, Klebsiella pneumonia, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species). The World Health Organization has recommended focusing on the discovery and development of new antibiotics with anti-gram-negative activity.

Summary of the Invention

Problems to be Solved by the Invention

[0003] Darobactin A is a ribosomally produced peptide antibiotic that selectively kills Gram-negative pathogenic bacteria, including bacteria from the ESKAPE panel (Imai, Y., Meyer, K. J., Iinishi, A. et al. A new antibiotic selectively kills Gram-negative pathogens. Nature 576, 459-464 (2019); International Publication No. WO 2020 / 018173). However, the production yield of darobactin A by known production strains has been insufficient, thereby limiting the possibility of performing structural modifications or isolating sufficient amounts of the compound required for potential semi-synthetic approaches.

Means for Solving the Problems

[0004] In one aspect, the present invention provides a compound of formula (I):

[0005] [Chemical formula] [wherein, R 1 , R 2 , R 4 and R 5 are each H, CH3, CH2OH, or the following groups:

[0006] [Chemical formula] -CH2-SR 1A (wherein, R 1A is independently selected from alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, alkylcycloalkyl, heteroalkylcycloalkyl, aryl, heteroaryl, aralkyl or heteroaralkyl groups, all of which groups may be optionally substituted), or -CH2-Ind (wherein Ind is an optionally substituted indole group) is independently selected from one of the following, R 3 is H, OH, SH, COOH, CONH2, or the following groups:

[0007] [Chemical formula] -SR 3A (wherein, R 3A is selected from alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, alkylcycloalkyl, heteroalkylcycloalkyl, aryl, heteroaryl, aralkyl or heteroaralkyl groups, all of which groups may be optionally substituted), or -Ind (wherein Ind is an optionally substituted indole group) is selected from one of the following, R 6 is a hydrogen atom or a methyl group (particularly a hydrogen atom), R7 is a hydrogen atom, a halogen atom, a hydroxy group or a methoxy group (especially a hydrogen atom), R 8 is independently selected from a halogen atom, a hydroxy group or a methoxy group as the case may be, m is an integer from 0 to 3 (especially 0 or 1, preferably 0), R 6A is a hydrogen atom or a methyl group (especially a hydrogen atom), R 7A is a hydrogen atom, a halogen atom, a hydroxy group or a methoxy group (especially a hydrogen atom), R 8A is independently selected from a halogen atom, a hydroxy group or a methoxy group as the case may be, p is an integer from 0 to 3 (especially 0 or 1, preferably 0), However, when R 4 is a group of the formula -CH2-OH or -CH2-CH2-CH2-NH-C(=NH)-NH2, R 5 is not a group of the formula -CH2-phenyl] To provide a compound of

[0008] Preferably, R 5 is H, CH3, CH2OH, or one of the following groups:

[0009]

Chemical formula

[0010] More preferably, R 5 is a group of the formula -CH2-Ind (wherein Ind is an optionally substituted indole group).

[0011] Even more preferably, R 5 is a group of the formula -CH3.

[0012] Even more preferably, R 5 is a group of the following formula.

[0013]

Chem.

[0014] Preferably thereon, the group of formula -CH2-Ind is of the following formula:

[0015]

Chem.

[0016] Particularly preferably, R 5 is of the following formula:

[0017]

Chem.

[0018] In some preferred embodiments, R 5 is of the following formula:

[0019]

Chem.

[0020] Even more preferably, R 5 is a group of the following formula

[0021]

Chemical formula

[0022] In some embodiments, the group of formula -CH2-SR 1A is preferably

[0023]

Chemical formula

[0024] Even more preferably, R 1 is selected from the following groups: -CH2-CONH2, -CH2-OH, -CH2-CH2-CONH2 and -CH2-CH(CH3)-OH; more preferably, R 1 is a group of -CH2-CONH2.

[0025] Preferably, R 2 is CH3, CH2OH, the following group:

[0026]

Chemical formula

[0027] Preferably thereon, R 2 is the following group: -CH3, -CH2-OH, -CH2-SH, -CH2-CONH2, -CH2-CH2-CONH2, -CH2-CH2-CH2-NH-C(=NH)-NH2 and -CH2-CH(CH3)-OH, for example, -CH3, -CH2-OH, -CH2-SH and -CH2-CH(CH3)-OH, or for example, -CH2-OH and -CH2-CH(CH3)-OH, and is selected from these.

[0028] Preferably thereon, R 2 is selected from the following groups: -CH2-OH, -CH2-CH(CH3)-OH and -CH3, or the group:[[]]

[0029]

Chemical formula

[0030] More preferably, R 3 is a hydrogen atom or is selected from the following groups: -CH2-CH2-CH2-NH2 and -CH2-CH2-NH-C(=NH)-NH2. In some preferred embodiments, R 3 is the group: -CH2-CH2-CH2-NH2 or -CH2-CH2-NH-C(=NH)-NH2, preferably the group: -CH2-CH2-CH2-NH2.

[0031] Preferably, R 4 is a hydrogen atom, CH3, CH2OH, the following group:[[]]

[0032]

Chemical formula

[0033] [Chemical formula] )] may represent one of them.

[0034] More preferably, R 4 is CH3, CH2OH, or the following group:

[0035] [Chemical formula] one of them, for example, CH3, CH2OH, -CH2-CH2-CH2-NH2, -CH2-CH2-NH-C(=NH)-NH2, -CH2-CH(CH3)-OH or

[0036] [Chemical formula] or is selected from, for example, CH3, CH2OH, -CH2-CH2-NH-C(=NH)-NH2 or -CH2-CH(CH3)-OH.

[0037] Furthermore preferably, R 4 is selected from the following groups: -CH2-OH, -CH3 and -CH2-CH2-CH2-NH-C(=NH)-NH2.

[0038] More preferably, R 4 is of the formula -CH2-SR 4A (in the formula, R 4Ais a group selected from a hydrogen atom, alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, alkylcycloalkyl, heteroalkylcycloalkyl, aryl, heteroaryl, aralkyl or heteroaralkyl group, all of these groups being optionally substitutable). The group of formula -CH2-SR 4A Preferred examples of the group of are the following groups.

[0039] [Chemical formula]

[0040] In some embodiments, R 6 is a methyl group or a hydrogen atom, preferably a hydrogen atom. In some embodiments, R 6A is a methyl group or a hydrogen atom, preferably a hydrogen atom. In some embodiments, R 6 and R 6A are each a hydrogen atom.

[0041] In some embodiments, R 7 is hydrogen or a halogen atom, preferably a hydrogen atom. In some embodiments, R 7A is hydrogen or a halogen atom, preferably a hydrogen atom. In some embodiments, R 7 and R 7A are each a hydrogen atom.

[0042] In some embodiments, R 6 , R 6A , R 7 and R 7A are each a hydrogen atom.

[0043] In some embodiments, R 8 is a halogen atom, preferably a fluorine or chlorine atom, more preferably a fluorine atom. In some embodiments, R 8A is a halogen atom, preferably a fluorine or chlorine atom, more preferably a fluorine source. In some embodiments, R 8 and R8A There is one of them.

[0044] In some embodiments, m is 0, 1, or 2, preferably 0 or 1, and more preferably 0.

[0045] In some embodiments, p is 0, 1 or 2, preferably 0 or 1, and more preferably 0.

[0046] In some embodiments, n is 0, 1, 2 or 3, preferably 0, 1 or 2, more preferably 0 or 1, and especially 0.

[0047] Furthermore preferably, the compound of formula (Ia):

[0048] [ka] (In the formula, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 6A , R 7A , R 8A , m and p are as defined above) or a salt thereof.

[0049] More preferably, the formula (Ia'):

[0050] [ka] (In the formula, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 6A , R 7A , R 8A , m and p are as defined above) It is a compound of formula (I) or a salt thereof.

[0051] Preferably thereon is a compound of formula (Ib):

[0052]

Chem.

[0053] More preferably, it is a compound of formula (Ib'):

[0054]

Chem.

[0055] Preferably thereon is a compound of formula (Ib''):

[0056]

Chem.

[0057] In some embodiments, the compound of formula (I) is of formula (1):

[0058]

Chem.

[0059] In some embodiments, the compound of formula (1) is of formula (1a) or formula (1a'):

[0060] [Chemical formula] (wherein R 2 and R 4 is as defined above) is a compound of formula (1) or a salt thereof (in particular, a pharmaceutically acceptable salt). [Mode for Carrying Out the Invention]

[0061] Compounds comprising suitable combinations of preferred embodiments of the compounds or salts thereof according to the present invention, for example, the residues R disclosed herein 1 , R 2 , R 3 , R 4 and R 5 in a preferred embodiment combination are particularly preferred. In other words, the present invention particularly encompasses all possible combinations of the residues shown above that result in stable compounds.

[0062] The most preferred compounds of the present invention are the compounds or salts thereof disclosed in the examples.

[0063] The term alkyl refers to a saturated, straight-chain or branched hydrocarbon group containing 1 to 20 carbon atoms, preferably 1 to 10 carbon atoms, particularly 1 to 6 (e.g., 1, 2, 3, or 4) carbon atoms, such as methyl (Me, CH3), ethyl (Et), n-propyl (nPr), iso-propyl (iPr), n-butyl (nBu), iso-butyl (iBu), sec-butyl (sBu), tert-butyl (tBu), n-pentyl, iso-pentyl, n-hexyl, 2,2-dimethylbutyl or n-octyl group.

[0064] Particularly preferred alkyl groups are C 1~6 alkyl groups, and on top of that, preferred alkyl groups are C 1~4 alkyl groups.

[0065] C 1~6 The expression "alkyl" refers to a saturated, straight-chain or branched hydrocarbon group containing 1 to 6 carbon atoms. C 1~4 The expression "alkyl" refers to a saturated, straight-chain or branched hydrocarbon group containing 1 to 4 carbon atoms. Examples are methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, sec-butyl or tert-butyl groups.

[0066] The expressions "alkenyl" and "alkynyl" refer to at least partially saturated, straight-chain or branched hydrocarbon groups containing 2 to 20 carbon atoms, preferably 2 to 10 carbon atoms, particularly 2 to 6 (e.g., 2, 3 or 4) carbon atoms, such as ethenyl (vinyl), propenyl (allyl), iso-propenyl, butenyl, ethynyl (ethynyl), propynyl (e.g., propargyl), butynyl, isoprenyl or hexa-2-enyl groups. Preferably, the alkenyl group has 1 or 2 (particularly preferably 1) double bond(s) (plural available), and the alkynyl group has 1 or 2 (particularly preferably 1) triple bond(s) (plural available).

[0067] Furthermore, the terms "alkyl", "alkenyl" and "alkynyl" refer to groups in which one or more hydrogen atoms are replaced by halogen atoms (preferably F or Cl), such as 2,2,2-trichloroethyl or trifluoromethyl groups, etc.

[0068] The expression "heteroalkyl" refers to an alkyl, alkenyl or alkynyl group in which one or more (preferably 1 to 8, particularly preferably 1, 2, 3 or 4) carbon atoms are replaced by an oxygen, nitrogen, phosphorus, boron, selenium, silicon or sulfur atom (preferably an oxygen, sulfur or nitrogen atom), or by an SO or SO2 group. Moreover, the expression "heteroalkyl" refers to a carboxylic acid or a group derived from a carboxylic acid, such as acyl, acylalkyl, alkoxycarbonyl, acyloxy, acyloxyalkyl, carboxyalkylamide or alkoxycarbonyloxy, etc. Moreover, the term "heteroalkyl" refers to a group in which one or more hydrogen atoms are replaced by a halogen atom (preferably F or Cl).

[0069] Preferably, the heteroalkyl group contains 1 to 12 carbon atoms and 1 to 8 heteroatoms selected from oxygen, nitrogen and sulfur (especially oxygen and nitrogen). Particularly preferably, the heteroalkyl group contains 1 to 6 (for example, 1, 2, 3 or 4) carbon atoms and 1, 2, 3 or 4 (especially 1, 2 or 3) heteroatoms selected from oxygen, nitrogen and sulfur (especially oxygen and nitrogen). C1-C 10 The term "heteroalkyl" refers to a heteroalkyl group containing 1 to 10 carbon atoms and 1, 2, 3, 4, 5 or 6 heteroatoms selected from O, S and / or N (especially O and / or N). The term "C1-C6 heteroalkyl" refers to a heteroalkyl group containing 1 to 6 carbon atoms and 1, 2, 3 or 4 heteroatoms selected from O, S and / or N (especially O and / or N). The term "C1-C4 heteroalkyl" refers to a heteroalkyl group containing 1 to 4 carbon atoms and 1, 2 or 3 heteroatoms selected from O, S and / or N (especially O and / or N).

[0070] More preferably, the expression "heteroalkyl" refers to an alkyl group (linear or branched) as defined above, in which one or more (preferably 1 to 6, particularly preferably 1, 2, 3 or 4) carbon atoms are replaced by an oxygen, sulfur or nitrogen atom or a CO group or a SO group or a SO2 group, and this group preferably contains 1 to 6 (e.g., 1, 2, 3 or 4) carbon atoms and 1, 2, 3 or 4 (particularly 1, 2 or 3) heteroatoms selected from oxygen, nitrogen and sulfur (especially oxygen and nitrogen), and this group may preferably be replaced by one or more (preferably 1 to 6, particularly preferably 1, 2, 3 or 4) fluorine, chlorine, bromine or iodine atoms or OH, =O, SH, =S, NH2, =NH, N3, CN or NO2 groups.

[0071] Examples of heteroalkyl groups are of the formula: R a -O-Y a -, R a -S-Y a -, R a -SO-Y a -, R a -SO2-Y a -, R a -N(R b )-SO2-Y a -, R a -SO2-N(R b )-Y a -, R a -N(R b )-Y a -, R a -CO-Y a -, R a -O-CO-Y a -, R a -CO-O-Y a -, R a -CO-N(R b )-Y a -, R a -N(R b )-CO-Y a -, R a -O-CO-N(R b )-Y a -, R a -N(R b)-CO-O-Y a -、R a -N(R b )-CO-N(R c )-Y a -、R a -O-CO-O-Y a -、R a -N(R b )-C(=NR d )-N(R c )-Y a -、R a -CS-Y a -、R a -O-CS-Y a -、R a -CS-O-Y a -、R a -CS-N(R b )-Y a -、R a -N(R b )-CS-Y a -、R a -O-CS-N(R b )-Y a -、R a -N(R b )-CS-O-Y a -、R a -N(R b )-CS-N(R c )-Y a -、R a -O-CS-O-Y a -、R a -S-CO-Y a -、R a -CO-S-Y a -、R a -S-CO-N(R b )-Y a -、R a -N(R b )-CO-S-Y a -、R a -S-CO-O-Y a -、R a -O-CO-S-Y a -、R a -S-CO-S-Y a -、R a -S-CS-Y a -、R a -CS-S-Ya -、R a -S-CS-N(R b )-Y a -、R a -N(R b )-CS-S-Y a -、R a -S-CS-O-Y a -、R a -O-CS-S-Y a - is a group, wherein R a is a hydrogen atom, a C1-C6 alkyl, a C2-C6 alkenyl or a C2-C6 alkynyl group, R b is a hydrogen atom, a C1-C6 alkyl, a C2-C6 alkenyl or a C2-C6 alkynyl group, R c is a hydrogen atom, a C1-C6 alkyl, a C2-C6 alkenyl or a C2-C6 alkynyl group, R d is a hydrogen atom, a C1-C6 alkyl, a C2-C6 alkenyl or a C2-C6 alkynyl group, and Y a is a bond, a C1-C6 alkylene, a C2-C6 alkenylene or a C2-C6 alkynylene group, and each heteroalkyl group contains at least one carbon atom, and one or more hydrogen atoms may be replaced by a fluorine or chlorine atom.

[0072] Specific examples of heteroalkyl groups are methoxy, trifluoromethoxy, ethoxy, n - propyloxy, iso - propyloxy, n - butoxy, tert - butyloxy, methoxymethyl, ethoxymethyl, - CH2CH2OH, - CH2OH, - SO2Me, - NHAc, methoxyethyl, 1 - methoxyethyl, 1 - ethoxyethyl, 2 - methoxyethyl or 2 - ethoxyethyl, methylamino, ethylamino, propylamino, isopropylamino, dimethylamino, diethylamino, isopropylethylamino, methylaminomethyl, ethylaminomethyl, diisopropylaminoethyl, methylthio, ethylthio, isopropylthio, enol ether, dimethylaminomethyl, dimethylaminoethyl, acetyl, propionyl, butyryloxy, acetyloxy, methoxycarbonyl, ethoxycarbonyl, propionyloxy, acetylamino or propionylamino, carboxymethyl, carboxyethyl or carboxypropyl, N - ethyl - N - methyl - carbamoyl or N - methylcarbamoyl. Further examples of heteroalkyl groups are nitrile (- CN), isonitrile, cyanato, thiocyanato, isocyanato, isothiocyanato and alkyl nitrile groups.

[0073] The expression "cycloalkyl" refers to a saturated or partially unsaturated cyclic group (e.g., a cycloalkenyl group) that contains one or more (preferably one or two) rings and 3 to 14 ring carbon atoms, preferably 3 to 10 (especially 3, 4, 5, 6, or 7) ring carbon atoms. Moreover, the expression "cycloalkyl" refers to a group in which one or more hydrogen atoms are replaced by fluorine, chlorine, bromine or iodine atoms or OH, =O, SH, =S, NH2, =NH, N3 or NO2 groups, and thus, for example, cyclic ketones such as cyclohexanone, 2-cyclohexenone or cyclopentanone and the like can be mentioned. Further specific examples of cycloalkyl groups are cyclopropyl, cyclobutyl, cyclopentyl, spiro[4,5]decanyl, norbornyl, cyclohexyl, cyclopentenyl, cyclohexadienyl, decalinyl, bicyclo[4.3.0]nonyl, tetralin, cyclopentylcyclohexyl, fluorocyclohexyl or cyclohex-2-enyl groups. Preferably, the expression "cycloalkyl" refers to a saturated cyclic group that contains one or more (preferably one or two) rings and 3 to 14 ring carbon atoms, preferably 3 to 10 (especially 3, 4, 5, 6, or 7) ring carbon atoms.

[0074] The expression "heterocycloalkyl" refers to a cycloalkyl group as defined above, wherein one or more (preferably 1, 2 or 3) ring carbon atoms are replaced by an oxygen, nitrogen, silicon, selenium, phosphorus or sulfur atom (preferably an oxygen, sulfur or nitrogen atom) or an SO group or an SO2 group. The heterocycloalkyl group preferably has 1 or 2 rings (multiple possible) and 3 to 10 (especially 3, 4, 5, 6 or 7) ring atoms (preferably selected from C, O, N and S). Moreover, the expression "heterocycloalkyl" refers to a group substituted by a fluorine, chlorine, bromine or iodine atom or an OH, =O, SH, =S, NH2, =NH, N3 or NO2 group. Examples are piperidyl, prolinyl, imidazolidinyl, piperazinyl, morpholinyl (e.g., -N(CH2CH2)2O), utropinyl, pyrrolidinyl, tetrahydrothiophenyl, tetrahydropyranyl, tetrahydrofuryl or 2-pyrazolinyl groups, and furthermore lactams, lactones, cyclic imides and cyclic anhydrides.

[0075] The expression "alkylcycloalkyl" refers to a group containing both a cycloalkyl and an alkyl, alkenyl or alkynyl group according to the above definition, for example, alkylcycloalkyl, cycloalkylalkyl, alkylcycloalkenyl, alkenylcycloalkyl and alkynylcycloalkyl groups. The alkylcycloalkyl group preferably contains a cycloalkyl group having 1 or 2 rings and 3 to 10 (especially 3, 4, 5, 6 or 7) ring carbon atoms, and 1 or 2 alkyl, alkenyl or alkynyl groups (especially an alkyl group) having 1 or 2 to 6 carbon atoms.

[0076] The expression "heteroalkylcycloalkyl" refers to an alkylcycloalkyl group as defined above, in which one or more (preferably 1, 2 or 3) carbon atoms are replaced by an oxygen, nitrogen, silicon, selenium, phosphorus or sulfur atom (preferably an oxygen, sulfur or nitrogen atom) or an SO or SO2 group. The heteroalkylcycloalkyl group preferably has one or two rings having 3 to 10 (especially 3, 4, 5, 6 or 7) ring atoms, and one or two alkyl, alkenyl, alkynyl or heteroalkyl groups (especially an alkyl or heteroalkyl group) having 1 or 2 to 6 carbon atoms. Examples of such groups are alkylheterocycloalkyl, alkylheterocycloalkenyl, alkenylheterocycloalkyl, alkynylheterocycloalkyl, heteroalkylcycloalkyl, heteroalkylheterocycloalkyl and heteroalkylheterocycloalkenyl, which are saturated or mono-, di- or tri-unsaturated cyclic groups.

[0077] The expression "aryl" refers to an aromatic group containing one or more rings and 6 to 14 ring carbon atoms, preferably 6 to 10 (especially 6) ring carbon atoms. Moreover, the expression "aryl" refers to a group substituted by a fluorine, chlorine, bromine or iodine atom or an OH, SH, NH2, N3 or NO2 group. Examples are the phenyl (Ph), naphthyl, biphenyl, 2-fluorophenyl, anilinyl, 3-nitrophenyl or 4-hydroxyphenyl group.

[0078] The term "heteroaryl" refers to an aromatic group containing one or more rings and one or more (preferably 1, 2, 3 or 4) oxygen, nitrogen, phosphorus or sulfur ring atoms (preferably O, S or N), and having 5 to 14 ring atoms, preferably 5 to 10 (especially 5 or 6 or 9 or 10) ring atoms. Moreover, the term "heteroaryl" refers to a group substituted by a fluorine, chlorine, bromine or iodine atom or an OH, SH, N3, NH2 or NO2 group. Examples are pyridyl (e.g., 4-pyridyl), imidazolyl (e.g., 2-imidazolyl), phenylpyrrolyl (e.g., 3-phenylpyrrolyl), thiazolyl, isothiazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, oxadiazolyl, thiadiazolyl, indolyl, indazolyl, tetrazolyl, pyrazinyl, pyrimidinyl, pyridazinyl, 4-hydroxypyridyl (4-pyridonyl), 3,4-hydroxypyridyl (3,4-pyridonyl), oxazolyl, isoxazolyl, triazolyl, tetrazolyl, isoxazolyl, indazolyl, indolyl, benzimidazolyl, benzoxazolyl, benzoisoxazolyl, benzothiazolyl, pyridazinyl, quinolinyl, isoquinolinyl, pyrrolyl, purinyl, carbazolyl, acridinyl, pyrimidyl, 2,3'-bifuryl, pyrazolyl (e.g., 3-pyrazolyl) and isoquinolinyl groups.

[0079] The term "aralkyl" refers to a group that, according to the above definition, includes both aryl and alkyl, alkenyl, alkynyl, and / or cycloalkyl groups. Examples include arylalkyl, arylalkenyl, arylalkynyl, arylcycloalkyl, arylcycloalkenyl, alkylarylcycloalkyl, and alkylarylcycloalkenyl groups. Specific examples of aralkyl include phenylcyclopentyl, cyclohexylphenyl, and groups derived from toluene, xylene, mesitylene, styrene, benzyl chloride, o-fluorotoluene, 1H-indene, tetralin, dihydronaphthalene, indanone, cumene, fluorene, and indane. The aralkyl group preferably includes one or two aromatic ring systems (especially one or two rings), each containing 6 to 10 carbon atoms, and one or two alkyl, alkenyl, and / or alkynyl groups containing 1 or 2 to 6 carbon atoms, and / or a cycloalkyl group containing 3, 4, 5, 6, or 7 ring carbon atoms.

[0080] The term "heteroaralkyl" refers to a group that, according to the above definition, includes both aryl and / or heteroaryl groups, and also alkyl, alkenyl, alkynyl, and / or heteroalkyl and / or cycloalkyl and / or heterocycloalkyl groups. The heteroaralkyl group preferably includes one or two aromatic ring systems (especially one or two rings), each containing 5 or 6 to 9 or 10 ring atoms (preferably selected from C, N, O, and S), and one or two alkyl, alkenyl, and / or alkynyl groups containing 1 or 2 to 6 carbon atoms, and / or one or two heteroalkyl groups containing 1 to 6 carbon atoms and 1, 2, or 3 heteroatoms selected from O, S, and N, and / or one or two cycloalkyl groups each containing 3, 4, 5, 6, or 7 ring carbon atoms, and / or one or two heterocycloalkyl groups each containing 3, 4, 5, 6, or 7 ring atoms containing 1, 2, 3, or 4 oxygen, sulfur, or nitrogen atoms.

[0081] Examples include aryl heteroalkyl, aryl heterocycloalkyl, aryl heterocycloalkenyl, arylalkyl heterocycloalkyl, arylalkenyl heterocycloalkyl, arylalkynyl heterocycloalkyl, arylalkyl heterocycloalkenyl, heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl, heteroaryl heteroalkyl, heteroaryl cycloalkyl, heteroaryl cycloalkenyl, heteroaryl - heterocycloalkyl, heteroaryl heterocycloalkenyl, heteroarylalkyl cycloalkyl, heteroaryl - alkyl heterocycloalkenyl, heteroaryl heteroalkyl cycloalkyl, heteroaryl heteroalkyl - cycloalkenyl and heteroaryl heteroalkyl heterocycloalkyl groups, which are saturated or mono-, di- or tri-unsaturated cyclic groups. Specific examples are tetrahydroisoquinolinyl, benzoyl, phthalidyl, 2- or 3-ethylindolyl, 4-methylpyridino, 2-, 3- or 4-methoxyphenyl, 4-ethoxyphenyl, 2-, 3- or 4-carboxyphenylalkyl groups.

[0082] As already mentioned above, the expressions cycloalkyl, heterocycloalkyl, alkylcycloalkyl, heteroalkylcycloalkyl, aryl, heteroaryl, aralkyl and heteroaralkyl also refer to groups substituted by fluorine, chlorine, bromine or iodine atoms or OH, =O, SH, =S, NH2, =NH, N3 or NO2 groups.

[0083] The term halogen refers to F, Cl, Br or I. Preferred halogens are F, Cl and Br. Particularly preferred halogens are F and Cl.

[0084] The term "optionally substituted" refers to a group that is unsubstituted or substituted by one or more (especially 1, 2 or 3, preferably 1 or 2) substituents.

[0085] When the base contains two or more substituents, such substituents are independently selected. That is, they can be the same or different.

[0086] Examples of substituents are fluorine, chlorine, bromine and iodine as well as OH, SH, NH2, -SO3H, -SO2NH2, -COOH, -COOMe, -COMe(Ac), -NHSO2Me, -SO2NMe2, -CH2NH2, -NHAc, -SO2Me, -CONH2, -CN, -NHCONH2, -NHC(NH)NH2, -NOHCH3, -N3 and -NO2 groups. Further examples of substituents are C1-C 10 alkyl, C2-C 10 alkenyl, C2-C 10 alkynyl, C1-C 10 heteroalkyl, C3-C 18 cycloalkyl, C1-C 17 heterocycloalkyl, C4-C 20 alkylcycloalkyl, C1-C 19 heteroalkylcycloalkyl, C6-C 18 aryl, C1-C 17 heteroaryl, C7-C 20 aralkyl and C1-C 19 heteroaralkyl groups, particularly C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 heteroalkyl, C3-C 10 cycloalkyl, C1-C9 heterocycloalkyl, C4-C 12 alkylcycloalkyl, C1-C 11 heteroalkylcycloalkyl, C6-C 10 aryl, C1-C9 heteroaryl, C7-C 12 aralkyl and C1-C 11 heteroaralkyl groups, more preferably C1-C6 alkyl and C1-C6 heteroalkyl groups.

[0087] When an aryl, heteroaryl, cycloalkyl, alkylcycloalkyl, heterocycloalkyl, alkylheterocycloalkyl, aralkyl or heteroaralkyl group contains two or more rings, such rings may be joined to each other via single or double bonds, or such rings may be cyclic, fused or bridged.

[0088] For these substitutions, the compounds of the present invention may contain one or more chiral centers. Accordingly, the present invention also includes both all pure enantiomers and all pure diastereomers, as well as mixtures thereof in any mixing ratio. Moreover, the present invention also includes all cis / trans isomers of the compounds of the present invention and mixtures thereof. Moreover, the present invention includes all tautomeric forms of the compounds of the present invention.

[0089] The present invention further provides a pharmaceutical composition comprising a compound according to the present invention or a salt thereof, and optionally one or more carrier substances and / or one or more adjuvants and / or one or more further active pharmaceutical ingredient(s).

[0090] Moreover, the present invention provides a compound (or a salt thereof) or a pharmaceutical composition described herein for use as a medicament, for example, for use in the prevention or treatment of bacterial infections, particularly for use in the prevention or treatment of bacterial infections caused by Gram-negative bacteria.

[0091] Also, the compounds and / or compositions described herein may be useful in the prophylactic treatment of infections caused by bacteria that are sensitive or multi-drug resistant, polymyxin-resistant mutants, carbapenem-resistant bacteria, methicillin-resistant Staphylococcus aureus, vancomycin-resistant Enterococcus or multi-drug resistant Neisseria gonorrhoeae.

[0092] Examples of Gram-negative bacteria include Escherichia coli, Pseudomonas aeruginosa, Candidatus liberibacter, Agrobacterium tumefaciens, Branhamella catarrhalis, Citrobacter diversus, Enterobacter aerogenes, Klebsiella pneumoniae, Proteus mirabilis, Salmonella typhimurium, Neisseria meningitidis, Serratia marcescens, Shigella sonnei, Shigella boydii, Neisseria gonorrhoeae, Acinetobacter baumannii, Salmonella enteriditis, Fusobacterium nucleatum, Veillonella parvula, Actinobacillus actinomycetemcomitans, Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Helicobacter pylori, Francisella tularensis, Yersinia pestis, Vibrio cholera, Morganella morganii, Ectwardsiella tarda, Campylobacter jejuni or HaemophilusInfluenza, Enterobacter cloacae, and many others, but not limited to these. Other notable groups of Gram-negative bacteria include cyanobacteria, spirochetes, green sulfur, and green non-sulfur bacteria.

[0093] The present invention further provides a compound (or a salt thereof) described herein or a pharmaceutical composition defined herein for the preparation of a medicament for use in the prevention or treatment of bacterial infections, particularly for use in the prevention or treatment of bacterial infections caused by Gram-negative bacteria.

[0094] Examples of salts of sufficiently basic compounds are salts of physiologically acceptable mineral acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, and phosphoric acid, or organic acids such as methanesulfonic acid, p-toluenesulfonic acid, lactic acid, acetic acid, trifluoroacetic acid, citric acid, succinic acid, fumaric acid, maleic acid, and salicylic acid. Further, sufficiently acidic compounds may form alkali or alkaline earth metal salts such as sodium, potassium, lithium, calcium, or magnesium salts; ammonium salts; or organic base salts such as 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.

[0095] The compounds described herein can be solvated, particularly hydrated. Solvation / hydration can occur during the production process or as a result of the hygroscopicity of the initially water-free compound. Solvates and / or hydrates can exist, for example, in solid or liquid form.

[0096] The therapeutic use of the compounds, salts, and formulations and pharmaceutical compositions described herein is also within the scope of the present invention.

[0097] In general, the compounds and pharmaceutical compositions described herein are administered by using established and accepted methods known in the art.

[0098] For oral administration, such therapeutically useful agents can be administered via the oral route in the following forms: for example, tablets, dragees, coated tablets, pills, semi-solids, soft or hard capsules, such as soft and hard gelatin capsules, aqueous or oily solutions, emulsions, suspensions, or syrups; via parenteral routes, for example, as injection solutions or suspensions, by intravenous, intramuscular, and subcutaneous injection; rectally as suppositories; by inhalation or insufflation, for example, as powder formulations, microcrystals or sprays (such as liquid aerosols); transdermally, for example, via a transdermal drug delivery system (TDDS), such as a plaster containing the active ingredient; or via one of the parenteral routes including intranasal. In the production of such tablets, pills, semi-solids, coated tablets, dragees, and, for example, hard gelatin capsules, the therapeutically useful formulations can be mixed with pharmaceutically inert inorganic or organic excipients, such as lactose, sucrose, glucose, gelatin, malt, silica gel, starch or their derivatives, talc, stearic acid or its salts, dried skim milk, etc. In the production of soft capsules, excipients such as vegetable, petroleum, animal or synthetic oils, waxes, fats and polyols can be used. In the production of solutions, emulsions or suspensions or syrups that are liquid, excipients such as water, alcohol, saline, glucose solution, polyols, glycerin, lipids, phospholipids, cyclodextrin, vegetable, petroleum, animal or synthetic oils can be used. Particularly preferably, they are lipids, more preferably phospholipids (preferably of natural origin, particularly preferably having a particle size of 300 - 350 nm), and preferably present in phosphate buffered saline (pH = 7 - 8, preferably 7.4). In suppositories, excipients such as vegetable, petroleum, animal or synthetic oils, waxes, fats and polyols can be used. In aerosol formulations, compressed gases suitable for this purpose, such as oxygen, nitrogen and carbon dioxide, can be used. Also, pharmaceutically useful agents can contain additives for preservation, stabilization, such as UV stabilizers, emulsifiers, sweeteners, fragrances, salts that change the osmotic pressure, buffers, coating additives and antioxidants.

[0099] Generally, although it may exceed the upper limit according to the instructions, for oral or parenteral administration to a human adult with a body weight of approximately 80 kg, a daily dosage of about 1 mg to about 10,000 mg, preferably about 5 mg to about 1,000 mg, should be appropriate. The daily dosage can be administered as a single dose, or in divided doses, or in the case of parenteral administration, it can be given as a continuous infusion or subcutaneous injection.

[0100] Moreover, according to a preferred embodiment, the present invention provides a method for treating a bacterial infection, comprising the step of administering a therapeutically effective amount of the compound or a salt thereof described herein to a subject in need of such treatment.

[0101] According to a more preferred embodiment, the present invention provides a method for treating, alleviating, or preventing a bacterial infection in a subject in need thereof, comprising the step of administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of at least one compound described herein, a salt thereof, or at least one of the (specific) compounds described herein, or a salt thereof. Administration of the compound can be local, for example, subcutaneous, transdermal, intrarectal, intravaginal, intranasal, intratracheal, intraocular, or intra-aural administration. Alternatively, administration can be systemic, for example, oral administration. In yet another alternative method, administration can be parenteral, intravenous, intramuscular, or intraperitoneal administration.

[0102] As used herein, the term "administration" can also include administering a combination of compounds. Thus, administration can be in a form such that a compound or a combination of compounds is dosed to an organism and delivered by the organism's circulatory system to a target site including, but not limited to, one or more cells, synapse junctions, and circulation. Additionally, administration can also mean placing a compound or a combination of compounds in direct contact with an organ, tissue, site, region, cell, or cell group, for example, but not limited to, direct injection of a combination of compounds.

[0103] In certain selected embodiments, combinations of compounds may be administered, which may also be considered as co-administering the individual compounds with each other. As used herein, "co-administration" refers to administering each of at least two compounds within a time frame in which the respective periods during which the biological activity or effect occurs overlap. Thus, the term co-administration includes both sequential and coextensive administration of the individual compounds, at least one of which is a compound of the invention. Accordingly, "administration" of a combination of compounds according to a part of the method of the invention includes both sequential and coextensive administration of the individual compounds of the invention. Similarly, the phrase "combination of compounds" indicates co-administration of the individual compounds, and the phrase "combination of compounds" does not mean that the compounds must necessarily be administered simultaneously or coextensively. In addition, the route of administration of the individual compounds need not be the same.

[0104] The present invention also relates to a formulation or pharmaceutical composition comprising at least one compound or a salt thereof according to the invention and at least one further (different) active pharmaceutical ingredient. The formulations of the invention can be used as medicaments, in particular for the treatment or prevention of bacterial infections caused by Gram-negative bacteria or Gram-negative and Gram-positive bacteria.

[0105] Preferably, in the formulation or pharmaceutical composition of the present invention, the additional active pharmaceutical ingredient is another antibiotic. Other antibiotics include β-lactam antibiotics such as penam, carbapenam, oxapenam, penem, carbapenem, monobactam, cephem, carbacephem, oxacephem and monobactam; aminoglycoside antibiotics such as amikacin, albekacin, astromicin, bekanamycin, dibekacin, framycetin, gentamicin, hygromycin B, isepamicin, kanamycin, neomycin, netilmicin, paromomycin, paromomycin sulfate, ribostamycin, sisomicin, spectinomycin, streptomycin, tobramycin and verdamycin; quinolone antibiotics such as ciprofloxacin, enoxacin, gatifloxacin, grepafloxacin, levofloxacin, lomefloxacin, moxifloxacin, nalidixic acid, norfloxacin, ofloxacin, sparfloxacin, temafloxacin and trovafloxacin; or glycopeptide antibiotics such as vancomycin, telavancin, bleomycin, ramoplanin and decaplanin; linezolid; or daptomycin, and may be selected from the group consisting of.

[0106] As used herein, the terms "treat" and "treatment" refer to delaying or reversing the worsening of a disease or infection. Treatment of a disease includes treating and / or reducing the symptoms of the disease or infection. The terms "prevent" and "prevention" refer to delaying the onset of a disease or the development of a disease, an infection or their symptoms. Prevention or prevention of a disease or infection may include stopping the onset of the disease, infection or their symptoms.

[0107] As used herein, the term "subject" can be an animal, vertebrate, mammal, rodent (e.g., guinea pig, hamster, rat, mouse), murine (e.g., mouse), canine (e.g., dog), feline (e.g., cat), equine (e.g., horse), primate, simian (e.g., monkey or ape), monkey (e.g., marmoset, baboon), ape (e.g., gorilla, chimpanzee, orangutan, gibbon) or human.

[0108] As used herein, the term "dosage unit" refers to a physically discrete unit, e.g., a capsule or tablet suitable as a unit dose for a subject. Each unit contains a predetermined amount of a compound of the invention or a salt thereof which has been found or is thought to yield the desired therapeutic effect and to provide the desired pharmacokinetic profile. The dosage unit consists of the compound of the invention or a salt thereof together with at least one pharmaceutically acceptable carrier, salt, excipient or combination thereof. The terms "dose" or "dosage" refer to the amount of active ingredient that an individual ingests or is administered at one time.

[0109] The term "therapeutically effective amount" refers to an amount sufficient to produce the desired biological effect in a subject. Thus, a therapeutically effective amount of a compound, when administered to a subject afflicted with or susceptible to a disease or infection, may be sufficient for the treatment or prevention of the disease or infection, and / or the delay in the onset or progression of the disease or infection, and / or the alleviation of one or more symptoms of the disease or infection. "Pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" refers herein to an API-free (where API refers to active pharmaceutical ingredient) substance used in the formulation of pharmaceutical preparations, e.g., disintegrants, binders, solubilizers and lubricants. These are generally safe for administration to humans.

[0110] The term "pharmaceutically acceptable" means approved by a regulatory agency of the federal or state government for use in animals, particularly humans, or included in the United States Pharmacopeia or other generally recognized pharmacopeias. The term "vehicle" refers to a diluent, adjuvant, excipient, or carrier with which the compounds of the invention are administered. Such pharmaceutical vehicles can be liquids such as, for example, water and oils.

[0111] In a further aspect, the invention relates to a compound of formula (Ic) or formula (Ic') or (Ic''):

[0112]

Chemical formula

[0113]

Chemical formula

[0114]

Chemical formula

[0115] In the production method of the present invention, the compounds of the present invention are produced by growing a recombinant host, such as a microorganism, such as a bacterium, such as Escherichia coli, Bacillus, Corynebacteria (e.g., Corynebacterium glutamicum), Lactobacillus (e.g., Lactococcus lactis) or Streptomyces (e.g., Streptomyces albus, Streptomyces lividans) species of bacteria, or yeast, such as Saccharomyces cerevisiae, Saccharomyces pombe, Pichia pastoris or Yarrowia lipolytica, under regulated conditions (e.g., as known in the art or as described below), and recovering the compound from the culture broth in a substantially pure form as described herein. In the production method of the present invention, the compounds are prepared by growing the respective bacterial host cells under the regulated conditions described below and recovering the compound from the culture broth in a substantially pure form as described herein.

[0116] The cultivation process can be carried out by liquid culture, for example, by growing each recombinant host (e.g., a microorganism, e.g., a bacterial host cell) in a medium containing one or several different carbon sources and one or various nitrogen sources. Also, salts are essential for growth and production. Suitable carbon sources are various mono-, di- and polysaccharides such as maltose, glucose, or carbon derived from amino acids such as peptone. The nitrogen source is nitrogen derived from ammonium, nitrate, urea, chitin, or amino acids. The following inorganic ions: Mg ions, Ca ions, Fe ions, Mn ions, Zn ions, K ions, sulfate ions, Cl ions, phosphate ions support growth or are essential for synthetic media. The preferred composition of the nutrient medium used in the production method of the present invention is described in more detail in the examples. In certain embodiments, the recombinant host is a microorganism, such as a bacterium, such as Escherichia coli, Bacillus, Corynebacterium (e.g., Corynebacterium glutamicum), Lactobacillus (e.g., Lactococcus lactis) or Streptomyces (e.g., Streptomyces albus, Streptomyces lividans) species of bacteria, or a yeast, such as Saccharomyces cerevisiae, Saccharomyces pombe, Pichia pastoris or Yarrowia lipolytica. In one embodiment, the recombinant host can be a bacterial host cell, such as an Escherichia coli or Lactobacillus cell, preferably an Escherichia coli cell, more preferably an Escherichia coli BL21(DE3) cell. Preferred embodiments include Escherichia coli BL21(DE3)pNOSOdarA-E-D9 (deposited with DSMZ on January 29, 2021; DSM33798) and Escherichia coli BL21(DE3)pNOSOdarA-E-D17 (deposited with DSMZ on January 29, 2021; DSM33799). The temperature for growth and production is from 15°C to 40°C, and the preferred temperature is from 25°C to 35°C, particularly 30°C. The pH of the culture solution is from 5 to 8, preferably from pH 6.9 to 7.2, more preferably about pH 7.1.

[0117] The compound can be recovered from the fermentation broth by resin absorption and eluted from the resin by washing with solvents of various polarities. Purification can be facilitated by chromatographic separation, for example, high performance liquid chromatography (HPLC) or reverse phase high performance liquid chromatography (RP-HPLC).

[0118] Compounds of formula (Ic), (Ic') or (Ic''), i.e., darobactin A and darobactin derivatives, are ribosomally synthesized and post-translationally modified peptide compounds (RiPPs). The artificial synthetic or recombinant biosynthetic gene cluster (BGC) encoding said peptide compound has the following predicted functions: darA or this modified variant: the darobactin propeptide or the propeptide of a darobactin derivative; darBCD: an ABC-type trans-envelope transporter; and darE: a radical S-adenosylmethionine (SAM) methyltransferase (which is proposed to catalyze a cyclization reaction to bind W3-K5 and W1-W3). The bacterial host cell contains at least one synthetic or recombinant nucleic acid sequence which encodes such a synthetic or recombinant BGC of a compound of formula (Ic), (Ic') or (Ic''), thereby enabling the synthesis of the compound according to formula (Ic), (Ic') or (Ic''). The synthetic or recombinant nucleic acid sequence used in the production method of the present invention may, in addition to the nucleic acid sequence encoding the BGC, also include control sequences, such as promoters and translation initiation and termination sequences, and sequences which facilitate stable maintenance in the host cell, i.e., sequences which provide the function of an origin of replication or which further facilitate integration by homologous recombination into the host cell chromosome or other DNA. The synthetic or recombinant nucleic acid sequence used in the production method of the present invention encodes the BGC of a compound of formula (Ic) (or formula (Ic') or formula (Ic'')), (i) at least 95%, 96%, 97%, 98%, 98.5%, 99% or 99.5% to 100% sequence identity to the full-length sequence of SEQ ID NO: 1, or (ii) a sequence which is completely complementary to any nucleic acid sequence of (i) It is a synthetic or recombinant nucleic acid sequence having

[0119] The compounds of the present invention described herein, namely, darobactin derivatives, are ribosomally synthesized and post-translationally modified peptide compounds (RiPPs). Such peptide compounds can be produced using any method. For example, the compounds can be produced by chemical synthesis. Alternatively, the peptide compounds described herein can be produced by standard recombinant techniques using a heterologous expression vector encoding the polypeptide. The expression vector can be introduced into a host cell, for example, by transformation or transfection, to express the encoded polypeptide, which can then be purified. Expression systems that can be used for small or large-scale production of polypeptides include microorganisms, such as bacteria (e.g., Escherichia coli, Bacillus, Corynebacterium (e.g., Corynebacterium glutamicum), Lactobacillus (e.g., Lactococcus lactis) or Streptomyces (e.g., Streptomyces albus, Streptomyces lividans) species) transformed with a recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vector containing the nucleic acid molecules described herein, and yeasts (e.g., Saccharomyces cerevisiae, Saccharomyces pombe, Pichia pastoris or Yarrowia lipolytica) transformed with a recombinant yeast expression vector containing the nucleic acid molecules described herein, but are not limited thereto. Also useful are expression systems including insect cell lines infected with a recombinant virus expression vector (e.g., baculovirus) containing the nucleic acid molecules described herein, and plant cell lines infected with a recombinant virus expression vector (e.g., tobacco mosaic virus) or transformed with a recombinant plasmid expression vector (e.g., Ti plasmid) containing the nucleic acid molecules described herein. Further, the peptide compounds of the present invention can be produced using a mammalian expression system that encompasses a recombinant expression construct containing a promoter derived from the genome of a mammalian cell (e.g., metallothionein promoter) or a promoter derived from a mammalian virus (e.g., adenovirus late promoter and cytomegalovirus promoter) together with the nucleic acids described herein. The peptide compounds of the present invention can have an N-terminal or C-terminal tag.

[0120] In one embodiment, formula (1’), (1b) or (1b’):

[0121] [Chemical formula] (wherein each of R 22 and R 44 is independently of each other H, CH3, CH2OH or the group:

[0122] [Chemical formula] represents) A method for the production of a compound of (a) preparing a recombinant host capable of producing the compound of formula (1’), (1b) or (1b’), said recombinant host comprising at least one synthetic or recombinant nucleic acid sequence encoding a biosynthetic gene cluster (BGC) of the compound, said BGC having (i) at least 95%, 96%, 97%, 98%, 98.5%, 99% or 99.5% to 100% sequence identity to the full-length sequence of SEQ ID NO: 44, or (ii) a sequence completely complementary to any nucleic acid sequence of (i) having, the step of (b) culturing the recombinant host for a time sufficient for the recombinant host to produce a compound of formula (1’), (1b) or (1b’), and (c) isolating the compound from the recombinant host or the culture supernatant, thereby producing a compound of formula (1’), (1b) or (1b’) is described herein.

[0123] In one embodiment, formula (II), (IIa) or (IIa’):

[0124] [Chemical formula] {wherein R2 and R 4 is each independently H, CH3, CH2OH or a group:

[0125] [Chemical formula] represents, R 3 is a group:

[0126] [Chemical formula] and is, R 5 is the following formula:

[0127] [Chemical formula] [In the formula, R 9 is a hydrogen or halogen atom, R 10 is a hydrogen atom or a methyl group, R 11 is independently selected, in case, from halogen atoms (especially fluorine or chlorine atoms), and n is an integer from 0 to 4 (especially 0 or 1, preferably 0)] group of, or group of formula -CH3, or the following formula:

[0128] [Chemical formula] is a group of} is a method for the production of a compound of, (a) preparing a recombinant host capable of producing the compound of formula (II) (or (IIa), (IIa') or (IIa'')), said recombinant host encapsulating at least one synthetic or recombinant nucleic acid encoding a biosynthetic gene cluster (BGC) of the compound, and this BGC is, (i) at least 95%, 96%, 97%, 98%, 98.5%, 99% or 99.5% to 100% sequence identity to the full-length sequence of SEQ ID NO: 86, or (ii) A sequence that is completely complementary to any nucleic acid sequence of (i) having, and (b) culturing the recombinant host for a time sufficient for the recombinant host to produce a compound of formula (II) (or (IIa), (IIa') or (IIa'')); (c) isolating the compound from the recombinant host or the culture supernatant, thereby producing a compound of formula (II) (or (IIa), (IIa') or (IIa'')) is described herein.

[0129] In some embodiments of the compounds of formula (II), (IIa), (IIa') or (IIa''), R 5 is of the formula:

[0130]

Chemical formula

[0131] In some embodiments of the compounds of formula (II), (IIa), (IIa') or (IIa''), R 2 is of the formula

[0132]

Chemical formula

[0133] In some embodiments of the compounds of formula (II), (IIa), (IIa') or (IIa''), R 4 is of the formula

[0134]

Chemical formula

[0135] In some embodiments of the compound of formula (II), (IIa), (IIa') or (IIa''), R 3 is the following group

[0136]

Chemical formula

[0137] In some embodiments of the compound of formula (II), (IIa), (IIa') or (IIa''), R 3 is the following group

[0138]

Chemical formula

[0139] In some preferred embodiments of the compound of formula (II), (IIa), (IIa') or (IIa''), R 3 is the following group

[0140]

Chemical formula

[0141] In some embodiments, the method for producing the compound of formula (II), (IIa), (IIa') or (IIa'') optionally includes the step of supplementing the fermentation broth in culture with one or more halogenated tryptophan(s), preferably, for example, commercially available 5-chloro-L-tryptophan, 7-chloro-L-tryptophan, 6-fluoro-L-tryptophan or 7-fluoro-L-tryptophan, i.e., halogenated L-tryptophan(s). The one or more halogenated tryptophan(s) can be replenished regularly at various concentrations known to those skilled in the art, and the total final concentration of the halogenated tryptophan(s) can be, for example, from 0.5 mM to 10 mM.

[0142] In some embodiments, the method for producing a compound of formula (II), (IIa), (IIa’) or (IIa’’) optionally comprises the step of supplementing the fermentation broth in culture with one or more halogenated tryptophan(s), preferably, for example, commercially available 5-chloro-L-tryptophan, 7-chloro-L-tryptophan, 6-fluoro-L-tryptophan or 7-fluoro-L-tryptophan, i.e., halogenated L-tryptophan(s). The one or more halogenated tryptophan(s) can be supplemented periodically at various concentrations known to those skilled in the art, and the total final concentration of the halogenated tryptophan(s) can be, for example, from 0.5 mM to 10 mM.

[0143] In some embodiments, the method for producing a compound of formula (II), (IIa), (IIa’) or (IIa’’) optionally comprises the step of culturing a host cell containing the vector pUC18-zeo-mx8-corP-trpAB described herein, and the step of co-supplementing the fermentation broth in culture with serine (preferably L-serine) and one or more halogenated indole(s). In some embodiments, the method for producing a compound of formula (II), (IIa), (IIa’) or (IIa’’) optionally comprises the step of culturing a recombinant host containing at least one synthetic or recombinant nucleic acid encoding the biosynthetic gene cluster (BGC) of the compound described herein, wherein the BGC further comprises the trpAB gene of pSTB7 37845 upstream of the darE gene in the BGC, and the step of supplementing the fermentation broth in the culture of the recombinant host with serine (preferably L-serine) and one or more halogenated indole(s). Serine and one or more halogenated indole(s) can be supplemented periodically at various concentrations known to those skilled in the art. For example, serine can be supplemented at a concentration of 0.5 mM to 15 mM (preferably 8 to 12 mM, particularly 10 mM), and the halogenated indole can be supplemented at a concentration of 5 to 10 mM.

[0144] As used herein, the term "recombinant host" is intended to refer to a host whose genome has been enhanced by at least one integrated DNA sequence. Such DNA sequences include, but are not limited to, genes that do not naturally occur, DNA sequences that are not normally transcribed into RNA or translated into protein ("expressed"), and other genes or DNA sequences that are desirable to introduce into a non-recombinant host. Typically, it is understood that the genome of the recombinant hosts described herein is enhanced by the stable introduction of one or more recombinant genes. However, self or replicating plasmids or vectors can also be used within the scope of the present invention. Moreover, the present invention can be carried out using plasmids or vectors with low copy numbers, e.g., single copy, or high copy numbers (exemplified herein). When using "introduced" or "enhanced" herein, it is known in the art to mean introduction or enhancement by human hand.

[0145] Generally, introduced DNA is not normally resident in the host that is the recipient of this DNA, but after isolating a DNA segment from a given host, introducing one or more additional copies of this DNA into the same host, e.g., to enhance the production of a gene product or to change the expression pattern of a gene, is within the scope of the present invention. In some cases, the introduction of DNA results in the modification or even replacement of an endogenous gene or DNA sequence, e.g., by homologous recombination or site-directed mutagenesis. Suitable recombinant hosts include microorganisms, e.g., bacteria (e.g., Escherichia coli, Bacillus, Corynebacterium (e.g., Corynebacterium glutamicum), Lactobacillus (e.g., Lactococcus lactis) or Streptomyces (e.g., Streptomyces albus, Streptomyces lividans) species), or yeast, e.g., Saccharomyces cerevisiae, Saccharomyces pombe, Pichia pastoris or Yarrowia lipolytica), as well as mammalian, insect or plant cell expression systems.

[0146] A synthetic or recombinant nucleic acid encoding a biosynthetic gene cluster (BGC) of a peptide compound described herein comprises a coding sequence of this polypeptide, which is operably linked in a sense orientation to one or more control regions suitable for the expression of the polypeptide. Since many microorganisms can express multiple gene products from polycistronic mRNA, multiple polypeptides can be expressed under the regulation of a single control region of such a microorganism, if necessary. When the control region and the coding sequence are arranged such that the control region is effective in controlling the transcription or translation of its sequence, the coding sequence and the control region are considered to be operably linked. Typically, the translation start site of the translation reading frame of the coding sequence is located 1 to about 50 nucleotides downstream of the control region of a monocistronic gene.

[0147] "Control region" refers to a nucleic acid having a nucleotide sequence that affects the initiation and rate of transcription or translation, and the stability and / or mobility of the transcription or translation product. Control regions include, but are not limited to, promoter sequences, enhancer sequences, response elements, protein recognition sites, inducible elements, protein-binding sequences, 5' and 3' untranslated regions (UTRs), transcription start sites, termination sequences, polyadenylation sequences, introns, and combinations thereof. Typically, a control region includes at least one core (basic) promoter. Also, a control region may include at least one regulatory element, such as an enhancer sequence, an upstream element, or an upstream activation region (UAR). The selection of the control regions to be included depends on several factors, including, but not limited to, efficiency, selectivity, inducibility, desired expression level, and preferential expression at a particular culture stage. It is routine for those skilled in the art to regulate the expression of a coding sequence by appropriately selecting and arranging control regions with respect to the coding sequence. It is understood that two or more control regions, such as introns, enhancers, upstream activation regions, transcription terminators, and inducible elements, may be present.

[0148] In many cases, the coding sequences of the polypeptides described herein are identified in species other than the recombinant host. That is, they are heterologous nucleic acids. The term "heterologous nucleic acid" as used herein refers to a nucleic acid introduced into a recombinant host, where the nucleic acid is not naturally present in the host. Thus, if the recombinant host is a microorganism, the coding sequence can be from another prokaryotic or eukaryotic microorganism, plant, or animal. However, in some cases, the heterologous nucleic acid can be host-derived and can include a sequence (or a portion thereof) that is reintroduced into that organism. A native sequence can often be distinguished from a naturally occurring sequence by the presence of non-native sequences linked to the native sequence in an exogenous nucleic acid, for example, non-native regulatory sequences adjacent to the native sequence in a recombinant nucleic acid construct. In addition, stably transformed exogenous nucleic acids typically integrate at positions other than where the native sequence is found.

[0149] Due to the degeneracy of the genetic code, it is understood that a number of nucleic acids can encode a particular peptide compound, i.e., for many amino acids, there are two or more nucleotide triplets that act as codons for the amino acid. Thus, the codons in the coding sequence of a given peptide compound can be modified using a codon bias table appropriate for the host (e.g., a microorganism) to obtain optimal expression in a particular host. Such modified sequences, like isolated nucleic acids, can exist as purified molecules and can be incorporated into vectors or viruses for use in the construction of modules for recombinant nucleic acid constructs.

[0150] The recombinant host described herein expresses a darobactin derivative, for example, a peptide compound of formula (I), (Ia) or (Ia') as described above. In one aspect, the invention relates to a recombinant host encapsulating a heterologous nucleic acid encoding a darobactin derivative described herein. In particular, the recombinant host described herein encapsulates a heterologous nucleic acid encoding a peptide compound of formula (I), (Ia) or (Ia'), wherein said heterologous nucleic acid is operably linked to a control region enabling expression in said recombinant host. In certain embodiments, the heterologous nucleic acid is a synthetic or recombinant BGC encoding said peptide compound. Such synthetic or recombinant BGCs contain darA (modified) and darE, which genes have the following predicted functions: modified darA variant (i.e., the propeptide of the darobactin derivative described herein) and darE: radical S-adenosylmethionine (SAM) methyltransferase (proposed to catalyze a cyclization reaction and bind to W3-K5 and W1-W3). In one embodiment, the synthetic or recombinant BGC further contains darBCD having the (predicted) function of an ABC-type trans-envelope transporter.

[0151] A recombinant host, for example, a microorganism, for example, a bacterial host cell or a yeast cell, contains at least one synthetic or recombinant nucleic acid sequence, which encodes a synthetic or recombinant BGC of a compound of such formula (I), (Ia) or (Ia'), thereby enabling the synthesis of a compound according to formula (I), (Ia) or (Ia'). The synthetic or recombinant nucleic acid sequences utilized in the production methods of the invention may, in addition to the nucleic acid sequence encoding the BGC, also include control sequences, such as promoters and translation initiation and termination sequences, and further sequences facilitating stable maintenance in the host cell, i.e., sequences providing the function of an origin of replication or facilitating integration by homologous recombination into the host cell chromosome or other DNA. The synthetic or recombinant nucleic acid sequences utilized in the production methods of the invention encode a BGC of a compound of formula (1) (or formula (1a) or formula (1a')), (i) At least 95%, 96%, 97%, 98%, 98.5%, 99% or 99.5% to 100% sequence identity to the full-length sequence of SEQ ID NO: 44, or (ii) A sequence that is completely complementary to any nucleic acid sequence of (i) A synthetic or recombinant nucleic acid sequence having the same.

[0152] As used herein, the term "identity" refers to the property of a sequence that measures their similarity or relatedness. Identity is evaluated by dividing the number of identical residues by the total number of residues and multiplying this quotient by 100.

[0153] As used herein, the terms "nucleic acid" or "nucleic acid sequence" refer to oligonucleotides, nucleotides, polynucleotides, or fragments of any of these, DNA of genomic or synthetic origin, which may be single-stranded or double-stranded and may represent sense or antisense strands of natural or synthetic origin. "Oligonucleotide" includes either single-stranded polydeoxynucleotides or two complementary polydeoxynucleotide strands, which may be chemically synthesized. Such synthetic oligonucleotides do not have a 5' phosphate and thus do not ligate to another oligonucleotide without adding phosphate with ATP in the presence of kinase. Synthetic oligonucleotides can ligate to dephosphorylated fragments. The "coding sequence" of a particular polypeptide or protein or the "nucleotide sequence encoding" it is a nucleic acid sequence that is transcribed and translated into a polypeptide or protein when placed under the control of appropriate regulatory sequences. The nucleic acids used to practice the present invention can be isolated from a variety of sources expressed / created by genetic modification, amplification, and / or recombination. Techniques for the manipulation of nucleic acids, such as subcloning, probe labeling (e.g., using Klenow polymerase, nick translation, random primer labeling using amplification), sequencing, hybridization, etc., are well described in the scientific and patent literature, see, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor, N.Y., VoIs. 1-3 (1989); Current Protocols in Molecular Biology, Ausubel ed., John Wiley & Sons, Inc., New York (1997); Laboratory Techniques In Biochemistry And Molecular Biology: Hybridization With Nucleic Acid Probes, Part I. Theory and Nucleic Acid Preparation, Tijssen ed., Elsevier, N.Y. (1993).The nucleic acid encoding the polypeptide of the present invention is constructed at an appropriate stage with a leader sequence capable of directing the secretion of the translated polypeptide or a fragment thereof.

[0154] As used herein, the term "isolated" means removing a substance, such as a nucleic acid, polypeptide, vector, cell, from its original environment, for example, from the natural environment if it exists naturally. For example, a polynucleotide or polypeptide that exists naturally in a living animal is not isolated, but the same polynucleotide or polypeptide separated from some or all of the substances coexisting in the natural system is isolated. Such a polynucleotide may be part of a vector and / or such a polynucleotide or peptide may be part of a composition, and such a vector or composition is still isolated in that it is not part of its natural environment.

[0155] As used herein, the term "synthetic" means that a substance, such as a nucleic acid, is synthesized in vitro by well-known chemical synthesis techniques as described, for example, in Adams (1983) J. Am. Chem. Soc. 105:661; Belousov (1997) Nucleic Acids Res. 25:3440-3444; Frenkel (1995) Free Radic. Biol. Med. 19:373-380; Blommers (1994) Biochemistry 33:7886-7896; Narang (1979) Meth. Enzymol. 68:90; Brown (1979) Meth. Enzymol. 68:109; Beaucage (1981) Tetra. Lett. 22:1859.

[0156] The term "recombinant" means that a nucleic acid is adjacent to a "backbone" nucleic acid that is not adjacent in its natural environment. The backbone molecules according to the invention include nucleic acids used for the maintenance or manipulation of nucleic acid inserts of interest, such as nucleic acids for cloning and expression vectors, such as plasmids. The recombinant peptide compounds of the invention made from such nucleic acids can be individually isolated or cloned and tested for the desired activity. Any suitable recombinant expression system, including bacterial expression systems, can be used.

[0157] Also provided is a vector comprising at least one heterologous nucleic acid, such as a synthetic or recombinant BGC encoding a peptide compound described herein. The vector can be a cloning vector, an expression vector or an artificial chromosome.

[0158] As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is ligated. Vectors, including cloning and expression vectors, contain the heterologous nucleic acids or functional equivalents thereof described herein. The heterologous nucleic acids described herein can be incorporated into recombinant replicable vectors, such as cloning or expression vectors. Nucleic acids can be replicated using a vector in a compatible host cell. Accordingly, the invention also provides a method for producing a polynucleotide of the invention, comprising introducing the polynucleotide of the invention into a replicable vector, introducing the vector into a compatible host cell, and growing the host cell under conditions that result in replication of the vector. Vectors can be recovered from host cells. Suitable host cells are generally known to those of skill in the art, and examples of suitable host cells are described herein. The vectors into which the expression cassettes or heterologous nucleic acids described herein are inserted can be any vector amenable to recombinant DNA procedures, and the choice of vector often depends on the host cell into which it is to be introduced. A variety of cloning and expression vectors for use with prokaryotic and eukaryotic hosts are described by Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor, N.Y., (1989).

[0159] The vectors according to the invention may be self-replicating vectors, i.e., vectors that exist as extrachromosomal entities, whose replication is independent of chromosomal replication, such as, for example, plasmids. Also provided herein is an isolated and purified plasmid comprising at least one synthetic or recombinant nucleic acid sequence having (i) at least 95%, 96%, 97%, 98%, 98.5%, 99% or 99.5% to 100% sequence identity to the full-length sequence of SEQ ID NO: 1, or (ii) a sequence that is fully complementary to any nucleic acid sequence of (i) .

[0160] In one embodiment, provided herein is an isolated and purified plasmid comprising at least one synthetic or recombinant nucleic acid sequence encoding an artificial BGC encoding a peptide compound of formula (II) (or (IIa), (IIa') or (IIa'')) described herein, (i) having at least 95%, 96%, 97%, 98%, 98.5%, 99% or 99.5% - 100% sequence identity to the full-length sequence of SEQ ID NO: 86, or (ii) a sequence that is completely complementary to any nucleic acid sequence of (i). Alternatively, the vector can be a vector that, when introduced into a host cell, integrates into the host cell genome and replicates with the chromosome(s) into which it has integrated.

[0161] In a preferred embodiment, provided herein is an isolated and purified plasmid comprising at least one synthetic or recombinant nucleic acid sequence encoding an artificial BGC encoding a peptide compound of formula (1') (or (1b) or (1b')) described herein, (i) having at least 95%, 96%, 97%, 98%, 98.5%, 99% or 99.5% - 100% sequence identity to the full-length sequence of SEQ ID NO: 44, or (ii) a sequence that is completely complementary to any nucleic acid sequence of (i). Alternatively, the vector can be a vector that, when introduced into a host cell, integrates into the host cell genome and replicates with the chromosome(s) into which it has integrated.

[0162] The term "plasmid" refers to a circular double-stranded DNA loop capable of ligating additional DNA segments. A plasmid may have a bacterial origin of replication or may contain an autonomous replication sequence (ARS) derived from the yeast chromosome, thereby enabling it to replicate in the bacteria, yeast, or host cell into which it is introduced. Alternatively, a plasmid may be one that, when introduced into a host cell, integrates into the host cell genome and is replicated along with the genomic DNA(s) into which it has integrated. Plasmids can be used to replicate synthetic or recombinant nucleic acids utilized in the present invention in compatible host cells. Plasmids may be recovered from host cells, and suitable host cells are described below. Moreover, a plasmid may be capable of directing the expression of a gene to which it is operably linked. The plasmid into which the synthetic or recombinant nucleic acid sequence (expression cassette) utilized in the present invention is inserted may be any plasmid that is conveniently amenable to recombinant DNA procedures, and the choice of plasmid often depends on the host cell into which it is introduced. A variety of cloning and expression plasmids for use with bacterial and yeast hosts are described by Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor, N.Y., (1989). The plasmids according to the present invention can be used in vitro, for example, to transfect or transform host cells. The plasmids of the present invention may contain one, two or more, for example, 3, 4 or 5 synthetic or recombinant nucleic acid sequences, such as those utilized in high expression in the production method of the present invention.The isolated and purified plasmids of the present invention contain the synthetic or recombinant nucleic acid sequences used in the production method of the present invention in a form suitable for the expression of nucleic acids in bacterial host cells (e.g., Escherichia coli, Bacillus, Corynebacterium (e.g., Corynebacterium glutamicum), Lactobacillus (e.g., Lactococcus lactis) or Streptomyces (e.g., Streptomyces albus, Streptomyces lividans) species of bacteria, or yeasts, e.g., Saccharomyces cerevisiae, Saccharomyces pombe, Pichia pastoris or Yarrowia lipolytica), which means that the plasmid may contain one or more control sequences operably linked to the nucleic acid sequence to be expressed, which are selected based on the host cell used for expression. Particularly useful examples of host cells include bacterial cells of the Escherichia coli, Bacillus, Corynebacterium, Lactobacillus or Streptomyces species, e.g., Escherichia coli BL21(DE3), and yeast cells of the Saccharomyces cerevisiae, Pichia pastoris or Yarrowia lipolytica species, e.g., Pichia pastoris GS115, Pichia pastoris X-33, Pichia pastoris KM71 and Yarrowia lipolytica Po1h(CLIB882). The plasmids of the present invention described herein can be designed for the expression of a BGC encoding a peptide compound or the peptide compound of interest in a host cell. Suitable host cells are further considered in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990). Representative examples of suitable hosts are generally known in the art and examples are described herein. Also, culture media and conditions suitable for the host cell are known in the art.

[0163] In a plasmid, for example, the isolated and purified plasmid of the present invention, "operably linked" means that the nucleic acid sequence of interest (i.e., BGC) is linked to the control sequence(s) in such a way that expression of this nucleic acid sequence is enabled (e.g., in an in vitro transcription / translation system or, when the vector is introduced into a host cell, in the host cell), that is, the term "operably linked" is intended to refer to the proximity of the recited components in a relationship such that they can function in their intended manner. Control sequences, such as a promoter, enhancer or other expression control signals, that are "operably linked" to a coding sequence are arranged to achieve expression of the coding sequence under conditions compatible with the regulatory sequences, or the sequences cooperate to function for their intended purpose, e.g., the transcription is initiated at the promoter and proceeds by the DNA sequence encoding the polypeptide.

[0164] The term "control sequence" or "regulatory sequence" is intended to include promoters, operators, enhancers, attenuators and other expression regulatory elements (e.g., polyadenylation signals). Such control sequences are described, for example, in Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990). The term control or regulatory sequence includes sequences that direct constitutive expression of a nucleotide sequence in many types of host cells and sequences that direct expression of a nucleotide sequence (e.g., genus-specific control sequences) in only certain host cells.

[0165] Thus, a plasmid or expression construct for a given host cell comprises, in order from the 5'-end to the 3'-end, the following elements operably linked to each other with respect to the coding strand of the sequence encoding the polypeptide for production by the method of the invention: (i) a promoter sequence capable of directing the transcription of a nucleotide sequence encoding a polypeptide in a given host cell, (ii) optionally, a signal sequence capable of directing the secretion of the polypeptide from the given host cell into the culture medium, (iii) optionally, a sequence encoding the C-terminus, N-terminus or an internal epitope tag sequence or a combination of the above sequences enabling the purification, detection or labeling of the polypeptide, (iv) a nucleic acid sequence of the invention encoding a polypeptide of the invention, and preferably, (v) a transcription termination region (terminator) capable of terminating transcription downstream of the nucleotide sequence encoding the polypeptide.

[0166] Particularly known bacterial promoters include lad, ntpII, lacZ, pTuf, Eftu, T3, T7, SP6, K1F, tac, tet, gpt, P BAD , lambda P R , P L and trp. Examples of Saccharomyces cerevisiae promoters include P ADH1 , P GAL1 , P CYC1 , P GPD (also called P TDH3 ), P ZEV P ACT1 P HXK1 , P YGR243 , P HXT4 , P HXT7 P TEF1 , P TPI1 , P PGK1 , P TDH3 and P PYK1 . In a preferred embodiment, the Saccharomyces cerevisiae promoter is P ADH1 . Examples of Pichia pastoris promoters include P AOX1 , P GAP , P ADH3 , P PGK1 , P DAS , PFLD1 and P PEX8 are mentioned, and in a preferred embodiment, P AOX1 , P GAP , P ADH3 and P PGK1 are included. Examples of the Yarowia lipolytica promoter include PmLEU2, pPOX2, pEYK1, pLIP2, pXPR2, and pLEU2, and in a preferred embodiment, it includes PmLEU2. Examples of the Streptomyces albus and Streptomyces lividans promoters include KasOp, psf ermEp, SF14p, PtipA, PkasO* R15 and Ptac. In a preferred embodiment, the Streptomyces albus and Streptomyces lividans promoters are KasOp, psf, and ermEp.

[0167] The selection of suitable plasmids and promoters is abundantly available within the scope of ordinary skill in the art. Downstream of the synthetic or recombinant nucleic acid sequence utilized in the production method of the present invention, a 3' untranslated region containing one or more transcription termination sites (e.g., terminators) may be present. The origin of the terminator is not so important. The terminator can be, for example, derived from a DNA sequence encoding a peptide compound. Preferably, the terminator is endogenous to the host cell (in which the nucleotide sequence encoding the polypeptide is expressed). In the transcription region, a ribosome binding site for translation may be present. The coding portion of the mature transcript expressed by the construct includes an initiation translation AUG (or TUG or GUG in prokaryotes) and a stop codon appropriately positioned at the end of the peptide compound to be translated or processed.

[0168] Furthermore, the enhancement of the expression of the polynucleotide or heterologous nucleic acid of the present invention described herein can be achieved by the selection of heterologous control regions, such as promoters, secretion leaders, and / or terminator regions, which can act to increase expression and, optionally, the secretion level of the protein of interest from the expression host and / or to provide inducible regulation of the expression of the peptide compound. It will be understood by those skilled in the art that plasmid design may depend on factors such as the choice of host cell to be transformed, the expression level of the desired protein, etc. The plasmids of the present invention can be introduced into recombinant hosts, such as microorganisms (e.g., bacterial host cells), thereby producing the peptides encoded by the nucleic acids described herein. Preferably, the plasmids of the present invention are capable of self-replication in microorganisms, such as bacteria (e.g., Escherichia coli, Bacillus, Corynebacterium (e.g., Corynebacterium glutamicum), Lactobacillus (e.g., Lactococcus lactis) or Streptomyces (e.g., Streptomyces albus, Streptomyces lividans) species of bacteria, or yeasts, such as Saccharomyces cerevisiae, Saccharomyces pombe, Pichia pastoris or Yarrowia lipolytica). Preferably, the plasmids of the present invention are capable of self-replication in Lactobacillus or Escherichia coli, preferably in bacteria of the Escherichia coli species.

[0169] The plasmids of the present invention can be designed for the expression of the target BGC or peptide compound in bacterial cells, such as Escherichia coli or Bacillus strains, such as Lactobacillus. Suitable host cells are further discussed in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990). Representative examples of suitable hosts are described later. Suitable culture media and conditions for the above host cells are known in the art.

[0170] As shown above, the term "regulatory sequence" or "control sequence" is defined herein to include at least any component that may be necessary and / or advantageous for the expression of the peptide compounds described herein. Any regulatory sequence may be native or foreign to the nucleic acid sequence encoding the BGC for the production of the peptide compounds described herein. Such regulatory sequences can include, but are not limited to, promoters, leaders, optimal translation initiation sequences (such as those described in Kozak, 1991, J. Biol. Chem. 266:19867-19870), secretion signal sequences, propeptide sequences, polyadenylation sequences, transcription terminators. At a minimum, regulatory sequences typically include a promoter, as well as transcription and translation stop signals. A stably transformed microorganism is a microorganism into which one or more DNA fragments have been introduced to maintain, replicate and segregate the introduced molecule in the growth medium. Stable transformation can result from multiple or single chromosomal integrations (multiple possible) or one or more extrachromosomal elements, such as one or more plasmid vectors. By plasmid vectors, it is possible to direct the expression of peptide compounds encoded by specific DNA fragments. Expression can be constitutive or controlled by an inducible (or repressible) promoter that allows for high-level transcription of a functionally related DNA fragment encoding a specific peptide compound.

[0171] In addition, the plasmids of the present invention may contain a selectable marker gene that allows for the selection of the transformed bacterial strain, for example, a gene that confers resistance to drugs such as chloramphenicol, erythromycin, kanamycin, neomycin, apramycin, tetracycline, zeocin, hygromycin, as well as ampicillin and other penicillin derivatives, such as carbenicillin. Also, the selectable marker may include biosynthetic genes, such as biosynthetic genes in the histidine, tryptophan and leucine biosynthetic pathways.

[0172] Appropriate polynucleotide sequences can be inserted into plasmids by a variety of procedures. Generally, the polynucleotide sequence is ligated into the desired position of the plasmid after digestion of the insert and the vector with an appropriate restriction endonuclease. Alternatively, blunt ends of both the insert and the vector can be ligated. A variety of cloning techniques are disclosed in Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc. 1997 and Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press (1989). Also, polynucleotide sequences can be cloned using homologous recombination techniques, including in vitro as well as in vivo recombination. Such procedures and others are considered to be within the scope of those skilled in the art.

[0173] The present invention further provides a recombinant host comprising a heterologous nucleic acid encoding a darobactin derivative of the present invention, namely a compound of formula (I), (Ia) or (Ia'). In certain embodiments, the recombinant host is a microorganism, such as a bacterium, such as Escherichia coli, Bacillus, Corynebacterium (e.g., Corynebacterium glutamicum), Lactobacillus (e.g., Lactococcus lactis) or Streptomyces (e.g., Streptomyces albus, Streptomyces lividans) species of bacteria, or a yeast, such as Saccharomyces cerevisiae, Saccharomyces pombe, Pichia pastoris or Yarrowia lipolytica. The heterologous nucleic acid can be part of a vector, such as a plasmid, contained in the microorganism. A vector, such as an expression vector, such as a plasmid, can be introduced into a microorganism, such as a bacterial host cell or a yeast host cell of the species described herein, by any of a variety of techniques known to those skilled in the art, including transformation or transfection. Also provided by the present invention is a bacterial host cell, i.e., a transformed cell, comprising at least one synthetic or recombinant nucleic acid sequence encoding a biosynthetic gene cluster for the production of a compound of formula (Ic) (or formula (Id) or (Ie)) or a plasmid of the present invention. The host cell can be any bacterial host cell known to those skilled in the art, such as an Escherichia coli or Lactobacillus cell, preferably an Escherichia coli cell, more preferably an Escherichia coli BL21(DE3) cell. The plasmid can be introduced into the bacterial host cell using any of a variety of techniques including transformation or transfection. Specific methods include calcium phosphate transfection, DEAE-dextran mediated transfection, lipofection or electroporation (Davis, L., Dibner, M., Battey, I., Basic Methods in Molecular Biology (1986)).

[0174] Optionally, the modified host cell can be cultured in a conventional nutrient medium that has been optionally modified for activation of the promoter, selection of transformants, or amplification of synthetic or recombinant nucleic acids utilized in the production method of the present invention. After transformation of a suitable host strain and growth of the host strain to an appropriate cell density, the selected promoter may be introduced by appropriate means (e.g., temperature change or chemical induction), and the cells can be cultured for a further period to produce the desired peptide compound or its derivative. Culture conditions, such as temperature, pH, etc., will be apparent to those skilled in the art, and examples are described herein. When cultured under suitable conditions, the host cell can produce darobactin A or a darobactin derivative, i.e., a peptide compound according to formula (Ic) (or (Ic') or (Ic'')), or any of the other compounds described herein (in particular, the darobactin derivatives of the present invention), which would not be produced or would be produced at a significantly lower level if the modifications according to the present invention were not made. The cells can be harvested by centrifugation and disrupted by physical or chemical means, and the resulting crude extract can be maintained for further purification. Microbial cells utilized for protein expression can be disrupted by any convenient method, including freeze-thaw cycles, sonication, mechanical disruption, or use of cell lysing agents. Such methods are well known to those skilled in the art. The expressed peptide compound or its derivative can be recovered and purified from the recombinant cell culture by methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography, and lectin chromatography. Optionally, high performance liquid chromatography (HPLC) can be used in the final purification step.

[0175] As used herein, the terms "protein," "polypeptide," and "peptide" refer to an organic compound consisting of two or more amino acid residues arranged in a linear or cyclic structure, wherein the individual amino acids in the organic compound are linked by peptide bonds, i.e., amide bonds formed between adjacent amino acid residues. By convention, the primary structure of a protein is reported to start from the amino terminus (N) to the carboxy terminus (C).

Brief Description of the Drawings

[0176]

Figure 1

Table 1

Figure 2

Examples

[0177] Preparation of the expression vector pNOSOdarA-E-DA The plasmid expression vector pNOSOdarA-E-DA (deposited with DSMZ on February 10, 2021; DSM33801) was prepared from the pNOSO vector and a synthetic sequence containing the BGC for the production of darobactin A. Both the synthetic sequence containing pNOSO and BGC were designed in silico and then chemically synthesized and constructed using cloning techniques based on restriction / ligation. The pNOSO vector contains p15Aori, a kanamycin resistance cassette (KanR), CEN6 / ARS4 (yeast ori), and URA3 (yeast counter-selection marker). The synthetic sequence contains the lactose-dependent repressor gene (lacI), the T7 promoter (PT7 )), the constitutive promoter (P ntpII ), the termination cassette (t D1 ) and the BGC of darobactin A. In addition, point mutations were introduced into pNOSO and the synthetic BGC to create unique R sites for cloning purposes. After chemical synthesis of the artificial synthetic sequence containing the vector pNOSO and the darobactin BGC, the pNOSOdarA-E-DA expression construct was prepared by restriction / ligation-based cloning techniques. The CEN6 / ARS4 sequence, the URA3 sequence and the relE gene were removed during the preparation of the plasmid expression vector pNOSOdarA-E-DA. The details of the vector construction are illustrated in Figure 1. For details of the R site, see Table 1 above.

[0178] Preparation of the artificial darA gene fragment and the plasmid expression vector In the preparation of a new artificial darobactin derivative with an amino acid sequence changed compared to darobactin A, the propeptide sequence of the darA gene was modified in silico by nucleotide and / or codon exchange. An artificial darA gene fragment containing the modified peptide sequence was chemically synthesized and cloned into pNOSOdarA-E-DA by using restriction / ligation-based cloning techniques to replace darA with the modified darA gene fragment. Using this strategy, plasmid expression vectors with the propeptide sequence of the darA gene changed were prepared (for example, from pNOSOdarA-E-DB to pNOSOdarA-E-DE, and from pNOSOdarA-E-D1 to pNOSOdarA-E-D17). Table 2 shows the modified propeptide DNA sequences of the artificial darA gene fragments and their corresponding propeptide amino acid sequences that can be used for the generation of darobactin derivatives.

[0179]

Table 2

[0180] The accuracy of the prepared plasmids was verified by agarose gel electrophoresis and DNA sequencing after restriction hydrolysis. The naming of the plasmid expression vectors corresponded to the nomenclature of the darobactin derivatives. For example, pNOSOdarA-E-D9 contained the propeptide sequence of darobactin D9 (i.e., WNWSKSW), and pNOSOdarA-E-D17 contained the propeptide sequence of darobactin derivative 17 (i.e., WNWSKSA). Both of these were deposited with the DSMZ on February 10, 2021; DSM33802 (pNOSOdarA-E-D9) and DSM33803 (pNOSOdarA-E-D17).

[0181] Heterologous expression and production For the heterologous production of darobactin A (DA) and darobactin derivatives, the above plasmid expression vectors were transformed into Escherichia coli BL21(DE3) cells by electroporation, and each transformed host cell was grown under controlled conditions as described below, for example.

[0182] Fermentation / culture of host cells Day 1 Seeding of a single clone of Escherichia coli BL21(DE3) cells containing the plasmid pNOSOdarA-E-DA or pNOSOdarA-E modified with the darA gene in 10 ml of LB medium containing 30 μg / ml of kanamycin (Kan30)

[0183] Day 2 Seeding of 50 ml of FM medium (1.254% K2HPO4, 0.231% KH2PO4, 0.4% D(+)-glucose, 0.1% NH4Cl, 1.2% yeast extract, 0.5% NaCl, and 0.0492% MgSO4(*7H2O); pH 7.1) containing Kan30 and 1 mg / l of vitamin B12 together with 1% (v / v) of the preculture Incubation at 30 °C and 180 rpm (Infors HT) for 3 days

[0184] Separation and maintenance of compounds Day 5 Collect the supernatant by centrifugation at 4°C for 10 minutes at 8,000 g. Incubate the supernatant with 0.05% XAD16N resin (Sigma Aldrich) (w / v) under stirring for 2 hours. Recover the XAD16N resin and elute the bound compounds from the XAD16N resin with 10 ml of 80% methanol under shaking for 1.5 hours. Dry the eluate in a rotary evaporator. Solubilize the evaporated extract in 0.5 ml of 50 / 50 methanol / water mixture. Verify the production of the target peptide compound, e.g., darobactin A or darobactin derivatives, by HPLC-MS analysis; the characteristic ions of darobactin in tandem MS mode are m / z 160.075 and prominent [M - H2O + 2H] 2+ ions.

[0185] In the case of the production of the compound for antimicrobial testing by the minimum inhibitory concentration assay described below, the antibiotic was not added to the expression culture as a selection marker.

[0186] HPLC purification was performed on a Waters Autopurifier (Eschborn, Germany) high-pressure gradient system equipped with a 2998 photodiode array detector linked to a 2545 binary gradient module, an SFO system fluid organizer, a 2767 sample manager, and a 3100 single quadrupole mass spectrometer operating in positive ion mode. The MS source and voltage settings were as follows: mass range, m / z 300 - 1000; scan time, 1 second; points per dalton, 4; capillary voltage, 3.5 kV; cone voltage, 30 V; extraction voltage 3 V; RF lens, 0.1 V; source temperature 120°C, desolvation temperature, 250°C; desolvation gas flow, 400 L / h; cone gas flow, 50 L / h; ion counting threshold, 30. Separation was performed on a Waters X-Bridge prep C-18 5 μm ODB, 150 × 19 mm column, using ACN + 0.1% FA as B and H2O + 0.1% FA as A, and a flow rate of 25 ml / min. Compound detection was at [M + 2H] 2+It was carried out by detection. The second purification step was performed on a Phenomenex Kinetex 5μm biphenyl 100A 250×21.2mm on the same instrument, using ACN + 0.1% FA as B, and H2O + 0.1% FA as A, and a flow rate of 25 ml / min. If necessary, a Waters CSH phenylhexyl 250mm×10mm 5μm dp column on a Dionex Ultimate 3000 SDLC low-pressure gradient system was used for further purification of the target peptide compound, with the eluate H2O + 0.1% FA as A, and ACN + 0.1% FA as B, a flow rate of 5 ml / min, and a column maintained at a constant temperature of 30°C. The target peptide compound, for example, darobactin A or a darobactin derivative, can be detected by UV absorption at 254 nm.

[0187] After the above protocol, darobactin A was heterologously produced, for example, by the expression of a modified artificial plasmid expression vector containing a propeptide sequence A (see, for example, Figure 2) in Escherichia coli BL21(DE3) cells and obtained as a slightly yellow solid.

[0188] Exemplary compounds Examples of exemplary compounds obtained by heterologous expression of the modified BGC according to the present invention are shown in Table 3 below.

[0189]

Table 3-1

Table 3-2

Table 3-3

Table 3-4

Table 3-5

Table 3-6

[0190] Evaluation of the Antimicrobial Activity of Darobactin A and Darobactin Derivative 9 All microorganisms were handled according to standard procedures recommended by the depositor. Strains were obtained from the German Collection of Cell Cultures and Microorganisms (Klebsiella pneumoniae DSM-30104, Acinetobacter baumannii DSM-30008), the American Type Culture Collection (Staphylococcus aureus ATCC-29213, Escherichia coli ATCC-25922, Enterococcus faecalis ATCC-29212), or were part of our in-house strain collection (Pseudomonas aeruginosa PAO1).

[0191] All samples were examined according to standard procedures, and the minimum inhibitory concentration (MIC) was established by the microbroth dilution method. Bacteria were grown on solid medium (CASO agar), single colonies were picked up by a sterile cotton swab, resuspended in saline (0.9% NaCl) to obtain a McFarland 0.5. The bacterial suspension was diluted 1:100 with cation-adjusted Mueller-Hinton broth to approximately 10 6A final inoculum of CFU / mL was obtained. Serial dilutions of the test sample were prepared in a total volume of 75 μL in a sterile 96-well microtiter plate with cation-adjusted Mueller-Hinton broth. The prepared cell suspension was added (75 μL), and the plate was incubated at either 30 °C or 37 °C under static conditions for at least 24 hours. The plate was then visually inspected, and the MIC was determined as the lowest concentration at which no visible growth was observed. Extracts of Escherichia coli BL21(DE3) cells containing sterile fermentation medium (FM medium) and the plasmid expression vector pNOSO without BGC were used as negative controls. The results of darobactin A and darobactin derivative 9 generated according to the present invention, compared with the data published by Imai et al. (already cited), are summarized in Table 4 below. The negative control showed no inhibitory effect against the test pathogenic strains (data not shown), thus confirming that the antimicrobial activity can be attributed to the test compounds.

[0192]

Table 4

[0193] As demonstrated above, darobactin A produced by the method of the present invention has at least the same antimicrobial activity against Gram-negative bacteria as that reported by Imai et al. Thus, the production method of the present invention provides a feasible and efficient method for selectively producing the target active compound in high yield. Moreover, the novel darobactin D9 has an activity comparable to or higher than that of darobactin A against the Gram-negative bacteria tested.

[0194] Evaluation of the Antimicrobial Activity of Darobactin Derivatives The crude extracts of the darobactin derivatives obtained by the above heterologous expression and separation protocols were tested for their activities against the exemplified Gram-negative bacteria by a minimum inhibitory concentration (MIC) assay. The presence of the darobactin derivatives in the crude extracts was verified by confirming the expected mass by LC-MS.

[0195] In the MIC assay, 10 μl of the crude extract of each test sample diluted with 50% methanol / H2O was added to the wells of the first column of a 96-well plate. After evaporating the methanol / H2O solvent until only approximately 4 μl of the solvent remained in each well of the first column, 71 μl of cation-adjusted Mueller-Hinton broth was added to each of the wells to make each well of the first column of the 96-well plate have a volume of 75 μl. Then, 75 μl of cation-adjusted Mueller-Hinton broth was added to each well to mix the samples contained in the first column with 75 μl of the medium / extract mixture. Serial dilutions of each mixture in the first column were prepared in the corresponding lower columns of the 96-well plate. The bacterial suspension of each strain to be tested was diluted 1:100 with cation-adjusted Mueller-Hinton broth to obtain a final inoculum of approximately 10 6 CFU / mL. 75 μl of each bacterial suspension to be tested was added to each crude extract sample in the wells of the 96-well plate, and the plate was incubated at either 30 °C or 37 °C under static conditions for at least 24 hours. The plate was then visually inspected, and the MIC was determined as the lowest concentration at which no visible growth was observed. To quantify the activity of the obtained darobactin derivatives, their relative activities against the activity of darobactin A were determined. The amount of darobactin derivative present in the extraction test sample was automatically calculated by DataAnalysis4.2 software and calculated based on the area under the curve (AUC) of the mass peak extracted from each LC-MS measurement value of the crude extract sample. The AUC of the mass peak of darobactin A (DA) was used as a calibration standard. That is, the ratio of the AUC of the darobactin derivative to that of darobactin A was correlated with the amount of the compound present in the sample. The results of the activities of the tested darobactin derivatives against the exemplified Gram-negative bacteria, when compared with the activity of darobactin A, are summarized in Table 5 below. A value of "+++" indicates that the derivative has the same antimicrobial activity as darobactin A, values from 0 (inactive) to "++" indicate lower activity than darobactin A, and values from "++++" to "++++++" indicate higher activity than darobactin A.

[0196]

Table 5

[0197] As demonstrated above, the darobactin derivatives have excellent antimicrobial activity against Gram-negative bacteria, and some show selectivity against specific pathogens.

[0198] Artificial darobactin 9 (D9) derivative In the preparation of a new artificial daptomycin 9 (D9) derivative with an altered amino acid sequence, the propeptide sequence of the darA gene was modified in silico by specific nucleotide and / or codon substitutions. Using the plasmid expression vector pNOSOdarA-E-D9 (DSM33802) containing the propeptide sequence of D9 (i.e., WNWSKSW) as a template, an artificial darA gene fragment containing the modified propeptide sequence was prepared by overlap extension polymerase chain reaction (OE-PCR). To insert the required mutations into the propeptide sequence, specific reverse primers (designated as pr2darXX_rv with XX indicating the number of the daptomycin derivative, e.g., pr2dar22_rv contains the substitutions (mismatches compared to the template sequence) required for the DNA propeptide core sequence of daptomycin derivative 22) were designed, which contain substitutions and complementary sequences with primer pr3dartrp_fw at their 5’ ends. The first DNA fragment was prepared using one of the primers pr1dar_fw and pr2darXX_rv. Primer pr1dar_fw binds to the region located in the T7lac promoter of the plasmid expression vector pNOSOdarA-E-D9 (DSM33802), and primer pr2darXX_rv binds to the core region of darA, contains the substitutions required to encode the desired daptomycin derivative, and has a 13-base pair region overlapping with primer pr3dartrp_fw. The second DNA fragment was prepared using primers pr3dartrp_fw and pr4dar_rv. Primer pr4dar_rv binds in front of the termination region at the end of the intergenic region between darA and darB of the plasmid expression vector pNOSOdarA-E-D9, and primer pr3dartrp_fw binds to the core region of darA. Then, each first DNA fragment (containing the mutations) was combined with the second DNA fragment, and a third PCR reaction was carried out with primers pr1dar_fw and pr4dar_rv, thereby generating a fusion fragment containing the modified darA propeptide core sequence and unique restriction sites at the 3’ and 5’ ends, respectively, for PCR-based overlap extension. The primer sequences are listed in Table 6, and the substitutions are shown in boldface.

[0199]

Table 6

[0200] The product of the 「repeated elongation」 PCR reaction was gel-purified, and the resulting DNA fragment was cloned into plasmid pNOSOdarA-E-D9 by replacing the sequence encoding darobactin derivative D9 with a modified darA gene fragment using a cloning technique based on restriction / ligation. Using this strategy, plasmid expression vectors with altered propeptide sequences of the darA gene were constructed (e.g., from pNOSOdarA-E-D22 to pNOSOdarA-E-D25, from pNOSOdarA-E-D30 to pNOSOdarA-E-D34, and from pNOSOdarA-E-D36 to pNOSOdarA-E-D39). Table 7 shows the modified propeptide DNA sequences of the modified darA gene fragments and their corresponding propeptide amino acid core sequences. Nucleotide substitutions compared to the D9-encoding darA core sequence are shown in bold. The naming of the plasmid expression vectors corresponds to the nomenclature of the darobactin derivatives; for example, pNOSOdarA-E-D22 contains the propeptide sequence of darobactin 22 (D22). The accuracy of the plasmid sequences was verified by agarose gel electrophoresis and Sanger sequencing at LGS genomics after restriction hydrolysis.

[0201]

Table 7

[0202] Heterologous expression and production For the heterologous production of darobactin derivatives, one of the above plasmid expression vectors, namely, pNOSOdarA-E-D22 to pNOSOdarA-E-D25, pNOSOdarA-E-D30 to pNOSOdarA-E-D34, and pNOSOdarA-E-D36 to pNOSOdarA-E-D39, was separately transformed into Escherichia coli BL21(DE3) cells by electroporation, and the respective recombinant strains were cultured to examine their darobactin production profiles as follows.

[0203] Fermentation / Culture of Host Cells Day 1 Seeding of a single clone of Escherichia coli BL21(DE3) cells containing the plasmid pNOSOdarA-E modified with the darA gene in 10 ml of LB medium containing 30 μg / ml of kanamycin (Kan30), and incubation overnight at 30 °C and 180 rpm (Infors HT)

[0204] Day 2 Seeding of 50 ml of FM medium (1.254% K2HPO4, 0.231% KH2PO4, 0.4% D(+)-glucose, 0.1% NH4Cl, 1.2% yeast extract, 0.5% NaCl, and 0.0492% MgSO4( * 7H2O); pH 7.1) containing Kan30 and 1 mg / l of vitamin B12 together with 1% (v / v) of the preculture Incubation at 30 °C and 180 rpm (Infors HT) for 3 days

[0205] Separation and Maintenance of Compounds Day 5 Recover the supernatant by centrifugation at 8,000 g for 10 minutes at 4 °C Incubate the supernatant with 0.05% XAD16N resin (Sigma Aldrich) (w / v) with stirring for 2 hours Recover the XAD16N resin and elute the bound compounds from the XAD16N resin with 10 ml of 80% methanol with shaking for 1.5 hours Dry the eluate in a rotary evaporator Solubilization of the evaporated extract in 0.5 ml of a 50 / 50 methanol / water mixture or 0.5 ml of 100% water HPLC-MS analysis to verify the production of the peptide compound of interest, for example, darobactin A or a darobactin derivative; characteristic ions of darobactin in tandem MS mode are m / z 160.075 and prominent [M-H2O+2H] 2+ ions.

[0206] In the case of the production of the compound for antimicrobial testing by the minimum inhibitory concentration assay described below, the antibiotic was not added to the expression culture as a selectable marker.

[0207] HPLC purification was performed on a Waters Autopurifier (Eschborn, Germany) high-pressure gradient system equipped with a 2545 binary gradient module, an SFO system fluid organizer, a 2767 sample manager, and a 2998 photodiode array detector operating in positive ion mode and connected to a 3100 single quadrupole mass spectrometer. The MS source and voltage settings were as follows: mass range, m / z 300 - 1000; scan time, 1 second; points per dalton, 4; capillary voltage, 3.5 kV; cone voltage, 30 V; extraction voltage 3 V; RF lens, 0.1 V; source temperature 120 °C, desolvation temperature, 250 °C; desolvation gas flow, 400 L / h; cone gas flow, 50 L / h; ion counting threshold, 30. Separation was carried out on a Waters X-Bridge prep C-18 5 μm ODB, 150×19 mm column, using ACN + 0.1% FA as B and H2O + 0.1% FA as A, and a flow rate of 25 ml / min. Compound detection was [M+2H] 2+It was carried out by detection. The second purification step was performed on a Phenomenex Kinetex 5μ biphenyl 100A 250×21.2 mm on the same equipment, using ACN + 0.1% FA as B, and H2O + 0.1% FA as A, and a flow rate of 25 ml / min. If necessary, a Waters CSH phenylhexyl 250 mm×10 mm 5μm dp column on a Dionex Ultimate 3000SDLC low-pressure gradient system was used for further purification of the target peptide compound, with eluent H2O + 0.1% FA as A, and ACN + 0.1% FA as B, a flow rate of 5 ml / min, and a column temperature of 30°C. The target peptide compound, for example, darobactin A or a darobactin derivative, can be detected by UV absorption at 254 nm.

[0208] After the above protocol, darobactin 22 was heterologously produced, for example, by the expression of a modified artificial plasmid expression vector pNOSOdarA-E-D22 containing the propeptide sequence D22 (see, for example, Table 2 above) in Escherichia coli BL21(DE3) cells.

[0209] Example compounds Examples of compounds obtained by heterologous expression of modified BGCs according to the present invention are shown in Tables 8 and 9 below. Darobactin derivatives having a sulfur-containing amino acid were found to possess a protecting group (as determined by nuclear magnetic resonance (NMR) spectroscopy).

[0210]

Table 8-1

Table 8-2

Table 8-3

Table 8-4

Table 8-5

Table 8-6

[0211]

Table 9A-1

Table 9A-2

[0212] Darobactins D32 to D34 and D32* to D34* shown in Table 9B can be prepared from the protected compounds PD32 to PD34* by following a standard protocol for the cleavage of disulfide bonds using dibutylamine (DTBA), tris(2-carboxyethyl)phosphine hydrochloride (TCEP-HCl), or mercaptoethanol or dithiothreitol (DTT). Such standard procedures are generally known in the art and are described, for example, by Kirley, Terence L. et al. (2016), Selective disulfide reduction for labeling and enhancement of Fab antibody fragments, Biochemical and Biophysical Research Communications 480(4), pp. 752-757 (DOI: 10.1016 / j.bbrc.2016.10.128); Lukesh, John C. et al. (2012), A Potent, Versatile Disulfide-Reducing Agent from Aspartic Acid, Journal of the American Chemical Society 134(9), pp. 4057-4059 (DOI: 10.1021 / ja211931f); and Yu, Shukun et al. (2020), Comparative study of protease hydrolysis reaction demonstrating Normalized Peptide Bond Cleavage Frequency and Protease Substrate Broadness Index, PLoS ONE 15(9), e0239080 (DOI: 10.1371 / journal.pone.0239080).

[0213]

Table 9B-1

Table 9B-2

[0214] Preparation of halogenated darobactin derivatives by supplementation with halogenated tryptophan during heterologous expression and production For the heterologous production of halogenated darobactin derivatives, for example, commercially available 5-chloro-L-tryptophan, 7-chloro-L-tryptophan, 6-fluoro-L-tryptophan or 7-fluoro-L-tryptophan can be periodically supplemented at various concentrations (the total final concentration can be, for example, 0.5 mM to 10 mM) to the fermentation broth during the cultivation of Escherichia coli BL21-Gold(DE3) (Agilent Technologies). Escherichia coli had been transformed with pNOSOdarA-E-DX. Here, X is an abbreviation of any expression plasmid prepared. The culture and supplementation periods were carried out as described below.

[0215] Fermentation / Cultivation of Host Cells Day 1 Seeding of a single clone of Escherichia coli BL21-Gold(DE3) cells (Agilent Technologies) containing the plasmid pNOSOdarA-E-DX modified with the darA gene in 10 ml of LB medium containing 30 μg / ml of kanamycin (Kan30), and incubation overnight at 30 °C, 180 rpm (Infors HT)

[0216] Day 2 Seeding of 20 ml of FM medium (1.254% K2HPO4, 0.231% KH2PO4, 0.4% D(+)-glucose, 0.1% NH4Cl, 1.2% yeast extract, 0.5% NaCl and 0.0492% MgSO4(*7H2O); pH 7.1) containing Kan30 and 1 mg / l of vitamin B12 together with 1% (v / v) of the preculture Preparation of a halogenated tryptophan stock solution by appropriate mass dissolution in dH20. For example, dissolve 10 mg of 6-fluoro-L-tryptophan in 1.2 ml of dH20 to reach an approximate final concentration of 2.1 mM in 20 mL of the production culture, and store the solution at 4 °C Add 0.4 μM (final concentration) of IPTG to an OD of 0.8 - 1.2600 Induce the T7lac promoter and perform the first replenishment with 1 / 6 (v / v) of the prepared halogenated tryptophan stock solution Two hours after the first replenishment, perform the second replenishment with 1 / 6 (v / v) of the prepared halogenated tryptophan stock solution

[0217] Day 3 Eighteen hours after the last replenishment, replenish with 1 / 6 (v / v) of the prepared halogenated tryptophan stock solution Six hours after the last replenishment, replenish with 1 / 6 (v / v) of the prepared halogenated tryptophan stock solution

[0218] Day 4 Eighteen hours after the last replenishment, replenish with 1 / 6 (v / v) of the prepared halogenated tryptophan stock solution Six hours after the last replenishment, replenish with 1 / 6 (v / v) of the prepared halogenated tryptophan stock solution and reach a total concentration of, for example, 0.5 mM to 10 mM according to the selected stock solution concentration Perform complete culturing at 30 °C and 180 rpm (Infors HT)

[0219] An alternative to the use of commercially available halogenated tryptophans for the supply to produce halogenated darobactin is the use of Escherichia coli pUC18-zeo-mx8-corP-trpAB that generates halogenated tryptophan from serine and halogenated indole. The vector pUC18-zeo-mx8-corP-trpAB was prepared from the trpAB genes of pUC18-zeo-mx8-corP (Pogorevc, D. et al Production optimization and biosynthesis revision of corallopyronin A, a potent anti-filarial antibiotic. Metab. Eng. 55, 201~211; 10.1016 / j.ymben.2019.07.010 (2019)) and pSTB7 37845 (ATCC) after restriction digestion with NsiI and NdeI, and then ligation was carried out according to standard molecular biology techniques. Co-expression of pNOSOdarA-E-DX and pUC18-zeo-mx8-corP-trpAB in Escherichia coli BL21-Gold(DE3) (Agilent Technologies) causes the production of halogenated tryptophan, for example, by the supply of 10 mM serine and 5~10 mM of the desired indole. This system is flexible and the trpAB gene can also be cloned into the darobactin BGC of upstream darE via unique restriction sites SpeI and PacI (R 11 and R 12 , shown in Table 1). The trpAB gene encodes tryptophan synthase TrpA and TrpB that catalyze the production of tryptophan from serine and indole. This enables the modification of darobactin with all commercially available or synthetically produced indoles that can be incorporated into darobactin during translation.

[0220] Separation and maintenance of compounds Day 5 In theory, after replenishing halogenated tryptophan, the appropriate halogenated tryptophan can be incorporated at each position where tryptophan is expected according to the genetic code. For example, derivatives of darobactin 9 having 6-fluoro-L-tryptophan can possess 6-fluoro-L-tryptophan at the 1st, 3rd, or 7th position, the 1st and 3rd, the 1st and 7th, the 3rd and 7th positions, or the 1st, 3rd, and 7th positions of each core peptide amino acid (see Figure 2). The nomenclature of such halogenated compounds follows the pattern of the position of the halogenated tryptophan in the amino acids of the core peptide after "darobactin derivative" (e.g., darobactin 9)-"position of fluorine on tryptophan according to IUPAC" (e.g., 6F of 6-fluoro-L-tryptophan). As an example, darobactin 9-6F1 represents darobactin 9 having 6-fluoro-L-tryptophan at the 1st position. In the case of multiple halogenated tryptophans, the positions are separated by hyphenation. For example, darobactin 9-6F1-7 represents darobactin 9 having 6-fluoro-L-tryptophan at the 1st and 7th positions. To confirm which new derivatives were generated, the compounds were extracted and analyzed by HPLC-MS and HPLC-MS-MS described in detail below.

[0221] The supernatant is recovered by centrifugation at 8,000 g for 10 minutes at 4 °C Incubation of the supernatant with 2 ml (w / v) of XAD16N resin (Sigma Aldrich) under stirring for 2 hours Recovery of the XAD16N resin and elution of the bound compounds from the XAD16N resin with 10 ml of 80% methanol under shaking for 1.5 hours Drying of the eluate in a rotary evaporator Solubilization of the evaporated extract in 0.2 ml of 50 / 50 methanol / water mixture or 0.2 ml of 100% water HPLC-MS analysis to verify the production of the peptide compound of interest, e.g., darobactin 9-F1 or a darobactin derivative; the characteristic ions of darobactin in tandem MS mode are m / z 160.075 and prominent [M-H2O+2H] 2+ ions.

[0222] As an example, the production of the darobactin 9-6F derivative was carried out by supplementing 6-fluoro-L-tryptophan during heterologous expression and production according to the described protocol. According to HPLC-MS and MS-MS analysis, darobactin 9-6F1, darobactin 9-6F7, and darobactin 9-6F1-7 were produced in large amounts. In some embodiments, preferably, darobactin 9-6F1 and 9-6F7, particularly darobactin 9-6F1, were produced. Further examples of the production of the halogenated darobactin derivatives described herein are darobactin 9-7F1, darobactin 9-7F7, darobactin 9-5Cl1, and 9-5Cl7. Confirmation of production was achieved by the mass, isotope pattern, and MS-MS fragmentation pattern showing the incorporation of halogenated tryptophan, either after or before purification by the described procedure.

[0223] Purification of Halogenated Darobactin HPLC purification was performed on a Waters Autopurifier (Eschborn, Germany) high-pressure gradient system equipped with a 2545 binary gradient module, an SFO system fluid organizer, a 2767 sample manager, and a 2998 photodiode array detector connected to a 3100 single quadrupole mass spectrometer operating in positive ion mode. The MS source and voltage settings were as follows: mass range, m / z 300 - 1000; scan time, 1 second; points per dalton, 4; capillary voltage, 3.5 kV; cone voltage, 30 V; extraction voltage 3 V; RF lens, 0.1 V; source temperature 120 °C, desolvation temperature, 250 °C; desolvation gas flow, 400 L / h; cone gas flow, 50 L / h; ion count threshold, 30. Separation was performed on a Waters XBridge® PrepC-18 5 μm ODB™, 150 × 19 mm column, using ACN + 0.1% FA as B, and H2O + 0.1% FA as A, and a flow rate of 25 ml / min. Compound detection was performed by the detection of 2+ [M + 2H].

[0224] The second purification step was performed on a Waters XSelect® peptide CSH C18 OBD™ Prep 130 Å, 5 μm, 10 mm × 250 mm column on a Thermo Scientific™ Dionex™ UltiMate™ 3000 SDLC low-pressure gradient system. The eluate was used with H2O + 0.1% FA as A, and ACN + 0.1% FA as B, at a flow rate of 6 ml / min, and a column maintained at a constant temperature of 45 °C was used for further purification of the target peptide compound. The target peptide compound, for example, darobactin 9-6F1, can be detected by UV absorption at 254 nm or 280 nm.

[0225] Two isomers, darobactin 9-6F1 and darobactin 9-6F7, eluted very similarly to each other. The first eluting isomer is the relatively minor 6F7 variant, followed by darobactin 9-6F1, which is the major isomer. Examples of compounds obtained by heterologous expression and production of modified BGC according to the present invention are shown in Table 10 below.

[0226]

Table 10-1

Table 10-2

[0227] Evaluation of the antimicrobial activity of darobactin derivatives The crude extracts containing each darobactin derivative obtained by the above heterologous expression and purification protocol were examined in a standard microbroth dilution assay for their activity against the exemplified Gram-negative bacteria. The presence of darobactin derivatives in the crude extracts was verified by confirming the mass predicted by LC-HRMS.

[0228] All microorganisms were handled according to standard procedures recommended by the depositor. Strains were obtained from the German Collection of Cell Cultures and Microorganisms (Klebsiella pneumoniae DSM-30104, Acinetobacter baumannii DSM-30007 and Acinetobacter baumannii DSM-30008), the American Type Culture Collection (Escherichia coli ATCC-25922), or were part of our in-house strain collection (Pseudomonas aeruginosa PAO1, PA14 and PA14ΔmexAB).

[0229] In the MIC assay, 10 μl of the crude extract of each test sample diluted with 50% methanol / H2O was added to the wells of the first column of a 96-well plate. After evaporating the methanol / H2O solvent until only about 4 μl of the solvent remained in each well of the first column, 71 μl of cation-adjusted Mueller-Hinton broth medium was added to each of the wells so that each well of the first column of the 96-well plate had a volume of 75 μl. Then, 75 μl of cation-adjusted Mueller-Hinton broth medium was added to each well to mix the samples contained in the first column with 75 μl of the medium / extract mixture. Serial dilutions of each mixture were prepared from 150 μl of the mixture in the first column into the corresponding lower columns of the 96-well plate. The bacterial suspension of each strain to be tested was diluted 1:100 with cation-adjusted Mueller-Hinton broth to approximately 10 6A final seeding material of CFU / mL was obtained. 75 μL of each bacterial suspension to be tested was added to each crude extraction sample in the wells of a 96-well plate, and the plate was incubated at either 30 °C or 37 °C for at least 24 hours under static conditions. The plate was then visually inspected, and the MIC was determined as the lowest concentration at which no visible growth was observed. To quantify the activity of the obtained darobactin derivatives, their relative activities with respect to the activity of darobactin A were determined. The amount of darobactin derivatives present in the extraction test samples was automatically calculated by Data Analysis 4.2 software and calculated based on the area under the curve (AUC) of the mass peak extracted from each LC-MS measurement value of the crude extraction samples. The concentration factor of the test extract was calculated by dividing the concentration (100× resulting from the extraction process from 50 mL of the culture to 0.5 mL of the extract) by the dilution factor at which no visible growth of each strain was observed anymore. For example, if the crude extraction containing darobactin derivatives in column 1 (of a standard 96-well plate) caused growth inhibition of each strain from well A1 to E1 but no longer in F1, a concentration factor score of 0.42× was assigned to the darobactin derivative. If the crude extraction containing darobactin derivatives in column 2 caused growth inhibition only from well A2 to B2 but no longer in C2, we assigned a concentration factor score of 3.34×, which means that the crude extraction in column 1 showed strong antibacterial activity (assuming similar compound concentrations).

[0230] The results of the activities of the tested darobactin derivatives against the exemplified Gram-negative bacteria, when compared with the activities of darobactin A and D9, are summarized in Table 11 below.

[0231]

Table 11-1

Table 11-2

[0232] Selected examples (darobactin 9, 22, 23, 22 *, 23 * ) was tested for these antibacterial activities in the above microbroth dilution assay. The compounds were tested by serial dilution (0.03 - 64 μg / mL). The determined MICs are shown in Table 12, which demonstrates the excellent activities of the new derivatives against Acinetobacter baumannii and Pseudomonas aeruginosa.

[0233]

Table 12

[0234] The activity data of the exemplified darobactin derivatives 31, 32, 36 - 38, 32*, 36* - 38*, 9 - 6F1 and 9 - 6F7 are summarized in Table 13 below. The compounds were tested for their antibacterial activities in the above microbroth dilution assay. In particular, the compounds were tested by serial dilution (0.03 - 64 μg / mL). The determined MICs demonstrate comparable or excellent microbial activities of the new derivatives.

[0235]

Table 13

[0236] As demonstrated above, the darobactin derivatives described herein have excellent antimicrobial activities against Gram - negative bacteria, and some show selectivity against specific pathogens. The antibacterial activities of specific darobactin derivatives are superior to clinical isolates of Acinetobacter baumannii, as shown in Table 14 below.

[0237]

Table 14

Deposit Number

[0238] DSM33798 DSM33799 DSM33801 DSM33802 DSM33803 DSM30104 DSM30008 ATCC29213 ATCC25922 ATCC29212 DSM30007

Claims

1. Formula (I): 【Chemical 1】 [wherein, R １ 、 R ２ and R ４ are H, CH ３ 、 CH ２ OH, the following groups: 【Chemical 2】 -CH ２ -SR １Ａ (wherein, R １Ａ is independently selected from an alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, alkylcycloalkyl, heteroalkylcycloalkyl, aryl, heteroaryl, aralkyl or heteroaralkyl group, and all of these groups may optionally be substituted), or -CH ２ -Ind (wherein Ind is an optionally substituted indole group), independently selected from among one of R ３ is H, OH, SH, COOH, CONH ２ , the following groups: [Chemical Formula 3] -SR ３Ａ (wherein R ３Ａ is selected from an alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, alkylcycloalkyl, heteroalkylcycloalkyl, aryl, heteroaryl, aralkyl or heteroaralkyl group, all of these groups being optionally substituted), or -Ind (wherein Ind is an optionally substituted indole group) is selected from one of R ５ is a group of the formula -CH ２ -Ind (wherein Ind is an optionally substituted indole group), and R ６ is a hydrogen atom or a methyl group, R ７ is a hydrogen atom, a halogen atom, a hydroxy group or a methoxy group, R ８ is a halogen atom, m is an integer from 0 to 3, R ６Ａ is a hydrogen atom or a methyl group, R ７Ａ is a hydrogen atom, a halogen atom, a hydroxy group or a methoxy group, R ８Ａ is a halogen atom, p is an integer from 0 to 3, and the group of -CH2-Ind is the following formula: 【Chemical Formula 5】 (wherein R9 is a hydrogen atom, a halogen atom, a hydroxy group or a methoxy group, R10 is a hydrogen atom or a methyl group, R11 is independently selected from a halogen atom, a hydroxy group or a methoxy group, and n is an integer from 0 to 4) is a group of] the compound or a salt thereof.

2. The compound or a salt thereof according to Claim 1, wherein R9 is a hydrogen atom or a halogen atom, R10 is a hydrogen atom, R11 is a halogen atom, and n is an integer from 0 to 4.

3. -CH ２ -SR １Ａ The group of 【Chemical Formula 6】 The compound or a salt thereof according to Claim 1 or 2, which is

4. R １ is the following group: -CH ２ -CONH ２ -, -CH ２ -OH, -CH ２ -CH ２ -CONH ２ and -CH ２ -CH(CH ３ ), the compound or a salt thereof according to any one of claims 1 to 3, selected from

5. R ２ is the following group: -CH ２ -OH and -CH ３ , or the following group: [Chemical Formula 7] The compound or a salt thereof according to any one of Claims 1 to 4, which is selected from one of

6. R ３ is a hydrogen atom or the following group: -CH ２ -CH ２ -CH ２ -NH ２ and -CH ２ -CH ２ -NH-C(=NH)-NH ２ The compound according to any one of claims 1 to 5, or a salt thereof, selected from

7. R ４ is the following group: -CH ２ -OH, -CH ３ and -CH ２ -CH ２ -CH ２ -NH-C(=NH)-NH ２ ; the following groups: [Chemical Formula 8] which is selected from one of R ４ is a group of formula -CH ２ -SR ４Ａ (wherein R ４Ａ is selected from a hydrogen atom, alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, alkylcycloalkyl, heteroalkylcycloalkyl, aryl, heteroaryl, aralkyl or heteroaralkyl group, all of these groups being optionally substitutable), the compound or a salt thereof according to any one of claims 1 to 6.

8. R ５ is given by the following formula: 【Chemical Formula 10】 [wherein, R ９ is a hydrogen atom, R １０ is a hydrogen atom, R １１ is a halogen atom, and n is an integer of 0 to 4] The compound or a salt thereof according to any one of Claims 1 to 7, which is a group of

9. R ６ 、 R ６Ａ 、 R ７ and R ７Ａ or a salt thereof, wherein each of R ６ 、 R ６Ａ 、 R ７ and R ７Ａ is a hydrogen atom, of the compound according to any one of claims 1 to 8

10. R ８ and / or R ８Ａ The compound or a salt thereof according to any one of claims 1 to 9, wherein R and / or R is a halogen atom.

11. Structural formula (Ia): 【Chemical Formula 11】 (wherein, R １ , R ２ , R ３ , R ４ , R ５ , R ６ , R ７ , R ８ , R ６Ａ , R ７Ａ , R ８Ａ , m and p are as defined in any one of the preceding claims) The compound or a salt thereof according to any one of Claims 1 to 10, which has

12. The compound or a salt thereof according to any one of Claims 1 to 11, and optionally containing one or more carrier substances and / or one or more adjuvants and / or one or more further active pharmaceutical ingredient(s), a pharmaceutical composition.

13. The pharmaceutical composition according to Claim 12, for use as a medicament.

14. The pharmaceutical composition according to Claim 12, for use in the prevention or treatment of bacterial infections.

15. The pharmaceutical composition according to Claim 12, for use in the prevention or treatment of bacterial infections caused by Gram-negative bacteria.

16. A method for producing the compound according to any one of Claims 1 to 11, wherein the compound is of formula (II): 【Chemical Formula 12】 {wherein, R ２ and R ４ are each independently H, CH ３ , CH ２ OH or a group: 【Chemical 13】 represents R ３ is the base: 【Chemical 14】 and R ５ is the following formula: 【Chemical Formula 15】 [wherein, R ９ is a hydrogen or halogen atom, R １０ is a hydrogen atom or a methyl group, R １１ is a halogen atom, and n is an integer of 0 to 4], and R8 is a halogen atom, m is an integer from 0 to 3, R8A is a halogen atom, m is an integer from 0 to 3} is a compound of the method is (a) Preparing a recombinant host capable of producing the compound of formula (II), wherein the recombinant host contains at least one synthetic or recombinant nucleic acid encoding the biosynthetic gene cluster (BGC) of the compound, and the BGC is (i) at least 95%, 96%, 97%, 98%, 98.5%, 99% or 99.5% to 100% sequence identity to the full-length sequence of SEQ ID NO: 86, or (ii) a sequence completely complementary to any nucleic acid sequence of (i) having, a step; (b) culturing the recombinant host for a time sufficient for the recombinant host to produce the compound of formula (II); (c) isolating the compound from the recombinant host or the culture supernatant, thereby producing the compound of formula (II) and A method comprising:

17. A recombinant host comprising a heterologous nucleic acid encoding the compound of formula (II) according to claim 16.

18. A microorganism, bacterium or yeast, such as a species of Escherichia coli, such as Escherichia coli BL21(DE3) (in particular DSM 33798 and / or DSM 33799); Corynebacterium, such as Corynebacterium glutamicum; Bacillus; Lactobacillus, such as Lactococcus lactis; or Streptomyces, such as Streptomyces albus and Streptomyces lividans species of bacterial cells, or Saccharomyces cerevisiae, Saccharomyces pombe, Pichia pastoris or Yarrowia lipolytica species of yeast cells, the recombinant host according to claim 17.

19. A vector comprising at least one nucleic acid defined in claim 16, which is an isolated and purified plasmid capable of self-replicating in bacteria or yeast.

20. A vector comprising at least one nucleic acid defined in claim 16, which is an isolated and purified plasmid capable of self-replicating in bacteria.

21. A vector comprising at least one nucleic acid defined in claim 16, which is an isolated and purified plasmid capable of self-replicating in bacteria of the genus Lactobacillus.

22. A vector comprising at least one nucleic acid defined in claim 16, which is an isolated and purified plasmid capable of self-replicating in Lactococcus actis.

23. A vector comprising at least one nucleic acid defined in claim 16, which is an isolated and purified plasmid capable of self-replicating in bacteria of Escherichia coli.

24. A vector comprising at least one nucleic acid defined in claim 16, which is an isolated and purified plasmid capable of self-replicating in the species of Escherichia coli.

25. A vector comprising at least one nucleic acid defined in claim 16, which is an isolated and purified plasmid capable of self-replicating in bacteria of DSM 33802 and / or DSM 33803.

26. Use of a compound according to any one of claims 1 to 11 or a pharmaceutically acceptable salt thereof for the preparation of a medicament.

27. Use of a compound according to any one of claims 1 to 11 or a pharmaceutically acceptable salt thereof for the preparation of a medicament for the prevention or treatment of a bacterial infection.

28. Use of a compound according to any one of claims 1 to 11 or a pharmaceutically acceptable salt thereof for the preparation of a medicament for the prevention or treatment of a bacterial infection caused by Gram-negative bacteria.

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