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WO2025158077A1 - Lipid degraders to trigger ferroptosis in cancer - Google Patents

Lipid degraders to trigger ferroptosis in cancer

Info

Publication number
WO2025158077A1
WO2025158077A1 PCT/EP2025/051999 EP2025051999W WO2025158077A1 WO 2025158077 A1 WO2025158077 A1 WO 2025158077A1 EP 2025051999 W EP2025051999 W EP 2025051999W WO 2025158077 A1 WO2025158077 A1 WO 2025158077A1
Authority
WO
WIPO (PCT)
Prior art keywords
moiety
conjugate
general formula
compound
fentomycin
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/EP2025/051999
Other languages
French (fr)
Inventor
Raphaël RODRIGUEZ
Tatiana CAÑEQUE
Ludovic COLOMBEAU
Sebastian Müller
Antoine VERSINI
Romain SASTOURNÉ-HALETOU
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Centre National de la Recherche Scientifique CNRS
Institut National de la Sante et de la Recherche Medicale INSERM
Institut Curie
Universite Paris Sciences et Lettres
Original Assignee
Centre National de la Recherche Scientifique CNRS
Institut National de la Sante et de la Recherche Medicale INSERM
Institut Curie
Universite Paris Sciences et Lettres
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Centre National de la Recherche Scientifique CNRS, Institut National de la Sante et de la Recherche Medicale INSERM, Institut Curie, Universite Paris Sciences et Lettres filed Critical Centre National de la Recherche Scientifique CNRS
Publication of WO2025158077A1 publication Critical patent/WO2025158077A1/en
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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Classifications

    • 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
    • A61K47/545—Heterocyclic compounds
    • 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
    • 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
    • A61K47/554—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 a steroid plant sterol, glycyrrhetic acid, enoxolone or bile acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the present invention is in the field of medicine.
  • the present invention relates to new conjugates, namely new chimeric small molecules, and their use as drugs.
  • the chimeric small molecules of the invention are bifunctional compounds that act as lipid degraders.
  • the invention relates to these new chimeric small molecules for use in a method for the treatment of cancer, preferably for use in a targeted treatment of Cancer Stem Cells (CSC)ZDrug Tolerant Persister (DTP) cancer cells/cancer cells in the mesenchymal state and/or for the treatment and prevention of cancer metastases.
  • CSC Cancer Stem Cells
  • DTP Drug Tolerant Persister
  • Cancer is a prevalent disease that spreads all over the world.
  • the World Health Organization (WHO) reported around 9.6 million deaths in 2018 due to cancer.f l] Cancer is a result of an abnormal development of cells derived from normal cells, and therefore it is challenging to selectively and specifically target cancerous cells over healthy cells in the body.
  • the etiology of cancer is complex, with various factors contributing to the appearance of cancerous cells, including genetic, epigenetic or metabolic alterations. Importantly, the initiating and driving factors of a particular cancer often differ.
  • CSC Cancer Stem Cells
  • DTP Drug-Tolerant Persister
  • CSCs cancer stem-like cells and tumor-initiating cells
  • TICs tumor-initiating cells
  • CSCs had been identified in leukemia, but were then found to be present in many other cancers including brain, prostate, pancreatic and breast.
  • the proportion of CSCs is variable and often denotes a fraction of the tumor but can constitute the bulk of the cancer, in particular in melanoma.
  • CSCs have an enhanced addiction to iron in their mesenchymal state.[14,15,16] This dependency for iron was observed for cancer stem -like cells of various indications including breast cancer, ovarian cancer and glioma. [15, 17, 18]
  • Ferroptosis is a specific type of non-apoptotic cell death resulting from the unrestrained occurrence of oxidized, in particular peroxidized, phospholipids, resulting from iron-mediated production of lethal oxygen radicals.
  • ferroptosis is a regulated oxidative cell death that can occur as a result of lipid peroxidation. This type of cell death was first coined in 2012 after observations describing a process different from apoptosis, necrosis or other known cell death pathways. [19] Two main axis were identified to be responsible for the regulation of ferroptosis.
  • the glutathione -dependent selenoenzyme also known as glutathione peroxidase-4 (GPX4), which plays a major role in protecting cells from ferroptosis.
  • Another enzyme has been identified to protect the cells from ferroptosis known as ferroptosis suppressor protein 1 (FSP 1).
  • Ferroptosis is mediated by the presence of reactive oxygen species (ROS) that cause the peroxidation of lipids.
  • ROS reactive oxygen species
  • Ferroptosis can either be prevented (for instance by iron chelation or radical trapping) or promoted (for instance by iron activation or inhibition of ferroptosis suppressors) by means of small molecule intervention.
  • CSCs cancer stem cells
  • DTP drug-tolerant persister
  • targeting lysosomal iron may provide new therapeutic opportunities to selectively impact CSC maintenance [14, 23, 24],
  • the inventors have developed new chimeric small molecules comprising a lysosomotropic molecule and a ligand known to promote iron catalysed oxidation of phospholipids and other substrates [25,26],
  • the lysosomotropic molecule is, in particular, a lipophilic moiety that drives the accumulation in lysosomes via endocytosis.
  • the lysosomotropic molecule drives the iron ligand inside of lysosomes, in particular via endocytosis, to promote a Fenton-type chemistry and lipid oxidation, such as peroxidation, in these cell organelles.
  • Fentomycin targets lysosomes and shows good efficacy in vitro by inducing lysosomal reactive oxygen species ( Figure 1).
  • the inventors have also shown that Fentomycin induces ferroptosis and targets iron rich cancer cells in vivo.
  • a first object of the invention is a conjugate for use in the treatment of cancer, the conjugate being of the following general formula (I):
  • A is a first moiety selected from metal chelating agents
  • B is a second moiety selected from lysosomotropic agents.
  • L is absent or is a linker that binds moieties A and B together, preferably L is a linker that binds moieties A and B together.
  • the conjugate of the invention is for use in a targeted treatment of Cancer Stem Cells (CSCs).
  • the first moiety A selected from ligands of the White-Chen Type preferably the first moiety A is White -Chen ligand.
  • the first moiety A is of the following general formula (II): wherein:
  • Ri is selected from the group consisting of NRARB, ORC, C(O)ORD, C(O)RE, S(O)RF, S(O)2RG, CN, halo-(C1-C6-alkyl), -C(O)-(C1-C6)alkyl-halo, halogen, NO2, and SO2, R . to RG being independently of each other H or a (C1-C6)alkyl, preferably Ri is N(CH 3 ) 2 ;
  • R2 is a hydrogen atom or (C1-C6)alkyl, preferably hydrogen atom ;
  • each Z is, independently of each other, a hydrogen or deuterium atom.
  • the first moiety A is of the general formula (Ila) (namely White-Chen ligand type): wherein
  • Ri is selected from the group consisting of NRARB, ORC, C(O)ORD, C(O)RE, S(O)RF, S(O)2RG, halo- (C1-C6-alkyl), -C(O)-(C1-C6)alkyl-halo, halogen, CN, NO2, and SO2, RA to RG being independently of each other H or a (C1-C6)alkyl, preferably Ri is N(CHs)2;
  • R2 is a hydrogen atom or (C1-C6)alkyl, preferably hydrogen atom;
  • X and Y are, independently of each other, a (C1-C6)alkyl; or X and Y can be linked together to form a heterocycloalkyl or heteroaryl, preferably a pyrrolidine; and each Z is, independently of each other, a hydrogen or deuterium atom; and wherein the wavy line represents the link to L.
  • the first moiety A is of the general formula (lib) (namely White-Chen ligand type): wherein
  • Ri is selected from the group consisting of NRARB, ORC, C(O)ORD, C(O)RE, S(O)RF, S(0)2RG, CN, halo-(C1-C6-alkyl), -C(O)-(C1-C6)alkyl-halo, halogen, NO2, and SO2, RA to RG being independently of each other H or a (C1-C6)alkyl, preferably Ri is N(CH3)2;
  • R2 is a hydrogen atom or (C1-C6)alkyl, preferably hydrogen atom; and each Z is, independently of each other, a hydrogen or deuterium atom; and wherein the wavy line represents the link to L.
  • the first moiety A is of the general formula (lie): wherein
  • Ri is N(CHS) 2 ;
  • Rs is an hydrogen atom or (C1-C6)alkyl, preferably hydrogen atom; and wherein the wavy line represents the link to L.
  • the first moiety A is selected from ligands of the Nordlander-Costas Type, preferably the first moiety A is Nordlander-Costas ligand, more preferably the first moiety A is of the following formula (lie): wherein
  • Rs is a hydrogen atom or (C1-C6)alkyl preferably methyl
  • X and Y are, independently of each other, a (C1-C6)alkyl; or X and Y can be linked together to form a heterocycloalkyl or heteroaryl, preferably pyrrolidine; and each Z is, independently of each other, a hydrogen or deuterium atom; and wherein the wavy line represents the link to L.
  • the first moiety A is of the general formula (Ilf): wherein each Z is, independently of each other, a hydrogen or deuterium atom, and wherein the wavy line represents the link to L.
  • the first moiety A is:
  • the first moiety A is selected from the group consisting of:
  • the second moiety B is selected from the group consisting of chloroquine, hydroxychloroquine, azithromycin, marmycin, steroids such as cholesterol and cholestanol, ezurpimtrostat hydrochloride, desmethylchloroquine, hydroxychloroquine phosphate, desmethyl- hydroxychloroquine, anthraquinones, methyl tetraphene dione, angucyclines, ammonium chloride (NH4CI), amantadine, methylamine, fluoxetine, imipramine, latrepirdine, tamoxifen, chlorpromazine, amitriptyline, verapamil, Triton WR 1339 (Tyloxapol), Suramin, metformin, Erythromycin, Amitriptyline, Imipramine, 4-aminoquinoline, amiodarone, amodiaquine, clindamycin, N-(3-[(2,4- dinitrophenyl)
  • the second moiety B is selected from the group consisting of chloroquine, hydroxychloroquine, azithromycin, marmycin, functionalized fatty acids, steroids, cholesterol, cholestanol, ezurpimtrostat hydrochloride, desmethylchloroquine, hydroxychloroquine phosphate, desmethyl-hydroxychloroquine, anthraquinones, methyl tetraphene dione, angucyclines.
  • the second moiety B is selected from the group consisting of chloroquine, hydroxychloroquine, azithromycin, marmycin, ezurpimtrostat hydrochloride, desmethylchloroquine, hydroxychloroquine phosphate, desmethyl-hydroxychloroquine, anthraquinones, methyl tetraphene dione, and angucyclines.
  • the second moiety B is selected from the group consisting of chloroquine, hydroxychloroquine, azithromycin, marmycin, cholesterol, cholestanol, and ezurpimtrostat
  • the second moiety B is selected from the group consisting of marmycin, steroids such as cholesterol and cholestanol, anthraquinones, and angucyclines.
  • the second moiety B is selected from the group consisting of marmycin, cholesterol, cholestanol, anthraquinones, and angucyclines. More particularly, the second moiety B is selected from the group consisting of marmycin A, cholesterol, and cholestanol.
  • the lysosomotropic agent is marmycin and wherein the linker is bound to said lysosomotropic agent via the hydroxyl of the sugar moiety of marmycin.
  • the lysosomotropic agent is marmycin A of formula (Bl): wherein the wavy line represents the link to L.
  • the lysosomotropic agent is cholesterol and wherein the linker is bound to said lysosomotropic agent via the hydroxyl group.
  • the lysosomotropic agent is cholesterol of formula (B2): wherein the wavy line represents the link to L.
  • the lysosomotropic agent is cholestanol and wherein the linker is bound to said lysosomotropic agent via the hydroxyl group.
  • the lysosomotropic agent is cholestanol of formula (B3): wherein the wavy line represents the link to L.
  • the lysosomotropic agent is of formula (B4): wherein the wavy line represents the link to L.
  • the lysosomotropic agent is of formula (B5): wherein the wavy line represents the link to L.
  • the linker is an aliphatic hydrocarbon chain comprising from 2 to 20 carbon atoms, and being optionally terminated at one or both ends and/or interrupted by at least one group independently selected from -C(O)NH-,-NHC(O)-, -C(O)O-, -OC(O)-, -NH-, -S-, -O-, -C(O) -, -S(O)-, and -S(O) 2 -.
  • the linker is an aliphatic hydrocarbon chain comprising from 2 to 16 carbon atoms, and being optionally terminated at one or both ends and/or interrupted by at least one group independently selected from -C(O)NH-,-NHC(O)-, -C(O)O-, -OC(O)-, -NH-, -S-, -O-, -C(O) -, -S(O)-, and -S(O) 2 -.
  • the linker is an aliphatic hydrocarbon chain comprising from 2 to 14 carbon atoms, and being optionally terminated at one or both ends and/or interrupted by at least one group independently selected from -C(O)NH-,-NHC(O)-, -C(O)O-, -OC(O)-, -NH-, -S-, -O-, -C(O) -, -S(O)-, and -S(O) 2 -.
  • the linker is an aliphatic hydrocarbon chain comprising from 2 to 12 carbon atoms and being optionally terminated at one or both ends and/or interrupted by at least one group independently selected from -C(O)NH-,-NHC(O)-, -C(O)O-, -OC(O)-, -NH-, -S-, -O-, -C(O) -S(O)-, and -S(O) 2 -.
  • the conjugate of the invention is selected from the group consisting of compounds of general formula (III), (III’), (IV), (VII), (VII’), (VIII), (VIII’), (IX) (IX’), (X), (X’), (XI), (xr), (xii), (xir), (xiii), (xiir), (xiv), (xiv’), (xv), (xv’), (xvi), (xvr), (xvii), (XVIF), (XVIII), (XVIII’), (XIX), (XIX’), (XX), and (XX’); preferably (III), (III’), (IV), (VII), (VII’), (VIII), (viir), (ix) (ix’), (X), (X’), (xi), (xr), (xii), (xiir), (xiv), (xiv’), (xviivii), (XVIF), (XVIII), (XVIII
  • the conjugate of the invention is Fentomycin of general formula (III) or Pre- Fentomycin of general formula (IV).
  • a second object of the invention relates to a conjugate of the following general formula (I): A-L-B (I) wherein:
  • A is a first moiety selected from metal chelating agents
  • B is a second moiety selected from lysosomotropic agent
  • L is absent or is a linker that binds moieties A and B together, preferably L is a linker that binds moieties A and B together.
  • A, L and B are as defined above.
  • a third object of the invention relates to the compound Fentomycin of general formula (III), or a pharmaceutically acceptable salt and/or solvate thereof.
  • a fourth object of the invention relates to the compound Pre-Fentomycin of general formula (IV), or a pharmaceutically acceptable salt and/or solvate thereof.
  • Another object of the invention relates to the compound Fentomycin-1 of general formula (III’), or a pharmaceutically acceptable salt and/or solvate thereof.
  • Another object of the invention relates to the compound Fentomycin-2 of general formula (VII) or a compound of formula (VII’), or a pharmaceutically acceptable salt and/or solvate thereof.
  • Another object of the invention relates to the compound Fentomycin-3 of general formula (VIII) or a compound of formula (VIII’), or a pharmaceutically acceptable salt and/or solvate thereof.
  • Another object of the invention relates to the compound Fentomycin-5 of general formula (IX) or a compound of formula (IX’), or a pharmaceutically acceptable salt and/or solvate thereof.
  • the conjugate of the invention is Fentomycin-6 of general formula (X) or a compound of formula (X’) as disclosed herein.
  • the conjugate of the invention is Fentomycin-7 of general formula (XI) or a compound of formula (XI’) as disclosed herein.
  • the conjugate of the invention is Fentomycin-8 of general formula (XII) or a compound of formula (XIF) as disclosed herein.
  • the conjugate of the invention is Fentomycin-9 of general formula (XIII) or a compound of formula (XIIF) as disclosed herein.
  • the conjugate of the invention is Fentomycin-10 of general formula (XIV) or a compound of formula (XIV’) as disclosed herein.
  • the conjugate of the invention is Fentomycin-11 of general formula (XV) or a compound of formula (XV’) as disclosed herein.
  • the conjugate of the invention is Fentomycin-12 of general formula (XVI) or a compound of formula (XVI’) as disclosed herein.
  • the conjugate of the invention is Fentomycin-13 of general formula (XVII) or a compound of formula (XVIF) as disclosed herein.
  • the conjugate of the invention is Fentomycin-14 of general formula (XVIII) or a compound of formula (XVIIF) as disclosed herein.
  • the conjugate of the invention is Fentomycin-15 of general formula (XIX) or a compound of formula (XIX’) as disclosed herein.
  • the conjugate of the invention is Fentomycin-16 of general formula (XX) or a compound of formula (XX’) as disclosed herein.
  • Another object of the invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a conjugate according to the invention, or a compound according to the invention, or a pharmaceutically acceptable salt and/or solvate thereof, and at least one pharmaceutically acceptable excipient.
  • Another object of the invention relates to nanoparticle comprising a conjugate according to the invention, or a compound according to the invention, or a pharmaceutically acceptable salt and/or solvate thereof.
  • a further objection of the invention relates to a conjugate according to the invention, or a compound according to the invention, or a pharmaceutically acceptable salt and/or solvate thereof, or a pharmaceutical composition according to the invention, or a nanoparticle according to the invention, for use as a drug.
  • a further objection of the invention relates to a conjugate according to the invention, or a compound according to the invention, or a pharmaceutically acceptable salt and/or solvate thereof, or a pharmaceutical composition according to the invention, or a nanoparticle according to the invention, for use for the treatment of cancer.
  • a further objection of the invention relates to a method for the treatment of cancer in a subject in need thereof, comprising administering a therapeutic effective amount of a conjugate according to the invention, or a compound according to the invention, or a pharmaceutical composition according to the invention, or a nanoparticle according to the invention, to said subject.
  • a further objection of the invention relates to the use of a conjugate according to the invention, or a compound according to the invention, or a pharmaceutical composition according to the invention, or a nanoparticle according to the invention, for the manufacture of a medicament for use as anti-tumoral agent or in a method for the treatment of cancer.
  • a further objection of the invention relates to a method for the treatment of a disease in a subject in need thereof, comprising administering a therapeutic effective amount of a conjugate according to the invention, or a compound according to the invention, or a pharmaceutical composition according to the invention, or a nanoparticle according to the invention, to said subject.
  • a further objection of the invention relates to the use of a conjugate according to the invention, or a compound according to the invention, or a pharmaceutical composition according to the invention, or a nanoparticle according to the invention, for the manufacture of a medicament.
  • Another object of the invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a conjugate according to the invention, or a compound of formula (III) or (IV) according to the invention or a pharmaceutically acceptable salt and/or solvate thereof, and at least one pharmaceutically acceptable excipient.
  • Another object of the invention relates to nanoparticle comprising a conjugate according to the invention, or a compound of formula (III) or (IV) according to the invention or a pharmaceutically acceptable salt and/or solvate thereof.
  • a further objection of the invention relates to a conjugate according to the invention, or a compound of formula (III) or (IV) according to the invention or a pharmaceutically acceptable salt and/or solvate thereof, or a pharmaceutical composition according to the invention, or a nanoparticle according to the invention, for use as a drug.
  • Figure 1 Rational design of the lysosomal iron activator Fentomycin, which targets lysosomes and shows good efficacy in vitro.
  • a Chemical formulae of Marmycin A, the iron activator ligand, Fentomycin and a clickable ligand, ligand- ‘click’, which can be subjected to click chemistry with a fluorophore for cellular imaging
  • b Fluorescence images of HT1080 cells treated with Fentomycin, Marmycin and LysoTracker and fluorescence images of labeled clickable ligand and LysoTracker.
  • Fentomycin induces Ferroptosis.
  • a Oxidized lipids of liposomes in the presence of iron and Fentomycin.
  • b Flow cytometry of BODYPY 581/591 Cl l fluorescence of HT1080 cells treated with Fentomycin, Marmycin A or ligand- ‘click’,
  • c Flow cytometry of BODYPY 581/591 Cl l fluorescence of HT1080 cells treated with Fentomycin and ferroptosis inhibitors
  • d Lipidomics of oxidized lipids in PDAC053T cells treated with Fentomycin for 6 h or 24 h.
  • e Viability curves of PDAC cell lines treated with Fentomycin or the standard of care (Irinotecan, 5-FU or Oxaliplatin)
  • f Viability curves of PDAC organoids treated with Fentomycin or the standard of care (Irinotecan, 5-FU or Oxaliplatin)
  • ICP-MS inductively-coupled plasma mass spectrometry
  • Figure 5 a, Flow cytometry of BODIPY-C11 581/591 in cells treated with 1 pM of Fentomycin-3 and -6 to -16 for 6 h in PDAC053T cells, b, quantification of mean fluorescent intensity (MFI) of BODIPY-C11 581/591 of the data of Figure 5a.
  • MFI mean fluorescent intensity
  • agent is used herein to denote a chemical compound (such as an organic or inorganic compound (including, such as, a compound of the present invention), a mixture of chemical compounds), a biological macromolecule (such as a nucleic acid, an antibody, including parts thereof as well as humanized, chimeric and human antibodies and monoclonal antibodies, a protein or portion thereof, e.g., a peptide, a lipid, a carbohydrate), or an extract made from biological materials such as bacteria, plants, fungi, or animal (particularly mammalian) cells or tissues.
  • Agents include, for example, agents which are known with respect to structure, and those which are not known with respect to structure.
  • aliphatic hydrocarbon chain refers to straight or branched hydrocarbon chain. Aliphatic hydrocarbon chain may be saturated such as alkyl group, or unsaturated such as alkenyl or alkynyl group.
  • alkyl refers to a monovalent linear or branched saturated hydrocarbon chain.
  • (Ci-C3)alkyl more specifically means methyl, ethyl, n- propyl, or isopropyl.
  • (C1-C6)alkyl more specifically means methyl, ethyl, n-propyl, isopropyl, n-butyl, iso-butyl, sec-butyl, tert-butyl, pentyl or linear or branched hexyl.
  • alkenyl refers to a straight or branched monovalent unsaturated hydrocarbon chain comprising at least one double bond including, but not limited to, ethenyl, propenyl, butenyl, pentenyl, hexenyl and the like.
  • alkynyl refers to a straight or branched monovalent unsaturated hydrocarbon chain comprising at least one triple bond including, but not limited to, ethynyl, propynyl, butynyl, pentynyl, hexynyl and the like.
  • an alkynyl group as used in the present disclosure comprises one triple bond.
  • cycloalkyl corresponds to a saturated or unsaturated mono-, bi- or tri-cyclic alkyl group comprising between 3 and 20 atoms of carbons. It also includes fused, bridged, or spiro-connected cycloalkyl groups.
  • cycloalkyl includes for instance cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl, preferably cyclopropyl.
  • spirocycloalkyl includes for instance a spirocyclopentyl.
  • the term “cycloalkyl” corresponds to a saturated monocycloalkyl group comprising between 3 and 7 atoms of carbons.
  • heterocycloalkyl corresponds to a saturated or unsaturated cycloalkyl group as above defined further comprising at least one heteroatom such as nitrogen, oxygen, or sulphur atom. It also includes fused, bridged, or spiro-connected heterocycloalkyl groups.
  • heterocycloalkyl groups include, but are not limited to 3-dioxolane, benzo [1,3] dioxolyl, pyrazolinyl, pyranyl, thiomorpholinyl, pyrazolidinyl, piperidyl, piperazinyl, 1,4-dioxanyl, imidazolinyl, pyrrolinyl, pyrrolidinyl, piperidinyl, imidazolidinyl, morpholinyl, 1,4-dithianyl, pyrrolidinyl, oxozolinyl, oxazolidinyl, isoxazolinyl, isoxazolidinyl, thiazolinyl, thiazolidinyl, isothiazolinyl, isothiazolidinyl, dihydropyranyl, tetrahydro-2H-pyranyl, tetrahydrofuranyl, and tetrahydrothioph
  • aryl corresponds to a mono- or bi-cyclic aromatic hydrocarbons having from 6 to 12 carbon atoms.
  • aryl includes phenyl, biphenyl, or naphthyl.
  • heteroaryl refers to an aromatic, mono- or poly-cyclic group comprising between 5 and 14 atoms, including at least one heteroatom such as nitrogen, oxygen or sulphur atom.
  • Examples of such mono- and poly-cyclic heteroaryl group may be: pyridyl, pyridinyl, thiazolyl, thiophenyl, furanyl, pyrrolyl, pyrazolyl, imidazolyl, triazolyl, tetrazolyl, benzofuranyl, thianaphthalenyl, indolyl, indolinyl, quinolinyl, isoquinolinyl, benzimidazolyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, triazinyl, thianthrenyl, isobenzofuranyl, chromenyl, xanthenyl, phenoxanthinyl, isothiazo
  • halogen refers to a fluorine, chlorine, bromine, or iodine atom, preferably a fluorine, chlorine or bromine, and more preferably a chlorine or a fluorine.
  • halo-(C1-C6 -alkyl) or (C1-C6-alkyl)-halo or (Ci.C6)alkyl-halo it refers to a (C1-C6)-alkyl as defined above substituted with one, two or three halogen. Examples of these groups can be -CH2F, - CHF2 or -CF3, preferably -CF3.
  • the term “pharmaceutically acceptable” is intended to mean what is useful to the preparation of a pharmaceutical composition, and what is generally safe and nontoxic, for a pharmaceutical use.
  • pharmaceutically acceptable salt is intended to mean, in the framework of the present invention, a salt of a compound which is pharmaceutically acceptable, as defined above, and which possesses the pharmacological activity of the corresponding compound.
  • the pharmaceutically acceptable salts comprise:
  • acid addition salts formed with inorganic acids such as hydrochloric, hydrobromic, sulfuric, nitric and phosphoric acid and the like; or formed with organic acids such as acetic, benzenesulfonic, fumaric, formic, glucoheptonic, gluconic, glutamic, glycolic, hydroxynaphtoic, 2- hydroxyethanesulfonic, lactic, maleic, malic, mandelic, methanesulfonic, muconic, 2- naphtalenesulfonic, propionic, succinic, dibenzoyl-L- tartaric, tartaric, p-toluenesulfonic, trimethylacetic, and trifluoroacetic acid and the like, and
  • organic bases comprise diethanolamine, ethanolamine. N- methylglucamine, triethanolamine, tromethamine and the like.
  • Acceptable inorganic bases comprise aluminium hydroxide, calcium hydroxide, potassium hydroxide, sodium carbonate and sodium hydroxide.
  • stereoisomers are isomeric compounds that have the same molecular formula and sequence of bonded atoms, but differ in the 3D-dimensional orientations of their atoms in space.
  • the stereoisomers include enantiomers, diastereoisomers, cis-trans and E-Z isomers, conformers, and anomers.
  • the stereoisomers include diastereoisomers and enantiomers.
  • the “tautomers” are isomeric compounds that differ only in the position of the protons and the electrons.
  • solvates include conventional solvates such as those formed during the last step of the preparation of the compounds of the invention due to the presence of solvents. It can be for example an hydrate or an alcoholate such as an ethanolate.
  • the terms “subject”, “individual” or “patient” are interchangeable and refer to an animal, preferably to a mammal, even more preferably to a human, including adult and child.
  • the term “subject” can also refer to non-human animals, in particular mammals such as dogs, cats, horses, cows, pigs, sheeps and non-human primates, among others.
  • treatment denotes curative, symptomatic, and preventive treatment.
  • Pharmaceutical compositions, kits, products and combined preparations of the invention can be used in humans with a disease or disorder.
  • compositions, kits, products and combined preparations of the invention will not necessarily cure the patient but will delay or slow the progression or prevent further progression of the disease or disorder, and/or ameliorating thereby the patients’ condition.
  • the pharmaceutical composition of the invention is administered in a therapeutically effective amount.
  • treatment of a disease or disorder or the like is mentioned with reference to the pharmaceutical composition of the invention, there is meant: a) a method for treating a disease or disorder, said method comprising administering a therapeutically effective amount of a compound of the invention or of a pharmaceutical composition comprising said compound to a subject in need of such treatment; b) the use of a compound of the invention or of a pharmaceutical composition comprising said compound for the treatment of a disease or disorder; c) the use of a compound of the invention or of a pharmaceutical composition comprising said compound for the manufacture of a medicament for the treatment of a disease or disorder; and/or d) a compound of the invention or of a pharmaceutical composition comprising said compound for use in the treatment a disease or disorder.
  • the term “therapeutic effect” refers to an effect induced by an active ingredient, or a pharmaceutical composition according to the invention, capable to prevent or to delay the appearance or development of a disease or disorder, or to cure or to attenuate the effects of a disease or disorder.
  • terapéuticaally effective amount or “active amount” or “effective amount”, it is meant the quantity of the pharmaceutical composition of the invention which prevents, removes or reduces the deleterious effects of a disease or disorder in mammals, including humans, alone or in combination with the other active ingredients of the pharmaceutical composition, kit, product or combined preparation. It is understood that the administered dose may be lower for each compound in the composition to the “therapeutic effective amount” define for each compound used alone or in combination with other treatments than the combination described here.
  • the “therapeutic effective amount” of the composition will be adapted by those skilled in the art according to the patient, the pathology, the mode of administration, etc.
  • pharmaceutically acceptable excipient refers to any ingredient except active ingredients which are present in a pharmaceutical composition. Its addition may be aimed to confer a particular consistency or other physical or gustative properties to the final product. A pharmaceutically acceptable excipient must be devoid of any interaction, in particular chemical, with the active ingredients.
  • the invention relates to a conjugate of the following general formula (I):
  • A is a first moiety selected from metal chelating agents
  • B is a second moiety selected from lysosomotropic agents.
  • L is absent or is a linker that binds moieties A and B together, preferably L is a linker that binds moieties A and B together.
  • the invention also relates to a conjugate of the following general formula (I):
  • A is a first moiety selected from metal chelating agents
  • B is a second moiety selected from lysosomotropic agents.
  • L is a linker that binds moieties A and B together.
  • the invention also relates to said conjugate for use in the treatment of cancer.
  • the conjugate of the invention is for use in a targeted treatment of Cancer Stem Cells (CSCs).
  • CSCs Cancer Stem Cells
  • the first moiety A is a lysosomal agent inducing intra-lysosomal activity and/or modulation.
  • the first moiety A is a lysosomal metal ion activator and/or modulator.
  • the first moiety A is selected from metal chelating agents.
  • metal chelating agents means agents which are able to chelate a metal by forming a complex and/or which promote or catalyze oxidation of lipids. Chelating agents are generally classified based upon the target metal such as iron, copper, mercury or lead.
  • metal chelating agents means agents which are able to bind to and activate iron and able to promote or catalyze oxidation of lipids.
  • the first moiety A is selected from metal ion activator agents, preferably a lysosomal metal ion activator.
  • the first moiety A is a metal cation chelating agent, preferably a metal cation activator agent.
  • the first moiety A is an iron chelating agent or a copper chelating agent, preferably an iron chelating agent.
  • the first moiety A is an iron activator agent or a copper activator agent, preferably an iron activator agent.
  • the first moiety A can be of the following general formula (II):
  • Ri is selected from the group consisting of NRARB, ORC, C(O)ORD, C(O)RE, S(O)RF, S(O)2RG, CN, halo-(C1-C6-alkyl), -C(O)-(C1-C6)alkyl-halo, halogen, NO2, and SO2, RA to RG being independently of each other H or a (C1-C6)alkyl, preferably Ri is N(CH3)2;
  • R2 is a hydrogen atom or (C1-C6)alkyl,; and each Z is, independently of each other, a hydrogen or deuterium atom.
  • the first moiety A can be of the following general formula (II):
  • Ri is selected from the group consisting of NRARB, ORC, C(O)ORD, C(O)RE, S(O)RF, S(O)2RG, CN, halo-(C1-C6-alkyl), -C(O)-(C1-C6)alkyl-halo, halogen, NO2, and SO2, RA to RG being independently of each other H or a (C1-C6)alkyl, preferably Ri is N(CH 3 ) 2 ;
  • R2 is a hydrogen atom or (C1-C6)alkyl,; and each Z is, independently of each other, a hydrogen or deuterium atom.
  • Z is an hydrogen atom.
  • R2 is a hydrogen atom or a methyl, more preferably a hydrogen atom.
  • Ri is selected from the group consisting of NRARB, ORC, C(O)ORD, C(O)RE, S(O)RF, S(O) 2 RG, CN, halo-(C1 -alkyl), -C(O)-(C1)alkyl-halo, halogen, , NO2 and SO2, RA to RG being independently of each other H or a (C1-C6)alkyl.
  • Ri is selected from the group consisting of NRARB, ORC, C(O)ORD, C(O)RE, S(O)RF, S(O) 2 RG, CN, CF 3 , -C(O)CF 3 , halogen, NO2 and SO2, RA to RG being independently of each other H or a (Ci-C 6 )alkyl.
  • Ri is selected from the group consisting of NRARB, ORC, C(O)ORD, C(O)RE, S(O)RF, S(O) 3 RG, CN, halo-(C1 -alkyl), halogen, and SO2, RA to RG being independently of each other H or a (C1- C 6 )alkyl.
  • R1 is selected from the group consisting of NRARB, ORC, C(O)ORD, C(O)RE, S(O)RF, S(O) 3 RG, CN, halogen, and SO2, RA to RG being independently of each other H or a (C1-C6)alkyl.
  • Ri is selected from the group consisting of -NH2, - (N(CH 3 ) 2 , -OH, -OCH 3 , -C(O)OH, -C(O)OCH 3 , -C(O)H, -C(O)CH 3 , -S(O)H, -S(O) 2 H, -CN, halogen, - CH2-CI, CH 3 -Br, CH2-F, CH2-I, -CF 3 , -C(O)CF 3 , -NO2 and -SO2. More preferably, in the compound of general formula (II), Ri is (N(CH 3 ) 3 .
  • Ri is selected from the group consisting of -NH2, - (N(CH 3 ) 2 , -OH, -OCH 3 , -C(O)OH, -C(O)OCH 3 , -C(O)H, -C(O)CH 3 , -S(O)H, -S(O) 2 H, -CN, halogen, - CH2-CI, CH 3 -Br, CH2-F, CH2-I, -CF 3 and -SO2. More preferably, in the compound of general formula (II), Ri is (N(CH 3 ) 2 .
  • Z is an hydrogen atom
  • R2 is a hydrogen atom
  • Ri is (N(CH 3 ) 2 .
  • the first moiety A is selected from ligands of the White-Chen Type, preferably the first moiety A is White -Chen ligand.
  • the White-Chen ligand is also known as 2-[[2-[l-(pyridin-2- ylmethyl)pyrrolidin-2-yl]pyrrolidin-l-yl]methyl]pyridine based ligand, and referred to the compound of the following formula (V):
  • ligands of the White-Chen Type referred to iron ligands that were developed by the Christina White group in the following articles: M. S. Chen, M. C. White, Science 2007, 318, 783-787 [25]; andM. S. Chen, M. C White, Science 2010, 327, 566-571 [27],
  • a ligand of the White-Chen Type is a ligand that forms part of an organometallic complex with iron able to catalyze selectively aliphatic C-H oxidations in conditions that are similar to that found in the lysosomal compartment. This family of ligands forms stable complexes with iron.
  • ligands of the White-Chen Type according to the invention can be of the general formula (Ila) as disclosed below: wherein
  • Ri is selected from the group consisting of NRARB, ORC, C(O)ORD, C(O)RE, S(O)RF, S(O)2RG, CN, halo-(C1-C6-alkyl), -C(O)-(C1-C6)alkyl-halo, halogen, NO2, and SO2, RA to RG being independently of each other H or a (C1-C6)alkyl, preferably Ri is N(CH3)2;
  • R2 is a hydrogen atom or (C1-C6)alkyl
  • X and Y are, independently of each other, a (C1-C6)alkyl; or X and Y can be linked together to form a heterocycloalkyl or heteroaryl, preferably a pyrrolidine; and each Z is, independently of each other, a hydrogen or deuterium atom; and wherein the wavy line represents the link to L.
  • the first moiety A is of the general formula (lib) : wherein
  • Ri is selected from the group consisting of NRARB, ORC, C(O)ORD, C(O)RE, S(O)RF, S(O)2RG, CN, halo-(C1-C6-alkyl), -C(O)-(C1-C6)alkyl-halo, halogen, NO2, and SO2, RA to RG being independently of each other H or a (C1-C6)alkyl, preferably Ri is N(CH3)2;
  • R2 is a hydrogen atom or (C1-C6)alkyl; and each Z is, independently of each other, a hydrogen or deuterium atom; and wherein the wavy line represents the link to L.
  • Z is an hydrogen atom.
  • R2 is a hydrogen atom or a methyl, more preferably a hydrogen atom.
  • Ri is selected from the group consisting of NRARB, ORC, C(O)ORD, C(O)R E , S(O)R F , S(O) 2 RG, CN, halo-(Ci-alkyl), -C(O)-(Ci)alkyl-halo, halogen, , NO2 and SO2, RA to RG being independently of each other H or a (C1-C6)alkyl.
  • Ri is selected from the group consisting of NRARB, OR C , C(O)OR D , C(O)R E , S(O)R E , S(O) 2 RG, CN, CF 3 , -C(O)CF 3 , halogen, NO 2 and SO 2 , RA to RG being independently of each other H or a (C1-C6)alkyl.
  • Ri is selected from the group consisting of NRARB, OR C , C(O)OR D , C(O)R E , S(O)R E , S(O) 2 RG, CN, halo-(C1 -alkyl), halogen, and SO 2 , RA to RG being independently of each other H or a (C1-C6)alkyl.
  • Ri is selected from the group consisting of NRARB, ORC, C(O)ORD, C(O)R E , S(O)R F , S(O)2RG, CN, halogen, and SO2, RA to RG being independently of each other H or a (C1-C6)alkyl.
  • Ri is selected from the group consisting of NH 2 , (N(CH 3 ) 2 , OH, OCH 3 , C(O)OH, C(O)OCH 3 , C(O)H, C(O)CH 3 , S(O)H, S(O) 2 H, CN, halogen, -CH2-CI, CH 2 -Br, CH 2 -F, CH2-I, -CF 3 , -C(O)CF 3 , NO 2 , and SO 2 .
  • Ri is selected from the group consisting of NH 2 , (N(CH 3 ) 2 , OH, OCH 3 , C(O)OH, C(O)OCH 3 , C(O)H, C(O)CH 3 , S(O)H, S(O) 2 H, CN, halogen, -CH 2 -C1, CH 2 -Br, CH 2 -F, CH 2 -I, -CF 3 and SO 2 . More preferably, in the compound of general formula (Ila) or (lib), Ri is (N(CH 3 ) 2 .
  • Z is an hydrogen atom
  • R 2 is a hydrogen atom
  • Ri is (N(CH 3 ) 2 .
  • the first moiety A is of the general formula (lie): (lie), wherein
  • Ri is N(CH 3 ) 2 ;
  • R 2 is an hydrogen atom or (C1-C6)alkyl; and wherein the wavy line represents the link to L.
  • R 2 is preferably a hydrogen atom or a methyl, more preferably a hydrogen atom.
  • the first moiety A is of the following formula (lid) :
  • the first moiety A is selected from ligands of the Nordlander-Costas Type, preferably the first moiety A is Nordlander-Costas ligand.
  • the terms “ligands of the Nordlander-Costas Type” referred to iron ligands that are developed by Costas and Norlander in the following paper : Chem. Commun., 2014, 50, 1408 (DOI: 10.1039/c3cc47830k) [28],
  • the new tetradentate ligand replaces the pyridyl arm by an N- methyl benzimidazolyl substituent.
  • the sp2 character and the rigidity of the latter substituent should provide a well-defined steric demand, intermediate between the a-H and the a-Me groups of a pyridine.
  • the relative donor capacities of pyridine and benzimidazole can be estimated to be very similar by comparing the pKa values of their conjugate acids (5.22 for pyridine, 5.41 for benzimidazole and 5.57 for a-Me pyridine), and therefore differences in reactivity among this set of complexes can be traced to steric factors.
  • ligands of the Nordlander-Costas Type according to the invention can be of the general formula (lie) as disclosed herein: wherein
  • Rs is a hydrogen atom or (C1-C6)alkyl preferably methyl
  • X and Y are, independently of each other, a (C1-C6)alkyl; or X and Y can be linked together to form a heterocycloalkyl or heteroaryl, preferably a pyrrolidine; and each Z is, independently of each other, a hydrogen or deuterium atom; and wherein the wavy line represents the link to L.
  • the first moiety A is of the general formula (Ilf): wherein each Z is, independently of each other, a hydrogen or deuterium atom; and wherein the wavy line represents the link to L.
  • Z is an hydrogen atom.
  • the Nordlander-Costas ligand refers to the compound of the following formula (Ilg): wherein the wavy line represents the link to L.
  • the first moiety A is: wherein the wavy line represents the link to L.
  • the first moiety A is selected from the group consisting of:
  • the first moiety A is selected from the group consisting of: In another specific embodiment, the first moiety A is selected from the group consisting of: The first moiety A allows to activate the metal ion to promote lipid peroxidation and ferroptosis by Fenton reaction.
  • the second moiety B is selected from lysosomotropic agents.
  • lysosomotropic agents is used to designate all substances that are taken up selectively into lysosomes, in vitro and in vivo, irrespective of their chemical nature or mechanism of uptake.
  • lysosomotropic agents can be weak bases that penetrate in the membrane and accumulate inside lysosomes as protonated form and increase the intracellular pH.
  • Lysosomotropic agent can also integrate in the cell membrane and be taken up by endocytosis, accumulating in lysosomes. Therefore, lysosomotropic agent can drive the attached iron chelating agent in these lysosomes to promote lipid oxidation and induce ferroptosis. Lysosomotropic agents exert their effects on the cell via lysosomes.
  • the moiety B allows to guide the cellular localization of Fentomycin to lysosome, to induce reactive oxygen species in endolysosomes promoting oxidative degradation of membrane lipids leading to ferroptosis.
  • Suitable lysosomotropic agents can include, but are not limited to, the chloroquine, hydroxychloroquine, azithromycin, marmycin, steroids such as cholesterol and cholestanol, ezurpimtrostat hydrochloride, desmethylchloroquine, hydroxychloroquine phosphate, desmethyl-hydroxychloroquine, anthraquinones, methyl tetraphene dione, angucyclines, ammonium chloride (NFLC1), amantadine, methylamine, fluoxetine, imipramine, latrepirdine, tamoxifen, chlorpromazine, amitriptyline, verapamil, Triton WR 1339 (Tyloxapol), Suramin, metformin, Erythromycin, Amitriptyline, Imipramine, 4-aminoquinoline, amiodarone, amodiaquine, clindamycin, N-(3-[(2,4-dinitrophic acid,
  • the second moiety B is selected from the group consisting of chloroquine, hydroxychloroquine, azithromycin, marmycin, cholesterol, cholestanol, ezurpimtrostat hydrochloride, desmethylchloroquine, hydroxychloroquine phosphate, desmethyl-hydroxychloroquine, anthraquinones, methyl tetraphene dione, angucyclines.
  • the second moiety B is selected from the group consisting of chloroquine, hydroxychloroquine, azithromycin, marmycin, ezurpimtrostat hydrochloride, desmethylchloroquine, hydroxychloroquine phosphate, desmethyl-hydroxychloroquine, anthraquinones, methyl tetraphene dione, and angucyclines.
  • the second moiety B is selected from the group consisting of chloroquine, hydroxychloroquine, marmycin, cholesterol, cholestanol, and ezurpimtrostat hydrochloride, as illustrated below:
  • the second moiety B is selected from the group consisting of:
  • the second moiety B is selected from the group consisting of marmycin, cholesterol, cholestanol, and ezurpimtrostat hydrochloride.
  • the second moiety B is marmycin such as marmycin A or marmycin B, preferably marmycin A.
  • the linker is preferably bound to said lysosomotropic agent via the hydroxyl of the sugar moiety of marmycin.
  • the second moiety B is of the following formula (Bl), referring to marmycin A:
  • the second moiety B is cholesterol or cholestanol.
  • the linker is preferably bound to said lysosomotropic agent via the hydroxyl group.
  • the second moiety B is of the following formula (B2) or (B3) or (B4) or (B5):
  • the second moiety B is of the following formula (B2) or (B3), referring to cholesterol or cholestanol:
  • the conjugate A-L-B comprises:
  • a second moiety B which is marmycin and wherein the linker is bound to said moiety B via the hydroxyl of the sugar moiety of marmycin.
  • the conjugate A-L-B comprises:
  • a second moiety B which is marmycin and wherein the linker is bound to said moiety B via the hydroxyl of the sugar moiety of marmycin.
  • the conjugate A-L-B comprises: - A first moiety A of the general formula (lib) : (lib), wherein
  • Ri is N(CH 3 ) 2 ;
  • R 2 is a hydrogen atom or (C1-C6)alkyl, preferably hydrogen atom; and wherein the wavy line represents the link to L, and
  • a second moiety B which is marmycin and wherein the linker is bound to said moiety B via the hydroxyl of the sugar moiety of marmycin.
  • the conjugate A-L-B comprises:
  • a second moiety B which is marmycin and wherein the linker is bound to said moiety B via the hydroxyl of the sugar moiety of marmycin.
  • the conjugate A-L-B comprises:
  • a second moiety B which is marmycin and wherein the linker is bound to said moiety B via the hydroxyl of the sugar moiety of marmycin.
  • the conjugate A-L-B comprises:
  • a second moiety B which is marmycin and wherein the linker is bound to said moiety B via the hydroxyl of the sugar moiety of marmycin.
  • the conjugate A-L-B comprises:
  • a second moiety B which is marmycin and wherein the linker is bound to said moiety B via the hydroxyl of the sugar moiety of marmycin.
  • the conjugate A-L-B comprises:
  • a second moiety B which is marmycin and wherein the linker is bound to said moiety B via the hydroxyl of the sugar moiety of marmycin.
  • the conjugate A-L-B comprises:
  • a second moiety B which is marmycin and wherein the linker is bound to said moiety B via the hydroxyl of the sugar moiety of marmycin.
  • marmycin is preferably marmycin A, in particular of formula (Bl).
  • the conjugate A-L-B comprises:
  • a second moiety B which is cholesterol of formula (B2).
  • the conjugate A-L-B comprises:
  • a second moiety B which is cholesterol of formula (B2).
  • the conjugate A-L-B comprises:
  • a second moiety B which is cholesterol of formula (B2).
  • the conjugate A-L-B comprises:
  • a second moiety B which is cholesterol of formula (B2).
  • the conjugate A-L-B comprises:
  • a second moiety B which is cholesterol of formula (B2).
  • the conjugate A-L-B comprises:
  • a second moiety B which is cholesterol of formula (B2).
  • the conjugate A-L-B comprises:
  • a second moiety B which is cholesterol of formula (B2).
  • the conjugate A-L-B comprises:
  • a second moiety B which is cholesterol of formula (B2).
  • the conjugate A-L-B comprises:
  • a second moiety B which is cholesterol of formula (B2).
  • the conjugate A-L-B comprises:
  • a second moiety B which is cholestanol of formula (B3).
  • the conjugate A-L-B comprises:
  • a second moiety B which is cholestanol of formula (B3).
  • the conjugate A-L-B comprises:
  • a second moiety B which is cholestanol of formula (B3).
  • the conjugate A-L-B comprises:
  • a second moiety B which is cholestanol of formula (B3).
  • the conjugate A-L-B comprises:
  • a second moiety B which is cholestanol of formula (B3).
  • the conjugate A-L-B comprises:
  • a second moiety B which is cholestanol of formula (B3).
  • the conjugate A-L-B comprises:
  • a second moiety B which is cholestanol of formula (B3).
  • the conjugate A-L-B comprises:
  • a second moiety B which is cholestanol of formula (B3).
  • the conjugate A-L-B comprises:
  • a second moiety B which is cholestanol of formula (B3).
  • the conjugate A-L-B comprises:
  • a second moiety B which is cholesterol of formula (B4).
  • the conjugate A-L-B comprises:
  • a second moiety B which is cholesterol of formula (B4).
  • the conjugate A-L-B comprises:
  • a second moiety B which is cholesterol of formula (B4).
  • the conjugate A-L-B comprises:
  • a second moiety B which is cholesterol of formula (B4).
  • the conjugate A-L-B comprises:
  • a second moiety B which is cholesterol of formula (B4).
  • the conjugate A-L-B comprises:
  • a second moiety B which is cholesterol of formula (B4).
  • the conjugate A-L-B comprises:
  • a second moiety B which is cholesterol of formula (B4).
  • the conjugate A-L-B comprises:
  • a second moiety B which is cholesterol of formula (B4).
  • the conjugate A-L-B comprises:
  • a second moiety B which is cholesterol of formula (B4).
  • the conjugate A-L-B comprises:
  • a second moiety B which is cholestanol of formula (B5).
  • the conjugate A-L-B comprises:
  • a second moiety B which is cholestanol of formula (B5).
  • the conjugate A-L-B comprises:
  • a second moiety B which is cholestanol of formula (B5).
  • the conjugate A-L-B comprises:
  • a second moiety B which is cholestanol of formula (B5).
  • the conjugate A-L-B comprises:
  • a second moiety B which is cholestanol of formula (B5).
  • the conjugate A-L-B comprises:
  • a second moiety B which is cholestanol of formula (B5).
  • the conjugate A-L-B comprises:
  • a second moiety B which is cholestanol of formula (B5).
  • the conjugate A-L-B comprises:
  • a second moiety B which is cholestanol of formula (B5).
  • the conjugate A-L-B comprises:
  • a second moiety B which is cholestanol of formula (B5).
  • L is a linker that binds moieties A and B together.
  • linker refers to a single covalent bond or a moiety comprising series of stable covalent bonds, the moiety often incorporating 1-40 plural valent atoms selected from the group consisting of C, N, O, S and P, that covalently attach a reactive group or bioactive group to the probe of the invention.
  • the number of plural valent atoms in a linker may be, for example, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 20, 25, 30 or a larger number up to 40 or more.
  • a linker may be linear or non-linear; some linkers have pendant side chains or pendant functional groups (or both). Examples of such pendant moieties are hydrophilicity modifiers, for example solubilizing groups like, e.g. sulfo (- SO3H or -SO3-), carboxy (-COOH or -COO-), hydroxy.
  • L is composed of any combination of single, double, triple or aromatic carboncarbon bonds, carbon-nitrogen bonds, nitrogen-nitrogen bonds, carbon-oxygen bonds and carbon-sulfur bonds.
  • Linkers may by way of example consist of a combination of moieties selected from alkyl, - C(O)NH-, -C(O)O-, -NH-, -S-, -O-, -C(O) -, -S(O)-, -S(O)2-.; -O-, 5- or 6- membered monocyclic rings and optional pendant functional groups, for example sulfo, hydroxy and carboxy.
  • L is selected from the group consisting of a single bond, -C(O)NH-, -C(O)O-, -NH-, -S-, -O-, -C(O) -, -S(O)-, -S(O)2-.; -O-, and 5- or 6- membered monocyclic rings.
  • L is -O-.
  • the reactive group may be reacted with a substance reactive therewith, whereby the linker becomes bonded to a bioactive group.
  • the linker typically contains a residue of a reactive group (such as for example the carbonyl group of an ester after reaction with a nucleophile; a triazolo group resulting from a click reaction between an azide and an alkyne; an amide link resulting from a reaction between an amine and an acid; a thiourea resulting from the coupling between an amine and an isothiocyanate; a -O-C(O)- moiety remaining for example after reaction of an activated carbonate with a nucleophile, etc.).
  • a reactive group such as for example the carbonyl group of an ester after reaction with a nucleophile; a triazolo group resulting from a click reaction between an azide and an alkyne; an amide link resulting from a reaction between an amine and an acid; a thiourea
  • the linker is an aliphatic hydrocarbon chain comprising from 2 to 20 carbon atoms and being optionally terminated at one or both end and/or interrupted by at least one group independently selected from -C(O)NH-,-NHC(O)-, -C(O)O-, -OC(O)-, -NH-, -S-, -O-, -C(O) -, triazolyl, -S(O)-, and -S(O)2-.
  • the linker is an aliphatic hydrocarbon chain comprising from 2 to 16 carbon atoms and being optionally terminated at one or both end and/or interrupted by at least one group independently selected from -C(O)NH-,-NHC(O)-, -C(O)O-, -OC(O)- , -NH-, -S-, -O-, -C(O) -, triazolyl, -S(O)-, and -S(O) 2 -.
  • the linker is an aliphatic hydrocarbon chain comprising from 2 to 14 carbon atoms and being optionally terminated at one or both end and/or interrupted by at least one group independently selected from -C(O)NH-,-NHC(O)-, -C(O)O-, -OC(O)- , -NH-, -S-, -O-, -C(O) -, triazolyl, -S(O)-, and -S(O) 2 -.
  • the linker is an aliphatic hydrocarbon chain comprising from 2 to 12 carbon atoms and being optionally terminated at one or both end and/or interrupted by at least one group independently selected from -C(O)NH-,-NHC(O)-, -C(O)O-, -OC(O)- , -NH-, -S-, -O-, -C(O) triazolyl, -S(O)-, and -S(O) 2 -.
  • the linker is selected from: -(CH2)t-, -(CH2-O) P -, -((CH2)t-O) p -, -(CH2)t-C(O)-, -C(O)- (CH 2 )t-C(O)-, (CH 2 )t-NH-(CH 2 ) q -C(O)-, -(CH 2 ) n -NH-CO-(CH 2 ) m -NH-, -(CH 2 ) n -NH-CO-(CH 2 ) m -CO-, - (CH 2 )n-NH-CO-(CH2-CH 2 -O) m -NH-, -(CH 2 )n-NH-CO-(CH2-CH 2 -O) m -CO-, -(CH 2 ) n -CO-NH-(CH 2 ) m - CO-, -(CH 2 )n-CO-NH-(CH 2
  • the linker is selected from: -(CH 2 ) t -, -(CH 2 -O) P -, -((CH 2 ) t -O) p -, -(CH 2 ) t -C(O)-, -C(O)- (CH 2 ) t -C(O)-, (CH 2 ) t -NH-(CH 2 ) q -C(O)-, -(CH 2 ) n -NH-CO-(CH 2 ) m -NH-, -(CH 2 ) n -NH-CO-(CH 2 ) m -CO-, - (CH 2 )n-NH-CO-(CH2-CH 2 -O) m -NH-, -(CH 2 )n-NH-CO-(CH2-CH 2 -O) m -CO-, -(CH 2 ) n -CO-NH-(CH 2 ) m -CO-
  • the linker is selected from: -(CH2)t-, -(CH2-O) P -, -((CH2)t-O) p -, -(CH2)t-C(O)-, -C(O)- (CH 2 ) t -C(O)-, (CH 2 ) t -NH-(CH 2 ) q -C(O)-, -(CH 2 ) n -NH-CO-(CH 2 ) m -NH-, -(CH 2 ) n -NH-CO-(CH 2 ) m -CO-, - (CH 2 )n-NH-CO-(CH2-CH 2 -O) m -NH-, -(CH 2 )n-NH-CO-(CH2-CH 2 -O) m -CO-, -(CH 2 ) n -CO-NH-(CH 2 ) m - CO-, -(CH 2 )n-CO-NH-
  • the linker L is of the following formula (VI):
  • Y is bound to the moiety A, and Y is selected from the group consisting of -(CH2) n -NH-, -(CH2) n -CO-, -(CH 2 -CH 2 -O) n -NH-, and -(CH 2 -CH 2 -O) n -CO-;
  • X is bound to the moiety B, and X is selected from the group consisting of -NH-(CH2) m -CO-, -CO- (CH 2 ) m -NH-, -CO-(CH 2 ) m -CO-, -NH-(CH 2 -CH 2 -O) m -CO-, -CO-(CH 2 -CH 2 -O) m -NH-, and -CO-(CH 2 - CH 2 -O) m -CO-; n is an integer from 2 to 14, preferably from 2 to 12; and and m is an integer from 2 to 6.
  • the linker L is of the following formula (VI):
  • Y is bound to the moiety A, and Y is selected from the group consisting of -(CH2) n -NH-, -(CH2) n -CO-, -(CH 2 -CH 2 -O) n -NH-, and -(CH 2 -CH 2 -O) n -CO-;
  • X is bound to the moiety B, and X is selected from the group consisting of -NH-(CH2) m -CO-, -CO- (CH 2 ) m -NH-, -CO-(CH 2 ) m -CO-, -NH-(CH 2 -CH 2 -O) m -CO-, -CO-(CH 2 -CH 2 -O) m -NH-, and -CO-(CH 2 - CH2-0)m-C0-; and n and m are, independently of each other, an integer from 2 to 6.
  • the linker L can be selected from the group consisting of: -CH2-CH2-, -CH2-CH2-CH2- CH 2 -, -CH2-CH2-O-CH2-CH2-, -CH2-CH2-O-CH2-CH2-, -CH2-CH2-NH-CH2-CH2-, -CH 2 - CH2-CH2-CH2-NH-CH2-CH2-, -CH2-CH2-CH2-NH-CH2-CH2-, -CH2-CH2-CH2-CH2-NH-CH2-CH2-CH2-CH2-, -CH 2 -CH 2 -C(O)-, -CH 2 - CH 2 -CH 2 -CH 2 -C(O)-, -C(O)-CH 2 -CH 2 -C(O)-, -C(O)-CH2-CH 2 -CH2-CH 2 -C(O)-, -CH 2 -CH 2 -NHC(O)- CH 2 -CH 2 -C(O)-, -CH2-CH2-CH2-CH 2
  • the linker L can be selected from the group consisting of: -CH2-CH2-, -CH2-CH2-CH2- CH2-, -CH2-CH2-O-CH2-CH2-, -CH2-CH2-O-CH2-CH2-, -CH2-CH2-NH-CH2-CH2-, -CH 2 - CH2-CH2-CH2-NH-CH2-CH2-, -CH2-CH2-CH2-NH-CH2-CH2-, -CH2-CH2-CH2-CH2-NH-CH2-CH2-CH2-CH2-, -CH 2 -CH 2 -C(O)-, -CH 2 - CH 2 -CH 2 -CH 2 -C(O)-, -C(O)-CH 2 -CH 2 -C(O)-, -C(O)-CH2-CH 2 -CH2-CH 2 -C(O)-, -CH 2 -CH2-CH 2 -C(O)-, -CH 2 -CH2-CH 2 -C(O)-, -CH 2 -
  • the linker L can be -(CH2) n -CH2-NHC(O)-CH2-CH2-C(O)-, wherein n’ is an integer from 1 to 17, preferably n’ is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17, more preferably n’ is 3, 4, 5, 6, 7, 8, 9, 10 or 11.
  • the conjugate A-L-B comprises:
  • a second moiety B which is marmycin and wherein the linker is bound to said moiety B via the hydroxyl of the sugar moiety of marmycin;
  • a linker L which is -(CH 2 ) n -CH2-NHC(O)-CH2-CH 2 -C(O)-, wherein n’ is an integer from 1 to 17.
  • the conjugate A-L-B comprises:
  • a second moiety B which is marmycin and wherein the linker is bound to said moiety B via the hydroxyl of the sugar moiety of marmycin; and A linker L which is -(CH2) n -CH2-NHC(O)-CH2-CH2-C(O)-, wherein n’ is an integer from 1 to 17.
  • the conjugate A-L-B comprises:
  • a second moiety B which is marmycin and wherein the linker is bound to said moiety B via the hydroxyl of the sugar moiety of marmycin;
  • a linker L which is -(CH2) n -CH2-NHC(O)-CH2-CH2-C(O)-, wherein n’ is an integer from 1 to 17.
  • the conjugate A-L-B comprises:
  • a second moiety B which is marmycin and wherein the linker is bound to said moiety B via the hydroxyl of the sugar moiety of marmycin;
  • a linker L which is -(CH 2 ) n -CH2-NHC(O)-CH2-CH 2 -C(O)-, wherein n’ is an integer from 1 to 17.
  • the conjugate A-L-B comprises:
  • a second moiety B which is marmycin and wherein the linker is bound to said moiety B via the hydroxyl of the sugar moiety of marmycin;
  • a linker L which is -(CH2) n -CH2-NHC(O)-CH2-CH2-C(O)-, wherein n’ is an integer from 1 to 17.
  • the conjugate A-L-B comprises:
  • a second moiety B which is marmycin and wherein the linker is bound to said moiety B via the hydroxyl of the sugar moiety of marmycin;
  • a linker L which is -(CH2) n -CH2-NHC(O)-CH2-CH2-C(O)-, wherein n’ is an integer from 1 to 17.
  • the conjugate A-L-B comprises:
  • a second moiety B which is marmycin and wherein the linker is bound to said moiety B via the hydroxyl of the sugar moiety of marmycin;
  • a linker L which is -(CH 2 ) n -CH2-NHC(O)-CH2-CH 2 -C(O)-, wherein n’ is an integer from 1 to 17.
  • the conjugate A-L-B comprises:
  • a second moiety B which is marmycin and wherein the linker is bound to said moiety B via the hydroxyl of the sugar moiety of marmycin; and A linker L which is -(CH2) n -CH2-NHC(O)-CH2-CH2-C(O)-, wherein n’ is an integer from 1 to 17.
  • the conjugate A-L-B comprises:
  • a second moiety B which is marmycin and wherein the linker is bound to said moiety B via the hydroxyl of the sugar moiety of marmycin;
  • a linker L which is -(CH2) n -CH2-NHC(O)-CH2-CH2-C(O)-, wherein n’ is an integer from 1 to 17.
  • marmycin is preferably marmycin A, in particular of formula (Bl).
  • the conjugate A-L-B comprises:
  • a second moiety B which is cholesterol of formula (B2);
  • a linker L which is -(CH2) n -CH2-NHC(O)-CH2-CH2-C(O)-, wherein n’ is an integer from 1 to 17.
  • the conjugate A-L-B comprises:
  • a second moiety B which is cholesterol of formula (B2);
  • a linker L which is -(CH2) n -CH2-NHC(O)-CH2-CH2-C(O)-, wherein n’ is an integer from 1 to 17.
  • the conjugate A-L-B comprises:
  • a second moiety B which is cholesterol of formula (B2);
  • a linker L which is -(CH2) n -CH2-NHC(O)-CH2-CH2-C(O)-, wherein n’ is an integer from 1 to 17.
  • the conjugate A-L-B comprises:
  • a second moiety B which is cholesterol of formula (B2);
  • a linker L which is -(CH 2 ) n -CH2-NHC(O)-CH2-CH 2 -C(O)-, wherein n’ is an integer from 1 to 17.
  • the conjugate A-L-B comprises:
  • a second moiety B which is cholesterol of formula (B2);
  • a linker L which is -(CH2) n -CH2-NHC(O)-CH2-CH2-C(O)-, wherein n’ is an integer from 1 to 17.
  • the conjugate A-L-B comprises:
  • a linker L which is -(CH2) n -CH2-NHC(O)-CH2-CH2-C(O)-, wherein n’ is an integer from 1 to 17.
  • the conjugate A-L-B comprises:
  • a second moiety B which is cholesterol of formula (B2);
  • a linker L which is -(CH2) n -CH2-NHC(O)-CH2-CH2-C(O)-, wherein n’ is an integer from 1 to 17.
  • the conjugate A-L-B comprises:
  • a second moiety B which is cholesterol of formula (B2);
  • a linker L which is -(CH 2 ) n -CH2-NHC(O)-CH2-CH 2 -C(O)-, wherein n’ is an integer from 1 to 17.
  • the conjugate A-L-B comprises:
  • a second moiety B which is cholesterol of formula (B2);
  • a linker L which is -(CH2) n -CH2-NHC(O)-CH2-CH2-C(O)-, wherein n’ is an integer from 1 to 17.
  • the conjugate A-L-B comprises:
  • a second moiety B which is cholestanol of formula (B3);
  • a linker L which is -(CH2) n -CH2-NHC(O)-CH2-CH2-C(O)-, wherein n’ is an integer from 1 to 17.
  • the conjugate A-L-B comprises:
  • a second moiety B which is cholestanol of formula (B3);
  • a linker L which is -(CH2) n -CH2-NHC(O)-CH2-CH2-C(O)-, wherein n’ is an integer from 1 to 17.
  • the conjugate A-L-B comprises:
  • a second moiety B which is cholestanol of formula (B3);
  • a linker L which is -(CH2) n -CH2-NHC(O)-CH2-CH2-C(O)-, wherein n’ is an integer from 1 to 17.
  • the conjugate A-L-B comprises:
  • a second moiety B which is cholestanol of formula (B3); and A linker L which is -(CH2) n -CH2-NHC(O)-CH2-CH2-C(O)-, wherein n’ is an integer from 1 to 17.
  • the conjugate A-L-B comprises:
  • a second moiety B which is cholestanol of formula (B3);
  • a linker L which is -(CH2) n -CH2-NHC(O)-CH2-CH2-C(O)-, wherein n’ is an integer from 1 to 17.
  • the conjugate A-L-B comprises:
  • a second moiety B which is cholestanol of formula (B3);
  • a linker L which is -(CH 2 ) n -CH2-NHC(O)-CH2-CH 2 -C(O)-, wherein n’ is an integer from 1 to 17.
  • the conjugate A-L-B comprises:
  • a second moiety B which is cholestanol of formula (B3);
  • a linker L which is -(CH2) n -CH2-NHC(O)-CH2-CH2-C(O)-, wherein n’ is an integer from 1 to 17.
  • the conjugate A-L-B comprises:
  • a second moiety B which is cholestanol of formula (B3);
  • a linker L which is -(CH2) n -CH2-NHC(O)-CH2-CH2-C(O)-, wherein n’ is an integer from 1 to 17.
  • the conjugate A-L-B comprises:
  • a second moiety B which is cholestanol of formula (B3);
  • a linker L which is -(CH2) n -CH2-NHC(O)-CH2-CH2-C(O)-, wherein n’ is an integer from 1 to 17.
  • the conjugate A-L-B comprises:
  • a second moiety B which is of formula (B4);
  • a linker L which is -(CH2) n -CH2-NHC(O)-CH2-CH2-C(O)-, wherein n’ is an integer from 1 to 17.
  • the conjugate A-L-B comprises:
  • a second moiety B which is of formula (B4); and A linker L which is -(CH2) n -CH2-NHC(O)-CH2-CH2-C(O)-, wherein n’ is an integer from 1 to 17.
  • the conjugate A-L-B comprises:
  • a second moiety B which is of formula (B4);
  • a linker L which is -(CH2) n -CH2-NHC(O)-CH2-CH2-C(O)-, wherein n’ is an integer from 1 to 17.
  • the conjugate A-L-B comprises:
  • a second moiety B which is of formula (B4);
  • a linker L which is -(CH 2 ) n -CH2-NHC(O)-CH2-CH 2 -C(O)-, wherein n’ is an integer from 1 to 17.
  • the conjugate A-L-B comprises:
  • a second moiety B which is of formula (B4);
  • a linker L which is -(CH2) n -CH2-NHC(O)-CH2-CH2-C(O)-, wherein n’ is an integer from 1 to 17.
  • the conjugate A-L-B comprises:
  • a second moiety B which is of formula (B4);
  • a linker L which is -(CH2) n -CH2-NHC(O)-CH2-CH2-C(O)-, wherein n’ is an integer from 1 to 17.
  • the conjugate A-L-B comprises:
  • a second moiety B which is of formula (B4);
  • a linker L which is -(CH2) n -CH2-NHC(O)-CH2-CH2-C(O)-, wherein n’ is an integer from 1 to 17.
  • the conjugate A-L-B comprises:
  • a second moiety B which is of formula (B4);
  • a linker L which is -(CH 2 ) n -CH2-NHC(O)-CH2-CH 2 -C(O)-, wherein n’ is an integer from 1 to 17.
  • the conjugate A-L-B comprises:
  • a second moiety B which is of formula (B4);
  • a linker L which is -(CH2) n -CH2-NHC(O)-CH2-CH2-C(O)-, wherein n’ is an integer from 1 to 17.
  • the conjugate A-L-B comprises:
  • a second moiety B which is of formula (B4);
  • a linker L which is -(CH2)t wherein t is an integer from 1 to 20.
  • the conjugate A-L-B comprises:
  • a second moiety B which is of formula (B5);
  • a linker L which is -(CH 2 ) n -CH2-NHC(O)-CH2-CH 2 -C(O)-, wherein n’ is an integer from 1 to 17.
  • the conjugate A-L-B comprises:
  • a second moiety B which is of formula (B5);
  • a linker L which is -(CH2) n -CH2-NHC(O)-CH2-CH2-C(O)-, wherein n’ is an integer from 1 to 17.
  • the conjugate A-L-B comprises:
  • a second moiety B which is of formula (B5);
  • a linker L which is -(CH2) n -CH2-NHC(O)-CH2-CH2-C(O)-, wherein n’ is an integer from 1 to 17.
  • the conjugate A-L-B comprises:
  • a second moiety B which is of formula (B5);
  • a linker L which is -(CH2) n -CH2-NHC(O)-CH2-CH2-C(O)-, wherein n’ is an integer from 1 to 17.
  • the conjugate A-L-B comprises:
  • a second moiety B which is of formula (B5);
  • a linker L which is -(CH 2 ) n -CH2-NHC(O)-CH2-CH 2 -C(O)-, wherein n’ is an integer from 1 to 17.
  • the conjugate A-L-B comprises:
  • a second moiety B which is of formula (B5);
  • a linker L which is -(CH2) n -CH2-NHC(O)-CH2-CH2-C(O)-, wherein n’ is an integer from 1 to 17.
  • the conjugate A-L-B comprises: A first moiety A of the general formula (lie),
  • a second moiety B which is of formula (B5);
  • a linker L which is -(CH2) n -CH2-NHC(O)-CH2-CH2-C(O)-, wherein n’ is an integer from 1 to 17.
  • the conjugate A-L-B comprises:
  • a second moiety B which is of formula (B5);
  • a linker L which is -(CH2) n -CH2-NHC(O)-CH2-CH2-C(O)-, wherein n’ is an integer from 1 to 17.
  • the conjugate A-L-B comprises:
  • a second moiety B which is of formula (B5);
  • a linker L which is -(CH 2 ) n -CH2-NHC(O)-CH2-CH 2 -C(O)-, wherein n’ is an integer from 1 to 17.
  • the conjugate A-L-B comprises:
  • a second moiety B which is of formula (B5);
  • a linker L which is -(CILjt, wherein t is an integer from 1 to 20.
  • the conjugate of the invention is selected from the group consisting of Fentomycin of general formula (III), Fentomycin-1 of general formula (III ), Pre-Fentomycin of general formula (IV), Fentomycin-2 of general formula (VII), a compound of formula (VIF), Fentomycin-3 of general formula (VIII), a compound of formula (VIII’), Fentomycin-5 of general formula (IX), a compound of formula (IX’), Fentomycin-6 of general formula (X), a compound of formula (X’), Fentomycin-7 of general formula (XI), a compound of formula (XI’), Fentomycin-8 of general formula (XII), a compound of formula (XIF), Fentomycin-9 of general formula (XIII), a compound of formula (XIII’), Fentomycin-10 of general formula (XIV), a compound of formula (XIV’), Fentomycin-11 of general formula (XV), a compound of formula (XV’), Fentomycin- 12
  • the conjugate of the invention is selected from the group consisting of Fentomycin of general formula (III), Fentomycin-1 of general formula (III ), Pre-Fentomycin of general formula (IV), Fentomycin-2 of general formula (VII), a compound of formula (VIF), Fentomycin-3 of general formula (VIII), a compound of formula (VIII’), Fentomycin-5 of general formula (IX) , a compound of formula (IX’), Fentomycin-6 of general formula (X), a compound of formula (X’), Fentomycin-7 of general formula (XI), a compound of formula (XI’), Fentomycin-8 of general formula (XII), a compound of formula (XIF), Fentomycin-9 of general formula (XIII), a compound of formula (XIII’), Fentomycin-10 of general formula (XIV), a compound of formula (XIV’), Fentomycin-13 of general formula (XVII), a compound of formula (XVIF), and Fenentomycin of general
  • the conjugate of the invention is selected from the group consisting of Fentomycin of general formula (III), Fentomycin-1 of general formula (III ), Pre-Fentomycin of general formula (IV), Fentomycin-2 of general formula (VII), Fentomycin-3 of general formula (VIII), Fentomycin-5 of general formula (IX), Fentomycin-6 of general formula (X), Fentomycin-7 of general formula (XI), Fentomycin-8 of general formula (XII), Fentomycin-9 of general formula (XIII), Fentomycin-10 of general formula (XIV), Fentomycin-11 of general formula (XV), Fentomycin-12 of general formula (XVI), Fentomycin-13 of general formula (XVII), Fentomycin-14 of general formula (XVIII), Fentomycin-15 of general formula (XIX), and Fentomycin-16 of general formula (XX); or a stereoisomers thereof or a pharmaceutically acceptable salt and/or solvate thereof.
  • the conjugate of the invention is selected from the group consisting of Fentomycin of general formula (III), Fentomycin-1 of general formula (III ), Pre-Fentomycin of general formula (IV), Fentomycin-2 of general formula (VII), Fentomycin-3 of general formula (VIII), Fentomycin-5 of general formula (IX), Fentomycin-6 of general formula (X), Fentomycin-7 of general formula (XI), Fentomycin-8 of general formula (XII), Fentomycin-9 of general formula (XIII), Fentomycin- 10 of general formula (XIV), Fentomycin- 13 of general formula (XVII), and Fentomycin- 14 of general formula (XVIII); or a stereoisomers thereof or a pharmaceutically acceptable salt and/or solvate thereof.
  • the conjugate of the invention is selected from the group consisting of Fentomycin of general formula (III), Fentomycin-1 of general formula (III ), Pre-Fentomycin of general formula (IV), Fentomycin-2 of general formula (VII), Fentomycin-3 of general formula (VIII), and Fentomycin-5 of general formula (IX); or a stereoisomers thereof or a pharmaceutically acceptable salt and/or solvate thereof.
  • the conjugate of the invention is selected from the group consisting of Fentomycin of general formula (III), Fentomycin-1 of general formula (III ), Pre-Fentomycin of general formula (IV), Fentomycin-2 of general formula (VII), Fentomycin-3 of general formula (VIII), and Fentomycin-5 of general formula (IX)
  • the conjugate of the invention is Fentomycin of general formula (III) as disclosed herein, or Pre-Fentomycin of general formula (IV) as disclosed herein, or a stereoisomer thereof.
  • the conjugate of the invention is Fentomycin of general formula (III) as disclosed herein.
  • the conjugate of the invention is Fentomycin-1 of general formula (III’) as disclosed herein.
  • the conjugate of the invention is Pre-Fentomycin of general formula (IV) as disclosed herein.
  • the conjugate of the invention is Fentomycin-2 of general formula (VII) or a compound of formula (VIF) as disclosed herein.
  • the conjugate of the invention is Fentomycin-3 of general formula (VIII) or a compound of formula (VIIF) -as disclosed herein.
  • the conjugate of the invention is Fentomycin-5 of general formula (IX) or a compound of formula (IX’) as disclosed herein.
  • the conjugate of the invention is Fentomycin-6 of general formula (X) or a compound of formula (X’) as disclosed herein.
  • the conjugate of the invention is Fentomycin-7 of general formula (XI) or a compound of formula (XF) as disclosed herein.
  • the conjugate of the invention is Fentomycin-8 of general formula (XII) or a compound of formula (XIF) as disclosed herein.
  • the conjugate of the invention is Fentomycin-9 of general formula (XIII) or a compound of formula (XIIF) as disclosed herein.
  • the conjugate of the invention is Fentomycin- 10 of general formula (XIV) or a compound of formula (XIV’) as disclosed herein.
  • the conjugate of the invention is Fentomycin- 11 of general formula (XV) or a compound of formula (XV’) as disclosed herein.
  • the conjugate of the invention is Fentomycin-12 of general formula (XVI) or a compound of formula (XVI’) as disclosed herein.
  • the conjugate of the invention is Fentomycin- 13 of general formula (XVII) or a compound of formula (XVIF) as disclosed herein.
  • the conjugate of the invention is Fentomycin- 14 of general formula (XVIII) or a compound of formula (XVIIF) as disclosed herein.
  • the conjugate of the invention is Fentomycin- 15 of general formula (XIX) or a compound of formula (XIX’) as disclosed herein.
  • the conjugate of the invention is Fentomycin- 16 of general formula (XX) or a compound of formula (XX’) as disclosed herein.
  • a further object of the invention relates to the compound Fentomycin of general formula (III), or a pharmaceutically acceptable salt and/or solvate thereof, or a stereoisomer thereof: wherein R1 is H or Cl.
  • a further object of the invention relates to the compound Fentomycin-1 of general formula (IIP), or a pharmaceutically acceptable salt and/or solvate thereof, or a stereoisomer thereof: (iir).
  • a further object of the invention relates to the compound Pre-Fentomycin of general formula (IV), or a pharmaceutically acceptable salt and/or solvate thereof, or a stereoisomer thereof:
  • a further object of the invention relates to the compound Fentomycin-2 of general formula (VII) or a compound of formula (VII’), or a pharmaceutically acceptable salt and/or solvate thereof, or a stereoisomer thereof:
  • a further object of the invention relates to the compound Fentomycin-3 of general formula (VIII) or a compound of formula (VIII’), or a pharmaceutically acceptable salt and/or solvate thereof, or a stereoisomer thereof: .
  • a further object of the invention relates to the compound Fentomycin-5 of general formula (IX) or a compound of formula (IX’), or a pharmaceutically acceptable salt and/or solvate thereof, or a stereoisomer thereof:
  • a further object of the invention relates to the compound Fentomycin-6 of general formula (X) or a compound of formula (X’), or a pharmaceutically acceptable salt and/or solvate thereof, or a stereoisomer thereof:
  • a further object of the invention relates to the compound Fentomycin-7 of general formula (XI) or a compound of formula (XI’), or a pharmaceutically acceptable salt and/or solvate thereof, or a stereoisomer thereof:
  • a further object of the invention relates to the compound Fentomycin-8 of general formula (XII) or a compound of formula (XII’), or a pharmaceutically acceptable salt and/or solvate thereof, or a stereoisomer thereof: .
  • a further object of the invention relates to the compound Fentomycin-9 of general formula (XIII) or a compound of formula (XIII’), or a pharmaceutically acceptable salt and/or solvate thereof, or a stereoisomer thereof: ).
  • a further object of the invention relates to the compound Fentomycin-10 of general formula (XIV) or (XIV’), or a pharmaceutically acceptable salt and/or solvate thereof, or a stereoisomer thereof:
  • a further object of the invention relates to the compound Fentomycin- 11 of general formula (XV) or a compound of formula (XV’), or a pharmaceutically acceptable salt and/or solvate thereof, or a stereoisomer thereof:
  • a further object of the invention relates to the compound Fentomycin-12 of general formula (XVI) or a compound of formula (XVI’), or a pharmaceutically acceptable salt and/or solvate thereof, or a stereoisomer thereof: .
  • a further object of the invention relates to the compound Fentomycin-13 of general formula (XVII) or a compound of formula (XVII’), or a pharmaceutically acceptable salt and/or solvate thereof, or a stereoisomer thereof:
  • a further object of the invention relates to the compound Fentomycin-14 of general formula (XVIII) or a compound of formula (XVIII’), or a pharmaceutically acceptable salt and/or solvate thereof, or a stereoisomer thereof: .
  • a further object of the invention relates to the compound Fentomycin-15 of general formula (XIX) or a compound of formula (XIX’), or a pharmaceutically acceptable salt and/or solvate thereof, or a stereoisomer thereof:
  • a further object of the invention relates to the compound Fentomycin-16 of general formula (XX) or a compound of formula (XX’), or a pharmaceutically acceptable salt and/or solvate thereof, or a stereoisomer thereof:
  • Another object of the invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a conjugate according to the invention, or a compound of formula (III), (III’), (IV), (VII), (VII’), (VIII), (VIII’), (IX) (ix’), (X), (X’), (xi), (xr), (xii), (xir), (xiii), (xiir), (xiv), (xiv’), (xv), (xv’), (xvi), (xvr), (XVII), (XVII’), (XVIII), (XVIII’), (XIX), (XIX’), (XX), and (XX’); preferably (III), (III’), (IV), (VII), (vir), (viii), (VIIF), (ix) (ix’), (X), (X’), (xi), (xr), (xii), (xir), (xiir), (xiv), (xiv
  • Another object of the invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a conjugate according to the invention, or a compound of formula (III), (III’), (IV), (VII), (VIII), (IX), (X), (XI), (XII), (XIII), (XIV), (XV), (XVI), (XVII), and (XVIII); preferably (III), (III’), (IV), (VII), (VIII), (IX), (X), (XI), (XII), (XIII), (XIV), (XVII), and (XVIII); according to the invention or a pharmaceutically acceptable salt and/or solvate thereof, and at least one pharmaceutically acceptable excipient, or a stereoisomer thereof.
  • Another object of the invention relates to nanoparticle comprising a conjugate according to the invention, or a compound of formula (III), (III’), (IV), (VII), (VII’), (VIII), (VIII’), (IX) (IX’), (X), (X’), (xi), (xr), (xii), (xir), (xiii), (xiir), (xiv), (xiv’), (xv), (xv’), (xvi), (xvr), (xvii), (xvir), (XVIII), (XVIII’), (XIX), (XIX’), (XX), and (XX’); preferably (III), (III’), (IV), (VII), (VII’), (VIII), (viir), (ix) (ix’), (X), (X’), (xi), (xr), (xii), (xiir), (xiv), (xiv’), (xiv’), (xiv
  • Another object of the invention relates to nanoparticle comprising a conjugate according to the invention, or a compound of formula (III), (III’), (IV), (VII), (VIII), (IX), (X), (XI), (XII), (XIII), (XIV), (XV), (XVI), (XVII), and (XVIII); preferably (III), (III’), (IV), (VII), (VIII), (IX), (X), (XI), (XII), (XIII), (XIV), (XVII), and (XVIII); according to the invention or a pharmaceutically acceptable salt and/or solvate thereof, or a stereoisomer thereof.
  • Another object of the invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a conjugate according to the invention, or a compound of formula (III), (III’), (IV), (VII), (VIII), and (IX), according to the invention or a pharmaceutically acceptable salt and/or solvate thereof, and at least one pharmaceutically acceptable excipient.
  • Another object of the invention relates to nanoparticle comprising a conjugate according to the invention, or a compound of formula (III), (III’), (IV), (VII), (VIII), and (IX), according to the invention or a pharmaceutically acceptable salt and/or solvate thereof.
  • Another object of the invention relates to a pharmaceutical composition comprising a conjugate according to the invention, or a compound of formula (III) or (IV) according to the invention or a pharmaceutically acceptable salt and/or solvate thereof, and at least one pharmaceutically acceptable excipient.
  • Another object of the invention relates to nanoparticle comprising a conjugate according to the invention, or a compound of formula (III) or (IV) according to the invention or a pharmaceutically acceptable salt and/or solvate thereof.
  • compositions contemplated herein may include a pharmaceutically acceptable carrier in addition to the active ingredient(s).
  • pharmaceutically acceptable carrier is meant to encompass any carrier (e.g., support, substance, solvent, etc.) which does not interfere with effectiveness of the biological activity of the active ingredient(s) and that is not toxic to the host to which it is administered.
  • the active compounds(s) may be formulated in a unit dosage form for injection in vehicles such as saline, dextrose solution, serum albumin and Ringer's solution.
  • the pharmaceutical composition can be formulated as solutions in pharmaceutically compatible solvents or as emulsions, suspensions or dispersions in suitable pharmaceutical solvents or vehicle, or as pills, tablets or capsules that contain solid vehicles in a way known in the art.
  • Formulations of the present invention suitable for oral administration may be in the form of discrete units as capsules, sachets, tablets or lozenges, each containing a predetermined amount of the active ingredient; in the form of a powder or granules; in the form of a solution or a suspension in an aqueous liquid or non-aqueous liquid; or in the form of an oil-in-water emulsion or a water-in-oil emulsion.
  • Formulations suitable for parental administration conveniently comprise a sterile oily or aqueous preparation of the active ingredient which is preferably isotonic with the blood of the recipient. Every such formulation can also contain other pharmaceutically compatible and nontoxic auxiliary agents, such as, e.g. stabilizers, antioxidants, binders, dyes, emulsifiers or flavoring substances.
  • the formulations of the present invention comprise an active ingredient in association with a pharmaceutically acceptable carrier therefore and optionally other therapeutic ingredients.
  • the carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulations and not deleterious to the recipient thereof.
  • compositions are advantageously applied by injection or intravenous infusion of suitable sterile solutions or as oral dosage by the digestive tract or by direct injection into the lymph node (i.e., intra- lymphatically).
  • lymph node i.e., intra- lymphatically.
  • Methods for the safe and effective administration of most of these chemotherapeutic agents are known to those skilled in the art. In addition, their administration is described in the standard literature.
  • compositions as disclosed herein may further comprise an additional active ingredient or drug.
  • the present invention relates to a conjugate as disclosed herein or a compound of general formula (III), (III’), (IV), (VII), (VIF), (VIII), (VIIF), (IX) (IX’), (X), (X’), (XI), (XT), (XII), (XIT), (XIII), (XIIF), (XIV), (XIV’), (XV), (XV’), (XVI), (XVI’), (XVII), (XVIF), (XVIII), (XVIIF), (XIX), (XIX’), (XX), and (XX’); preferably (III), (III’), (IV), (VII), (VII’), (VIII), (VIII’), (IX) (IX’), (X), (X’), (XI), (XII’), (XIII), (XIII’), (XIV), (XIV’), (XVII), (XVII’), (XVII’), (XVII’),
  • the present invention relates to the use of a conjugate as disclosed herein or a compound of general formula (III), (III’), (IV), (VII), (VII’), (VIII), (VIII’), (IX) (IX’), (X), (X’), (XI), (XI’), (XII’), (XIII), (XIII’), (XIV), (XIV’), (XV), (XV’), (XVI), (XVI’), (XVII), (XVII’), (XVIII), (XVIII’), (XIX), (XIX’), (XX), and (XX’); preferably (III), (III’), (IV), (VII), (VII’), (VIII), (VIII’), (IX) (IX’), (XX), and (XX’); preferably (III), (III’), (IV), (VII), (VII’), (VIII), (VIII’), (IX) (IX’), (X), (
  • the invention further relates to the method for the treatment of a disease in a subject in need thereof, comprising administering a therapeutic effective amount of a conjugate as disclosed herein or a compound of general formula (III), (III’), (IV), (VII), (VII’), (VIII), (VIII’), (IX) (IX’), (X), (X’), (XI), (XT), (XII), (XIT), (XIII), (XIII’), (XIV), (XIV’), (XV), (XV’), (XVI), (XVI’), (XVII), (XVII’), (XVIII), (XVIII’), (XIX), (XIX’), (XX), and (XX’); preferably (III), (III’), (IV), (VII), (VII’), (VIII), (VIII’), (IX) (IX’), (X), (XT), (XII), (XIT), (XIII),
  • the present invention relates to a conjugate as disclosed herein or a compound of general formula (III), (III’), (IV), (VII), (VIII), (IX), (X), (XI), (XII), (XIII), (XIV), (XV), (XVI), (XVII), and (XVIII), (XIX), and (XX); preferably (III), (III’), (IV), (VII), (VIII), (IX), (X), (XI), (XII), (XIII), (XIV), (XVII), and (XVIII); as disclosed herein, or a pharmaceutical composition as disclosed herein, or a nanoparticle as disclosed herein, for use as a drug.
  • the present invention relates to the use of a conjugate as disclosed herein or a compound of general formula (III), (III’), (IV), (VII), (VIII), (IX), (X), (XI), (XII), (XIII), (XIV), (XV), (XVI), (XVII), and (XVIII), (XIX), and (XX); preferably (III), (III’), (IV), (VII), (VIII), (IX), (X), (XI), (XII), (XIII), (XIV), (XVII), and (XVIII); as disclosed herein, or a pharmaceutical composition as disclosed herein, or a nanoparticle as disclosed herein, for the manufacture of a medicament.
  • the invention further relates to the method for the treatment of a disease in a subject in need thereof, comprising administering a therapeutic effective amount of a conjugate as disclosed herein or a compound of general formula (III), (III’), (IV), (VII), (VIII), (IX), (X), (XI), (XII), (XIII), (XIV), (XV), (XVI), (XVII), and (XVIII), (XIX), and (XX); preferably (III), (III’), (IV), (VII), (VIII), (IX), (X), (XI),
  • the present invention relates to a conjugate as disclosed herein or a compound of general formula (III), (III’), (IV), (VII), (VIII), and (IX), as disclosed herein, or a pharmaceutical composition as disclosed herein, or a nanoparticle as disclosed herein, for use as a drug.
  • the present invention relates to the use of a conjugate as disclosed herein or a compound of general formula (III), (III’), (IV), (VII), (VIII), and (IX), as disclosed herein, or a pharmaceutical composition as disclosed herein, or a nanoparticle as disclosed herein, for the manufacture of a medicament.
  • the invention further relates to the method for the treatment of a disease in a subject in need thereof, comprising administering a therapeutic effective amount of a conjugate as disclosed herein or a compound of general formula (III), (III’), (IV), (VII), (VIII), and (IX), as disclosed herein, or a pharmaceutical composition as disclosed herein, or a nanoparticle as disclosed herein, to said subject.
  • the present invention relates to the use of a conjugate as disclosed herein or a compound of general formula (III) or (IV) as disclosed herein, or a pharmaceutical composition as disclosed herein, or a nanoparticle as disclosed herein, for the manufacture of a medicament.
  • the invention further relates to the method for the treatment of a disease in a subject in need thereof, comprising administering a therapeutic effective amount of a conjugate as disclosed herein or a compound of general formula (III) or (IV) as disclosed herein, or a pharmaceutical composition as disclosed herein, or a nanoparticle as disclosed herein, to said subject.
  • the present invention also relates to a conjugate as disclosed herein or a compound of general formula (III), (III’), (IV), (VII), (VIT), (VIII), (VIIT), (IX) (IX’), (X), (X’), (XI), (XT), (XII), (XII’), (XIII), (XIII’), (XIV), (XIV’), (XV), (XV’), (XVI), (XVI’), (XVII), (XVII’), (XVIII), (XVIII’), (XIX), (XIX’), (XX), and (XX’); preferably (III), (III’), (IV), (VII), (VIT), (VIII), (VIII’), (IX) (IX’), (X), (X’), (XI), (XT), (XII), (XIT), (XIII), (XIII’), (XIV), (XIV’), (XVII), (XVII’), (
  • the present invention relates to the use of a conjugate as disclosed herein or a compound of general formula (III), (III’), (IV), (VII), (VIT), (VIII), (VIII’), (IX) (IX’), (X), (X’), (XI), (XT), (XII), (XIT), (XIII), (XIII’), (XIV), (XIV’), (XV), (XV’), (XVI), (XVI’), (XVII), (XVII’), (XVIII), (XVIII’), (XIX), (XIX’), (XX), and (XX’); preferably (III), (III’), (IV), (VII), (VIT), (VIII), (VIII’), (IX) (IX’), (X), (X), (XIII), (XIV), (XIV’), (XVII), (XVII’), (XVII’), (XVII’), (XX), and (XX’);
  • the invention further relates to the method for the treatment of a subject suffering of cancer, comprising administering a therapeutic effective amount of a conjugate as disclosed herein or a compound of general formula (III), (III’), (IV), (VII), (VII’), (VIII), (VIII’), (IX) (IX’), (X), (X’), (XI), (XI’), (XII’), (XIII), (XIII’), (XIV), (XIV’), (XV), (XV’), (XVI), (XVI’), (XVII), (XVII’), (XVIII), (XVIII’), (XIX), (XIX’), (XX), and (XX’); preferably (III), (III’), (IV), (VII), (VII’), (VIII), (VIII’), (IX) (IX’), (XIX’), (XX), and (XX’); preferably (III), (III’), (IV), (VII), (
  • the present invention also relates to a conjugate as disclosed herein or a compound of general formula (III), (III’), (IV), (VII), (VIII), (IX), (X), (XI), (XII), (XIII), (XIV), (XV), (XVI), (XVII), and (XVIII), (XIX), and (XX); preferably (III), (III’), (IV), (VII), (VIII), (IX), (X), (XI), (XII), (XIII), (XIV), (XVII), and (XVIII); as disclosed herein, or a pharmaceutical composition as disclosed herein, or a nanoparticle as disclosed herein, for use in a method for the treatment of cancer.
  • the present invention relates to the use of a conjugate as disclosed herein or a compound of general formula (III), (III’), (IV), (VII), (VIII), (IX), (X), (XI), (XII), (XIII), (XIV), (XV), (XVI), (XVII), and (XVIII), (XIX), and (XX); preferably (III), (III’), (IV), (VII), (VIII), (IX), (X), (XI), (XII), (XIII), (XIV), (XVII), and (XVIII); as disclosed herein, or a pharmaceutical composition as disclosed herein, or a nanoparticle as disclosed herein, for the manufacture of a medicament useful as anti-tumoral agent or useful in a method for the treatment of cancer.
  • the invention further relates to the method for the treatment of a subject suffering of cancer, comprising administering a therapeutic effective amount of a conjugate as disclosed herein or a compound of general formula (III), (III’), (IV), (VII), (VIII), (IX), (X), (XI), (XII), (XIII), (XIV), (XV), (XVI), (XVII), and (XVIII), (XIX), and (XX); preferably (III), (III’), (IV), (VII), (VIII), (IX), (X), (XI), (XII), (XIII), (XIV), (XVII), and (XVIII); as disclosed herein, or a pharmaceutical composition as disclosed herein, or a nanoparticle as disclosed herein, to said subject.
  • the present invention also relates to a conjugate as disclosed herein or a compound of general formula (III), (III’), (IV), (VII), (VIII), and (IX), as disclosed herein, or a pharmaceutical composition as disclosed herein, or a nanoparticle as disclosed herein, for use in a method for the treatment of cancer.
  • the present invention relates to the use of a conjugate as disclosed herein or a compound of general formula (III), (III’), (IV), (VII), (VIII), and (IX), as disclosed herein, or a pharmaceutical composition as disclosed herein, or a nanoparticle as disclosed herein, for the manufacture of a medicament useful as anti-tumoral agent or useful in a method for the treatment of cancer.
  • the invention further relates to the method for the treatment of a subject suffering of cancer, comprising administering a therapeutic effective amount of a conjugate as disclosed herein or a compound of general formula (III), (III’), (IV), (VII), (VIII), and (IX), as disclosed herein, or a pharmaceutical composition as disclosed herein, or a nanoparticle as disclosed herein, to said subject.
  • the present invention also relates to a conjugate as disclosed herein or a compound of general formula (III) or (IV) as disclosed herein, or a pharmaceutical composition as disclosed herein, or a nanoparticle as disclosed herein, for use in a method for the treatment of cancer.
  • the present invention relates to the use of a conjugate as disclosed herein or a compound of general formula (III) or (IV) as disclosed herein, or a pharmaceutical composition as disclosed herein, or a nanoparticle as disclosed herein, for the manufacture of a medicament useful as anti-tumoral agent or useful in a method for the treatment of cancer.
  • the invention further relates to the method for the treatment of a subject suffering of cancer, comprising administering a therapeutic effective amount of a conjugate as disclosed herein or a compound of general formula (III) or (IV) as disclosed herein, or a pharmaceutical composition as disclosed herein, or a nanoparticle as disclosed herein, to said subject.
  • CSCs Cancer Stem Cells
  • the conjugate as disclosed herein or the compound of general formula (III), (III’), (IV), (VII), (VIII), and (IX) as disclosed herein, or the pharmaceutical composition as disclosed herein, or the nanoparticle as disclosed herein, are for use in a targeted treatment of Cancer Stem Cells (CSCs).
  • CSCs Cancer Stem Cells
  • the conjugate as disclosed herein or the compound of general formula (III) or (IV) as disclosed herein, or the pharmaceutical composition as disclosed herein, or the nanoparticle as disclosed herein are for use in a targeted treatment of Cancer Stem Cells (CSCs).
  • CSCs Cancer Stem Cells
  • cancer refers to any cancer that may affect any one of the following tissues or organs: breast; liver; kidney; heart, mediastinum, pleura; floor of mouth; lip; salivary glands; tongue; gums; oral cavity; palate; tonsil; larynx; trachea; bronchus, lung; pharynx, hypopharynx, oropharynx, nasopharynx; esophagus; digestive organs such as stomach, intrahepatic bile ducts, biliary tract, pancreas, small intestine, colon; rectum; urinary organs such as bladder, gallbladder, ureter; rectosigmoid junction; anus, anal canal; skin; bone; joints, articular cartilage of limbs; eye and adnexa; brain; peripheral nerves, autonomic nervous system; spinal cord, cranial nerves, meninges; and various parts of the central nervous system; connective, sub
  • cancer comprises leukemias, seminomas, melanomas, teratomas, lymphomas, non-Hodgkin lymphoma, neuroblastomas, gliomas, adenocarcinoma, mesothelioma (including pleural mesothelioma, peritoneal mesothelioma, pericardial mesothelioma and end stage mesothelioma), rectal cancer, endometrial cancer, thyroid cancer (including papillary thyroid carcinoma, follicular thyroid carcinoma, medullary thyroid carcinoma, undifferentiated thyroid cancer, multiple endocrine neoplasia type 2A, multiple endocrine neoplasia type 2B, familial medullary thyroid cancer, pheochromocytoma and paraganglioma), skin cancer (including malignant melanoma, basal cell carcinoma, squamous cell carcinoma, Kaposi’s s
  • the cancer can be selected in the group consisting of rectal cancer, colorectal cancer, stomach cancer, head and neck cancer, thyroid cancer, cervical cancer, uterine cancer, breast cancer, in particular triple negative breast cancer, ovarian cancer, brain cancer, in particular glioblastoma and neuroblastoma, lung cancer, in particular small-cell lung cancer and non-small-cell lung cancer, skin cancer, bladder cancer, blood cancer, renal cancer, liver cancer, prostate cancer, multiple myeloma, pancreatic cancer and endometrial cancer.
  • the cancer is a pancreatic cancer.
  • the compounds of the present invention are of particular interest for targeting persister cancer cells, cancer-stem cells (CSCs), cancer stem-like cells, drug-tolerant cancer cells, and therapy-resistent cancer cells, and for targeting epithelial-mesenchymal transition, targeting epithelial- mesenchymal plasticity.
  • CSCs cancer-stem cells
  • the compounds of the present invention can be used as inhibitor of cell plasticity in cancer, for instance by blocking epithelial-mesenchymal transition. They can be used to desensitize cancer cells to cytotoxic agents, especially those of the standard of care.
  • the present invention relates to a compound or pharmaceutical composition for use in the treatment of a subject having a cancer resistant or susceptible to become resistant to a cytotoxic agent.
  • said compound or pharmaceutical composition can be used in combination with said cytotoxic agent. It further relates to a method for reversing or decreasing or delaying a resistance of cancer cells to a cytotoxic agent in a subject having a cancer, comprising administering a therapeutic amount of a compound or a composition of the present invention to said subject, thereby reversing or decreasing or delaying the resistance to said cytotoxic agent, especially a chemotherapeutic agent.
  • the compound or pharmaceutical composition is used in combination with radiotherapy and/or another drug, preferably an antitumoral drug, more preferable a drug selecting from the group consisting of chemotherapy, targeted therapy, hormonotherapy and immunotherapy such as immune checkpoint therapy.
  • another drug preferably an antitumoral drug, more preferable a drug selecting from the group consisting of chemotherapy, targeted therapy, hormonotherapy and immunotherapy such as immune checkpoint therapy.
  • targeted therapy refers to targeted therapy agents, drugs designed to interfere with specific molecules necessary for tumor growth and progression.
  • targeted therapy agents such as therapeutic monoclonal antibodies target specific antigens found on the cell surface, such as transmembrane receptors or extracellular growth factors.
  • Small molecules can penetrate the cell membrane to interact with targets inside a cell. Small molecules are usually designed to interfere with the enzymatic activity of the target protein such as for example proteasome inhibitor, tyrosine kinase or cyclin-dependent kinase inhibitor, histone deacetylase inhibitor.
  • Targeted therapy may also use cytokines.
  • Examples of such targeted therapy include with no limitations: Ado-trastuzumab emtansine (HER2), Afatinib (EGFR (HER1/ERBB1), HER2), Aldesleukin (Proleukin), alectinib (ALK), Alemtuzumab (CD52), axitinib (kit, PDGFRbeta, VEGFR1/2/3), Belimumab (BAFF), Belinostat (HDAC), Bevacizumab (VEGF ligand), Blinatumomab (CD19/CD3), bortezomib (proteasome), Brentuximab vedotin (CD30), bosutinib (ABL), brigatinib (ALK), cabozantinib (FLT3, KIT, MET, RET, VEGFR2), Canakinumab (IL-1 beta), carfilzomib (proteasome), ceritinib (ALK
  • antineoplastic agents refers to a cancer therapeutic treatment using chemical or biochemical substances, in particular using one or several antineoplastic agents or chemotherapeutic agents.
  • Chemotherapeutic agents include, but are not limited to alkylating agents such as thiotepa and cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); a camptothecin (including the synthetic analogue topotecan); bryostatin; callystatin; CC-
  • calicheamicin especially calicheamicin gammall and calicheamicin omegall ; dynemicin, including dynemicin A; bisphosphonates, such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromophores, aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxy doxorubicin
  • hormone therapy refers to a cancer treatment having for purpose to block, add or remove hormones.
  • the female hormones estrogen and progesterone can promote the growth of some breast cancer cells.
  • the term “immunotherapy” refers to a cancer therapeutic treatment using the immune system to reject cancer.
  • the therapeutic treatment stimulates the patient's immune system to attack the malignant tumor cells.
  • Immune checkpoint therapy such as checkpoint inhibitors include, but are not limited to programmed death- 1 (PD-1) inhibitors, programmed death ligand- 1 (PD-L1) inhibitors, programmed death ligand-2 (PD-L2) inhibitors, lymphocyte -activation gene 3 (LAG3) inhibitors, T-cell immunoglobulin and mucin-domain containing protein 3 (TIM-3) inhibitors, T cell immunoreceptor with Ig and ITIM domains (TIGIT) inhibitors, B- and T-lymphocyte attenuator (BTLA) inhibitors, V-domain Ig suppressor of T-cell activation (VISTA) inhibitors, cytotoxic T-lymphocyte-associated protein 4 (CTLA4) inhibitors, Indoleamine 2,3-dioxygenase (IDO) inhibitors, killer immunoglobulin-like receptors (KIR) inhibitors, KIR2L3 inhibitors, KIR3DL2 inhibitors and carcinoembryonic antigen- related cell adhesion molecule 1 (CEACAM-
  • checkpoint inhibitors include antibodies anti-PDl, anti-PD-Ll, anti-CTLA-4, anti-TIM-3, anti-LAG3.
  • Immune checkpoint therapy also includes co-stimulatory antibodies delivering positive signals through immune-regulatory receptors including but not limited to ICOS, CD 137, CD27, OX-40 and GITR.
  • Example of anti-PDl antibodies include, but are not limited to, nivolumab, cemiplimab (REGN2810 or REGN-2810), tislelizumab (BGB-A317), tislelizumab, spartalizumab (PDR001 or PDR-001), ABBV- 181, JNJ-63723283, BI 754091, MAG012, TSR-042, AGEN2034, pidilizumab, nivolumab (ONO-4538, BMS-936558, MDX1106, GTPL7335 or Opdivo), pembrolizumab (MK-3475, MK03475, lambrolizumab, SCH-900475 or Keytruda) and antibodies described in International patent applications W02004004771, W02004056875, W02006121168, WO2008156712, W02009014708,
  • Example ofanti-PD-Ll antibodies include, but are not limited to, LY3300054, atezolizumab, durvalumab and avelumab.
  • Example of anti-CTLA-4 antibodies include, but are not limited to, ipilimumab (see, e.g., US patents US6,984,720 and US8,017,114), tremelimumab (see, e.g., US patents US7, 109,003 and US8, 143,379), single chain anti- CTLA4 antibodies (see, e.g., International patent applications WO1997020574 and WO2007123737) and antibodies described in US patent US8,491,895.
  • Example of anti-VISTA antibodies are described in US patent application US20130177557.
  • Example of inhibitors of the LAG3 receptor are described in US patent US5,773,578.
  • Example of KIR inhibitor is IPH4102 targeting KIR3DL2.
  • radiotherapy refers to radiation therapies including, but not limited to external beam radiotherapy (such as superficial X-rays therapy, orthovoltage X-rays therapy, megavoltage X- rays therapy, radiosurgery, stereotactic radiation therapy, Fractionated stereotactic radiation therapy, cobalt therapy, electron therapy, fast neutron therapy, neutron-capture therapy, proton therapy, intensity modulated radiation therapy (IMRT), 3-dimensional conformal radiation therapy (3D-CRT) and the like); brachytherapy; unsealed source radiotherapy; tomotherapy; and the like.
  • IMRT intensity modulated radiation therapy
  • 3D-CRT 3-dimensional conformal radiation therapy
  • Radiotherapy may be proton radiotherapy or proton minibeam radiation therapy.
  • Proton radiotherapy is an ultra-precise form of radiotherapy that uses proton beams (Prezado Y, Jouvion G, Guardiola C, Gonzalez W, Juchaux M, Bergs J, Nauraye C, Labiod D, De Marzi L, Pouzoulet F, Patriarca A, Dendale R. Tumor Control in RG2 Glioma-Bearing Rats: A Comparison Between Proton Minibeam Therapy and Standard Proton Therapy.
  • Radiotherapy may also be FLASH radiotherapy (FLASH-RT) or FLASH proton irradiation.
  • FLASH radiotherapy involves the ultra-fast delivery of radiation treatment at dose rates several orders of magnitude greater than those currently in routine clinical practice (ultra-high dose rate) (Favaudon V, Fouillade C, Vozenin MC. The radiotherapy FLASH to save healthy tissues. Med Sci (Paris) 2015; 31: 121-123. DOI:
  • compositions contemplated herein may include a pharmaceutically acceptable carrier in addition to the active ingredient(s).
  • pharmaceutically acceptable carrier is meant to encompass any carrier (e.g., support, substance, solvent, etc.) which does not interfere with effectiveness of the biological activity of the active ingredient(s) and that is not toxic to the host to which it is administered.
  • the active compounds(s) may be formulated in a unit dosage form for injection in vehicles such as saline, dextrose solution, serum albumin and Ringer's solution.
  • AI2O3 Aluminum oxide Fe(OTf)2: Iron(II) trifluoromethanesulfonate aq.: Aqueous HATU : 1 -[Bis(dimethylamino)methylene] - 1H- Ar: Argon 40 l,2,3-triazolo[4,5-6]pyridinium 3-oxid AU: Arbitrary units hexafluorophosphate BINAP: 2,2 ’ -bis(diphenylphosphino)- 1 , 1 ’ - HBF4: Tetrafluoroboric acid binaphthalene HBTU: A, A, JV' JV'-Tetramethyl-O-( 1H-
  • BOC2O Di-tert-butyl dicarbonate benzotriazol- 1 -y
  • I juroni um CAM Cerium ammonium molybdate 45 hexafluorophosphate
  • CS2CO3 Cesium carbonate
  • HC1 Hydrogen chloride or hydrochloric acid
  • Cui Copper iodide
  • HPLC High performance liquid
  • DCM Dichloromethane chromatography
  • DFO Deferoxamine
  • HRMS High resolution mass spectrometry
  • DIPEA A'-A'-Diisopropylcthylaminc 50
  • K2CO3 Potassium carbonate
  • DMAP Dimethylaminopyridine
  • LRMS Low resolution mass spectrometry
  • DMF Dimethylformamide
  • MeCN Acetonitrile
  • DMSO Dimethylsulfoxide
  • MeOH Methanol
  • TMS Trimethylsilyl dichloride
  • TMSC1 Trimethylsilyl chloride
  • Reaction products were purified by flash column chromatography on silica gel 60 (230-400 mesh, Merck and co.) or aluminium oxide (activated neutral, Sigma- Aldrich), by Combiflash® Rf, or by preparative HPLC Quaternary Gradient 2545 equipped with a Photodiode Array detector (Waters) fitted with a reverse phase column (XBridge BEH C18 OBD Prep column 5 pm 30x 150 mm). NMR spectroscopy was performed on Bruker 300, 400 or 500 MHz apparatus. Spectra were run in CD3OD or CDCE, at 298 K. ’H chemical shifts 5 are expressed in ppm using the residual non-deuterated solvent as internal standard and the coupling constants J are specified in Hz.
  • HBF 4 (48% wt/wt), MeCN, 82 °C, 2 h, 13%.
  • succinic anhydride DMAP, anhydrous pyridine, 90 °C, 40 h, 36%.
  • dimethylamine HC1 salt NaOH, H 2 O, sealed tube, 155 °C, 16 h, 91%.
  • SOCE SOCE, DCM, rt, 16 h, 93%.
  • cholesteryl hemisuccinate is adapted from a previously described procedure 68 .
  • Cholesterol (50) (387 mg, 1 mmol) and succinic anhydride (51) (1 g, 10 mmol) were dissolved in 10 mL of anhydrous pyridine.
  • DMAP 61 mg, 0.5 mmol
  • the reaction mixture was stirred at 90 °C for 24 h under argon.
  • the dark brown coloured reaction mixture was cooled to rt and a solution of HC1 (I N) was added dropwise to precipitate the product.
  • the resulting white precipitate was filtered and thoroughly washed with distilled water.
  • Monocholestanylsuccinate ChtaMS The synthesis of monocholestanyl succinate is adapted from a previously described procedure [39], Cholestanol (389 mg, 1 mmol) and succinic anhydride (1 g, 10 mmol) were dissolved in 10 mL of dry pyridine. Then, DMAP (61 mg, 0.5 mmol) was added, and the reactional mixture was stirred at 90 °C for 24 h under an inert atmosphere. After completion of the reaction, the dark brown colored reaction mixture was cooled to room temperature and a solution of 1 M HC1 was added dropwise to precipitate the product. The resulting white precipitate was thoroughly washed with distilled water.
  • Fentomycin 7 (XI) was synthesized via procedure B (4 mg, 18%) using 10 (12 mg, 0.025 mmol), HBTU (47 mg, 0.025 mmol) and DIPEA (0.13 mL, 0.75 mmol) in anhydrous THF (0.4 mL) and 8 (10 mg, 0.025 mmol) in anhydrous DMF (0.4 mL).
  • Fentomycin 8 (XII) was synthesized via procedure B (4 mg,
  • Fentomycin 9 (XIII) Fentomycin 9 (XIII) was synthesized via procedure D (3 mg,
  • Fentomycin 10 (XIV) was synthesized via procedure D (3 mg, 16%) using 8 (10 mg, 0.025 mmol), 17 (9 mg, 0.017 mmol) and Na 3 CO 3 (5 mg, 0.050 mmol) in anhydrous MeCN (0.1 mL).
  • Fentomycin 11 (XV) Fentomycin 11 (XV) was synthesized via procedure E (8 mg, 7%) using 20 (62 mg, 0.18 mmol), 9 (83 mg, 0.21 mmol) and NaH (5 mg, 0.21 mmol) in anhydrous DMF (0.8 mL).
  • 1H NMR (500 MHz, Methanol-d4) ⁇ : 8.68 (s, 1H, formate), 8.28 (d, J 5.8 Hz, 1H), 8.07 (d,
  • Fentomycin 13 (XVII) was synthesized via procedure D (3 mg, 16%) using 25 (20 mg, 0.044 mmol), 15 (15 mg, 0.029 mmol) and Na2CO3 (9 mg, 0.058 mmol) in anhydrous MeCN (0.18 mL).
  • Fentomycin 16 (XX) Fentomycin 16 (XX) was synthesized via procedure E (8 mg, 7%) using 28 (7 mg, 0.018 mmol), 11 (8 mg, 0.021 mmol) and NaH (0.5 mg, 0.021 mmol) in anhydrous DMF (0.1 mL).
  • PDAC085T, PDAC090T, PDAC053T, PDAC211T and PDAC030T cells were grown in serum-free ductal medium: DMEM/F12 supplemented with 0.61g/500mL nicotinamide (Sigma- Aldrich, 3376), 2.50g/ 500mL glucose (Sigma-Aldrich, G6152), 1:200 ITS+ (Coming, 354352), 1:20 Nu-serum IV (Coming, 355104), 100 ng/ml cholera toxin, 1 pM dexamethasone (Sigma-Aldrich, D4902), 50 nM 3,3’,5-triiodo-L-thyronine (Sigma-Aldrich, T6397) and 1 x PenStrep.
  • DMEM/F12 supplemented with 0.61g/500mL nicotinamide (Sigma- Aldrich, 3376), 2.50g/ 500mL glucose (Sigma
  • Human FC 1242, mouse 4a and human hMIA-2D cells were a generous gift from D. Tuveson and were grown in DMEM (Thermo Fisher Scientific, 10566016) supplemented with 10% FBS (v/v) (Thermo Fisher Scientific, 10270106) and 1 x PenStrep (Invitrogen #15070-063).
  • HT-1080 cells were obtained from ATCC and grown in DMEM (Thermo Fisher Scientific, 10566016) supplemented with 10% FBS (v/v) (Thermo Fisher Scientific, 10270106) and 1 x PenStrep (Invitrogen #15070-063).
  • Fisher Scientific AF-100-15-1MG were added to new medium when cells were split or plated.
  • mice All mouse experiments complied with all relevant ethical regulations and were performed according to protocols approved by the Institutional Animal Care and Use Committee at Harvard T.H. Chan School of Public Health (protocol IS00003460). No formal randomization techniques were used; however, animals were allocated randomly to treatment groups and specimens were processed in an arbitrary order. For all experiments, the maximum permitted tumor diameter was 2.0 cm and this limit was not exceeded in any experiment. For all experiments, mice were kept on normal chow and fed ad-libitum. For human samples, all patients provided written informed consent for use of tumor samples and the study was approved by institutional regulatory board (DATA 190160).
  • Cells were then cultured in RPMI-1640 Glutamax/10%FBS,l% PenSTREP for 1 h and analyzed by flow cytometry, or treated with 1 pM Fentomycin and relevant ferroptosis inhibitors for 24 h and subsequently analyzed by flow cytometry.
  • Cells were plated on cover slips 24 h prior to the experiment. Then, cells were treated with 1 pM Fentomycin, 1 pM Marmycin A or 1 pM ligand- ‘click’ and LysoTracker Deep Red (Thermo Fisher Scientific, L12492, 1: 10000) or CellRox (Thermo Fisher Scientific, C10422, 1: 10000) according to the manufacturer’s protocol. Subsequently, cells were washed three times with l x PBS and fixed with 2% paraformaldehyde (w/v) in l x PBS for 12 min, and then washed three times with l x PBS.
  • w/v paraformaldehyde
  • Cell viability curves were established by plating 4,000 cells/well in 96-well plates using CellTiter-Blue viability assay according to the manufacturer’s protocol. Cells were treated with serial dilutions of irinotecan (Sigma-Aldrich, 11406), gemcitabine (Sigma-Aldrich, G6423), 5 -fluorouracil (Sigma- Aldrich, F6627-1G) or oxaliplatin (Biotechne, 2623) for 72 h.
  • irinotecan Sigma-Aldrich, 11406
  • gemcitabine Sigma-Aldrich, G6423
  • 5 -fluorouracil Sigma- Aldrich, F6627-1G
  • oxaliplatin Biotechne, 2623
  • CellTiter-Blue reagent Promega, G8081 was added after 72 h treatment and cells were incubated for 2 h before recording fluorescence intensities (E ex 560 nm; E em 590 nm) using a Perkin Elmer Wallac 1420 Victor2 Microplate Reader.
  • Cell viability was also assessed using a CellTiterGlo 2.0 (Promega, G9241) or CellTiter blue (Promega, G8081) kit according to the manufacturer’s protocol in a 96-well plate.
  • 4000 cells HT-1080, PDAC053T or 4T1 were seeded per well in clear-bottom and darkened 96-well plates (Greiner, 655090, lot E23063EG) 24 h prior to the experiment.
  • Cells were then pre-treated for 2 h with Lip-1 (10 pM), cLip-1 (10 pM), ferrostatin- 1 (Fer-1, SML0583, 10 pM), deferoxamine (DFO, Sigma-Aldrich, D9533, 100 pM), deferasirox (DFX, Cayman chemical, 16753, 10 pM), deferiprone (Def, Sigma-Aldrich, Y0001976, 100 pM), a-tocopherol (Toe, Sigma-Aldrich, PHR1031, 100 pM), Vitamin K3 (Sigma- Aldrich, M5625-25G, 10 pM), Z-VAD-FMK (Enzo Life Sciences, ALX-260-020-M005, 50 pM) or Necrostatin-1 (Nec-l, Sigma-Aldrich, N9037, 20 pM).
  • Lip-1 (10 pM
  • Fento-1 (10 pM, 6 h) was added. Samples were processed as detailed in the manufacturer’s protocols and data were recorded on a SpectraMax ID3 plate reader (Molecular Devices). For standard-of-care cell viability measurements, cells were plated at 2000 cells per well 24 h prior to the experiment. Cells were incubated with serial dilutions of Fento-1, irinotecan (Sigma-Aldrich, 11406), 5-FU (Alfa Aesar, A13456-06) or oxaliplatin (Bio-Techne, 2623) for 72 h. CellTiter blue assay: Cell viability was assessed using a CellTiter blue assay and data recorded on a SpectraMax ID3 plate reader (Molecular Devices).
  • Fluorescent probes CellRox (Thermo Fisher Scientific, C10491) and Bodipy-Cl l (Thermo Fisher Scientific, D3861) were used according to the manufacturer’s protocol.
  • Cells were harvested with Trypsin (if adherent) or directly (tumor samples) then washed twice with ice-cold 1 x PBS and suspended in incubation buffer prior to being analyzed by flow cytometry.
  • Antibody staining of cells was achieved by incubation with human TruStain FcX (Biolegend, 422302) for 15 mins at room temperature with subsequent incubation of antibodies for 20 min at 4°C and then washed with 1 x PBS.
  • CD44-AF647 Novus Biologicals, NB500-481AF647
  • CD45-BV510 BioLegend, 368526
  • CD31-BV605 BioLegend, 303122
  • FAP-750 R&D, FAB3715S
  • CD71-PE R&D, FAB2474P
  • SytoxBlue Thermo Fisher Scientific, SI 13478 was added according to the manufacturer’s instructions to identify dead cells. Live tumor cells were defined as SytoxBlue neg /CD45 neg /CD3 l neg /FAP neg cells.
  • CD45-BV510 BioLegend, 103138
  • CD31-BV605 BioLegend, 102427
  • IA/IE-APC-Cy7 BioLegend, 107628
  • CD44-AF647 BioLegend, 103018
  • Live tumor cells were defined as Sytoxblue neg CD45 neg /CD31 neg /I-A/I-E pos cells.
  • Data were recorded on an Attune (Thermo Fisher Scientific) and processed using Cell Quest (BD Biosciences) and FlowJo (FLOWJO, LLC).
  • PDAC053T cells were seeded in 6-well plates at the density of 1.5x 10 5 cells/well in 2 mL culture medium.
  • LDH assay (Sigma Aldrich, 11644793001) and CellTiterGlo (Promega, G9241) were performed according to manufacturer’s instructions in 96-well plates.
  • Liposomal structures were prepared using the traditional lipid film hydration method: 100 mL of a stock solution (Img/mL CH3CI) of 18: 1 (A9-Cis) PC (DOPC, Avanti Polar Lipids) were dissolved in 400 mL of CH3CI and transferred into a round-bottom flask. Organic solvent was removed under vacuum in a rotary evaporator for 15 min at 200 revolutions per minute (rpm) at 37 °C in a water bath. Afterwards, the lipid film was dried with a vacuum pump overnight. Then was hydrated with 1 mL of 0.1 mM Acetate buffer (pH 4.5) and vortexing every 5 min for 20 min. Liposomes were extruded by passing the suspension through 2 polycarbonate membranes (pore size 0.2 mm) 20 times and keep at -4 °C.
  • a stock solution Img/mL CH3CI
  • 1 (A9-Cis) PC DOPC, Avanti Polar Lipids
  • ICP-MS Inductively coupled plasma mass spectrometry
  • 100,000 cells per condition were plated in 6-well plates 24 h prior to the experiment. Cells were treated with 1 pM fentomycin for 6h or 24 h. Cells were subsequently washed with lx PBS and then with 150 mM ammonium bicarbonate. Cells were then scraped and resuspended in 150 mM ammonium bicarbonate and centrifuged at 300 g for 5 min. The supernatant was removed and cells were resuspended in 1 mL of 150 mM ammonium bicarbonate. The solutions were centrifuged at 12,000 RPM for 10 min and the supernatant was removed. 200 ⁇ L of 150 mM sodium bicarbonate was added to the pellet and samples were flash frozen in liquid nitrogen.
  • lipidomics analysis was performed on the same day for all the replicates.
  • the 200 ⁇ L cell lysates were spiked with 1.4 ⁇ L of internal standard lipid mixture containing 300 pmol of phosphatidylcholine 17:0-17:0, 50 pmol of phosphatidylethanolamine 17:0- 17:0, 30 pmol of phosphatidylinositol 16:0-16:0, 50 pmol of phosphatidylserine 17:0-17:0, 30 pmol of phosphatidylglycerol 17:0-17:0 and 30 pmol of phosphatidic acid 17:0-17:0 and subjected to lipid extraction at 4 °C.
  • Automatic gain control (AGC) was set to 1E6 and maximum ion injection time (IT) was set to 50ms.
  • BDPOs were obtained from endoscopic ultrasound-guided fine-needle aspirations (EUS-FNA) from patients with PDAC included under the PaCaOmics clinical trial (ClinicalTrials.gov: NCT01692873) after approval by the Paoli-Calmettes hospital ethics committee and following patient informed consent. Cultures were established as previously described [38], Briefly, PDAC biopsies were slightly digested with the Tumor Dissociation Kit (Miltenyi Biotec) at 37°C for 5 minutes. The pancreatic tissue slurry was transferred into a tissue strainer 100 pm and was placed into 12-well plates coated with 150 ⁇ L GFRmatrigel (Coming, Boulogne-Billancourt, France).
  • the samples cultured with Pancreatic Organoid Feeding Media consisted of Advanced DMEM/F12 supplemented with 10 mM HEPES (Thermo Fisher Scientifics, Courtaboeuf, France); 1 x Glutamax (Thermo-Fisher Scientifics); penicillin/streptomycin (Thermo-Fisher Scientifics); 100 ng/mL Animal -Free Recombinant Human FGF10 (Peprotech, Peprotech, Neuilly-Sur-Seine, France); 50 ng/mL Animal-Free Recombinant Human EGF (Peprotech); 100 ng/mL Recombinant Human Noggin (Biotechne, Bio-Techne, Rennes, France); Wnt3a-conditioned medium (30% v/v); RSPO1 -conditioned medium (10% v/v); 10 nM human Gastrin 1 (Sigma- Aldrich Lyon, France); 10 mM nicotinamide (Sigma- Ald
  • BDPOs were disaggregated with accutase (Thermo Fisher Scientific) and re-plated as needed. Chemograms on BDPO
  • BDPOs were disaggregated with accutase (Thermo Fisher Scientific), and 1,000 cells/well were plated in two 96-well round bottom ultra-low plates (Coming) with the medium described above. 24 hours later, one plate was used directly for RNA preparation (Time 0 transcriptome) and on the other the medium was supplemented with increasing concentrations of each drug, 72 hours later cell viability was measured with CellTiter-Glo 3D (Promega) reagent quantified using the plate reader Tristar LB941 (Berthold Technologies). Values were normalized and expressed as the percentage of the control (vehicle), which represents 100% of normalized fluorescence. Increasing concentrations of drugs were used. Each experiment was repeated at least twice.
  • Intranodal murine metastasis models for determining Fentomycin efficacy in vivo Murine breast cancer cells (4T1) were transplanted into 6 to 8-week-old female Balb/c mice (syngeneic with the 4T1 model). To perform injections into lymph nodes, the lymphatics were first traced by injecting 2% Evans Blue Dye (product E2129, Sigma- Aldrich) into the foot pedal 5 minutes before performing intranodal injections. After injecting Evans Blue Dye, the mice were anesthetized using isoflurane and a small (5-10 mm) incision was made in the region of the popliteal lymph node.
  • Evans Blue Dye product E2129, Sigma- Aldrich
  • the lymph node was located based on Evans Blue staining, immobilized with forceps, and 10,000 cells suspended in Phosphate-buffered saline (PBS) were injected in a volume of 10 ⁇ L into the popliteal lymph node using a 27 gauge Hamilton syringe. Injection into the lymph node was confirmed by visible swelling of the lymph node. The incision was closed using surgical glue (product 1469SB, 3M VetBond Tissue Adhesive) and the mice were closely monitored for signs of pain or distress.
  • PBS Phosphate-buffered saline
  • Fentomycin is a chimera of Marmycin A and an iron-activating ligand, tethered by a linker (Fig la).
  • Fig la an iron-activating ligand
  • Fig la an alkyne substituted ligand
  • Fig la Fentomycin is naturally fluorescent, like its component Marmycin A, and readily accumulates in lysosomes (Fig lb), whereas the linker- ‘click’, labelled with a fluorophore via click chemistry, shows a pan-cellular staining. This shows that the Marmycin A subunit of Fentomycin guides its cellular localization. Fentomycin induces lysosomal reactive oxygen species (Fig 1c). Importantly, it shows low micromolar efficacy on cell viability with different human and mouse cancer cells tested in vitro, whereas Marmycin A and the ligand- ‘click’ are largely ineffective (Fig Id).
  • Fento-1 The activity of structural variants of Fento-1 were then explored (Fig 4a).
  • Fento-2 which contains a longer linker separating marmycin from the Chen- White ligand, exhibited a lower potency against PDAC and sarcoma cells compared to Fento-1. This supported the notion that Fento-1 exerts its activity by promoting proximity between a reactive iron catalyst and membrane lipids (Fig 4b, c).

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Abstract

The present invention relates to a conjugate for use in the treatment of cancer, the conjugate comprising a first moiety A selected from metal chelating agents, a second moiety B selected from lysosomotropic agents; and a linker L that binds moieties A and B together. The invention also relates to their use in the pharmaceutical field, in particular in a method for the treatment of cancer.

Description

Lipid degraders to trigger ferroptosis in cancer
Field of the invention
The present invention is in the field of medicine. The present invention relates to new conjugates, namely new chimeric small molecules, and their use as drugs. The chimeric small molecules of the invention are bifunctional compounds that act as lipid degraders. In particular, the invention relates to these new chimeric small molecules for use in a method for the treatment of cancer, preferably for use in a targeted treatment of Cancer Stem Cells (CSC)ZDrug Tolerant Persister (DTP) cancer cells/cancer cells in the mesenchymal state and/or for the treatment and prevention of cancer metastases.
Background of the invention
Cancer is a prevalent disease that spreads all over the world. The World Health Organization (WHO) reported around 9.6 million deaths in 2018 due to cancer.f l] Cancer is a result of an abnormal development of cells derived from normal cells, and therefore it is challenging to selectively and specifically target cancerous cells over healthy cells in the body. The etiology of cancer is complex, with various factors contributing to the appearance of cancerous cells, including genetic, epigenetic or metabolic alterations. Importantly, the initiating and driving factors of a particular cancer often differ.
Subpopulations of cancer cells that can denote Cancer Stem Cells (CSC) or Drug-Tolerant Persister (DTP) cells have been shown to maintain tumor growth through self-renewal processes and cause cancer metastases. Non-stem cancer cells can also exhibit a cell plasticity giving rise to cells with self-renewal properties, through mechanisms including epithelial-to-mesenchymal transition, noradrenergic-to-mesenchymal transition, melanocytic -to-mesenchymal, endothelial-to-mesenchymal and others. Henceforth these populations of cells with tumor-renewal capacity will be donated as CSCs for simplification. These cells represent, in general, a small fraction of the bulk of a solid tumors and have the capacity of tumor seeding, resulting in metastatic dissemination. [2,3] The paradigm of CSCs, also termed cancer stem-like cells and tumor-initiating cells (TICs), defines the existence of a long-lived population of cancer cells prone to self-renewal that fuel tumor growth. CSCs often represent a fraction of the cellular content of tumors, and they have been associated with cancer recurrence after chemotherapy or radiation therapy, tumor dormancy, and metastasis. CSCs had been identified in leukemia, but were then found to be present in many other cancers including brain, prostate, pancreatic and breast. [4- 12] The proportion of CSCs is variable and often denotes a fraction of the tumor but can constitute the bulk of the cancer, in particular in melanoma. [13]
It has recently been shown that CSCs have an enhanced addiction to iron in their mesenchymal state.[14,15,16] This dependency for iron was observed for cancer stem -like cells of various indications including breast cancer, ovarian cancer and glioma. [15, 17, 18]
Ferroptosis is a specific type of non-apoptotic cell death resulting from the unrestrained occurrence of oxidized, in particular peroxidized, phospholipids, resulting from iron-mediated production of lethal oxygen radicals. In other words, ferroptosis is a regulated oxidative cell death that can occur as a result of lipid peroxidation. This type of cell death was first coined in 2012 after observations describing a process different from apoptosis, necrosis or other known cell death pathways. [19] Two main axis were identified to be responsible for the regulation of ferroptosis. [20] The glutathione -dependent selenoenzyme, also known as glutathione peroxidase-4 (GPX4), which plays a major role in protecting cells from ferroptosis. Another enzyme has been identified to protect the cells from ferroptosis known as ferroptosis suppressor protein 1 (FSP 1). [21 ,22] Ferroptosis is mediated by the presence of reactive oxygen species (ROS) that cause the peroxidation of lipids. Ferroptosis can either be prevented (for instance by iron chelation or radical trapping) or promoted (for instance by iron activation or inhibition of ferroptosis suppressors) by means of small molecule intervention.
Thus, despite previous advances, there is a great need to efficiently target CSCs. Indeed, there is as off yet no small molecule in the clinic that can target specifically CSCs/persister cancer cells.
Summary of the invention
The inventors envisaged that inducing ferroptotic cell death is a potential strategy for targeting CSCs (Cancer stem cells) and/or persister cancer cells and/or drug-tolerant persister (DTP) cells. In particular, the inventors have found that targeting lysosomal iron may provide new therapeutic opportunities to selectively impact CSC maintenance [14, 23, 24],
The inventors have developed new chimeric small molecules comprising a lysosomotropic molecule and a ligand known to promote iron catalysed oxidation of phospholipids and other substrates [25,26], The lysosomotropic molecule is, in particular, a lipophilic moiety that drives the accumulation in lysosomes via endocytosis. According to the inventors, the lysosomotropic molecule drives the iron ligand inside of lysosomes, in particular via endocytosis, to promote a Fenton-type chemistry and lipid oxidation, such as peroxidation, in these cell organelles.
The inventors have shown that a conjugate according to the invention named Fentomycin targets lysosomes and shows good efficacy in vitro by inducing lysosomal reactive oxygen species (Figure 1). The inventors have also shown that Fentomycin induces ferroptosis and targets iron rich cancer cells in vivo.
A first object of the invention is a conjugate for use in the treatment of cancer, the conjugate being of the following general formula (I):
A-L-B (I) wherein:
A is a first moiety selected from metal chelating agents;
B is a second moiety selected from lysosomotropic agents; and
L is absent or is a linker that binds moieties A and B together, preferably L is a linker that binds moieties A and B together. In a particular embodiment, the conjugate of the invention is for use in a targeted treatment of Cancer Stem Cells (CSCs).
In a particular embodiment, the first moiety A selected from ligands of the White-Chen Type, preferably the first moiety A is White -Chen ligand.
In a specific embodiment, the first moiety A is of the following general formula (II): wherein:
• Ri is selected from the group consisting of NRARB, ORC, C(O)ORD, C(O)RE, S(O)RF, S(O)2RG, CN, halo-(C1-C6-alkyl), -C(O)-(C1-C6)alkyl-halo, halogen, NO2, and SO2, R . to RG being independently of each other H or a (C1-C6)alkyl, preferably Ri is N(CH3)2;
• R2 is a hydrogen atom or (C1-C6)alkyl, preferably hydrogen atom ;
• and each Z is, independently of each other, a hydrogen or deuterium atom.
In a specific embodiment, the first moiety A is of the general formula (Ila) (namely White-Chen ligand type): wherein
Ri is selected from the group consisting of NRARB, ORC, C(O)ORD, C(O)RE, S(O)RF, S(O)2RG, halo- (C1-C6-alkyl), -C(O)-(C1-C6)alkyl-halo, halogen, CN, NO2, and SO2, RA to RG being independently of each other H or a (C1-C6)alkyl, preferably Ri is N(CHs)2;
R2 is a hydrogen atom or (C1-C6)alkyl, preferably hydrogen atom;
X and Y are, independently of each other, a (C1-C6)alkyl; or X and Y can be linked together to form a heterocycloalkyl or heteroaryl, preferably a pyrrolidine; and each Z is, independently of each other, a hydrogen or deuterium atom; and wherein the wavy line represents the link to L.
In a specific embodiment, the first moiety A is of the general formula (lib) (namely White-Chen ligand type): wherein
Ri is selected from the group consisting of NRARB, ORC, C(O)ORD, C(O)RE, S(O)RF, S(0)2RG, CN, halo-(C1-C6-alkyl), -C(O)-(C1-C6)alkyl-halo, halogen, NO2, and SO2, RA to RG being independently of each other H or a (C1-C6)alkyl, preferably Ri is N(CH3)2;
R2 is a hydrogen atom or (C1-C6)alkyl, preferably hydrogen atom; and each Z is, independently of each other, a hydrogen or deuterium atom; and wherein the wavy line represents the link to L.
In a specific embodiment, the first moiety A is of the general formula (lie): wherein
Ri is N(CHS)2 ; and
Rs is an hydrogen atom or (C1-C6)alkyl, preferably hydrogen atom; and wherein the wavy line represents the link to L.
In another specific embodiment, the first moiety A is selected from ligands of the Nordlander-Costas Type, preferably the first moiety A is Nordlander-Costas ligand, more preferably the first moiety A is of the following formula (lie): wherein
Rs is a hydrogen atom or (C1-C6)alkyl preferably methyl;
X and Y are, independently of each other, a (C1-C6)alkyl; or X and Y can be linked together to form a heterocycloalkyl or heteroaryl, preferably pyrrolidine; and each Z is, independently of each other, a hydrogen or deuterium atom; and wherein the wavy line represents the link to L.
In another specific embodiment, the first moiety A is of the general formula (Ilf): wherein each Z is, independently of each other, a hydrogen or deuterium atom, and wherein the wavy line represents the link to L.
In another specific embodiment, the first moiety A is:
wherein the wavy line represents the link to L.
In another specific embodiment, the first moiety A is selected from the group consisting of:
In a particular embodiment, the second moiety B is selected from the group consisting of chloroquine, hydroxychloroquine, azithromycin, marmycin, steroids such as cholesterol and cholestanol, ezurpimtrostat hydrochloride, desmethylchloroquine, hydroxychloroquine phosphate, desmethyl- hydroxychloroquine, anthraquinones, methyl tetraphene dione, angucyclines, ammonium chloride (NH4CI), amantadine, methylamine, fluoxetine, imipramine, latrepirdine, tamoxifen, chlorpromazine, amitriptyline, verapamil, Triton WR 1339 (Tyloxapol), Suramin, metformin, Erythromycin, Amitriptyline, Imipramine, 4-aminoquinoline, amiodarone, amodiaquine, clindamycin, N-(3-[(2,4- dinitrophenyl)-amino]-propyl)-N-(3-aminopropyl-methylamine) dihydrochloride (DAMP), monensin, monodansylcadaverine, perhexilene, phenylalanine methyl ester, primaquine, quinacrine, thioridazine, tilorone, tributylamine, ketotifen fumarate, glycerol, sucrose, Trehalose, Resveratrol, PVP, and Gold sodium thiomalate.
In particular, the second moiety B is selected from the group consisting of chloroquine, hydroxychloroquine, azithromycin, marmycin, functionalized fatty acids, steroids, cholesterol, cholestanol, ezurpimtrostat hydrochloride, desmethylchloroquine, hydroxychloroquine phosphate, desmethyl-hydroxychloroquine, anthraquinones, methyl tetraphene dione, angucyclines.
In particular, the second moiety B is selected from the group consisting of chloroquine, hydroxychloroquine, azithromycin, marmycin, ezurpimtrostat hydrochloride, desmethylchloroquine, hydroxychloroquine phosphate, desmethyl-hydroxychloroquine, anthraquinones, methyl tetraphene dione, and angucyclines.
In a particular embodiment, the second moiety B is selected from the group consisting of chloroquine, hydroxychloroquine, azithromycin, marmycin, cholesterol, cholestanol, and ezurpimtrostat
In another particular embodiment, the second moiety B is selected from the group consisting of marmycin, steroids such as cholesterol and cholestanol, anthraquinones, and angucyclines.
In particular, the second moiety B is selected from the group consisting of marmycin, cholesterol, cholestanol, anthraquinones, and angucyclines. More particularly, the second moiety B is selected from the group consisting of marmycin A, cholesterol, and cholestanol.
Preferably, the lysosomotropic agent is marmycin and wherein the linker is bound to said lysosomotropic agent via the hydroxyl of the sugar moiety of marmycin.
Preferably, the lysosomotropic agent is marmycin A of formula (Bl): wherein the wavy line represents the link to L.
Preferably, the lysosomotropic agent is cholesterol and wherein the linker is bound to said lysosomotropic agent via the hydroxyl group.
Preferably, the lysosomotropic agent is cholesterol of formula (B2): wherein the wavy line represents the link to L.
Preferably, the lysosomotropic agent is cholestanol and wherein the linker is bound to said lysosomotropic agent via the hydroxyl group.
Preferably, the lysosomotropic agent is cholestanol of formula (B3): wherein the wavy line represents the link to L. In another embodiment, the lysosomotropic agent is of formula (B4): wherein the wavy line represents the link to L.
In another embodiment, the lysosomotropic agent is of formula (B5): wherein the wavy line represents the link to L.
In a particular embodiment, the linker is an aliphatic hydrocarbon chain comprising from 2 to 20 carbon atoms, and being optionally terminated at one or both ends and/or interrupted by at least one group independently selected from -C(O)NH-,-NHC(O)-, -C(O)O-, -OC(O)-, -NH-, -S-, -O-, -C(O) -, -S(O)-, and -S(O)2-.
In a particular embodiment, the linker is an aliphatic hydrocarbon chain comprising from 2 to 16 carbon atoms, and being optionally terminated at one or both ends and/or interrupted by at least one group independently selected from -C(O)NH-,-NHC(O)-, -C(O)O-, -OC(O)-, -NH-, -S-, -O-, -C(O) -, -S(O)-, and -S(O)2-.
In a particular embodiment, the linker is an aliphatic hydrocarbon chain comprising from 2 to 14 carbon atoms, and being optionally terminated at one or both ends and/or interrupted by at least one group independently selected from -C(O)NH-,-NHC(O)-, -C(O)O-, -OC(O)-, -NH-, -S-, -O-, -C(O) -, -S(O)-, and -S(O)2-.
In a particular embodiment, the linker is an aliphatic hydrocarbon chain comprising from 2 to 12 carbon atoms and being optionally terminated at one or both ends and/or interrupted by at least one group independently selected from -C(O)NH-,-NHC(O)-, -C(O)O-, -OC(O)-, -NH-, -S-, -O-, -C(O) -S(O)-, and -S(O)2-.
In a particular embodiment, the conjugate of the invention is selected from the group consisting of compounds of general formula (III), (III’), (IV), (VII), (VII’), (VIII), (VIII’), (IX) (IX’), (X), (X’), (XI), (xr), (xii), (xir), (xiii), (xiir), (xiv), (xiv’), (xv), (xv’), (xvi), (xvr), (xvii), (XVIF), (XVIII), (XVIII’), (XIX), (XIX’), (XX), and (XX’); preferably (III), (III’), (IV), (VII), (VII’), (VIII), (viir), (ix) (ix’), (X), (X’), (xi), (xr), (xii), (xir), (xiii), (xiir), (xiv), (xiv’), (xvii), (xvir), (XVIII), and (XVIII’), as defined below, or a stereoisomers thereof or a pharmaceutically acceptable salt and/or solvate thereof.
In a particular embodiment, the conjugate of the invention is Fentomycin of general formula (III) or Pre- Fentomycin of general formula (IV).
A second object of the invention relates to a conjugate of the following general formula (I): A-L-B (I) wherein:
A is a first moiety selected from metal chelating agents;
B is a second moiety selected from lysosomotropic agent; and
L is absent or is a linker that binds moieties A and B together, preferably L is a linker that binds moieties A and B together.
In a particular embodiment, A, L and B are as defined above.
A third object of the invention relates to the compound Fentomycin of general formula (III), or a pharmaceutically acceptable salt and/or solvate thereof.
A fourth object of the invention relates to the compound Pre-Fentomycin of general formula (IV), or a pharmaceutically acceptable salt and/or solvate thereof.
Another object of the invention relates to the compound Fentomycin-1 of general formula (III’), or a pharmaceutically acceptable salt and/or solvate thereof.
Another object of the invention relates to the compound Fentomycin-2 of general formula (VII) or a compound of formula (VII’), or a pharmaceutically acceptable salt and/or solvate thereof.
Another object of the invention relates to the compound Fentomycin-3 of general formula (VIII) or a compound of formula (VIII’), or a pharmaceutically acceptable salt and/or solvate thereof.
Another object of the invention relates to the compound Fentomycin-5 of general formula (IX) or a compound of formula (IX’), or a pharmaceutically acceptable salt and/or solvate thereof.
In a particular embodiment, the conjugate of the invention is Fentomycin-6 of general formula (X) or a compound of formula (X’) as disclosed herein. In a particular embodiment, the conjugate of the invention is Fentomycin-7 of general formula (XI) or a compound of formula (XI’) as disclosed herein.
In a particular embodiment, the conjugate of the invention is Fentomycin-8 of general formula (XII) or a compound of formula (XIF) as disclosed herein.
In a particular embodiment, the conjugate of the invention is Fentomycin-9 of general formula (XIII) or a compound of formula (XIIF) as disclosed herein.
In a particular embodiment, the conjugate of the invention is Fentomycin-10 of general formula (XIV) or a compound of formula (XIV’) as disclosed herein.
In a particular embodiment, the conjugate of the invention is Fentomycin-11 of general formula (XV) or a compound of formula (XV’) as disclosed herein.
In a particular embodiment, the conjugate of the invention is Fentomycin-12 of general formula (XVI) or a compound of formula (XVI’) as disclosed herein.
In a particular embodiment, the conjugate of the invention is Fentomycin-13 of general formula (XVII) or a compound of formula (XVIF) as disclosed herein.
In a particular embodiment, the conjugate of the invention is Fentomycin-14 of general formula (XVIII) or a compound of formula (XVIIF) as disclosed herein.
In a particular embodiment, the conjugate of the invention is Fentomycin-15 of general formula (XIX) or a compound of formula (XIX’) as disclosed herein.
In a particular embodiment, the conjugate of the invention is Fentomycin-16 of general formula (XX) or a compound of formula (XX’) as disclosed herein.
Another object of the invention relates to a pharmaceutical composition comprising a conjugate according to the invention, or a compound according to the invention, or a pharmaceutically acceptable salt and/or solvate thereof, and at least one pharmaceutically acceptable excipient.
Another object of the invention relates to nanoparticle comprising a conjugate according to the invention, or a compound according to the invention, or a pharmaceutically acceptable salt and/or solvate thereof.
A further objection of the invention relates to a conjugate according to the invention, or a compound according to the invention, or a pharmaceutically acceptable salt and/or solvate thereof, or a pharmaceutical composition according to the invention, or a nanoparticle according to the invention, for use as a drug.
A further objection of the invention relates to a conjugate according to the invention, or a compound according to the invention, or a pharmaceutically acceptable salt and/or solvate thereof, or a pharmaceutical composition according to the invention, or a nanoparticle according to the invention, for use for the treatment of cancer.
A further objection of the invention relates to a method for the treatment of cancer in a subject in need thereof, comprising administering a therapeutic effective amount of a conjugate according to the invention, or a compound according to the invention, or a pharmaceutical composition according to the invention, or a nanoparticle according to the invention, to said subject.
A further objection of the invention relates to the use of a conjugate according to the invention, or a compound according to the invention, or a pharmaceutical composition according to the invention, or a nanoparticle according to the invention, for the manufacture of a medicament for use as anti-tumoral agent or in a method for the treatment of cancer.
A further objection of the invention relates to a method for the treatment of a disease in a subject in need thereof, comprising administering a therapeutic effective amount of a conjugate according to the invention, or a compound according to the invention, or a pharmaceutical composition according to the invention, or a nanoparticle according to the invention, to said subject.
A further objection of the invention relates to the use of a conjugate according to the invention, or a compound according to the invention, or a pharmaceutical composition according to the invention, or a nanoparticle according to the invention, for the manufacture of a medicament.
Another object of the invention relates to a pharmaceutical composition comprising a conjugate according to the invention, or a compound of formula (III) or (IV) according to the invention or a pharmaceutically acceptable salt and/or solvate thereof, and at least one pharmaceutically acceptable excipient.
Another object of the invention relates to nanoparticle comprising a conjugate according to the invention, or a compound of formula (III) or (IV) according to the invention or a pharmaceutically acceptable salt and/or solvate thereof.
A further objection of the invention relates to a conjugate according to the invention, or a compound of formula (III) or (IV) according to the invention or a pharmaceutically acceptable salt and/or solvate thereof, or a pharmaceutical composition according to the invention, or a nanoparticle according to the invention, for use as a drug.
Brief description of the figures
Figure 1 | Rational design of the lysosomal iron activator Fentomycin, which targets lysosomes and shows good efficacy in vitro. a, Chemical formulae of Marmycin A, the iron activator ligand, Fentomycin and a clickable ligand, ligand- ‘click’, which can be subjected to click chemistry with a fluorophore for cellular imaging, b, Fluorescence images of HT1080 cells treated with Fentomycin, Marmycin and LysoTracker and fluorescence images of labeled clickable ligand and LysoTracker. c, Fluorescence images PDAC053T cells of Fentomycin and CellRox showing that Fentomycin induces lysosomal reactive oxygen species (ROS), d, Viability curves of cells with Fentomycin, Marmycin A or ligand- ‘click’. Cell types are indicated on each graph.
Figure 2 | Fentomycin induces Ferroptosis. a, Oxidized lipids of liposomes in the presence of iron and Fentomycin. b, Flow cytometry of BODYPY 581/591 Cl l fluorescence of HT1080 cells treated with Fentomycin, Marmycin A or ligand- ‘click’, c, Flow cytometry of BODYPY 581/591 Cl l fluorescence of HT1080 cells treated with Fentomycin and ferroptosis inhibitors, d, Lipidomics of oxidized lipids in PDAC053T cells treated with Fentomycin for 6 h or 24 h. SFA (saturated fatty acids), MUFA (monounsaturated fatty acids), PUFA (polyunsaturated fatty acids). Two-sided Mann-Whitney test, error bars = s.e.m. e, Cell viability of HT1080 cells treated with 10 pM Fentomycin and ferroptosis inhibitors for 6 h. f, Lactate dehydrogenase (LDH) release assay of HT1080 cells treated with 10 pM Fentomycin and ferroptosis inhibitors for 6 h. g, Cell viability of HT1080 cells treated with 10 pM Fentomycin and ferroptosis inhibitors for 6 h. For c and e - g Kruskal- Wallis test with Dunn’s post-test. Box plots: boxes represent interquartile range and median and whiskers indicate the minimum and maximum values. Lip-1 = Liproxstatin-1, cLip-1 = clickable Liproxstatin-1, Fer-1 = Ferrostatin 1, DFO = deferoxamine, DFX = deferasirox, Toe = tocopherol, Vit K3 = vitamin K3, Z-VAD = Z-VAD-FMK, Nec = necrostatin.
Figure 3 | Fentomycin targets iron rich cancer cells in vivo. a, Flow cytometry of BODYPY 581/591 Cl l fluorescence of dissociated fresh human PDAC tumor cells treated with Fentomycin and ferroptosis inhibitors (n = 10-11). b, Flow cytometry of CD44 of dissociated fresh human PDAC cells (n = 10-13). c, Flow cytometry of BODYPY 581/591 Cl l fluorescence of dissociated fresh human undifferentiated pleomorphic sarcoma tumor cells treated with Fentomycin and ferroptosis inhibitors (n = 10-11). d, Flow cytometry of CD44 of dissociated fresh human undifferentiated pleomorphic sarcoma cells (n = 10-13). e, Viability curves of PDAC cell lines treated with Fentomycin or the standard of care (Irinotecan, 5-FU or Oxaliplatin), f, Viability curves of PDAC organoids treated with Fentomycin or the standard of care (Irinotecan, 5-FU or Oxaliplatin), g, inductively-coupled plasma mass spectrometry (ICP-MS) of iron of tumors in untreated (n = 10) and fentomycin-treated (n = 4) mice. Two-sided Mann-Whitney test, h, Flow cytometry of CD44 of tumors in untreated (n = 10) and Fentomycin-treated (n = 4) mice, i, Body weight of mice with tumors resulting from 4T1 cells injected into the lymphatic system. Vehicle (n = 10) and Fentomycin (n = 4) treatment, j, Tumor size over time of tumors resulting from 4T1 cells injected into the lymphatic system. Vehicle (n = 10 ) and Fentomycin (n = 4). Two-way ANOVA, error bars = s.e.m. For a - d Kruskal-Wallis test with Dunn’s post-test. Box plots: boxes represent interquartile range and median and whiskers indicate the minimum and maximum values.
Figure 4 | Fentomycin analogues. a, Molecular structures, b, Viability of cells treated for 6 h or 72 h. Data are mean ± s.d. c, Flow cytometry of BODIPY-C11 581/591 in cells treated for 6 h. Representative of n = 3 independent experiments, d, Colony-formation assay of DTP cancer cells selected from Doxo (150 nM, 72h) treatment, then treated with Fento-1 (5 pM) or DMSO (0.2%) for 72 h. n = 3 independent experiments. Data are mean ± s.d.
Figure 5 | a, Flow cytometry of BODIPY-C11 581/591 in cells treated with 1 pM of Fentomycin-3 and -6 to -16 for 6 h in PDAC053T cells, b, quantification of mean fluorescent intensity (MFI) of BODIPY-C11 581/591 of the data of Figure 5a.
Detailed description of the invention
Definition
The term “agent” is used herein to denote a chemical compound (such as an organic or inorganic compound (including, such as, a compound of the present invention), a mixture of chemical compounds), a biological macromolecule (such as a nucleic acid, an antibody, including parts thereof as well as humanized, chimeric and human antibodies and monoclonal antibodies, a protein or portion thereof, e.g., a peptide, a lipid, a carbohydrate), or an extract made from biological materials such as bacteria, plants, fungi, or animal (particularly mammalian) cells or tissues. Agents include, for example, agents which are known with respect to structure, and those which are not known with respect to structure.
The term “aliphatic hydrocarbon chain”, as used in the invention, refers to straight or branched hydrocarbon chain. Aliphatic hydrocarbon chain may be saturated such as alkyl group, or unsaturated such as alkenyl or alkynyl group.
The term “alkyl”, as used in the invention, refers to a monovalent linear or branched saturated hydrocarbon chain. For example, the term “(Ci-C3)alkyl” more specifically means methyl, ethyl, n- propyl, or isopropyl. The term “(C1-C6)alkyl” more specifically means methyl, ethyl, n-propyl, isopropyl, n-butyl, iso-butyl, sec-butyl, tert-butyl, pentyl or linear or branched hexyl.
The term “alkenyl”, as used in the invention, refers to a straight or branched monovalent unsaturated hydrocarbon chain comprising at least one double bond including, but not limited to, ethenyl, propenyl, butenyl, pentenyl, hexenyl and the like.
The term “alkynyl”, as used in the invention, refers to a straight or branched monovalent unsaturated hydrocarbon chain comprising at least one triple bond including, but not limited to, ethynyl, propynyl, butynyl, pentynyl, hexynyl and the like. Preferably, an alkynyl group as used in the present disclosure comprises one triple bond.
The term “cycloalkyl” corresponds to a saturated or unsaturated mono-, bi- or tri-cyclic alkyl group comprising between 3 and 20 atoms of carbons. It also includes fused, bridged, or spiro-connected cycloalkyl groups. The term “cycloalkyl” includes for instance cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl, preferably cyclopropyl. The term “spirocycloalkyl” includes for instance a spirocyclopentyl. In a particular aspect, the term “cycloalkyl” corresponds to a saturated monocycloalkyl group comprising between 3 and 7 atoms of carbons.
The term “heterocycloalkyl” corresponds to a saturated or unsaturated cycloalkyl group as above defined further comprising at least one heteroatom such as nitrogen, oxygen, or sulphur atom. It also includes fused, bridged, or spiro-connected heterocycloalkyl groups. Representative heterocycloalkyl groups include, but are not limited to 3-dioxolane, benzo [1,3] dioxolyl, pyrazolinyl, pyranyl, thiomorpholinyl, pyrazolidinyl, piperidyl, piperazinyl, 1,4-dioxanyl, imidazolinyl, pyrrolinyl, pyrrolidinyl, piperidinyl, imidazolidinyl, morpholinyl, 1,4-dithianyl, pyrrolidinyl, oxozolinyl, oxazolidinyl, isoxazolinyl, isoxazolidinyl, thiazolinyl, thiazolidinyl, isothiazolinyl, isothiazolidinyl, dihydropyranyl, tetrahydro-2H-pyranyl, tetrahydrofuranyl, and tetrahydrothiophenyl. The term “heterocycloalkyl” may also refer to a 5-10 membered bridged heterocyclyl such as 7- oxabicyclo[2,2, l]heptanyl.
The term “aryl” corresponds to a mono- or bi-cyclic aromatic hydrocarbons having from 6 to 12 carbon atoms. For instance, the term “aryl” includes phenyl, biphenyl, or naphthyl.
The term “heteroaryl” as used herein corresponds to an aromatic, mono- or poly-cyclic group comprising between 5 and 14 atoms, including at least one heteroatom such as nitrogen, oxygen or sulphur atom. Examples of such mono- and poly-cyclic heteroaryl group may be: pyridyl, pyridinyl, thiazolyl, thiophenyl, furanyl, pyrrolyl, pyrazolyl, imidazolyl, triazolyl, tetrazolyl, benzofuranyl, thianaphthalenyl, indolyl, indolinyl, quinolinyl, isoquinolinyl, benzimidazolyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, triazinyl, thianthrenyl, isobenzofuranyl, chromenyl, xanthenyl, phenoxanthinyl, isothiazolyl, isoxazolyl, pyrazinyl, pyridazinyl, indolizinyl, isoindolyl, indazolyl, purinyl, quinolizinyl, phtalazinyl, naphthyridinyl, quinoxalinyl, quinazolinyl, cinnolinyl, pteridinyl, carbazolyl, [3-carbolinyl, phenanthridinyl, acridinyl, pyrimidyl, pyrimidinyl, phenanthrolinyl, phenazinyl, phenothiazinyl, furazanyl, phenoxazinyl, isochromanyl, chromanyl, imidazolidinyl, imidazolinyl, pyrazolidinyl, pyrazolinyl, indolinyl, isoindolinyl, oxazolidinyl, benzotriazolyl, benzisoxazolyl, oxindolyl, benzoxazolinyl, benzothienyl, benzothiazolyl, isatinyl, dihydropyridyl, , s- triazinyl, oxazolyl, or thiofuranyl.
By “-CO-“ or “-C(O)-“, it refers to an oxo group. By “-SO-“ or “-S(O)-“, it refers to a sulfinyl group. By “-SC>2-“ or “-S(C>2)-“, it refers to a sulfonyl group.
The term “halogen” or “halo” refers to a fluorine, chlorine, bromine, or iodine atom, preferably a fluorine, chlorine or bromine, and more preferably a chlorine or a fluorine. By halo-(C1-C6 -alkyl) or (C1-C6-alkyl)-halo or (Ci.C6)alkyl-halo, it refers to a (C1-C6)-alkyl as defined above substituted with one, two or three halogen. Examples of these groups can be -CH2F, - CHF2 or -CF3, preferably -CF3.
For the purpose of the invention, the term “pharmaceutically acceptable” is intended to mean what is useful to the preparation of a pharmaceutical composition, and what is generally safe and nontoxic, for a pharmaceutical use.
The term “pharmaceutically acceptable salt” is intended to mean, in the framework of the present invention, a salt of a compound which is pharmaceutically acceptable, as defined above, and which possesses the pharmacological activity of the corresponding compound.
The pharmaceutically acceptable salts comprise:
(1) acid addition salts formed with inorganic acids such as hydrochloric, hydrobromic, sulfuric, nitric and phosphoric acid and the like; or formed with organic acids such as acetic, benzenesulfonic, fumaric, formic, glucoheptonic, gluconic, glutamic, glycolic, hydroxynaphtoic, 2- hydroxyethanesulfonic, lactic, maleic, malic, mandelic, methanesulfonic, muconic, 2- naphtalenesulfonic, propionic, succinic, dibenzoyl-L- tartaric, tartaric, p-toluenesulfonic, trimethylacetic, and trifluoroacetic acid and the like, and
(2) salts formed when an acid proton present in the compound is either replaced by a metal ion, such as an alkali metal ion, an alkaline-earth metal ion, or an aluminium ion; or coordinated with an organic or inorganic base. Acceptable organic bases comprise diethanolamine, ethanolamine. N- methylglucamine, triethanolamine, tromethamine and the like. Acceptable inorganic bases comprise aluminium hydroxide, calcium hydroxide, potassium hydroxide, sodium carbonate and sodium hydroxide.
The “stereoisomers” are isomeric compounds that have the same molecular formula and sequence of bonded atoms, but differ in the 3D-dimensional orientations of their atoms in space. The stereoisomers include enantiomers, diastereoisomers, cis-trans and E-Z isomers, conformers, and anomers. In a specific aspect of the disclosure, the stereoisomers include diastereoisomers and enantiomers.
The “tautomers” are isomeric compounds that differ only in the position of the protons and the electrons.
The “solvates” of the present disclosure include conventional solvates such as those formed during the last step of the preparation of the compounds of the invention due to the presence of solvents. It can be for example an hydrate or an alcoholate such as an ethanolate.
As used herein, the terms “subject”, “individual” or “patient” are interchangeable and refer to an animal, preferably to a mammal, even more preferably to a human, including adult and child. However, the term "subject" can also refer to non-human animals, in particular mammals such as dogs, cats, horses, cows, pigs, sheeps and non-human primates, among others. Within the context of the present disclosure, the term treatment denotes curative, symptomatic, and preventive treatment. Pharmaceutical compositions, kits, products and combined preparations of the invention can be used in humans with a disease or disorder. The pharmaceutical compositions, kits, products and combined preparations of the invention will not necessarily cure the patient but will delay or slow the progression or prevent further progression of the disease or disorder, and/or ameliorating thereby the patients’ condition. In treating the disease or disorder, the pharmaceutical composition of the invention is administered in a therapeutically effective amount.
Whenever within this whole specification "treatment of a disease or disorder" or the like is mentioned with reference to the pharmaceutical composition of the invention, there is meant: a) a method for treating a disease or disorder, said method comprising administering a therapeutically effective amount of a compound of the invention or of a pharmaceutical composition comprising said compound to a subject in need of such treatment; b) the use of a compound of the invention or of a pharmaceutical composition comprising said compound for the treatment of a disease or disorder; c) the use of a compound of the invention or of a pharmaceutical composition comprising said compound for the manufacture of a medicament for the treatment of a disease or disorder; and/or d) a compound of the invention or of a pharmaceutical composition comprising said compound for use in the treatment a disease or disorder.
As used herein, the term “therapeutic effect” refers to an effect induced by an active ingredient, or a pharmaceutical composition according to the invention, capable to prevent or to delay the appearance or development of a disease or disorder, or to cure or to attenuate the effects of a disease or disorder.
By "therapeutically effective amount" or “active amount” or “effective amount”, it is meant the quantity of the pharmaceutical composition of the invention which prevents, removes or reduces the deleterious effects of a disease or disorder in mammals, including humans, alone or in combination with the other active ingredients of the pharmaceutical composition, kit, product or combined preparation. It is understood that the administered dose may be lower for each compound in the composition to the “therapeutic effective amount” define for each compound used alone or in combination with other treatments than the combination described here. The “therapeutic effective amount” of the composition will be adapted by those skilled in the art according to the patient, the pathology, the mode of administration, etc.
As used herein, the term "pharmaceutically acceptable excipient" refers to any ingredient except active ingredients which are present in a pharmaceutical composition. Its addition may be aimed to confer a particular consistency or other physical or gustative properties to the final product. A pharmaceutically acceptable excipient must be devoid of any interaction, in particular chemical, with the active ingredients.
In the context of the invention, the ranges of values expressed by “from X to XX” or “between X and XX” comprise the upper and lower limits. Conjugate of the invention
The invention relates to a conjugate of the following general formula (I):
A-L-B (I) wherein:
A is a first moiety selected from metal chelating agents;
B is a second moiety selected from lysosomotropic agents; and
L is absent or is a linker that binds moieties A and B together, preferably L is a linker that binds moieties A and B together.
The invention also relates to a conjugate of the following general formula (I):
A-L-B (I) wherein:
A is a first moiety selected from metal chelating agents;
B is a second moiety selected from lysosomotropic agents; and
L is a linker that binds moieties A and B together.
The invention also relates to said conjugate for use in the treatment of cancer. In particular, the conjugate of the invention is for use in a targeted treatment of Cancer Stem Cells (CSCs).
According to the invention, the first moiety A is a lysosomal agent inducing intra-lysosomal activity and/or modulation. In one embodiment, the first moiety A is a lysosomal metal ion activator and/or modulator.
In one embodiment, the first moiety A is selected from metal chelating agents. In the context of the invention, the terms “metal chelating agents” means agents which are able to chelate a metal by forming a complex and/or which promote or catalyze oxidation of lipids. Chelating agents are generally classified based upon the target metal such as iron, copper, mercury or lead.
In particular, the terms “metal chelating agents” means agents which are able to bind to and activate iron and able to promote or catalyze oxidation of lipids.
In a specific embodiment, the first moiety A is selected from metal ion activator agents, preferably a lysosomal metal ion activator.
In a particular embodiment of the invention, the first moiety A is a metal cation chelating agent, preferably a metal cation activator agent.
In a particular embodiment of the invention, the first moiety A is an iron chelating agent or a copper chelating agent, preferably an iron chelating agent. In a more particular embodiment of the invention, the first moiety A is an iron activator agent or a copper activator agent, preferably an iron activator agent. In a specific embodiment, the first moiety A can be of the following general formula (II):
Ri is selected from the group consisting of NRARB, ORC, C(O)ORD, C(O)RE, S(O)RF, S(O)2RG, CN, halo-(C1-C6-alkyl), -C(O)-(C1-C6)alkyl-halo, halogen, NO2, and SO2, RA to RG being independently of each other H or a (C1-C6)alkyl, preferably Ri is N(CH3)2;
R2 is a hydrogen atom or (C1-C6)alkyl,; and each Z is, independently of each other, a hydrogen or deuterium atom.
In a specific embodiment, the first moiety A can be of the following general formula (II):
Ri is selected from the group consisting of NRARB, ORC, C(O)ORD, C(O)RE, S(O)RF, S(O)2RG, CN, halo-(C1-C6-alkyl), -C(O)-(C1-C6)alkyl-halo, halogen, NO2, and SO2, RA to RG being independently of each other H or a (C1-C6)alkyl, preferably Ri is N(CH3)2;
R2 is a hydrogen atom or (C1-C6)alkyl,; and each Z is, independently of each other, a hydrogen or deuterium atom.
Preferably, in the compound of general formula (II), Z is an hydrogen atom.
Preferably, in the compound of general formula (II), R2 is a hydrogen atom or a methyl, more preferably a hydrogen atom.
Preferably, Ri is selected from the group consisting of NRARB, ORC, C(O)ORD, C(O)RE, S(O)RF, S(O)2RG, CN, halo-(C1 -alkyl), -C(O)-(C1)alkyl-halo, halogen, , NO2 and SO2, RA to RG being independently of each other H or a (C1-C6)alkyl.
Preferably, Ri is selected from the group consisting of NRARB, ORC, C(O)ORD, C(O)RE, S(O)RF, S(O)2RG, CN, CF3, -C(O)CF3, halogen, NO2 and SO2, RA to RG being independently of each other H or a (Ci-C6)alkyl.
Preferably, Ri is selected from the group consisting of NRARB, ORC, C(O)ORD, C(O)RE, S(O)RF, S(O)3RG, CN, halo-(C1 -alkyl), halogen, and SO2, RA to RG being independently of each other H or a (C1- C6)alkyl.
Preferably, R1 is selected from the group consisting of NRARB, ORC, C(O)ORD, C(O)RE, S(O)RF, S(O)3RG, CN, halogen, and SO2, RA to RG being independently of each other H or a (C1-C6)alkyl.
Preferably, in the compound of general formula (II), Ri is selected from the group consisting of -NH2, - (N(CH3)2, -OH, -OCH3, -C(O)OH, -C(O)OCH3, -C(O)H, -C(O)CH3, -S(O)H, -S(O)2H, -CN, halogen, - CH2-CI, CH3-Br, CH2-F, CH2-I, -CF3, -C(O)CF3, -NO2 and -SO2. More preferably, in the compound of general formula (II), Ri is (N(CH3)3.
Preferably, in the compound of general formula (II), Ri is selected from the group consisting of -NH2, - (N(CH3)2, -OH, -OCH3, -C(O)OH, -C(O)OCH3, -C(O)H, -C(O)CH3, -S(O)H, -S(O)2H, -CN, halogen, - CH2-CI, CH3-Br, CH2-F, CH2-I, -CF3 and -SO2. More preferably, in the compound of general formula (II), Ri is (N(CH3)2.
Preferably, in the compound of general formula (II), Z is an hydrogen atom, R2 is a hydrogen atom and Ri is (N(CH3)2. In a specific embodiment, the first moiety A is selected from ligands of the White-Chen Type, preferably the first moiety A is White -Chen ligand.
In the context of the invention, the White-Chen ligand is also known as 2-[[2-[l-(pyridin-2- ylmethyl)pyrrolidin-2-yl]pyrrolidin-l-yl]methyl]pyridine based ligand, and referred to the compound of the following formula (V):
In the context of the invention, the terms “ligands of the White-Chen Type” referred to iron ligands that were developed by the Christina White group in the following articles: M. S. Chen, M. C. White, Science 2007, 318, 783-787 [25]; andM. S. Chen, M. C White, Science 2010, 327, 566-571 [27], In particular, a ligand of the White-Chen Type is a ligand that forms part of an organometallic complex with iron able to catalyze selectively aliphatic C-H oxidations in conditions that are similar to that found in the lysosomal compartment. This family of ligands forms stable complexes with iron.
In a specific embodiment, ligands of the White-Chen Type according to the invention can be of the general formula (Ila) as disclosed below: wherein
Ri is selected from the group consisting of NRARB, ORC, C(O)ORD, C(O)RE, S(O)RF, S(O)2RG, CN, halo-(C1-C6-alkyl), -C(O)-(C1-C6)alkyl-halo, halogen, NO2, and SO2, RA to RG being independently of each other H or a (C1-C6)alkyl, preferably Ri is N(CH3)2;
R2 is a hydrogen atom or (C1-C6)alkyl;
X and Y are, independently of each other, a (C1-C6)alkyl; or X and Y can be linked together to form a heterocycloalkyl or heteroaryl, preferably a pyrrolidine; and each Z is, independently of each other, a hydrogen or deuterium atom; and wherein the wavy line represents the link to L. In a specific embodiment, the first moiety A is of the general formula (lib) : wherein
Ri is selected from the group consisting of NRARB, ORC, C(O)ORD, C(O)RE, S(O)RF, S(O)2RG, CN, halo-(C1-C6-alkyl), -C(O)-(C1-C6)alkyl-halo, halogen, NO2, and SO2, RA to RG being independently of each other H or a (C1-C6)alkyl, preferably Ri is N(CH3)2;
R2 is a hydrogen atom or (C1-C6)alkyl; and each Z is, independently of each other, a hydrogen or deuterium atom; and wherein the wavy line represents the link to L.
Preferably, in the compound of general formula (Ila) or (lib), Z is an hydrogen atom.
Preferably, in the compound of general formula (Ila) or (lib), R2 is a hydrogen atom or a methyl, more preferably a hydrogen atom.
Preferably, in the compound of general formula (Ila) or (lib), Ri is selected from the group consisting of NRARB, ORC, C(O)ORD, C(O)RE, S(O)RF, S(O)2RG, CN, halo-(Ci-alkyl), -C(O)-(Ci)alkyl-halo, halogen, , NO2 and SO2, RA to RG being independently of each other H or a (C1-C6)alkyl.
Preferably, in the compound of general formula (Ila) or (lib), Ri is selected from the group consisting of NRARB, ORC, C(O)ORD, C(O)RE, S(O)RE, S(O)2RG, CN, CF3, -C(O)CF3, halogen, NO2 and SO2, RA to RG being independently of each other H or a (C1-C6)alkyl.
Preferably, in the compound of general formula (Ila) or (lib), Ri is selected from the group consisting of NRARB, ORC, C(O)ORD, C(O)RE, S(O)RE, S(O)2RG, CN, halo-(C1 -alkyl), halogen, and SO2, RA to RG being independently of each other H or a (C1-C6)alkyl.
Preferably, in the compound of general formula (Ila) or (lib), Ri is selected from the group consisting of NRARB, ORC, C(O)ORD, C(O)RE, S(O)RF, S(O)2RG, CN, halogen, and SO2, RA to RG being independently of each other H or a (C1-C6)alkyl.
Preferably, in the compound of general formula (Ila) or (lib), Ri is selected from the group consisting of NH2, (N(CH3)2, OH, OCH3, C(O)OH, C(O)OCH3, C(O)H, C(O)CH3, S(O)H, S(O)2H, CN, halogen, -CH2-CI, CH2-Br, CH2-F, CH2-I, -CF3, -C(O)CF3, NO2, and SO2. Preferably, in the compound of general formula (Ila) or (lib), Ri is selected from the group consisting of NH2, (N(CH3)2, OH, OCH3, C(O)OH, C(O)OCH3, C(O)H, C(O)CH3, S(O)H, S(O)2H, CN, halogen, -CH2-C1, CH2-Br, CH2-F, CH2-I, -CF3 and SO2. More preferably, in the compound of general formula (Ila) or (lib), Ri is (N(CH3)2. Preferably, in the compound of general formula (Ila) or (lib), Z is an hydrogen atom, R2 is a hydrogen atom and Ri is (N(CH3)2.
In a specific embodiment, the first moiety A is of the general formula (lie): (lie), wherein
Ri is N(CH3)2 ; and
R2 is an hydrogen atom or (C1-C6)alkyl; and wherein the wavy line represents the link to L. Preferably, in the compound of general formula (lie), R2 is preferably a hydrogen atom or a methyl, more preferably a hydrogen atom.
Preferably, the first moiety A is of the following formula (lid) : In another specific embodiment, the first moiety A is selected from ligands of the Nordlander-Costas Type, preferably the first moiety A is Nordlander-Costas ligand.
In the context of the invention, the terms “ligands of the Nordlander-Costas Type” referred to iron ligands that are developed by Costas and Norlander in the following paper : Chem. Commun., 2014, 50, 1408 (DOI: 10.1039/c3cc47830k) [28], The new tetradentate ligand replaces the pyridyl arm by an N- methyl benzimidazolyl substituent. The sp2 character and the rigidity of the latter substituent should provide a well-defined steric demand, intermediate between the a-H and the a-Me groups of a pyridine. On the other hand, the relative donor capacities of pyridine and benzimidazole can be estimated to be very similar by comparing the pKa values of their conjugate acids (5.22 for pyridine, 5.41 for benzimidazole and 5.57 for a-Me pyridine), and therefore differences in reactivity among this set of complexes can be traced to steric factors.
In a specific embodiment, ligands of the Nordlander-Costas Type according to the invention can be of the general formula (lie) as disclosed herein: wherein
Rs is a hydrogen atom or (C1-C6)alkyl preferably methyl;
X and Y are, independently of each other, a (C1-C6)alkyl; or X and Y can be linked together to form a heterocycloalkyl or heteroaryl, preferably a pyrrolidine; and each Z is, independently of each other, a hydrogen or deuterium atom; and wherein the wavy line represents the link to L.
In another specific embodiment, the first moiety A is of the general formula (Ilf): wherein each Z is, independently of each other, a hydrogen or deuterium atom; and wherein the wavy line represents the link to L.
Preferably, in the compound of general formula (Ilf), Z is an hydrogen atom.
In the context of the invention, the Nordlander-Costas ligand refers to the compound of the following formula (Ilg): wherein the wavy line represents the link to L.
In another specific embodiment, the first moiety A is: wherein the wavy line represents the link to L.
In another specific embodiment, the first moiety A is selected from the group consisting of:
In another specific embodiment, the first moiety A is selected from the group consisting of: In another specific embodiment, the first moiety A is selected from the group consisting of: The first moiety A allows to activate the metal ion to promote lipid peroxidation and ferroptosis by Fenton reaction.
According to the invention, the second moiety B is selected from lysosomotropic agents.
In the context of the invention, the terms “lysosomotropic agents” is used to designate all substances that are taken up selectively into lysosomes, in vitro and in vivo, irrespective of their chemical nature or mechanism of uptake. In particular, lysosomotropic agents can be weak bases that penetrate in the membrane and accumulate inside lysosomes as protonated form and increase the intracellular pH. Lysosomotropic agent can also integrate in the cell membrane and be taken up by endocytosis, accumulating in lysosomes. Therefore, lysosomotropic agent can drive the attached iron chelating agent in these lysosomes to promote lipid oxidation and induce ferroptosis. Lysosomotropic agents exert their effects on the cell via lysosomes.
In the context of the invention, the moiety B allows to guide the cellular localization of Fentomycin to lysosome, to induce reactive oxygen species in endolysosomes promoting oxidative degradation of membrane lipids leading to ferroptosis.
Suitable lysosomotropic agents according to the invention can include, but are not limited to, the chloroquine, hydroxychloroquine, azithromycin, marmycin, steroids such as cholesterol and cholestanol, ezurpimtrostat hydrochloride, desmethylchloroquine, hydroxychloroquine phosphate, desmethyl-hydroxychloroquine, anthraquinones, methyl tetraphene dione, angucyclines, ammonium chloride (NFLC1), amantadine, methylamine, fluoxetine, imipramine, latrepirdine, tamoxifen, chlorpromazine, amitriptyline, verapamil, Triton WR 1339 (Tyloxapol), Suramin, metformin, Erythromycin, Amitriptyline, Imipramine, 4-aminoquinoline, amiodarone, amodiaquine, clindamycin, N-(3-[(2,4-dinitrophenyl)-amino]-propyl)-N-(3-aminopropyl-methylamine)dihydrochloride (DAMP), monensin, monodansylcadaverine, perhexilene, phenylalanine methyl ester, primaquine, quinacrine, thioridazine, tilorone, tributylamine, ketotifen fumarate, glycerol, sucrose, Trehalose, Resveratrol, PVP, and Gold sodium thiomalate.
In a specific embodiment, the second moiety B is selected from the group consisting of chloroquine, hydroxychloroquine, azithromycin, marmycin, cholesterol, cholestanol, ezurpimtrostat hydrochloride, desmethylchloroquine, hydroxychloroquine phosphate, desmethyl-hydroxychloroquine, anthraquinones, methyl tetraphene dione, angucyclines.
In a specific embodiment, the second moiety B is selected from the group consisting of chloroquine, hydroxychloroquine, azithromycin, marmycin, ezurpimtrostat hydrochloride, desmethylchloroquine, hydroxychloroquine phosphate, desmethyl-hydroxychloroquine, anthraquinones, methyl tetraphene dione, and angucyclines. In a particular embodiment, the second moiety B is selected from the group consisting of chloroquine, hydroxychloroquine, marmycin, cholesterol, cholestanol, and ezurpimtrostat hydrochloride, as illustrated below:
In a more specific embodiment, the second moiety B is selected from the group consisting of:
Azithromycin Marmycin
Ezurpimtrostat hydrochloride methyl tetraphene dione , and
In a particular embodiment, the second moiety B is selected from the group consisting of marmycin, cholesterol, cholestanol, and ezurpimtrostat hydrochloride. In a more specific embodiment, the second moiety B is marmycin such as marmycin A or marmycin B, preferably marmycin A.
In the conjugate according to the invention, when the lysosomotropic agent is marmycin, the linker is preferably bound to said lysosomotropic agent via the hydroxyl of the sugar moiety of marmycin.
In particular, the second moiety B is of the following formula (Bl), referring to marmycin A:
In another specific embodiment, the second moiety B is cholesterol or cholestanol.
In the conjugate according to the invention, when the lysosomotropic agent is cholesterol or cholestanol, the linker is preferably bound to said lysosomotropic agent via the hydroxyl group.
In particular, the second moiety B is of the following formula (B2) or (B3) or (B4) or (B5):
In particular, the second moiety B is of the following formula (B2) or (B3), referring to cholesterol or cholestanol:
In a preferred embodiment of the invention, the conjugate A-L-B comprises:
A first moiety A of the general formula (II), and
A second moiety B which is marmycin and wherein the linker is bound to said moiety B via the hydroxyl of the sugar moiety of marmycin. In a preferred embodiment of the invention, the conjugate A-L-B comprises:
A first moiety A of the general formula (Ila), and
A second moiety B which is marmycin and wherein the linker is bound to said moiety B via the hydroxyl of the sugar moiety of marmycin.
In a preferred embodiment of the invention, the conjugate A-L-B comprises: - A first moiety A of the general formula (lib) : (lib), wherein
Ri is N(CH3)2 ; and
R2 is a hydrogen atom or (C1-C6)alkyl, preferably hydrogen atom; and wherein the wavy line represents the link to L, and
A second moiety B which is marmycin and wherein the linker is bound to said moiety B via the hydroxyl of the sugar moiety of marmycin.
In a preferred embodiment of the invention, the conjugate A-L-B comprises:
A first moiety A of the general formula (lie), and
A second moiety B which is marmycin and wherein the linker is bound to said moiety B via the hydroxyl of the sugar moiety of marmycin.
In a preferred embodiment of the invention, the conjugate A-L-B comprises:
A first moiety A of the general formula (lid), and
A second moiety B which is marmycin and wherein the linker is bound to said moiety B via the hydroxyl of the sugar moiety of marmycin.
In a preferred embodiment of the invention, the conjugate A-L-B comprises:
A first moiety A of the general formula (V), and
A second moiety B which is marmycin and wherein the linker is bound to said moiety B via the hydroxyl of the sugar moiety of marmycin.
In a preferred embodiment of the invention, the conjugate A-L-B comprises:
A first moiety A of the general formula (lie), and
A second moiety B which is marmycin and wherein the linker is bound to said moiety B via the hydroxyl of the sugar moiety of marmycin.
In a preferred embodiment of the invention, the conjugate A-L-B comprises:
A first moiety A of the general formula (Ilf), and
A second moiety B which is marmycin and wherein the linker is bound to said moiety B via the hydroxyl of the sugar moiety of marmycin.
In a preferred embodiment of the invention, the conjugate A-L-B comprises:
A first moiety A of the general formula (Ilg), and
A second moiety B which is marmycin and wherein the linker is bound to said moiety B via the hydroxyl of the sugar moiety of marmycin.
In these embodiments, marmycin is preferably marmycin A, in particular of formula (Bl).
In a preferred embodiment of the invention, the conjugate A-L-B comprises:
A first moiety A of the general formula (II), and
A second moiety B which is cholesterol of formula (B2). In a preferred embodiment of the invention, the conjugate A-L-B comprises:
A first moiety A of the general formula (Ila), and
A second moiety B which is cholesterol of formula (B2).
In a preferred embodiment of the invention, the conjugate A-L-B comprises:
A first moiety A of the general formula (lib), and
A second moiety B which is cholesterol of formula (B2).
In a preferred embodiment of the invention, the conjugate A-L-B comprises:
A first moiety A of the general formula (lie), and
A second moiety B which is cholesterol of formula (B2).
In a preferred embodiment of the invention, the conjugate A-L-B comprises:
A first moiety A of the general formula (lid), and
A second moiety B which is cholesterol of formula (B2).
In a preferred embodiment of the invention, the conjugate A-L-B comprises:
A first moiety A of the general formula (V), and
A second moiety B which is cholesterol of formula (B2).
In a preferred embodiment of the invention, the conjugate A-L-B comprises:
A first moiety A of the general formula (lie), and
A second moiety B which is cholesterol of formula (B2).
In a preferred embodiment of the invention, the conjugate A-L-B comprises:
A first moiety A of the general formula (Ilf), and
A second moiety B which is cholesterol of formula (B2).
In a preferred embodiment of the invention, the conjugate A-L-B comprises:
A first moiety A of the general formula (Ilg), and
A second moiety B which is cholesterol of formula (B2).
In a preferred embodiment of the invention, the conjugate A-L-B comprises:
A first moiety A of the general formula (II), and
A second moiety B which is cholestanol of formula (B3).
In a preferred embodiment of the invention, the conjugate A-L-B comprises:
A first moiety A of the general formula (Ila), and
A second moiety B which is cholestanol of formula (B3).
In a preferred embodiment of the invention, the conjugate A-L-B comprises:
A first moiety A of the general formula (lib), and
A second moiety B which is cholestanol of formula (B3).
In a preferred embodiment of the invention, the conjugate A-L-B comprises:
A first moiety A of the general formula (lie), and
A second moiety B which is cholestanol of formula (B3). In a preferred embodiment of the invention, the conjugate A-L-B comprises:
A first moiety A of the general formula (lid), and
A second moiety B which is cholestanol of formula (B3).
In a preferred embodiment of the invention, the conjugate A-L-B comprises:
A first moiety A of the general formula (V), and
A second moiety B which is cholestanol of formula (B3).
In a preferred embodiment of the invention, the conjugate A-L-B comprises:
A first moiety A of the general formula (lie), and
A second moiety B which is cholestanol of formula (B3).
In a preferred embodiment of the invention, the conjugate A-L-B comprises:
A first moiety A of the general formula (Ilf), and
A second moiety B which is cholestanol of formula (B3).
In a preferred embodiment of the invention, the conjugate A-L-B comprises:
A first moiety A of the general formula (Ilg), and
A second moiety B which is cholestanol of formula (B3).
In a preferred embodiment of the invention, the conjugate A-L-B comprises:
A first moiety A of the general formula (II), and
A second moiety B which is cholesterol of formula (B4).
In a preferred embodiment of the invention, the conjugate A-L-B comprises:
A first moiety A of the general formula (Ila), and
A second moiety B which is cholesterol of formula (B4).
In a preferred embodiment of the invention, the conjugate A-L-B comprises:
A first moiety A of the general formula (lib), and
A second moiety B which is cholesterol of formula (B4).
In a preferred embodiment of the invention, the conjugate A-L-B comprises:
A first moiety A of the general formula (lie), and
A second moiety B which is cholesterol of formula (B4).
In a preferred embodiment of the invention, the conjugate A-L-B comprises:
A first moiety A of the general formula (lid), and
A second moiety B which is cholesterol of formula (B4).
In a preferred embodiment of the invention, the conjugate A-L-B comprises:
A first moiety A of the general formula (V), and
A second moiety B which is cholesterol of formula (B4).
In a preferred embodiment of the invention, the conjugate A-L-B comprises:
A first moiety A of the general formula (lie), and
A second moiety B which is cholesterol of formula (B4). In a preferred embodiment of the invention, the conjugate A-L-B comprises:
A first moiety A of the general formula (Ilf), and
A second moiety B which is cholesterol of formula (B4).
In a preferred embodiment of the invention, the conjugate A-L-B comprises:
A first moiety A of the general formula (Ilg), and
A second moiety B which is cholesterol of formula (B4).
In a preferred embodiment of the invention, the conjugate A-L-B comprises:
A first moiety A of the general formula (II), and
A second moiety B which is cholestanol of formula (B5).
In a preferred embodiment of the invention, the conjugate A-L-B comprises:
A first moiety A of the general formula (Ila), and
A second moiety B which is cholestanol of formula (B5).
In a preferred embodiment of the invention, the conjugate A-L-B comprises:
A first moiety A of the general formula (lib), and
A second moiety B which is cholestanol of formula (B5).
In a preferred embodiment of the invention, the conjugate A-L-B comprises:
A first moiety A of the general formula (lie), and
A second moiety B which is cholestanol of formula (B5).
In a preferred embodiment of the invention, the conjugate A-L-B comprises:
A first moiety A of the general formula (lid), and
A second moiety B which is cholestanol of formula (B5).
In a preferred embodiment of the invention, the conjugate A-L-B comprises:
A first moiety A of the general formula (V), and
A second moiety B which is cholestanol of formula (B5).
In a preferred embodiment of the invention, the conjugate A-L-B comprises:
A first moiety A of the general formula (lie), and
A second moiety B which is cholestanol of formula (B5).
In a preferred embodiment of the invention, the conjugate A-L-B comprises:
A first moiety A of the general formula (Ilf), and
A second moiety B which is cholestanol of formula (B5).
In a preferred embodiment of the invention, the conjugate A-L-B comprises:
A first moiety A of the general formula (Ilg), and
A second moiety B which is cholestanol of formula (B5).
According to the invention, L is a linker that binds moieties A and B together. In the context of the invention, the term “linker” refers to a single covalent bond or a moiety comprising series of stable covalent bonds, the moiety often incorporating 1-40 plural valent atoms selected from the group consisting of C, N, O, S and P, that covalently attach a reactive group or bioactive group to the probe of the invention. The number of plural valent atoms in a linker may be, for example, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 20, 25, 30 or a larger number up to 40 or more. A linker may be linear or non-linear; some linkers have pendant side chains or pendant functional groups (or both). Examples of such pendant moieties are hydrophilicity modifiers, for example solubilizing groups like, e.g. sulfo (- SO3H or -SO3-), carboxy (-COOH or -COO-), hydroxy.
In one embodiment, L is composed of any combination of single, double, triple or aromatic carboncarbon bonds, carbon-nitrogen bonds, nitrogen-nitrogen bonds, carbon-oxygen bonds and carbon-sulfur bonds. Linkers may by way of example consist of a combination of moieties selected from alkyl, - C(O)NH-, -C(O)O-, -NH-, -S-, -O-, -C(O) -, -S(O)-, -S(O)2-.; -O-, 5- or 6- membered monocyclic rings and optional pendant functional groups, for example sulfo, hydroxy and carboxy.
In another embodiment, L is selected from the group consisting of a single bond, -C(O)NH-, -C(O)O-, -NH-, -S-, -O-, -C(O) -, -S(O)-, -S(O)2-.; -O-, and 5- or 6- membered monocyclic rings. In a specific embodiment, L is -O-.
The reactive group may be reacted with a substance reactive therewith, whereby the linker becomes bonded to a bioactive group. In this case, the linker typically contains a residue of a reactive group (such as for example the carbonyl group of an ester after reaction with a nucleophile; a triazolo group resulting from a click reaction between an azide and an alkyne; an amide link resulting from a reaction between an amine and an acid; a thiourea resulting from the coupling between an amine and an isothiocyanate; a -O-C(O)- moiety remaining for example after reaction of an activated carbonate with a nucleophile, etc.).
In a particular embodiment of the invention, the linker is an aliphatic hydrocarbon chain comprising from 2 to 20 carbon atoms and being optionally terminated at one or both end and/or interrupted by at least one group independently selected from -C(O)NH-,-NHC(O)-, -C(O)O-, -OC(O)-, -NH-, -S-, -O-, -C(O) -, triazolyl, -S(O)-, and -S(O)2-.
In another particular embodiment of the invention, the linker is an aliphatic hydrocarbon chain comprising from 2 to 16 carbon atoms and being optionally terminated at one or both end and/or interrupted by at least one group independently selected from -C(O)NH-,-NHC(O)-, -C(O)O-, -OC(O)- , -NH-, -S-, -O-, -C(O) -, triazolyl, -S(O)-, and -S(O)2-.
In another particular embodiment of the invention, the linker is an aliphatic hydrocarbon chain comprising from 2 to 14 carbon atoms and being optionally terminated at one or both end and/or interrupted by at least one group independently selected from -C(O)NH-,-NHC(O)-, -C(O)O-, -OC(O)- , -NH-, -S-, -O-, -C(O) -, triazolyl, -S(O)-, and -S(O)2-.
In another particular embodiment of the invention, the linker is an aliphatic hydrocarbon chain comprising from 2 to 12 carbon atoms and being optionally terminated at one or both end and/or interrupted by at least one group independently selected from -C(O)NH-,-NHC(O)-, -C(O)O-, -OC(O)- , -NH-, -S-, -O-, -C(O) triazolyl, -S(O)-, and -S(O)2-.
In particular, the linker is selected from: -(CH2)t-, -(CH2-O)P-, -((CH2)t-O)p-, -(CH2)t-C(O)-, -C(O)- (CH2)t-C(O)-, (CH2)t-NH-(CH2)q-C(O)-, -(CH2)n-NH-CO-(CH2)m-NH-, -(CH2)n-NH-CO-(CH2)m-CO-, - (CH2)n-NH-CO-(CH2-CH2-O)m-NH-, -(CH2)n-NH-CO-(CH2-CH2-O)m-CO-, -(CH2)n-CO-NH-(CH2)m- CO-, -(CH2)n-CO-NH-(CH2-CH2-O)m-CO-, -(CH2-CH2-O)n-NH-CO-(CH2)m-NH-, -(CH2-CH2-O)n-NH- CO-(CH2)m-CO-, -(CH2-CH2-O)n-NH-CO-(CH2-CH2-O)m-NH-, -(CH2-CH2-O)n-NH-CO-(CH2-CH2- O)m-CO-, -(CH2-CH2-O)n-CO-NH-(CH2)m-CO-, and -(CH2-CH2-O)n-CO-NH-(CH2-CH2-O)m-CO-;-, wherein p, q and t are, independently of each other, an integer from 2 to 20, preferably from 2 to 16, preferably from 2 to 14, more preferably from 2 to 12; wherein n is an integer from 2 to 14, preferably from 2 to 12; and wherein m is an integer from 2 to 6.
In particular, the linker is selected from: -(CH2)t-, -(CH2-O)P-, -((CH2)t-O)p-, -(CH2)t-C(O)-, -C(O)- (CH2)t-C(O)-, (CH2)t-NH-(CH2)q-C(O)-, -(CH2)n-NH-CO-(CH2)m-NH-, -(CH2)n-NH-CO-(CH2)m-CO-, - (CH2)n-NH-CO-(CH2-CH2-O)m-NH-, -(CH2)n-NH-CO-(CH2-CH2-O)m-CO-, -(CH2)n-CO-NH-(CH2)m- CO-, -(CH2)n-CO-NH-(CH2-CH2-O)m-CO-, -(CH2-CH2-O)n-NH-CO-(CH2)m-NH-, -(CH2-CH2-O)n-NH- CO-(CH2)m-CO-, -(CH2-CH2-O)n-NH-CO-(CH2-CH2-O)m-NH-, -(CH2-CH2-O)n-NH-CO-(CH2-CH2- O)m-CO-, -(CH2-CH2-O)n-CO-NH-(CH2)m-CO-, and -(CH2-CH2-O)n-CO-NH-(CH2-CH2-O)m-CO-;-, wherein p, q and t are, independently of each other, an integer from 2 to 14; wherein n is an integer from 2 to 12; and wherein m is an integer from 2 to 6.
In particular, the linker is selected from: -(CH2)t-, -(CH2-O)P-, -((CH2)t-O)p-, -(CH2)t-C(O)-, -C(O)- (CH2)t-C(O)-, (CH2)t-NH-(CH2)q-C(O)-, -(CH2)n-NH-CO-(CH2)m-NH-, -(CH2)n-NH-CO-(CH2)m-CO-, - (CH2)n-NH-CO-(CH2-CH2-O)m-NH-, -(CH2)n-NH-CO-(CH2-CH2-O)m-CO-, -(CH2)n-CO-NH-(CH2)m- CO-, -(CH2)n-CO-NH-(CH2-CH2-O)m-CO-, -(CH2-CH2-O)n-NH-CO-(CH2)m-NH-, -(CH2-CH2-O)n-NH- CO-(CH2)m-CO-, -(CH2-CH2-O)n-NH-CO-(CH2-CH2-O)m-NH-, -(CH2-CH2-O)n-NH-CO-(CH2-CH2- O)m-CO-, -(CH2-CH2-O)n-CO-NH-(CH2)m-CO-, and -(CH2-CH2-O)n-CO-NH-(CH2-CH2-O)m-CO-;-, wherein p, q and t are, independently of each other, an integer from 2 to 12, and wherein n and m are, independently of each other, an integer from 2 to 6.
In a particular embodiment of the invention, the linker L is of the following formula (VI):
-Y-X- (VI), wherein:
Y is bound to the moiety A, and Y is selected from the group consisting of -(CH2)n-NH-, -(CH2)n-CO-, -(CH2-CH2-O)n-NH-, and -(CH2-CH2-O)n-CO-;
X is bound to the moiety B, and X is selected from the group consisting of -NH-(CH2)m-CO-, -CO- (CH2)m-NH-, -CO-(CH2)m-CO-, -NH-(CH2-CH2-O)m-CO-, -CO-(CH2-CH2-O)m-NH-, and -CO-(CH2- CH2-O)m-CO-; n is an integer from 2 to 14, preferably from 2 to 12; and and m is an integer from 2 to 6.
In a particular embodiment of the invention, the linker L is of the following formula (VI):
-Y-X- (VI), wherein:
Y is bound to the moiety A, and Y is selected from the group consisting of -(CH2)n-NH-, -(CH2)n-CO-, -(CH2-CH2-O)n-NH-, and -(CH2-CH2-O)n-CO-;
X is bound to the moiety B, and X is selected from the group consisting of -NH-(CH2)m-CO-, -CO- (CH2)m-NH-, -CO-(CH2)m-CO-, -NH-(CH2-CH2-O)m-CO-, -CO-(CH2-CH2-O)m-NH-, and -CO-(CH2- CH2-0)m-C0-; and n and m are, independently of each other, an integer from 2 to 6.
In particular, the linker L can be selected from the group consisting of: -CH2-CH2-, -CH2-CH2-CH2- CH2-, -CH2-CH2-O-CH2-CH2-, -CH2-CH2-O-CH2-CH2-O-CH2-CH2-, -CH2-CH2-NH-CH2-CH2-, -CH2- CH2-CH2-CH2-NH-CH2-CH2-, -CH2-CH2-CH2-CH2-NH-CH2-CH2-CH2-CH2-, -CH2-CH2-C(O)-, -CH2- CH2-CH2-CH2-C(O)-, -C(O)-CH2-CH2-C(O)-, -C(O)-CH2-CH2-CH2-CH2-C(O)-, -CH2-CH2-NHC(O)- CH2-CH2-C(O)-, -CH2-CH2-CH2-CH2-NHC(O)-CH2-CH2-C(O)-, -(CH2)9-CH2-NHC(O)-CH2-CH2- C(O)-, -(CH2)11-CH2-NHC(O)-CH2-CH2-C(O)-, -(CH2)13-CH2-NHC(O)-CH2-CH2-C(O)-, and -(CH2)I7- CH2-NHC(O)-CH2-CH2-C(O)-.
In particular, the linker L can be selected from the group consisting of: -CH2-CH2-, -CH2-CH2-CH2- CH2-, -CH2-CH2-O-CH2-CH2-, -CH2-CH2-O-CH2-CH2-O-CH2-CH2-, -CH2-CH2-NH-CH2-CH2-, -CH2- CH2-CH2-CH2-NH-CH2-CH2-, -CH2-CH2-CH2-CH2-NH-CH2-CH2-CH2-CH2-, -CH2-CH2-C(O)-, -CH2- CH2-CH2-CH2-C(O)-, -C(O)-CH2-CH2-C(O)-, -C(O)-CH2-CH2-CH2-CH2-C(O)-, -CH2-CH2-NHC(O)- CH2-CH2-C(O)-, and -CH2-CH2-CH2-CH2-NHC(O)-CH2-CH2-C(O)-.
In a particular embodiment, the linker L can be -(CH2)n -CH2-NHC(O)-CH2-CH2-C(O)-, wherein n’ is an integer from 1 to 17, preferably n’ is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17, more preferably n’ is 3, 4, 5, 6, 7, 8, 9, 10 or 11.
In a preferred embodiment of the invention, the conjugate A-L-B comprises:
A first moiety A of the general formula (II),
A second moiety B which is marmycin and wherein the linker is bound to said moiety B via the hydroxyl of the sugar moiety of marmycin; and
A linker L which is -(CH2)n -CH2-NHC(O)-CH2-CH2-C(O)-, wherein n’ is an integer from 1 to 17.
In a preferred embodiment of the invention, the conjugate A-L-B comprises:
A first moiety A of the general formula (Ila),
A second moiety B which is marmycin and wherein the linker is bound to said moiety B via the hydroxyl of the sugar moiety of marmycin; and A linker L which is -(CH2)n -CH2-NHC(O)-CH2-CH2-C(O)-, wherein n’ is an integer from 1 to 17.
In a preferred embodiment of the invention, the conjugate A-L-B comprises:
A first moiety A of the general formula (lib),
A second moiety B which is marmycin and wherein the linker is bound to said moiety B via the hydroxyl of the sugar moiety of marmycin; and
A linker L which is -(CH2)n -CH2-NHC(O)-CH2-CH2-C(O)-, wherein n’ is an integer from 1 to 17.
In a preferred embodiment of the invention, the conjugate A-L-B comprises:
A first moiety A of the general formula (lie),
A second moiety B which is marmycin and wherein the linker is bound to said moiety B via the hydroxyl of the sugar moiety of marmycin; and
A linker L which is -(CH2)n -CH2-NHC(O)-CH2-CH2-C(O)-, wherein n’ is an integer from 1 to 17.
In a preferred embodiment of the invention, the conjugate A-L-B comprises:
A first moiety A of the general formula (lid),
A second moiety B which is marmycin and wherein the linker is bound to said moiety B via the hydroxyl of the sugar moiety of marmycin; and
A linker L which is -(CH2)n -CH2-NHC(O)-CH2-CH2-C(O)-, wherein n’ is an integer from 1 to 17.
In a preferred embodiment of the invention, the conjugate A-L-B comprises:
A first moiety A of the general formula (V),
A second moiety B which is marmycin and wherein the linker is bound to said moiety B via the hydroxyl of the sugar moiety of marmycin; and
A linker L which is -(CH2)n -CH2-NHC(O)-CH2-CH2-C(O)-, wherein n’ is an integer from 1 to 17.
In a preferred embodiment of the invention, the conjugate A-L-B comprises:
A first moiety A of the general formula (lie),
A second moiety B which is marmycin and wherein the linker is bound to said moiety B via the hydroxyl of the sugar moiety of marmycin; and
A linker L which is -(CH2)n -CH2-NHC(O)-CH2-CH2-C(O)-, wherein n’ is an integer from 1 to 17.
In a preferred embodiment of the invention, the conjugate A-L-B comprises:
A first moiety A of the general formula (Ilf),
A second moiety B which is marmycin and wherein the linker is bound to said moiety B via the hydroxyl of the sugar moiety of marmycin; and A linker L which is -(CH2)n -CH2-NHC(O)-CH2-CH2-C(O)-, wherein n’ is an integer from 1 to 17.
In a preferred embodiment of the invention, the conjugate A-L-B comprises:
A first moiety A of the general formula (Ilg),
A second moiety B which is marmycin and wherein the linker is bound to said moiety B via the hydroxyl of the sugar moiety of marmycin; and
A linker L which is -(CH2)n -CH2-NHC(O)-CH2-CH2-C(O)-, wherein n’ is an integer from 1 to 17.
In these embodiments, marmycin is preferably marmycin A, in particular of formula (Bl).
In a preferred embodiment of the invention, the conjugate A-L-B comprises:
A first moiety A of the general formula (II),
A second moiety B which is cholesterol of formula (B2); and
A linker L which is -(CH2)n -CH2-NHC(O)-CH2-CH2-C(O)-, wherein n’ is an integer from 1 to 17.
In a preferred embodiment of the invention, the conjugate A-L-B comprises:
A first moiety A of the general formula (Ila),
A second moiety B which is cholesterol of formula (B2); and
A linker L which is -(CH2)n -CH2-NHC(O)-CH2-CH2-C(O)-, wherein n’ is an integer from 1 to 17.
In a preferred embodiment of the invention, the conjugate A-L-B comprises:
A first moiety A of the general formula (lib),
A second moiety B which is cholesterol of formula (B2); and
A linker L which is -(CH2)n -CH2-NHC(O)-CH2-CH2-C(O)-, wherein n’ is an integer from 1 to 17.
In a preferred embodiment of the invention, the conjugate A-L-B comprises:
A first moiety A of the general formula (lie),
A second moiety B which is cholesterol of formula (B2); and
A linker L which is -(CH2)n -CH2-NHC(O)-CH2-CH2-C(O)-, wherein n’ is an integer from 1 to 17.
In a preferred embodiment of the invention, the conjugate A-L-B comprises:
A first moiety A of the general formula (lid),
A second moiety B which is cholesterol of formula (B2); and
A linker L which is -(CH2)n -CH2-NHC(O)-CH2-CH2-C(O)-, wherein n’ is an integer from 1 to 17.
In a preferred embodiment of the invention, the conjugate A-L-B comprises:
A first moiety A of the general formula (V), A second moiety B which is cholesterol of formula (B2); and
A linker L which is -(CH2)n -CH2-NHC(O)-CH2-CH2-C(O)-, wherein n’ is an integer from 1 to 17.
In a preferred embodiment of the invention, the conjugate A-L-B comprises:
A first moiety A of the general formula (lie),
A second moiety B which is cholesterol of formula (B2); and
A linker L which is -(CH2)n -CH2-NHC(O)-CH2-CH2-C(O)-, wherein n’ is an integer from 1 to 17.
In a preferred embodiment of the invention, the conjugate A-L-B comprises:
A first moiety A of the general formula (Ilf),
A second moiety B which is cholesterol of formula (B2); and
A linker L which is -(CH2)n -CH2-NHC(O)-CH2-CH2-C(O)-, wherein n’ is an integer from 1 to 17.
In a preferred embodiment of the invention, the conjugate A-L-B comprises:
A first moiety A of the general formula (Ilg),
A second moiety B which is cholesterol of formula (B2); and
A linker L which is -(CH2)n -CH2-NHC(O)-CH2-CH2-C(O)-, wherein n’ is an integer from 1 to 17.
In a preferred embodiment of the invention, the conjugate A-L-B comprises:
A first moiety A of the general formula (II),
A second moiety B which is cholestanol of formula (B3); and
A linker L which is -(CH2)n -CH2-NHC(O)-CH2-CH2-C(O)-, wherein n’ is an integer from 1 to 17.
In a preferred embodiment of the invention, the conjugate A-L-B comprises:
A first moiety A of the general formula (Ila),
A second moiety B which is cholestanol of formula (B3); and
A linker L which is -(CH2)n -CH2-NHC(O)-CH2-CH2-C(O)-, wherein n’ is an integer from 1 to 17.
In a preferred embodiment of the invention, the conjugate A-L-B comprises:
A first moiety A of the general formula (lib),
A second moiety B which is cholestanol of formula (B3); and
A linker L which is -(CH2)n -CH2-NHC(O)-CH2-CH2-C(O)-, wherein n’ is an integer from 1 to 17.
In a preferred embodiment of the invention, the conjugate A-L-B comprises:
A first moiety A of the general formula (lie),
A second moiety B which is cholestanol of formula (B3); and A linker L which is -(CH2)n -CH2-NHC(O)-CH2-CH2-C(O)-, wherein n’ is an integer from 1 to 17.
In a preferred embodiment of the invention, the conjugate A-L-B comprises:
A first moiety A of the general formula (lid),
A second moiety B which is cholestanol of formula (B3); and
A linker L which is -(CH2)n -CH2-NHC(O)-CH2-CH2-C(O)-, wherein n’ is an integer from 1 to 17.
In a preferred embodiment of the invention, the conjugate A-L-B comprises:
A first moiety A of the general formula (V),
A second moiety B which is cholestanol of formula (B3); and
A linker L which is -(CH2)n -CH2-NHC(O)-CH2-CH2-C(O)-, wherein n’ is an integer from 1 to 17.
In a preferred embodiment of the invention, the conjugate A-L-B comprises:
A first moiety A of the general formula (lie),
A second moiety B which is cholestanol of formula (B3); and
A linker L which is -(CH2)n -CH2-NHC(O)-CH2-CH2-C(O)-, wherein n’ is an integer from 1 to 17.
In a preferred embodiment of the invention, the conjugate A-L-B comprises:
A first moiety A of the general formula (Ilf),
A second moiety B which is cholestanol of formula (B3); and
A linker L which is -(CH2)n -CH2-NHC(O)-CH2-CH2-C(O)-, wherein n’ is an integer from 1 to 17.
In a preferred embodiment of the invention, the conjugate A-L-B comprises:
A first moiety A of the general formula (Ilg),
A second moiety B which is cholestanol of formula (B3); and
A linker L which is -(CH2)n -CH2-NHC(O)-CH2-CH2-C(O)-, wherein n’ is an integer from 1 to 17.
In a preferred embodiment of the invention, the conjugate A-L-B comprises:
A first moiety A of the general formula (II),
A second moiety B which is of formula (B4); and
A linker L which is -(CH2)n -CH2-NHC(O)-CH2-CH2-C(O)-, wherein n’ is an integer from 1 to 17.
In a preferred embodiment of the invention, the conjugate A-L-B comprises:
A first moiety A of the general formula (Ila),
A second moiety B which is of formula (B4); and A linker L which is -(CH2)n -CH2-NHC(O)-CH2-CH2-C(O)-, wherein n’ is an integer from 1 to 17.
In a preferred embodiment of the invention, the conjugate A-L-B comprises:
A first moiety A of the general formula (lib),
A second moiety B which is of formula (B4); and
A linker L which is -(CH2)n -CH2-NHC(O)-CH2-CH2-C(O)-, wherein n’ is an integer from 1 to 17.
In a preferred embodiment of the invention, the conjugate A-L-B comprises:
A first moiety A of the general formula (lie),
A second moiety B which is of formula (B4); and
A linker L which is -(CH2)n -CH2-NHC(O)-CH2-CH2-C(O)-, wherein n’ is an integer from 1 to 17.
In a preferred embodiment of the invention, the conjugate A-L-B comprises:
A first moiety A of the general formula (lid),
A second moiety B which is of formula (B4); and
A linker L which is -(CH2)n -CH2-NHC(O)-CH2-CH2-C(O)-, wherein n’ is an integer from 1 to 17.
In a preferred embodiment of the invention, the conjugate A-L-B comprises:
A first moiety A of the general formula (V),
A second moiety B which is of formula (B4); and
A linker L which is -(CH2)n -CH2-NHC(O)-CH2-CH2-C(O)-, wherein n’ is an integer from 1 to 17.
In a preferred embodiment of the invention, the conjugate A-L-B comprises:
A first moiety A of the general formula (lie),
A second moiety B which is of formula (B4); and
A linker L which is -(CH2)n -CH2-NHC(O)-CH2-CH2-C(O)-, wherein n’ is an integer from 1 to 17.
In a preferred embodiment of the invention, the conjugate A-L-B comprises:
A first moiety A of the general formula (Ilf),
A second moiety B which is of formula (B4); and
A linker L which is -(CH2)n -CH2-NHC(O)-CH2-CH2-C(O)-, wherein n’ is an integer from 1 to 17.
In a preferred embodiment of the invention, the conjugate A-L-B comprises:
A first moiety A of the general formula (Ilg),
A second moiety B which is of formula (B4); and
A linker L which is -(CH2)n -CH2-NHC(O)-CH2-CH2-C(O)-, wherein n’ is an integer from 1 to 17. In a preferred embodiment of the invention, the conjugate A-L-B comprises:
A first moiety A of the general formula (II),
A second moiety B which is of formula (B4); and
A linker L which is -(CH2)t wherein t is an integer from 1 to 20.
In a preferred embodiment of the invention, the conjugate A-L-B comprises:
A first moiety A of the general formula (II),
A second moiety B which is of formula (B5); and
A linker L which is -(CH2)n -CH2-NHC(O)-CH2-CH2-C(O)-, wherein n’ is an integer from 1 to 17.
In a preferred embodiment of the invention, the conjugate A-L-B comprises:
A first moiety A of the general formula (Ila),
A second moiety B which is of formula (B5); and
A linker L which is -(CH2)n -CH2-NHC(O)-CH2-CH2-C(O)-, wherein n’ is an integer from 1 to 17.
In a preferred embodiment of the invention, the conjugate A-L-B comprises:
A first moiety A of the general formula (lib),
A second moiety B which is of formula (B5); and
A linker L which is -(CH2)n -CH2-NHC(O)-CH2-CH2-C(O)-, wherein n’ is an integer from 1 to 17.
In a preferred embodiment of the invention, the conjugate A-L-B comprises:
A first moiety A of the general formula (lie),
A second moiety B which is of formula (B5); and
A linker L which is -(CH2)n -CH2-NHC(O)-CH2-CH2-C(O)-, wherein n’ is an integer from 1 to 17.
In a preferred embodiment of the invention, the conjugate A-L-B comprises:
A first moiety A of the general formula (lid),
A second moiety B which is of formula (B5); and
A linker L which is -(CH2)n -CH2-NHC(O)-CH2-CH2-C(O)-, wherein n’ is an integer from 1 to 17.
In a preferred embodiment of the invention, the conjugate A-L-B comprises:
A first moiety A of the general formula (V),
A second moiety B which is of formula (B5); and
A linker L which is -(CH2)n -CH2-NHC(O)-CH2-CH2-C(O)-, wherein n’ is an integer from 1 to 17.
In a preferred embodiment of the invention, the conjugate A-L-B comprises: A first moiety A of the general formula (lie),
A second moiety B which is of formula (B5); and
A linker L which is -(CH2)n -CH2-NHC(O)-CH2-CH2-C(O)-, wherein n’ is an integer from 1 to 17.
In a preferred embodiment of the invention, the conjugate A-L-B comprises:
A first moiety A of the general formula (Ilf),
A second moiety B which is of formula (B5); and
A linker L which is -(CH2)n -CH2-NHC(O)-CH2-CH2-C(O)-, wherein n’ is an integer from 1 to 17.
In a preferred embodiment of the invention, the conjugate A-L-B comprises:
A first moiety A of the general formula (Ilg),
A second moiety B which is of formula (B5); and
A linker L which is -(CH2)n -CH2-NHC(O)-CH2-CH2-C(O)-, wherein n’ is an integer from 1 to 17.
In a preferred embodiment of the invention, the conjugate A-L-B comprises:
A first moiety A of the general formula (II),
A second moiety B which is of formula (B5); and
A linker L which is -(CILjt, wherein t is an integer from 1 to 20.
In a particular embodiment, the conjugate of the invention is selected from the group consisting of Fentomycin of general formula (III), Fentomycin-1 of general formula (III ), Pre-Fentomycin of general formula (IV), Fentomycin-2 of general formula (VII), a compound of formula (VIF), Fentomycin-3 of general formula (VIII), a compound of formula (VIII’), Fentomycin-5 of general formula (IX), a compound of formula (IX’), Fentomycin-6 of general formula (X), a compound of formula (X’), Fentomycin-7 of general formula (XI), a compound of formula (XI’), Fentomycin-8 of general formula (XII), a compound of formula (XIF), Fentomycin-9 of general formula (XIII), a compound of formula (XIII’), Fentomycin-10 of general formula (XIV), a compound of formula (XIV’), Fentomycin-11 of general formula (XV), a compound of formula (XV’), Fentomycin- 12 of general formula (XVI), a compound of formula (XVI’), Fentomycin- 13 of general formula (XVII), a compound of formula (XVII’), Fentomycin-14 of general formula (XVIII), a compound of formula (XVIII’), Fentomycin-15 of general formula (XIX) a compound of formula (XIX’), Fentomycin- 16 of general formula (XX) and a compound of formula (XX’); or a stereoisomers thereof or a pharmaceutically acceptable salt and/or solvate thereof:
(XI)
617
zs
In a particular embodiment, the conjugate of the invention is selected from the group consisting of Fentomycin of general formula (III), Fentomycin-1 of general formula (III ), Pre-Fentomycin of general formula (IV), Fentomycin-2 of general formula (VII), a compound of formula (VIF), Fentomycin-3 of general formula (VIII), a compound of formula (VIII’), Fentomycin-5 of general formula (IX) , a compound of formula (IX’), Fentomycin-6 of general formula (X), a compound of formula (X’), Fentomycin-7 of general formula (XI), a compound of formula (XI’), Fentomycin-8 of general formula (XII), a compound of formula (XIF), Fentomycin-9 of general formula (XIII), a compound of formula (XIII’), Fentomycin-10 of general formula (XIV), a compound of formula (XIV’), Fentomycin-13 of general formula (XVII), a compound of formula (XVIF), and Fentomycin-14 of general formula (XVIII), a compound of formula (XVIIF); or a stereoisomers thereof or a pharmaceutically acceptable salt and/or solvate thereof.
In a particular embodiment, the conjugate of the invention is selected from the group consisting of Fentomycin of general formula (III), Fentomycin-1 of general formula (III ), Pre-Fentomycin of general formula (IV), Fentomycin-2 of general formula (VII), Fentomycin-3 of general formula (VIII), Fentomycin-5 of general formula (IX), Fentomycin-6 of general formula (X), Fentomycin-7 of general formula (XI), Fentomycin-8 of general formula (XII), Fentomycin-9 of general formula (XIII), Fentomycin-10 of general formula (XIV), Fentomycin-11 of general formula (XV), Fentomycin-12 of general formula (XVI), Fentomycin-13 of general formula (XVII), Fentomycin-14 of general formula (XVIII), Fentomycin-15 of general formula (XIX), and Fentomycin-16 of general formula (XX); or a stereoisomers thereof or a pharmaceutically acceptable salt and/or solvate thereof. In a particular embodiment, the conjugate of the invention is selected from the group consisting of Fentomycin of general formula (III), Fentomycin-1 of general formula (III ), Pre-Fentomycin of general formula (IV), Fentomycin-2 of general formula (VII), Fentomycin-3 of general formula (VIII), Fentomycin-5 of general formula (IX), Fentomycin-6 of general formula (X), Fentomycin-7 of general formula (XI), Fentomycin-8 of general formula (XII), Fentomycin-9 of general formula (XIII), Fentomycin- 10 of general formula (XIV), Fentomycin- 13 of general formula (XVII), and Fentomycin- 14 of general formula (XVIII); or a stereoisomers thereof or a pharmaceutically acceptable salt and/or solvate thereof.
In a particular embodiment, the conjugate of the invention is selected from the group consisting of Fentomycin of general formula (III), Fentomycin-1 of general formula (III ), Pre-Fentomycin of general formula (IV), Fentomycin-2 of general formula (VII), Fentomycin-3 of general formula (VIII), and Fentomycin-5 of general formula (IX); or a stereoisomers thereof or a pharmaceutically acceptable salt and/or solvate thereof.
In a particular embodiment, the conjugate of the invention is selected from the group consisting of Fentomycin of general formula (III), Fentomycin-1 of general formula (III ), Pre-Fentomycin of general formula (IV), Fentomycin-2 of general formula (VII), Fentomycin-3 of general formula (VIII), and Fentomycin-5 of general formula (IX)
In a particular embodiment, the conjugate of the invention is Fentomycin of general formula (III) as disclosed herein, or Pre-Fentomycin of general formula (IV) as disclosed herein, or a stereoisomer thereof.
In a particular embodiment, the conjugate of the invention is Fentomycin of general formula (III) as disclosed herein.
In a particular embodiment, the conjugate of the invention is Fentomycin-1 of general formula (III’) as disclosed herein.
In a particular embodiment, the conjugate of the invention is Pre-Fentomycin of general formula (IV) as disclosed herein.
In a particular embodiment, the conjugate of the invention is Fentomycin-2 of general formula (VII) or a compound of formula (VIF) as disclosed herein.
In a particular embodiment, the conjugate of the invention is Fentomycin-3 of general formula (VIII) or a compound of formula (VIIF) -as disclosed herein.
In a particular embodiment, the conjugate of the invention is Fentomycin-5 of general formula (IX) or a compound of formula (IX’) as disclosed herein.
In a particular embodiment, the conjugate of the invention is Fentomycin-6 of general formula (X) or a compound of formula (X’) as disclosed herein.
In a particular embodiment, the conjugate of the invention is Fentomycin-7 of general formula (XI) or a compound of formula (XF) as disclosed herein. In a particular embodiment, the conjugate of the invention is Fentomycin-8 of general formula (XII) or a compound of formula (XIF) as disclosed herein.
In a particular embodiment, the conjugate of the invention is Fentomycin-9 of general formula (XIII) or a compound of formula (XIIF) as disclosed herein.
In a particular embodiment, the conjugate of the invention is Fentomycin- 10 of general formula (XIV) or a compound of formula (XIV’) as disclosed herein.
In a particular embodiment, the conjugate of the invention is Fentomycin- 11 of general formula (XV) or a compound of formula (XV’) as disclosed herein.
In a particular embodiment, the conjugate of the invention is Fentomycin-12 of general formula (XVI) or a compound of formula (XVI’) as disclosed herein.
In a particular embodiment, the conjugate of the invention is Fentomycin- 13 of general formula (XVII) or a compound of formula (XVIF) as disclosed herein.
In a particular embodiment, the conjugate of the invention is Fentomycin- 14 of general formula (XVIII) or a compound of formula (XVIIF) as disclosed herein.
In a particular embodiment, the conjugate of the invention is Fentomycin- 15 of general formula (XIX) or a compound of formula (XIX’) as disclosed herein.
In a particular embodiment, the conjugate of the invention is Fentomycin- 16 of general formula (XX) or a compound of formula (XX’) as disclosed herein.
Compounds of the invention
A further object of the invention relates to the compound Fentomycin of general formula (III), or a pharmaceutically acceptable salt and/or solvate thereof, or a stereoisomer thereof: wherein R1 is H or Cl.
A further object of the invention relates to the compound Fentomycin-1 of general formula (IIP), or a pharmaceutically acceptable salt and/or solvate thereof, or a stereoisomer thereof: (iir).
A further object of the invention relates to the compound Pre-Fentomycin of general formula (IV), or a pharmaceutically acceptable salt and/or solvate thereof, or a stereoisomer thereof: A further object of the invention relates to the compound Fentomycin-2 of general formula (VII) or a compound of formula (VII’), or a pharmaceutically acceptable salt and/or solvate thereof, or a stereoisomer thereof: A further object of the invention relates to the compound Fentomycin-3 of general formula (VIII) or a compound of formula (VIII’), or a pharmaceutically acceptable salt and/or solvate thereof, or a stereoisomer thereof: . A further object of the invention relates to the compound Fentomycin-5 of general formula (IX) or a compound of formula (IX’), or a pharmaceutically acceptable salt and/or solvate thereof, or a stereoisomer thereof: A further object of the invention relates to the compound Fentomycin-6 of general formula (X) or a compound of formula (X’), or a pharmaceutically acceptable salt and/or solvate thereof, or a stereoisomer thereof: A further object of the invention relates to the compound Fentomycin-7 of general formula (XI) or a compound of formula (XI’), or a pharmaceutically acceptable salt and/or solvate thereof, or a stereoisomer thereof: A further object of the invention relates to the compound Fentomycin-8 of general formula (XII) or a compound of formula (XII’), or a pharmaceutically acceptable salt and/or solvate thereof, or a stereoisomer thereof: . A further object of the invention relates to the compound Fentomycin-9 of general formula (XIII) or a compound of formula (XIII’), or a pharmaceutically acceptable salt and/or solvate thereof, or a stereoisomer thereof: ). A further object of the invention relates to the compound Fentomycin-10 of general formula (XIV) or (XIV’), or a pharmaceutically acceptable salt and/or solvate thereof, or a stereoisomer thereof: A further object of the invention relates to the compound Fentomycin- 11 of general formula (XV) or a compound of formula (XV’), or a pharmaceutically acceptable salt and/or solvate thereof, or a stereoisomer thereof: A further object of the invention relates to the compound Fentomycin-12 of general formula (XVI) or a compound of formula (XVI’), or a pharmaceutically acceptable salt and/or solvate thereof, or a stereoisomer thereof: . A further object of the invention relates to the compound Fentomycin-13 of general formula (XVII) or a compound of formula (XVII’), or a pharmaceutically acceptable salt and/or solvate thereof, or a stereoisomer thereof: A further object of the invention relates to the compound Fentomycin-14 of general formula (XVIII) or a compound of formula (XVIII’), or a pharmaceutically acceptable salt and/or solvate thereof, or a stereoisomer thereof: . A further object of the invention relates to the compound Fentomycin-15 of general formula (XIX) or a compound of formula (XIX’), or a pharmaceutically acceptable salt and/or solvate thereof, or a stereoisomer thereof: A further object of the invention relates to the compound Fentomycin-16 of general formula (XX) or a compound of formula (XX’), or a pharmaceutically acceptable salt and/or solvate thereof, or a stereoisomer thereof: Pharmaceutical compositions
Another object of the invention relates to a pharmaceutical composition comprising a conjugate according to the invention, or a compound of formula (III), (III’), (IV), (VII), (VII’), (VIII), (VIII’), (IX) (ix’), (X), (X’), (xi), (xr), (xii), (xir), (xiii), (xiir), (xiv), (xiv’), (xv), (xv’), (xvi), (xvr), (XVII), (XVII’), (XVIII), (XVIII’), (XIX), (XIX’), (XX), and (XX’); preferably (III), (III’), (IV), (VII), (vir), (viii), (VIIF), (ix) (ix’), (X), (X’), (xi), (xr), (xii), (xir), (xiii), (xiir), (xiv), (xiv’), (XVII), (XVII’), (XVIII), and (XVIII’); according to the invention or a pharmaceutically acceptable salt and/or solvate thereof, and at least one pharmaceutically acceptable excipient, or a stereoisomer thereof. Another object of the invention relates to a pharmaceutical composition comprising a conjugate according to the invention, or a compound of formula (III), (III’), (IV), (VII), (VIII), (IX), (X), (XI), (XII), (XIII), (XIV), (XV), (XVI), (XVII), and (XVIII); preferably (III), (III’), (IV), (VII), (VIII), (IX), (X), (XI), (XII), (XIII), (XIV), (XVII), and (XVIII); according to the invention or a pharmaceutically acceptable salt and/or solvate thereof, and at least one pharmaceutically acceptable excipient, or a stereoisomer thereof.
Another object of the invention relates to nanoparticle comprising a conjugate according to the invention, or a compound of formula (III), (III’), (IV), (VII), (VII’), (VIII), (VIII’), (IX) (IX’), (X), (X’), (xi), (xr), (xii), (xir), (xiii), (xiir), (xiv), (xiv’), (xv), (xv’), (xvi), (xvr), (xvii), (xvir), (XVIII), (XVIII’), (XIX), (XIX’), (XX), and (XX’); preferably (III), (III’), (IV), (VII), (VII’), (VIII), (viir), (ix) (ix’), (X), (X’), (xi), (xr), (xii), (xir), (xiii), (xiir), (xiv), (xiv’), (xvii), (xvir), (XVIII), and (XVIII’); according to the invention or a pharmaceutically acceptable salt and/or solvate thereof, or a stereoisomer thereof.
Another object of the invention relates to nanoparticle comprising a conjugate according to the invention, or a compound of formula (III), (III’), (IV), (VII), (VIII), (IX), (X), (XI), (XII), (XIII), (XIV), (XV), (XVI), (XVII), and (XVIII); preferably (III), (III’), (IV), (VII), (VIII), (IX), (X), (XI), (XII), (XIII), (XIV), (XVII), and (XVIII); according to the invention or a pharmaceutically acceptable salt and/or solvate thereof, or a stereoisomer thereof.
Another object of the invention relates to a pharmaceutical composition comprising a conjugate according to the invention, or a compound of formula (III), (III’), (IV), (VII), (VIII), and (IX), according to the invention or a pharmaceutically acceptable salt and/or solvate thereof, and at least one pharmaceutically acceptable excipient.
Another object of the invention relates to nanoparticle comprising a conjugate according to the invention, or a compound of formula (III), (III’), (IV), (VII), (VIII), and (IX), according to the invention or a pharmaceutically acceptable salt and/or solvate thereof.
Another object of the invention relates to a pharmaceutical composition comprising a conjugate according to the invention, or a compound of formula (III) or (IV) according to the invention or a pharmaceutically acceptable salt and/or solvate thereof, and at least one pharmaceutically acceptable excipient. Another object of the invention relates to nanoparticle comprising a conjugate according to the invention, or a compound of formula (III) or (IV) according to the invention or a pharmaceutically acceptable salt and/or solvate thereof.
The pharmaceutical compositions contemplated herein may include a pharmaceutically acceptable carrier in addition to the active ingredient(s). The term "pharmaceutically acceptable carrier" is meant to encompass any carrier (e.g., support, substance, solvent, etc.) which does not interfere with effectiveness of the biological activity of the active ingredient(s) and that is not toxic to the host to which it is administered. For example, for parental administration, the active compounds(s) may be formulated in a unit dosage form for injection in vehicles such as saline, dextrose solution, serum albumin and Ringer's solution.
The pharmaceutical composition can be formulated as solutions in pharmaceutically compatible solvents or as emulsions, suspensions or dispersions in suitable pharmaceutical solvents or vehicle, or as pills, tablets or capsules that contain solid vehicles in a way known in the art. Formulations of the present invention suitable for oral administration may be in the form of discrete units as capsules, sachets, tablets or lozenges, each containing a predetermined amount of the active ingredient; in the form of a powder or granules; in the form of a solution or a suspension in an aqueous liquid or non-aqueous liquid; or in the form of an oil-in-water emulsion or a water-in-oil emulsion. Formulations suitable for parental administration conveniently comprise a sterile oily or aqueous preparation of the active ingredient which is preferably isotonic with the blood of the recipient. Every such formulation can also contain other pharmaceutically compatible and nontoxic auxiliary agents, such as, e.g. stabilizers, antioxidants, binders, dyes, emulsifiers or flavoring substances. The formulations of the present invention comprise an active ingredient in association with a pharmaceutically acceptable carrier therefore and optionally other therapeutic ingredients. The carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulations and not deleterious to the recipient thereof. The pharmaceutical compositions are advantageously applied by injection or intravenous infusion of suitable sterile solutions or as oral dosage by the digestive tract or by direct injection into the lymph node (i.e., intra- lymphatically). Methods for the safe and effective administration of most of these chemotherapeutic agents are known to those skilled in the art. In addition, their administration is described in the standard literature.
The pharmaceutical or veterinary composition as disclosed herein may further comprise an additional active ingredient or drug.
Therapeutic uses
Accordingly, the present invention relates to a conjugate as disclosed herein or a compound of general formula (III), (III’), (IV), (VII), (VIF), (VIII), (VIIF), (IX) (IX’), (X), (X’), (XI), (XT), (XII), (XIT), (XIII), (XIIF), (XIV), (XIV’), (XV), (XV’), (XVI), (XVI’), (XVII), (XVIF), (XVIII), (XVIIF), (XIX), (XIX’), (XX), and (XX’); preferably (III), (III’), (IV), (VII), (VII’), (VIII), (VIII’), (IX) (IX’), (X), (X’), (XI), (XI’), (XII), (XII’), (XIII), (XIII’), (XIV), (XIV’), (XVII), (XVII’), (XVIII), and (XVIII’); as disclosed herein, or a pharmaceutical composition as disclosed herein, or a nanoparticle as disclosed herein, for use as a drug.
The present invention relates to the use of a conjugate as disclosed herein or a compound of general formula (III), (III’), (IV), (VII), (VII’), (VIII), (VIII’), (IX) (IX’), (X), (X’), (XI), (XI’), (XII), (XII’), (XIII), (XIII’), (XIV), (XIV’), (XV), (XV’), (XVI), (XVI’), (XVII), (XVII’), (XVIII), (XVIII’), (XIX), (XIX’), (XX), and (XX’); preferably (III), (III’), (IV), (VII), (VII’), (VIII), (VIII’), (IX) (IX’), (X), (X’),
(XI), (XI’), (XII), (XIT), (XIII), (XIII’), (XIV), (XIV’), (XVII), (XVII’), (XVIII), and (XVIII’); as disclosed herein, or a pharmaceutical composition as disclosed herein, or a nanoparticle as disclosed herein, for the manufacture of a medicament.
The invention further relates to the method for the treatment of a disease in a subject in need thereof, comprising administering a therapeutic effective amount of a conjugate as disclosed herein or a compound of general formula (III), (III’), (IV), (VII), (VII’), (VIII), (VIII’), (IX) (IX’), (X), (X’), (XI), (XT), (XII), (XIT), (XIII), (XIII’), (XIV), (XIV’), (XV), (XV’), (XVI), (XVI’), (XVII), (XVII’), (XVIII), (XVIII’), (XIX), (XIX’), (XX), and (XX’); preferably (III), (III’), (IV), (VII), (VII’), (VIII), (VIII’), (IX) (IX’), (X), (X’), (XI), (XT), (XII), (XIT), (XIII), (XIII’), (XIV), (XIV’), (XVII), (XVII’), (XVIII), and (XVIII’); as disclosed herein, or a pharmaceutical composition as disclosed herein, or a nanoparticle as disclosed herein, to said subject.
Accordingly, the present invention relates to a conjugate as disclosed herein or a compound of general formula (III), (III’), (IV), (VII), (VIII), (IX), (X), (XI), (XII), (XIII), (XIV), (XV), (XVI), (XVII), and (XVIII), (XIX), and (XX); preferably (III), (III’), (IV), (VII), (VIII), (IX), (X), (XI), (XII), (XIII), (XIV), (XVII), and (XVIII); as disclosed herein, or a pharmaceutical composition as disclosed herein, or a nanoparticle as disclosed herein, for use as a drug.
The present invention relates to the use of a conjugate as disclosed herein or a compound of general formula (III), (III’), (IV), (VII), (VIII), (IX), (X), (XI), (XII), (XIII), (XIV), (XV), (XVI), (XVII), and (XVIII), (XIX), and (XX); preferably (III), (III’), (IV), (VII), (VIII), (IX), (X), (XI), (XII), (XIII), (XIV), (XVII), and (XVIII); as disclosed herein, or a pharmaceutical composition as disclosed herein, or a nanoparticle as disclosed herein, for the manufacture of a medicament.
The invention further relates to the method for the treatment of a disease in a subject in need thereof, comprising administering a therapeutic effective amount of a conjugate as disclosed herein or a compound of general formula (III), (III’), (IV), (VII), (VIII), (IX), (X), (XI), (XII), (XIII), (XIV), (XV), (XVI), (XVII), and (XVIII), (XIX), and (XX); preferably (III), (III’), (IV), (VII), (VIII), (IX), (X), (XI),
(XII), (XIII), (XIV), (XVII), and (XVIII); as disclosed herein, or a pharmaceutical composition as disclosed herein, or a nanoparticle as disclosed herein, to said subject. Accordingly, the present invention relates to a conjugate as disclosed herein or a compound of general formula (III), (III’), (IV), (VII), (VIII), and (IX), as disclosed herein, or a pharmaceutical composition as disclosed herein, or a nanoparticle as disclosed herein, for use as a drug.
The present invention relates to the use of a conjugate as disclosed herein or a compound of general formula (III), (III’), (IV), (VII), (VIII), and (IX), as disclosed herein, or a pharmaceutical composition as disclosed herein, or a nanoparticle as disclosed herein, for the manufacture of a medicament.
The invention further relates to the method for the treatment of a disease in a subject in need thereof, comprising administering a therapeutic effective amount of a conjugate as disclosed herein or a compound of general formula (III), (III’), (IV), (VII), (VIII), and (IX), as disclosed herein, or a pharmaceutical composition as disclosed herein, or a nanoparticle as disclosed herein, to said subject. Accordingly, the present invention relates to a conjugate as disclosed herein or a compound of general formula (III) or (IV) as disclosed herein, or a pharmaceutical composition as disclosed herein, or a nanoparticle as disclosed herein, for use as a drug.
The present invention relates to the use of a conjugate as disclosed herein or a compound of general formula (III) or (IV) as disclosed herein, or a pharmaceutical composition as disclosed herein, or a nanoparticle as disclosed herein, for the manufacture of a medicament.
The invention further relates to the method for the treatment of a disease in a subject in need thereof, comprising administering a therapeutic effective amount of a conjugate as disclosed herein or a compound of general formula (III) or (IV) as disclosed herein, or a pharmaceutical composition as disclosed herein, or a nanoparticle as disclosed herein, to said subject.
The present invention also relates to a conjugate as disclosed herein or a compound of general formula (III), (III’), (IV), (VII), (VIT), (VIII), (VIIT), (IX) (IX’), (X), (X’), (XI), (XT), (XII), (XII’), (XIII), (XIII’), (XIV), (XIV’), (XV), (XV’), (XVI), (XVI’), (XVII), (XVII’), (XVIII), (XVIII’), (XIX), (XIX’), (XX), and (XX’); preferably (III), (III’), (IV), (VII), (VIT), (VIII), (VIII’), (IX) (IX’), (X), (X’), (XI), (XT), (XII), (XIT), (XIII), (XIII’), (XIV), (XIV’), (XVII), (XVII’), (XVIII), and (XVIII’); as disclosed herein, or a pharmaceutical composition as disclosed herein, or a nanoparticle as disclosed herein, for use in a method for the treatment of cancer.
The present invention relates to the use of a conjugate as disclosed herein or a compound of general formula (III), (III’), (IV), (VII), (VIT), (VIII), (VIII’), (IX) (IX’), (X), (X’), (XI), (XT), (XII), (XIT), (XIII), (XIII’), (XIV), (XIV’), (XV), (XV’), (XVI), (XVI’), (XVII), (XVII’), (XVIII), (XVIII’), (XIX), (XIX’), (XX), and (XX’); preferably (III), (III’), (IV), (VII), (VIT), (VIII), (VIII’), (IX) (IX’), (X), (X’), (XI), (XT), (XII), (XIT), (XIII), (XIII’), (XIV), (XIV’), (XVII), (XVII’), (XVIII), and (XVIII’); as disclosed herein, or a pharmaceutical composition as disclosed herein, or a nanoparticle as disclosed herein, for the manufacture of a medicament useful as anti-tumoral agent or useful in a method for the treatment of cancer.
The invention further relates to the method for the treatment of a subject suffering of cancer, comprising administering a therapeutic effective amount of a conjugate as disclosed herein or a compound of general formula (III), (III’), (IV), (VII), (VII’), (VIII), (VIII’), (IX) (IX’), (X), (X’), (XI), (XI’), (XII), (XII’), (XIII), (XIII’), (XIV), (XIV’), (XV), (XV’), (XVI), (XVI’), (XVII), (XVII’), (XVIII), (XVIII’), (XIX), (XIX’), (XX), and (XX’); preferably (III), (III’), (IV), (VII), (VII’), (VIII), (VIII’), (IX) (IX’), (X), (X’), (XI), (XI’), (XII), (XII’), (XIII), (XIII’), (XIV), (XIV’), (XVII), (XVII’), (XVIII), and (XVIII’); as disclosed herein, or a pharmaceutical composition as disclosed herein, or a nanoparticle as disclosed herein, to said subject.
The present invention also relates to a conjugate as disclosed herein or a compound of general formula (III), (III’), (IV), (VII), (VIII), (IX), (X), (XI), (XII), (XIII), (XIV), (XV), (XVI), (XVII), and (XVIII), (XIX), and (XX); preferably (III), (III’), (IV), (VII), (VIII), (IX), (X), (XI), (XII), (XIII), (XIV), (XVII), and (XVIII); as disclosed herein, or a pharmaceutical composition as disclosed herein, or a nanoparticle as disclosed herein, for use in a method for the treatment of cancer.
The present invention relates to the use of a conjugate as disclosed herein or a compound of general formula (III), (III’), (IV), (VII), (VIII), (IX), (X), (XI), (XII), (XIII), (XIV), (XV), (XVI), (XVII), and (XVIII), (XIX), and (XX); preferably (III), (III’), (IV), (VII), (VIII), (IX), (X), (XI), (XII), (XIII), (XIV), (XVII), and (XVIII); as disclosed herein, or a pharmaceutical composition as disclosed herein, or a nanoparticle as disclosed herein, for the manufacture of a medicament useful as anti-tumoral agent or useful in a method for the treatment of cancer.
The invention further relates to the method for the treatment of a subject suffering of cancer, comprising administering a therapeutic effective amount of a conjugate as disclosed herein or a compound of general formula (III), (III’), (IV), (VII), (VIII), (IX), (X), (XI), (XII), (XIII), (XIV), (XV), (XVI), (XVII), and (XVIII), (XIX), and (XX); preferably (III), (III’), (IV), (VII), (VIII), (IX), (X), (XI), (XII), (XIII), (XIV), (XVII), and (XVIII); as disclosed herein, or a pharmaceutical composition as disclosed herein, or a nanoparticle as disclosed herein, to said subject.
The present invention also relates to a conjugate as disclosed herein or a compound of general formula (III), (III’), (IV), (VII), (VIII), and (IX), as disclosed herein, or a pharmaceutical composition as disclosed herein, or a nanoparticle as disclosed herein, for use in a method for the treatment of cancer. The present invention relates to the use of a conjugate as disclosed herein or a compound of general formula (III), (III’), (IV), (VII), (VIII), and (IX), as disclosed herein, or a pharmaceutical composition as disclosed herein, or a nanoparticle as disclosed herein, for the manufacture of a medicament useful as anti-tumoral agent or useful in a method for the treatment of cancer.
The invention further relates to the method for the treatment of a subject suffering of cancer, comprising administering a therapeutic effective amount of a conjugate as disclosed herein or a compound of general formula (III), (III’), (IV), (VII), (VIII), and (IX), as disclosed herein, or a pharmaceutical composition as disclosed herein, or a nanoparticle as disclosed herein, to said subject.
The present invention also relates to a conjugate as disclosed herein or a compound of general formula (III) or (IV) as disclosed herein, or a pharmaceutical composition as disclosed herein, or a nanoparticle as disclosed herein, for use in a method for the treatment of cancer. The present invention relates to the use of a conjugate as disclosed herein or a compound of general formula (III) or (IV) as disclosed herein, or a pharmaceutical composition as disclosed herein, or a nanoparticle as disclosed herein, for the manufacture of a medicament useful as anti-tumoral agent or useful in a method for the treatment of cancer.
The invention further relates to the method for the treatment of a subject suffering of cancer, comprising administering a therapeutic effective amount of a conjugate as disclosed herein or a compound of general formula (III) or (IV) as disclosed herein, or a pharmaceutical composition as disclosed herein, or a nanoparticle as disclosed herein, to said subject.
In a more particular aspect of the invention, the conjugate as disclosed herein or the compound of general formula (III), (III’), (IV), (VII), (VII’), (VIII), (VIIT), (IX) (IX’), (X), (X’), (XI), (XT), (XII), (XIT), (XIII), (XIIT), (XIV), (XIV’), (XV), (XV’), (XVI), (XVI’), (XVII), (XVII’), (XVIII), (XVIII’), (XIX), (XIX’), (XX), and (XX’); preferably (III), (III’), (IV), (VII), (VIT), (VIII), (VIII’), (IX) (IX’), (X), (X’), (XI), (XT), (XII), (XIT), (XIII), (XIII’), (XIV), (XIV’), (XVII), (XVII’), (XVIII), and (XVIII’); as disclosed herein, or the pharmaceutical composition as disclosed herein, or the nanoparticle as disclosed herein, are for use in a targeted treatment of Cancer Stem Cells (CSCs).
In a more particular aspect of the invention, the conjugate as disclosed herein or the compound of general formula (III), (III’), (IV), (VII), (VIII), (IX), (X), (XI), (XII), (XIII), (XIV), (XV), (XVI), (XVII), and (XVIII), (XIX), and (XX); preferably (III), (III’), (IV), (VII), (VIII), (IX), (X), (XI), (XII), (XIII), (XIV), (XVII), and (XVIII); as disclosed herein, or the pharmaceutical composition as disclosed herein, or the nanoparticle as disclosed herein, are for use in a targeted treatment of Cancer Stem Cells (CSCs). In a more particular aspect of the invention, the conjugate as disclosed herein or the compound of general formula (III), (III’), (IV), (VII), (VIII), and (IX) as disclosed herein, or the pharmaceutical composition as disclosed herein, or the nanoparticle as disclosed herein, are for use in a targeted treatment of Cancer Stem Cells (CSCs).
In a more particular aspect of the invention, the conjugate as disclosed herein or the compound of general formula (III) or (IV) as disclosed herein, or the pharmaceutical composition as disclosed herein, or the nanoparticle as disclosed herein, are for use in a targeted treatment of Cancer Stem Cells (CSCs).
As used herein, the term “cancer” refers to any cancer that may affect any one of the following tissues or organs: breast; liver; kidney; heart, mediastinum, pleura; floor of mouth; lip; salivary glands; tongue; gums; oral cavity; palate; tonsil; larynx; trachea; bronchus, lung; pharynx, hypopharynx, oropharynx, nasopharynx; esophagus; digestive organs such as stomach, intrahepatic bile ducts, biliary tract, pancreas, small intestine, colon; rectum; urinary organs such as bladder, gallbladder, ureter; rectosigmoid junction; anus, anal canal; skin; bone; joints, articular cartilage of limbs; eye and adnexa; brain; peripheral nerves, autonomic nervous system; spinal cord, cranial nerves, meninges; and various parts of the central nervous system; connective, subcutaneous and other soft tissues; retroperitoneum, peritoneum; adrenal gland; thyroid gland; endocrine glands and related structures; female genital organs such as ovary, uterus, cervix uteri; corpus uteri, vagina, vulva; male genital organs such as penis, testis and prostate gland; hematopoietic and reticuloendothelial systems; blood; lymph nodes; thymus.
The term “cancer” according to the invention comprises leukemias, seminomas, melanomas, teratomas, lymphomas, non-Hodgkin lymphoma, neuroblastomas, gliomas, adenocarcinoma, mesothelioma (including pleural mesothelioma, peritoneal mesothelioma, pericardial mesothelioma and end stage mesothelioma), rectal cancer, endometrial cancer, thyroid cancer (including papillary thyroid carcinoma, follicular thyroid carcinoma, medullary thyroid carcinoma, undifferentiated thyroid cancer, multiple endocrine neoplasia type 2A, multiple endocrine neoplasia type 2B, familial medullary thyroid cancer, pheochromocytoma and paraganglioma), skin cancer (including malignant melanoma, basal cell carcinoma, squamous cell carcinoma, Kaposi’s sarcoma, keratoacanthoma, moles, dysplastic nevi, lipoma, angioma and dermatofibroma), nervous system cancer, brain cancer (including astrocytoma, medulloblastoma, glioma, lower grade glioma, ependymoma, germinoma (pinealoma), glioblastoma multiform, oligodendroglioma, schwannoma, retinoblastoma, congenital tumors, spinal cord neurofibroma, glioma or sarcoma), skull cancer (including osteoma, hemangioma, granuloma, xanthoma or osteitis deformans), meninges cancer (including meningioma, meningiosarcoma or gliomatosis), head and neck cancer (including head and neck squamous cell carcinoma and oral cancer (such as, e.g., buccal cavity cancer, lip cancer, tongue cancer, mouth cancer or pharynx cancer)), lymph node cancer, gastrointestinal cancer, liver cancer (including hepatoma, hepatocellular carcinoma, cholangiocarcinoma, hepatoblastoma, angiosarcoma, hepatocellular adenoma and hemangioma), colon cancer, stomach or gastric cancer, esophageal cancer (including squamous cell carcinoma, larynx, adenocarcinoma, leiomyosarcoma or lymphoma), colorectal cancer, intestinal cancer, small bowel or small intestines cancer (such as, e.g., adenocarcinoma lymphoma, carcinoid tumors, Kaposi’s sarcoma, leiomyoma, hemangioma, lipoma, neurofibroma or fibroma), large bowel or large intestines cancer (such as, e.g., adenocarcinoma, tubular adenoma, villous adenoma, hamartoma or leiomyoma), pancreatic cancer (including ductal adenocarcinoma, insulinoma, glucagonoma, gastrinoma, carcinoid tumors or vipoma), ear, nose and throat (ENT) cancer, breast cancer (including HER2-enriched breast cancer, luminal A breast cancer, luminal B breast cancer and triple negative breast cancer), cancer of the uterus (including endometrial cancer such as endometrial carcinomas, endometrial stromal sarcomas and malignant mixed Mullerian tumors, uterine sarcomas, leiomyosarcomas and gestational trophoblastic disease), ovarian cancer (including dysgerminoma, granulosa-theca cell tumors and Sertoli-Leydig cell tumors), cervical cancer, vaginal cancer (including squamous-cell vaginal carcinoma, vaginal adenocarcinoma, clear cell vaginal adenocarcinoma, vaginal germ cell tumors, vaginal sarcoma botryoides and vaginal melanoma), vulvar cancer (including squamous cell vulvar carcinoma, verrucous vulvar carcinoma, vulvar melanoma, basal cell vulvar carcinoma, Bartholin gland carcinoma, vulvar adenocarcinoma and erythroplasia of Queyrat), genitourinary tract cancer, kidney cancer (including clear renal cell carcinoma, chromophobe renal cell carcinoma, papillary renal cell carcinoma, adenocarcinoma, Wilm’s tumor, nephroblastoma, lymphoma or leukemia), adrenal cancer, bladder cancer, urethra cancer (such as, e.g., squamous cell carcinoma, transitional cell carcinoma or adenocarcinoma), prostate cancer (such as, e.g., adenocarcinoma or sarcoma) and testis cancer (such as, e.g., seminoma, teratoma, embryonal carcinoma, teratocarcinoma, choriocarcinoma, sarcoma, interstitial cell carcinoma, fibroma, fibroadenoma, adenomatoid tumors or lipoma), lung cancer (including small cell lung carcinoma (SCLC), non-small cell lung carcinoma (NSCLC) including squamous cell lung carcinoma, lung adenocarcinoma (LUAD), and large cell lung carcinoma, bronchogenic carcinoma, alveolar carcinoma, bronchiolar carcinoma, bronchial adenoma, lung sarcoma, chondromatous hamartoma and pleural mesothelioma), sarcomas (including Askin's tumor, sarcoma botryoides, chondrosarcoma, Ewing's sarcoma, malignant hemangioendothelioma, malignant schwannoma, osteosarcoma and soft tissue sarcomas), soft tissue sarcomas (including alveolar soft part sarcoma, angiosarcoma, cystosarcoma phyllodes, dermatofibrosarcoma protuberans, desmoid tumor, desmoplastic small round cell tumor, epithelioid sarcoma, extraskeletal chondrosarcoma, extraskeletal osteosarcoma, fibrosarcoma, gastrointestinal stromal tumor (GIST), hemangiopericytoma, hemangiosarcoma, Kaposi's sarcoma, leiomyosarcoma, liposarcoma, lymphangiosarcoma, lymphosarcoma, malignant peripheral nerve sheath tumor (MPNST), neurofibrosarcoma, plexiform fibrohistiocytic tumor, rhabdomyosarcoma, synovial sarcoma and undifferentiated pleomorphic sarcoma, cardiac cancer (including sarcoma such as, e.g., angiosarcoma, fibrosarcoma, rhabdomyosarcoma or liposarcoma, myxoma, rhabdomyoma, fibroma, lipoma and teratoma), bone cancer (including osteogenic sarcoma, osteosarcoma, fibrosarcoma, malignant fibrous histiocytoma, chondrosarcoma, Ewing’s sarcoma, malignant lymphoma and reticulum cell sarcoma, multiple myeloma, malignant giant cell tumor chordoma, osteochondroma, osteocartilaginous exostoses, benign chondroma, chondroblastoma, chondromyxoid fibroma, osteoid osteoma and giant cell tumors), hematologic and lymphoid cancer, blood cancer (including acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, myeloproliferative diseases, multiple myeloma and myelodysplasia syndrome), Hodgkin’s disease, non-Hodgkin’s lymphoma and hairy cell and lymphoid disorders, and the metastases thereof.
Optionally, the cancer can be selected in the group consisting of rectal cancer, colorectal cancer, stomach cancer, head and neck cancer, thyroid cancer, cervical cancer, uterine cancer, breast cancer, in particular triple negative breast cancer, ovarian cancer, brain cancer, in particular glioblastoma and neuroblastoma, lung cancer, in particular small-cell lung cancer and non-small-cell lung cancer, skin cancer, bladder cancer, blood cancer, renal cancer, liver cancer, prostate cancer, multiple myeloma, pancreatic cancer and endometrial cancer. In a very particular aspect, the cancer is a pancreatic cancer.
In a particular aspect, the compounds of the present invention are of particular interest for targeting persister cancer cells, cancer-stem cells (CSCs), cancer stem-like cells, drug-tolerant cancer cells, and therapy-resistent cancer cells, and for targeting epithelial-mesenchymal transition, targeting epithelial- mesenchymal plasticity. In particular, the compounds of the present invention can be used as inhibitor of cell plasticity in cancer, for instance by blocking epithelial-mesenchymal transition. They can be used to desensitize cancer cells to cytotoxic agents, especially those of the standard of care. Optionally, the present invention relates to a compound or pharmaceutical composition for use in the treatment of a subject having a cancer resistant or susceptible to become resistant to a cytotoxic agent. Optionally, said compound or pharmaceutical composition can be used in combination with said cytotoxic agent. It further relates to a method for reversing or decreasing or delaying a resistance of cancer cells to a cytotoxic agent in a subject having a cancer, comprising administering a therapeutic amount of a compound or a composition of the present invention to said subject, thereby reversing or decreasing or delaying the resistance to said cytotoxic agent, especially a chemotherapeutic agent.
Optionally, the compound or pharmaceutical composition is used in combination with radiotherapy and/or another drug, preferably an antitumoral drug, more preferable a drug selecting from the group consisting of chemotherapy, targeted therapy, hormonotherapy and immunotherapy such as immune checkpoint therapy.
As used herein, the term “targeted therapy” refers to targeted therapy agents, drugs designed to interfere with specific molecules necessary for tumor growth and progression. For example, targeted therapy agents such as therapeutic monoclonal antibodies target specific antigens found on the cell surface, such as transmembrane receptors or extracellular growth factors. Small molecules can penetrate the cell membrane to interact with targets inside a cell. Small molecules are usually designed to interfere with the enzymatic activity of the target protein such as for example proteasome inhibitor, tyrosine kinase or cyclin-dependent kinase inhibitor, histone deacetylase inhibitor. Targeted therapy may also use cytokines. Examples of such targeted therapy include with no limitations: Ado-trastuzumab emtansine (HER2), Afatinib (EGFR (HER1/ERBB1), HER2), Aldesleukin (Proleukin), alectinib (ALK), Alemtuzumab (CD52), axitinib (kit, PDGFRbeta, VEGFR1/2/3), Belimumab (BAFF), Belinostat (HDAC), Bevacizumab (VEGF ligand), Blinatumomab (CD19/CD3), bortezomib (proteasome), Brentuximab vedotin (CD30), bosutinib (ABL), brigatinib (ALK), cabozantinib (FLT3, KIT, MET, RET, VEGFR2), Canakinumab (IL-1 beta), carfilzomib (proteasome), ceritinib (ALK), Cetuximab (EGFR), cofimetinib (MEK), Crizotinib (ALK, MET, ROS1), Dabrafenib (BRAF), Daratumumab (CD38), Dasatinib (ABL), Denosumab (RANKL), Dinutuximab (B4GALNT1 (GD2)), Elotuzumab (SLAMF7), Enasidenib (IDH2), Erlotinib (EGFR), Everolimus (mTOR), Gefitinib (EGFR), Ibritumomab tiuxetan (CD20), Sonidegib (Smoothened), Sipuleucel-T, Siltuximab (IL-6), Sorafenib (VEGFR, PDGFR, KIT, RAF),(Tocilizumab (IL-6R), Temsirolimus (mTOR), Tofacitinib (JAK3), Trametinib (MEK), Tositumomab (CD20), Trastuzumab (HER2), Vandetanib (EGFR), Vemurafenib (BRAF), Venetoclax (BCL2), Vismodegib (PTCH, Smoothened), Vorinostat (HDAC), Ziv-aflibercept (PIGF, VEGFA/B), Olaparib (PARP inhibitor). As used herein, the term “antitumor chemotherapy” or “chemotherapy” refers to a cancer therapeutic treatment using chemical or biochemical substances, in particular using one or several antineoplastic agents or chemotherapeutic agents. Chemotherapeutic agents include, but are not limited to alkylating agents such as thiotepa and cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); a camptothecin (including the synthetic analogue topotecan); bryostatin; callystatin; CC- 1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW- 2189 and CB1-TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlomaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as the enediyne antibiotics (e.g. , calicheamicin, especially calicheamicin gammall and calicheamicin omegall ; dynemicin, including dynemicin A; bisphosphonates, such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromophores, aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxy doxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5 -fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; methylhydrazine derivatives including N-methylhydrazine (MIH) and procarbazine; PSK polysaccharide complex); razoxane; rhizoxin; sizofuran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2"-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, e.g., paclitaxel and doxetaxel; gemcitabine; 6-thioguanine; mercaptopurine; platinum coordination complexes such as cisplatin, oxaliplatin and carboplatin; vinblastine; platinum; etoposide (VP- 16); ifosfamide; mitoxantrone; vincristine; vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (e.g., CPT-1 1); topoisomerase inhibitor RFS 2000; difluoromethylomithine (DMFO); retinoids such as retinoic acid; capecitabine; anthracyclines, nitrosoureas, antimetabolites, epipodophylotoxins, enzymes such as L-asparaginase; anthracenediones; hormones and antagonists including adrenocorticosteroid antagonists such as prednisone and equivalents, dexamethasone and aminoglutethimide; progestin such as hydroxyprogesterone caproate, medroxyprogesterone acetate and megestrol acetate; estrogen such as diethylstilbestrol and ethinyl estradiol equivalents; antiestrogen such as tamoxifen; androgens including testosterone propionate and fluoxymesterone/equivalents; antiandrogens such as flutamide, gonadotropin-releasing hormone analogs and leuprolide; and non-steroidal antiandrogens such as flutamide; and pharmaceutically acceptable salts, acids or derivatives of any of the above..
As used herein, the term “hormonal therapy” refers to a cancer treatment having for purpose to block, add or remove hormones. For instance, in breast cancer, the female hormones estrogen and progesterone can promote the growth of some breast cancer cells.
As used herein, the term “immunotherapy” refers to a cancer therapeutic treatment using the immune system to reject cancer. The therapeutic treatment stimulates the patient's immune system to attack the malignant tumor cells.
Immune checkpoint therapy such as checkpoint inhibitors include, but are not limited to programmed death- 1 (PD-1) inhibitors, programmed death ligand- 1 (PD-L1) inhibitors, programmed death ligand-2 (PD-L2) inhibitors, lymphocyte -activation gene 3 (LAG3) inhibitors, T-cell immunoglobulin and mucin-domain containing protein 3 (TIM-3) inhibitors, T cell immunoreceptor with Ig and ITIM domains (TIGIT) inhibitors, B- and T-lymphocyte attenuator (BTLA) inhibitors, V-domain Ig suppressor of T-cell activation (VISTA) inhibitors, cytotoxic T-lymphocyte-associated protein 4 (CTLA4) inhibitors, Indoleamine 2,3-dioxygenase (IDO) inhibitors, killer immunoglobulin-like receptors (KIR) inhibitors, KIR2L3 inhibitors, KIR3DL2 inhibitors and carcinoembryonic antigen- related cell adhesion molecule 1 (CEACAM-1) inhibitors. In particular, checkpoint inhibitors include antibodies anti-PDl, anti-PD-Ll, anti-CTLA-4, anti-TIM-3, anti-LAG3. Immune checkpoint therapy also includes co-stimulatory antibodies delivering positive signals through immune-regulatory receptors including but not limited to ICOS, CD 137, CD27, OX-40 and GITR.
Example of anti-PDl antibodies include, but are not limited to, nivolumab, cemiplimab (REGN2810 or REGN-2810), tislelizumab (BGB-A317), tislelizumab, spartalizumab (PDR001 or PDR-001), ABBV- 181, JNJ-63723283, BI 754091, MAG012, TSR-042, AGEN2034, pidilizumab, nivolumab (ONO-4538, BMS-936558, MDX1106, GTPL7335 or Opdivo), pembrolizumab (MK-3475, MK03475, lambrolizumab, SCH-900475 or Keytruda) and antibodies described in International patent applications W02004004771, W02004056875, W02006121168, WO2008156712, W02009014708,
W02009114335, WO2013043569 and WO2014047350. Example ofanti-PD-Ll antibodies include, but are not limited to, LY3300054, atezolizumab, durvalumab and avelumab. Example of anti-CTLA-4 antibodies include, but are not limited to, ipilimumab (see, e.g., US patents US6,984,720 and US8,017,114), tremelimumab (see, e.g., US patents US7, 109,003 and US8, 143,379), single chain anti- CTLA4 antibodies (see, e.g., International patent applications WO1997020574 and WO2007123737) and antibodies described in US patent US8,491,895. Example of anti-VISTA antibodies are described in US patent application US20130177557. Example of inhibitors of the LAG3 receptor are described in US patent US5,773,578. Example of KIR inhibitor is IPH4102 targeting KIR3DL2.
As used herein, the term “radiotherapy” refers to radiation therapies including, but not limited to external beam radiotherapy (such as superficial X-rays therapy, orthovoltage X-rays therapy, megavoltage X- rays therapy, radiosurgery, stereotactic radiation therapy, Fractionated stereotactic radiation therapy, cobalt therapy, electron therapy, fast neutron therapy, neutron-capture therapy, proton therapy, intensity modulated radiation therapy (IMRT), 3-dimensional conformal radiation therapy (3D-CRT) and the like); brachytherapy; unsealed source radiotherapy; tomotherapy; and the like. Gamma rays are another form of photons used in radiotherapy. Gamma rays are produced spontaneously as certain elements (such as radium, uranium, and cobalt 60) release radiation as they decompose, or decay. In some embodiments, radiotherapy may be proton radiotherapy or proton minibeam radiation therapy. Proton radiotherapy is an ultra-precise form of radiotherapy that uses proton beams (Prezado Y, Jouvion G, Guardiola C, Gonzalez W, Juchaux M, Bergs J, Nauraye C, Labiod D, De Marzi L, Pouzoulet F, Patriarca A, Dendale R. Tumor Control in RG2 Glioma-Bearing Rats: A Comparison Between Proton Minibeam Therapy and Standard Proton Therapy. Int J Radiat Oncol Biol Phys. 2019 Jun l;104(2):266- 271. doi: 10. 1016/j .ijrobp.2019.01.080; Prezado Y, Jouvion G, Patriarca A, Nauraye C, Guardiola C, Juchaux M, Lamirault C, Labiod D, Jourdain L, Sebrie C, Dendale R, Gonzalez W, Pouzoulet F. Proton minibeam radiation therapy widens the therapeutic index for high-grade gliomas. Sci Rep. 2018 Nov 7;8(1): 16479. doi: 10. 1038/s41598-018-34796-8). Radiotherapy may also be FLASH radiotherapy (FLASH-RT) or FLASH proton irradiation. FLASH radiotherapy involves the ultra-fast delivery of radiation treatment at dose rates several orders of magnitude greater than those currently in routine clinical practice (ultra-high dose rate) (Favaudon V, Fouillade C, Vozenin MC. The radiotherapy FLASH to save healthy tissues. Med Sci (Paris) 2015; 31: 121-123. DOI:
10.1051/medsci/20153102002); Patriarca A., Fouillade C. M., Martin F., Pouzoulet F., Nauraye C., et al. Experimental set-up for FLASH proton irradiation of small animals using a clinical system. Int J Radiat Oncol Biol Phys, 102 (2018), pp. 619-626. doi: 10.1016/j.ijrobp.2018.06.403. Epub 2018 Jul 11). The pharmaceutical compositions contemplated herein may include a pharmaceutically acceptable carrier in addition to the active ingredient(s). The term "pharmaceutically acceptable carrier" is meant to encompass any carrier (e.g., support, substance, solvent, etc.) which does not interfere with effectiveness of the biological activity of the active ingredient(s) and that is not toxic to the host to which it is administered. For example, for parental administration, the active compounds(s) may be formulated in a unit dosage form for injection in vehicles such as saline, dextrose solution, serum albumin and Ringer's solution.
Further aspects and advantages of the present invention will be described in the following examples, which should be regarded as illustrative and not limiting.
Examples
List of abbreviations
AI2O3: Aluminum oxide Fe(OTf)2: Iron(II) trifluoromethanesulfonate aq.: Aqueous HATU : 1 -[Bis(dimethylamino)methylene] - 1H- Ar: Argon 40 l,2,3-triazolo[4,5-6]pyridinium 3-oxid AU: Arbitrary units hexafluorophosphate BINAP: 2,2 ’ -bis(diphenylphosphino)- 1 , 1 ’ - HBF4: Tetrafluoroboric acid binaphthalene HBTU: A, A, JV' JV'-Tetramethyl-O-( 1H-
BOC2O: Di-tert-butyl dicarbonate benzotriazol- 1 -y I juroni um CAM: Cerium ammonium molybdate 45 hexafluorophosphate CS2CO3: Cesium carbonate HC1: Hydrogen chloride or hydrochloric acid Cui: Copper iodide HPLC: High performance liquid DCM: Dichloromethane chromatography DFO: Deferoxamine HRMS: High resolution mass spectrometry
DIPEA: A'-A'-Diisopropylcthylaminc 50 K2CO3: Potassium carbonate DMAP: Dimethylaminopyridine LRMS: Low resolution mass spectrometry DMF: Dimethylformamide MeCN: Acetonitrile DMSO: Dimethylsulfoxide MeOH: Methanol
DOPC: l,2-dioleoyl-5«-glycero-3- MgSOy Magnesium sulfate phosphocholine 55 M0MC1: Chloromethyl methyl ether EtOAc: Ethyl acetate MS: Mass spectrometry EtOH: Ethanol NaHCOy Sodium bicarbonate eq.: Molar equivalent Na2CO3: Sodium carbonate ESI-MS: Electrospray ionisation mass NH4CI: Ammonium chloride spectrometry 60 NMR multiplicity: s (singlet), bs (broad FA: Formic acid singlet), d (doublet), dd (doublet of doublets), ddd (doublet of doublet of doublets), dt (doublet sat.: Saturated of triplets), dq (doublet of quadruplets), td SiO2: Silica (triplet of doublets), q (quadruplet), t (triplet), SOC12: Thionyl chloride quint (quintet), m (multiplet) 20 TBABr: Tetrabutylammonium bromide NMR: Nuclear magnetic resonance TBAF: Tetrabutylammonium fluoride
NaOAc: Sodium acetate TEA: Triethylamine
NaOH: Sodium hydroxide TFA: Trifluoroacetic acid
PTSA: p-Toluenesulfonic acid Tf2O: Trifluoromethanesulfonic anhydride
Pd(OAc)2: Palladium(II) acetate 25 THF: Tetrahydrofuran Pd(PPh3)2Cl2: TLC: Thin layer chromatography
Bis(triphenylphosphine)palladium(II) TMS: Trimethylsilyl dichloride TMSC1: Trimethylsilyl chloride
POCh: Phosphorous trichloride UPLC: Ultra-high performance liquid
Py: Pyridine 30 chromatography rpm: Revolution per minute UV: Ultraviolet rt: Room temperature
Example 1 : Chemical Synthesis of Fentomycin
General information.
Starting materials were purchased from Sigma-Aldrich or TCI Chemicals and used without further purification. Solvents were dried under standard conditions. Reactions were monitored by thin layer chromatography using aluminum plates coated with silica gel or aluminium oxide neutral from Merck (60 F254). TEC plates were visualized by UV or by treatment with a ninhydrin solution and heating. Reaction products were purified by flash column chromatography on silica gel 60 (230-400 mesh, Merck and co.) or aluminium oxide (activated neutral, Sigma- Aldrich), by Combiflash® Rf, or by preparative HPLC Quaternary Gradient 2545 equipped with a Photodiode Array detector (Waters) fitted with a reverse phase column (XBridge BEH C18 OBD Prep column 5 pm 30x 150 mm). NMR spectroscopy was performed on Bruker 300, 400 or 500 MHz apparatus. Spectra were run in CD3OD or CDCE, at 298 K. ’H chemical shifts 5 are expressed in ppm using the residual non-deuterated solvent as internal standard and the coupling constants J are specified in Hz. The following abbreviations are used: s, singlet; d, doublet; dd, doublet of doublets; dt, doublet of triplets; t, triplet; quint., quintet; m, multiplet. 13C chemical shifts 5 are expressed in ppm using the solvent as internal standard.
The purity of final compounds, determined to be >98% by UPLC-MS, and low-resolution mass spectra (LRMS) were recorded on a Waters Acquity H-class equipped with a Photodiode array detector and SQ Detector 2 (UPLC-MS) fitted with a reverse phase column (Acquity UPLC BEH C18 1.7 pm, 2.1x50 mm). HRMS were recorded on a Thermo Scientific Q-Exactive Plus equipped with a Robotic TriVersa NanoMate Advion.
Scheme 1 | Chemical synthesis of Fentomycin. (a) 2, 4, toluene, 100 °C, 16 h, then concentrated to dryness; MeOH, rt, 4 h, then K2CO3, rt, 1 h, 33%. (b) Tf2O, Et3N, CH2C12, -78 °C to rt, 6 h, 78%. (c) 6, 12, Cui (20 mol%), K2CO3, toluene, 160 °C, sealed tube, 72 h, 33%. (d) BF3 Et2O, CH2C12, -78 °C to rt, 6 h, 62%. (e) Benzaldehyde dimethylacetal, p-Toluenesulfonic acid monohydrate (PTSA), dimethylformamide (DMF), 40 °C (30 mbar), 16 h, 98%. (f) (iPr^EtN, M0MC1, CH2CI2, rt, 72 h, 91%. (g) sec-Butyllithium (1.6 M, hexane), THF, -40 °C, 1.5 h, 61%. (h) O-benzylhydroxylamine HC1 salt, NaOAc, EtOH, rt, 2 h, 90%. (i) Anhydrous CeCE, methyllithium (3M, diethoxymethane), THF, -78 °C, 1 h; then 11, -78 °C to 0 °C, 2 h, 76%. (j) H2, Pd/C (10 mol%), MeOH, rt, 16 h, 88%. (k) 48% wt/wt aq. HBF4, MeCN, 82 °C, 2 h, 18%. (1) succinic anhydride, DMAP, anhydrous pyridine, 90 °C, 40 h, 36%. (m) dimethylamine HC1 salt, NaOH, H2O, sealed tube, 155 °C, 16 h, 91%. (n) SOCE, CH2CI2, rt, 16 h 93%. (o) Na2CO3, tetra-butylammonium bromide (TBABr) anhydrous MeCN, 90 °C, 16 h, 27%. (p) Pd(OAc)2, BINAP, CS2CO3, dioxane, reflux, overnight, 66%. (q) SOCE, CH2CI2, 0 °C to rt, 16 h, 56 %. (r) Na2CO3, TBABr, MeCN, reflux under Ar, 16 h, 66%. (s) TFA, CH2CI2, 0 °C then rt, 2 h, 69%. (t) HBTU, DIPEA, THF, rt, 4 h 86%.
Experimental procedures 2-((2S,2'S)-[2,2'-bipyrrolidin]-l-ylmethyl)-N,N-dimethylpyridin-4-amine (17a)
[29] and 2,2'-((2S,2'S)-[2,2'-bipyrrolidine]-l,r-diylbis(methylene))bis(N,N-dimethylpyridin-4-amine) (17b) [30] 2-Chloromethyl-4-dimethylaminopyridine (16) [31] (0.79 g, 4.63 mmol), (S,S)-2,2- bipyrrolidine (0.65 g, 4.63 mmol) were mixed in a 100 mb Schlenk in anhydrous acetonitrile (45 mb). Na2CO3 (1.96 g, 18.54 mmol) and tetra-butylammonium bromide (TBABr) (0.29 g, 0.93 mmol) were added directly as solids and the resulting mixture was heated at reflux under Ar for 16 h. After cooling to room temperature, the resulting brown mixture was filtered and the filter cake was washed with dichloromethane. The combined filtrates were evaporated under reduced pressure. To the resulting residue, IN NaOH (10 mb) was added and the mixture was extracted with dichloromethane (3 x 10 mb). The combined organic layers were dried over anhydrous MgSCh, filtered and the solvent was removed under reduced pressure. The residue was purified by Combiflash® in AI2O3 neutral using DCM:MeOH (gradient 0 to 5% MeOH). Product 17a was obtained as a brown oil (0.38 g, 27%). Data is in accordance with the literature [29], 1H NMR (500 MHz, Mcthanol-d4) δ 8.06 (d, J = 7.0 Hz, 1H), 6.62 (d, J = 5.1 Hz, 2H), 3.98 (dd, J = 15.2, 2.4 Hz, 1H), 3.77 (d, J = 15.2 Hz, 1H), 3.41 (q, J = 8.2 Hz, 1H), 3.26 - 3.24 (m, 1H), 3.09 - 2.96 (m, 9H), 2.63 (dt, J = 11.0, 7.0 Hz, 1H), 2.15 - 2.04 (m, 2H), 2.04 - 1.88 (m, 3H), 1.88 - 1.74 (m, 1H), 1.72 - 1.51 (m, 2H) ppm. Product 17b was obtained as a side product of the reaction and as brown semisolid
(0.13 g, 10%). 1H NMR (400 MHz, Methanol-d4) δ 7.95 (d, J = 6.1 Hz, 2H), 6.72 (d, J = 2.6 Hz, 2H), 6.48 (dd, J = 6.1, 2.7 Hz, 2H), 4.16 (d, J = 14.2 Hz, 2H), 3.42 (d, J = 14.2 Hz, 2H), 3.06 - 2.98 (m, 2H), 2.95 (s, 12H), 2.82 - 2.72 (m, 2H), 2.34 - 2.22 (m, 2H), 1.92 - 1.87 (m, 2H), 1.80 - 1.65 (m, 6H). tert-butyl (4-((2-(hydroxymethyl)pyridin-4-yl)amino)butyl)carbamate (18) Boc In a dry Schlenk were added under Ar (4-chloropyridin-2-yl)methanol (1 g, 6.97 mmol), tert-butyl(4-aminobutyl)carbamate (1.6 mb, 8.36 mmol), Pd(OAc)2 (0.08 g, 0.35 mmol), BINAP (0.22 g, 0.35 mmol) and CS2CO3 (3.40 g, 10.45 mmol), then anhydrous dioxane (70 mL) was added and the reaction was stirred at reflux overnight. After cooling at room temperature, the solvent was removed under reduced pressure and the residue was purified by flash chromatography in SiO2 using DCM:MeOH:Et3N (9: 1:5%). 1.35 g (66%) of the product was obtained as a brown oil.1H NMR (500 MHz, Methanol-d4) δ 7.90 (d, J = 5.8 Hz, 1H), 6.69 (s, 1H), 6.42 (d, J = 5.8 Hz, 1H), 4.52 (s, 2H), 3.17 (t, J = 6.8 Hz, 2H), 3.07 (t, J = 6.8 Hz, 2H), 1.63 - 1.60 (m, 2H), 1.59 - 1.54 (m, 2H), 1.43 (s, 9H) ppm. 13C NMR (126 MHz, Methanol-d4) δ 161.5, 158.6, 156.9, 148.7, 106.9, 104.5, 79.9, 65.3, 42.9, 41.00, 28.8 (3C), 28.5, 27.1 ppm. HRMS(ESI-MS) m/z calculated for C15H26N3O3 [M+H]+ 296.1969, found 296.1967. tert-butyl (4-((2-(chloromethyl)pyridin-4-yl)amino)butyl)carbamate (19) Boc
To a Schlenk tube containing 18 (1.35 g, 4.58 mmol) was added anhydrous dichloromethane (45 mL) and the solution was cooled at 0 °C, then was added dropwise SOCI2 (0.50 mL, 6.86 mmol). The reaction was allowed to reach room temperature and was stirred overnight. After this time the reaction was carefully quenched with a solution of sat. aq. Na2CO3 and extracted with dichloromethane (3 x 20 mL). The combined organic phases were dried over MgSO4, filtered and concentrated to dryness under reduced pressure and purified by Combiflash® chromatography using DCM:MeOH (1:0 to 1:0.1 MeOH) to afford 19 as a brown oil (0.80 g, 56%). 1H NMR (400 MHz, Methanol-d4) δ 7.94 (d, J = 5.9 Hz, 1H), 6.66 (d, J = 2.1 Hz, 1H), 6.46 (dd, J = 5.9, 2.3 Hz, 1H), 4.48 (s, 2H), 3.15 (t, J = 6.7 Hz, 2H), 3.07 (t, J = 6.6 Hz, 2H), 1.69 - 1.49 (m, 4H), 1.42 (s, 9H) ppm. 13C NMR (126 MHz, Chloroform-d) 5 156.9, 156.3, 154.5, 149.7 (2C), 106.8, 106.3, 50.9, 47.2, 42.5, 28.5 (3C), 27.9, 26.2 ppm. HRMS(ESI- MS) m/z calculated for C15H25CIN3O2 [M+H]+ 314.1630, found 314.1630. tert-butyl (4-((2-(((2S,2'S)-r-((4-(dimethylamino)pyridin-2-yl)methyl)-[2,2'-bipyrrolidin]-l- yl)methyl)pyridin-4-yl)amino)butyl)carbamate (20) 0.34 mmol) and 17a (0.09 g, 0.34 mmol) were mixed in anhydrous acetonitrile (4 mL) in a 10 mb Schlenk. Na2CO3 (0.14 g, 1.37 mmol) and TBABr (0.02 g,
0.07 mmol) were added directly as solids and the resulting mixture was heated at reflux under Ar for 16 h. After cooling to room temperature, the resulting brown mixture was filtered and the filter cake was washed with dichloromethane. The combined filtrates were evaporated under reduced pressure. To the resulting residue a solution of sat. aq. Na2CO3 (5 mL) was added and the mixture was extracted with dichloromethane (3 x 5 mL). The combined organic layers were dried over anhydrous MgSO4. filtered and the solvent was removed under reduced pressure. The residue was purified by Combiflash® in AI2O3 using DCM:MeOH (1:0 to 1:0.5). The product was obtained as a brown oil (0.124 g, yield 66 %). ’H NMR (400 MHz, Chloroform-J) δ 8.10 (d, J = 6.0 Hz, 1H), 8.05 (d, J = 5.7 Hz, 1H), 6.65 (d, J = 2.6 Hz, 1H), 6.55 (d, J = 2.4 Hz, 1H), 6.32 (dd, J = 5.9, 2.7 Hz, 1H), 6.24 (dd, J = 5.8, 2.4 Hz, 1H), 4.82 (bs, 1H), 4.29 (bs, 1H), 4.07 (dd, J = 17.6, 14.2 Hz, 2H), 3.37 (dd, J = 14.1, 12.4 Hz, 2H), 3.18 - 3.06 (m, 4H), 3.06 - 2.96 (m, 2H), 2.93 (s, 6H), 2.81 - 2.73 (m, 2H), 2.68 (bs, 1H), 2.29 - 2.16 (m, 2H), 1.86 - 1.51 (m, 12H), 1.42 (s, 9H) ppm. 13C NMR (101 MHz, Chloroform-d) δ 160.4, 160.3, 156.2, 154.9, 154.1, 149.2, 148.9, 106.4, 105.7, 105.2, 105.1, 79.3, 65.7, 65.7, 61.7, 61.4, 55.4, 55.4, 42.4, 40.3, 39.2
(2C), 28.5 (3C), 27.8, 26.4, 26.4, 26.3, 23.7 (2C) ppm. HRMS(ESI-MS) m/z calculated for C3iH5oN702[M+H]+ 552.4021, found 552.4025. N1-(2-(((2S,2'S)-l'-((4-(dimethylamino)pyridin-2-yl)methyl)-[2,2'-bipyrrolidin]-l-yl)methyl)pyridin-
4-yl)butane-l,4-diamine (21) Over a solution of 20 (0.084 g, 0.1525 mmol) in 2 mL of anhydrous dichloromethane at 0 °C, was added TFA (0.059 mL, 0.762 mmol). The reaction was allowed to reach room temperature and then was stirred for 2 h. After this time the solvent was removed under vacuum and the residue redissolved in dichloromethane, washed with IN NaOH (2 mL) and extracted with dichloromethane (3 x 2 mL). Combined organic layers were dried over MgSO4, filtered and the solvent removed under vacuum. The residue was purified by flash chromatography in AI2O3 using DCM:MeOH:Et3N (9: 1:5%). Product 21 was obtained as a brown oil (0.047 g, yield 69 %). 1H NMR (500 MHz, Methanol-d4) δ 7.96 (d, J = 6.1 Hz, 1H), 7.86 (d, J = 5.9 Hz, 1H), 6.72 (d, J = 2.6 Hz, 1H),
6.65 (d, J = 2.4 Hz, 1H), 6.49 (dd, J = 6.2, 2.7 Hz, 1H), 6.37 (dd, J = 5.9, 2.4 Hz, 1H), 4.16 - 4.06 (m, 2H), 3.43 - 3.34 (m, 2H), 3.12 - 3.04 (m, 2H), 3.04 - 2.97 (m, 2H), 2.96 (s, 6H), 2.80 - 2.71 (m, 2H),
2.64 (t, J = 7.0 Hz, 2H), 2.32 - 2. 19 (m, 2H), 1.93 - 1.83 (m, 2H), 1.77 - 1.65 (m, 6H), 1.65 - 1.56 (m, 2H), 1.56 - 1.48 (m, 2H) ppm. 13C NMR (126 MHz, Methanol-d4) δ 160.5, 160.4, 156.8, 156.6, 148.7 (2C), 106.9, 106.8, 106.6, 106.5, 67.8, 67.6, 62.3, 62.1, 56.4 (2C), 43.1, 42.3, 39.2 (2C), 31.3, 27.8 (2C),
27.3, 24.6, 24.6 ppm. HRMS(ESI-MS) m/z calculated for C26H42N7[M+H]+ 452.3496, found 452.3498. Fentomycin formate salt (22) A solution of Marmysunate (14) [32] (3.2 mg, 0.0062 mmol), HBTU (11.8 mg, 0.0312 mmol) and DIPEA (32.4 mL, 0.1860 mmol) in 100 mL of anhydrous tetrahydrofuran was stirred at room temperature for Ih before the addition of 21 (14.1 mg, 0.0312 mmol) in 100 mL of anhydrous tetrahydrofuran. The reaction was stirred 4h at room temperature until TLC in AI2O3 (DCM:MeOH, 9.5:0.5) showed a total conversion, then solvent was removed under vacuum and the residue was purified using by preparative HPLC using H2O:CH3 : Formic acid (95:5:0. 1 to 0: 100:0. 1) to afford 5.0 mg (86 %) of Fentomycin as a red solid after freeze drying.1H NMR (500 MHz, Methanol-d4 ) δ 9.64 (s, IH), 9.44 (d, J = 9.0 Hz, IH), 8.54 (bs, IH), 8.24 (d, J = 8.6 Hz, IH), 8.09 (d, J = 8.6 Hz, IH), 7.90 (dd, J = 6.3, 0.6 Hz, IH), 7.82 (d, J = 6.5 Hz, IH), 7.70 (d, J = 1.8 Hz, IH), 7.53 (dd, J = 9.0, 1.8 Hz, IH), 7.51 - 7.43 (m, 2H), 6.66 - 6.61(m, 2H), 6.59 - 6.52 (m, 2H), 4.84 (dd, J = 3.3, 1.9 Hz, IH), 4.74 (d, J = 9.6 Hz, 1H), 4.12 (d, J = 15.1 Hz, IH), 4.10 (d, J = 15.4 Hz, 1H), 3.65 (d, J = 15.1 Hz, 1H), 3.62 (d, J = 15.4 Hz, IH), 3.33 (dq, J = 9.6, 6.1 Hz, IH), 3.20 (t, J = 6.7 Hz, 2H,), 3.13 (t, J = 6.7 Hz, 2H), 3.08 - 2.98 (m, 8H), 2.93 - 2.83 (m, 2H), 2.82 - 2.65 (m, 2H), 2.58 (t, J = 6.5 Hz, 2H), 2.55 - 2.47 (m, 5H), 2.26 (ddd, J = 13.4, 3.3, 1.3 Hz, IH), 2.00 - 1.69 (m, 7H), 1.68 - 1.51 (m, 6H), 1.38 (s, 3H), 1.04 (d, J = 6.1 Hz, 3H). 13C NMR (126 MHz, Methanol-d4) δ 187.3, 186.4, 169.9, 174.0, 173.7, 158.6, 157.9, 155.0(2C), 149.9, 144.0, 143.5, 140.2, 137.9, 137.5, 136.9, 135.9, 135.7, 133.1, 129.6, 129.6, 129.2, 128.9, 128.4, 123.3, 116.7, 111.8, 107.2(2C), 106.6(2C), 80.7, 70.4, 68.9, 68.8, 67.8, 59.8, 59.2, 55.9(2C), 52.3, 43.2, 39.7(3C), 35.8, 31.2, 30.5, 28.2, 28.2, 28.0, 26.6, 25.3, 24.9, 24.8, 21.6, 18.5. HRMS(ESI-MS) m/z calculated for C56H67N8O6[M+H]+ 947.5184, found 947.5174.
Synthesis of Click-ligand (26)
Methyl 3-((2-(((2S,2'S)-r-((4-(dimethylamino)pyridin-2-yl)methyl)-[2,2'-bipyrrolidin]-l- yl)methyl)pyridin-4-yl)(methyl)amino)propanoate (24) dry Schlenk were added 23 [33] (0.30 g, 1.24 mmol), 17a (0.34 g, 1.24 mmol) and anhydrous acetonitrile (12 mL). Then were added Na2CO2 (0.52 g, 4.99 mmol) and TBABr (0.08 g, 0.25 mmol). The resulting mixture was heated at reflux under Ar for 16 hours. After cooling to room temperature, the resulting brown mixture was filtered and the filter cake was washed with dichloromethane. The combined filtrates were evaporated under reduced pressure. To the resulting residue, a solution of sat. aq. Na2CO2 (5 mL) was added and the mixture was extracted with dichloromethane (3 x 5 mL). The combined organic layers were dried over anhydrous MgSO4, filtered and the solvent was removed under reduced pressure. Subsequently, the residue was purified by Combiflash® in AI2O3 neutral using DCM:MeOH (gradient 0 to 5% MeOH). Product was obtained as a brown oil (0.228 g, yield 38 %). 1H NMR (500 MHz, Methanol-d4) δ 7.97 (d, J = 6.2 Hz, 1H), 7.94 (d, J = 6.3 Hz, 1H), 6.76 (d, J = 2.7 Hz, 1H), 6.70 (d, J = 2.7 Hz, 1H), 6.56 - 6.49 (m, 2H), 4.22 - 4.12 (m, 2H), 3.67 (t, J = 7.0 Hz, 1H), 3.65 (s, 3H), 3.51 (d, J = 14.2 Hz, 2H), 3.10 - 2.99 (m, 3H), 2.96 (s, 6H), 2.94 (s, 3H), 2.88 - 2.78 (m, 2H), 2.56 (t, J = 7.0 Hz, 2H), 2.44 - 2.31 (m, 2H), 2.00 - 1.87 (m, 2H), 1.85 - 1.64 (m, 6H) ppm. 13C NMR (126 MHz, Methanol-d4) δ 173.7, 160.1, 159.6, 156.9, 155.7, 148.8, 148.3, 106.7 (2C), 106.6 (2C), 68.4, 68.3, 62.4, 62.3, 56.3 (2C), 52.3, 48.2, 39.2 (2C), 37.8, 32.3, 28.2 (2C), 24.8, 24.7 ppm. HRMS(ESI-MS) m/z calculated for C27H4iN6O2[M+H]+ 481.3286, found 481.3288.
3-((2-(((2S,2'S)-T-((4-(dimethylamino)pyridin-2-yl)methyl)-[2,2'-bipyrrolidin]-l-yl)methyl)pyridin-4- yl)(methyl)amino)propanoic acid (25) solution of 24 (214 mg, 0.446 mmol) in 5 mL of a mixture MeOH/IN NaOH
(1: 1) was stirred at room temperature overnight. Then the reaction was neutralized using a solution of aq. IN HC1. Solvent was removed under reduced pressure; the resultant residue was washed with MeOH and the salts filtered off. The filtrate was evaporated under reduced pressure. The product was purified by preparative HPLC using H2O:CH3CN:Formic acid (95:5:0. 1 to 0: 100:0. 1). The product was obtained as a white solid (80 mg, yield 38 %). ’H NMR (500 MHz, Methanol-d4 δ 77 .98 (d, J = 6.9 Hz, 1H), 7.96 (d, J = 6.9 Hz, 1H), 6.86 (d, J = 2.7 Hz, 1H), 6.82 - 6.78 (m, 2H), 6.76 (dd, J = 6.9, 2.8 Hz, 1H), 4.35 - 4.21 (m, 2H), 3.87 - 3.79 (m, 2H), 3.76 (td, J = 7.1, 1.6 Hz, 2H), 3.17 - 3.09 (m, 11H), 3.04 - 2.93 (m, 2H), 2.67 - 2.58 (m, 2H), 2.44 (t, J = 7.1 Hz, 2H), 2.08 - 1.98 (m, 2H), 1.95 - 1.79 (m, 4H), 1.74 - 1.65 (m, 2H) ppm. 13C NMR (126 MHz, Methanol-^) 5 179.0, 158.2, 157.6, 154.6, 143.4, 143.3, 107.4, 107.2, 106.8, 106.6, 69.4, 69.3, 59.7, 59.5, 56.1, 56.0, 50.5, 39.9 (3C), 38.4, 35.9, 28.6, 28.5, 24.9, 24.8 ppm. HRMS(ESI-MS) m/z calculated for 467.3129, found 467.3242. Ligand-click (26) solution of 25 (10 mg, 0.0214 mmol), HBTU (24.4 mg, 0.0644 mmol) and
DIPEA (11.2 mL, 0.0644 mmol) in 1 mL of anhydrous tetrahydrofuran was stirred at room temperature for 3 h before the addition of propargylamine (4.1 mL, 0.0644 mmol). The reaction was stirred overnight, then solvent was removed under vacuum and the residue was purified by preparative HPLC equipped with a C18-reverse phase column using H2O:CH3CN:Formic acid (95:5:0.1 to 0: 100:0.1). to afford 1.5 mg (14 %) of product as an oil.1H NMR (500 Methanol-d4 ) δ 8.47 (s, 2H), 8.00 (dd, J = 13.4, 6.7 Hz, 2H), 6.84 (d, J = 2.8 Hz, 1H), 6.83 - 6.71 (m, 3H), 4.25 (dd, J = 15.3, 1.9 Hz, 2H), 3.92 (d, J = 2.5 Hz, 2H), 3.84 - 3.76 (m, 4H), 3.19 - 3.09 (m, 8H), 3.08 (s, 3H), 3.07 - 3.01 (m, 2H), 2.72 - 2.61 (m, 2H), 2.59 (t, J = 2.5 Hz, 1H), 2.50 (t, J = 6.7 Hz, 2H), 2.09 - 1.98 (m, 2H), 1.97 - 1.90 (m, 2H), 1.90 - 1.79 (m, 2H), 1.78 - 1.68 (m, 2H). 13C NMR (126 MHz, Methanol-^) 5 172.7, 169.2 (HCOO ), 158.3, 157.5, 154.9, 154.5, 144.4, 143.4, 107.6, 107.3, 107.2, 106.7, 80.4, 72.4, 69.0, 68.9, 59.8, 59.4, 55.9, 55.8, 49.0, 39.9 (2C), 38.3, 34.1, 29.5, 28.2, 28.1, 24.8 (2C). HRMS(ESI-MS) m/z calculated for C29H42N7O[M+H]+ 504.3445, found 504.3450.
Total synthesis scheme of Fentomycin-2 (Fento-2)
Synthesis of Fento-2
Scheme 6 Scheme 6. (a) 17, 19, toluene, 100 °C, 16 h, then concentrated to dryness, MeOH, rt, 4 h, then K2CO3, rt, 1 h, 33%. (b) Tf2O, TEA, DCM, -78 °C to rt, 6 h, 78%. (c) 21, 27, Cui, K2CO3, toluene, 160 °C, sealed tube, 72 h, 33%. (d) BF3 Et2O, DCM, -78 °C to rt, 6 h, 62%. (e) Benzaldehyde dimethylacetal, PTSA, DMF, 40 °C (30 mbar), 16 h, 98%. (f) DIPEA, M0MC1, DCM, rt, 72 h, 91%. (g) sec- Butyllithium (1.6 M in hexane), THF, -40 °C, 1.5 h, 61%. (h) O-benzylhydroxylamine HC1 salt, NaOAc, EtOH, rt, 2 h, 90%. (i) Anhydrous CeCE, methyllithium (3 M in diethoxymethane), THF, -78 °C, 1 h; then 26, -78 °C to 0 °C, 2 h, 76%. (j) H2, Pd/C, MeOH, rt, 16 h, 88%. (k) aq. HBF4 (48% wt/wt), MeCN, 82 °C, 2 h, 13%. (1) succinic anhydride, DMAP, anhydrous pyridine, 90 °C, 40 h, 36%. (m) dimethylamine HC1 salt, NaOH, H2O, sealed tube, 155 °C, 16 h, 91%. (n) SOCE, DCM, rt, 16 h, 93%. (o) Na2CO3, TBABr, anhydrous MeCN, 90 °C, 16 h, 27%. (p) Pd(OAc)2, BINAP, CS2CO3, dioxane, reflux, overnight, 46%. (q) SOC12, Py, DCM, 0 °C to rt, 2 h, 27%. (r) NaOH (1 N), DCM/H2O, 90 °C, overnight, 42%. (s) TFA, DCM, 0 °C then rt, 3 h, 69%. (t) HBTU, DIPEA, anhydrous THF, rt, overnight, 15%.
Ze/7- Butyl (12-{[2-(hydroxymethyl)pyridin-4-yl]amino}dodecyl)carbamate (45) dry Schlenk flask were added (4-chloropyridin-2-yl)methanol (30) (0.200 g, 1.394 mmol), tert-butyl(12-aminododecyl)carbamate (44) (0.502 g, 1.673 mmol), Pd(0Ac)2 (0.016 g, 0.069 mmol), BINAP (0.043 g, 0.069 mmol) and CS2CO3 (0.681 g, 2.090 mmol) under argon. Then anhydrous dioxane (15 mL) was added and the reaction was stirred at reflux overnight. After cooling to rt, the mixture was concentrated to dryness under reduced pressure and the residue was purified by CombiFlash on SiO2 using DCM:MeOH (100:0 to 80:20) to afford 45 as a brown oil (0.261 g, 46%). Further purification by preparative HPLC using H2O:MeCN:FA (95:5:0.1 to 0: 100:0. 1) was performed for NMR characterisation.1H NMR (500 MHz, mcthanol-d4) δ: 8.55 (bs, formate), 7.93 (d, J= 6.7 Hz, 1H), 6.75 (d, J= 2.2 Hz, 1H), 6.65 (dd, J= 6.7, 2.2 Hz, 1H), 4.63 (s, 2H), 3.25 (t, J= 7.0 Hz, 2H), 3.01 (t, J= 7.0 Hz, 2H), 1.65 (quint., J = 7.0 Hz, 2H), 1.46 (bs, 13H), 1.32 (bs, 14H) ppm. 13C NMR (126 MHz, methanol-d4) δ: 170.3 (formate), 159.1, 158.6, 157.4, 142.7, 107.6, 104.4, 79.8, 62.4, 43.7, 41.4, 31.0, 30.7 (4C), 30.4 (2C), 29.6, 28.8 (3C), 28.0, 27.9 ppm. HRMS (ESI-MS) m/z calculated for C23H42N3O3 [M+H]+ 408.3226, found 408.3223. re/7- Butyl (12-((2-(chloromethyl)pyridin-4-yl)amino)dodecyl)carbamate (46) To a Schlenk tube containing 45 (0.094 g, 0.231 mmol) and pyridine (0.075 mL, 0.924 mmol) was added anhydrous DCM (10 mL) and the solution was cooled at 0 °C, then SOCI2 (0.025 mL, 0.346 mmol) was added dropwise. The reaction was allowed to reach rt and was stirred for 2 h. After this time the reaction was carefully quenched with a sat. aq. solution of NaA’CL and extracted with DCM (3x 10 mL). The combined organic layers were dried over MgSO4, filtered and concentrated to dryness under reduced pressure and the residue was purified by CombiFlash on SiO2 using DCM:MeOH (100:0 to 90: 10) to afford 46 as a light yellow oil (0.026 g, 27%). 1H NMR (500 MHz, Methanol-d4) δ: 7.95 (d, J= 6.0 Hz, 1H), 6.69 (d, J= 2.4 Hz, 1H), 6.51 (dd, J= 6.1, 2.4 Hz, 1H), 4.51 (s, 2H), 3.16 (t, J= 7.1 Hz, 2H), 3.01 (t, J = 7.1 Hz, 2H), 1.62 (quint., J = 7.2 Hz, 2H), 1.44 (bs, 13H), 1.31 (bs, 14H) ppm. 13C NMR (126 MHz, methanol-J4) δ : 158.6, 157.5, 156.5, 148.4, 107.5 (2C), 79.8, 46.5, 43.4, 41.4, 31.0, 30.7 (4C), 30.5, 30.4, 29.8, 28.8 (3C), 28.1, 27.9 ppm. HRMS (ESI-MS) m/z calculated for [M+H]+ 426.2887, found 426.2886. tert-butyl {12-[(2-{[(2S,2'S)-l'-{[4-(dimethylamino)pyridin-2-yl]methyl}[2,2'-bipyrrolidin]-l- yl]methyl}pyridin-4-yl)amino]dodecyl}carbamate (47)
In a 10 mL Schlenk flask were mixed 46 (0.017 g, 0.040 mmol) and 33a (0.011 g, 0.040 mmol) in DCM/H2O (1 mL, 1: 1). Then an aq. solution of NaOH (1 N, 0.200 mL) was added and the resulting mixture was heated at 90 °C overnight. After cooling to rt, a sat. aq. solution of NaA’CL (1 mL) was added over the resulting brown mixture, which was extracted with DCM (3x 2 mL). The combined organic layers were dried over anhydrous MgSO4, filtered and concentrated to dryness under reduced pressure. The residue was purified by CombiFlash on neutral AI2O3 using DCM:MeOH (100:0 to 95:5) to afford 47 as a brown oil (0.011 g, 42%). 1H NMR (500 MHz, Methanol-d4) δ : 7.96 (d, J = 6.2 Hz, 1H), 7.84 (d, J= 6.1 Hz, 1H), 6.70 (d, J= 2.6 Hz, 1H), 6.63 (d, J= 2.4 Hz, 1H), 6.52 (dd, J= 6.2, 2.7 Hz, 1H), 6.40 (dd, J= 6.1, 2.4 Hz, 1H), 4.14 (d, J= 14.1 Hz, 1H), 4.12 (d, J= 14.2 Hz, 1H), 3.49 (d, J = 14.1 Hz, 1H), 3.46 (d, J= 14.4 Hz, 1H), 3.12 - 2.99 (m, 6H), 2.97 (s, 6H), 2.85 - 2.80 (m, 2H), 2.42 - 2.34 (m, 2H), 1.96 - 1.90 (m, 2H), 1.81 - 1.66 (m, 6H), 1.60 - 1.53 (m, 2H), 1.47 - 1.41 (m, 11H), 1.33 - 1.28 (m, 16H) ppm.13C NMR (126 MHz, Methanol-d4) δ: 159.8 (2C), 158.7, 157.1 (2C), 148.6, 148.1 106.9 (2C), 106.8 (2C), 79.9, 68.4, 68.1, 62.4, 62.0, 56.4 (2C), 43.4, 41.5, 39.4 (2C), 31.1, 30.9 (4C), 30.7, 30.6, 30.1, 29.0 (3C), 28.3, 28.2 (2C), 28.0, 24.9, 24.8. ppm. HRMS (ESI-MS) m/z calculated for C39H56N7O2 [M+H]+ 664.5278, found 664.5281. N 1-(2-{[(2iS,2'iS)-l'-{[4-(dimethylamino)pyridin-2-yl]methyl}[2,2'-bipyrrolidin]-l- yl]methyl}pyridin-4-yl)dodecane-l,12-diamine (48)
Over a solution of 47 (0.011 g, 0.017 mmol) in anhydrous DCM (0.5 mL) at 0 °C, was added TFA (0.026 mL, 0.34 mmol). The reaction was allowed to reach rt and then was stirred for 3 h. After this time the mixture was concentrated to dryness under reduced pressure and the residue was redissolved in DCM, washed with an aq. solution of NaOH (1 N, 1 mL) and extracted with DCM (3x 1 mL). Combined organic layers were dried over anhydrous MgSO4. filtered and concentrated to dryness under reduced pressure. The residue was purified by preparative HPLC using FLOMeCNTA (95:5:0.1 to 0: 100:0.1) to afford 48 as a light brown oil (0.006 g, 69%). (500 MH1Hz, N MMetRhanol-d4) δ: 8.53 (bs, formate), 8.01 (d, J= 6.8 Hz, 1H), 7.72 (d, J= 6.8 Hz, 1H), 6.83 - 6.76 (m, 2H), 6.71 (d, J= 2.3 Hz, 1H), 6.66 (dd, J = 6.8, 2.5 Hz, 1H), 4.25 (d, J= 15.4 Hz, 1H), 4.21 (d, J= 15.6 Hz, 1H), 3.76 (d, J= 15.4 Hz, 1H), 3.69 (d, J = 15.6 Hz, 1H), 3.23 (t, J = 7.1 Hz, 2H), 3.16 (s, 6H), 3.12 - 3.03 (m, 2H), 3.00 - 2.93 (m, 2H), 2.90 (t, J= 7.6 Hz, 2H), 2.59 - 2.47 (m, 2H), 2.05 - 1.93 (m, 2H), 1.93 - 1.78 (m, 4H), 1.78 - 1.69 (m, 2H), 1.68 - 1.59 (m, 4H), 1.45 - 1.28 (m, 16H) ppm. 13C NMR (126 MHz, Methanol-d4) 5: 170 (formate), 159.4, 158.6, 154.4 (2C), 142.8, 142.4, 107.4 (2C), 106.7 (2C), 68.8, 68.7, 59.1, 58.4, 56.2, 56.1, 43.9, 40.9, 40.1 (2C), 30.9 (2C), 30.8, 30.7, 30.6, 30.4, 29.7, 28.8, 28.2 (3C), 27.6, 24.9 (2C) ppm. HRMS (ESI-MS) m/z calculated for C34H58N7 [M+H]+ 564.4754, found 564.4752. A solution of marmysunate (29)35,66 (8.0 mg, 0.016 mmol), HBTU (11.8 mg, 0.0312 mmol) and DIPEA (28.0 μL, 0.159 mmol) in anhydrous THF (100 μL) was stirred 1 h at rt before the addition of 48 (9.0 mg, 0.016 mmol) in anhydrous THF (100 μL). The reaction was stirred 4 h at rt until TLC in AI2O3 (DCM:MeOH, 95:5) showed a total conversion, then the mixture was concentrated to dryness under reduced pressure and the residue was purified by preparative HPLC using H2O:MeCN:FA (95:5:0.1 to 0: 100:0.1) to afford Fento-2 (49) as a red solid (5 mg, 15%). (500 MHz, m1Hct NhaMnoRl-Uy) 5: 9.69 (bs, 1H), 9.52 (d, J= 8.9 Hz, 1H), 8.36 (d, J= 8.6 Hz, 1H), 8. 19 (d, J= 8.6 Hz, 1H), 7.94 (d, J= 6.4 Hz, 1H), 7.85 (d, J= 6.4 Hz, 1H), 7.77 (s, 1H), 7.59 (dd, J= 8.8, 1.1 Hz, 1H), 7.52 (bs, 2H), 6.69 - 6.63 (m, 2H), 6.63 - 6.53 (m, 2H), 4.89 - 4.82 (m under solvent, 1H), 4.76 (d, J= 9.5 Hz, 1H), 4.17 (d, J= 14.8 Hz 1H), 4.16 (d, J= 15.4 Hz, 1H), 3.70 (t, J= 15.0 Hz, 2H), 3.40 - 3.32 (m, 1H), 3.20 - 3.00 (m, 12H), 2.98 - 2.89 (m, 2H), 2.83 - 2.64 (m, 2H), 2.64 - 2.51 (m, 7H), 2.31 - 2.21 (m, 1H), 2.03 - 1.93 (m, 2H), 1.93 - 1.83 (m, 3H), 1.83 - 1.73 (m, 2H), 1.72 - 1.61 (m, 2H), 1.61 - 1.51 (m, 2H), 1.40 - 1.37 (m, 3H), 1.36 - 1.10 (m, 18H), 1.04 (d, J= 6.1 Hz, 3H) ppm. 13C NMR (126 MHz, methanol-<74) δ: 187.6, 186.7, 174.0, 173.7, 158.7, 158.0, 155.6, 155.3, 150.1, 144.9, 144.1, 140.3, 138.2, 137.8, 137.2, 136.2, 135.9, 133.2, 129.9, 129.8, 129.3, 129.1, 128.7 123.6, 116.9, 112.0, 107.3 (2C), 106.9 (2C), 80.8, 70.6, 69.2, 69.1, 68.0, 60.4, 59.6, 56.0 (2C), 52.5, 43.7, 40.6, 39.8 (2C), 36.0, 31.4, 30.7 (5C), 30.6 (2C), 30.4, 29.8, 28.5, 28.4, 28.1, 28.0, 25.5, 25.0 (2C), 21.7, 18.6 ppm. 2D NMR COSY, HSQC, HMBC (spectra in methanol-<4 provided). HRMS (ESI-MS) m/z calculated for C64H83N8O6 [M+H]+ 1059.6436, found 1059.6433.
Total synthesis scheme of Fentomycin-3 (Fento-3)
Synthesis of Fento-3
Scheme 7. (a) DMAP, anhydrous pyridine, 90 °C, 24 h, 66%. (b) dimethylamine HC1 salt, NaOH, H2O, sealed tube, 155 °C, 16 h, 91%. (c) SOCE, DCM, rt, 16 h, 93%. (d) Na2CO3, TBABr, anhydrous MeCN, 90 °C, 16 h, 27%. (e) Pd(OAc)2, BINAP, CS2CO3, dioxane, reflux, overnight, 66%. (f) SOC12, DCM, 0 °C to rt, 16 h, 56%. (g) Na2CO3, TBABr, MeCN, reflux under Ar, 16 h, 66%. (h) TFA, DCM, 0 °C then rt, 2 h, 69%. (i) HBTU, DIPEA, THF, rt, 6 h, 70%.
Cholesteryl hemisuccinate (52)
The synthesis of cholesteryl hemisuccinate is adapted from a previously described procedure68. Cholesterol (50) (387 mg, 1 mmol) and succinic anhydride (51) (1 g, 10 mmol) were dissolved in 10 mL of anhydrous pyridine. Then, DMAP (61 mg, 0.5 mmol) was added, and the reaction mixture was stirred at 90 °C for 24 h under argon. After completion of the reaction, the dark brown coloured reaction mixture was cooled to rt and a solution of HC1 (I N) was added dropwise to precipitate the product. The resulting white precipitate was filtered and thoroughly washed with distilled water. The obtained residue was dried under reduced pressure and purified by CombiFlash in SiO2 using DCM:MeOH (100:0 to 80:20) to afford 52 as a white solid (320 mg, 66 %). Spectral data is in agreement with the literature. ’H NMR (400 MHz, chloroform-d) δ: 5.40 - 5.34 (m, 1H), 4.69 - 4.57 (m, 1H), 2.73 - 2.64 (m, 2H), 2.64 - 2.57 (m, 2H), 2.36 - 2.28 (m, 2H), 2.05 - 1.91 (m, 2H), 1.91 - 1.76 (m, 3H), 1.67-1.05 (m, 19H), 1.04 - 0.95 (m, 6H), 0.93 - 0.89 (3H, m), 0.89 - 0.79 (m, 6H), 0.71 - 0.64 (m, 3H) ppm. 13C NMR (101 MHz, chloroform-J) δ: 177.4, 171.7, 139.7, 122.9, 74.7, 56.8, 56.3, 50.1, 42.5, 39.9, 39.7, 38.2, 37.1, 36.7, 36.3, 35.9, 32.0 (2C), 29.4, 29.0, 28.4, 28.2, 27.8, 24.4, 24.0, 23.0, 22.7, 21.2, 19.4, 18.9, 12.0 ppm. HRMS (ESI-MS) m/z calculated for C31H49O4 [M-H]’ 485.3625, found 485.3639.
A solution of 52 (9.8 mg, 0.02 mmol), HBTU (38 mg, 0.1 mmol) and DIPEA (105 μL, 0.62 mmol) in anhydrous THF (350 μL) was stirred 1 h at rt before the addition of 38 ( 18 mg, 0.04 mmol) in anhydrous THF (350 μL). The reaction was stirred 6 h at rt until TLC (DCM:MeOH, 95:5) showed a total conversion, then the mixture was concentrated to dryness under reduced pressure and the residue was purified by preparative HPLC using ITOMeCNTA (95:5:0.1 to 0: 100:0.1) to afford Fento-3 (53) as a white solid (13.0 mg, 70%). 1H (5 N00M MRHz, Methanol-d4) δ: 7.99 (dd, J= 5.2, 2.2 Hz, 1H), 7.89 (d, J= 6.6 Hz, 1H), 6.80 - 6.71(m, 2H), 6.71 - 6.60 (m, 2H), 5.34 (d, J= 4.9 Hz, 1H), 4.56 - 4.45 (m, 1H), 4.26 (d, J= 15.3 Hz, 1H), 4.20 (d, J = 15.6 Hz, 1H), 3.77 (d, J= 15.3 Hz, 1H), 3.71 (d, J= 15.5 Hz, 1H), 3.28 - 3.18 (m, 4H), 3.15 (s, 6H), 3.12 - 3.04 (m, 2H), 3.00 - 2.91 (m, 2H), 2.64 - 2.51 (m, 4H), 2.47 (t, J= 6.7 Hz, 2H), 2.35 - 2.24 (m, 2H), 2.10 - 1.77 (m, 11H), 1.77 - 1.24 (m, 17H), 1.24 - 1.05 (m, 7H), 1.05 - 0.98 (m, 5H), 0.98 - 0.91 (m, 4H), 0.89 (d, J = 6.6 Hz, 3H), 0.88 (d, J = 6.6 Hz, 3H), 0.71 (s, 3H) ppm. 13C NMR (126 MHz, methanol-J4) δ: 174.6, 174.0, 159.2, 158.5, 154.9, 154.8, 143.4, 142.9, 141.2, 123.8, 107.5 (2C), 106.7 (2C), 75.7, 69.0, 68.9, 59.5, 59.0, 58.3, 57.7, 56.2 (2C), 51.8, 43.6, 43.5, 41.3, 40.8, 40.0 (2C), 39.9, 39.3, 38.4, 37.9, 37.5, 37.3, 33.3, 33.2, 31.6, 30.9, 29.5, 29.3, 28.9, 28.3 (2C), 28.1, 26.8, 25.5, 25.1, 25.0 (2C), 23.3, 23.1, 22.3, 19.9, 19.4, 12.5 ppm. 2D NMR COSY, HSQC, HMBC (spectra in me th anol <6 provided). HRMS (ESI-MS) m/z calculated for C57H90N7O3 [M+H]+ 920.7100, found 920.7099.
Total synthesis scheme of Fentomycin-4 (Fento-4)
Synthesis of Fento-4
Scheme 8. (a) benzene, reflux, overnight, 84%. (b) dimethylamine HC1 salt, NaOH, H2O, sealed tube, 155 °C, 16 h, 91%. (c) SOCh, DCM, rt, 16 h, 93%. (d) Na2CO3, TBABr, anhydrous MeCN, 90 °C, 16 h, 27%. (e) Pd(OAc)2, BINAP, CS2CO3, dioxane, reflux, overnight, 66%. (f) SOC12, DCM. 0 °C to rt, 16 h, 56%. (g) Na2CO3, TBABr, MeCN, reflux under Ar, 16 h, 66%. (h) TFA, DCM, 0 °C then rt, 2 h, 69%. (i) HBTU, DIPEA, THF, overnight, 20%.
4-morpholino-4-oxobutanoic acid (55)
To a solution of morpholine 54 (1.0 g, 11.48 mmol) in benzene (23 mL) was added succinic anhydride 51 (1.4 g, 13.98 mmol). After stirring at reflux overnight, the mixture was concentrated under reduced pressure and the crude was precipitated in w-hcxanc/EtOAc (4 mL, 1 :2) to afford 55 as a white solid (1.8 g, 84%). Spectral data is in agreement with the literature69,70. (400 MHz, chloro1fHor NmM-JR) 5: 7.80 (bs, 1H), 3.77 - 3.59 (m, 4H), 3.59 - 3.50 (m, 2H), 3.50 - 3.29 (m, 2H), 2.75 - 2.62 (m, 2H), 2.62 - 2.50 (m, 2H).
Fento-4 (56)
A solution of 55 (8.7 mg, 0.046 mmol), HBTU (34.0 mg, 0.092 mmol) and DIPEA (80 μL, 0.46 mmol) in anhydrous THF (1 mL) was stirred at rt for 2 h under argon. Then, a solution of 38 (21.0 mg, 0.045 mmol) in anhydrous THF (0.5 mL) was added dropwise. After stirring at rt overnight, the mixture was concentrated under reduced pressure. The crude was purified by preparative HPLC using H2O:MeCN:FA (90: 10:0.1 to 80:20:0.1) to afford Fento-4 (56) as a white solid (formate salt, 6.0 mg, 20%). 1H NMR (500 MHz, Methanol-d4) δ : 8.73 - 8.21 (m, formate), 8.01 (dd, J = 6.0, 1.4 Hz, 1H), 7.93 (d, J= 6.5 Hz, 1H), 6.88 - 6.79 (m, 2H), 6.77 - 6.67 (m, 2H), 4.25 (d, J= 15,6 Hz, 1H), 4.21 (d, J = 15.7 Hz, 1H), 3.74 (d, J= 15.6 Hz, 1H), 3.71 - 3.59 (m, 5H), 3.59 - 3.50 (m, 4H), 3.28 (t, J= 7.0 Hz, 2H), 3.22 (t, J= 6.8 Hz, 2H), 3.19 (s, 6H), 3.12 - 3.03 (m, 2H), 2.99 - 2.90 (m, 2H), 2.67 (t, J= 6.7 Hz, 2H), 2.56 - 2.42 (m, 4H), 2.04 - 1.92 (m, 2H), 1.92 - 1.73 (m, 6H), 1.72 - 1.55 (m, 4H) ppm. 13C NMR (126 MHz, methanol-J4) δ: 175.0, 172.9, 168.8 (formate), 159.7, 158.9, 154.0 (2C), 142.0 (2C), 107.5 Ill
(2C), 106.6 (2C), 68.6, 68.4, 67.8 (2C), 58.6, 58.1, 56.4, 56.2, 47.2, 43.6, 43.5, 40.2 (2C), 39.9, 31.8, 29.1, 28.0, 27.9 (2C), 26.8, 24.9 (2C) ppm. HRMS (ESI-MS) m/z calculated for C34H53N8O3 [M+H]+ 621.4241, found 621.4229.
Total synthesis scheme of Fentomycin-5 (Fento-5)
Synthesis of Fento-5
Scheme 9. (a) 17, 19, toluene, 100 °C, 16 h, then concentrated to dryness, MeOH, rt, 4 h, then K2CO3, rt, 1 h, 33%. (b) Tf2O, TEA, DCM, -78 °C to rt, 6 h, 78%. (c) 21, 27, Cui, K2CO3, toluene, 160 °C, sealed tube, 72 h, 33%. (d) BF3 Et2O, DCM, -78 °C to rt, 6 h, 62%. (e) Benzaldehyde dimethylacetal, PTSA, DMF, 40 °C (30 mbar), 16 h, 98%. (f) DIPEA, M0MC1, DCM, rt, 72 h, 91%. (g) sec- Butyllithium (1.6 M in hexane), THF, -40 °C, 1.5 h, 61%. (h) O-benzylhydroxylamine HC1 salt, NaOAc, EtOH, rt, 2 h, 90%. (i) Anhydrous CeCE, methyllithium (3 M in diethoxymethane), THF, -78 °C, 1 h; then 26, -78 °C to 0 °C, 2 h, 76%. (j) H2, Pd/C, MeOH, rt, 16 h, 88%. (k) aq. HBF4 (48% wt/wt), MeCN, 82 °C, 2 h, 13%. (1) succinic anhydride, DMAP, anhydrous pyridine, 90 °C, 40 h, 36%. (m) NaOH (IN), DCM/H2O, rt overnight, 49%. (n) K2CO3, MeCN, rt, overnight, 70%. (o) SOCI2, DCM, 0 °C to rt, 2 h, 57%. (p) NaOH (1 N), DCM/H2O, rt, overnight, 68%. (q) TFA, anhydrous DCM, 0 °C to rt, 48 h, 69%. (r) HBTU, DIPEA, THF, rt, 4 h, 26%.
Scheme 9 (25,2'iS)-l-[(l-methyl-l/7-benzimidazol-2-yl)methyl]-2,2'-bipyrrolidine (58a)
Over a solution of 2-(chloromethyl)-l-methyl-lH-l,3-benzodiazole hydrochloride (57) (0.200 g, 0.922 mmol) in DCM/H2O (1: 1, 10 mL) were added an aq. solution of NaOH (IN, 5 mL) and (S,S)-2,2- bipyrrolidine (32) (0.258 g, 1.843 mmol). The resulting mixture was stirred at rt overnight. After this time the resulting brown mixture was extracted with DCM (3x 10 mL). The combined organic layers were dried over anhydrous MgSO4, filtered and concentrated to dryness under reduced pressure. The residue was purified by CombiFlash on SiCL using DCM:MeOH:TEA (80:20:5) to afford 58a as a light brown oil (0.127 g, 49%). Spectral data is in agreement with the literature71. (500 MHz, 1H NMR Methanol-d4) δ: 7.60 (dd, J= 7.6, 1.0 Hz, 1H), 7.45 (d, J= 7.8 Hz, 1H), 7.31 - 7.18 (m, 2H), 4.30 (d, J = 14.0 Hz, 1H), 3.86 (s, 3H), 3.77 (d, J= 14.0 Hz, 1H), 3.14 (q, J= 7.4 Hz, 1H), 2.95 - 2.88 (m, 1H), 2.87 - 2.74 (m, 3H), 2.55 - 2.47 (m, 1H), 2.03 - 1.93 (m, 1H), 1.85 - 1.64 (m, 5H), 1.62 - 1.52 (m, 1H), 1.52 - 1.41 (m, 1H) ppm.
(25,2'iS)-l,l'-bis[(l-methyl-l/7-benzimidazol-2-yl)methyl]-2,2'-bipyrrolidine (Nordlander-Costas, 58b) was obtained as a side product of the reaction as a light brown oil (0.065 g, 16%). 1 H NMR (500 MHz, Methanol-d4) δ: 7.57 (d, J = 7.8 Hz, 2H), 7.42 (d, J = 7.8 Hz, 2H), 7.29 - 7.19 (m, 4H), 5.47 (s, DCM), 4.22 (d, J= 13.7 Hz, 2H), 3.81 (s, 6H), 3.67 - 3.58 (m, 2H), 2.87 - 2.75 (m, 2H), 2.75 - 2.63 (m, 2H), 2.41 - 2.24 (m, 2H), 1.87 - 1.69 (m, 6H), 1.68 - 1.58 (m, 2H) ppm. 13C NMR (126 MHz, methanol-^) δ: 153.9 (2C), 142.3 (2C), 137.2 (2C), 124.0 (2C), 123.4 (2C), 119.4 (2C), 110.8 (2C), 66.8 (2C), 56.6 (2C), 52.9 (2C), 30.5 (2C), 27.3 (2C), 25.0 (2C) ppm. HRMS (ESI-MS) m/z calculated for C26H33N6 [M+H]+ 429.2767, found 429.2758. tert-Butyl {4-[2-(hydroxymethyl)-TH-benzimidazol-l-yl]butyl}carbamate (61) dry 50 mL round bottom flask were added (lH-l,3-benzodiazol-2-yl)methanol
(59) (0.200 g, 1.351 mmol), tert-butyl A'-(4-bromobuty I (carbamate (60) (0.681 g, 2.703 mmol) and K2CO3 (0.746 g, 5.405 mmol) under argon. Then anhydrous MeCN (10 mL) was added. The reaction mixture was stirred at rt overnight. After this time the reaction mixture was concentrated to dryness under reduced pressure. The residue was purified by CombiFlash on SiCL using DCM:MeOH (100:0 to 90: 10) to afford 61 as a white powder (0.300 g, 70%). (500 MHz1,H m NetMhaRnol -<A) 5: 7.62 (d, J= 7.8 Hz, 1H), 7.53 (d, J= 8 Hz, 1H), 7.30 - 7.22 (m, 2H), 5.47 (s, DCM), 4.85 (s, 2H), 4.34 (t, J= 7.5 Hz, 2H), 3.07 (t, J = 6.7 Hz, 2H), 1.95 - 1.83 (m, 2H), 1.60 - 1.48 (m, 2H), 1.41 (s, 9H) ppm. 13C NMR (126 MHz, methanol-J4) δ : 158.6, 154.5, 142.6, 136.5, 124.2, 123.4, 119.8, 111.5, 79.9, 57.9, 54.9 (DCM), 44.7, 40.7, 28.8 (3C), 28.3, 28. 1 ppm. HRMS (ESI-MS) m/z calculated for CI7H26N3O3 [M+H]+ 320.1974, found 320.1970. tert- Butyl {4-[2-(chloromethyl)-l//-benzimidazol-l-yl|butyl}carbamate (62) Schlenk tube containing 61 (0.300 g, 0.940 mmol) in anhydrous DCM (10 mL) was added pyridine (0.152 mL, 1.881 mmol) and the solution was cooled at 0 °C, then SOCI2 (0.102 mL, 1.412 mmol) was added dropwise. The reaction was allowed to reach rt and was stirred for 2h. After this time the reaction was carefully quenched with a sat. aq. solution of Na2CO3 and extracted with DCM (3x 10 mL). The combined organic layers were dried over MgSO4, filtered and concentrated to dryness under reduced pressure and the residue was purified by CombiFlash on SiO? using DCM:MeOH (100:0 to 90: 10) to afford 62 as a colourless oil (0.181 g, 57%). (500 MHz, 1 mHe NthManRol-<4) 5: 7.64 (d, J = 7.9 Hz, 1H), 7.56 (d, J= 8.1 Hz, 1H), 7.37 - 7.25 (m, 2H), 4.95 (s, 2H), 4.38 - 4.31 (m, 2H), 3.14 - 3.07 (m, 2H), 1.96 - 1.86 (m, 2H), 1.62 - 1.53 (m, 2H), 1.42 (s, 9H). 13C NMR (126 MHz, methanol- d4) 8: 158.6, 150.8, 142.7, 136.3, 124.9, 124.0, 120.1, 111.9, 79.9, 45.0, 40.6, 36.9, 28.8 (3C), 28.3, 28.0. HRMS (ESI-MS) m/z calculated for CnfLsCIbLCh [M+H]+ 338.1635, found 338.1630. tert- Butyl {4-[2-({(2A,2'A)-l'-[(l-methyl-LH-benzimidazol-2-yl)methyl] [2,2'-bipyrrolidin]-l- yl}methyl)-l/7-benzimidazol-l-yl]butyl}carbamate (63)
In a 10 mL Schlenk flask were mixed 62 (0.059 g, 0.176 mmol) and 58a (0.050 g, 0.176 mmol) in DCM/H2O (1: 1, 2 mL). Then an aq. solution of NaOH (1 N, 0.88 mL) was added and the resulting mixture was stirred at rt overnight. After this time, a sat. aq. solution of Na2CO3 (1 mL) was added and the mixture was extracted with DCM (3x2 mL). The combined organic layers were dried over anhydrous MgSO4, filtered and concentrated to dryness under reduced pressure. The residue was purified by CombiFlash on neutral A12O3 using w-hexane:EtOAc (1: 1) to afford 63 as a light yellow oil (0.071 g, 68%). 1H NMR (500 MHz, methylene chloride <6) δ: 7.68 - 7.66 (m, 2H), 7.38 - 7.34 (m, 2H), 7.29 - 7.20 (m, 4H), 4.31 - 4.27 (m, 1H), 4.22 - 4.19 (m, 3H), 3.81 (s, 3H), 3.68 (d, J= 13.3 Hz, 1H), 3.56 (d, J= 13.3 Hz, 1H), 3.21 - 3.11 (m, 2H), 2.87 - 2.77 (m, 2H), 2.74 (t, J = 7.7 Hz, 1H), 2.71 - 2.63 (m, 1H), 2.37 - 2.22 (m, 2H), 1.93 - 1.50 (m, 12H), 1.43 (s, 9H) ppm. 13C NMR (126 MHz, methylene chloride <6) δ: 156.4, 153.1, 152.7, 143.0, 142.8, 136.9, 136.2, 122.7 (2C), 122.0 (2C), 119.8, 119.7, 110.2, 109.7, 79.2, 65.9, 65.5, 55.9 (2C), 53.0, 52.8, 44.0, 40.5 (Ji3C-i4N = 16 HZ), 30.4, 28.7 (3C), 28.2, 27.5, 26.7, 26.5, 24.6, 24.4 ppm. HRMS (ESI-MS) m/z calculated for C34H48N7O2 [M+H]+ 586.3869, found 586.3869.
4- [2-({(2iS,2'iS)-l'-[(l-methyl-l/f-benzimidazol-2-yl)methyl] [2,2'-bipyrrolidin]-l-yl}methyl)-LH- benzimidazol-l-yl]butan-l-amine (64)
Over a solution of 63 (0.045 g, 0.077 mmol) in anhydrous DCM (1 mL) at 0 °C, was added TFA (0.118 mL, 1.536 mmol). The reaction was allowed to reach rt and then was stirred for 48 h. After this time the mixture was concentrated to dryness under reduced pressure and the residue was redissolved in DCM (2 mL), washed with an aq. solution ofNaOH (1 N, 2 mL) and extracted with DCM (3 x2 mL). Combined organic layers were dried over anhydrous MgSO4, filtered and concentrated to dryness under reduced pressure. The residue was purified by CombiFlash on AI2O3 using DCM:MeOH:TEA (90: 10:5) to afford 64 as a brown oil (0.026 g, 69%). Further purification by preparative HPLC using H2O:MeCN:FA (95:5:0.1 to 0: 100:0.1) was performed for NMR characterisation. (500 MHz, m1eHth NanMolR-<4) 5: 8.53 (bs, formate), 7.49 - 7.43 (m, 2H), 7.43 - 7.36 (m, 2H), 7.31 - 7.23 (m, 2H), 7.23 - 7.13 (m, 2H), 4.49 > 4.34 (m, 3H), 4.27 - 4.14 (m, 2H), 4.00 (d, J= 14.8 Hz, 1H), 3.79 (s, 3H), 3.26 (bs, 2H), 3.18 - 2.95 (m, 3H), 2.93 (t, J= 6.7 Hz, 2H), 2.83 (bs, 1H), 2.17 - 1.99 (m, 3H), 1.98 - 1.63 (m, 9H).13C NMR (126 MHz, Methanol-d4) δ: 169.7 (formate), 152.6, 152.1, 142.3 (2C), 137.2, 136.5, 124.7 (2C), 124.0 (2C), 119.6, 119.5, 111.5, 111.2, 69.3 69.1, 56.2 (2C), 53.4, 53.1, 44.4, 40.5, 30.7, 28.7, 28.4, 27.9, 26.3, 25.4, 25.4 ppm. HRMS (ESI-MS) m/z calculated for C29H40N7 [M+H]+ 486.3345, found 486.3343. Fento-5 (65)
A solution of marmysunate (29)35,66 (5.5 mg, 0.0107 mmol), HBTU (7.9 mg, 0.0214 mmol) and DIPEA (18.6 μL, 0.107 mmol) in anhydrous THF (200 μL) was stirred 1 h at rt before the addition of 64 (5.2 mg, 0.0107 mmol) in anhydrous THF (200 μL). The reaction was stirred 4 h at rt until TLC in AI2O3 (DCM:MeOH, 95:5) showed a total conversion, then the mixture was concentrated to dryness under reduced pressure and the residue was purified by preparative HPLC using H2O:MeCN:FA (95:5:0.1 to 0: 100:0.1) to afford Fento-5 (65) as a red solid (2.7 mg, 26%). 'HNMR (500 MHz, methylene chloride- d2) 8: 9.59 (bs, 1H), 9.52 (d, J = 8.8 Hz, 1H), 8.30 (d, J= 8.7 Hz, 1H), 8.04 (d, J = 8.6 Hz, 1H), 7.69 (bs, 1H), 7.64 (d, J= 7.8 Hz, 1H), 7.62 - 7.52 (m, 3H), 7.48 (d, J= 7.3 Hz, 1H), 7.34 - 7.24 (m, 3H), 7.24 - 7.11 (m, 3H), 4.80 (bs, 1H), 4.69 (d, J= 9.5 Hz, 1H), 4.38 - 4.29 (m, 1H), 4.27 - 4.16 (m, 2H), 4.16 - 4.06 (m, 1H), 3.96 - 3.84 (m, 2H), 3.70 (s, 3H), 3.39 - 3.20 (m, 3H), 3.15 - 2.95 (m, 4H), 2.81 - 2.61 (m, 4H), 2.60 - 2.49 (m, 5H), 2.27 - 2.17 (m, 1H), 2.03 - 1.61 (m, 11H), 1.61 - 1.51 (m, 2H), 1.34 - 1.28 (m, 3H), 1.05 - 0.96 (d, J = 6.1 Hz, 3H) ppm. 13C NMR (126 MHz, methylene chloride <6) 5: 186.8, 186.0, 172.6, 171.8, 152.3, 151.4, 149.2, 142.4, 142.0, 139.4, 137.0, 136.9, 136.4, 136.3, 135.8,
135.3, 135.0, 132.6, 129.3, 129.0, 128.6, 128.3, 127.6, 123.3, 123.2, 122.8, 122.6, 122.5, 119.7, 119.6, 116.1, 111.4, 110.3, 109.9, 79.7, 69.7, 66.9 (2C), 55.8 (2C), 55.6, 53.0 (2C), 51.6, 44.0, 39.3, 35.3, 31.1,
30.3, 30.2 (2C), 27.6 (2C), 27.5, 25.2, 24.9, 24.7, 21.8, 18.3 ppm. 2D NMR COSY, HSQC, HMBC (spectra in methylene chloridc-dS provided). HRMS (ESI-MS) m/z calculated for Cv:,H„sNsO„ [M+H]+ 981.5027, found 981.5026.
Synthesis of Fentomycin 6 (X)
Scheme 2. Synthesis of Fentomycin 6 (X). (x) succinic acid, DMAP, anhydrous pyridine, 90 °C, 24 h, xx%. (y) HBTU, DIPEA, THF, rt, o n., xx%.
Monocholestanylsuccinate ChtaMS The synthesis of monocholestanyl succinate is adapted from a previously described procedure [39], Cholestanol (389 mg, 1 mmol) and succinic anhydride (1 g, 10 mmol) were dissolved in 10 mL of dry pyridine. Then, DMAP (61 mg, 0.5 mmol) was added, and the reactional mixture was stirred at 90 °C for 24 h under an inert atmosphere. After completion of the reaction, the dark brown colored reaction mixture was cooled to room temperature and a solution of 1 M HC1 was added dropwise to precipitate the product. The resulting white precipitate was thoroughly washed with distilled water. The obtained product was dried under reduced pressure and purified by Combiflash in SiO2 using DCM:MeOH (100:0 to 80:20) to afford ChtaMS as a white solid (320 mg, 66 %). 1H NMR (400 MHz, chloroform-^) δ: 4.71 (m, 1H), 2.66 (m, 2H), 2.58 (m, 2H), 1.96 (dt, J = 2.9, 12.4 Hz, 1H), 1.86-1.42 (m, 9H), 1.42-1.20 (m, 9H), 1.20-0.93 (m, 10H), 0.93-0.83 (10H, m), 0.81 (m, 3H), 0.67-0.60 (m, 4H) ppm. 13C NMR (101 MHz, chloroform-^) δ: 177.6, 171.8, 74.5, 56.6, 56.4,
54.3, 44.8, 42.8, 40.1, 39.7, 36.9, 36.3, 35.9, 35.6 (2C), 34.1, 32.1, 29.4, 29.1, 28.7, 28.4, 28.1, 27.5,
24.3, 24.0, 23.0, 22.7, 21.3, 18.8, 12.4, 12.2 ppm. HRMS(ESI-MS) m/z calculated for C31H51O4 [M-H]+ 487.3782, found 487.3797.
Fentomycin 6 (X)
A solution of ChtaMS (15 mg, 0.031 mmol), HBTU (59 mg, 0.155 mmol) and DIPEA (162 μL, 0.93 mmol) in anhydrous THF (500 μL) was stirred 1 h at room temperature before the addition of 21 (14 mg, 0.031 mmol) in anhydrous DMF (500 μL). The reaction was stirred overnight at room temperature until TLC (DCM:MeOH, 95:5) showed a total conversion, then the mixture was concentrated to dryness under reduced pressure and the residue was purified by preparative HPLC using H2O:MeCN:FA (95:5:0. 1 to 0: 100:0. 1) to afford Fentomycin 6 (X) as a white solid after freeze drying (7.0 mg, 25%). ’H NMR (500 MHz, Methanol-d4) δ : 8.02 - 7.95 (d, J= 6.5 Hz, 1H), 7.93 - 7.84 (d, J= 6.5 Hz, 1H), 6.78 - 6.69 (m, 2H), 6.69 - 6.57 (m, 2H), 4.69 - 4.57 (m, 1H), 4.30 - 4.13 (t, J= 15.3 Hz, 2H), 3.82 - 3.75 (d, J= 15.1 Hz, 1H), 3.75 - 3.69 (d, J= 15.3 Hz, 1H), 3.26 - 3.16 (t, J= 6.5 Hz, 4H), 3.16 - 3.04 (m, 8H), 3.03 - 2.93 (m, 2H), 2.69 - 2.59 (m, 2H), 2.59 - 2.52 (t, J= 6.6 Hz, 2H), 2.52 - 2.41 (t, J= 6.6 Hz, 2H), 2.1 - 1.95 (m, 3H), 1.95 - 1.44 m, 19H), 1.44 - 1.21 m, 9H), 1.21 - 0.95(m, 10H), 0.95 - 0.91 (d, J= 6.4 Hz, 3H), 0.91 - 0.85 (m, 7H), 0.85 - 0.78 (s, 3H), 0.68 (s, 3H), 0.67 - 0.58 (m, 1H) ppm. 13C NMR (126 MHz, Methanol-d4) δ : 174.6, 174.0, 158.8, 158.2, 155.4 (2C), 144.5, 143.9, 107.4 (2C), 107.9 (2C), 75.5, 69.3, 69.2, 60.2, 59.6, 58.0, 57.8, 56.1 (2C), 55.8, 46.2, 43.9, 43.4, 41.5, 40.8, 39.9 (3C), 38.1, 37.5, 37.3, 37.0, 36.7, 35.3, 33.4, 31.6, 30.9, 30.0, 29.4, 29.3, 28.7, 28.5 (2C), 28.1, 26.9, 25.4, 25.1 (3C), 23.3; 23.1, 22.4, 19.3, 12.8, 12.6 ppm. 2D NMR COSY, HSQC, HMBC (spectra in methanol <6 provided). HRMS(ESI+MS) m/z calculated for CsvH^NyOs [M+H]+ 922.7256, found 922.7257.
Synthesis of Fentomycin 7 (XI)
Scheme 3. Synthesis of modified BPMEN ligand with linker, (a) dimethylamine hydrochloride salt, NaOH, H2O, sealed tube, 150 °C, 2 days, 93%. (b) SOC12, DCM, rt, overnight, 69%. (c) Na2CO3, anhydrous MeCN, 90 °C, 24 h, 23%. (d) Pd(OAc)2, BINAP, Cs2CO3, DMF, reflux, overnight, 34%. (e) SOC12, DCM, 0 °C to rt, 16 h, 32%. (f) Na2CO3, MeCN, reflux under Ar, 24 h, 15%. (g) TFA, DCM, 0 °C then rt, 2 h, 82%.
(4-(dimethylamino)pyridin-2-yl)methanol (2) Over a sealed tube containing 1 (500 mg, 3.5 mmol) and Me2NH.HCl (1.42 g, 17.4 mmol) was added water (1 mL). Then, NaOH (696 mg, 17.4 mmol) was quickly added to the tube and the mixture was stirred at 150 °C for 2 days. After cooling to rt, the aqueous layer was extracted with DCM (2 x 5 mL). Combined organic layers were washed with brine (10 mL), dried over anhydrous MgSO4, fdtered and concentrated to dryness under reduced pressure to afford 2 as a pale brown solid (496 mg, 93%). Data is in agreement with the literature [ ]. 'H NMR (500 MHz, chloroform-<7) δ: 8.20 - 8.10
(m, 1H), 6.52 - 6.46 (m, 1H), 7.26 - 7.19 (m, 1H), 5.21 (s, 1H), 4.71 (s, 2H), 3.08 (s, 6H) ppm. LRMS (ESI-MS) m/z calculated for C8HI2N2O [M+H]+ 153.10, found 153.20.
2-chloromethyl-4-dimethylaminopyridine (3) A o,
TO a stirred solution of 2 (475 mg, 3.1 mmol) in anhydrous DCM (22 mL) was added SOC12 (0.46 mL, 6.2 mmol) dropwise at 0 °C. After addition, the resulting mixture was stirred at rt overnight. The reaction was quenched with Na2CO3 aqueous solution (10 mL) and the aqueous layer was extracted with DCM (2 x 10 mL). Combined organic layers were washed with brine (20 mL), dried over anhydrous MgSO4, filtered and concentrated to dryness under reduced pressure. The residue was purified by CombiFlash on SiO2 using DCM:MeOH (100:0 to 90: 10) to afford 3 as a yellow solid (364 mg, 69%). Data is in agreement with the literature [41] . (500 MH1Hz, N MMetRhanol-d4) δ: 8.32 - 8.28 (m, 1H), 6.79 - 6.75 (m, 1H), 6.60 - 6.55 (m, 1H), 4.53 (s, 2H), 3.04 (s, 6H) ppm. LRMS (ESI-MS) m/z calculated for C8HHC1N2 [M+H]+ 171.06, found 171.20.
N1-((4-(dimethylamino)pyridine-2-yl)methyl)-N1,N2-dimethylethane-l,2-diamine (4) stirred solution of 3 (350 mg, 2.1 mmol) and Na2CO3 (1.1 g, 10.3 mmol) in anhydrous MeCN (10 mL) was added N,N’ -Dimethylethylenediamine (1.1 mL, 10.3 mmol). After addition, the mixture was stirred at 90 °C under argon for 4 h. After cooling to rt, the mixture was filtered and concentrated to dryness under reduced pressure. The residue was purified by CombiFlash on Si O2 using DCM:MeOH:Et3N (100:0:5 to 80:20:5) to afford 4 as a yellow oil (104 mg, 23%). 1 H NMR (500 MHz, Methanol-d4) δ : 8.04 - 8.01 (m, 1H), 6.74 - 6.71 (m, 1H), 6.58 - 6.54 (m, 1H), 3.53 (s, 2H), 3.04 (s, 6H), 2.73 (t, J= 6.2 Hz, 2H), 2.59 (t, J= 6.2 Hz, 2H), 2.40 (s, 3H), 2.26 (s, 3H) ppm. LRMS (ESI-MS) m/z calculated for CI2H22N4 [M+H]+ 223.18, found 223.67. tert-butyl (4-((2-(hydroxymethyl)pyridine-4-yl)amino)butyl)carbamate (5)
( JL Over a Schlenk tube under argon containing 1 (500 mg, 3.5 mmol), tert-butyl (4- aminobutyl)carbamate (791 mg, 4.2 mmol), Pd(OAc)2 (39 mg, 0.18 mmol), Cs2CO3 (1.7 g, 5.3 mmol) and BINAP (112 mg, 0.18 mmol) was added anhydrous DMF (35 mL). After addition, the mixture was stirred at 70 °C overnight. After cooling to rt, the mixture was concentrated to dryness under reduced pressure. The residue was purified by CombiFlash on SiO2 using DCM:MeOH (100:0 to 80:20) to afford 5 as a yellow oil (348 mg, 34%). 1 H NMR (500 MHz, Methanol-d4) δ: 7.90 (d, J= 5.8 Hz, 1H), 6.69 (s, 1H), 6.42 (d, J= 5.8 Hz, 1H), 4.52 (s, 2H), 3.17 (t, J= 6.8 Hz, 2H), 3.07 (t, J= 6.8 Hz, 2H), 1.63 - 1.60 (m, 2H), 1.59 - 1.54 (m, 2H), 1.43 (s, 9H) ppm. LRMS (ESI-MS) m/z calculated for C I5H25N3O3 [M+H]+ 296.19, found 296.24. tert-butyl (4-((2-(chloromethyl)pyridine-4-yl)amino)butyl)carbamate (6)
Cx c, To a stirred solution of 5 (230 mg, 0.78 mmol) in anhydrous DCM (6 mL) was added SOC12 (0.12 mL, 1.6 mmol) dropwise at 0 °C. After addition, the mixture was stirred at rt overnight. The reaction was quenched with Na2CC>3 aqueous solution (5 mL) and the aqueous layer was extracted with DCM (2 x 10 mL). Combined organic layers were washed with brine (20 mL), dried over anhydrous MgSCfi, filtered and concentrated to dryness under reduced pressure. The residue was purified by CombiFlash on SiO2 using DCM:MeOH (100:0 to 90: 10) to afford 6 as a yellow solid (77 mg, 32%). Data is in agreement with the literature. (400 M1HH NzM, mRethanol-<4) δ: 7.94 (d, J= 5.9 Hz, 1H), 6.66 (d, J= 2.1 Hz, 1H), 6.46 (dd, J= 5.9, 2.3 Hz, 1H), 4.48 (s, 2H), 3.15 (t, J = 6.7 Hz, 2H), 3.07 (t, J = 6.6 Hz, 2H), 1.69 - 1.49 (m, 4H), 1.42 (s, 9H) ppm. LRMS (ESI-MS) m/z calculated for CisH^CMCL [M+H]+ 314.16, found 314.24.
Ze/7-butyl (4-((2-(((2-(((4-(dimethylamino)pyridine-2- yl)methyl)(methyl)amino)ethyl)(methyl)amino)methyl)pyridine-4-yl)amino)butyl)carbamate formic acid salt (7) solution of 4 (55 mg, 0.3 mmol) and Na2CO3 (53 mg, 0.5 mmol) in anhydrous MeCN (3 mL)was added a solution of 6 (77 mg, 0.3 mmol) in anhydrous MeCN (2 mL). After addition, the mixture was stirred at 90 °C overnight. After cooling to rt, the mixture was filtered and concentrated to dryness under reduced pressure. The residue was purified by preparative HPLC using H2O:MeCN:FA (90: 10:0.1 to 50:50:0.1) to afford 7 as a yellow oil (18 mg, 15%). 'H NMR (500 MHz, Methanol-d4) δ: 8.42 (s, 2H, formate), 7.99 - 7.94 (m, 1H), 6.91 - 6.89 (m, 1H), 6.89 - 6.85 (m, 2H), 6.79 - 6.74 (m, 2H), 3.79 (s, 2H), 3.73 (s, 2H), 3.31 (m, 2H), 3.21 (s, 6H), 3.08 (t, J= 6.9 Hz, 2H), 2.75 (tt, J= 3.1, 1.8 Hz, 4H), 2.25 (s, 3H), 2.32 (s, 3H), 1.71 - 1.63 (m, 2H), 1.61 - 1.53 (m, 2H), 1.43 (s, 9H) ppm. 13C NMR (126 MHZ, Methanol-d4) 8: 168.36 (formate), 159.87, 159.02, 158.63, 152.30, 141.08, 107.43, 107.19, 79.94, 59.96, 59.70, 55.65, 55.59, 43.53, 42.69 (2C), 40.74, 40.18 (2C), 28.78 (3C), 28.40, 26.65 ppm. LRMS (ESI-MS) m/z calculated for C27H45N7O2 [M+H]+ 500.37, found 500.41. N1-(2-(((2-(((4-(dimethylamino)pyridine-2- yl)methyl)(methyl)amino)ethyl)(methyl)amino)methyl)pyridine-4-yl)butane-l,4-diamine (8) solution of 7 (103 mg, 0.21 mmol) in anhydrous DCM (3 mL) was added TFA (0.24 mL, 3.1 mmol) at 0 °C. After addition, the mixture was stirred at rt for 2 h until total conversion. The reaction was quenched with NaOH (1 M) to reach pH 14. Then, the aqueous layer was extracted with DCM (3 x 10 mL). Combined organic layers were washed with brine (20 mL), dried over anhydrous MgSO4, filtered and concentrated to dryness under reduced pressure to afford 8 as a yellow solid (69 mg, 82%). 1H N (5M00R MHz, Methanol-d4) δ: 7.99 (d, J= 6.1 Hz, 1H), 7.89 (d, J= 6.0 Hz, 1H), 6.77 (d, J= 2.7 Hz, 1H), 6.65 (d, J= 2.4 Hz, 1H), 6.54 (dd, J= 6.1, 2.7 Hz, 1H), 6.42 (dd, J= 6.0, 2.4 Hz, 1H), 3.54 (s, 2H), 3.49 (s, 2H), 3.12 (t, J= 6.8 Hz, 2H), 2.99 (s, 6H), 2.65 (t, J= 6.8 Hz, 2H), 2.50 (s, 4H), 2.26 (d, J = 4.5 Hz, 6H), 1.67 - 1.57 (m, 2H), 1.57 - 1.49 (m, 2H) ppm. 13C NMR (126 MHz, Methanol-d4) δ: 159.22, 159.14, 156.91, 156.71, 148.97 (2C), 107.00 (2C), 106.71 (2C), 64.82, 64.64, 56.15 (2C), 43.31 (2C), 43.15, 42.28, 39.23 (2C), 31.25, 27.37 ppm. LRMS (ESI-MS) m/z calculated for C22H37N7 [M+H]+ 400.31, found 400.22.
Scheme 4. Synthesis of Fentomycin 7 (XI). (a) succinic acid, DMAP, anhydrous pyridine, 90 °C, 24 h, 87%. (b) HBTU, DIPEA, THF, rt, o.n., 18%.
Procedure A To a solution of sterol (1 eq.) in anhydrous pyridine (10 mL/mmol) was added successively under stirring succinic acid (10 eq.) and DMAP (0.5 eq). After addition, the mixture was stirred at 90 °C overnight. After cooling to rt, H2O (5 mL) and DCM (5 mL) were added and the aqueous layer was extracted with DCM (3 x 20 mL). Combined organic layers were washed with 10% HC1 solution (10 mL), brine (30 mL), dried over anhydrous MgSO4, filtered and concentrated to dryness under reduced pressure to afford the title compound as a white solid.
Procedure B
A solution of carboxylic acid (1 eq.), HBTU (5 eq.) and DIPEA (30 eq.) in anhydrous THF (16 mL/mmol) was stirred 1 h at room temperature before the addition of 8 (1 eq.) in anhydrous DMF (16 mL/mmol). The reaction was stirred overnight at room temperature Then, the mixture was concentrated to dryness under reduced pressure and the residue was purified by preparative HPLC using H2O:MeCN:FA (95:5:0.1 to 0: 100:0.1) to afford the title compound as a red solid after freeze drying.
4-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)- 2,3,4,7,8,9,10,ll,12,13,14,15,16,17-tetradecahydro-lH-cyclopent[a]phenanthren-3-yl)oxy)-4- oxobutanoic acid (10)
Compound 10 was synthesized via procedure A (545 mg, 87%) using 9 (500 mg, 1.3 mmol), succinic acid (1.29 g, 12.9 mmol) and DMAP (79 mg, 0.65 mmol) in anhydrous pyridine (13 mL). 'HNMR (400 MHz, chloroform-J) δ: 5.42 - 5.34 (m, 1H), 4.70 - 4.56 (m, 1H), 2.72 - 2.64 (m, 2H), 2.64 - 2.57 (m, 2H), 2.35 - 2.28 (m, 2H), 2.05 - 1.91 (m, 2H), 1.91 - 1.76 (m, 3H), 1.67 - 1.06 (m, 19H), 1.02 (s, 3H), 0.93 - 0.89 (m, 3H), 0.89 - 0.84 (m, 6H), 0.67 (s, 3H) ppm. 13C NMR (101 MHz, chloroform-J) 5: 176.84, 171.71, 139.68, 122.90, 74.72, 56.84, 56.29, 50.16, 42.46, 39.88, 39.66, 38.16, 37.10, 36.73, 36.33, 35.94, 32.05, 32.00, 29.40, 28.95, 28.37, 28.16, 27.85, 24.43, 23.98, 22.97, 22.71, 21.18, 19.45, 18.86, 12.00 ppm.
Fentomycin 7 (XI)
Fentomycin 7 (XI) was synthesized via procedure B (4 mg, 18%) using 10 (12 mg, 0.025 mmol), HBTU (47 mg, 0.025 mmol) and DIPEA (0.13 mL, 0.75 mmol) in anhydrous THF (0.4 mL) and 8 (10 mg, 0.025 mmol) in anhydrous DMF (0.4 mL). (510H0 N MMHRz, mcthanol-<A) δ: 8.03 (d, J = 6.6 Hz, 1H), 7.95 (d, J = 6.6 Hz, 1H), 6.80 (s, 1H), 6.75 - 6.64 (m, 3H), 5.35 (d, J= 5.1 Hz, 1H), 4.51 (tt, J =
10.9, 5.5 Hz, 1H), 3.69 (d, J= 13.9 Hz, 4H), 3.24 (dt, J= 17.1, 6.7 Hz, 4H), 3.12 (s, 6H), 2.71 (s, 4H), 2.59 (t, J= 6.8 Hz, 2H), 2.46 (t, J= 6.7 Hz, 2H), 2.32 (d, J= 12.7 Hz, 6H), 2.08 - 2.01 (m, 1H), 2.00 - 1.92 (m, 1H), 1.92 - 1.79 (m, 3H), 1.71 - 1.08 (m, 22H), 1.03 (s, 4H), 0.94 (d, J = 6.6 Hz, 3H), 0.88 (dd,J= 6.6, 1.9 Hz, 6H), 0.71 (s, 3H) ppm. 13C NMR (126 MHz, Methanol-d4) δ: 174.42, 173.94, 158.81 (2C), 155.64 (2C), 144.99 (2C), 141.04, 123.63, 107.29 (2C), 107.11 (2C), 75.62, 61.82, 60.77, 58.12,
57.57, 55.59 (2C), 51.61, 43.49 (2C), 42.82, 41.11 (3C), 40.69, 39.72 (2C), 39.15, 38.23, 37.76, 37.36, 37.11, 33.19 (2C), 33.02, 31.46, 30.73, 29.30, 29.16, 28.79, 27.99, 26.74, 25.30, 24.93, 23.17, 22.93, 22.13, 19.75, 19.23, 12.30 ppm. HRMS (ESI+) m/z calculated for CAHssNyCh [M+H]+ 868.6714, found 868.6785.
Synthesis of Fentomycin 8 (XII)
Scheme 5. Synthesis of Fentomycin 8 (XII). (a) succinic acid, DMAP, anhydrous pyridine, 90 °C, 24 h, 47%. (b) HBTU, DIPEA, THF, rt, o.n., 18%.
4-(((3S,8R,9S,10S,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-hexadecahydro- lH-cyclopent[a]phenanthren-3-yl)oxy)-4-oxobutanoic acid (13) Compound 13 was synthesized via procedure A (118 mg, 47%) using 12 (200 mg, 0.51 mmol), succinic acid (510 mg, 5.1 mmol) and DMAP (31 mg, 0.26 mmol) in anhydrous pyridine (5 mL). 'HNMR (400 MHz, chloroform-J) δ: 4.70 - 4.56 (m, 1H), 2.72 - 2.64 (m, 2H), 2.64 - 2.57 (m, 2H), 2.35 - 2.28 (m, 2H), 2.05 - 1.91 (m, 2H), 1.91 - 1.76 (m, 3H), 1.67 - 1.06 (m, 20H), 1.02 (s, 4H), 0.93 - 0.89 (m, 3H), 0.89 - 0.84 (m, 6H), 0.67 (s, 3H) ppm. 13C NMR (126 MHz, chloroform-J) δ: 177.72, 171.81, 74.52, 56.56, 56.42, 54.36, 44.79, 42.74, 40.13, 39.66, 36.87, 36.31,
35.95, 35.59, 34.07 (2C), 32.13, 29.42, 29.10, 28.74, 28.39, 28.16, 27.54, 24.36, 23.99, 22.96, 22.71, 21.36, 18.82, 12.37, 12.22 ppm.
Fentomycin 8 (XII)
Fentomycin 8 (XII) was synthesized via procedure B (4 mg,
18%) using 13 (12 mg, 0.025 mmol), HBTU (47 mg, 0.025 mmol) and DIPEA (0.13 mL, 0.75 mmol) in anhydrous THF (0.4 mL) and 8 (10 mg, 0.025 mmol) in anhydrous DMF (0.4 mL). 'H NMR (500 MHz, Methanol-d4) δ: 8.57 (s, 1H, formate), 8.03 (d, J= 6.8 Hz, 1H), 7.95 (d, J= 6.7 Hz, 1H), 6.84 (d, J= 2.7 Hz, 1H), 6.79 (dd, J= 6.9, 2.8 Hz, 1H), 6.75 - 6.67 (m, 2H), 4.64 (tt, J= 11.0, 4.8 Hz, 1H), 3.73
(d, J= 19.7 Hz, 4H), 3.31 - 3.18 (m, 5H), 3.16 (s, 6H), 2.73 (s, 4H), 2.57 (t, J= 6.8 Hz, 2H), 2.46 (t, J = 6.7 Hz, 2H), 2.33 (d, J= 14.3 Hz, 6H), 2.00 (dt, J= 12.6, 3.4 Hz, 1H), 1.90 - 0.96 (m, 22H), 0.95 - 0.85 (m, 9H), 0.84 (s, 3H), 0.68 (s, 3H) ppm. 13C NMR (126 MHz, Methanol-d4) δ : 174.44, 173.90, 168.72 (formate), 159.22, 158.39, 154.25 (2C), 143.38 (2C), 107.24 (4C), 75.34, 61.06, 60.29, 57.70 (2C), 55.56, 46.01, 43.76, 43.40 (2C), 42.80, 41.37, 40.68, 39.91 (2C), 39.74 (2C), 37.92, 37.35, 37.12,
36.83, 36.58, 35.11, 33.21, 31.49, 30.79, 29.81, 29.31, 29.15, 28.51, 27.94, 26.68, 25.23, 24.94, 23.18, 22.93, 22.30, 19.19, 12.63, 12.51 ppm. HRMS (ESI+) m/z calculated for C53H87N7O3 [M+H]+ 870.6870, found 870.6946. Synthesis of Fentomycin 9 (XIII)
Scheme 6. Synthesis of Fentomycin 9 (XIII). (a) TsCl, anhydrous pyridine, rt, overnight, 71%. (b) Na2CO3, anhydrous MeCN, 90 °C, overnight, 15%.
Procedure C
To a solution of alcohol (1 eq.) in anhydrous pyridine (3 mL/mmol) at 0 °C was added under stirring p- toluenesulfonyl chloride (2 eq.) in anhydrous pyridine (0.3 mL/mmol). After addition, the mixture was stirred at rt overnight. The solvent was then removed under reduced pressure to afford a white solid. The solid was taken up in a minimum amount of chloroform and then precipitated via addition of methanol. The solid was filtered and washed with methanol then acetonitrile and dried under vacuum to afford the title compound as a white solid.
Procedure D
To a stirred solution of the amine (1 eq.) and Na2CO3 (2 eq.) in anhydrous MeCN (4 mL/mmol) was added the tosylate derivative (1 eq.). After addition, the mixture was stirred at 90 °C under argon overnight. After cooling to rt, the mixture was filtered and concentrated to dryness under reduced pressure and the residue was purified by preparative HPLC using H2O:MeCN:FA (95:5:0. 1 to 0: 100:0. 1) to afford the title compound as a white solid after freeze drying.
(3S,8S,9R,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)- 2,3,4,7,8,9,10,ll,12,13,14,15,16,17-tetradecahydro-lH-cyclopenta[a]phenanthren-3-yl 4- methylbenzenesulfonate (15) Compound 15 was synthesized via procedure C (494 mg, 71%) using 9 (500 mg, 1.3 mmol), and TsCl (493 mg, 2.6 mmol) in anhydrous pyridine (0.8 mL). 'H NMR (500 MHz, chloroform-J) δ: 7.84 - 7.77 (m, 2H), 7.33 (tt, J= 12, 1.0 Hz, 2H), 4.32 (tt, J = 11.4, 4.7
Hz, 1H), 2.45 (d, J = 4.6 Hz, 3H), 2.29 - 2.27 (m, 1H), 1.60 - 0.94 (m, 42H), 0.67 (d, J= 11.4 Hz, 3H) ppm. 13C NMR (126 MHz, chloroform-J) δ: 144.52, 138.89, 134.69, 129.83, 127.67, 123.58, 116.50, 82.51, 56.69, 56.14, 49.95, 42.34, 39.70, 39.55, 38.91, 36.92, 36.39, 36.21, 35.80, 31.89, 31.79, 28.67,
28.24, 28.05, 24.29, 23.84, 22.85, 22.60, 21.67 (2C), 21.03, 19.19, 18.74, 11.87 ppm.
Fentomycin 9 (XIII) Fentomycin 9 (XIII) was synthesized via procedure D (3 mg,
16%) using 8 (10 mg, 0.025 mmol), 15 (9 mg, 0.017 mmol) and Na2CO3 (5 mg, 0.050 mmol) in anhydrous MeCN (0.1 mL). 'HNMR (500 MHz, Methanol-d4) δ: 8.55 (s, 2H, formate), 8.05 (d, J= 6.8
Hz, 1H), 7.99 (d, J= 6.6 Hz, 1H), 6.84 (d, J= 2.7 Hz, 1H), 6.80 - 6.73 (m, 2H), 6.70 (dd, J= 6.6, 2.3
Hz, 1H), 5.49 (s, 1H), 3.72 (d, J= 22.2 Hz, 4H), 3.44 (d, J= 5.0 Hz, 1H), 3.15 (s, 6H), 3.13 - 2.96 (m, 3H), 2.78 (d, J= 4.2 Hz, 1H), 2.74 (s, 4H), 2.32 (d, J= 16.4 Hz, 6H), 2.12 - 1.09 (m, 32H), 1.06 (d, J= 3.3 Hz, 3H), 0.95 (dd, J= 6.6, 2.2 Hz, 3H), 0.92 - 0.85 (m, 6H), 0.82 - 0.78 (m, 3H) ppm. 13C NMR (126 MHz, Methanol-d4) δ: 170.05 (2C), 158.75, 158.26, 154.56, 143.92 (2C), 107.27 (2C), 62.89, 61.23,
60.84, 57.51, 55.49, 43.99, 43.68, 43.03, 42.73, 41.22, 40.68, 39.85, 37.30, 37.11, 35.72, 34.34, 32.41,
31.16, 29.33, 29.16, 27.49, 26.90, 25.40, 25.13, 24.92, 24.38, 23.57, 23.18, 22.92, 19.39, 19.16, 13.71, 12.71 ppm. HRMS (ESI+) m/z calculated for C49H8IN7 [M+H]+ 768.6553, found 768.6627. Synthesis of Fentomycin 10 (XIV)
Scheme 7. Synthesis of Fentomycin 10 (XIV). (a) TsCl, anhydrous pyridine, rt, overnight, 71%. (b) Na2CO3, anhydrous MeCN, 90 °C, overnight, 15%.
(3S,8R,9S,10S,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-hexadecahydro-lH- cyclopenta[a]phenanthren-3-yl 4-methylbenzenesulfonate (17) Compound 17 was synthesized via procedure C (494 mg, 71%) using 9 (500 mg, 1.3 mmol), and TsCl (493 mg, 2.6 mmol) in anhydrous pyridine (0.8 mL). 'H NMR (500 MHz, chloroform-J) δ: 7.84 - 7.77 (m, 2H), 7.33 (tt, J= 12, 1.0 Hz, 2H), 2.45 (d, J= 4.6 Hz, 3H),
2.29 - 2.27 (m, 1H), 1.60 - 0.94 (m, 42H), 0.67 (d, J = 11.4 Hz, 3H) ppm. 13C NMR (126 MHz, chloroform-J) δ: 144.43, 134.95, 129.85 (2C), 127.74 (2C), 82.75, 56.51, 56.38, 54.23, 44.95, 42.70,
40.06, 39.65, 36.93, 36.29, 35.91, 35.51, 35.34, 35.00, 32.03, 28.54, 28.35, 28.15, 24.31, 23.95, 22.95, 22.70 (2C), 21.78, 21.32, 18.79, 12.26 ppm.
Fentomycin 10 (XIV)
Fentomycin 10 (XIV) was synthesized via procedure D (3 mg, 16%) using 8 (10 mg, 0.025 mmol), 17 (9 mg, 0.017 mmol) and Na3CO3 (5 mg, 0.050 mmol) in anhydrous MeCN (0.1 mL). 'H NMR (500 MHz, Methanol-d4) δ: 8.54 (s, 2H, formate), 8.04 (d, J= 6.8 Hz, 1H), 7.98 (d, J= 6.6 Hz, 1H), 6.84 (d, J = 2.7 Hz, 1H), 6.81 - 6.67 (m, 3H), 5.49 (s, 1H), 3.86 (s, 1H), 3.79 - 3.68 (m, 4H), 3.48 - 3.41 (m, 2H), 3.16 (d, J= 2.1 Hz, 6H), 3.05 (s, 1H), 2.74 (s, 4H), 2.38 - 2.28 (m, 6H), 2.07 - 0.97 (m, 32H), 0.93 (d, J= 6.5 Hz, 3H), 0.90 - 0.84 (m, 9H), 0.70 (s, 3H) ppm. 13C NMR (126 MHz, Methanol-d4) 5: 168.62, 163.02, 157.54 (2C), 156.95 (2C), 142.15 (2C), 105.84 (2C), 56.58, 56.32, 54.30, 54.13, 43.54, 42.36, 41.84, 41.32, 39.97, 39.27, 38.48, 35.94, 35.70, 35.46, 35.34, 32.42, 31.67, 31.15, 28.15, 27.87, 27.75, 26.67, 25.33, 24.37, 23.76, 23.53, 21.76, 21.52, 20.46, 17.78, 15.48, 11.08, 10.32 ppm. HRMS (ESI+) m/z calculated for C49H83N7 [M+H]+ 770.6710, found 770.6780.
Synthesis of Fentomycin 11 (XV)
Scheme 8. Synthesis of modified BPMEN ligand with chlorine, (a) dimethylamine hydrochloride, NaOH, H2O, sealed tube, 150 °C, 2 days, 93%. (b) SOCI2, DCM, rt, overnight, 69%. (c) Na3CO3, anhydrous MeCN, 90 °C, 24 h, 23%. (d) SOCI2, DCM, rt, overnight, 38%. (e) NaOH, DCM/H2O, rt, overnight, 38%.
4-chloro-2-(chloromethyl)pyridine (19) a stirred solution of 1 (1 g, 6.9 mmol) in anhydrous DCM (50 mL) was added SOCI2 (1 mL, 13.9 mmol) dropwise at 0 °C. After addition, the resulting mixture was stirred at rt overnight. The reaction was quenched with Na2CO3 aqueous solution (25 mL) and the aqueous layer was extracted with DCM (2 x 10 mL). Combined organic layers were washed with brine (20 mL), dried over anhydrous MgSO4, filtered and concentrated to dryness under reduced pressure. The residue was purified by CombiFlash on SiO2 using n-Hex:EtOAc (100:0 to 80:20) to afford 19 as a yellow oil (422 mg, 38%). Data is in agreement with the literature [42], (500 M1HHz N, cMhlRoroform-J) δ: 8.46 (d, J= 5.4 Hz, 1H), 7.51 (d, J= 1.9 Hz, 1H), 7.27 - 7.22 (m, 1H), 4.64 (s, 2H) ppm. 13C NMR (126 MHz, chloroforme d: 158.30, 150.37, 145.23, 123.54, 123.29, 46.07 ppm. LRMS (ESI-MS) m/z calculated for C6H5C12N [M+H]+ 161.98, found 161.47.
N1-((4-chloropyridin-2-yl)methyl)-N2-((4-(dimethylamino)pyridine-2-yl)methyl)-N1,N2- dimethylethane-l,2-diamine (20) solution of 19 (119 mg, 0.54 mmol) and 4 (86 mg, 0.54 mmol) in DCM (3.5 mL) was added a IM NaOH solution (3.5 mL). After addition, the resulting mixture was stirred at rt overnight.
The reaction was quenched with IM NaOH solution to reach pH 14 and the aqueous layer was extracted with DCM (3 x 5 mL). Combined organic layers were washed with brine (10 mL), dried over anhydrous MgSO4, filtered and concentrated to dryness under reduced pressure. The residue was purified by preparative HPLC using H2O:MeCN:FA (100:0:0.1 to 80:20:0.1) to afford the title compound as a brown oil after freeze drying (72 mg, 38%). (4001 MHH NzM, mRethanol -<A) δ: 8.42 (dd, J= 5.5, 0.6
Hz, 1H), 8.03 (d, J= 6.6 Hz, 1H), 7.59 (dd, J= 2.1, 0.6 Hz, 1H), 7.37 (dd, J= 5.5, 2.1 Hz, 1H), 6.82 (d, J= 2.7 Hz, 1H), 6.67 (dd, J= 6.5, 2.8 Hz, 1H), 3.71 (s, 2H), 3.64 (s, 2H), 3.09 (s, 6H), 2.72 - 2.60 (m, 4H), 2.29 (d, J= 7.5 Hz, 6H) ppm. 13C NMR (126 MHz, Methanol-d4) δ: 169.80, 161.09, 158.90, 153.47, 151.32, 146.35, 141.38, 125.41, 124.29, 107.24, 106.64, 63.38, 59.30, 55.61, 55.31, 42.87, 42.68, 40.10 ppm. LRMS (ESI-MS) m/z calculated for CI8H26C1N5 [M+H]+ 348.19, found 348.27.
Scheme 9. Synthesis of Fentomycin 11 (XV). (a) NaH, DMF, 70 °C, overnight, 7%.
Procedure E
To a mixture of 20 (1 eq), the sterol (1.2 eq) and NaH (1.2 eq) under argon was added dry DMF (4 mL/mmol). After addition, the mixture was stirred at 70 °C overnight. After cooling to rt, the mixture was concentrated to dryness under reduced pressure and the residue was purified by preparative HPLC using H2O:MeCN:FA (95:5:0.1 to 0: 100:0.1) to afford the title compound as a white solid after freeze drying.
Fentomycin 11 (XV) Fentomycin 11 (XV) was synthesized via procedure E (8 mg, 7%) using 20 (62 mg, 0.18 mmol), 9 (83 mg, 0.21 mmol) and NaH (5 mg, 0.21 mmol) in anhydrous DMF (0.8 mL). 1H NMR (500 MHz, Methanol-d4) δ: 8.68 (s, 1H, formate), 8.28 (d, J= 5.8 Hz, 1H), 8.07 (d,
J= 7.0 Hz, 1H), 7.04 (d, J= 2.6 Hz, 1H), 6.86 (ddd, J= 11. 1, 5.5, 3.2 Hz, 3H), 5.42 (dt, J= 4.0, 1.8 Hz, 1H), 4.26 (tq, J= 9.6, 4.8 Hz, 1H), 3.83 - 3.74 (m, 4H), 3.20 (s, 6H), 2.80 - 2.72 (m, 4H), 2.40 (s, 3H), 2.31 (s, 3H), 2.12 - 1.10 (m, 25H), 1.07 (s, 3H), 0.96 (d, J= 6.5 Hz, 3H), 0.89 (dd, J= 6.7, 1.9 Hz, 6H), 0.73 (s, 3H) ppm. 13C NMR (126 MHz, Methanol-d4) δ : 166.65 (formate), 159.95, 158.71, 153.82, 151.24, 142.06, 140.86, 123.91, 112.30, 111.59, 107.23, 106.77, 78.34, 63.52, 59.67, 58.18, 57.62, 55.40, 55.09, 51.69, 43.52, 42.77, 42.71, 41.14, 40.69, 40.07, 39.28, 38.08, 37.86, 37.38, 37.12, 33.20, 33.06, 29.32, 29.16, 28.82, 25.31, 24.96, 23.20, 22.95, 22.17, 19.82, 19.27, 12.33 ppm. HRMS (ESI+) m/z calculated for C45H7IN5O [M+H]+ 698.5659, found 698.5731.
Synthesis of Fentomycin 12 (XVI)
Scheme 10. Synthesis of Fentomycin 12 (XVI). (a) NaH, DMF, 70 °C, overnight, 7%. Fentomycin 12 (XVI) was synthesized via procedure E (5 mg, 5%) using 20 (62 mg, 0.18 mmol), 11 (83 mg, 0.21 mmol) and NaH (5 mg, 0.21 mmol) in anhydrous DMF (0.8 mL). 1H NMR (500 MHz, Methanol-d4) δ: 8.61 (s, 1H, formate), 8.27 (d, J= 5.9 Hz, 1H), 8.07 (d, J= 6.9 Hz, 1H), 7.03 (d, J = 2.5 Hz, 1H), 6.90 - 6.81 (m, 3H), 4.38 (tt, J= 10.6, 4.8 Hz, 1H), 3.77 (d, J = 4.5 Hz, 4H), 3.19 (s, 6H), 2.79 - 2.71 (m, 4H), 2.39 (s, 3H), 2.31 (s, 3H), 2.07 - 0.97 (m, 32H), 0.94 (d, J= 6.5 Hz, 3H), 0.88 (dd, J= 6.7, 1.8 Hz, 9H), 0.70 (s, 3H) ppm. 13C NMR (126 MHz, Methanol-d4) 5: 167.02 (formate), 160.16, 158.86, 154.12, 151.34, 142.33, 112.48, 111.79, 107.41, 106.91, 78.15, 63.74, 59.79, 58.08, 57.92, 55.97, 55.59, 55.27, 46.01, 43.97, 42.95, 42.90, 41.59, 40.86, 40.23, 37.97,
37.53, 37.30, 37.02, 36.87, 35.26, 33.43, 29.97, 29.49, 29.33, 28.77, 25.41, 25.12, 23.35, 23.10, 22.53, 19.37, 12.87, 12.67 ppm. HRMS (ESI+) m/z calculated for C45H73N5O [M+H]+ 700.5815, found 700.5884.
Scheme 11. Synthesis of Fentomycin 13 (XVII). (a) TsCl, anhydrous pyridine, rt, 24 h, 71%. (b) Na2CO3, anhydrous MeCN, 90 °C, 24 h, 15%. Fentomycin 13 (XVII)
Fentomycin 13 (XVII) was synthesized via procedure D (3 mg, 16%) using 25 (20 mg, 0.044 mmol), 15 (15 mg, 0.029 mmol) and Na2CO3 (9 mg, 0.058 mmol) in anhydrous MeCN (0.18 mL).
1H NMR , Methanol-d4) δ: 7.99 (d, J= 6.6 Hz, 1H),
7.90 (d, J = 6.3 Hz, 1H), 6.75 - 6.67 (m, 2H), 6.64 (s, 1H), 6.62 - 6.57 (m, 1H), 4.25 (d, J= 14.8 Hz, lH), 4.18 (d, J= 15.1 Hz, 1H), 3.79 (d, J= 14.8 Hz, 1H), 3.72 (d, J= 15.1 Hz, 1H), 3.24 (t, J= 5.0 Hz, 2H), 3.19 - 2.92 (m, 10H), 2.81 - 2.58 (m, 4H), 2.40 (s, 1H), 2.12 - 1.11 (m, 37H), 1.05 (s, 3H), 0.95 (dd, J= 6.6, 2.2 Hz, 3H), 0.88 (dt, J= 6.6, 1.8 Hz, 6H), 0.79 (d, J = 7.2 Hz, 3H) ppm. 13C NMR (126 MHz, Methanol-d4) δ: 169.97, 157.76, 143.40, 107.01, 69.33, 69.21, 62.85, 60.29, 57.51, 57.41, 55.85,
55.72, 43.98, 43.68, 42.89, 41.20, 40.69, 39.62, 37.30, 37.11, 35.98, 34.34, 32.45, 31.16, 29.33, 29.16,
28.47, 27.48, 26.99, 25.42, 25.14, 24.92, 24.57, 23.56, 23.17, 22.92, 19.40, 19.16, 13.66, 12.69 ppm.
HRMS (ESI+) m/z calculated for C53H85N7 [M+H]+ 820.6866, found 820.6934.
Synthesis of Fentomycin 14 (XVIII) Scheme 12. Synthesis of Fentomycin 14 (XVIII). (a) TsCl, anhydrous pyridine, rt, 24 h, 71%. (b) Na2CO3, anhydrous MeCN, 90 °C, 24 h, 15%.
Fentomycin 14 (XVIII) Fentomycin 14 (XVIII) was synthesized via procedure
D (3 mg, 16%) using 25 (20 mg, 0.044 mmol), 17 (15 mg, 0.029 mmol) and Na7CO3 (9 mg, 0.058 mmol) in anhydrous MeCN (0.18 mL). 1 (H50 N0M MRHz, Methanol-d4) δ: 8.00 (d, J= 6.6 Hz, 1H), 7.92 (d, J= 6.4 Hz, 1H), 6.75 (dq, J= 12.6, 2.9 Hz, 2H), 6.68 (d, J= 6.7 Hz, 1H), 6.63 (d, J= 6.2 Hz, 1H), 4.25 (d, J= 15.0 Hz, 1H), 4.20 (d, J = 15.2 Hz, 1H), 3.85 (s, 1H), 3.78 (d, J = 15.0 Hz, 1H), 3.71 (dd, J =
15.3, 4.0 Hz, 1H), 3.45 - 3.37 (m, 1H), 3.29 - 3.23 (m, 2H), 3.11 (d, J= 2.8 Hz, 6H), 3.05 - 2.94 (m, 2H), 2.61 (dt, J= 25.3, 8.5 Hz, 2H), 2.35 (s, 2H), 2.11 - 0.98 (m, 40H), 0.93 (d, J= 6.5 Hz, 3H), 0.89 - 0.83 (m, 9H), 0.70 (d, J = 1.2 Hz, 3H) ppm. 13C NMR (126 MHz, Methanol-d4) δ : 169.68, 158.04, 144.66, 107.26, 107.04, 69.07, 68.81, 57.94, 57.73, 55.84, 55.46, 47.05, 44.67, 43.76, 43.24, 41.38, 40.68, 39.72, 37.35, 37.10, 36.70, 33.60, 30.52, 29.56, 29.15, 28.25, 26.79, 25.77, 25.15, 24.93, 24.62,
23.54, 23.17, 22.92, 21.86, 19.19, 12.49 ppm. HRMS (ESI+) m/z calculated for C53H87N7 [M+H]+ 822.7023, found 822.7092.
Synthesis of Fentomycin 15 (XIX)
Scheme 13. Synthesis of modified White-Chen ligand with chlorine, (a) dimethylamine hydrochloride, NaOH, H2O, sealed tube, 150 °C, 2 days, 93%. (b) SOC12, DCM, rt, 16 h, 69%. (c)
Na2COs, anhydrous MeCN, 90 °C, 24 h, 32%. (d) SOC12, DCM, rt, overnight, 38%. (e) NaOH, DCM/H2O, rt, overnight, 36%.
2-(((2S,2’S)-l’-((4-chloropyridin-2-yl)methyl)-[2,2’-bipyrrolidin]-l-yl)methyl)-N,N- dimethylpyridin-4-amine (28) solution of 19 (34 mg, 0.21 mmol) and 23 (60 mg, 0.21 mmol) in DCM (1.5 mb) was added a IM NaOH solution (1.5 mb). After addition, the resulting mixture was stirred at rt overnight. The reaction was quenched with IM NaOH to reach pH 14 solution and the aqueous layer was extracted with DCM (3 x 5 mb). Combined organic layers were washed with brine (10 mb), dried over anhydrous MgSO4, filtered and concentrated to dryness under reduced pressure. The residue was purified by preparative HPLC using H2O:MeCN:FA (100:0:0.1 to 80:20:0.1) to afford the title compound as a brown oil after freeze drying (30 mg, 36%). (500 MHz1,H m NetMhaRnol -rift 5: 8.42 (d, J = 5.5 Hz, 1H), 8.00 (d, J = 6.9 Hz, 1H), 7.50 (d, J = 2.0 Hz, 1H), 7.38 (dd, J= 5.4, 2.0 Hz, 1H), 6.80 - 6.73 (m, 2H), 4.39 - 4.27 (m, 2H), 3.96 (d, J= 14.8 Hz, 1H), 3.86 (d, J= 15.7 Hz, 1H), 3.25 - 3.17 (m, 1H), 3.14 - 3.04 (m, 9H), 2.81 - 2.66 (m, 2H), 2.09 (dq, J= 12.9, 8.0 Hz, 2H), 2.02 - 1.90 (m, 2H), 1.90 - 1.79 (m, 2H), 1.67 (ddp, J= 12.7, 8.4, 3.8 Hz, 2H) ppm. 13C NMR (126 MHz, methanol-rift 5: 160.52, 158.37, 154.05, 151.22, 146.48, 142.73, 124.95, 124.40, 107.31, 106.37, 69.73, 69.65, 62.08, 59.30, 55.85, 55.54, 39.88 (2C), 28.73, 28.55, 24.82, 24.77 ppm. LRMS (ESI-MS) m/z calculated for C22H30CIN5 [M+H]+ 400.22, found 400.30.
Scheme 14. Synthesis of Fentomycin 15 (XIX). (a) NaH, DMF, 70 °C, overnight, 7%. mg, 7%) using 28 (7 mg, 0.018 mmol), 9 (8 mg, 0.021 mmol) and NaH (0.5 mg, 0.021 mmol) in anhydrous DMF (0.1 mL). 1H ( N50M0R MHz, methanol-<4) δ: 8.54 (s, 1H, formate), 8.25 (d, J = 5.9 Hz, 1H), 7.93 (d, J= 6.5 Hz, 1H), 7.00 (d, J= 2.4 Hz, 1H), 6.85 (dd, J= 5.9, 2.5 Hz, 1H), 6.68 (dt, J =
8.8, 2.7 Hz, 2H), 5.39 (dd, J= 4.7, 2.6 Hz, 1H), 4.35 (d, J= 14.1 Hz, 1H), 4.23 (d, J= 15.7 Hz, 1H), 4.05 (tq, J= 9.2, 4.5 Hz, 1H), 4.00 - 3.86 (m, 2H), 3.27 - 3.04 (m, 11H), 2.90 (dt, J= 11.0, 7. 1 Hz, 1H), 2.82 (dt, J = 11.0, 7.0 Hz, 1H), 2.45 - 2.31 (m, 2H), 2.10 (dtd, J= 20.3, 7.9, 3.9 Hz, 3H), 2.05 - 1.95 (m, 3H), 1.94 - 1.77 (m, 5H), 1.71 - 1.45 (m, 9H), 1.45 - 1.26 (m, 5H), 1.25 - 1.00 (m, 11H), 0.96 (d, J= 6.5 Hz, 4H), 0.89 (dd, J= 6.6, 1.9 Hz, 7H), 0.73 (s, 3H) ppm. 13C NMR (126 MHz, Methanol-d4) 5: 166.63, 157.46, 150.95, 146.12, 143.14, 140.88, 123.85, 112.29, 111.68, 107.26, 106.47, 83.80, 78.48, 69.75, 69.29, 62.56, 60.83, 58.19, 57.64, 55.82, 55.44, 51.69, 43.53, 41.15, 40.69, 39.55, 39.14, 38.08, 37.85, 37.38, 37.12, 33.21, 33.07, 29.31, 29.17, 28.88, 28.75, 28.51, 25.31, 25.09, 25.04, 24.94, 23.18,
22.93, 22.15, 19.76, 19.24, 12.46, 12.29 ppm. HRMS (ESI+) m/z calculated for C49H75N5O [M+H]+ 750.5972, found 750.6043.
Synthesis of Fentomycin 16 (XX)
Scheme 15. Synthesis of Fentomycin 16 (XX). (a) NaH, DMF, 70 °C, overnight, 7%.
Fentomycin 16 (XX) Fentomycin 16 (XX) was synthesized via procedure E (8 mg, 7%) using 28 (7 mg, 0.018 mmol), 11 (8 mg, 0.021 mmol) and NaH (0.5 mg, 0.021 mmol) in anhydrous DMF (0.1 mL). (500 MHz, 1H NMR Methanol-d4) δ: 8.56 (s, 1H, formate), 8.21 (d, J = 5.8 Hz, 1H), 7.97 (d, J = 6.4 Hz, 1H), 7.01 (d, J= 2.5 Hz, 1H), 6.83 (dd, J = 5.9, 2.5 Hz, 1H), 6.71 - 6.64 (m, 2H), 4.30 (d, J = 14.4 Hz, 1H), 4.27 - 4.19 (m, 2H), 3.86 (dd, J = 22.3, 14.9 Hz, 2H), 3.23 - 2.99 (m, 11H), 2.82 - 2.70 (m, 2H), 2.13 - 1.90 (m, 5H),
1.90 - 1.48 (m, 14H), 1.46 - 1.23 (m, 10H), 1.22 - 1.07 (m, 5H), 1.09 - 0.98 (m, 2H), 0.94 (d, J = 6.6 Hz, 3H), 0.91 - 0.84 (m, 10H), 0.70 (s, 4H) ppm. 13CNMR(126 MHz, Methanol-d4 ) δ: 166.83 (formate), 159.23, 157.60, 155.89, 150.98, 145.72, 112.37, 111.83, 107.42, 106.58, 78.14, 70.04, 69.51, 62.56, 60.61, 57.93, 57.79, 55.77, 55.36, 45.78, 43.80, 41.43, 40.69, 39.65, 37.75, 37.35, 37.13, 36.83, 36.67, 34.99, 33.26, 29.80, 29.32, 29.16, 28.83, 28.63, 28.58, 25.23, 25.13, 25.07, 24.96, 23.18, 22.93, 22.34,
19.19, 12.65, 12.49 ppm. HRMS (ESI+) m/z calculated for C49H77N5O [M+H]+ 752.6128, found 752.6199.
Example 2: Chemical synthesis of Pre-Fentomycin dry schlenk were added under Ar 21 (3.0 mg, 0.0082 mmol),
Pd(dba)2 (0.2 mg, 0.0004 mmol), dppf (0.4 mg, 0.0008 mmol) and CS2CO3 (2.9 mg, 0.0090 mmol). Then was added 6 (5.2 mg, 0.0115 mmol) dissolved in 1 mL of anhydrous dioxane and the mixture was stirred at 80 °C overnight. Once the reaction was cooled at room temperature the solvent was removed under vacuum. The residue was dissolved in dichloromethane (3mL) and washed with a solution of sat. Na2CO3 (3mL) and extracted with dichloromethane (3 x 3 mL). The combined organic layers were dried over anhydrous MgSCE and the solvent was removed under reduced pressure. The residue was purified using a preparative HPLC equipped with a CIS-reverse phase column (gradient acetonitrile/water/formic acid acid 5:95:0.1 to acetonitrile/water/formic acid 100:0:0.1) to afford 2.5 mg (42%) of the product as a violet solid. (501H0 M NMHzR, Methanol-<A) δ 9.51 (d, J = 9.0 Hz, 1H), 8.30 (d, J = 8.5 Hz, 1H), 8.16 (d, J = 8.6 Hz, 1H), 7.87 (d, J = 6.4 Hz, 1H), 7.81 - 7.73 (m, 2H), 7.64 - 7.57 (m, 2H), 7.51 (dd, J = 7.3, 1.0 Hz, 1H), 7.18 (dd, J = 8.7, 1.1 Hz, 1H), 6.61 - 6.48 (m, 4H), 4.14 - 4.01 (m, 2H), 3.71 - 3.60 (m, 2H), 3.46 (t, J = 6.3 Hz, 2H), 3.26 (t, J = 6.6 Hz, 2H), 3.07 - 2.98 (m, 2H), 2.95 (s, 6H), 2.90 - 2.79 (m, 2H), 2.66 - 2.57 (m, 2H), 2.55 (s, 3H), 1.98 - 1.88 (m, 2H), 1.88 - 1.77 (m, 6H), 1.77 - 1.65 (m, 2H), 1.58 - 1.46 (m, 2H). 13C NMR (126 MHz, Methanol-J4) 5 187.8, 186.2, 157.9, 157.3, 156.1 (2C), 152.4, 146.2, 145.5, 140.2, 137.9, 137.6, 136.70, 136.1, 135.7, 133.1, 129.7, 129.6, 129.2, 128.9, 123.4, 118.7, 116.68, 113.1, 107.1 (2C), 106.9 (2C), 69.4 (2C), 61.1, 60.6, 55.8, 55.5, 43.1, 43.0, 39.4 (2C), 28.6 (2C), 27.2, 27.0, 25.0, 24.9, 21.5. HRMS(ESI-MS) m/z calculated for C45H52N7O2 [M+H]+ 722.4177, found 722.7188
Example 3: Biological results
Materials and methods
Cell culture
PDAC085T, PDAC090T, PDAC053T, PDAC211T and PDAC030T cells were grown in serum-free ductal medium: DMEM/F12 supplemented with 0.61g/500mL nicotinamide (Sigma- Aldrich, 3376), 2.50g/ 500mL glucose (Sigma-Aldrich, G6152), 1:200 ITS+ (Coming, 354352), 1:20 Nu-serum IV (Coming, 355104), 100 ng/ml cholera toxin, 1 pM dexamethasone (Sigma-Aldrich, D4902), 50 nM 3,3’,5-triiodo-L-thyronine (Sigma-Aldrich, T6397) and 1 x PenStrep. Human FC 1242, mouse 4a and human hMIA-2D cells were a generous gift from D. Tuveson and were grown in DMEM (Thermo Fisher Scientific, 10566016) supplemented with 10% FBS (v/v) (Thermo Fisher Scientific, 10270106) and 1 x PenStrep (Invitrogen #15070-063). HT-1080 cells were obtained from ATCC and grown in DMEM (Thermo Fisher Scientific, 10566016) supplemented with 10% FBS (v/v) (Thermo Fisher Scientific, 10270106) and 1 x PenStrep (Invitrogen #15070-063).
Primary human pancreatic PDAC053T cells were grown in semm-free ductal medium: DMEM/F12 (Gibco, 10565018), supplemented with 0.61g/500mL nicotinamide (Sigma-Aldrich, 3376), 2.50g/500mL glucose (Sigma- Aldrich, G6152), 1:200 ITS+ (Coming, 354352), 1:20 Nu-semm IV (Coming, 355104), 100 ng/ml cholera toxin, 1 pM dexamethasone (Sigma-Aldrich, D4902), 50 nM 3,3’,5-triiodo-L-thyronine (Sigma-Aldrich, T6397) and penicillin/streptomycin. Fresh bovine pituitary extract (Gibco, 13028-014, 22.7 ng/mL) and 50 ng/mL animal-free recombinant human EGF (Thermo
Fisher Scientific, AF-100-15-1MG) were added to new medium when cells were split or plated.
Ethical considerations
All mouse experiments complied with all relevant ethical regulations and were performed according to protocols approved by the Institutional Animal Care and Use Committee at Harvard T.H. Chan School of Public Health (protocol IS00003460). No formal randomization techniques were used; however, animals were allocated randomly to treatment groups and specimens were processed in an arbitrary order. For all experiments, the maximum permitted tumor diameter was 2.0 cm and this limit was not exceeded in any experiment. For all experiments, mice were kept on normal chow and fed ad-libitum. For human samples, all patients provided written informed consent for use of tumor samples and the study was approved by institutional regulatory board (DATA 190160).
Tumor dissociation
Fresh human tumors were dissociated using a gentle MACS Miltenyi Octo dissociator at 37°C using a human tumor dissociation kit (Miltenyi, 130-095-929) according to the manufacturer’s instructions. In brief, tumors were sliced into small pieces with a scalpel and then digested using the pre-described enzyme mix in RPMI-1640 Glutamax (Fisher Scientific, 11554516) in gentleMACS C tubes (Miltenyi, 130-093-237). Mouse tumors were dissociated using the same procedure but with the mouse tumor dissociation kit (Miltenyi, 130-096-730) according to the manufacturer’s instructions. Cells were then cultured in RPMI-1640 Glutamax/10%FBS,l% PenSTREP for 1 h and analyzed by flow cytometry, or treated with 1 pM Fentomycin and relevant ferroptosis inhibitors for 24 h and subsequently analyzed by flow cytometry.
Fluorescence microscopy
Cells were plated on cover slips 24 h prior to the experiment. Then, cells were treated with 1 pM Fentomycin, 1 pM Marmycin A or 1 pM ligand- ‘click’ and LysoTracker Deep Red (Thermo Fisher Scientific, L12492, 1: 10000) or CellRox (Thermo Fisher Scientific, C10422, 1: 10000) according to the manufacturer’s protocol. Subsequently, cells were washed three times with l x PBS and fixed with 2% paraformaldehyde (w/v) in l x PBS for 12 min, and then washed three times with l x PBS. For Fentomycin and Marmycin A, cover slips were then mounted using VECTASHIELD containing DAPI and were sealed with nail varnish Express manucure (Maybelline, 16P201). For fluorescent labeling of the ligand- ‘click’, after fixation, cells were permeabilized with 0.1% Triton X-100 (v/v) in 1 x PBS for 5 min and washed three times with 1 x PBS. Then, cells were blocked in 2% BSA, 0.2% Tween-20 (w/v) /I x PBS (blocking buffer) for 20 min at RT. The click reaction cocktail was prepared from Click- iT EdU Imaging Kits (ThermoFisher Scientific, C10337) according to the manufacturer’s protocol. Briefly, 868 μL of 1 x Click-iT reaction buffer was mixed with 40 μL Q1SO4 solution, 2 μL Alexa Fluor azide and 90 μL reaction buffer additive (sodium ascorbate) to reach a final volume of 1 mb. Cover slips were incubated with 50 μL of the click reaction cocktail in the dark at RT for 30 minutes, then washed three times with 1 x PBS. Cover slips were mounted using VECTASHIELD containing DAPI and were sealed with nail varnish Express manucure (Maybelline, 16P201).
Cell viability curves
Cell viability curves were established by plating 4,000 cells/well in 96-well plates using CellTiter-Blue viability assay according to the manufacturer’s protocol. Cells were treated with serial dilutions of irinotecan (Sigma-Aldrich, 11406), gemcitabine (Sigma-Aldrich, G6423), 5 -fluorouracil (Sigma- Aldrich, F6627-1G) or oxaliplatin (Biotechne, 2623) for 72 h. CellTiter-Blue reagent (Promega, G8081) was added after 72 h treatment and cells were incubated for 2 h before recording fluorescence intensities (Eex 560 nm; Eem 590 nm) using a Perkin Elmer Wallac 1420 Victor2 Microplate Reader.
Cell viability was also assessed using a CellTiterGlo 2.0 (Promega, G9241) or CellTiter blue (Promega, G8081) kit according to the manufacturer’s protocol in a 96-well plate. In brief, 4000 cells (HT-1080, PDAC053T or 4T1) were seeded per well in clear-bottom and darkened 96-well plates (Greiner, 655090, lot E23063EG) 24 h prior to the experiment. Cells were then pre-treated for 2 h with Lip-1 (10 pM), cLip-1 (10 pM), ferrostatin- 1 (Fer-1, SML0583, 10 pM), deferoxamine (DFO, Sigma-Aldrich, D9533, 100 pM), deferasirox (DFX, Cayman chemical, 16753, 10 pM), deferiprone (Def, Sigma-Aldrich, Y0001976, 100 pM), a-tocopherol (Toe, Sigma-Aldrich, PHR1031, 100 pM), Vitamin K3 (Sigma- Aldrich, M5625-25G, 10 pM), Z-VAD-FMK (Enzo Life Sciences, ALX-260-020-M005, 50 pM) or Necrostatin-1 (Nec-l, Sigma-Aldrich, N9037, 20 pM). Subsequently, Fento-1 (10 pM, 6 h) was added. Samples were processed as detailed in the manufacturer’s protocols and data were recorded on a SpectraMax ID3 plate reader (Molecular Devices). For standard-of-care cell viability measurements, cells were plated at 2000 cells per well 24 h prior to the experiment. Cells were incubated with serial dilutions of Fento-1, irinotecan (Sigma-Aldrich, 11406), 5-FU (Alfa Aesar, A13456-06) or oxaliplatin (Bio-Techne, 2623) for 72 h. CellTiter blue assay: Cell viability was assessed using a CellTiter blue assay and data recorded on a SpectraMax ID3 plate reader (Molecular Devices).
Flow cytometry
Fluorescent probes CellRox (Thermo Fisher Scientific, C10491) and Bodipy-Cl l (Thermo Fisher Scientific, D3861) were used according to the manufacturer’s protocol. Cells were harvested with Trypsin (if adherent) or directly (tumor samples) then washed twice with ice-cold 1 x PBS and suspended in incubation buffer prior to being analyzed by flow cytometry. Antibody staining of cells was achieved by incubation with human TruStain FcX (Biolegend, 422302) for 15 mins at room temperature with subsequent incubation of antibodies for 20 min at 4°C and then washed with 1 x PBS. The following antibody panels were used for dissociated human tumors: CD44-AF647 (Novus Biologicals, NB500-481AF647), CD45-BV510 (BioLegend, 368526), CD31-BV605 (BioLegend, 303122), FAP-750 (R&D, FAB3715S) and CD71-PE (R&D, FAB2474P). SytoxBlue (Thermo Fisher Scientific, SI 1348) was added according to the manufacturer’s instructions to identify dead cells. Live tumor cells were defined as SytoxBlueneg/CD45neg/CD3 lneg/FAPneg cells. The following antibody panels were used for dissociated mouse tumors: CD45-BV510 (BioLegend, 103138), CD31-BV605 (BioLegend, 102427), IA/IE-APC-Cy7 (BioLegend, 107628) and CD44-AF647 (BioLegend, 103018). Live tumor cells were defined as SytoxbluenegCD45neg/CD31 neg /I-A/I-Epos cells. Data were recorded on an Attune (Thermo Fisher Scientific) and processed using Cell Quest (BD Biosciences) and FlowJo (FLOWJO, LLC).PDAC053T cells were seeded in 6-well plates at the density of 1.5x 105 cells/well in 2 mL culture medium. On the next day, cells were treated with 1 pM of fentomycins for 5 h. Subsequently, 2 pM of BODIPY-C11 581/591 were added for 1 h. After a total incubation of 6 h with the small molecules, cells were washed with l x PBS, harvested with trypsin, washed with media and lx PBS an resuspended in ice-cold lx PBS with 10%FBS. Data were recorded on an AttuneTM NxT Flow Cytometer (Thermo Fisher Scientific). All data were analysed with FlowJo software v. 10.10.0.
LDH assay and Cell Titer Go assay
LDH assay (Sigma Aldrich, 11644793001) and CellTiterGlo (Promega, G9241) were performed according to manufacturer’s instructions in 96-well plates.
Liposome preparation
Liposomal structures were prepared using the traditional lipid film hydration method: 100 mL of a stock solution (Img/mL CH3CI) of 18: 1 (A9-Cis) PC (DOPC, Avanti Polar Lipids) were dissolved in 400 mL of CH3CI and transferred into a round-bottom flask. Organic solvent was removed under vacuum in a rotary evaporator for 15 min at 200 revolutions per minute (rpm) at 37 °C in a water bath. Afterwards, the lipid film was dried with a vacuum pump overnight. Then was hydrated with 1 mL of 0.1 mM Acetate buffer (pH 4.5) and vortexing every 5 min for 20 min. Liposomes were extruded by passing the suspension through 2 polycarbonate membranes (pore size 0.2 mm) 20 times and keep at -4 °C.
Liposome oxidation by Fentomycin
Control experiment (» = 3): 200 mL of the liposome solution were added into an Eppendorf and warmed at 37 °C with 800 rpm. Then were added 5 mL of an aqueous solution of Fe(OTf)2 (1.4 mg/1554,52 mL) and 13 uL of 0.1 mM Acetate buffer (pH 4.5). At t = 0 min 13 mL of an aqueous solution of H2O2 (10 mL H2O2 (30%)/1000 mL) were added.
Fentomycin oxidation (n = 3): 200 mL of the liposome solution were added into an Eppendorf and warmed at 37 °C with 800 rpm. Then were added 13 mL of a solution ImM of Fentomycin in DMSO and 5 mL of an aqueous solution of Fe(OTf)2 (1.4 mg/1554,52 mL). At t= 0 min 13 mL of an aqueous solution of H2O2 (10 mL H2O2 (30%)/1000 mL) were added.
The kinetic process of DOPC oxidation was recorded with a QExactive mass spectrometer (Thermo Fisher Scientific) equipped with a TriVersa NanoMate ion source (Advion Biosciences). Samples were injected at 0.5 h, 1 h, 2 h, 3 h, 4 h, 7h and 24 h reaction time.
Inductively coupled plasma mass spectrometry (ICP-MS)
Glass vials equipped with Teflon septa were cleaned with nitric acid 65% (w/v) (VWR, Suprapur, 1.00441.0250), washed with ultrapure water (Sigma- Aldrich, 1012620500) and dried. Small tumor junks were lyophilized and weighed. Samples were subsequently mixed with nitric acid 65% (w/v) overnight followed by heating at 80 °C for 2 h. Samples were diluted with ultrapure water (Sigma- Aldrich, 1012620500) to a final concentration of 0.475 N nitric acid and transferred to metal -free centrifuge vials (VWR, 89049-172) for subsequent ICP-MS analysis. 56Fe concentrations were measured using an Agilent 7900 ICP-QMS in low -resolution mode. Sample introduction was achieved with a micro-nebulizer (MicroMist, 0.2 mL/min) through a Scott spray chamber. Isotopes were measured using a collision-reaction interface with helium gas (5 mL/min) to remove polyatomic interferences. Scandium and indium internal standards were injected after inline mixing with the samples to control the absence of signal drift and matrix effects. A mix of certified standards was measured at concentrations spanning those of the samples to convert count measurements to concentrations in the solution. Uncertainties on sample concentrations were calculated using algebraic propagation of ICP-MS blank and sample counts uncertainties. Values were normalized by dry weight. Lipidomics
100,000 cells per condition were plated in 6-well plates 24 h prior to the experiment. Cells were treated with 1 pM fentomycin for 6h or 24 h. Cells were subsequently washed with lx PBS and then with 150 mM ammonium bicarbonate. Cells were then scraped and resuspended in 150 mM ammonium bicarbonate and centrifuged at 300 g for 5 min. The supernatant was removed and cells were resuspended in 1 mL of 150 mM ammonium bicarbonate. The solutions were centrifuged at 12,000 RPM for 10 min and the supernatant was removed. 200 μL of 150 mM sodium bicarbonate was added to the pellet and samples were flash frozen in liquid nitrogen. Cells were prepared in 5 independent biological replicates and lipidomics analysis was performed on the same day for all the replicates. For lipidomics analysis, the 200 μL cell lysates were spiked with 1.4 μL of internal standard lipid mixture containing 300 pmol of phosphatidylcholine 17:0-17:0, 50 pmol of phosphatidylethanolamine 17:0- 17:0, 30 pmol of phosphatidylinositol 16:0-16:0, 50 pmol of phosphatidylserine 17:0-17:0, 30 pmol of phosphatidylglycerol 17:0-17:0 and 30 pmol of phosphatidic acid 17:0-17:0 and subjected to lipid extraction at 4 °C. The sample was then extracted with 1 mL of chloroform-methanol (10: 1) for 2 h. The lower organic phase was collected, and the aqueous phase was re-extracted with 1 mL of chloroformmethanol (2: 1) for 1 h. The lower organic phase was collected and evaporated in a SpeedVac vacuum concentrator. Lipid extracts were dissolved in 100 μL of infusion mixture consisting of 7.5 mM ammonium acetate dissolved in propanol: chloroform: methanol [4: 1:2 (vol/vol)]. Samples were analyzed by direct infusion in a QExactive mass spectrometer (Thermo Fisher Scientific) equipped with a TriVersa NanoMate ion source (Advion Biosciences). 5 μL of sample were infused with gas pressure and voltage set to 1.25 psi and 0.95 kV, respectively. PC, PE, PEO, PCOx and PEOx were detected in the 10: 1 extract, by positive ion mode FTMS as protonated aducts by scanning m/z= 580-1000 Da, at Rm/z=2oo=28O 000 with lock mass activated at a common background (m/z=680.48022) for 30 s. Every scan is the average of 2 micro-scans, automatic gain control (AGC) was set to 1E6 and maximum ion injection time (IT) was set to 50 ms. PG and PGOx were detected as deprotonated adducts in the 10: 1 extract, by negative ion mode FTMS by scanning m/z= 420-1050 Da, at Rm/z=2oo=28O 000 with lock mass activated at a common background (m/z=529.46262) for 30 s. Every scan is the average of 2 microscans. Automatic gain control (AGC) was set to 1E6 and maximum ion injection time (IT) was set to 50ms. PA, PAOx, PI, PIOx, PS and PSOx were detected in the 2: 1 extract, by negative ion mode FTMS as deprotonated ions by scanning m/z= 400-1100 Da, at Rm/z=2oo=28O 000 with lock mass activated at a common background (m/z=529.46262) for 30 s. Every scan is the average of 2 micro-scans, automatic gain control (AGC) was set to 1E6 and maximum ion IT was set to 50 ms. All data was acquired in centroid mode. All lipidomics data were analyzed with the lipid identification software, LipidXplorer. Tolerance for MS and identification was set to 2 ppm. Data were normalized to internal standards.
Biopsy-derived pancreatic organoid (BDPO) generation
BDPOs were obtained from endoscopic ultrasound-guided fine-needle aspirations (EUS-FNA) from patients with PDAC included under the PaCaOmics clinical trial (ClinicalTrials.gov: NCT01692873) after approval by the Paoli-Calmettes hospital ethics committee and following patient informed consent. Cultures were established as previously described [38], Briefly, PDAC biopsies were slightly digested with the Tumor Dissociation Kit (Miltenyi Biotec) at 37°C for 5 minutes. The pancreatic tissue slurry was transferred into a tissue strainer 100 pm and was placed into 12-well plates coated with 150 μL GFRmatrigel (Coming, Boulogne-Billancourt, France). The samples cultured with Pancreatic Organoid Feeding Media (POFM) consisted of Advanced DMEM/F12 supplemented with 10 mM HEPES (Thermo Fisher Scientifics, Courtaboeuf, France); 1 x Glutamax (Thermo-Fisher Scientifics); penicillin/streptomycin (Thermo-Fisher Scientifics); 100 ng/mL Animal -Free Recombinant Human FGF10 (Peprotech, Peprotech, Neuilly-Sur-Seine, France); 50 ng/mL Animal-Free Recombinant Human EGF (Peprotech); 100 ng/mL Recombinant Human Noggin (Biotechne, Bio-Techne, Rennes, France); Wnt3a-conditioned medium (30% v/v); RSPO1 -conditioned medium (10% v/v); 10 nM human Gastrin 1 (Sigma- Aldrich Lyon, France); 10 mM nicotinamide (Sigma- Aldrich); 1.25 mM N- acetylcysteine (Sigma- Aldrich); 1 x B27 (Invitrogen, (Invitrogen, Villebon sur Yvette, France); 500 nM A83-01 (Tocris, Noyal Chatillon sur Seiche, France); 10.5 pM Y27632 (Tocris). The plates were incubated at 37 °C in a 5% CO2 incubator, and the media were changed every 3 or 4 days. For routine passages BDPOs were disaggregated with accutase (Thermo Fisher Scientific) and re-plated as needed. Chemograms on BDPO
BDPOs were disaggregated with accutase (Thermo Fisher Scientific), and 1,000 cells/well were plated in two 96-well round bottom ultra-low plates (Coming) with the medium described above. 24 hours later, one plate was used directly for RNA preparation (Time 0 transcriptome) and on the other the medium was supplemented with increasing concentrations of each drug, 72 hours later cell viability was measured with CellTiter-Glo 3D (Promega) reagent quantified using the plate reader Tristar LB941 (Berthold Technologies). Values were normalized and expressed as the percentage of the control (vehicle), which represents 100% of normalized fluorescence. Increasing concentrations of drugs were used. Each experiment was repeated at least twice.
Intranodal murine metastasis models for determining Fentomycin efficacy in vivo Murine breast cancer cells (4T1) were transplanted into 6 to 8-week-old female Balb/c mice (syngeneic with the 4T1 model). To perform injections into lymph nodes, the lymphatics were first traced by injecting 2% Evans Blue Dye (product E2129, Sigma- Aldrich) into the foot pedal 5 minutes before performing intranodal injections. After injecting Evans Blue Dye, the mice were anesthetized using isoflurane and a small (5-10 mm) incision was made in the region of the popliteal lymph node. The lymph node was located based on Evans Blue staining, immobilized with forceps, and 10,000 cells suspended in Phosphate-buffered saline (PBS) were injected in a volume of 10 μL into the popliteal lymph node using a 27 gauge Hamilton syringe. Injection into the lymph node was confirmed by visible swelling of the lymph node. The incision was closed using surgical glue (product 1469SB, 3M VetBond Tissue Adhesive) and the mice were closely monitored for signs of pain or distress. Once tumors were palpable in at least 75 % of the mice (~ 1 week after inj ection), 10 μL of volume of Fentomycin delivered intralymphatically into the tumor-bearing lymph node every second day until the experimental endpoint. Intranodal tumor diameters were measured thrice weekly with calipers until any tumor in the mouse cohort reached 2.0 cm in its largest diameter which was the pre-determined experimental endpoint for these experiments. At that point, all mice in the cohort were killed, per approved protocol, for analysis of intranodal tumor diameter, tumor mass, mouse mass, and metastatic disease burden. Metastatic disease burden was evaluated by bioluminescence imaging of visceral organs of the luciferase-tagged 4T1 cells. Tumor samples were snap frozen for metabolomics and lipidomics, and frozen in 10% DMSO in FBS for subsequent cellular analyses.
Results:
Fentomycin is a chimera of Marmycin A and an iron-activating ligand, tethered by a linker (Fig la). We also synthesized an alkyne substituted ligand (ligand- ‘click’) in order to label it in cells via click chemistry (Fig la). Fentomycin is naturally fluorescent, like its component Marmycin A, and readily accumulates in lysosomes (Fig lb), whereas the linker- ‘click’, labelled with a fluorophore via click chemistry, shows a pan-cellular staining. This shows that the Marmycin A subunit of Fentomycin guides its cellular localization. Fentomycin induces lysosomal reactive oxygen species (Fig 1c). Importantly, it shows low micromolar efficacy on cell viability with different human and mouse cancer cells tested in vitro, whereas Marmycin A and the ligand- ‘click’ are largely ineffective (Fig Id).
Using reconstituted liposomes and iron, an increased oxidation of lipids was observed in the presence of Fentomycin in vitro, a hallmark of ferroptosis (Fig 2a). Fentomycin, but not Marmycin A or the ligand- ‘click’, increases BODIPY 581/591 Cl l oxidation, a surrogate for lipid oxidation and hallmark of ferroptosis (Fig 2b). This can be reversed in the presence of the ferroptosis inhibitors Liproxstatin-1 (Lipl) and clickable Liproxstatin-1 (cLip-1), the iron chelator deferiprone (Def) and the antioxidant tocopherol (Toe). In line with this, oxidized lipids were observed in cells treated with Fentomycin, which are in the range of 20-40% of total corresponding lipids (Fig 2c). Cell viability induced by Fentomycin could be rescued with ferroptosis inhibitors, iron chelators and antioxidants, but failed to be rescued by the apoptosis inhibitor ZVAD-FMK (ZVAD) or the necroptosis inhibitor necrostatin (Nec). In line with this, these molecules also reduced fentomycin-induced lactodehydrogenase (LDH) release (Fig 2d). Taken together, these data show that fentomycin induces ferroptosis.
Using dissociated human tumors of pancreatic ductal adenocarcinoma (PDAC) it could be shown that BODIPY 581/591 Cl l was oxidized in the presence of fentomycin, specifically in the tumor cell population (SYTOXblueneg/ CD45neg/ CD3 lneg/FAPneg cells) of the dissociated heterogenic tumors (Fig 3a), and reduced the number of CD44hlgh cells in this cancer cell population (Fig 3b). This is indicative of a reduction of iron-rich plastic cells that usually have a persister cell character responsible for metastatic dissemination. Similar data could be obtained using freshly dissociated human undifferentiated pleomorphic sarcoma cells (Fig 3c, d), showing that fentomycin can target and eliminate persister cancer cells in different indications of human cancers. Using primary human PDAC cells and organoids, fentomycin exhibited more efficacy than Irinotecan, Oxaliplatin or 5-FU (Fig 3e, f), which constitute the current standard of care for PDAC chemotherapy. Using an intranodal murine metastasis model using 4T1 mouse cells, it was observed that Fentomycin treatment reduced iron levels in tumors (Fig 3g), reduced the CD44hlgh population of cancer cells (Fig 3h) and had no effect on bodyweight, indicative of no acute toxicity (Fig 3i). Strikingly, tumor size was significantly reduced in fentomycin-treated mice, showing an in vivo efficacy of fentomycin on tumor growth (Fig 3j).
The activity of structural variants of Fento-1 were then explored (Fig 4a). Fento-2, which contains a longer linker separating marmycin from the Chen- White ligand, exhibited a lower potency against PDAC and sarcoma cells compared to Fento-1. This supported the notion that Fento-1 exerts its activity by promoting proximity between a reactive iron catalyst and membrane lipids (Fig 4b, c). In contrast, replacing marmycin by cholesterol, an apolar natural product known to intercalate between phospholipids at the plasma membrane [34], and which plays a role in the mesenchymal state of cancer cells [35], led to the non-fluore scent analogue Fento-3 that exhibited a potency comparable to that of Fento-1, illustrating the versatile nature of this strategy (Fig 4b, c). As a control, replacing the apolar warhead by a more polar, yet neutral moiety such as morpholine, yielded Fento-4, which was biologically inactive (Fig 4b, c). Finally, replacing the Chen-White ligand by the structurally more hindered Nordlander-Costas [36] iron-activating ligand yielded Fento-5, which conserved cytotoxic and membrane lipid-oxidation properties, albeit with a delayed cell death response compared to Fento-1. This reflected a distinct reactivity of the iron catalyst towards organic substrates [37] (Fig 4b, c). In a colony-formation assay, Fento-1 eradicated DTP of triple -negative breast cancer SUM159 cells overexpressing CD44 that survived treatment with doxorubicin (Doxo) (Fig 4d). Altogether, these data indicated that pharmacological activation of lysosomal iron can trigger ferroptosis.
Fentomycins -3 and -6 to -16 have been evaluated fortheir peroxidation ability. The results of Figure 5 showed that these compounds induced peroxidation. References in sections “Background of the invention'1'’ and “Summary of the invention'1'’
Tian, AL., Wu, Q., Liu, P. et al. Lysosomotropic agents including azithromycin, chloroquine and hydroxychloroquine activate the integrated stress response. Cell Death Dis 12, 6 (2021). https://doi.org/10.1038/s41419-020-03324-w
Brun S, Bassissi F, Serdjebi C, Novello M, Tracz J, Autelitano F, Guillemot M, Fabre P, Courcambeck J, Ansaldi C, Raymond E, Halfon P. GNS561, a new lysosomotropic small molecule, for the treatment of intrahepatic cholangiocarcinoma. Invest New Drugs. 2019 Dec;37(6): 1135-1145. doi: 10.1007/S10637-019-00741-3.
[1] “https://www.who.int/,” can be found under https://www.who.int/, n.d.
[2] J. P. Thiery, H. Acloque, R. Y. J. Huang, M. A. Nieto, Cell 2009, 139, 871-890.
[3] T. Shibue, R. A. Weinberg, Nat. Rev. Clin. Oncol. 2017, 14, 611-629.
[4] T. Lapidot, C. Sirard, J. Vormoor, B. Murdoch, T. Hoang, J. Caceres-Cortes, M. Minden, B. Paterson, M. A. Caligiuri, J. E. Dick, Nature 1994, 367, 645-648.
[5] D. Bonnet, J. E. Dick, Nat. Med. 1997, 3, 730-737.
[6] T. N. Ignatova, V. G. Kukekov, E. D. Laywell, O. N. Suslov, F. D. Vrionis, D. A. Steindler, Glia 2002, 39, 193-206.
[7] S. K. Singh, I. D. Clarke, M. Terasaki, V. E. Bonn, C. Hawkins, J. Squire, P. B. Dirks, Cancer Res. 2003, 63, 5821-5828.
[8] C. Li, D. G. Heidt, P. Dalerba, C. F. Burant, L. Zhang, V. Adsay, M. Wicha, M. F. Clarke, D. M. Simeone, Cancer Res. 2007, 67, 1030-1037.
[9] P. C. Hermann, S. L. Huber, T. Herrler, A. Aicher, J. W. Ellwart, M. Guba, C. J. Bruns, C. Heeschen, Cell Stem Cell 2007, 1, 313-323.
[10] Y. Shiozawa, J. E. Berry, M. R. Eber, Y. Jung, K. Yumoto, F. C. Cackowski, H. J. Yoon, P. Parsana, R. Mehra, J. Wang, S. McGee, E. Lee, S. Nagrath, K. J. Pienta, R. S. Taichman, Oncotarget 2016, 7, 41217-41232.
[11] F. Yang, J. Xu, L. Tang, X. Guan, Cell. Mol. Life Sci. 2017, 74, 951-966.
[12] J. Stingl, C. Caldas, Nat. Rev. Cancer 2007, 7, 791-799.
[13] A. Kreso, J. E. Dick, Cell Stem Cell 2014, 14, 275-291.
[14] T. T. Mai, A. Hamai, A. Hienzsch, T. Caneque, S. Muller, J. Wicinski, O. Cabaud, C. Leroy, A. David, V. Acevedo, A. Ryo, C. Ginestier, D. Birnbaum, E. Charafe-Jauffret, P. Codogno, M. Mehrpour, R. Rodriguez, Nat. Chem. 2017, 9, 1025-1033.
[15] D. Basuli, L. Tesfay, Z. Deng, B. Paul, Y. Yamamoto, G. Ning, W. Xian, F. McKeon, M. Lynch, C. P. Crum, P. Hegde, M. Brewer, X. Wang, L. D. Miller, N. Dyment, F. M. Torti, S. V. Torti, Oncogene 2017, 36, 4089-4099.
[16] S. Muller, F. Sindikubwabo, T. Caneque, A. Lafon, A. Versini, B. Lombard, D. Loew, T.-D. Wu, C. Ginestier, E. Charafe-Jauffret, A. Durand, C. Vallot, S. Baulande, N. Servant, R. Rodriguez, Nat. Chem. 2020, 12, 929-938. [17] D. L. Schonberg, T. E. Miller, Q. Wu, W. A. Flavahan, N. K. Das, J. S. Hale, C. G. Hubert,
S.C. Mack, A. M. Jarrar, R. T. Karl, A. M. Rosager, A. M. Nixon, P. J. Tesar, P. Hamerlik, B.W. Kristensen, C. Horbinski, J. R. Connor, P. L. Fox, J. D. Lathia, J. N. Rich, Cancer Cell 2015, 28, 441- 455.
[18] S. V. Torti, F. M. Torti, Nat. Rev. Cancer 2013, 13, 342-355.
[19] S. J. Dixon, K. M. Lemberg, M. R. Lamprecht, R. Skouta, E. M. Zaitsev, C. E. Gleason, D. N. Patel, A. J. Bauer, A. M. Cantley, W. S. Yang, B. Morrison, B. R. Stockwell, Cell 2012, 149, 1060- 1072.
[20] M. Conrad, D. A. Pratt, Nat. Chem. Biol. 2019, 15, 1137-1147.
[21] S. Doll, F. P. Freitas, R. Shah, M. Aldrovandi, M. C. da Silva, I. Ingold, A. Goya Grocin,
T.N. Xavier da Silva, E. Panzilius, C. H. Scheel, A. Mourao, K. Buday, M. Sato, J. Wanninger, T. Vignane, V. Mohana, M. Rehberg, A. Flatley, A. Schepers, A. Kurz, D. White, M. Sauer, M. Sattler, E. W. Tate, W. Schmitz, A. Schulze, V. O’Donnell, B. Proneth, G. M. Popowicz, D. A. Pratt, J. P. F. Angeli, M. Conrad, Nature 2019, 575, 693-698
[22] Kirill Bersuker, Joseph M Hendricks, Zhipeng Li, Leslie Magtanong, Breanna Ford, Peter H Tang, Melissa A Roberts, Bingqi Tong, Thomas J Maimone, Roberto Zoncu, Michael C Bassik, Daniel K Nomura, Scott J Dixon, James A Olzmann; Nature 2019 Nov; 575; 688-692
[23] A. Versini, L. Colombeau, A. Hienzsch, C. Gaillet, P. Retailleau, S. Debieu, S. Muller, T. Caneque, R. Rodriguez, Chem. Eur. J. 2020, 26, 7416
[24] A. L. Turcu, A. Versini, N. Khene, C. Gaillet, T. Caneque, S. Muller, R. Rodriguez, Chem. Eur. J. 2020, 26, 7369
[25] Chen, M.S. & White, M.C. A predictably selective aliphatic C-H oxidation reaction for complex molecule synthesis. Science 318, 783-787 (2007).
[26] Chen, K. & Que, L., Jr. Stereospecific alkane hydroxylation by non-heme iron catalysts: mechanistic evidence for an Fe(V)=O active species. J. Am. Chem. Soc. 123, 6327-6337 (2001).
[27] Chen MS, White MC. Combined effects on selectivity in Fe-catalyzed methylene oxidation. Science. 2010 Jan 29;327(5965):566-71. doi: 10.1126/science.l l83602.
[28] Mitra M, Lloret-Fillol J, Haukka M, Costas M, Nordlander E. Evidence that steric factors modulate reactivity of tautomeric iron-oxo species in stereospecific alkane C-H hydroxylation. Chem Commun (Camb). 2014 Feb 11;50(12): 1408-10. doi: 10.1039/c3cc47830k.
[29] Olaf Cusso, Marco Cianfanelli, Xavi Ribas, Robertas J. M. Klein Gebbink, and Miquel Costas, Iron Catalyzed Highly Enantioselective Epoxidation of Cyclic Aliphatic Enones with Aqueous H2O2, J. Am. Chem. Soc. 2016, 138, 2732-2738.
[30] Olaf Cusso, Isaac Garcia-Bosch, Xavi Ribas, Julio Lloret-Fillol, and Miquel Costas, Asymmetric Epoxidation with H2O2 by Manipulating the Electronic Properties of Non-heme Iron Catalysts, JACS, 2013 735, 14871-14878. [31] Wenfang Wang, Daqian Xu, Qiangsheng Sun, Wei Sun, Efficient Aliphatic C-H Bond Oxidation Catalyzed by Manganese Complexes with Hydrogen Peroxide, Chem. Asian J. 2018, 13, 2458-2464.
[32] a)Mariani, A.; Mai, T. T.; Zacharioudakis, E.; Hienzsch, A.; Bartoli, A.; Caneque, T.; Rodriguez, R. Iron-dependent lysosomal dysfunction mediated by a natural product hybrid, Chem. Commun. 2016, 52, 1358-1360. b)Caneque, T.; Gomes, F.; Mai, T.; Maestri, G.; Malacria, M.; Rodriguez, R. Synthesis of marmycin A and investigation into its cellular activity, Nature Chem. 2015, 7, 744-751.
[33] Yoshifumi Amamoto, Yuki Aoi, Nozomu Nagashima, Hiroki Suto, Daisuke Yoshidome, Yasuhiro Arimura, Akihisa Osakabe, Daiki Kato, Hitoshi Kurumizaka, Shigehiro A. Kawashima, Kenzo Yamatsugu, and Motomu Kanai, Synthetic Posttranslational Modifications: Chemical Catalyst-Driven Regioselective Histone Acylation of Native Chromatin, J. Am. Chem. Soc. 2017, 139, 7568-7576.
[34] Sezgin, E., Levental, I., Mayor, S. & Eggeling, C. The mystery of membrane organization: composition, regulation and roles of lipid rafts. Nat. Rev. Mol. Cell Biol. 18, 361-374 (2017).
[35] Abdulla, N., Vincent, C.T. & Kaur, M. Mechanistic Insights Delineating the Role of Cholesterol in Epithelial Mesenchymal Transition and Drug Resistance in Cancer. Front. Cell Dev. Biol. 9, 728325 (2021).
[36] Mitra, M. et al. Highly enantioselective epoxidation of olefins by H(2)O(2) catalyzed by a nonheme Fe(ii) catalyst of a chiral tetradentate ligand. Dalton Trans 48, 6123-6131 (2019).
[37] Ftirstner, A. Iron Catalysis in Organic Synthesis: A Critical Assessment of What It Takes To Make This Base Metal a Multitasking Champion. ACS Cent. Sci. 2, 778-789 (2016).
[38] Fraunhoffer NA, Abuelafia AM, Bigonnet M, Gayet O, Roques J, Telle E, Santofimia-Castano P, Borrello MT, Chuluyan E, Dusetti N, lovanna J. Evidencing a Pancreatic Ductal Adenocarcinoma Subpopulation Sensitive to the Proteasome Inhibitor Carfilzomib. Clin Cancer Res. 2020 Oct 15;26(20):5506-5519. doi: 10.1158/1078-0432.CCR-20-1232.
[39] Zhou A, Du J, Jiao M, Xie D, Wang Q, Xue L, Ju C, Hua Z, Zhang C. Co-delivery of TRAIL and siHSP70 using hierarchically modular assembly formulations achieves enhanced TRAIL-resistant cancer therapy. J Control Release. 2019 Jun 28;304: 111-124.
[40] Choukairi Afailal N, Borrell M, Cianfanelli M, Costas M. Dearomative syn-Dihydroxylation of Naphthalenes with a Biomimetic Iron Catalyst. J Am Chem Soc. 2024 Jan 10;146(l):240-249.
[41] Wang W, Xu D, Sun Q, Sun W. Efficient Aliphatic C-H Bond Oxidation Catalyzed by Manganese Complexes with Hydrogen Peroxide. Chem Asian J. 2018 Sep 4;13(17):2458-2464.
[42] Wang J., Gong Y ., Sun D. and Gong H., Nickel-catalyzed reductive benzylation of tertiary alkyl halides with benzyl chlorides and chloroformates, Org. Chem. Front., 2021,8, 2944-2948.

Claims

1. A conjugate for use in the treatment of cancer, the conjugate being of the following general formula (I):
A-L-B (I) wherein:
A is a first moiety selected from metal chelating agents;
B is a second moiety selected from lysosomotropic agents; and
L is absent or is a linker that binds moieties A and B together, preferably L is a linker that binds moieties A and B together..
2. The conjugate for use according to claim 1, for use in a targeted treatment of Cancer Stem Cells (CSCs).
3. The conjugate for use according to claim 1 or 2, wherein the first moiety A is selected from ligands of the White-Chen Type, preferably the first moiety A is White-Chen ligand.
4. The conjugate for use according to any one of claims 1 to 3, wherein the first moiety A is of the following general formula (II):
• Ri is selected from the group consisting of NRARB, ORC, C(O)ORD, C(O)RE, S(O)RF, S(0)2RG, CN, halo-(C1-C6-alkyl), -C(O)-(C1-C6)alkyl-halo, halogen, NO2, and SO2, RA to RG being independently of each other H or a (C1-C6)alkyl, preferably Ri is N(CH3)2; R.2 is a hydrogen atom or (C1-C6)alkyl, preferably hydrogen atom ; and each Z is, independently of each other, a hydrogen or deuterium atom.
5. The conjugate for use according to any one of claims 1 to 4, wherein the first moiety A is of the following general formula (Ila): wherein
• Ri is selected from the group consisting of NRARB, ORC, C(O)ORD, C(O)RE, S(O)RF, S(O)2RG, CN, halo-(C1-C6-alkyl), -C(O)-(C1-C6)alkyl-halo, halogen, NO2, and SO2, RA to RG being independently of each other H or a (C1-C6)alkyl, preferably Ri is N(CH3)2;
• R2 is a hydrogen atom or (C1-C6)alkyl preferably hydrogen atom;
• X and Y are, independently of each other, a (C1-C6)alkyl; or X and Y can be linked together to form a heterocycloalkyl or heteroaryl, preferably a pyrrolidine; and
• each Z is, independently of each other, a hydrogen or deuterium atom; and wherein the wavy line represents the link to L.
6. The conjugate for use according to any one of claims 1 to 5, wherein the first moiety A is of the following general formula (lib): wherein
• Ri is selected from the group consisting of NRARB, ORC, C(O)ORD, C(O)RE, S(O)RF, S(O)SRG, CN, halo-(C1-C6-alkyl), -C(O)-(C1-C6)alkyl-halo, halogen, NO2, and SO2, RA to RG being independently of each other H or a (C1-C6)alkyl, preferably Ri is N(CHs)2;
• R2 is a hydrogen atom or (C1-C6)alkyl preferably hydrogen atom; and
• each Z is, independently of each other, a hydrogen or deuterium atom; and wherein the wavy line represents the link to L.
7. The conjugate for use according to claim 1 or 2, wherein the first moiety A is selected from ligands of the Nordlander-Costas Type, preferably the first moiety A is Nordlander-Costas ligand.
8. The conjugate for use according to any one of claims 1, 2 and 7, wherein the first moiety A is of the following general formula (lie): wherein
• Rs is a hydrogen atom or (C1-C6)alkyl preferably methyl;
• X and Y are, independently of each other, a (C1-C6)alkyl; or X and Y can be linked together to form a heterocycloalkyl or heteroaryl, preferably pyrrolidine; and
• each Z is, independently of each other, a hydrogen or deuterium atom; and wherein the wavy line represents the link to L.
9. The conjugate for use according to any one of claims 1, 2 and 7 to 8, wherein the first moiety A is of the following general formula ( wherein each Z is, independently of each other, a hydrogen or deuterium atom, and wherein the wavy line represents the link to L.
10. The conjugate for use according to claims 1 or 2, wherein the first moiety A is of the following formula (lid) wherein the wavy line represents the link to L.
11. The conjugate for use according to claim 1 or 2, wherein the first moiety A is selected from the group consisting of:
12. The conjugate for use according to any one of claims 1 to 11, wherein the second moiety B is selected from the group consisting of chloroquine, hydroxychloroquine, azithromycin, marmycin, steroids such as cholesterol and cholestanol, ezurpimtrostat hydrochloride, desmethylchloroquine, hydroxychloroquine phosphate, desmethyl-hydroxychloroquine, anthraquinones, methyl tetraphene dione, angucyclines, ammonium chloride (NH4CI), amantadine, methylamine, fluoxetine, imipramine, latrepirdine, tamoxifen, chlorpromazine, amitriptyline, verapamil, Triton WR 1339 (Tyloxapol), Suramin, metformin, Erythromycin, Amitriptyline, Imipramine, 4-aminoquinoline, amiodarone, amodiaquine, clindamycin, N-(3-[(2,4-dinitrophenyl)-amino]-propyl)-N-(3-aminopropyl- methylaminejdihydrochloride (DAMP), monensin, monodansylcadaverine, perhexilene, phenylalanine methyl ester, primaquine, quinacrine, thioridazine, tilorone, tributylamine, ketotifen fumarate, glycerol, sucrose, Trehalose, Resveratrol, PVP, and Gold sodium thiomalate.
13. The conjugate for use according to any one of claims 1 to 12, wherein the second moiety B is selected from the group consisting of chloroquine, hydroxychloroquine, azithromycin, marmycin, cholesterol, cholestanol, and ezurpimtrostat hydrochloride.
14. The conjugate for use according to any one of claims 1 to 13, wherein the second moiety B is selected from the group consisting of marmycin, cholesterol, cholestanol, anthraquinones, and angucyclines.
15. The conjugate for use according to any one of claims 1 to 14, wherein the lysosomotropic agent is marmycin A of formula (B 1) or cholesterol of formula (B2) or cholestanol of formula (B3) or compound of formula (B4), or compound (B5):
wherein the wavy line represents the link to L.
16. The conjugate for use according to claim 1 or 2, wherein said conjugate is selected from the group of the following compounds: wherein Ri is H or Cl,
,
17. A conjugate of the following general formula (I):
A-L-B (I) wherein: A is a first moiety selected from metal chelating agents;
B is a second moiety selected from lysosomotropic agent; and
L is absent or is a linker that binds moieties A and B together, preferably L is a linker that binds moieties A and B together.
18. The conjugate of claim 17, wherein A, L and B are as defined in any one of claims 1 to 16.
19. A Compound selected from the group consisting of: wherein Ri is H or Cl,
981
20. A pharmaceutical composition comprising a conjugate according to claim 17 or 18, or a compound according to claim 19 or a pharmaceutically acceptable salt and/or solvate thereof, and at least one pharmaceutically acceptable excipient.
21. A Nanoparticle comprising a conjugate according to claim 17 or 18 or a compound according to claim 19 or a pharmaceutically acceptable salt and/or solvate thereof.
22. A conjugate according to claim 17 or 18, or a compound according to claim 19 or a pharmaceutically acceptable salt and/or solvate thereof, or a pharmaceutical composition according to claim 20, or a nanoparticle according to claim 21, for use as a drug.
23. A conjugate according to claim 17 or 18, or a compound according to claim 19 or a pharmaceutically acceptable salt and/or solvate thereof, or a pharmaceutical composition according to claim 20, or a nanoparticle according to claim 21, for use in the treatment of cancer.
24. A method for the treatment of cancer in a subject in need thereof, comprising administering a therapeutic effective amount of a conjugate as defined in any one of claims 1 to 18, or a compound as defined in claim 19, or a pharmaceutical composition as defined in claim 20, or a nanoparticle as defined in claim 21, to said subject.
25. Use of a conjugate as defined in any one of claims 1 to 18, or a compound as defined in claim 19, or a pharmaceutical composition as defined in claim 20, or a nanoparticle as defined in claim 21 , for the manufacture of a medicament for use as anti-tumoral agent or in a method for the treatment of cancer.
26. A method for the treatment of a disease in a subject in need thereof, comprising administering a therapeutic effective amount of a conjugate as defined in claim 17 or 18, or a compound as defined in claim 19, or a pharmaceutical composition as defined in claim 20, or a nanoparticle as defined in claim 21, to said subject.
27. Use of a conjugate as defined in claim 17 or 18, or a compound as defined in claim 19, or a pharmaceutical composition as defined in claim 20, or a nanoparticle as defined in claim 21, for the manufacture of a medicament.
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Citations (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997020574A1 (en) 1995-12-04 1997-06-12 The Regents Of The University Of California Blockade of t lymphocyte down-regulation associated with ctla-4 signaling
US5773578A (en) 1990-01-08 1998-06-30 Institut National De La Sante Et De La Recherche Medicale Proteins produced by human lymphocytes, DNA sequence encoding these proteins and their pharmaceutical and biological use
WO2004004771A1 (en) 2002-07-03 2004-01-15 Ono Pharmaceutical Co., Ltd. Immunopotentiating compositions
WO2004056875A1 (en) 2002-12-23 2004-07-08 Wyeth Antibodies against pd-1 and uses therefor
US6984720B1 (en) 1999-08-24 2006-01-10 Medarex, Inc. Human CTLA-4 antibodies
US7109003B2 (en) 1998-12-23 2006-09-19 Abgenix, Inc. Methods for expressing and recovering human monoclonal antibodies to CTLA-4
WO2006121168A1 (en) 2005-05-09 2006-11-16 Ono Pharmaceutical Co., Ltd. Human monoclonal antibodies to programmed death 1(pd-1) and methods for treating cancer using anti-pd-1 antibodies alone or in combination with other immunotherapeutics
WO2007123737A2 (en) 2006-03-30 2007-11-01 University Of California Methods and compositions for localized secretion of anti-ctla-4 antibodies
WO2008156712A1 (en) 2007-06-18 2008-12-24 N. V. Organon Antibodies to human programmed death receptor pd-1
WO2009014708A2 (en) 2007-07-23 2009-01-29 Cell Genesys, Inc. Pd-1 antibodies in combination with a cytokine-secreting cell and methods of use thereof
WO2009114335A2 (en) 2008-03-12 2009-09-17 Merck & Co., Inc. Pd-1 binding proteins
US8017114B2 (en) 1999-08-24 2011-09-13 Medarex, Inc. Human CTLA-4 antibodies and their uses
WO2013043569A1 (en) 2011-09-20 2013-03-28 Vical Incorporated Synergistic anti-tumor efficacy using alloantigen combination immunotherapy
US20130177557A1 (en) 2010-03-26 2013-07-11 Randolph J. Noelle Vista regulatory t cell mediator protein, vista binding agents and use thereof
WO2014047350A1 (en) 2012-09-20 2014-03-27 Morningside Technology Ventures Ltd. Oncolytic virus encoding pd-1 binding agents and uses of the same

Patent Citations (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5773578A (en) 1990-01-08 1998-06-30 Institut National De La Sante Et De La Recherche Medicale Proteins produced by human lymphocytes, DNA sequence encoding these proteins and their pharmaceutical and biological use
WO1997020574A1 (en) 1995-12-04 1997-06-12 The Regents Of The University Of California Blockade of t lymphocyte down-regulation associated with ctla-4 signaling
US7109003B2 (en) 1998-12-23 2006-09-19 Abgenix, Inc. Methods for expressing and recovering human monoclonal antibodies to CTLA-4
US8491895B2 (en) 1998-12-23 2013-07-23 Amgen Fremont Inc. Methods of treating cancer with human monoclonal antibodies to CTLA-4
US8143379B2 (en) 1998-12-23 2012-03-27 Amgen Fremont Inc. Human monoclonal antibodies to CTLA-4
US6984720B1 (en) 1999-08-24 2006-01-10 Medarex, Inc. Human CTLA-4 antibodies
US8017114B2 (en) 1999-08-24 2011-09-13 Medarex, Inc. Human CTLA-4 antibodies and their uses
WO2004004771A1 (en) 2002-07-03 2004-01-15 Ono Pharmaceutical Co., Ltd. Immunopotentiating compositions
WO2004056875A1 (en) 2002-12-23 2004-07-08 Wyeth Antibodies against pd-1 and uses therefor
WO2006121168A1 (en) 2005-05-09 2006-11-16 Ono Pharmaceutical Co., Ltd. Human monoclonal antibodies to programmed death 1(pd-1) and methods for treating cancer using anti-pd-1 antibodies alone or in combination with other immunotherapeutics
WO2007123737A2 (en) 2006-03-30 2007-11-01 University Of California Methods and compositions for localized secretion of anti-ctla-4 antibodies
WO2008156712A1 (en) 2007-06-18 2008-12-24 N. V. Organon Antibodies to human programmed death receptor pd-1
WO2009014708A2 (en) 2007-07-23 2009-01-29 Cell Genesys, Inc. Pd-1 antibodies in combination with a cytokine-secreting cell and methods of use thereof
WO2009114335A2 (en) 2008-03-12 2009-09-17 Merck & Co., Inc. Pd-1 binding proteins
US20130177557A1 (en) 2010-03-26 2013-07-11 Randolph J. Noelle Vista regulatory t cell mediator protein, vista binding agents and use thereof
WO2013043569A1 (en) 2011-09-20 2013-03-28 Vical Incorporated Synergistic anti-tumor efficacy using alloantigen combination immunotherapy
WO2014047350A1 (en) 2012-09-20 2014-03-27 Morningside Technology Ventures Ltd. Oncolytic virus encoding pd-1 binding agents and uses of the same

Non-Patent Citations (57)

* Cited by examiner, † Cited by third party
Title
A. KRESOJ. E. DICK, CELL STEM CELL, vol. 14, 2014, pages 275 - 291
A. L. TURCUA. VERSININ. KHENEC. GAILLETT. CAFIEQUES. MULLERR. RODRIGUEZ, CHEM. EUR. J, vol. 26, 2020, pages 7369
A. VERSINIL. COLOMBEAUA. HIENZSCHC. GAILLETP. RETAILLEAUS. DEBIEUS. MILLERT. CAFIEQUER. RODRIGUEZ, CHEM. EUR. J., vol. 26, 2020, pages 7416
ABDULLA, N.VINCENT, C.TKAUR, M: "Mechanistic Insights Delineating the Role of Cholesterol in Epithelial Mesenchymal Transition and Drug Resistance in Cancer", FRONT. CELL DEV. BIOL, vol. 9, 2021, pages 728325
BRUN SBASSISSI FSERDJEBI CNOVELLO MTRACZ JAUTELITANO FGUILLEMOT MFABRE PCOURCAMBECK JANSALDI C: "GNS561, a new lysosomotropic small molecule, for the treatment of intrahepatic cholangiocarcinoma", INVEST NEW DRUGS, vol. 37, no. 6, December 2019 (2019-12-01), pages 1135 - 1145, XP036942091, DOI: 10.1007/s10637-019-00741-3
CANEQUE, T.GOMES, F.MAI, T.MAESTRI, G.MALACRIA, M.RODRIGUEZ, R: "Synthesis of marmycin A and investigation into its cellular activity", NATURE CHEM, vol. 7, 2015, pages 744 - 751, XP093185237, DOI: 10.1038/nchem.2302
CHEN MSWHITE MC: "Combined effects on selectivity in Fe-catalyzed methylene oxidation", SCIENCE, vol. 327, no. 5965, 29 January 2010 (2010-01-29), pages 566 - 71
CHEN, K.QUE, L., JR: "Stereospecific alkane hydroxylation by non-heme iron catalysts: mechanistic evidence for an Fe(V)=O active species", J. AM. CHEM. SOC., vol. 123, 2001, pages 6327 - 6337, XP093153060, DOI: 10.1021/ja010310x
CHOUKAIRI AFAILALNBORRELL MCIANFANELLI MCOSTAS M: "Dearomative syn-Dihydroxylation of Naphthalenes with a Biomimetic Iron Catalyst", J AM CHEM SOC., vol. 146, no. 1, 10 January 2024 (2024-01-10), pages 240 - 249
COSTASNORLANDER, CHEM. COMMUN., vol. 50, 2014, pages 1408
D. BASULIL. TESFAYZ. DENGB. PAULY. YAMAMOTOG. NINGW. XIANF. MCKEONM. LYNCHC. P. CRUM, ONCOGENE, vol. 36, 2017, pages 4089 - 4099
D. BONNETJ. E. DICK, NAT. MED., vol. 3, 1997, pages 730 - 737
D. L. SCHONBERGT. E. MILLERQ. WUW. A. FLAVAHANN. K. DASJ. S. HALEC. G. HUBERTS.C. MACKA. M. JARRARR. T. KARL, CANCER CELL, vol. 28, 2015, pages 441 - 455
F. YANGJ. XUL. TANGX. GUAN, CELL. MOL. LIFE SCI., vol. 74, 2017, pages 951 - 966
FAVAUDON VFOUILLADE CVOZENIN MC: "The radiotherapy FLASH to save healthy tissues", MED SCI, vol. 31, 2015, pages 121 - 123
FRAUNHOFFER NAABUELAFIA AMBIGONNET MGAYET OROQUES JTELLE ESANTOFIMIA-CASTAFTO PBORRELLO MTCHULUYAN EDUSETTI N: "Evidencing a Pancreatic Ductal Adenocarcinoma Subpopulation Sensitive to the Proteasome Inhibitor Carfilzomib", CLIN CANCER RES, vol. 26, no. 20, 15 October 2020 (2020-10-15), pages 5506 - 5519
FÜRSTNER, A: "Iron Catalysis in Organic Synthesis: A Critical Assessment of What It Takes To Make This Base Metal a Multitasking Champion", ACS CENT. SCI., vol. 2, 2016, pages 778 - 789
J. P. THIERYH. ACLOQUER. Y. J. HUANGM. A. NIETO, CELL, vol. 139, 2009, pages 871 - 890
J. STINGLC. CALDAS, NAT. REV. CANCER, vol. 7, 2007, pages 791 - 799
KIRILL BERSUKERJOSEPH M HENDRICKSZHIPENG LILESLIE MAGTANONGBREANNA FORDPETER H TANGMELISSA A ROBERTSBINGQI TONGTHOMAS J MAIMONEROB, NATURE, vol. 575, November 2019 (2019-11-01), pages 688 - 692
LIU ZU D ET AL: "Design, Synthesis and Evaluation of N-Basic Substituted 3-Hydroxypyridin-4-ones: Orally Active Iron Chelators with Lysosomotrophic Potential", vol. 52, no. 3, 18 February 2010 (2010-02-18), GB, pages 263 - 272, XP055854546, ISSN: 0022-3573, Retrieved from the Internet <URL:https://watermark.silverchair.com/0022357001773940.pdf?token=AQECAHi208BE49Ooan9kkhW_Ercy7Dm3ZL_9Cf3qfKAc485ysgAAAuQwggLgBgkqhkiG9w0BBwagggLRMIICzQIBADCCAsYGCSqGSIb3DQEHATAeBglghkgBZQMEAS4wEQQMt75oawejVOg4bN7NAgEQgIIClxL6FvFjIXlSuWn1bK2RUl57q3dZTOywiuWJ6g1SIWjRb9Q6cvWmeJeZGmVhS04oD1ppDJbywIkVa4MPOHc> DOI: 10.1211/0022357001773940 *
M. CONRADD. A. PRATT, NAT. CHEM. BIOL., vol. 15, 2019, pages 1137 - 1147
M. S. CHENM. C. WHITE, SCIENCE, vol. 327, 2010, pages 566 - 571
M. S. CHENM. C. WHITE: "A predictably selective aliphatic C-H oxidation reaction for complex molecule synthesis", SCIENCE, vol. 318, 2007, pages 783 - 787
M. SIMEONE, CANCER RES., vol. 67, 2007, pages 1030 - 1037
MARIANI A. ET AL: "Iron-dependent lysosomal dysfunction mediated by a natural product hybrid", vol. 52, no. 7, 24 November 2015 (2015-11-24), UK, pages 1358 - 1360, XP093185335, ISSN: 1359-7345, Retrieved from the Internet <URL:https://pubs.rsc.org/en/content/articlepdf/2016/cc/c5cc09255h> DOI: 10.1039/C5CC09255H *
MARIANI, A.MAI, T. T.ZACHARIOUDAKIS, E.HIENZSCH, A.BARTOLI, A.CANEQUE, T.RODRIGUEZ, R: "Iron-dependent lysosomal dysfunction mediated by a natural product hybrid", CHEM. COMMUN., vol. 52, 2016, pages 1358 - 1360, XP093185335, DOI: 10.1039/C5CC09255H
MARTA GONZ�LEZ-B�RTULOS ET AL: "Pro-Oxidant Activity of Amine-Pyridine-Based Iron Complexes Efficiently Kills Cancer and Cancer Stem-Like Cells", PLOS ONE, vol. 10, no. 9, 14 September 2015 (2015-09-14), pages e0137800, XP055300833, DOI: 10.1371/journal.pone.0137800 *
MITRA MLLORET-FILLOL JHAUKKA MCOSTAS MNORDLANDER E: "Evidence that steric factors modulate reactivity of tautomeric iron-oxo species in stereospecific alkane C-H hydroxylation", CHEM COMMUN (CAMB, vol. 50, no. 12, 11 February 2014 (2014-02-11), pages 1408 - 10
MITRA, M. ET AL.: "Highly enantioselective epoxidation of olefins by H(2)O(2) catalyzed by a non-heme Fe(ii) catalyst of a chiral tetradentate ligand", DALTON TRANS, vol. 48, 2019, pages 6123 - 6131
OLAF CUSSOISAAC GARCIA-BOSCHXAVI RIBASJULIO LLORET-FILLOLMIQUEL COSTAS: "Asymmetric Epoxidation with H 0 by Manipulating the Electronic Properties of Non-heme Iron Catalysts", JACS, vol. 135, 2013, pages 14871 - 14878
OLAF CUSSOMARCO CIANFANELLIXAVI RIBASROBERTUS J. MKLEIN GEBBINKMIQUEL COSTAS: "Iron Catalyzed Highly Enantioselective Epoxidation of Cyclic Aliphatic Enones with Aqueous H 0", J. AM. CHEM. SOC., vol. 138, 2016, pages 2732 - 2738
P. C. HERMANNS. L. HUBERT. HERRLERA. AICHERJ. W. ELLWARTM. GUBAC. J. BRUNSC. HEESCHEN, CELL STEM CELL, vol. 1, 2007, pages 313 - 323
PALMA ELISA ET AL: "Unravelling the antitumoral potential of novel bis(thiosemicarbazonato) Zn(II) complexes: structural and cellular studies", JBIC. JOURNAL OF BIOLOGICAL INORGANIC CHEMISTRY, SPRINGER, DE, vol. 24, no. 1, 24 November 2018 (2018-11-24), pages 71 - 89, XP036708528, ISSN: 0949-8257, [retrieved on 20181124], DOI: 10.1007/S00775-018-1629-6 *
PATRIARCA A.FOUILLADE C. M.MARTIN F.POUZOULET F.NAURAYE C. ET AL.: "Experimental set-up for FLASH proton irradiation of small animals using a clinical system", INT J RADIAT ONCOL BIOL PHYS, vol. 102, 11 July 2018 (2018-07-11), pages 619 - 626, XP085474451, DOI: 10.1016/j.ijrobp.2018.06.403
PRAGYA ARORA ET AL: "Pivotal Role of Salicylates in Tuning the Formation and Reactivity of Mn(V)=O's", EUROPEAN JOURNAL OF INORGANIC CHEMISTRY, WILEY-VCH VERLAG , WENHEIM, DE, vol. 27, no. 17, 6 March 2024 (2024-03-06), pages n/a, XP072651656, ISSN: 1434-1948, DOI: 10.1002/EJIC.202400079 *
PREZADO YJOUVION GGUARDIOLA CGONZALEZ WJUCHAUX MBERGS JNAURAYE CLABIOD DDE MARZI LPOUZOULET F: "Tumor Control in RG2 Glioma-Bearing Rats: A Comparison Between Proton Minibeam Therapy and Standard Proton Therapy", INT J RADIAT ONCOL BIOL PHYS, vol. 104, no. 2, 1 June 2019 (2019-06-01), pages 266 - 271
PREZADO YJOUVION GPATRIARCA ANAURAYE CGUARDIOLA CJUCHAUX MLAMIRAULT CLABIOD DJOURDAIN LSEBRIE C: "Proton minibeam radiation therapy widens the therapeutic index for high-grade gliomas", SCI REP, vol. 8, no. 1, 7 November 2018 (2018-11-07), pages 16479
RODRIGUEZ RAPHA�L ET AL: "Activation of lysosomal iron triggers ferroptosis in cancer", RESEARCH SQUARE, 8 April 2024 (2024-04-08), pages 1 - 34, XP093182963, Retrieved from the Internet <URL:https://www.researchsquare.com/article/rs-4165774/v1.pdf?c=1712579074000> *
S. DOLLF. P. FREITASR. SHAHM. ALDROVANDIM. C. DA SILVAI. INGOLDA. GOYA GROCINT.N. XAVIER DA SILVAE. PANZILIUSC. H. SCHEEL, NATURE, vol. 575, 2019, pages 693 - 698
S. J. DIXONK. M. LEMBERGM. R. LAMPRECHTR. SKOUTAE. M. ZAITSEVC. E. GLEASOND. N. PATELA. J. BAUERA. M. CANTLEYW. S. YANG, CELL, vol. 149, 2012, pages 1060 - 1072
S. K. SINGHI. D. CLARKEM. TERASAKIV. E. BONNC. HAWKINSJ. SQUIREP. B. DIRKS, CANCER RES., vol. 63, 2003, pages 5821 - 5828
S. MILLERF. SINDIKUBWABOT. CAFIEQUEA. LAFONA. VERSINIB. LOMBARDD. LOEWT.-D. WUC. GINESTIERE. CHARAFE-JAUFFRET, NAT. CHEM., vol. 12, 2020, pages 929 - 938
S. V. TORTIF. M. TORTI, NAT. REV. CANCER, vol. 13, 2013, pages 342 - 355
SEZGIN, E.LEVENTAL, I.MAYOR, SEGGELING, C: "The mystery of membrane organization: composition, regulation and roles of lipid rafts", NAT. REV. MOL. CELL BIOL., vol. 18, 2017, pages 361 - 374
SIWARUTT BOONYARATTANAKALIN ET AL: "Synthesis of an Artificial Cell Surface Receptor that Enables Oligohistidine Affinity Tags to Function as Metal-Dependent Cell-Penetrating Peptides", JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, vol. 128, no. 2, 1 January 2006 (2006-01-01), pages 386 - 387, XP055075422, ISSN: 0002-7863, DOI: 10.1021/ja056126j *
SOLER MARTA ET AL: "Design, Preparation, and Characterization of Zn and Cu Metallopeptides Based On Tetradentate Aminopyridine Ligands Showing Enhanced DNA Cleavage Activity", INORGANIC CHEMISTRY, vol. 54, no. 22, 27 October 2015 (2015-10-27), Easton , US, pages 10542 - 10558, XP093065394, ISSN: 0020-1669, DOI: 10.1021/acs.inorgchem.5b01680 *
T. LAPIDOTC. SIRARDJ. VORMOORB. MURDOCHT. HOANGJ. CACERES-CORTES, MMINDEN, BPATERSON, MA. CALIGIURIJ. E. DICK, NATURE, vol. 367, 1994, pages 645 - 648
T. N. IGNATOVAV. G. KUKEKOVE. D. LAYWELLO. N. SUSLOVF. D. VRIONISD. A. STEINDLER, GLIA, vol. 39, 2002, pages 193 - 206
T. SHIBUER. A. WEINBERG, NAT. REV. CLIN. ONCOL., vol. 14, 2017, pages 611 - 629
T. T. MAIA. HAMAIA. HIENZSCHT. CAFIEQUES. MILLERJ. WICINSKIO. CABAUDC. LEROYA. DAVIDV. ACEVEDO, NAT. CHEM., vol. 9, 2017, pages 1025 - 1033
TIAN, AL.WU, Q.LIU, P ET AL.: "Lysosomotropic agents including azithromycin, chloroquine and hydroxychloroquine activate the integrated stress response", CELL DEATH DIS, vol. 12, 2021, pages 6, Retrieved from the Internet <URL:https://doi.org/10.1038/s41419-020-03324-w>
WANG J.GONG Y.SUN DGONG H: "Nickel-catalyzed reductive benzylation of tertiary alkyl halides with benzyl chlorides and chloroformates", ORG. CHEM. FRONT., vol. 8, 2021, pages 2944 - 2948
WANG WXU DSUN QSUN W: "Efficient Aliphatic C-H Bond Oxidation Catalyzed by Manganese Complexes with Hydrogen Peroxide", CHEM ASIAN J., vol. 13, no. 17, 4 September 2018 (2018-09-04), pages 2458 - 2464, XP072428396, DOI: 10.1002/asia.201800068
Y. SHIOZAWAJ. E. BERRYM. R. EBERY. JUNGK. YUMOTOF. C. CACKOWSKIH. J. YOONP. PARSANAR. MEHRAJ. WANG, ONCOTARGET, vol. 7, 2016, pages 41217 - 41232
YOSHIFUMI AMAMOTOYUKI AOINOZOMU NAGASHIMAHIROKI SUTODAISUKE YOSHIDOMEYASUHIRO ARIMURAAKIHISA OSAKABEDAIKI KATOHITOSHI KURUMIZAKASH: "Synthetic Posttranslational Modifications: Chemical Catalyst-Driven Regioselective Histone Acylation of Native Chromatin", J. AM. CHEM. SOC., vol. 139, 2017, pages 7568 - 7576, XP055429451, DOI: 10.1021/jacs.7b02138
ZHOU ADU JJIAO MXIE DWANG QXUE LJU CHUA ZZHANG C: "Co-delivery of TRAIL and siHSP70 using hierarchically modular assembly formulations achieves enhanced TRAIL-resistant cancer therapy", J CONTROL RELEASE, vol. 304, 28 June 2019 (2019-06-28), pages 111 - 124, XP085733377, DOI: 10.1016/j.jconrel.2019.05.013

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