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WO2024209005A1 - Conjugation of catechol containing siderophore with cargo molecules - Google Patents

Conjugation of catechol containing siderophore with cargo molecules Download PDF

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Publication number
WO2024209005A1
WO2024209005A1 PCT/EP2024/059245 EP2024059245W WO2024209005A1 WO 2024209005 A1 WO2024209005 A1 WO 2024209005A1 EP 2024059245 W EP2024059245 W EP 2024059245W WO 2024209005 A1 WO2024209005 A1 WO 2024209005A1
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Prior art keywords
group
conjugate
siderophore
cargo
het
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French (fr)
Inventor
Ruben Christiaan HARTKOORN
Thibault CARADEC
Ravil PETROV
Ernesto ANOZ CARBONELL
Xavier TRIVELLI
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Centre National de la Recherche Scientifique CNRS
Institut Pasteur de Lille
Institut National de la Sante et de la Recherche Medicale INSERM
Centre Hospitalier Universitaire de Lille
Institut Pasteur
Universite de Lille
Original Assignee
Centre National de la Recherche Scientifique CNRS
Institut Pasteur de Lille
Institut National de la Sante et de la Recherche Medicale INSERM
Centre Hospitalier Universitaire de Lille
Institut Pasteur
Universite de Lille
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Publication of WO2024209005A1 publication Critical patent/WO2024209005A1/en
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/55—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound the modifying agent being also a pharmacologically or therapeutically active agent, i.e. the entire conjugate being a codrug, i.e. a dimer, oligomer or polymer of pharmacologically or therapeutically active compounds
    • A61K47/552—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound the modifying agent being also a pharmacologically or therapeutically active agent, i.e. the entire conjugate being a codrug, i.e. a dimer, oligomer or polymer of pharmacologically or therapeutically active compounds one of the codrug's components being an antibiotic
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
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    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/55—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound the modifying agent being also a pharmacologically or therapeutically active agent, i.e. the entire conjugate being a codrug, i.e. a dimer, oligomer or polymer of pharmacologically or therapeutically active compounds
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    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
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    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
    • A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
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    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
    • A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
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    • C07D223/06—Heterocyclic compounds containing seven-membered rings having one nitrogen atom as the only ring hetero atom not condensed with other rings with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
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    • C07D277/10—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings having one double bond between ring members or between a ring member and a non-ring member with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached to ring carbon atoms
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    • C07D491/12—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00 in which the condensed system contains three hetero rings
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    • C07D499/44—Compounds with an amino radical acylated by carboxylic acids, attached in position 6
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    • C07D499/58—Compounds with an amino radical acylated by carboxylic acids, attached in position 6 with a carbon chain, substituted by hetero atoms or by carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, attached to the carboxamido radical substituted in alpha-position to the carboxamido radical
    • C07D499/64—Compounds with an amino radical acylated by carboxylic acids, attached in position 6 with a carbon chain, substituted by hetero atoms or by carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, attached to the carboxamido radical substituted in alpha-position to the carboxamido radical by nitrogen atoms

Definitions

  • the present invention relates to conjugates of catechol-based siderophores with cargo molecules, such as antibiotics or fluorophores, and to the preparation and uses thereof.
  • the bacterial outer cell wall in particular the one of some species of bacteria, such as Gramnegative bacteria, provides a daunting barrier to exogenic compounds.
  • the permeability properties of this barrier have a major impact on the susceptibility of the bacteria to antibiotics. Indeed, by blocking the penetration of antibiotics, some bacteria are intrinsically resistant to antibiotics. Overcoming the low-permeability barrier of bacteria remains a challenge in the development of new antibiotics.
  • One method for overcoming the issue of penetration into bacteria is to hijack the active transport mechanisms of the bacteria, in particular the active transport of iron.
  • siderophores-antibiotic conjugates have been developed for “Trojan horse” antibiotic delivery.
  • the active transport of iron is fundamental to bacterial survival.
  • bacteria produce siderophores. These organic compounds chelate and scavenge iron from the host's body and are then actively transported into the bacteria.
  • a large number of bacterial siderophores are known. They generally contain structures like hydroxamates, catechols, carboxylates, and various heterocycles.
  • antibiotics i.e. siderophores-antibiotic conjugates, such as sideromycins, are able to bypass this penetration barrier by hijacking the active siderophore: iron uptake systems of bacteria for their own active uptake and delivering the drug inside the target bacterial cell.
  • sideromycins a small number of natural sideromycins have been described, namely, albomycin, ferrimycin, microcin E492m and salmycin.
  • the present invention relates to a conjugate comprising a siderophore moiety and a cargo moiety, wherein the siderophore moiety comprises a siderophore (Sid) bearing a substituted or unsubstituted catechol group (Cat), said Cat group having the following formula: with Ri and R 2 being independently selected from the group consisting of a hydrogen atom, a Ci-C 6 alkyl group, a C 2 -C 6 alkenyl group, a Ci-C 6 alkoxy group, a halogen atom, a Ci-C 6 hydroxyalkyl group, a hydroxy-C 2 -C 6 alkyloxy group, an aryl group, a heteroaryl group, a non- aromatic heterocycle, a Ci-C 6 alky l-ary I and an aryl-Ci-C 6 alkyl group, preferably a hydrogen atom, a C1-C4 alkyl group or a halogen atom, more preferably a
  • the present invention relates to a process for preparing a conjugate as disclosed herein.
  • the process comprises the step of reacting a siderophore bearing a catechol group of the following formula: with R1 and R 2 being independently selected from the group consisting of a hydrogen atom, a Ci-C 6 alkyl group, a C 2 -C 6 alkenyl group, a Ci-C 6 alkoxy group, a halogen atom, a Ci-C 6 hydroxyalkyl group, a hydroxy-C 2 -C 6 alkyloxy group, an aryl group, a heteroaryl group, a nonaromatic heterocycle, a Ci-Ce alky l-ary I and an aryl-Ci-Ce alkyl group, preferably a hydrogen atom, a C1-C4 alkyl group or a halogen atom, more preferably a hydrogen atom or a halogen atom; with a cargo molecule bearing a substituted or unsub
  • the invention also relates to a composition comprising the conjugate of the invention and an acceptable excipient.
  • the invention also relates to a conjugate or composition as disclosed herein for use in medicine, in particular in the treatment of a bacterial infection or for use in the diagnosis of a bacterial infection.
  • Figure 1 Fluorescence microscopy of D. fulvum cultivated into iron-limiting conditions (in presence of 350 pM of DIP) and treated with 1 pM of TAMRA3515 (A) and chlorodactyloferrin-TAMRA3515 conjugate (B).
  • C D. fulvum was treated with chlorodactyloferrin-TAMRA3515 (1 pM) together with unlabelled chlorodactyloferrin (100 pM).
  • Figure 2 Fluorescence microscopy of D. aurantiacum cultivated into iron-limiting conditions (in presence of 350 pM of DIP) and treated with 1 pM of TAMRA3515 (A) and chlorodactyloferrin-TAMRA3515 conjugate (B).
  • Figure 3 Fluorescence microscopy of D. matsukiense cultivated into iron-limiting conditions (in presence of 350 pM of DIP) and treated with 1 pM of TAMRA3515 (A) and chlorodactyloferrin-TAMRA3515 conjugate (B).
  • Figure 4 Fluorescence microscopy of D. roseum cultivated into iron-limiting conditions (in presence of 350 pM of DIP) and treated with 1 pM of TAMRA3515 (A) and chlorodactyloferrin-TAMRA3515 conjugate (B).
  • Figure 5 Fluorescence microscopy of D. vinaceum cultivated into iron-limiting conditions (in presence of 350 pM of DIP) and treated with 1 pM of TAMRA3515 (A) and chlorodactyloferrin-TAMRA3515 conjugate (B).
  • Figure 6 Fluorescence microscopy of S. coelicolor cultivated into iron-limiting conditions (in presence of 350 pM of DIP) and treated with 1 pM of TAMRA3515 (A) and chlorodactyloferrin-TAMRA3515 conjugate (B).
  • Figure 7 Fluorescence microscopy of S. subtilis cultivated into iron-limiting conditions (in presence of 350 pM of DIP) and treated with 1 pM of TAMRA3515 (A) and chlorodactyloferrin-TAMRA3515 conjugate (B).
  • Figure 8 Fluorescence microscopy of E. coli WT (A) and AfepA (B) cultivated into iron-limiting conditions (in presence of 100 pM of DIP) and treated with 1 pM of enterobactin-TAMRA3515 conjugate.
  • Figure 9 Fluorescence microscopy of iron-deprived A. baumannii (in presence of 100 pM of DIP) treated with 1 pM TAMRA3515 (A) and 1 pM acinetobactin-TAMRA3515 conjugate (B).
  • Figure 10 Schematic representation of a conjugate according to the present invention.
  • FIG. 11 (A) Disk diffusion assay of RPII151 alone and that of the reaction mixture following conjugation of enterobactin and RPII151 (using iodine) on E. coli grown on high and low iron solid media (M9 and M9 + 100 pM DIP). These data show a zone of growth inhibition only for the reaction mixture when bacteria were grown under low iron conditions. (B) A HPLC-MS UV chromatogram of the product of the enterobactin/RPII151 iodine based conjugation reaction showing remaining residual, RPII151 as well as the peak of the enterobactin-RPII151 conjugate 24.
  • the present invention stems from the identification and isolation of a novel sideromycin-like molecule from a bacterial culture, more specifically from the identification of a conjugate between a newly identified catecholate-based siderophore (called herein chlorodactyloferrin) and pyridomycin (see section “EXAMPLES”). Based on their findings, the inventors have designed a new series of sideromycin-like molecules that were found to be actively delivered into bacteria by hijacking the siderophore uptake systems. Thanks to their unique structure, i.e.
  • the proposed conjugates allow the delivery of molecules of interest (called herein “cargo”), such as for instance therapeutic agents or fluorophores, to a wide spectrum of pathogenic and non-pathogenic bacteria that naturally produce and/or use catecholate-containing siderophores for iron uptake.
  • Cargo molecules of interest
  • the present invention relates to a conjugate comprising a siderophore moiety and a cargo moiety, wherein the siderophore moiety comprises a siderophore (Sid) bearing a substituted or unsubstituted catechol group (Cat), said Cat group having the following formula: with Ri and R 2 being independently selected from the group consisting of a hydrogen atom, a Ci-Ce alkyl group, a C2-C6 alkenyl group, a Ci-Ce alkoxy group, a halogen atom, a Ci-Ce hydroxyalkyl group, a hydroxy-C 2 -C 6 alkyloxy group, an aryl group, a heteroaryl group, a nonaromatic heterocycle, a Ci-C 6 alky l-ary I and an aryl-Ci-C 6 alkyl group, preferably a hydrogen atom, a C1-C4 alkyl group or a halogen atom, more preferably a
  • the siderophore moiety comprises a siderophore (Sid) bearing a substituted or unsubstituted catechol group (Cat) and the cargo moiety comprises a cargo molecule (Cargo) bearing a substituted or unsubstituted heteraryl group (Het).
  • the Het group comprises one or more nitrogen atoms.
  • the siderophore moiety and cargo moiety are covalently linked through the Cat and Het groups.
  • conjugate refers to a compound or construct comprising the above-mentioned elements, i.e. a siderophore moiety and a cargo moiety, which are coupled, i.e. , conjugated or bonded, to each other via covalent interactions.
  • halogen refers to fluorine, chlorine, bromine or iodine.
  • Ci-C 6 alkyl refers to a saturated, branched or straight hydrocarbon chain comprising from one to six carbon atoms, such as methyl, ethyl, n-propyl or isopropyl, n-butyl, sec-butyl, iso-butyl, tert-butyl, n-pentyl, isopentyl, n-hexyl.
  • C2-C 6 alkenyl designates a branched or straight hydrocarbon chain comprising from two to six carbon atoms and containing one or more double bonds, including di-enes, tri-enes, such as vinyl, propen-1-yl, propen-2-yl, but-1-en-1-yl, but-1-en-2- yl, but-2-en-1-yl.
  • Ci-C 6 hydroxyalkyl designates a Ci-C 6 alkyl group as defined above, which Ci-C 6 alkyl group is substituted with one or more hydroxy groups.
  • Examples of Ci-Ce hydroxyalkyl groups include 2-hydroxy-ethyl, 3-hydroxy-propyl, 4-hydroxy-butyl, 5- hydroxy-pentyl and 6-hydroxy-hexyl.
  • Ci-C 6 alkoxy designates a radical — O-Ci-C 6 alkyl which Ci-C 6 alkyl group is as disclosed above.
  • Examples of Ci-C 6 alkoxy include methoxy, ethoxy, propyloxy, /so-propyloxy, butyloxy, /so-butyloxy, terf-butyloxy, sec-butyloxy, pentyloxy, iso- pentyloxy, hexyloxy.
  • hydroxy-C 2 -C 6 alkyloxy designates a C 2 -C 6 alkoxy, which C 2 -C 6 alkoxy group is substituted with one or more hydroxy groups.
  • aryl refers to aromatic carbocyclic groups having a single ring (e.g., phenyl), multiple rings (e.g., biphenyl), or multiple fused rings in which at least one is aromatic (e.g., 1 ,2,3,4-tetrahydronaphthyl, naphthyl, anthryl, or phenanthryl).
  • non-aromatic carbocycle refers to cyclic groups, saturated or unsaturated, containing carbon atoms as ring atoms.
  • heteroaryl refers to aryl groups as defined herein comprising at least one heteroatom as a ring atom. Suitable heteroatoms include oxygen, sulfur, nitrogen, phosphorus, selenium and the like.
  • a heteroaryl is a cyclic aromatic radical having from five to ten ring atoms of which at least one ring atom is selected from S, O, and N; zero, one, or two ring atoms are additional heteroatoms independently selected from S, O, and N; and the remaining ring atoms are carbon.
  • non-aromatic heterocycle refers to refer to cyclic groups, saturated or unsaturated, containing at least one heteroatom as a ring atom, in some cases, 1 to 3 heteroatoms as ring atoms, with the remainder of the ring atoms being carbon atoms. Suitable heteroatoms include oxygen, sulfur, nitrogen, phosphorus, selenium and the like. In some cases, the heterocycle may be 3- to 10-membered ring structures or 3- to 7-membered rings, in which the ring structures include one to four heteroatoms.
  • Ci-C 6 alkyl-aryl refers to a Ci-C 6 alkyl as defined herein substituted by an aryl as defined herein.
  • aryl-Ci-C 6 alkyl refers to an aryl as defined herein substituted by a Ci-C 6 alkyl as defined herein.
  • the present invention encompasses hydrates, solvates, or salts of the herein disclosed conjugates.
  • Preferred salts in the context of the present invention are physiologically acceptable salts of the conjugates.
  • the invention also encompasses salts which themselves are unsuitable for pharmaceutical applications but which can be used, for example, for the isolation or purification of the compounds according to the invention.
  • physiologically acceptable salt refers to a relatively non-toxic, inorganic or organic addition salt of the conjugates.
  • a suitable pharmaceutically acceptable salt may be, for example, an acid-addition salt of a compound of the conjugates, such as an acid-addition salt with an inorganic acid, such as hydrochloric, hydrobromic, hydroiodic, sulfuric, bisulfuric, phosphoric, or nitric acid, for example, or with an organic acid, such as formic, acetic, acetoacetic, pyruvic, trifluoroacetic, propionic, butyric, hexanoic, heptanoic, undecanoic, lauric, benzoic, salicylic, 2-(4-hydroxybenzoyl)-benzoic, camphoric, cinnamic, cyclopentanepropionic, digluconic, 3 -hydroxy-2 -naphthoic, nicotinic, pamoic, pectin
  • Solvates in the context of the invention are described as those forms of the compounds which form a complex in the solid or liquid state by coordination with solvent molecules. Hydrates are a specific form of the solvates in which the coordination is with water.
  • the present invention includes all possible stereoisomers of the conjugate of the present invention as single stereoisomer, or as any mixture of said stereoisomers, in any ratio.
  • Isolation of a single stereoisomer, e.g. a single enantiomer or a single diastereomer, of a conjugate can be achieved by any suitable state of the art method, such as chromatography, especially chiral chromatography, for example.
  • the siderophore moiety which is part of the conjugate of the present invention can be any siderophore (i.e. an iron binding molecule) bearing (comprising) one or more substituted or unsubstituted catechol groups of the formula shown above.
  • the siderophore moiety can be a natural catecholate-containing siderophore or a natural siderophore which has been chemically modified to introduce a substituted or unsubstituted catechol group.
  • the siderophore moiety can be a natural or semisynthetic siderophore moiety.
  • a natural catecholate-containing siderophore designates a molecule secreted by bacteria that tightly binds to iron and allows the bacteria to internalize the siderophore-iron complex.
  • natural catecholate-containing siderophores include, but are not limited to, enterobactin, salmochelin, acinetobactin, fimsbactin, dactyloferrin, chlorodacyloferrin, agrobactin, alterobactin, amonabactin, anguibactin, azotochelin, bacillibactin, benarthin, chrysobactin, corynebactin, cyclic trichrisobactin, dibenarthin, dichrysobactin, divanchrobactin, fluvibactin, fuscachelin, heterobactin, JBIR-16, mirubactin, myxochelin, nigribactin, parabactin, pistillarin
  • the siderophore moiety comprises a substituted or unsubstituted catechol group of the following formula: with Ri being selected from the group consisting of a hydrogen atom, a Ci-C 6 alkyl group, a C 2 -C 6 alkenyl group, a Ci-C 6 alkoxy group, a halogen atom, a Ci-C 6 hydroxyalkyl group, a hydroxy-C 2 -C 6 alkyloxy group, an aryl group, a heteroaryl group, a non-aromatic heterocycle, a Ci-C 6 alkyl-aryl and an aryl-Ci-C 6 alkyl group, preferably a hydrogen atom, a C1-C4 alkyl group or a halogen atom.
  • Ri being selected from the group consisting of a hydrogen atom, a Ci-C 6 alkyl group, a C 2 -C 6 alkenyl group, a Ci-C 6 alkoxy group, a hal
  • R1 is a hydrogen atom or a halogen atom, such as chlorine.
  • the siderophore moiety can be selected in the group consisting of:
  • Ri and R 2 are as disclosed herein above and R 3 , when present, represents a C1-C18 alkyl.
  • the siderophore moiety is dactyloferrin, chlorodactyloferrin, enterobactin, acinetobactin or chloroacinetobactin.
  • the siderophore moiety is:
  • the cargo moiety which is part of the conjugate of the present invention can be any molecule bearing (comprising) a substituted or unsubstituted heteraryl group (Het), said Het group comprising one or more nitrogen atoms, transport of which in the targeted bacteria is desired.
  • Het heteraryl group
  • the cargo molecule may be a pharmaceutical agent, such as an antibiotic, or it may be a fluorophore.
  • the cargo moiety may be a pharmaceutical agent or fluorophore known to comprise a nitrogen-containing heteroaryl group (Het) as disclosed herein in its structure or it may be a known pharmaceutical agent or fluorophore which has been modified to incorporate a nitrogen-containing heteroaryl group (Het) as disclosed herein in its structure.
  • antibiotics comprising a nitrogen-containing heteroaryl group (Het) as disclosed herein or that may be modified to comprise a nitrogen-containing heteroaryl group as disclosed herein, include, but are not limited to, antibiotics of the following classes: rifamycins (such as rifampicin), fluoroquinolones, nitroimidazole (such as metronidazole), penicillins, monobactam (such as aztreonam), cephalosporin (such as cefepime and ceftazidime), carbapenams, pyridomycin, oxazolidinones, sulfonamides, macrolides, fabl inhibitors, such as fabl inhibitors related to Debio-1452.
  • antibiotics of the following classes include, but are not limited to, antibiotics of the following classes: rifamycins (such as rifampicin), fluoroquinolones, nitroimidazole (such as metronidazole), penicillin
  • suitable fluorophore comprising a nitrogen-containing heteroaryl group (Het) as disclosed herein or that may be modified to comprise a nitrogen-containing heteroaryl group as disclosed herein include, but are not limited to, TAMRA, coumarin, boron-dipyrromethene (BODIPY), fluorescein, dansyl, 7-nitrobenz-2-oxa-1 ,3-diazol-4-yl (NBD), and pyrene.
  • the Het group designates a heteroaryl group that comprises one or more nitrogen atoms.
  • the heteroaryl group may comprise one or more further heteroatoms selected from the group consisting of oxygen and sulfur.
  • the heteroaryl group may be monocyclic (typically 5- or 6-membered cycle) or polycyclic (typically 7- to 14-membered cycle).
  • the heteroaryl group may consist of a monocyclic heteroaryl comprising one or more nitrogen atoms and optionally one or more further heteroatoms selected from the group consisting of oxygen and sulfur, fused to a monocyclic aryl (e.g. phenyl) or to a monocyclic heteroaryl or to a monocyclic non-aromatic carbocycle or to a monocyclic non-aromatic heterocycle.
  • the Het group can be connected to the cargo molecule through any carbon atom or nitrogen contained within the Het group, except through the nitrogen atom that links the cargo moiety to the siderophore moiety.
  • the Het group consists of a monocyclic heteroaryl comprising one or more nitrogen atoms and optionally one or more further heteroatoms selected from the group consisting of oxygen and sulfur, fused to a monocyclic non-aromatic heterocycle and that said monocyclic non-aromatic heterocycle comprises one or more nitrogen atoms
  • the siderophore and the cargo moieties are attached to each other by a direct and covalent bond between the carbon atom marked * of the Cat group and a nitrogen atom of the monocyclic heteroaryl part of Het.
  • the Het group may be substituted on any available position, except on the nitrogen atom that links the cargo moiety to the siderophore moiety.
  • Suitable substituents include halogen, hydroxyl, -SH, -S-C1-C12 alkyl, C1-C12 alkoxy, -NH-C1-C12 alkyl, -N-(Ci-Ci2 alkyl)2 and C1-C12- alkyl.
  • Het group examples include, but are not limited to substituted or unsubstituted pyrrolyl, pyrazolyl, imidazolyl, pyrazolyl, imadozolyl, thiazolyl, thiadiazolyl, triazolyl, oxazolyl, isoxazolyl, isothiazolyl, thiazolyl, pyridinyl, pyrimidinyl, pyrazinyl, triazinyl, indolyl, benzimidazolyl, indazolyl, benzothiazolyl, benzoxazolyl, quinolinyl, isoquinolinyl, quinoxalinyl, and quinazoline.
  • the Het group may comprise any of the listed 5- or 6-membered heteroaryl fused to a 5-membered or 6-membered non aromatic heterocycle.
  • the Het group is selected from substituted or unsubstituted groups of the following formula: wherein X is O or NH.
  • the siderophore and the cargo moieties are attached to each other by a direct and covalent bond between the carbon atom marked * of the catechol group and the nitrogen marked * of the Het group. It is to be noted that the nitrogen marked * of Het group is generally positively charged once bonded to the carbon atom marked * of the catechol group.
  • the cargo moiety comprises a nitrogen-containing aryl group selected from substituted or unsubstituted groups of the following formula:
  • the conjugates of the present invention may be any of the following compounds:
  • X' when represented, designates a counter-ion (negatively charged).
  • counter-ions include halogen atoms, acetate or any counter-ions that provide a physiologically acceptable salt as described herein above.
  • the conjugates of the present invention may be any of the conjugates 1 to 24 disclosed in the “EXAMPLES” section.
  • the conjugates of the present invention may be prepared by a process comprising the step of reacting a siderophore bearing a catechol group of the following formula: with Ri and R 2 are as disclosed herein above; with a cargo molecule bearing a substituted or unsubstituted heteraryl group (Het), said Het group comprising one or more nitrogen atoms; in conditions that allow oxidation of the catechol group and subsequent addition of the cargo molecule.
  • Het substituted or unsubstituted heteraryl group
  • the Het group may be as disclosed herein above.
  • the Het group is selected from substituted or unsubstituted groups of the following formula: wherein X is O or NH. It is known that oxidation of catechol groups leads to the formation of reactive o-quinone that can undergo 1 ,4-Michael addition reaction (Adolphs et al., 1996); Saxena et al., 1978; Cavalieri et al., 2002; Mirobo et al., 2010).
  • Known methods for the synthesis of o-quinones from various classes of organic compounds includes the following reagents: sodium nitrite, hydrogen peroxide, potassium ferricyanide, silver oxide (Ag 2 O) (Cavalieri et al., 2002) alone or together with hydrogen peroxide at room temperature under batch and continuous-flow conditions (Derikvand et al., 2010); silver carbonate, sodium chlorate (NaCIO 3 ), sodium periodate (NalO 4 ), sodium or potassium dichromate (Na 2 Cr 2 O7/K 2 Cr 2 O 7 ) with sulfuric acid (H 2 SO 4 ) (Yang et al., 2018) or glacial acetic acid/dioxane (Kato et al., 1977), the nano-magnetic Fe 3 O 4 (NM-Fe 3 O 4 ) at room temperature (Sharghi et al., 2020), dichlorodicyanoquinone (DDQ), o-chloranil (Walker & Hi
  • Additional catechol oxidation agents include iodine, bromine, in particular in the presence of pyridine and its derivatives (Saxena et al., 1978).
  • Ferric iron has also been demonstrated to cause catechol oxidation in acidic conditions (in basic conditions catechols form coordination bonds with Fe 3+ that prevents further oxidation).
  • At acidic pH Fe 3+ has been shown to oxidize substituted and unsubstituted catechols and to react to produce o-quinones (Fullenkamp et al., 2014; Yang et al., 2014), which react with nucleophiles or proceeds with a concomitant catechol-catechol oligomerization due to oxidative homocoupling (e.g. (Napolitano et al., 2000)(Yang et al., 2014)).
  • the conditions that allow oxidation of the catechol group and subsequent addition of the cargo molecule may consist in reacting the cargo molecule and the siderophore in presence of an oxidizing agent or by electrochemistry.
  • the oxidation of the catechol group is performed with the use of oxidizing agents selected from the group consisting of Fe(lll), l 2 , Br 2 , Ag 2 O and NalO 4 or by electrochemistry.
  • oxidizing agents selected from the group consisting of Fe(lll), l 2 , Br 2 , Ag 2 O and NalO 4 or by electrochemistry.
  • the formed reactive o-quinone can then undergo 1 ,4-Michael addition reaction with the cargo molecule to provide the conjugates of the present invention.
  • the siderophore is reacted with the cargo molecule in presence of Fe 3+ ions.
  • the catechol group is oxidized allowing subsequent addition of the cargo molecule.
  • the reaction is performed in presence of 3 to 10 equivalents of Fe 3+ ions (with respect to the catechol group).
  • the Fe 3+ ions may originate from iron sulphate or iron trichloride.
  • the siderophore may be reacted with the cargo molecule in presence of iron sulphate or iron trichloride, typically in presence of 3 to 10 equivalents of iron sulphate or iron trichloride, to provide the conjugates of the present invention.
  • the reaction is typically performed in a solvent selected from the group consisting methanol, ethanol, acetonitrile, DMSO, water, acetonitrile, chloroform, ethyl acetate and mixtures thereof.
  • a solvent selected from the group consisting methanol, ethanol, acetonitrile, DMSO, water, acetonitrile, chloroform, ethyl acetate and mixtures thereof.
  • the reaction is typically performed at room temperature, but also proceeds at lower and higher temperatures.
  • the siderophore is reacted with the cargo molecule in presence of l 2 , Br 2 , Ag 2 O, NalO 4 or Cu(ll) to provide the conjugates of the present invention.
  • the catechol group is oxidized allowing subsequent addition of the cargo molecule.
  • the oxidation of the catechol group is performed by electrochemistry.
  • the isolation and purification of the conjugates may be performed in accordance with methods well-known for the man skilled in the art.
  • the conjugates of the present invention may be used in medicine, for example as a medicament or for detection/diagnosis purpose.
  • the conjugates of the present invention may be used for the delivery of pharmaceutical agents, for examples antibiotics, into targeted bacteria. More specifically, the conjugates of the invention, when comprising an antibiotic moiety as cargo moiety, may be used in the treatment of a bacterial infection.
  • the present invention relates to a conjugate as disclosed herein for use in the treatment of a bacterial infection.
  • the conjugates of the present invention may be used to target bacteria belonging to the following bacterial order: Enterobacterales, Mycobacteriales, Pseudomonadales, Burkholderiales, Bacillales and Lactobacillales.
  • the conjugates of the present invention can be used to prepare compositions, such as pharmaceutical compositions, for example, by combining the conjugates with an acceptable excipient.
  • Acceptable excipients include fillers and carriers, ointment bases, bases for suppositories, solvents, surfactants, emulsifiers, dispersants or wetting agents, buffers, acids and bases, isotonicity agents, adsorbent, viscosity-increasing agents, gel formers, thickeners and/or binders, disintegrants, coating materials and film formers for films or diffusion membranes, capsule materials, natural or synthetic polymers, plasticizers, penetration enhancers, stabilizers, preservatives, colourants, flavourings, sweeteners, flavour- and/or odour-masking agents.
  • the conjugates may be used in a neutral form or in the form of a salt or solvate.
  • compositions can be administered to a mammalian host, such as a human patient, in a variety of forms.
  • the forms can be specifically adapted to a chosen route of administration, e.g., oral or parenteral administration, by intravenous, intramuscular, topical or subcutaneous routes.
  • the invention provides therapeutic methods of treating bacterial infections in a mammal, which involve administering to a mammal having a bacterial infection an effective amount of a conjugate or composition described herein.
  • a mammal includes a primate, human, rodent, canine, feline, bovine, ovine, equine, swine, caprine, bovine and the like.
  • the bacterial infection can be a bacterial infection caused by a bacterium described herein.
  • the amount of a conjugate which constitutes an effective amount will vary depending on many factors, such as for instance the conjugate and its biological activity, the composition used for administration, the route of administration, the type of disorder being treated and its severity, and the age, body weight, general health, sex, and diet of the patient. Such an effective amount can be determined routinely by one of ordinary skill in the art having regard to their own knowledge.
  • This compound with m/z of 1001 was purified from a 10 L culture using cation exchange resin and preparative chromatography using a C18 column. Yield of the purified molecule were 5 mg/L.
  • the compound m/z of 1001 was tested in a Chrome Azurol S assay, and it was found to be able to chelate Fe 3+ better than desferrioxamine B. The non-chlorinated m/z 967 could also chelate Fe 3+ .
  • a combination of MALDI-TOF fragmentation, high resolution mass spectrometry and NMR analysis allowed for the elucidation of the chemical structure of both the chlorinated m/z of 1001 (named chlorodactyloferrin), and the non-chlorinated m/z of 967 (here named dactyloferrin).
  • the structures are as follow : The orientation of the stereo centers was determined using the information from the biosynthetic clusters, specifically, the presence or absence of epimerase domains in the NRPS modules. This elucidated structure fits with the structure of the biosynthetic cluster identified.
  • Chlorodactyloferrin could bind Fe 3+ or Ga 3+ as observed by LC-MS.
  • the chlorodactyloferrin - Ga 3+ complex was purified and analyzed by NMR to determine mechanism of iron coordination. To this extent, the catechol and the two hydroxamate groups were found to coordinate the metal ion.
  • the FeCI 3 based conjugation reaction was also found to occur between chlorodactyloferrin and pyridomycin analogues where the pyridine was conserved: 5CI-HPA- pyridomycin, 6CI-HPA- pyridomycin and pyridomycin precursor 558, but not with Phe-pyridomycin where the pyridine moiety was changed to a phenyl.
  • the pyridomycin precursors come from mutasynthesis from D. fulvum, and the conjugation products were confirmed only by mass spectrometry, not by NMR.
  • conjugate 18 was prepared by following the same synthetic procedure used to prepare enterobactin-TAMRA3515 (6”). Yield as a white solid: 0.3 mg (5%).
  • LC-MS analysis confirmed formation of the target product.
  • Chemical Formula: C33H34N7OnS 2 + calcd m/z 768.1725; obsd m/z 768.25.
  • conjugate 19 was prepared by following the same synthetic procedure used to prepare enterobactin-TAMRA3515 (6”). Yield as a white solid: 0.44 mg (10%).
  • LC-MS analysis confirmed formation of the target product.
  • conjugate 20 Conjugate of acinetobactin and 3-pyridyl-ciprofloxacin (20) Using acinetobactin (1 mg, 0.0029 mmol), 3-pyridyl-ciprofloxacin RPII67 (1.4 mg, 0.0029 mmol) and iodine (6.82 mg, 0.029 mmol) as starting compounds, conjugate 20 was prepared by following the same synthetic procedure used to prepare enterobactin-TAMRA3515 (6”). Yield as a white solid: 0.4 mg (15%). LC-MS analysis confirmed formation of the target product. C42H47FN 9 O9 + : calcd m/z 840.3476; obsd m/z 840.42.
  • conjugate 21 was prepared by following the same synthetic procedure used to prepare enterobactin-
  • conjugate 22 Conjugate of acinetobactin and pyridomycin (22) Using acinetobactin (2.15 mg, 0.0062 mmol), pyridomycin (3.36 mg, 0.0062 mmol) and iodine (1.46 mg, 0.0062 mmol) as starting compounds, conjugate 22 was prepared by following the same synthetic procedure used to prepare enterobactin-TAMRA3515 (6”). Yield as a white solid: 0.7 mg (12%). LC-MS analysis confirmed formation of the target product. C43H 4 9N 8 Oi3 + : calcd m/z 885.3414; obsd m/z 885.50.
  • conjugate 23 was prepared by following the same synthetic procedure used to prepare enterobactin-TAMRA3515 (6”). Yield as a white solid: 0.9 mg (18%).
  • LC-MS analysis confirmed formation of the target product.
  • Enterobactin (0.0668 mg, 0.0001 mmol) was coupled to 3-pyridyl-levofloxacin derivatives (0.0466 mg, 0.0001 mmol) by the addition of iodine (0.0759 mg, 0.0003 mmol). Reaction mixtures were diluted 1 :4 in DMSO and presence of the expected enterobactin-3-pyridyl- levofloxacin conjugates was confirmed by UPLC-MS. 1
  • the uptake of the siderophore-cargo conjugates was evaluated via (1) the uptake of siderophores conjugated to fluorophores followed by confocal microscopy and (2) determination of the antimicrobial activity of the antibiotic-siderophore conjugates.
  • the uptake of the chlorodactyloferrin-TAMRA3515 conjugate (1) by Dactylosporangiae and other related bacteria was also assessed.
  • the genomes of these bacteria are encoding homologous transport systems to the one used by D. fulvum for chlorodactyloferrin uptake. For that, these strains were incubated with both chlorodactyloferrin-TAMRA3515 and TAMRA3515, in the same conditions as previously reported.
  • the uptake of the enterobactin-TAMRA3515 conjugate (6) by E. coli was evaluated via imaging by super-resolution confocal microscopy iron-deprived bacteria (treated 2 h in presence of the iron chelator 2, 2’-dypiridyl, DIP, 100 JLLM) treated with the conjugate for 1 h.
  • the results are presented in Figure 8. All images were captured using the oil immersion objective 60x/1.40 of an inverted spinning-disk microscope Yokogawa CSU-W1 equipped with a Roper Scientific Live-SR module.
  • F fluorescence TAMRA filter set (excitation: 561 nm; emission: 595 nm, 50 nm bandwidth), BF: bright field, and C: composite. Shown pictures are representative of all the sample and reproducible across independent preparations.
  • siderophore-antibiotic conjugates were synthesised as previously described, including different combinations of catechol containing siderophores (chlorodactyloferrin and enterobactin) with both pyridyl-antibiotics (pyridomycin, p-lactam-Pyr, fluoroquinolone-Pyr, rifampicin-Pyr) and other “nitrogen containing heteroaromatic”-containing antibiotics (aztreonam, cefepime, ceftazidime, metronidazole).
  • catechol containing siderophores chlorodactyloferrin and enterobactin
  • pyridyl-antibiotics pyridomycin, p-lactam-Pyr, fluoroquinolone-Pyr, rifampicin-Pyr
  • other “nitrogen containing heteroaromatic”-containing antibiotics asztreonam, cefepime, ceftazi
  • conjugates 16 and 17 were evaluated against Escherichia coli BW25113 by the microtitre-dilution method in both normal and low iron-conditions (minimal media M9 with and without 100 pM 2,2’-dipyridyl, DIP, respectively).
  • conjugate 16 showed a superior minimal inhibitory concentration (MIC) of 0.11- 0.22 pM, compared to under normal iron-conditions where there was an MIC of 3.5 pM (Table 1).
  • MIC minimal inhibitory concentration
  • Conjugate 17 did not show a measurable antimicrobial activity below 5 pM in ether media, but the molecule did promote bacterial growth in the presence of DIP, indicating that conjugate 17 is taken up and can relieve iron stress.
  • enterobactin can also be activity take up by other bacterial species, the enterobactin- antibiotic conjugates 16 & 17 were also tested against A. baumannii ATCC 19606. In A.
  • Table 2 Bacterial antibiotic susceptibility of E. coli and A. baumannii in liquid meda with different iron availability, to conjugates 16 and 17. Compounds were tested in normal (M9 or Muller-Hinton, MH) and low iron conditions (M9 supplemented with 100 pM or Muller-Hinton with 10% FCS). * indicates that the compound is promoting growth in low iron conditions.
  • Table 3 Bacterial antibiotic susceptibility in liquid culture to conjugates 18-23. Compounds were tested in normal (Muller-Hinton, MH) and low iron conditions (Muller-Hinton with 10% FCS) against A. baumannii ATCC 19606. * indicates that the compound is promoting growth in low iron conditions.
  • the by disk diffusion assay was performed on solid media with low and high-iron media (M9 agar with or without 100 pM DIP). Zones of growth inhibition of reaction mixtures (enterobactin + free-fluoroquinolone + conjugate) were compared to those from mixtures of the unconjugated reactants (enterobactin + free fluoroquinolone), so that the total fluoroquinolone concentration in all the samples is consistent. Most of the reaction mixtures showed similar zone of inhibition as the reactants, suggesting that coupling of fluoroquinolones to enterobactin did not greatly improve activity.
  • Cavalieri EL Li KM, Balu N, Saeed M, Devanesan P, Higginbotham S, Zhao J, Gross ML, Rogan EG. 2002.
  • Catechol ortho-quinones The electrophilic compounds that form depurinating DNA adducts and could initiate cancer and other diseases. Carcinogenesis 23:1071-1077.

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Abstract

The present disclosure relates to conjugates of catechol-based siderophores with cargo molecules, such as antibiotics or fluorophores, and to the preparation and uses thereof.

Description

Figure imgf000002_0001
The present invention relates to conjugates of catechol-based siderophores with cargo molecules, such as antibiotics or fluorophores, and to the preparation and uses thereof.
BACKGROUND OF THE INVENTION
The bacterial outer cell wall, in particular the one of some species of bacteria, such as Gramnegative bacteria, provides a formidable barrier to exogenic compounds. The permeability properties of this barrier have a major impact on the susceptibility of the bacteria to antibiotics. Indeed, by blocking the penetration of antibiotics, some bacteria are intrinsically resistant to antibiotics. Overcoming the low-permeability barrier of bacteria remains a challenge in the development of new antibiotics.
One method for overcoming the issue of penetration into bacteria is to hijack the active transport mechanisms of the bacteria, in particular the active transport of iron. In this respect, siderophores-antibiotic conjugates have been developed for “Trojan horse” antibiotic delivery. The active transport of iron is fundamental to bacterial survival. To access iron during an infection, bacteria produce siderophores. These organic compounds chelate and scavenge iron from the host's body and are then actively transported into the bacteria. A large number of bacterial siderophores are known. They generally contain structures like hydroxamates, catechols, carboxylates, and various heterocycles.
«Trojan horse» antibiotics, i.e. siderophores-antibiotic conjugates, such as sideromycins, are able to bypass this penetration barrier by hijacking the active siderophore: iron uptake systems of bacteria for their own active uptake and delivering the drug inside the target bacterial cell. To date, a small number of natural sideromycins have been described, namely, albomycin, ferrimycin, microcin E492m and salmycin.
In addition to these natural sideromycins, chemically synthesized sideromycins have also been described, including the clinically used cefiderocol. The current literature on chemical preparation of novel sideromycins is inspired by either mimicking natural sideromycins, or by exploring parts of siderophores where cargo coupling is tolerated. For this second approach it has proven to be a challenge to design synthetic siderophores that have all the biological properties of a Trojan horse antibiotic (uptake and decoupling). Of the disclosed synthetic sideromycins that mimick natural sideromycins, only few examples are described where the antibiotics are coupled to a catechol group of the siderophore, with examples limited to the C- 5 position of the catechol group (mimicking the attachment of glucosylated enterobactins such as salmochelins) (Zheng et al., 2012). This is particularly problematic as many of the bacteria on the World Health Organization list that need to be targeted for antibiotic development use catechol containing siderophores. Furthermore, the majority of described synthetic sideromycins bear «non-natural» molecular linkers between the siderophore and the antibiotics. These linkers may have an unknown impact on the activity of the synthetic sideromycins.
In view of the serious public health problems that antibiotic resistance causes across the globe, it remains a need to provide new strategies for combatting bacterial infections. The potential of using siderophores in therapeutic development continues to attract significant interest though hurdles still need to be overcome. Therefore, it remains a need to provide new compounds that are able to overcome the issue of penetration of exogenic compounds into bacteria.
SUMMARY OF THE INVENTION
The present invention relates to a conjugate comprising a siderophore moiety and a cargo moiety, wherein the siderophore moiety comprises a siderophore (Sid) bearing a substituted or unsubstituted catechol group (Cat), said Cat group having the following formula:
Figure imgf000003_0001
with Ri and R2 being independently selected from the group consisting of a hydrogen atom, a Ci-C6 alkyl group, a C2-C6 alkenyl group, a Ci-C6 alkoxy group, a halogen atom, a Ci-C6 hydroxyalkyl group, a hydroxy-C2-C6 alkyloxy group, an aryl group, a heteroaryl group, a non- aromatic heterocycle, a Ci-C6 alky l-ary I and an aryl-Ci-C6 alkyl group, preferably a hydrogen atom, a C1-C4 alkyl group or a halogen atom, more preferably a hydrogen atom or a halogen atom; wherein the cargo moiety comprises a cargo molecule (Cargo) bearing a substituted or unsubstituted heteraryl group (Het), said Het group comprising one or more nitrogen atoms; wherein the siderophore and the cargo moieties are attached to each other by a direct and covalent bond between the carbon atom marked * of the Cat group and a nitrogen atom of the Het group, and wherein the conjugate has the following formula:
Sid-Cat-Het-Cargo.
The present invention relates to a process for preparing a conjugate as disclosed herein. The process comprises the step of reacting a siderophore bearing a catechol group of the following formula:
Figure imgf000004_0001
with R1 and R2 being independently selected from the group consisting of a hydrogen atom, a Ci-C6 alkyl group, a C2-C6 alkenyl group, a Ci-C6 alkoxy group, a halogen atom, a Ci-C6 hydroxyalkyl group, a hydroxy-C2-C6 alkyloxy group, an aryl group, a heteroaryl group, a nonaromatic heterocycle, a Ci-Ce alky l-ary I and an aryl-Ci-Ce alkyl group, preferably a hydrogen atom, a C1-C4 alkyl group or a halogen atom, more preferably a hydrogen atom or a halogen atom; with a cargo molecule bearing a substituted or unsubstituted heteraryl group (Het), said Het group comprising one or more nitrogen atoms; in conditions that allow oxidation of the catechol group and subsequent addition of the cargo molecule.
The invention also relates to a composition comprising the conjugate of the invention and an acceptable excipient.
The invention also relates to a conjugate or composition as disclosed herein for use in medicine, in particular in the treatment of a bacterial infection or for use in the diagnosis of a bacterial infection.
Further aspects of the invention are as disclosed herein and in the claims.
FIGURES
Figure 1 : Fluorescence microscopy of D. fulvum cultivated into iron-limiting conditions (in presence of 350 pM of DIP) and treated with 1 pM of TAMRA3515 (A) and chlorodactyloferrin-TAMRA3515 conjugate (B). In (C), D. fulvum was treated with chlorodactyloferrin-TAMRA3515 (1 pM) together with unlabelled chlorodactyloferrin (100 pM). Figure 2: Fluorescence microscopy of D. aurantiacum cultivated into iron-limiting conditions (in presence of 350 pM of DIP) and treated with 1 pM of TAMRA3515 (A) and chlorodactyloferrin-TAMRA3515 conjugate (B).
Figure 3: Fluorescence microscopy of D. matsukiense cultivated into iron-limiting conditions (in presence of 350 pM of DIP) and treated with 1 pM of TAMRA3515 (A) and chlorodactyloferrin-TAMRA3515 conjugate (B).
Figure 4: Fluorescence microscopy of D. roseum cultivated into iron-limiting conditions (in presence of 350 pM of DIP) and treated with 1 pM of TAMRA3515 (A) and chlorodactyloferrin-TAMRA3515 conjugate (B).
Figure 5: Fluorescence microscopy of D. vinaceum cultivated into iron-limiting conditions (in presence of 350 pM of DIP) and treated with 1 pM of TAMRA3515 (A) and chlorodactyloferrin-TAMRA3515 conjugate (B).
Figure 6: Fluorescence microscopy of S. coelicolor cultivated into iron-limiting conditions (in presence of 350 pM of DIP) and treated with 1 pM of TAMRA3515 (A) and chlorodactyloferrin-TAMRA3515 conjugate (B). Figure 7: Fluorescence microscopy of S. subtilis cultivated into iron-limiting conditions (in presence of 350 pM of DIP) and treated with 1 pM of TAMRA3515 (A) and chlorodactyloferrin-TAMRA3515 conjugate (B).
Figure 8: Fluorescence microscopy of E. coli WT (A) and AfepA (B) cultivated into iron-limiting conditions (in presence of 100 pM of DIP) and treated with 1 pM of enterobactin-TAMRA3515 conjugate.
Figure 9: Fluorescence microscopy of iron-deprived A. baumannii (in presence of 100 pM of DIP) treated with 1 pM TAMRA3515 (A) and 1 pM acinetobactin-TAMRA3515 conjugate (B).
Figure 10: Schematic representation of a conjugate according to the present invention.
Figure 11 : (A) Disk diffusion assay of RPII151 alone and that of the reaction mixture following conjugation of enterobactin and RPII151 (using iodine) on E. coli grown on high and low iron solid media (M9 and M9 + 100 pM DIP). These data show a zone of growth inhibition only for the reaction mixture when bacteria were grown under low iron conditions. (B) A HPLC-MS UV chromatogram of the product of the enterobactin/RPII151 iodine based conjugation reaction showing remaining residual, RPII151 as well as the peak of the enterobactin-RPII151 conjugate 24.
DESCRIPTION OF THE INVENTION
The present invention stems from the identification and isolation of a novel sideromycin-like molecule from a bacterial culture, more specifically from the identification of a conjugate between a newly identified catecholate-based siderophore (called herein chlorodactyloferrin) and pyridomycin (see section “EXAMPLES”). Based on their findings, the inventors have designed a new series of sideromycin-like molecules that were found to be actively delivered into bacteria by hijacking the siderophore uptake systems. Thanks to their unique structure, i.e. direct and covalent bonding of the catecholate-based siderophore to the molecule of interest to be delivered (through C-6 of the catechol group), the proposed conjugates allow the delivery of molecules of interest (called herein “cargo”), such as for instance therapeutic agents or fluorophores, to a wide spectrum of pathogenic and non-pathogenic bacteria that naturally produce and/or use catecholate-containing siderophores for iron uptake.
Thus, the present invention relates to a conjugate comprising a siderophore moiety and a cargo moiety, wherein the siderophore moiety comprises a siderophore (Sid) bearing a substituted or unsubstituted catechol group (Cat), said Cat group having the following formula:
Figure imgf000007_0001
with Ri and R2 being independently selected from the group consisting of a hydrogen atom, a Ci-Ce alkyl group, a C2-C6 alkenyl group, a Ci-Ce alkoxy group, a halogen atom, a Ci-Ce hydroxyalkyl group, a hydroxy-C2-C6 alkyloxy group, an aryl group, a heteroaryl group, a nonaromatic heterocycle, a Ci-C6 alky l-ary I and an aryl-Ci-C6 alkyl group, preferably a hydrogen atom, a C1-C4 alkyl group or a halogen atom, more preferably a hydrogen atom or a halogen atom; wherein the cargo moiety comprises a cargo molecule (Cargo) bearing a substituted or unsubstituted heteraryl group (Het), said Het group comprising one or more nitrogen atoms; wherein the siderophore and the cargo moieties are attached to each other by a direct and covalent bond between the carbon atom marked * of the Cat group and a nitrogen atom of the Het group, the conjugate having the following formula:
Sid-Cat-Het-Cargo.
A schematic representation of a conjugate according to the invention is presented in figure 10. As shown on the figure, the siderophore moiety comprises a siderophore (Sid) bearing a substituted or unsubstituted catechol group (Cat) and the cargo moiety comprises a cargo molecule (Cargo) bearing a substituted or unsubstituted heteraryl group (Het). The Het group comprises one or more nitrogen atoms. The siderophore moiety and cargo moiety are covalently linked through the Cat and Het groups.
The carbon atom marked * of the substituted or unsubstituted catechol group corresponds to the carbon atom in position 6 of the catechol group. The term “conjugate” as used herein refers to a compound or construct comprising the above-mentioned elements, i.e. a siderophore moiety and a cargo moiety, which are coupled, i.e. , conjugated or bonded, to each other via covalent interactions.
The term “halogen” as used herein refers to fluorine, chlorine, bromine or iodine.
The term “Ci-C6 alkyl” as used herein refers to a saturated, branched or straight hydrocarbon chain comprising from one to six carbon atoms, such as methyl, ethyl, n-propyl or isopropyl, n-butyl, sec-butyl, iso-butyl, tert-butyl, n-pentyl, isopentyl, n-hexyl.
The term “C2-C6 alkenyl” as used herein designates a branched or straight hydrocarbon chain comprising from two to six carbon atoms and containing one or more double bonds, including di-enes, tri-enes, such as vinyl, propen-1-yl, propen-2-yl, but-1-en-1-yl, but-1-en-2- yl, but-2-en-1-yl.
The term “Ci-C6 hydroxyalkyl” as used herein designates a Ci-C6 alkyl group as defined above, which Ci-C6 alkyl group is substituted with one or more hydroxy groups. Examples of Ci-Ce hydroxyalkyl groups include 2-hydroxy-ethyl, 3-hydroxy-propyl, 4-hydroxy-butyl, 5- hydroxy-pentyl and 6-hydroxy-hexyl.
The term “Ci-C6 alkoxy” as used herein designates a radical — O-Ci-C6 alkyl which Ci-C6 alkyl group is as disclosed above. Examples of Ci-C6 alkoxy include methoxy, ethoxy, propyloxy, /so-propyloxy, butyloxy, /so-butyloxy, terf-butyloxy, sec-butyloxy, pentyloxy, iso- pentyloxy, hexyloxy.
The term “hydroxy-C2-C6 alkyloxy” as used herein designates a C2-C6 alkoxy, which C2-C6 alkoxy group is substituted with one or more hydroxy groups.
The term “aryl” as used herein refers to aromatic carbocyclic groups having a single ring (e.g., phenyl), multiple rings (e.g., biphenyl), or multiple fused rings in which at least one is aromatic (e.g., 1 ,2,3,4-tetrahydronaphthyl, naphthyl, anthryl, or phenanthryl).
The term “non-aromatic carbocycle” as used herein refers to cyclic groups, saturated or unsaturated, containing carbon atoms as ring atoms. The terms “heteroaryl” as used herein refers to aryl groups as defined herein comprising at least one heteroatom as a ring atom. Suitable heteroatoms include oxygen, sulfur, nitrogen, phosphorus, selenium and the like. In some cases, a heteroaryl is a cyclic aromatic radical having from five to ten ring atoms of which at least one ring atom is selected from S, O, and N; zero, one, or two ring atoms are additional heteroatoms independently selected from S, O, and N; and the remaining ring atoms are carbon.
The term “non-aromatic heterocycle” as used herein refers to refer to cyclic groups, saturated or unsaturated, containing at least one heteroatom as a ring atom, in some cases, 1 to 3 heteroatoms as ring atoms, with the remainder of the ring atoms being carbon atoms. Suitable heteroatoms include oxygen, sulfur, nitrogen, phosphorus, selenium and the like. In some cases, the heterocycle may be 3- to 10-membered ring structures or 3- to 7-membered rings, in which the ring structures include one to four heteroatoms.
The term "Ci-C6 alkyl-aryl" as used herein, refers to a Ci-C6 alkyl as defined herein substituted by an aryl as defined herein.
The term “aryl-Ci-C6 alkyl " as used herein, refers to an aryl as defined herein substituted by a Ci-C6 alkyl as defined herein.
The present invention encompasses hydrates, solvates, or salts of the herein disclosed conjugates. Preferred salts in the context of the present invention are physiologically acceptable salts of the conjugates. However, the invention also encompasses salts which themselves are unsuitable for pharmaceutical applications but which can be used, for example, for the isolation or purification of the compounds according to the invention.
The term “physiologically acceptable salt" refers to a relatively non-toxic, inorganic or organic addition salt of the conjugates. A suitable pharmaceutically acceptable salt may be, for example, an acid-addition salt of a compound of the conjugates, such as an acid-addition salt with an inorganic acid, such as hydrochloric, hydrobromic, hydroiodic, sulfuric, bisulfuric, phosphoric, or nitric acid, for example, or with an organic acid, such as formic, acetic, acetoacetic, pyruvic, trifluoroacetic, propionic, butyric, hexanoic, heptanoic, undecanoic, lauric, benzoic, salicylic, 2-(4-hydroxybenzoyl)-benzoic, camphoric, cinnamic, cyclopentanepropionic, digluconic, 3 -hydroxy-2 -naphthoic, nicotinic, pamoic, pectinic, persulfuric, 3-phenylpropionic, picric, pivalic, 2-hydroxyethanesulfonate, itaconic, sulfamic, trifluoromethane sulfonic, dodecylsulfuric, ethansulfonic, benzenesulfonic, para-toluene sulfonic, methansulfonic, 2- naphthalenesulfonic, naphthalinedisulfonic, camphorsulfonic acid, citric, tartaric, stearic, lactic, oxalic, malonic, succinic, malic, adipic, alginic, maleic, fumaric, D-gluconic, mandelic, ascorbic, glucoheptanoic, glycerophosphoric, aspartic, sulfosalicylic, hemisulfuric, or thiocyanic acid, for example.
Solvates in the context of the invention are described as those forms of the compounds which form a complex in the solid or liquid state by coordination with solvent molecules. Hydrates are a specific form of the solvates in which the coordination is with water.
The present invention includes all possible stereoisomers of the conjugate of the present invention as single stereoisomer, or as any mixture of said stereoisomers, in any ratio. Isolation of a single stereoisomer, e.g. a single enantiomer or a single diastereomer, of a conjugate can be achieved by any suitable state of the art method, such as chromatography, especially chiral chromatography, for example.
Siderophore moiety
The siderophore moiety which is part of the conjugate of the present invention can be any siderophore (i.e. an iron binding molecule) bearing (comprising) one or more substituted or unsubstituted catechol groups of the formula shown above.
The siderophore moiety can be a natural catecholate-containing siderophore or a natural siderophore which has been chemically modified to introduce a substituted or unsubstituted catechol group. Thus, the siderophore moiety can be a natural or semisynthetic siderophore moiety.
A natural catecholate-containing siderophore designates a molecule secreted by bacteria that tightly binds to iron and allows the bacteria to internalize the siderophore-iron complex. Examples of natural catecholate-containing siderophores include, but are not limited to, enterobactin, salmochelin, acinetobactin, fimsbactin, dactyloferrin, chlorodacyloferrin, agrobactin, alterobactin, amonabactin, anguibactin, azotochelin, bacillibactin, benarthin, chrysobactin, corynebactin, cyclic trichrisobactin, dibenarthin, dichrysobactin, divanchrobactin, fluvibactin, fuscachelin, heterobactin, JBIR-16, mirubactin, myxochelin, nigribactin, parabactin, pistillarin, protochelin, serratiochelin, streptobactin, tribenarthin, trivanchrobactin, vanchrobactin, vibriobactin, vulnibactin. Examples of siderophore in which a substituted or unsubstituted catechol group may be introduced include, but are not limited to, mycobactin, pyochelin, versiniabactin and py overdin.
In some embodiments, the siderophore moiety comprises a substituted or unsubstituted catechol group of the following formula:
Figure imgf000011_0001
with Ri being selected from the group consisting of a hydrogen atom, a Ci-C6 alkyl group, a C2-C6 alkenyl group, a Ci-C6 alkoxy group, a halogen atom, a Ci-C6 hydroxyalkyl group, a hydroxy-C2-C6 alkyloxy group, an aryl group, a heteroaryl group, a non-aromatic heterocycle, a Ci-C6 alkyl-aryl and an aryl-Ci-C6 alkyl group, preferably a hydrogen atom, a C1-C4 alkyl group or a halogen atom.
More preferably, R1 is a hydrogen atom or a halogen atom, such as chlorine.
The siderophore moiety can be selected in the group consisting of:
Figure imgf000011_0002
Figure imgf000012_0001
Figure imgf000013_0001
wherein Ri and R2 are as disclosed herein above and R3, when present, represents a C1-C18 alkyl.
In some embodiments, the siderophore moiety is dactyloferrin, chlorodactyloferrin, enterobactin, acinetobactin or chloroacinetobactin.
In some embodiments, the siderophore moiety is:
Figure imgf000013_0002
Figure imgf000014_0001
Cargo moiety
The cargo moiety which is part of the conjugate of the present invention can be any molecule bearing (comprising) a substituted or unsubstituted heteraryl group (Het), said Het group comprising one or more nitrogen atoms, transport of which in the targeted bacteria is desired.
As a matter of examples, the cargo molecule may be a pharmaceutical agent, such as an antibiotic, or it may be a fluorophore. The cargo moiety may be a pharmaceutical agent or fluorophore known to comprise a nitrogen-containing heteroaryl group (Het) as disclosed herein in its structure or it may be a known pharmaceutical agent or fluorophore which has been modified to incorporate a nitrogen-containing heteroaryl group (Het) as disclosed herein in its structure.
Examples of suitable antibiotics comprising a nitrogen-containing heteroaryl group (Het) as disclosed herein or that may be modified to comprise a nitrogen-containing heteroaryl group as disclosed herein, include, but are not limited to, antibiotics of the following classes: rifamycins (such as rifampicin), fluoroquinolones, nitroimidazole (such as metronidazole), penicillins, monobactam (such as aztreonam), cephalosporin (such as cefepime and ceftazidime), carbapenams, pyridomycin, oxazolidinones, sulfonamides, macrolides, fabl inhibitors, such as fabl inhibitors related to Debio-1452.
Examples of suitable fluorophore comprising a nitrogen-containing heteroaryl group (Het) as disclosed herein or that may be modified to comprise a nitrogen-containing heteroaryl group as disclosed herein include, but are not limited to, TAMRA, coumarin, boron-dipyrromethene (BODIPY), fluorescein, dansyl, 7-nitrobenz-2-oxa-1 ,3-diazol-4-yl (NBD), and pyrene. As said above, the Het group designates a heteroaryl group that comprises one or more nitrogen atoms. The heteroaryl group may comprise one or more further heteroatoms selected from the group consisting of oxygen and sulfur. The heteroaryl group may be monocyclic (typically 5- or 6-membered cycle) or polycyclic (typically 7- to 14-membered cycle). When the heteroaryl group is polycyclic, it may consist of a monocyclic heteroaryl comprising one or more nitrogen atoms and optionally one or more further heteroatoms selected from the group consisting of oxygen and sulfur, fused to a monocyclic aryl (e.g. phenyl) or to a monocyclic heteroaryl or to a monocyclic non-aromatic carbocycle or to a monocyclic non-aromatic heterocycle. The Het group can be connected to the cargo molecule through any carbon atom or nitrogen contained within the Het group, except through the nitrogen atom that links the cargo moiety to the siderophore moiety.
When the Het group consists of a monocyclic heteroaryl comprising one or more nitrogen atoms and optionally one or more further heteroatoms selected from the group consisting of oxygen and sulfur, fused to a monocyclic non-aromatic heterocycle and that said monocyclic non-aromatic heterocycle comprises one or more nitrogen atoms, it is to be understood that the siderophore and the cargo moieties are attached to each other by a direct and covalent bond between the carbon atom marked * of the Cat group and a nitrogen atom of the monocyclic heteroaryl part of Het.
The Het group may be substituted on any available position, except on the nitrogen atom that links the cargo moiety to the siderophore moiety. Suitable substituents include halogen, hydroxyl, -SH, -S-C1-C12 alkyl, C1-C12 alkoxy, -NH-C1-C12 alkyl, -N-(Ci-Ci2 alkyl)2 and C1-C12- alkyl.
Examples of suitable Het group include, but are not limited to substituted or unsubstituted pyrrolyl, pyrazolyl, imidazolyl, pyrazolyl, imadozolyl, thiazolyl, thiadiazolyl, triazolyl, oxazolyl, isoxazolyl, isothiazolyl, thiazolyl, pyridinyl, pyrimidinyl, pyrazinyl, triazinyl, indolyl, benzimidazolyl, indazolyl, benzothiazolyl, benzoxazolyl, quinolinyl, isoquinolinyl, quinoxalinyl, and quinazoline. The Het group may comprise any of the listed 5- or 6-membered heteroaryl fused to a 5-membered or 6-membered non aromatic heterocycle.
In some embodiments, the Het group is selected from substituted or unsubstituted groups of the following formula:
Figure imgf000016_0001
wherein X is O or NH. In the conjugates comprising these Het groups, the siderophore and the cargo moieties are attached to each other by a direct and covalent bond between the carbon atom marked * of the catechol group and the nitrogen marked * of the Het group. It is to be noted that the nitrogen marked * of Het group is generally positively charged once bonded to the carbon atom marked * of the catechol group.
Preferably, the cargo moiety comprises a nitrogen-containing aryl group selected from substituted or unsubstituted groups of the following formula:
Figure imgf000016_0002
The conjugates of the present invention may be any of the following compounds:
Figure imgf000017_0001
Figure imgf000018_0001
Figure imgf000019_0001
Figure imgf000020_0001
Figure imgf000021_0001
Figure imgf000022_0001
Figure imgf000023_0001
Figure imgf000024_0001
Figure imgf000025_0001
Figure imgf000026_0001
Figure imgf000027_0001
Figure imgf000028_0001
Figure imgf000029_0001
Figure imgf000030_0001
\
5
Figure imgf000031_0001
Figure imgf000032_0001
Figure imgf000033_0001
Figure imgf000034_0001
Figure imgf000035_0001
Figure imgf000036_0001
Figure imgf000037_0001
Figure imgf000038_0001
Figure imgf000039_0001
When the conjugates are positively charged, X' , when represented, designates a counter-ion (negatively charged). Examples of counter-ions include halogen atoms, acetate or any counter-ions that provide a physiologically acceptable salt as described herein above. The conjugates of the present invention may be any of the conjugates 1 to 24 disclosed in the “EXAMPLES” section.
Preparation of the conjugates The conjugates of the present invention may be prepared by a process comprising the step of reacting a siderophore bearing a catechol group of the following formula:
Figure imgf000040_0001
with Ri and R2 are as disclosed herein above; with a cargo molecule bearing a substituted or unsubstituted heteraryl group (Het), said Het group comprising one or more nitrogen atoms; in conditions that allow oxidation of the catechol group and subsequent addition of the cargo molecule.
The Het group may be as disclosed herein above. In some embodiments, the Het group is selected from substituted or unsubstituted groups of the following formula:
Figure imgf000040_0002
wherein X is O or NH. It is known that oxidation of catechol groups leads to the formation of reactive o-quinone that can undergo 1 ,4-Michael addition reaction (Adolphs et al., 1996); Saxena et al., 1978; Cavalieri et al., 2002; Miserez et al., 2010).
Known methods for the synthesis of o-quinones from various classes of organic compounds includes the following reagents: sodium nitrite, hydrogen peroxide, potassium ferricyanide, silver oxide (Ag2O) (Cavalieri et al., 2002) alone or together with hydrogen peroxide at room temperature under batch and continuous-flow conditions (Derikvand et al., 2010); silver carbonate, sodium chlorate (NaCIO3), sodium periodate (NalO4), sodium or potassium dichromate (Na2Cr2O7/K2Cr2O7) with sulfuric acid (H2SO4) (Yang et al., 2018) or glacial acetic acid/dioxane (Kato et al., 1977), the nano-magnetic Fe3O4 (NM-Fe3O4) at room temperature (Sharghi et al., 2020), dichlorodicyanoquinone (DDQ), o-chloranil (Walker & Hiebert, 1967), ceric ammonium nitrate (CAN) on silica, horseradish peroxidase, (diacetoxyiodo)benzene, and phenyliodine(iii) bis(trifluoroacetate) (PIFA)/BF3 Et2O (Axford et al., 2008), nitric acid (Zherebtsov et al., 2021), copper (I or II) chloride (US5502214A), electrochemical oxidation (Nematollahi and Khoshsafar, 2009) (Ghorbani et al., 2020). Additional catechol oxidation agents include iodine, bromine, in particular in the presence of pyridine and its derivatives (Saxena et al., 1978). Ferric iron has also been demonstrated to cause catechol oxidation in acidic conditions (in basic conditions catechols form coordination bonds with Fe3+ that prevents further oxidation). At acidic pH, Fe3+ has been shown to oxidize substituted and unsubstituted catechols and to react to produce o-quinones (Fullenkamp et al., 2014; Yang et al., 2014), which react with nucleophiles or proceeds with a concomitant catechol-catechol oligomerization due to oxidative homocoupling (e.g. (Napolitano et al., 2000)(Yang et al., 2014)).
Therefore, the conditions that allow oxidation of the catechol group and subsequent addition of the cargo molecule may consist in reacting the cargo molecule and the siderophore in presence of an oxidizing agent or by electrochemistry.
Preferably, the oxidation of the catechol group is performed with the use of oxidizing agents selected from the group consisting of Fe(lll), l2, Br2, Ag2O and NalO4 or by electrochemistry. The formed reactive o-quinone can then undergo 1 ,4-Michael addition reaction with the cargo molecule to provide the conjugates of the present invention.
In some embodiments, the siderophore is reacted with the cargo molecule in presence of Fe3+ ions. In these conditions, the catechol group is oxidized allowing subsequent addition of the cargo molecule. Typically, the reaction is performed in presence of 3 to 10 equivalents of Fe3+ ions (with respect to the catechol group). The Fe3+ ions may originate from iron sulphate or iron trichloride. Thus, in some embodiments, the siderophore may be reacted with the cargo molecule in presence of iron sulphate or iron trichloride, typically in presence of 3 to 10 equivalents of iron sulphate or iron trichloride, to provide the conjugates of the present invention. The reaction is typically performed in a solvent selected from the group consisting methanol, ethanol, acetonitrile, DMSO, water, acetonitrile, chloroform, ethyl acetate and mixtures thereof. The reaction is typically performed at room temperature, but also proceeds at lower and higher temperatures.
In some embodiments, the siderophore is reacted with the cargo molecule in presence of l2, Br2, Ag2O, NalO4 or Cu(ll) to provide the conjugates of the present invention. In these conditions, the catechol group is oxidized allowing subsequent addition of the cargo molecule.
In some embodiments, the oxidation of the catechol group is performed by electrochemistry. The isolation and purification of the conjugates may be performed in accordance with methods well-known for the man skilled in the art.
Uses
The conjugates of the present invention may be used in medicine, for example as a medicament or for detection/diagnosis purpose.
For instance, the conjugates of the present invention may be used for the delivery of pharmaceutical agents, for examples antibiotics, into targeted bacteria. More specifically, the conjugates of the invention, when comprising an antibiotic moiety as cargo moiety, may be used in the treatment of a bacterial infection. Thus, in some aspects, the present invention relates to a conjugate as disclosed herein for use in the treatment of a bacterial infection.
The conjugates of the present invention may also be used to diagnose bacterial infections. In these embodiments, the conjugates of the present invention comprise a fluorophore as cargo moiety.
The conjugates of the present invention may be used to target bacteria belonging to the following bacterial order: Enterobacterales, Mycobacteriales, Pseudomonadales, Burkholderiales, Bacillales and Lactobacillales.
The conjugates of the present invention can be used to prepare compositions, such as pharmaceutical compositions, for example, by combining the conjugates with an acceptable excipient. Acceptable excipients include fillers and carriers, ointment bases, bases for suppositories, solvents, surfactants, emulsifiers, dispersants or wetting agents, buffers, acids and bases, isotonicity agents, adsorbent, viscosity-increasing agents, gel formers, thickeners and/or binders, disintegrants, coating materials and film formers for films or diffusion membranes, capsule materials, natural or synthetic polymers, plasticizers, penetration enhancers, stabilizers, preservatives, colourants, flavourings, sweeteners, flavour- and/or odour-masking agents. The conjugates may be used in a neutral form or in the form of a salt or solvate.
The pharmaceutical compositions can be administered to a mammalian host, such as a human patient, in a variety of forms. The forms can be specifically adapted to a chosen route of administration, e.g., oral or parenteral administration, by intravenous, intramuscular, topical or subcutaneous routes.
The invention provides therapeutic methods of treating bacterial infections in a mammal, which involve administering to a mammal having a bacterial infection an effective amount of a conjugate or composition described herein. A mammal includes a primate, human, rodent, canine, feline, bovine, ovine, equine, swine, caprine, bovine and the like. The bacterial infection can be a bacterial infection caused by a bacterium described herein. The amount of a conjugate which constitutes an effective amount will vary depending on many factors, such as for instance the conjugate and its biological activity, the composition used for administration, the route of administration, the type of disorder being treated and its severity, and the age, body weight, general health, sex, and diet of the patient. Such an effective amount can be determined routinely by one of ordinary skill in the art having regard to their own knowledge.
Embodiments of the present invention will now be described by way of the following examples which are provided for illustrative purposes only, and not intended to limit the scope of the disclosure.
EXAMPLES
L _ Identification of a new siderophore
Whole genome assembly of Dactylosporangium fulvum by PacBio sequencing, followed by AntiSMASH biosynthetic gene cluster identification confirmed the presence of a biosynthetic cluster responsible for the production of pyridomycin. Around half of this cluster (more than 20 genes) contained genes that were not associated with pyridomycin biosynthesis. Amongst the non-pyridomycin related genes were 2 large multimodular NRPS genes, an annotated halogenase and a number of annotated siderophore uptake transporters. RNAseq analysis showed that these genes were well expressed, with an expression dynamics different to that of the pyridomycin associated genes. Genetic inactivation of the first of these NRPS genes (Df2930) did not affect pyridomycin production by D. fulvum, but instead led to a loss of a number of LC-MS peaks, of which the predominant one had an m/z of 1001. Isotope distribution analysis of the MALDI-TOF peak at m/z of 1001 suggested it was chlorinated. An additional peak was observed at m/z of 967 which correspond to the non-chlorinated molecule. Mass fragmentation of these two molecules were related, with only a chlorination difference.
This compound with m/z of 1001 was purified from a 10 L culture using cation exchange resin and preparative chromatography using a C18 column. Yield of the purified molecule were 5 mg/L. The compound m/z of 1001 was tested in a Chrome Azurol S assay, and it was found to be able to chelate Fe3+ better than desferrioxamine B. The non-chlorinated m/z 967 could also chelate Fe3+. A combination of MALDI-TOF fragmentation, high resolution mass spectrometry and NMR analysis allowed for the elucidation of the chemical structure of both the chlorinated m/z of 1001 (named chlorodactyloferrin), and the non-chlorinated m/z of 967 (here named dactyloferrin). The structures are as follow :
Figure imgf000044_0001
Figure imgf000044_0002
The orientation of the stereo centers was determined using the information from the biosynthetic clusters, specifically, the presence or absence of epimerase domains in the NRPS modules. This elucidated structure fits with the structure of the biosynthetic cluster identified.
Chlorodactyloferrin could bind Fe3+ or Ga3+ as observed by LC-MS. The chlorodactyloferrin - Ga3+ complex was purified and analyzed by NMR to determine mechanism of iron coordination. To this extent, the catechol and the two hydroxamate groups were found to coordinate the metal ion.
The proximity of the chlorodactyloferrin and pyridomycin biosynthetic gene cluster led to the investigation of a potential conjugate of the two molecules. Indeed, analysis of cell pellets, and supernatants of fermentation reactions could identify a m/z of 1539 (chlorinated by isotopic profile), a mass equal to the sum of the masses of pyridomycin (m/z = 540) and chlorodactyloferrin (m/z of 1000). MALDI-TOF fragmentation showed this chlorodactyloferrin- pyridomycin conjugate to contained chlorodactyloferrin with a conjugated molecule on the chlorocatecholate moiety. This conjugate was also found to complex ferric iron giving a peak at m/z 1592.
Mutants of D. fulvum incapable of generating chlorodactyloferrin (ADf2930), were found not to produce chlorodactyloferrin or the conjugate, but did produced pyridomycin. Mutants for pyridomycin production (ApyrA) did not produce pyridomycin or the conjugate, but did produce chlorodactyloferrin. Interestingly, feeding of D. fulvum (ApyrA) culture with pyridomycin restored the production of the conjugate mass. This data suggested that pyridomycin and chlorodactyloferrin are produced independently, and are conjugated together after formation.
The possibility of chemical conjugation between chlorodactyloferrin and pyridomycin was then investigated. When mixing pyridomycin with chlorodactyloferrin no conjugate was formed. However, upon the addition of ferric iron (FeCI3, 10 eq), a product was formed with the mass of m/z=1539. This conjugation reaction was also observed to occur in the presence of copper(ll) chloride (CuCI2), but not did not occur with ferrous iron (FeSO4), gallium (III) trichloride (GaCh) or cobalt(lll) chloride (C0CI3). Purification of the chlorodacyloferrin-pyridomycin complex and structural elucidation by NMR confirmed that the conjugation occurred through the formation of a covalent bond between the siderophore catechol group (C6) and the nitrogen of the pyridomycin pyridine group forming a pyridinium product as shown below:
Figure imgf000046_0001
In addition, it was found that dactyloferrin (instead of chlorodactyloferrin) could also conjugate with pyridomycin, showing that the chlorine was not essential.
As determined by MALDI-TOF, the FeCI3 based conjugation reaction was also found to occur between chlorodactyloferrin and pyridomycin analogues where the pyridine was conserved: 5CI-HPA- pyridomycin, 6CI-HPA- pyridomycin and pyridomycin precursor 558, but not with Phe-pyridomycin where the pyridine moiety was changed to a phenyl. The pyridomycin precursors come from mutasynthesis from D. fulvum, and the conjugation products were confirmed only by mass spectrometry, not by NMR.
Figure imgf000046_0002
5CI-HPA- pyridomycin SCI-HPA- pyridomycin pyridomycin precursor 558 Phe-pyridomycin >
II. Conjugation of catechol containing siderophores with cargo molecules (antibiotics and fluorophores) The following examples illustrate the direct coupling of catecholate containing siderophores with antibiotics or other cargo molecules using catechol oxidation agents such as Fe(lll), l2, NalO4, Br2, Ag2O or electrochemistry.
1. Conjugate of chlorodactyloferrin and pyridyl-modified TAMA (pyridyl-TAMRA called herein TAMRA3515) (1)
Figure imgf000047_0001
Under nitrogen atmosphere, to an ice-cold solution of chlorodactyloferrin (10 mg, 0.01 mmol) and TAMRA3515 (5.21 mg, 0.01 mmol) in water/acetonitrile 1 :1 (1 ml), iron trichloride was added at 10 eguivalents (16.2 mg, 0.1 mmol). The reaction mixture was diluted with a minimum amount of water, filtered and deposited on a C18-column. Product (1) was eluted by applying a water-acetonitrile gradient as eluents. Deep red/purple solid was obtained after lyophilization. Yield: 3.2 mg (20.3 % as Fe3+ salt). C7iH88CIFeNi8Oi8+: calcd m/z 1572.6520; obsd m/z 1572.42. For NMR characterization analytical amounts of the product were dissolved in 1 M solution of Ga3+ and purified using reverse-phase chromatography with water-acetonitrile as eluents. NMR analysis is consistent with the shown structure.
Conjugate of dactyloferrin and pyridomycin (2)
Figure imgf000048_0001
Using dactyloferrin (10 mg, 0.01 mmol), pyridomycin (5.4 mg, 0.01 mmol) and iron trichloride (16.2 mg, 0.1 mmol) as starting compounds, compound (2) was prepared as a brownish solid according to the synthesis of TAMRA-chlorodactyloferrin conjugate. Yield: 3.8 mg (24.3 %); C67H93FeNi8O22+: calcd m/z 1592.87; obsd m/z 1592.67. NMR analysis is consistent with the proposed structure.
3. Conjugate of chlorodactyloferrin and pyridyl-modified norfloxacin (called herein
Figure imgf000048_0002
Using chlorodactyloferrin 1 (6 mg, 0.006 mmol), Fluoroquinolone-Pyr (2.54 mg, 0.006 mmol) and iron trichloride (9.7 mg, 0.06 mmol) as starting compounds, compound (3) was prepared as a brownish solid according to the synthesis of TAMRA-chlorodactyloferrin conjugate. Yield: 2.9 mg (32.7 %); C62H8iCIFFeNisOi8+: calcd m/z 1476.73; obsd m/z 1476.58. NMR analysis is consistent with the proposed structure. herein B-lactam-
Figure imgf000048_0003
Figure imgf000049_0001
Using chlorodactyloferrin (10 mg, 0.01 mmol), p-lactam-Pyr (3.3 mg, 0.01 mmol) and iron trichloride (16.3 mg, 0.1 mmol) as starting compounds, compound (4) was prepared as a brownish solid according to the synthesis of TAMRA-chlorodactyloferrin conjugate. Yield: 1 mg (7.2 %); C55H77CIFeNi7Oi8S+: calcd m/z 1387.67; obsd m/z 1387.50. NMR analysis is consistent with the proposed structure.
5. Conjugate of chlorodactyloferrin and pyridyl-modified rifampicin (called herein
Figure imgf000049_0002
Under nitrogen atmosphere, to an ice-cold solution of chlorodactyloferrin (10 mg, 0.01 mmol) and Rifamycin-Pyr (9 mg, 0.01 mmol) in water/acetonitrile 1 :1 (1 ml), iron trichloride was added at 100 equivalents (162 mg, 1 mmol). A sudden change of the color to dark red was observed, due to the complexation of chlorodactyloferrin with iron and the oxidation of Rifamycin-Pyr to its quinone form. Reduction of the Rifamycin-Pyr-quinone to the hydroquinone form was achieved by adding 100 equivalents of ascorbic acid (176 mg, 1 mmol). Purification of the conjugate (5) was performed as indicated for the synthesis of TAMRA-chlorodactyloferrin conjugate. Yield: 4.2 mg (21.5 %); CssH^iCIFeNigC : calcd m/z 1952.34; obsd m/z 1952.50. NMR analysis is consistent with the proposed structure.
6. Conjugate of enterobactin and TAMRA3515 (6) 6.1 Synthesis by electrochemistry
Figure imgf000050_0001
Synthesis of enterobactin-TAMRA3515 conjugate using Electrochemistry, reagents and conditions: (i) 0.1 M NaOAc in water/acetonitrile (1 :1 , v/v), 1.9 eq. enterobactin, 1 eq TAMRA, constant voltage of 0.85V (2 mA, 2F/mol), 7 hr, RT.
A solution of sodium acetate buffer (0.1 M; pH = 7.2) in water/acetonitrile (50:50; 4 mL) containing enterobactin (10 mg, 0.015 mmol) and TAMRA (4 mg, 0.008 mmol) was electrolyzed in an undivided cell at constant voltage of 0.85V (2 mA, 2F/mol) vs. Ag/AgCI for
7 hr. at room temperature. After electrolysis, the solution was acidified with 0.2 M hydrochloric acid and analyzed by LC-MS which confirmed formation of the target product (6) (same product as obtained by iodine oxidation, see below). C61H54N7O19+: calcd m/z 1188.3469; obsd m/z 1188.25.
6.2. Synthesis with iodine
Figure imgf000051_0001
Iodine (2.6 mg, 0.01 mmol) was added to an ice-cold solution of enterobactin (12.5 mg, 0.019 mmol) and TAMRA (5.3 mg, 0.01 mmol) in anhydrous DMSO (1 mL). The solution was allowed to stir for 2 hr while warming to room temperature. The reaction mixture was then diluted with a mixture of water-acetonitrile solution (10 mL, 7:3, v/v), filtered through a 1 pm filter and purified by reverse phase chromatography. Yield as a red-orange solid: 3.6 mg (30%). LC-MS analysis confirmed formation of the target product (6). CGI H54N7OI9+: calcd m/z 1188.3469; obsd m/z 1188.25. NMR analysis is consistent with the proposed structure.
7. Conjugate of enterobactin and pyridyl-modified dansyl (called herein Dansyl-Pyr) (7)
Figure imgf000052_0001
Using enterobactin (3 mg, 0.0045 mmol), Dansyl-Pyr (3 mg, 0.009 mmol) and iodine (1.1 mg, 0.0045 mmol) as starting compounds, compound (7) was prepared by following the same synthetic procedure used to prepare enterobactin-TAMRA3515. LC-MS analysis confirmed formation of the target product (7). C48H4sN6Oi7S+: calcd m/z 1009.2556; obsd m/z 1007.30.
8. Conjugate of enterobactin and pyridyl-modified coumarin 343 (called herein
Coumarine-Pyr) (8)
Figure imgf000053_0001
Using enterobactin (3 mg, 0.0045 mmol), Coumarine-Pyr (3.8 mg, 0.009 mmol) and iodine (1.1 mg, 0.0045 mmol) as starting compounds, compound (8) was prepared by following the same synthetic procedure used to prepare enterobactin-TAMRA3515. LC-MS analysis confirmed formation of the target product (8). C52H47N6Oi8+: calcd m/z 1043.2941 ; obsd m/z 1043.30. NMR analysis is consistent with the proposed structure.
9. Conjugate of enterobactin and dabcyl (9)
9.1 Synthesis of dabcyl-3-picolamide
Figure imgf000053_0002
Synthesis of dabcyl-3-picolamide Reagents and conditions: (a) 1.2 eq. 3- (aminomethyl)pyridine, DMF, 16h, RT, 80%. 3-(Aminomethyl)pyridine (0.29 mmol, 31 mg) was added in one portion to a solution of the succinate ester (0.24 mmol, 90 mg) in 7 mL DMF under nitrogen atmosphere at room temperature. After 16 h of stirring, the organic solvent was removed under reduced pressure. EtOAc (100 mL) was added to the obtained residue. The organic layer was washed with a
Figure imgf000054_0001
Using enterobactin (3 mg, 0.0045 mmol), dabcyl-Pyr (3.22 mg, 0.009 mmol) and iodine (1.1 mg, 0.0045 mmol) as starting compounds, compound (9) was prepared by following the same synthetic procedure used to prepare enterobactin-TAMRA3515. LC-MS analysis confirmed formation of the target product (9). C52H47N6Oi8+: calcd m/z 1027.3105; obsd m/z 1027.30. NMR analysis is consistent with the proposed structure. 10. Conjugate of enterobactin and metronidazole (10)
Figure imgf000055_0001
Using enterobactin (3 mg, 0.0045 mmol), metronidazole (1.54 mg, 0.009 mmol) and iodine (1.1 mg, 0.0045 mmol) as starting compounds, compound (10) was prepared by following the same synthetic procedure used to prepare enterobactin-TAMRA3515. LC-MS analysis confirmed formation of the target product. C52H47N6Oi8+: calcd m/z 839.70; obsd m/z 839.15. 11. Conjugate of enterobactin and aztreonam (11)
Figure imgf000055_0002
Using enterobactin (3 mg, 0.0045 mmol), aztreonam (3.91 mg, 0.009 mmol) and iodine (1.1 mg, 0.0045 mmol) as starting compounds, compound (11) was prepared by following the same synthetic procedure used to prepare enterobactin-TAMRA3515. LC-MS analysis confirmed formation of the target product. C43H43NsO23S2+: calcd m/z 1103.97; obsd m/z 1101.17.
12. Conjugate of enterobactin and cefepime (12)
Figure imgf000056_0001
Using enterobactin (3 mg, 0.0045 mmol), cefepime (4.31 mg, 0.009 mmol) and iodine (1.1 mg, 0.0045 mmol) as starting compounds, compound (12) was prepared by following the same synthetic procedure used to prepare enterobactin-TAMRA3515. LC-MS analysis confirmed formation of the target product. C49H5oNg02oS2+: calcd m/z 1148.2608; obsd m/z 1148.21.
13. Conjugate of enterobactin and ceftazidime (13)
Figure imgf000057_0001
Using enterobactin (3 mg, 0.0045 mmol), ceftazidime (4.9 mg, 0.009 mmol) and iodine (1.1 mg, 0.0045 mmol) as starting compounds, compound (13) was prepared by following the same synthetic procedure used to prepare enterobactin-TAMRA3515. LC-MS analysis confirmed formation of the target product. C52H48NgO22S2+: calcd m/z 1214.2350; obsd m/z 1214.16.
14. Conjugate of acinetobactin and TAMRA3515 (14)
14.1 Synthesis by electrochemistry
Figure imgf000058_0001
Synthesis of Acinetobactin-TAMRA3515 conjugate using Electrochemistry, reagents and conditions: (i) 0.1 M NaOAc in water/acetonitrile (1 :1 , v/v), constant voltage of 0.77 V (2 mA, 2F/mol), 7 hr., RT.
Using acinetobactin (7 mg, 0.02 mmol) and TAMRA3515 (10 mg, 0.02 mmol) as starting compounds, compound (14) was prepared as a red-orange solid according to the electrochemical synthesis of enterobactin-TAMRA3515 conjugate (14). Trace amounts could be detected by analytical LC-MS. C47H45N8O9 +: calcd m/z 865.3304; obsd m/z 865.3312.
14.2 Synthesis with FeCI3
Figure imgf000059_0001
Synthesis of Acinetobactin-TAMRA3515 conjugate using FeCI3 method, reagents and conditions: (i) 1 eq. acinetobactin, 10 eq. FeCI3, 1 eq. TAMRA3515, water-acetonitrile, 2 hr.
Using acinetobactin (10 JLLL, 10 mM soln in water-acetonitrile 1 :1), TAMRA3515 (10 JLLL, 10 mM soln in water-acetonitrile 1 :1) and iron trichloride (10 JLLL, 100 mM soln in water- acetonitrile 1 :1) as starting compounds, compound (14) was prepared. LC-MS analysis confirmed formation of the target product (14). C47H45N8O9 +: calcd m/z 865.3304; obsd m/z 865.34.
14.3 Synthesis with Iodine
Figure imgf000060_0001
Synthesis of Acinetobactin-TAMRA3515 conjugate using Iodine method, reagents and conditions: (i) 1 eq. acinetobactin, 1 eq. I2, 1 eq. TAMRA3515, water-acetonitrile, 2 hr.
Using acinetobactin (10 JLLL, 10 mM soln in water-acetonitrile 1 :1), TAMRA3515 (10 JLLL, 10 mM soln in water-acetonitrile 1 :1) and iodine (10 JLLL, 10 mM soln in water-acetonitrile 1 :1) as starting compounds, compound (14) was prepared. LC-MS analysis confirmed formation of the target product. C47H45N8O9 +: calcd m/z 865.3304; obsd m/z 865.34.
15. Conjugate of enterobactin and pyridomycin (15)
Figure imgf000061_0001
Using enterobactin (1.33 mg, 0.002 mmol), pyridomycin (1.08 mg, 0.002 mmol) and iodine (1.52 mg, 0.006 mmol) as starting compounds, compound 15 was prepared by following the same synthetic procedure used to prepare enterobactin-TAMRA3515 (6”). LC-MS analysis confirmed formation of the target product. C57H58N7C : calcd m/z 1208.3579; obsd m/z 1208.50.
16. Conjugate of enterobactin and 3-pyridyl-rifabutin (16)
Figure imgf000062_0001
Using enterobactin (1.33 mg, 0.002 mmol), 3-pyridyl-rifabutin (1.76 mg, 0.006 mmol) and iodine (1.52 mg, 0.006 mmol) as starting compounds, compound 16 was prepared by following the same synthetic procedure used to prepare enterobactin-TAMRA3515 (6”). Yield as a yellowish solid: 0.25 mg (8%). LC-MS analysis confirmed formation of the target product. CysHssNs : calcd m/z 1549.5570; obsd m/z 1549.50.
17. Conjugate of enterobactin and 3-pyridyl-rifabutin (17)
Figure imgf000063_0001
Using enterobactin (1.33 mg, 0.002 mmol), 3-pyr/c/y/-levofloxacin RPII146 (2.54 mg, 0.006 mmol) and iodine (1.52 mg, 0.006 mmol) as starting compounds, compound 17 was prepared by following the same synthetic procedure used to prepare enterobactin-TAMRA3515 (6”). Yield as a yellowish solid: 0.75 mg (34%). LC-MS analysis confirmed formation of the target product. C52H47FN7Oi9+ : calcd m/z 1092.2905; obsd m/z 1092.33.
18. Conjugate of acinetobactin and cefapirin (18)
Figure imgf000064_0001
Using acinetobactin (2.48 mg, 0.0072 mmol), cefapirin (3.19 mg, 0.0072 mmol) and iodine (1.69 mg, 0.0072 mmol) as starting compounds, conjugate 18 was prepared by following the same synthetic procedure used to prepare enterobactin-TAMRA3515 (6”). Yield as a white solid: 0.3 mg (5%). LC-MS analysis confirmed formation of the target product. Chemical Formula: C33H34N7OnS2 + : calcd m/z 768.1725; obsd m/z 768.25.
19. Conjugate of chloroacinetobactin and cefapirin (19)
Figure imgf000064_0002
Using chloroacinetobactin (2.09 mg, 0.0055 mmol), cefapirin (2.48 mg, 0.0055 mmol) and iodine (1.39 mg, 0.0055 mmol) as starting compounds, conjugate 19 was prepared by following the same synthetic procedure used to prepare enterobactin-TAMRA3515 (6”). Yield as a white solid: 0.44 mg (10%). LC-MS analysis confirmed formation of the target product. C33CIH34N7O1 iS2 + : calcd m/z 802.1363; obsd m/z 802.25.
20. Conjugate of acinetobactin and 3-pyridyl-ciprofloxacin (20)
Figure imgf000065_0001
Using acinetobactin (1 mg, 0.0029 mmol), 3-pyridyl-ciprofloxacin RPII67 (1.4 mg, 0.0029 mmol) and iodine (6.82 mg, 0.029 mmol) as starting compounds, conjugate 20 was prepared by following the same synthetic procedure used to prepare enterobactin-TAMRA3515 (6”). Yield as a white solid: 0.4 mg (15%). LC-MS analysis confirmed formation of the target product. C42H47FN9O9+ : calcd m/z 840.3476; obsd m/z 840.42.
21 . Conjugate of chloroacinetobactin and 3-pyridyl-ciprofloxacin (21)
Figure imgf000066_0001
Using chloroacinetobactin (1 mg, 0.0026 mmol), 3-pyridyl-ciprofloxacin PRII67 (1.3 mg, 0.0026 mmol) and iodine (6.65 mg, 0.026 mmol) as starting compounds, conjugate 21 was prepared by following the same synthetic procedure used to prepare enterobactin-
TAMRA3515 (6”). Yield as a white solid: 0.3 mg (13%). LC-MS analysis confirmed formation of the target product. C42CIH47FN9O9+ : calcd m/z 874.3086; obsd m/z 874.31.
22. Conjugate of acinetobactin and pyridomycin (22)
Figure imgf000066_0002
Using acinetobactin (2.15 mg, 0.0062 mmol), pyridomycin (3.36 mg, 0.0062 mmol) and iodine (1.46 mg, 0.0062 mmol) as starting compounds, conjugate 22 was prepared by following the same synthetic procedure used to prepare enterobactin-TAMRA3515 (6”). Yield as a white solid: 0.7 mg (12%). LC-MS analysis confirmed formation of the target product. C43H49N8Oi3+ : calcd m/z 885.3414; obsd m/z 885.50.
23. Conjugate of chloroacinetobactin and pyridomycin (23)
Figure imgf000067_0001
Using chloroacinetobactin (2 mg, 0.0055 mmol), pyridomycin (2.97 mg, 0.0055 mmol) and iodine (1.39 mg, 0.0055 mmol) as starting compounds, conjugate 23 was prepared by following the same synthetic procedure used to prepare enterobactin-TAMRA3515 (6”). Yield as a white solid: 0.9 mg (18%). LC-MS analysis confirmed formation of the target product. C43CIH49N8Oi3+ : calcd m/z 919.3024; obsd m/z 919.58. 24. Conjugate of enterobactin and 3-pyridyl-levofloxacin (24)
Figure imgf000068_0001
Enterobactin (0.0668 mg, 0.0001 mmol) was coupled to 3-pyridyl-levofloxacin derivatives (0.0466 mg, 0.0001 mmol) by the addition of iodine (0.0759 mg, 0.0003 mmol). Reaction mixtures were diluted 1 :4 in DMSO and presence of the expected enterobactin-3-pyridyl- levofloxacin conjugates was confirmed by UPLC-MS. 1
III. Biological data
The uptake of the siderophore-cargo conjugates was evaluated via (1) the uptake of siderophores conjugated to fluorophores followed by confocal microscopy and (2) determination of the antimicrobial activity of the antibiotic-siderophore conjugates.
1 . Uptake of siderophore-fluorophore conjugate
Siderophore-fluorophore conjugates were synthetized as previously described. This includes different combinations between catechol containing siderophores (chlorodactyloferrin, enterobactin and acinetobactin) and fluorophores with different physicochemical and optical properties (such as Coumarin-Pyr, Dabcyl-Pyr, Dansyl-Pyr and TAMRA-Pyr).
The results of the uptake of chlorodactyloferrin-TAMRA3515, enterobactin-TAMRA3515 and acinetobactin-TAMRA3515 bioconjugates by D. fulvum (and other phylogenetically-related species), E. coli and A. baumannii, respectively are hereby presented. However, obtained results are presumed to be extrapolatable to other siderophore-fluorophore conjugates and bacterial strains.
1.1 Uptake by D. fulvum D. fulvum wildtype under iron-limiting conditions (grown overnight in presence of the iron chelator 2,2’-dypiridyl, DIP, 350 pM) was incubated with chlorodactyloferrin-TAMRA3515 conjugate (1) (1 pM) for 4 h, and evaluated by super-resolution confocal microscopy.
The results are presented in Figure 1 .
All images were captured using the oil immersion objective 60x/1 .40 of an inverted spinningdisk microscope Yokogawa CSU-W1 equipped with a Roper Scientific Live-SR module. F: fluorescence TAMRA filter set (excitation: 561 nm; emission: 595 nm, 50 nm bandwidth), BF: bright field, and C: composite. Shown pictures are representative of all the sample and reproducible across independent preparations.
As it can be observed in Figure 1 , bacteria were fluorescently labelled, confirming the uptake of the siderophore-fluorophore conjugates. Even though a limitation of this approach would be the technical difficulty to distinguish between intracellular uptake and surface/receptor binding with no uptake, single optical sections of the samples show fluorescence signal labelling the bacteria intracellularly and not only at the surface.
When comparing Figure 1 (A) and (B), it can be observed that TAMRA3515 is not incorporated by D. fulvum, but its uptake is facilitated by the chlorodactyloferrin molecule. However, this intracellular accumulation could be the result of passive transport of the conjugate (in the hypothetical case it is having better permeability than the fluorophore alone) or active transport through the natural siderophore import system. To demonstrate that the conjugate is hijacking the chlorodactyloferrin transport system, a competition assay incubating D. fulvum both with the conjugate and the native siderophore at high excess (100- times more concentrated) was performed. The results are presented in figure 1 (C). Bacteria are still fluorescently labelled but with lower signal intensity, indicating that the uptake of the conjugate is outcompeted by the natural siderophore, and pointing to the same active transport mechanism for both compounds.
1.2 _ Uptake by Dactylosporanqiae and other related bacteria
The uptake of the chlorodactyloferrin-TAMRA3515 conjugate (1) by Dactylosporangiae and other related bacteria was also assessed. The genomes of these bacteria are encoding homologous transport systems to the one used by D. fulvum for chlorodactyloferrin uptake. For that, these strains were incubated with both chlorodactyloferrin-TAMRA3515 and TAMRA3515, in the same conditions as previously reported.
The results are presented in figures 2 to 7. All images were captured using the oil immersion objective 60x/1 .40 of an inverted spinningdisk microscope Yokogawa CSU-W1 equipped with a Roper Scientific Live-SR module. F: fluorescence TAMRA filter set (excitation: 561 nm; emission: 595 nm, 50 nm bandwidth), BF: bright field, and C: composite. Shown pictures are representative of all the sample and reproducible across independent preparations.
All the strains tested showed intense fluorescence signal when treated with the conjugate in comparison with the unconjugated-fluorophore, as shown in Figures 2 to 7. Additionally, no uptake was observed with other strains that are more distant phylogenetically, such as Mycobacterium tuberculosis, S. aureus or different Enterobacteriaceaes (data not shown). Altogether, our data points to an active and species-specific import system for the conjugates.
1.3 _ Uptake by E. coli
The uptake of the enterobactin-TAMRA3515 conjugate (6) by E. coli was evaluated via imaging by super-resolution confocal microscopy iron-deprived bacteria (treated 2 h in presence of the iron chelator 2, 2’-dypiridyl, DIP, 100 JLLM) treated with the conjugate for 1 h. The results are presented in Figure 8. All images were captured using the oil immersion objective 60x/1.40 of an inverted spinning-disk microscope Yokogawa CSU-W1 equipped with a Roper Scientific Live-SR module. F: fluorescence TAMRA filter set (excitation: 561 nm; emission: 595 nm, 50 nm bandwidth), BF: bright field, and C: composite. Shown pictures are representative of all the sample and reproducible across independent preparations.
As shown in Figure 8, all bacteria become fluorescently labelled, indicating the uptake of the siderophore-fluorophore conjugate. In the case of E. coli, the unconjugated fluorophore is also accumulated by the bacteria (data not shown), therefore it cannot be used as control to demonstrate the improved uptake of the TAMRA3515 molecule when conjugated to the siderophore. However, a similar experiment with an E. coli AfepA mutant (not harbouring the outer membrane component of the enterobactin transport system) was conducted. Even if some fluorescence was still detected (Figure 8 (B)), its signal intensity is lower in comparison to the WT and its intracellular distribution pattern was different (more intense at the bacterial poles). Overall, the data indicate that enterobactin-TAMRA3515 conjugate is imported thought the same machinery as natural enterobactin.
1.4 Uptake by A baumannii The uptake of acinetobactin-TAMRA3515 conjugate (14) by A. baumannii was also evaluated. Experiments were performed as with E. coli. Briefly, the wildtype strain under iron- limited conditions (incubated 4 h presence of DIP 100 pM) was treated with the 10 pM siderophore-fluorophore conjugate for 4 h and imaged individual bacteria through confocal microscopy.
The results are presented in Figure 9. All images were captured using the oil immersion objective 60x/1.40 of an inverted spinning-disk microscope Yokogawa CSU-W1 equipped with a Roper Scientific Live-SR module. F: fluorescence TAMRA filter set (excitation: 561 nm; emission: 595 nm, 50 nm bandwidth), BF: bright field, and C: composite. Shown pictures are representative of all the sample and reproducible across independent preparations.
Some bacteria were fluorescently labelled (Figure 9), confirming conjugate incorporation.
2. _ Antimicrobial activity of siderophore-antibiotic conjugates
Several siderophore-antibiotic conjugates were synthesised as previously described, including different combinations of catechol containing siderophores (chlorodactyloferrin and enterobactin) with both pyridyl-antibiotics (pyridomycin, p-lactam-Pyr, fluoroquinolone-Pyr, rifampicin-Pyr) and other “nitrogen containing heteroaromatic”-containing antibiotics (aztreonam, cefepime, ceftazidime, metronidazole).
The results for chlorodactyloferrin conjugates with home-synthetized 3-pyridy l-antibiotics (i.e. chlorodactyloferrin-pyridomycin (2), chlorodactyloferrin-p-lactam (4), chlorodactyloferrin- fluoroquinolone (3), chlorodactyloferrin-rifampicin (5)) against D. fulvum and related species are herein presented. Nonetheless, these results can be extrapolated to other siderophore- fluorophore conjugates and bacterial strains.
The antimicrobial activity of these conjugates against D. fulvum, D. vinaceum and S. coelicolor was assessed by the microdilution method in deep-well plates with a final volume of 400 pL and heavy shaking (to ensure the growth of strictly aerobic bacteria), and with visual readout after 1 day of incubation. Results are summarized in Table 1. The chlorodactyloferrin-p-lactam (4) and chlorodactyloferrin-rifampicin (5) are active against D. fulvum and D. vinaceum at similar concentrations as their respective antibiotics (p-lactam and rifampicin “Rif’). Nonetheless, these compounds are inactive against S. coelicolor at the tested concentrations. Both chlorodactyloferrin-fluoroquinolone (3) and chlorodactyloferrin- pyridomycin (2) conjugates, as well as their parental 3-pyridyl-antibiotics (fluoroquinolone “FQ” and pyridomycin “Pyr”), showed no antimicrobial activity below 10 pM in all the strains tested. However, some effect on S. coelicolor growth is observed at the highest concentrations tested (10-50 pM) for both the conjugates and the antibiotics alone. Of note is that while Beta-Lactam antibiotics (and the conjugates) targets extra-cytoplasmic proteins (penicillin bind in penecillin binding proteins (PBP)), rifampicin targets the DNA dependent RNA polymerase which is intracellular.
Figure imgf000072_0001
Table 1 : Antimicrobial activity of conjugates of the invention against D. fulvum, D. vinaceum and S. coelicolor
3. Antimicrobial activity of conjugates 16 and 17
The antimicrobial activity of conjugates 16 and 17 was evaluated against Escherichia coli BW25113 by the microtitre-dilution method in both normal and low iron-conditions (minimal media M9 with and without 100 pM 2,2’-dipyridyl, DIP, respectively). Under low iron conditions, conjugate 16 showed a superior minimal inhibitory concentration (MIC) of 0.11- 0.22 pM, compared to under normal iron-conditions where there was an MIC of 3.5 pM (Table 1). Thus finding supports active uptake of conjugate 16 by siderophore uptake pumps induced under ron stress. Conjugate 17 did not show a measurable antimicrobial activity below 5 pM in ether media, but the molecule did promote bacterial growth in the presence of DIP, indicating that conjugate 17 is taken up and can relieve iron stress. As enterobactin can also be activity take up by other bacterial species, the enterobactin- antibiotic conjugates 16 & 17 were also tested against A. baumannii ATCC 19606. In A. baumannii conjugates 16 & 17 showed a similar pattern as observed in E.coli, with conjugate 16 havng better activity under low iron conditions (MIC of 0.125 pM in Muller-Hinton broth 10% FCS) vs normal media (MIC - 1 pM in Muller-Hinton broth) and with conjugate 17 showing growth-promoting activity (Table 2), suggesting these compounds act in an irondependent manner.
Minimal Inhibitory Concentration, M
Figure imgf000073_0001
Table 2: Bacterial antibiotic susceptibility of E. coli and A. baumannii in liquid meda with different iron availability, to conjugates 16 and 17. Compounds were tested in normal (M9 or Muller-Hinton, MH) and low iron conditions (M9 supplemented with 100 pM or Muller-Hinton with 10% FCS). * indicates that the compound is promoting growth in low iron conditions.
4. Antimicrobial activity of conjugates 18-23
To target A. baumannii, antibiotic-conjugates with acinetobactin and chloro-acinetobactin (derivative of acinetobactin with chlorinated catechol group) 18-23 were generated using the here described iodine based conjugation reactions. Both acinetobactin and chloro- acinetobactin are taken up by A. baumannii and provide a growth advantage under low iron conditions.
The antimicrobial activity of conjugates 18-23 was determined against A. baumannii ATCC 19606 by the micro-titre dilution method in media with both normal and low iron-conditions (Muller Hinton media and Muller Hinton media with 10% FCS, respectively). Conjugates with cefapirin 18 and 19 were not active against A. baumannii below 200 pM, similarly to the parental antibiotic cefapirin. Similarly, neither the fluoroquinolone analogue RPII67, nor the generated acnetobacter conjugates (20) was active (MIC > 200 pM), however, when coupled to chloroacinetobactin (conjugate 21), the conjugate showed an MIC of 100 pM in low iron conditions, and no activity in normal iron condition. Acinetobactin-pyridomycin conjugate 22 and chloroacinetobactin-pyridomycin conjugate 23 had an MIC of 150 pM and 20 pM respectively in low iron media and >150 pM and 80 pM in normal iron media, respectively. Additionally, conjugates 21 , 22 and 23 showed clear growth promotion in low iron conditions, indicating that these compounds were efficiently taken up.
Figure imgf000074_0001
Table 3: Bacterial antibiotic susceptibility in liquid culture to conjugates 18-23. Compounds were tested in normal (Muller-Hinton, MH) and low iron conditions (Muller-Hinton with 10% FCS) against A. baumannii ATCC 19606. * indicates that the compound is promoting growth in low iron conditions.
5. Antimicrobial activity of conjugate 24
10 pL of the reaction mixture obtained in example 24 was loaded onto disks to evaluate the zone of inhibition of the crude conjugate and compared with the same amount of original unconjugated fluoroquinolone (10 pL of 1 pM).
The by disk diffusion assay was performed on solid media with low and high-iron media (M9 agar with or without 100 pM DIP). Zones of growth inhibition of reaction mixtures (enterobactin + free-fluoroquinolone + conjugate) were compared to those from mixtures of the unconjugated reactants (enterobactin + free fluoroquinolone), so that the total fluoroquinolone concentration in all the samples is consistent. Most of the reaction mixtures showed similar zone of inhibition as the reactants, suggesting that coupling of fluoroquinolones to enterobactin did not greatly improve activity. However, in the case of enterobacting conjugation reactions with the levofloxacin analogue RPII151 , the unconjugated compound showed no activity under any condition (no inhibition zone), while improved activity was detected for the conjugation reaction mixture containing enterobactin- RPII151 (conjugate 24) only under low iron conditions (Figure 11). This points to an irondependent import and subsequent antimicrobial activity of enterobactin-RPII151 (24).
BIBLIOGRAPHY
Adolphs M, Taraz K, Budzikiewicz H. 1996. Catecholate siderophores from Chryseomonas luteola. Zeitschrift fur Naturforsch Sect C - J Biosci 51 :281 -285. doi: 10.1515/znc-1996-5-603
Axford L.C., Kate E. Holden K.E , Hasse K, Martin G. Banwell M.G. , Steglich W, Wagler J. and Willis A.C., Attempts to Mimic Key Bond-Forming Events Associated with the Proposed Biogenesis of the Pentacyclic Lamellarins, Australian Journal of Chemistry 61 (2) 80-93, 2008
Cavalieri EL, Li KM, Balu N, Saeed M, Devanesan P, Higginbotham S, Zhao J, Gross ML, Rogan EG. 2002. Catechol ortho-quinones: The electrophilic compounds that form depurinating DNA adducts and could initiate cancer and other diseases. Carcinogenesis 23:1071-1077.
Derikvand F, Bigi F, Maggi R, Piscopo CG, Sartori G. 2010. Oxidation of hydroquinones to benzoquinones with hydrogen peroxide using catalytic amount of silver oxide under batch and continuous-flow conditions. J Catal 271 :99-103.
Fullenkamp DE, Barrett DG, Miller DR, Kurutz JW, Messersmith PB. 2014. PH-dependent cross-linking of catechols through oxidation via Fe 3+ and potential implications for mussel adhesion. RSC Adv 4:25127-25134.
Ghorbani A, Amani A, Nematollahi D, Sharafi-kolkeshvandi M. 2020. Electrochemically Induced Diels-Alder Reactions of Some Substituted o-quinones with 1 ,3-cyclopentadiene: An Interesting Finding in the Diels-Alder Reactions. J Electrochem Soc 167:155516. Kato T, Katagiri N, Nakano J, Kawamura H. 1977. Total synthesis of bikaverin involving the novel rearrangement of an ortho-quinone to a para-quinone. J Chem Soc Chem Commun 645-646.
Miserez A, Rubin D, Waite JH. 2010. Cross-linking chemistry of squid beak. J Biol Chem 285:38115-38124.
Napolitano A, Palumbo A, D’Ischia M. 2000. Oxidation of the neurotoxin 6-nitrodopamine and related 4-nitrocatechols under biomimetic conditions. Tetrahedron 56:5941-5945.
Nematollahi D, Khoshsafar H. 2009. Investigation of electrochemically induced Michael addition reactions. Oxidation of some dihydroxybenzene derivatives in the presence of azide ion. Tetrahedron 65:4742-4750.
Saxena JP, Purohit NL, Jain ML, Soni RP. 1978. Studies on the reaction of diiodides of heterocyclic tertiary bases with hydroquinone and catechol. Indian J Chem, Sect B 16B:165- 166.
Sharghi H, Aboonajmi J, Aberi M, Shekouhy M. 2020. Amino Acids: Nontoxic and Cheap Alternatives for Amines for the Synthesis of Benzoxazoles through the Oxidative Functionalization of Catechols. Adv Synth Catal 362:1064-1083.
Walker D. and Hiebert J.D, 2,3-Dichloro-5,6-dicyanobenzoquinone and Its Reactions, Chem. Rev. 1967, 67, 2, 153-195
Yang J, Cohen Stuart MA, Kamperman M. 2014. Jack of all trades: Versatile catechol crosslinking mechanisms. Chem Soc Rev 43:8271 -8298.
Yang S, Chu M, Miao Q. 2018. Connecting two phenazines with a four-membered ring: The synthesis, properties and applications of cyclobuta[1 ,2-: B:3,4- b ']diphenazines. J Mater Chem C 6:3651-3657.
Zherebtsovab M., Arsenyev M.V., Baranov E.V., Chesnokov S. A., Cherkasov V.K., Synthesis and structure of sterically hindered o-benzoquinone carboxylic acid, Volume 31 , Issue 2, March-April 2021 , Pages 268-270
Zheng T, Bullock JL, Nolan EM. 2012. Siderophore-mediated cargo delivery to the cytoplasm of escherichia coli and pseudomonas aeruginosa: Syntheses of monofunctionalized enterobactin scaffolds and evaluation of enterobactin-cargo conjugate uptake. J Am Chem
Soc 134:18388-18400. doi:10.1021/ja3077268

Claims

1 . A conjugate comprising a siderophore moiety and a cargo moiety, wherein the siderophore moiety comprises a siderophore (Sid) bearing a substituted or unsubstituted catechol group (Cat), said Cat group having the following formula:
Figure imgf000078_0001
with Ri and R2 being independently selected from the group consisting of a hydrogen atom, a Ci-C6 alkyl group, a C2-C6 alkenyl group, a Ci-C6 alkoxy group, a halogen atom, a Ci-C6 hydroxyalkyl group, a hydroxy-C2-C6 alkyloxy group, an aryl group, a heteroaryl group, a nonaromatic heterocycle, a Ci-Ce alky l-ary I and an aryl-Ci-Ce alkyl group, preferably a hydrogen atom, a C1-C4 alkyl group or a halogen atom, more preferably a hydrogen atom or a halogen atom; wherein the cargo moiety comprises a cargo molecule (Cargo) bearing a substituted or unsubstituted heteraryl group (Het), said Het group comprising one or more nitrogen atoms; wherein the siderophore and the cargo moieties are attached to each other by a direct and covalent bond between the carbon atom marked * of the Cat group and a nitrogen atom of the Het group, and wherein the conjugate has the following formula:
Sid-Cat-Het-Cargo.
2. The conjugate according to claim 1 wherein the siderophore moiety is a natural catecholate-containing siderophore or a natural siderophore which has been chemically modified to introduce the substituted or unsubstituted catechol group.
3. The conjugate according to claim 1 or 2 wherein the siderophore moiety is selected from the group consisting enterobactin, salmochelin, acinetobactin, fimsbactin, dactyloferrin, chlorodacyloferrin, agrobactin, alterobactin, amonabactin, anguibactin, azotochelin, bacillibactin, benarthin, chrysobactin, corynebactin, cyclic trichrisobactin, dibenarthin, dichrysobactin, divanchrobactin, fluvibactin, Fuscachelin, heterobactin, JBIR-16, mirubactin, myxochelin, nigribactin, parabactin, pistillarin, protochelin, serratiochelin, streptobactin, tribenarthin, trivanchrobactin, vanchrobactin, vibriobactin, vulnibactin, mycobactin, pyochelin, versiniabactin and pyoverdine, preferably enterobactin, acinetobactin, dactyloferrin and chlorodactyloferrin.
4. The conjugate according to any one of claims 1 to 3 wherein the cargo molecule is a pharmaceutical agent or a fluorophore.
5. The conjugate according to claim 4 wherein the pharmaceutical agent is an antibiotic, preferably selected from the group consisting of rifamycins, fluoroquinolones, nitroimidazole, penicillins, monobactam, cephalosporin, carbapenams, pyridomycin, oxazolidinones, sulfonamides, macrolides and fabl inhibitors.
6. The conjugate according to claim 4 wherein the fluorophore is selected from the group consisting of TAMRA, coumarin, boron-dipyrromethene (BODIPY), fluorescein, dansyl, 7- nitrobenz-2-oxa-1 ,3-diazol-4-yl and pyrene.
7. The conjugate according to any of claims 1 to 6 wherein the siderophore moiety comprises a substituted or unsubstituted catechol group (Cat) of the following formula:
Figure imgf000079_0001
with Ri being selected from the group consisting of a hydrogen atom, a Ci-C6 alkyl group, a C2-C6 alkenyl group, a Ci-C6 alkoxy group, a halogen atom, a Ci-C6 hydroxyalkyl group, a hydroxy-C2-C6 alkyloxy group, an aryl group, a heteroaryl group, a non-aromatic heterocycle, a Ci-C6 alkyl-aryl and an aryl-Ci-C6 alkyl group.
8. The conjugate according to any one of the preceding claims wherein the Het group is selected from substituted or unsubstituted groups of the following formula:
Figure imgf000080_0001
wherein X is O or NH, and wherein the siderophore and the cargo moieties are attached to each other by a direct and covalent bond between the carbon atom marked * of the catechol group and the nitrogen marked * of the Het group.
9. A process for preparing a conjugate according to any one of claims 1 to 8 comprising the step of reacting a siderophore bearing a catechol group of the following formula:
Figure imgf000080_0002
with Ri and R2 being independently selected from the group consisting of a hydrogen atom, a Ci-C6 alkyl group, a C2-C6 alkenyl group, a Ci-C6 alkoxy group, a halogen atom, a Ci-C6 hydroxyalkyl group, a hydroxy-C2-C6 alkyloxy group, an aryl group, a heteroaryl group, a nonaromatic heterocycle, a Ci-C6 alky l-ary I and an aryl-Ci-C6 alkyl group, preferably a hydrogen atom, a C1-C4 alkyl group or a halogen atom, more preferably a hydrogen atom or a halogen atom; with a cargo molecule bearing a substituted or unsubstituted heteraryl group (Het), said Het group comprising one or more nitrogen atoms; in conditions that allow oxidation of the catechol group and subsequent addition of the cargo molecule.
10. The process according to claim 9 wherein the conditions that allow oxidation of the catechol group and subsequent addition of the cargo molecule consist in reacting the cargo molecule and the siderophore in presence of an oxidizing agent or by electrochemistry.
11. The process according to claim 10 wherein the oxidizing agent is selected from the group consisting of Fe(lll), l2, Br2, Ag2O and NalO4.
12. A composition comprising a conjugate as recited in any one of claims 1 to 8 and an acceptable excipient.
13. A conjugate as recited in any one of claims 1 to 8 or a composition according to claim 12 for use in medicine.
14. A conjugate as recited in any one of claims 1 to 8 or a composition according to claim 12 for use in the treatment of a bacterial infection.
15. A conjugate as recited in any one of claims 1 to 8 or a composition according to claim
12 for use in the diagnosis of a bacterial infection.
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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5502214A (en) 1993-07-19 1996-03-26 Dana Farber Cancer Institute 1,2-benzoquinones and methods for making and using same
WO2015057958A2 (en) * 2013-10-16 2015-04-23 Massachusetts Institute Of Technology Enterobactin conjugates and uses thereof
WO2023023393A1 (en) * 2021-08-20 2023-02-23 University Of Notre Dame Du Lac Conjugates of monocyclic beta-lactams and siderophore mimetics

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5502214A (en) 1993-07-19 1996-03-26 Dana Farber Cancer Institute 1,2-benzoquinones and methods for making and using same
WO2015057958A2 (en) * 2013-10-16 2015-04-23 Massachusetts Institute Of Technology Enterobactin conjugates and uses thereof
WO2023023393A1 (en) * 2021-08-20 2023-02-23 University Of Notre Dame Du Lac Conjugates of monocyclic beta-lactams and siderophore mimetics

Non-Patent Citations (19)

* Cited by examiner, † Cited by third party
Title
ADOLPHS M. ET AL: "Catecholate Siderophores from Chryseomonas luteola", ZEITSCHRIFT FUER NATURFORSCHUNG. C, A JOURNAL OF BIOSCIENCES., vol. 51, no. 5-6, 1 June 1996 (1996-06-01), DE, pages 281 - 285, XP093081054, ISSN: 0939-5075, DOI: 10.1515/znc-1996-5-603 *
ADOLPHS MTARAZ KBUDZIKIEWICZ H: "Catecholate siderophores from Chryseomonas luteola", ZEITSCHRIFT FUR NATURFORSCH SECT C - J BIOSCI, vol. 51, 1996, pages 281 - 285, XP093081054, DOI: 10.1515/znc-1996-5-603
AXFORD L.C.KATE EHOLDEN K.EHASSE KMARTIN GBANWELL M.G.STEGLICH WWAGLER J.WILLIS A.C.: "Attempts to Mimic Key Bond-Forming Events Associated with the Proposed Biogenesis of the Pentacyclic Lamellarins", AUSTRALIAN JOURNAL OF CHEMISTRY, vol. 61, no. 2, 2008, pages 80 - 93
CAVALIERI ELLI KMBALU NSAEED MDEVANESAN PHIGGINBOTHAM SZHAO JGROSS MLROGAN EG: "Catechol ortho-quinones: The electrophilic compounds that form depurinating DNA adducts and could initiate cancer and other diseases", CARCINOGENESIS, vol. 23, 2002, pages 1071 - 1077, XP003008451, DOI: 10.1093/carcin/23.6.1071
DERIKVAND FBIGI FMAGGI RPISCOPO CGSARTORI G: "Oxidation of hydroquinones to benzoquinones with hydrogen peroxide using catalytic amount of silver oxide under batch and continuous-flow conditions", J CATAL, vol. 271, 2010, pages 99 - 103, XP026980670
FULLENKAMP DEBARRETT DGMILLER DRKURUTZ JWMESSERSMITH PB: "PH-dependent cross-linking of catechols through oxidation via Fe 3+ and potential implications for mussel adhesion", RSC ADV, vol. 4, 2014, pages 25127 - 25134
GHORBANI AAMANI ANEMATOLLAHI DSHARAFI-KOLKESHVANDI M: "Electrochemically Induced Diels-Alder Reactions of Some Substituted o-quinones with 1,3-cyclopentadiene: An Interesting Finding in the Diels-Alder Reactions", J ELECTROCHEM SOC, vol. 167, 2020, pages 155516
HAYASHI TSUKIHO ET AL: "Development of 1,3a,6a-triazapentalene-labeled enterobactin as a fluorescence quenching sensor of iron ion", TETRAHEDRON LETTERS, vol. 58, no. 20, 5 April 2017 (2017-04-05), pages 1961 - 1964, XP029992176, ISSN: 0040-4039, DOI: 10.1016/J.TETLET.2017.04.011 *
KATO TKATAGIRI NNAKANO JKAWAMURA H: "Total synthesis of bikaverin involving the novel rearrangement of an ortho-quinone to a para-quinone", J CHEM SOC CHEM COMMUN, 1977, pages 645 - 646
MISEREZ ARUBIN DWAITE JH: "Cross-linking chemistry of squid beak", J BIOL CHEM, vol. 285, 2010, pages 38115 - 38124
NAPOLITANO APALUMBO AD'ISCHIA M: "Oxidation of the neurotoxin 6-nitrodopamine and related 4-nitrocatechols under biomimetic conditions", TETRAHEDRON, vol. 56, 2000, pages 5941 - 5945, XP004229193, DOI: 10.1016/S0040-4020(00)00483-X
NEMATOLLAHI DKHOSHSAFAR H: "Investigation of electrochemically induced Michael addition reactions. Oxidation of some dihydroxybenzene derivatives in the presence of azide ion", TETRAHEDRON, vol. 65, 2009, pages 4742 - 4750, XP026106654, DOI: 10.1016/j.tet.2009.04.015
SAXENA JPPUROHIT NLJAIN MLSONI RP: "Studies on the reaction of diiodides of heterocyclic tertiary bases with hydroquinone and catechol", INDIAN J CHEM, SECT B, vol. 16B, 1978, pages 165 - 166
SHARGHI HABOONAJMI JABERI MSHEKOUHY M: "Amino Acids: Nontoxic and Cheap Alternatives for Amines for the Synthesis of Benzoxazoles through the Oxidative Functionalization of Catechols", ADV SYNTH CATAL, vol. 362, 2020, pages 1064 - 1083, XP072362114, DOI: 10.1002/adsc.201901096
WALKER D. AND HIEBERT J.D: "2,3-Dichloro-5,6-dicyanobenzoquinone and Its Reactions", REV., vol. 67, no. 2, 1967, pages 153 - 195, XP002325452, DOI: 10.1021/cr60246a002
YANG JCOHEN STUART MAKAMPERMAN M.: "Jack of all trades: Versatile catechol crosslinking mechanisms", CHEM SOC REV, vol. 43, 2014, pages 8271 - 8298, XP055253735, DOI: 10.1039/C4CS00185K
YANG SCHU MMIAO Q.: "Connecting two phenazines with a four-membered ring: The synthesis, properties and applications of cyclobuta[1,2-: B:3,4- b ']diphenazines", J MATER CHEM C, vol. 6, 2018, pages 3651 - 3657
ZHENG TBULLOCK JLNOLAN EM: "Siderophore-mediated cargo delivery to the cytoplasm of escherichia coli and pseudomonas aeruginosa: Syntheses of monofunctionalized enterobactin scaffolds and evaluation of enterobactin-cargo conjugate uptake", J AM CHEM SOC, vol. 134, 2012, pages 18388 - 18400, XP055149011, DOI: 10.1021/ja3077268
ZHEREBTSOVAB M.ARSENYEV M.V.BARANOV E.V.CHESNOKOV S.A.CHERKASOV V.K., SYNTHESIS AND STRUCTURE OF STERICALLY HINDERED O-BENZOQUINONE CARBOXYLIC ACID, vol. 31, March 2021 (2021-03-01), pages 268 - 270

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