EP3762038A1 - Compounds for modulation and as functional replacement of alpha-ketoglutaric acid (2og)-dependent oxygenases - Google Patents

Compounds for modulation and as functional replacement of alpha-ketoglutaric acid (2og)-dependent oxygenases

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Publication number
EP3762038A1
EP3762038A1 EP19750128.1A EP19750128A EP3762038A1 EP 3762038 A1 EP3762038 A1 EP 3762038A1 EP 19750128 A EP19750128 A EP 19750128A EP 3762038 A1 EP3762038 A1 EP 3762038A1
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Prior art keywords
compound
compound according
compounds
enzyme
formula
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EP19750128.1A
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German (de)
French (fr)
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Thomas Melchior Homann
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Individual
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/542—Carboxylic acids, e.g. a fatty acid or an amino acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/24—Heavy metals; Compounds thereof
    • A61K33/26—Iron; Compounds thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • 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
    • A61K47/62—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 a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F15/00—Compounds containing elements of Groups 8, 9, 10 or 18 of the Periodic Table
    • C07F15/02—Iron compounds
    • C07F15/025—Iron compounds without a metal-carbon linkage
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y114/00—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14)
    • C12Y114/11—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14) with 2-oxoglutarate as one donor, and incorporation of one atom each of oxygen into both donors (1.14.11)

Definitions

  • the invention relates to compounds as functional equivalents of the cosubstrate a-ketoglutaric acid (also known synonymously as 2-oxoglutarate or 20G) in 20G-dependent oxygenases (DIOs), in particular for their functional production in the presence of competitive inhibitors of DIOs, such as 2-hydroxyglutaric acid ( 2HG).
  • DIOs 20G-dependent oxygenases
  • A-Ketoglutarate (2-oxoglutarate-20G) -dependent dioxygenases are oxygen-dependent enzymes which contain a-ketoglutaric acid (2-oxoglutarate 20G, 2KG) as co-substrates and non-heme Fe (II) use as a co-factor. They catalyze a variety of oxidation reactions. These include (not exclusively) hydroxylation reactions, demethylations, ring expansions, ring closings and the introduction of double bonds. [1] [2, 3]
  • 20G-dependent dioxygenases are involved in basic functions in biosynthesis [6-8] In plants, 20G-dependent dioxygenases are involved in many different reactions in plant metabolism [9] These include (but not exclusively) flavonoid biosynthesis and ethylene biosynthesis [10] In mammals and humans, 20G-dependent dioxygenase have functional roles in biosynthesis (e.g. collagen biosynthesis [l 1] and L-camitine biosynthesis [l2]), post-translational modifications (e.g. protein hydroxylation [l3]), epigenetic regulations (e.g. Histone and DNA demethylation [l4]) as well as sensors of energy metabolism. [15].
  • biosynthesis e.g. collagen biosynthesis [l 1] and L-camitine biosynthesis [l2]
  • post-translational modifications e.g. protein hydroxylation [l3]
  • epigenetic regulations e.g. Histone and DNA demethylation [l4]
  • 20G-dependent dioxygenases catalyze oxidation reactions by incorporating a single oxygen atom into their substrates. This is always associated with the oxidation of cosubstrate 20G to succinate and carbon dioxide [16].
  • 20G-dependent dioxygenases are characterized by a common catalytic mechanism.
  • 20G and substrate bind to the active binding site [22-24]
  • 20G is directly coordinated with iron (II) (Ni II; Mn II) in the activity center, while the substrate is in close proximity, but not directly coordinating, binds to the metal.
  • the second step is the binding of molecular oxygen, which occupies a third position at the Fe (II) (Nill; MnII) center.
  • All 20G-dependent dioxygenases contain a conserved double-stranded ß-helix (DSBH), which forms a column with two ß-leaves [33, 34]
  • the active side contains a highly conserved 2-His-l-carboxylate (HXD / E ... H) amino acid residue triad motif in which the catalytically essential metal is fixed by two histidine residues and an aspartic acid or glutamic acid residue [35] However, they differ in the amino acid arrangement in the active center.
  • a-Ketoglutaric acid (20G) -dependent oxygenases catalyze a remarkably wide range of oxidative reactions. In humans and animals, these are hydroxylations and N-demethylations, which take place via hydroxylation reactions; in plants and microbes they catalyze reactions such as ring formations, rearrangements, desaturations and halogenations.
  • DIOs The biological function of the DIOs reflects the catalytic flexibility. After the role of DIOs in collagen biosynthesis was identified, it was shown that they also play a role in the development of plants and animals, the regulation of transcription, the modification / repair of nucleic acids (DNA, RNA), the fatty acid metabolism in the formation and stabilization of stem cells PS iPS) and the biosynthesis of secondary metabolites, including medically important antibiotics, play a role.
  • the present invention is intended to provide compounds for performing the function of the cosubstrate 20G in 20G-dependent oxygenases, and their use in the treatment of diseases which are associated with the 20G-dependent oxygenases (DIOs).
  • DIOs 20G-dependent oxygenases
  • the present invention provides compounds, the compounds being selected from the compounds of the formula (I) or formula (II)
  • Rl and R2 are oxygen (hydroxyl) or carboxyl groups, halogens, in particular fluorine, chlorine, or iodine, a single to multiple halogenated methyl group, in particular CH2F up to CF3; and
  • Cn represents a C atom, a hetero atom or the bridge to a heterocycle. which are characterized by the fact that they can take a reactive distance from the catalytic center and thus can form the corresponding transition states (TS) for the necessary function as cosubstrate.
  • TS transition states
  • Rl is hydrogen or a CH2R3 group, where R3 is hydrogen or oxygen (hydroxyl, carbonyl) or a shorter carbon chain (C1 to C4).
  • this can be part of a ring system, the ring size being between 3 to 5 atoms with at least one heteroatom.
  • C7 can consist of a carbon chain with up to 5 atoms and contain double bonds.
  • the compound may further comprise R2 as a carboxylic acid.
  • R2 can represent a hydrogen atom, a methyl group, an alkyl group with up to 6 carbon atoms, which may be branched saturated or unsaturated or even contain a hetero atom.
  • a bridge-forming cyclic structure is arranged between Rl and R2, with single or double bonds, and contains heteroatoms.
  • pharmaceutically acceptable salts and tautomers of the respective compound are used alone or in combination in previously defined mixing ratios.
  • Another object of the present invention is the use of one of the compounds described above as a medicament.
  • an object of the invention is the use of a compound as described above as a functional cosubstrate with other active ingredients in a medicament, wherein the other active ingredients can be selected, but are not limited to, from the group comprising chemotherapeutic agents, cytostatic agents (alkylating agents, Antimetabolites, topoisomerase inhibitors, mitotic inhibitors, antibiotics, antibodies, kinase inhibitors, proteosome inhibitors and supportive drugs for tumor therapy such as interferons, cytokines, tumor necrosis factor and IDH inhibitors)
  • chemotherapeutic agents cytostatic agents (alkylating agents, Antimetabolites, topoisomerase inhibitors, mitotic inhibitors, antibiotics, antibodies, kinase inhibitors, proteosome inhibitors and supportive drugs for tumor therapy such as interferons, cytokines, tumor necrosis factor and IDH inhibitors)
  • the invention further comprises the use of a compound as described above as a medicament for the prevention, treatment or aftercare of cancer, neurodegenerative diseases and of congenital or acquired metabolic disorders.
  • Another object of the present invention is the use of a compound of formula (I) or formula (II) which show the TS in the enzyme for the manufacture of a medicament for the prevention, treatment or aftercare of cancer, neurodegenerative diseases and congenital or acquired metabolic disorders.
  • the present invention also includes a medicament comprising a compound of formula (I) or formula (II).
  • FIG. 1 Binding relationships necessary for the function as cosubstrate
  • FIG. 2 TET2 enzyme, binding of the TET inhibitor NGA (N-oxalylglycine)
  • FIG. 3 4 binding ratios of 20G in the TET enzyme
  • FIG. 6 Functional replacement of the 20G cosubstrate by DKA
  • FIG. 7 Known therapies and the new pharmacological concept
  • FIG. 8 DKA toxicity
  • FIG. 9 Toxicity 20G
  • FIG. 10 Combination of IDH inhibitors with cosubstrates according to the
  • FIG. 11 Correlation of 2-hydroxyglutarate level with 5-hmdC levels in IDH1-MT
  • FIG. 12 Restoration of TET enzyme function by DKA in the presence
  • FIG. 13-18 Selected chemical compounds as a functional replacement
  • FIG. 19-27 Selected chemical compounds as a functional replacement for
  • FIG. 28 Restoration of functionality using DKA
  • FIG. 29 MTT cytotoxicity assays
  • FIG. 30, 31 apoptosis assays with 2,3-diketogulonic acid
  • FIG. 34 Western blot with HCT116 cells
  • the present invention relates to an alternative cosubstrate of ketoglutaric acid-dependent dioxygenases ([1] [6, 7] Tab. 1-2; Tab. 4) for their function production and regulation with the aim of therapeutic effects against cancer, neurodegenerative, and age-related diseases.
  • cosubstrate denotes low-molecular chemical compounds which are required in an enzymatic reaction in order to enable the actual substrate to be converted.
  • cosubstrate serve as a kind of "auxiliary molecules" that are reacted together with the substrate, but do not have their own catalytic effect.
  • the present invention relates to a new way of influencing the enzyme for disease therapies, the functional replacement of the native cosubstrate by one of our substances.
  • a regulation of the D10 by appropriate cosubstrate replacement (modulators) can optimize this, this goes so far that a targeted influence on epigenetic regulation, restoration and regulation of the cell metabolism and the associated further genetic regulation can be exerted.
  • DlO-dependent orphan diseases such as 2-hydroxyglutaric aciduria can also be treated.
  • the present invention provides compounds for the replacement and thus for the modulation or regulation of a-ketoglutaric acid (20G) -dependent oxygenases.
  • the compounds according to the invention can be used in the treatment of diseases which are dependent on the function (activity) of the a-ketoglutaric acid on dioxygenases (DlOs).
  • This Diseases include in particular cancer, Alzheimer's, Parkinson's disease, age-related diseases.
  • a variation of the compounds provided by the invention which are also referred to as cosubstrates in connection with the description of the invention, enables regulation and adaptation to the respective target structures in the case of an indication.
  • the invention provides the following compounds of the formula (I) and formula (II):
  • the compound of the formula (I) can be configured as follows:
  • R1 and R2 can be oxygen (hydroxyl) or carboxyl groups, halogens, in particular fluorine, choir, or iodine, a mono- to poly-halogenated methyl group, in particular CH2F up to CF3;
  • Rl can also simply be hydrogen or a CH 2 R 3 group, where R 3 is hydrogen or
  • Is oxygen hydroxyl, carbonyl
  • a shorter C chain (Ci to C 4 ).
  • the compound of formula (I) can be part of a ring system, the ring size containing 3 to 5 atoms with one or more heteroatoms.
  • C7 can consist of a carbon chain of up to 5 atoms and contain double bonds as shown in the following example:
  • This chain can also be integrated in a ring system as follows
  • Cn represents at least one carbon atom, a hetero atom or the bridge to a heterocycle which contains one or more heteronomes.
  • R2 represents a carboxylic acid
  • Cn can represent one, two or more C atoms as in 2-oxoadipic acid:
  • R2 and Cn can represent an unsaturated or saturated ring with or without heteroatoms.
  • the compound of formula (II) can be configured as follows:
  • R1 and R2 can be oxygen (hydroxyl) or carboxyl groups, halogens, in particular fluorine, choir, or iodine, a mono- to poly-halogenated methyl group, in particular CH2F up to CF3;
  • Rl can be simply bonded hydrogen or a CH 2 R 3 group, where R 3 can be hydrogen or oxygen (hydroxyl, carbonyl) a shorter C chain (Ci to C 4 ).
  • the compound of formula (II) can be part of a ring system, the ring size containing 3 to 5 atoms with one or more heteroatoms.
  • C7 can consist of a carbon chain of up to 5 atoms and contain double bonds, as shown by way of example:
  • This chain can also be integrated in a ring system:
  • Cn represents a carbon atom or a heteroatom.
  • R2 represents a carboxylic acid
  • Cn can represent one, two or more C atoms, such as in 2-oxoadipic acid (see above) or oxobutane dioate (see above).
  • Cn can be part or bridge to a heterocycle containing one or more heteronomes, e.g. in:
  • Rl can be a hydrogen atom, a methyl group or an alkyl group up to 5 carbon atoms which can be branched saturated or unsaturated or also contain heteroatoms.
  • R2 can also be a hydrogen atom, a methyl group, an alkyl group with up to 6 C atoms, which can be branched saturated or unsaturated or can also contain a hetero atom, as indicated in the following examples.
  • a bridge-forming cyclic structure can be arranged in formula (II), which can contain unsaturated or saturated or heteroatoms.
  • the cycle formed can be a 3-ring, a 4-ring, a 5-ring or a 6-ring.
  • the ring can contain one or more double bonds, as well as this alone or one or more heteroatoms of the same type or mixed, as indicated in the following examples.
  • the compounds can be used both as a substance, as a salt and in a buffered form.
  • the carboxylic acids can also be used esterified, the esterification also being possible with higher-chain alcohols (up to C12 atoms).
  • Preparations such as Creams, ointments, gels, nanoformulations, infusion solutions, tablets, capsules.
  • Vitamin C can act as a classic prodrug which can be metabolized in the body to form compounds according to formula (I) or according to formula (II) and to the structure according to formula (II) shown below. Esterification of the carboxyl group also leads to prodrugs with improved absorption and cell uptake. Further options for prodrug design are listed in [48].
  • prodrug and replacement cosubstrate are possible.
  • Administration can be oral, local, or by infusion.
  • CAS A large number of known structures (CAS) are available for the claimed structural elements, which, depending on the DIOs, can be available as modulators and thus influence the DIOs.
  • vitamin C and its derivatives such as 2-OaD-glucopyranosyl-l-ascorbic acid, which are used for 2,3-diketoglulonic acid (DKA) and also for 3,4,5-trihydroxy-2-oxopentanoic acid (III; 2KGL )
  • the D10s are reactivated and the competitive displacement of oncometabolites (hydroxyglutarate HG) from the active center is made possible. The function is restored and adjusted.
  • the compounds form a basis for the further development and modification of the basic formulas.
  • salts as used herein includes salts of the compound of general formulas (I) and (II), which are in relatively non-toxic (ie. Pharmaceutically acceptable) acids or bases Dependence on the particular substituents found are made on the compounds of the present invention.
  • base addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired base, either in pure form or in a suitable inert solvent.
  • Nonlimiting examples of pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amino or magnesium salt or a similar salt.
  • acid addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired acid, either in pure form or in a suitable inert solvent.
  • suitable inert solvent include those derived from inorganic acids such as hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, phosphoric acid, partially neutralized phosphoric acids, sulfuric acid, partially neutralized sulfuric, hydroiodic or phosphoric acid and the like as well as the salts, which are derived from relatively non-toxic organic acids such as acetic acid, propionic acid, isobutyric acid, maleic acid.
  • the starting form of the compound differs from the different salt forms in certain physical properties, such as that Solubility in polar solvents, however, the salts are otherwise equivalent to the starting form of the compound for the purposes of the present invention.
  • the compounds of the present invention can have chiral or asymmetrical carbon atoms (optical centers) and / or double bonds. The racemates, diastereomers, geometric isomers and individual optical isomers are encompassed by the present invention.
  • the compounds of the present invention can exist in unsolvated forms as well as in solvated forms, including hyperated forms. Generally, the solvated forms are equivalent to unsolvated forms and are also encompassed by the present invention.
  • the compounds of the present invention may further exist in multiple crystalline or amorphous forms.
  • the compounds of the present invention may also be in a so-called prodrug form.
  • Prodrugs of the compounds of the invention are those compounds that readily undergo chemical changes under physiological conditions to provide the compounds of the present invention.
  • prodrugs in the compounds of the present invention can be converted by chemical or biochemical methods in an ex vivo environment. For example, prodrugs can be slowly converted to the compounds of the present invention when placed, for example, in a transdermal patch reservoir with an appropriate enzyme or chemical reagent.
  • the compounds of the invention described herein can be administered in a suitable dose to mammals such as humans or pets.
  • pets are pigs, cows, buffalos, sheep, goats, rabbits, horses, donkeys, chickens, ducks, cats, dogs, real pigs or hamsters. Most preferably it is administered to humans.
  • the preferred mode of administration depends on the form of the compound of the invention (having the general formula (1)).
  • the compound represented by the general formula (1) can take the form of pharmaceutically acceptable salts, prodrugs, enantiomers, diastereomers, racemic mixtures, crystalline forms, non-crystalline forms, amorphous forms, unsolvated forms or solvates.
  • the compound of the invention can be administered orally, parenterally, such as subcutaneously, intraventrally, intramuscularly, intraperitoneally, intrathecally, intraocularly, transdermally, transmucosally, subdurally, locally or topically via iontophoresis, sublingually, by inhalation spray. Aerosol or rectally and the like in unit dosage formulations, which may further comprise conventional pharmaceutically acceptable excipients.
  • the compound of the invention for use in accordance with the present invention can be formulated as a pharmaceutical composition using one or more physiological carriers or excipients.
  • the pharmaceutical composition of the invention can take the form of tablets or capsules, for example, which are prepared in a conventional manner with pharmaceutically acceptable excipients such as binders (e.g. pregelatinized corn starch, polyvinylpyrrolidone, hydroxypropylmethyl cellulose), fillers ( e.g. lactose, microcrystalline cellulose, calcium hydrogen phosphate), lubricants (e.g. magnesium stearate, talc, silicon dioxide), disintegrants (e.g. pota starch, sodium starch glycolate) or wetting agents (e.g. sodium lauryl) sulfate).
  • binders e.g. pregelatinized corn starch, polyvinylpyrrolidone, hydroxypropylmethyl cellulose
  • fillers e.g. lactose, microcrystalline cellulose, calcium hydrogen phosphate
  • lubricants e.g. magnesium stearate, talc, silicon dioxide
  • disintegrants e.g. pota starch
  • the term “pharmaceutically acceptable” means that it is approved by a regulatory or other generally recognized pharmacopoeia for use in animals, and particularly in humans.
  • carrier refers to a diluent, adjuvant, excipient or vehicle with which the therapeutic agent is administered.
  • Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be used as liquid carriers, especially for injectable solutions.
  • Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium ion, dried skim milk, glycerin, propylene, glycol, water, ethanol and the like.
  • the composition can also contain small amounts of wetting or emulsifying agents or pH buffering agents.
  • These compositions may be in the form of ointments, solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations and the like. A preferred form is an ointment.
  • the composition can be formulated as a suppository with traditional binders and carriers such as triglycerides.
  • the oral formulation may contain standard carriers such as mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc. of pharmaceutical quality.
  • EW Martin describes examples of suitable pharmaceutical carriers in "Remington's Pharmaceutical Sciences”.
  • Such compositions contain a therapeutically effective amount of the above-mentioned compounds, preferably in purified form, together with an appropriate amount of carrier so as to provide the form for proper administration to the patient.
  • the formulation should correspond to the route of administration.
  • Liquid preparations for oral administration may, for example, be in the form of solutions, syrups or suspensions, or may be presented as a dry product for use with water or other suitable vehicle before use.
  • Such a liquid preparation can be hydrogenated in a conventional manner with pharmaceutically acceptable additives such as suspending agents (e.g. sorbitol, syrup, cellulose derivatives edible fetuses), emulsifiers (e.g. lecithin, acacia gum), non-aqueous vehicles (e.g. almond oil), oil, oily esters, ethyl alcohol, fractionated vegetable oils), preservatives (e.g. methyl or propyl p-hydroxycarbonates, soro acids) ).
  • the preparations can optionally also contain buffer salts, flavoring, coloring and sweetening agents.
  • Preparations for oral administration can be suitably formulated to provide controlled release of the pharmaceutical composition of the invention.
  • the pharmaceutical composition of the invention is conveniently delivered in the form of an aerosol spray presentation from a pressure pack or nebulizer using a suitable propellant (e.g. dichlorodifluoromethane, trichlorofluoromethane) B. dichlorotetrafluoroethane, carbon dioxide or other suitable Gas)
  • a suitable propellant e.g. dichlorodifluoromethane, trichlorofluoromethane
  • the dosage unit can be determined by providing a valve for dispensing a measured amount.
  • Capsules and cartridges of, for example, gelatin for use in an inhaler or insufflator can be formulated which contain a powder mixture of the pharmaceutical composition of the invention and a suitable powder base such as lactose or starch.
  • the pharmaceutical composition of the invention can be formulated for parenteral administration by injection, for example by bolus injection or continuous infusion.
  • the location of the injections is intravensal, intraperitoneal, or subcutaneous.
  • Formulations for injection can be presented in unit dosage form (e.g. in ampoules, in multiple dose containers) and with an additional preservative.
  • the pharmaceutical composition of the invention can take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles and can contain formulating agents such as suspending, stabilizing or dispersing agents.
  • the agent may be in powder form for constitution with a suitable vehicle (e.g., sterile pyrogen-free water) before use.
  • the compositions for intravenous administration are solutions in sterile isotonic aqueous buffers.
  • the composition may also include a solubilizing agent and a local anesthetic, such as lignocaine, to alleviate the pain at the injection site.
  • a solubilizing agent such as lignocaine
  • the ingredients are either separated or mixed together in unit dosage form, for example as a dry lyophilized powder or anhydrous concentrate in a hermetically sealed Container, such as an ampoule or pouch, that shows the amount of active agent. If the composition is to be administered by infusion, an infusion bottle containing sterile water or pharmaceutical grade saline can be omitted.
  • an ampoule When the composition is administered by injection, an ampoule can be provided with sterile water for injection or saline so that the ingredients can be mixed prior to administration.
  • sustained release dosage forms designed to release a drug at a predetermined rate to maintain a constant drug concentration for a drug over a period of time minimal side effects.
  • This can be achieved by a variety of formulations or devices, including microspheres, nanoparticles, liposomes and other polymer matrices such as drug-polymer conjugates such as hydrogels or biodegradable substances such as poly (lactic acid-co-glycolic acid) (PLGA), which are the active ingredient encapsulate.
  • PLGA poly (lactic acid-co-glycolic acid)
  • the pharmaceutical composition of the invention may also be provided in a package or dispenser, if desired, which may contain one or more unit dosage forms containing the agent.
  • the pack can include, for example, metal or plastic film, such as a blister pack.
  • the pack or dispenser may be accompanied with instructions for administration.
  • the pharmaceutical composition of the invention can be administered as the sole active ingredient or can be administered in combination with other active ingredients.
  • additional active agents should primarily be selected from agents that are related to the treatment of the same disease.
  • an additional active ingredient should be selected from the group of anti-obesity drugs.
  • anti-diabetic agents and also anti-NAFLD / NASH and anti-dyslipidemic drugs can be used as further active ingredients.
  • such an additional active ingredient should be selected from active ingredients that are associated with side effects such as body weight gain and antipsychotic treatments.
  • the compounds according to the invention or the compositions according to the invention can be used as a cosubstrate for the prevention, treatment and aftertreatment of tumor diseases.
  • the tumor disease is preferably a disease selected from the group comprising tumors of the ear, nose and throat region, including tumors of the inner nose, paranasal sinuses, nasopharynx, lips, oral cavity, oropharynx, larynx, hypopharynx, ear, salivary glands, and paragangliomas, lung tumors, comprising non-parvicellar bronchial carcinomas, parvicellular bronchial carcinomas, tumors of the mediastinum, tumors of the gastrointestinal tract, including tumors of the esophagus, stomach, pancreas, the Liver, gallbladder and biliary tract, small bowel, colon and intestinal carcinoma and anal carcinoma, urogenital tumors, including tumors of the kidneys, ureters, bladder, prostate gland, urethra, penis and
  • chemotherapeutic agents which can be selected from the group comprising antibodies, alkylating agents, platinum analogues, intercalation agents, antibiotics, mitosis suppressors, taxanes, topoisomerase suppressors, antimetabolites and / or L-asparaginase, hydroxycarbamide, mitotane and / or amanitine.
  • FIG. 1 shows the binding relationships in the enzyme which are necessary for the function as cosubstrate.
  • F1G Transistion state; TS) for the reaction in the enzyme of the cosubstrate or cosubstrate function replacement.
  • F1G also shows. 1 the environment in the active center of DlOs and the embedding and noncovalent fixation of the cosubstrate / cofactor complex 4 (cf. also facile 2-His-l-carboxylate triad [41] [42]).
  • a carbon atom with the same electronic properties 5 is also shown.
  • the compounds of the present invention according to formulas (1) and (11) can reach the target cell actively or passively via transporters (e.g. DKA, 2-OKG) or after corresponding derivatization.
  • transporters e.g. DKA, 2-OKG
  • R1 and R2 can be oxygen (hydroxyl) or carboxyl groups, halogens, in particular fluorine, choir, or iodine, a mono- to poly-halogenated methyl group, in particular CH2F up to CF3; and
  • Cn represents a C atom, a hetero atom or the bridge to a heterocycle.
  • the present invention is based on the discovery of compounds which restore the function of DIOs even in the presence of oncometabolites (HG) and thus far-reaching treatment options for diseases become available. So far, no compounds have been described that can mimic the physiological function of the 20G as a cosubstrate.
  • the present invention makes it possible to answer extensive and for the first time open questions about the DIOs and to convert them into new therapy options for DIO-dependent diseases.
  • the present invention is further based on docking experiments and molecular dynamic investigations with methods and algorithms according to Homann, which made it possible to show the actual conditions in the active site (binding or interaction site) of the D10 enzyme and by corresponding experiments in cell-free and cell systems to be able to prove.
  • the binding of the TET inhibitor NGA (N-oxalylglycine) is known from X-ray structure studies on the TET2 enzyme (cf. F1G. 2).
  • the binding ratios of 20G in the TET enzyme are hitherto unknown and were assumed to be analogous to FIG. 2.
  • FIG. 3 and Fig. 4 these binding relationships (in the TS) are shown for the first time. Based on FIGS. 1 and 2, it can be seen that the binding distances necessary for the enzyme reaction are observed.
  • the water is a necessary component in the enzyme complex and is also shown here.
  • FIG. 6 show the results of a cell-free assay for the function of DKA in the TET enzyme as a functional cofactor.
  • the investigations were carried out in a cell-free TET assay (in 50 mM HEPES buffer with 50mM NaCl 8mM Fe (NH4) 2 (SO i) 2 5mM ATP, 3mM DTT, 0.25pg TET enzyme and herring sperm DNA. After an 8 Constant incubation was carried out using mass spectrometry.
  • the native 20G was replaced by the functional coenzyme DKA) It was shown that DKA can take over the function of the native 20G and can serve as a replacement for the native cosubstrate.
  • Mutations in the genes encoding isocitrate dehydrogenase (IDH) require the reduction of 20G to the oncometabolites D-2-hydroxyglutarate (2HG), which leads to an inhibition of the demethylation of 5-methylcytosine (5mC) in the DNA 5-hydroxymethylcytosine (5hmC) by (TET), as well as methylated histone lysine (HK) residues (HKme) by Jumonji C domain demethylases (JMJC) and N6-methyladenosine (m6A) by FTO [44, 45]
  • IDH isocitrate dehydrogenase
  • 2HG is an oncometabolite that inhibits numerous demethylases, which leads to changes in genomic and transcriptomic methylation profiles as well as to changes in gene expression and genome topology [46]. Cancer and considering the total methylation of the genome aging processes.
  • the cornerstone of cancer therapy including the later success of epigenetic therapies, is the use of effective and rational drug combinations.
  • the focus is on the combination of epigenetically effective drugs (our 20G functional replacement cosubstrates could also be counted) with other therapies and the optimization of these.
  • the TET enzymes because of their central epigenetic role, represent a particular target for the replacement of the native 20G coenzyme.
  • HDAC histone deacetylases HDAC histone deacetylases
  • EZH2 Enhancer of zeste homolog 2 is a histone-lysine N-methyltransferase enzyme
  • DOT1L Disruptor of telomeric silencing l-like
  • BET Bromodomain and extra-terminal motif HDAC histone deacetylases
  • IDH 1 mutations are associated with an altered IDH 1 enzyme function, which induces the overproduction of neomorphic metabolite 2-hydroxyglutarate.
  • the 2-HG content was analyzed using LC-MS / MS analysis ( Figure 1). Intracellular 2-HG in IDH1R132H / + cells was 56 times higher than in IDH1 wild-type cells (8.5 nmoPmg protein or 0.15 nmol / mg protein; 56 times higher; p ⁇ 0.0001).
  • FIG. Figure 11 shows that 2-hydroxyglutarate levels correlate with 5-hmdC levels in IDH1-MT HCT116.
  • FIG. 12 shows that by using DKA alone as a functional cosubstrate, the function of the TET enzyme can be restored even in the presence of the competitive inhibitor 2HG.
  • FIG. 13-18 are other selected chemical compounds that can act as functional substitutes for cosubtrate 20G on TET and HIF in their conformation in the enzyme. Corresponding and cell experiments are listed below.
  • FIG. 13 shows DKA for the TET enzyme and in FIG. 13 to which atoms coordinate amino acids AS.
  • DKA is shown spatially and in FIG. 15 the spatial coordination of DKA in the enzyme.
  • the arrow in FIG. 15 indicates the distance of the Fe 2 + from the DKA with 2.2 ⁇ . It is shown that DKA is in the TS in the enzyme and that the specified conditions (FIG. 1) are also achieved here, so that a functional replacement of the cosubstrate 20G is made possible.
  • FIG. 16A shows 2-keto-gulonicacid (2-OKG) and shows how the 2-OKG in the TS lies in the enzyme and the specified conditions (FIG. 1) are also achieved here, so that a functional replacement of the cosubstrate 20G is made possible.
  • FIG. 16B the assay shows that 2-OGK has an effect depending on the dose.
  • FIG. 17 shows 4-methyl-5-oxohex-2-enedioic acid with Fe 2 + , indicating the amino acids that interact.
  • FIG. 18A shows AOF (2- (furan-2-yl) -2-oxoacetate with Fe 2 + and amino acids, the effect in FIG. 18B and the interaction between enzyme and AOF in FIG. 18C.
  • Figure 19-27 shows compounds for the HF enzyme.
  • the structure is shown and shown in FIG. 19B is in 2G19 is 2 - ((hydroxy (4-hydroxy-8-iodoisoquinolin-2-ium-3- yl) methylene) amino) acetates as an antagonist in hypoxia-induced factor (hypoxia-inducible factor prolyl hydroxylase, PHD2).
  • FIG. 20 is shown 20G in the enzyme PHD2 (according to the Homann method).
  • the position in the enzyme corresponds to the principle necessary for the function as a cosubstrate.
  • NGA is shown as an antagonist in the enzyme.
  • FIG. 22 shows the competitive antagonist 2 HG in the enzyme, which competitively interacts with 20G in the enzyme and leads to loss of function of the enzyme.
  • FIG. 23 shows 2-OKG in the enzyme as a functional coenzyme. The necessary principles for this function are observed.
  • FIG. 24 shows 3-bromo-2-oxopentanoate in the enzyme as a functional coenzyme.
  • FIG. 25 shows (E) -5-oxohex-2-enedioic acid in the enzyme as a functional coenzyme.
  • FIG. 26 shows 4- (S) -methyl-5-oxohex-2-enedioic acid in the enzyme as a functional coenzyme.
  • FIG. 27 shows DKA in the enzyme as a functional coenzyme.
  • TERT Tet Enzyme
  • FIG. 28 shows DKA - 2,3-diketogulonic acid from Examples 1 and 20G (2 KG - 2-keto-L-gulonic acid:
  • HCT116 IDH1 + / + and HCT116 IDH1R132H / + are from Horizon Discovery and were given to us for testing purposes.
  • the cells were in Dulbecco's Modified Eagle's Medium (DMEM) with 2 mM L-glutamine, supplemented with 10% fetal bovine serum (FBS), 45 lU / ml penicillin and 45 lU / ml streptomycin cultured.
  • DMEM Dulbecco's Modified Eagle's Medium
  • FBS fetal bovine serum
  • 45 lU / ml penicillin and 45 lU / ml streptomycin cultured were tested negative for mycoplasma infections within six months prior to use.
  • HCT1 16 cells were used in 96-well plates (TPP, Trasadingen, Switzerland). After 24 h, the indicated concentrations for 24, 48 and 72 h were added. The cells were then incubated with 100 pL MTT solution (0.5 mg / ml in PBS) for 4 h. After the supernatants were removed, 50 pL of dimethyl sulfoxide was added to dissolve the formazone salt and the optical density (OD) was measured with a microplate reader (Tecan, Crailsheim, Germany). The excitation was set to 540 nm. The positive controls were treated with 0.002% SDS. Cell viability ⁇ 75% predicts cytotoxic effects.
  • the level of apoptotic and dead cells was determined by flow cytometry using eBioscience TM Annexin V Apoptosis Detection Kit APC (Thermo Fisher, Darmstadt, Germany).
  • the cells were 2 x 105 HCT116 cells / wells in 6-well plates ( TPP, Trasadingen, Switzerland). After 24 hours, the cells were incubated with the substances in the stated concentrations for 72 hours. The cells were then washed and stained with Annexin V and propidium iodide according to the manufacturer's instructions. The cells were analyzed on a FACSCanto 11 (BD Biosciences, Heidelberg, Germany). The FlowJo software (Treestar, Ashland, USA) was used for the data analysis.
  • RNA was extracted according to the instructions of the RNA High Pure RNA Kit (Roche, Mannheim, Germany) and 0.5-5 pg (ideally 3 mg) of the RNA was extracted using the RevertAid Reverse Transcriptase (Thermo Fisher, Darmstadt, Germany) reverse transcribed according to the protocol.
  • the qRT-PCR was carried out with the Maxima SYBR Green qPCR Mix (ThermoFisher, Darmstadt, Germany) on a Lightcycler 480 II Real-Time PCR System (Roche, Mannheim, Germany). The quantification was performed using the DD Ct method and the GAPDH expression was used as an internal reference.
  • the melting curve analysis confirmed that all qRT-PCR products were generated in the form of double-stranded DNA.
  • the primers used are listed in Table 3.
  • Genomic DNA (20 pg) samples were hydrolyzed to 2'-deoxynucleosides with 2'-deoxynucleosides as described with micrococal nuclease from Staphylococcus aureus, bovine spleen phosphodiesterase and calf's intestinal alkaline phosphatase (all from Sigma-Aldrich, Taufkirchen, Germany), as described [62] with application modifications.
  • 10 pL of 50 nM 5-hmdC-d3 (Toronto Research Chemicals, Toronto, Canada) were added as an internal standard to the DNA digestion mixture and the incubation time of the two-stage hydrolysis was 1 hour each.
  • DNA hydrolyzates were then centrifuged (5 min, 16,000 x g) and 10 pL of the supernatants were used to quantify dC and 5-mdC stable labeled references.
  • the extraction of the protein pellets was repeated by adding a further 100 pL of methanol and centrifuging (1,400 rpm) for 5 min. After centrifugation at 16,000 xg for 10 min, both methanolic fractions were combined and evaporated to dryness under reduced pressure. The dried residues were reconstituted in 50 pL of water containing 0.0075% formic acid, which was ultrasonified for 10 min, followed by centrifugation for 5 min (1400 rpm) and centrifugation for 5 min at 16,000 x g.
  • the LC-MS / MS analyzes of the supernatants were carried out using an Agilent 1260 Infmity LC system in conjunction with an Agilent 6490 triple quadrupole mass spectrometer (both from Waldbronn, Germany) using an electrospray ion source in positive ion mode (ESI +) is connected. Chromatographic conditions and settings of the ESI source as described for the quantification of dC and 5-mdC [62]. An Agilent Poroshell 120 EC-C18 (2.7 pm, 3.0 x 150 mm) was used as the separation column, the injection volume was 5 pL.
  • FIG. 29 shows the results of an MTT cytotoxicity assay, which was carried out as follows: Incubation with DKA in increasing concentrations (100 mM-10 mM) for 24 h, 48 h and 72 h
  • FIG. 30 shows the results of an apoptosis assay performed as follows:
  • FIG. 31 shows the results of another apoptosis assay, which was carried out as follows:
  • FIG. 32 shows the results of hmdC measurements (various representations), which were carried out as follows:
  • FIG. 33 shows the results of hmdC measurements (various representations) which were carried out as follows:
  • FIG. 34 shows a Western blot with HCT116 cells, which was carried out as follows:
  • the examples showed that the claim of a cosubstrate replacement and modulation with an atoxic example substance such as DKG is successful.
  • the effects such as secondary apoptosis indicate a reconstruction of the cell metabolism.
  • TETs Due to the increased or changed energy requirements of tumor cells, they are no longer able to generate them from normal metabolic cycles and thus come into secondary apoptosis due to the treatment.
  • the production of activity of the TETs leads to demethylation of the cytosine and reversal of epigenetic changes associated with the regeneration of tumor suppressor genes.
  • Salminen, A., A. Kauppinen, and K. Kaarniranta 2-Oxoglutarate-dependent dioxygenases are sensors of energy metabolism, oxygen availability, and iron homeostasis: potential role in the regulation of aging process. Cellular and Molecular Life Sciences, 2015. 72 (20): p. 3897-3914.
  • PHD2 prolyl hydroxylase domain-containing protein 2
  • Rabe, P., et al. Roles of 2-oxoglutarate oxygenases and isopenicillin N synthase in 6-lactam biosynthesis. Natural Product Reports, 2018.
  • Vitamin C induces specific demethylation of H3K9me2 in mouse embryonic stem cells via Kdm3a / b. Epigenetics & Chromatin, 2017. 10 (1): p. 36th
  • hypoxia-inducible factor (HIF) -l inhibitors Recent advances in hypoxia-inducible factor (HIF) -l inhibitors.

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Abstract

The present invention relates to an alternative co-substrate of ketoglutaric acid-dependent dioxygenases for functional production and control of same, with the aim of achieving therapeutic effects against cancer, neurodegenerative diseases and age-related diseases. Epigenetically induced diseases caused by dysregulation and in particular also by metabolic dysfunction in the citric acid cycle are likewise targeted by this therapy.

Description

VERBINDUNGEN ZUR MODULATION UND ALS FUNKTIONELLER ERSATZ VON a- CONNECTIONS FOR MODULATION AND AS A FUNCTIONAL REPLACEMENT OF a-
KETOGLUTARSÄURE (20G)-ABHÄNGIGEN OXYGENASEN KETOGLUTARIC ACID (20G) DEPENDENT OXYGENASES
BESCHREIBUNG DESCRIPTION
Gebiet der Erfindung Field of the Invention
[0001] Die Erfindung betrifft Verbindungen als Funktionsäquivalente des Cosubstrats a- Ketoglutarsäure (synonym auch als 2-Oxoglutarat oder 20G bezeichnet) in 20G abhängigen Oxygenasen (DIOs) insbesondere zu deren Funktionsherstellung in Gegenwart von kompetitiven Hemmern der DIOs, wie beispielsweise 2-Hydroxyglutarsäure (2HG). The invention relates to compounds as functional equivalents of the cosubstrate a-ketoglutaric acid (also known synonymously as 2-oxoglutarate or 20G) in 20G-dependent oxygenases (DIOs), in particular for their functional production in the presence of competitive inhibitors of DIOs, such as 2-hydroxyglutaric acid ( 2HG).
Hintergrund der Erfindung Background of the Invention
[0002] a-Ketoglutarat- (2-Oxoglutarat-20G)-abhängige Dioxygenasen (DIOs auch als 20G- Oxygenasen bezeichnet) sind sauerstoffabhängige Enzyme die a-Ketoglutarsäure (2-oxoglutarate 20G, 2KG) als Co-Substrate und Nicht-Häm Fe(II) als Co-Faktor nutzen. Sie katalysieren eine Vielzahl von Oxidationsreaktionen. Dazu gehören (nicht ausschließlich) Hydroxylierungsreaktionen, Demethylierungen, Ringexpansionen, Ringschlüsse und Einführung von Doppelbindungen. [1] [2, 3] A-Ketoglutarate (2-oxoglutarate-20G) -dependent dioxygenases (DIOs also known as 20G-oxygenases) are oxygen-dependent enzymes which contain a-ketoglutaric acid (2-oxoglutarate 20G, 2KG) as co-substrates and non-heme Fe (II) use as a co-factor. They catalyze a variety of oxidation reactions. These include (not exclusively) hydroxylation reactions, demethylations, ring expansions, ring closings and the introduction of double bonds. [1] [2, 3]
[0003] 20G-abhängige Dioxygenasen sind an vielen biologischen Prozessen und Funktionen beteiligt. [0003] 20G dependent dioxygenases are involved in many biological processes and functions.
[4, 5] In Mikroorganismen wie Bakterien sind 20G-abhängige Dioxygenasen an grundlegenden Funktionen bei der Biosynthese beteiligt [6-8] In Pflanzen sind 20G-abhängige Dioxygenasen an vielen verschiedenen Reaktionen des Pflanzenstoffwechsels beteiligt [9] Dazu gehören (jedoch nicht ausschließlich) die Flavonoidbiosynthese und Ethylenbiosynthesen [10] Bei Säugetieren und Menschen haben 20G-abhängige Dioxygenase funktionelle Rollen in Biosynthesen (z.B. Kollagenbiosynthese[l 1] und L-Camitinbiosynthese[l2]), posttranslationale Modifikationen (z.B. Proteinhydroxylierung[l3]), epigenetische Regulationen (z.B. Histon und DNA-Demethylierung[l4]) sowie Sensoren des Energiestoffwechsels. [15] . [4, 5] In microorganisms such as bacteria, 20G-dependent dioxygenases are involved in basic functions in biosynthesis [6-8] In plants, 20G-dependent dioxygenases are involved in many different reactions in plant metabolism [9] These include (but not exclusively) flavonoid biosynthesis and ethylene biosynthesis [10] In mammals and humans, 20G-dependent dioxygenase have functional roles in biosynthesis (e.g. collagen biosynthesis [l 1] and L-camitine biosynthesis [l2]), post-translational modifications (e.g. protein hydroxylation [l3]), epigenetic regulations (e.g. Histone and DNA demethylation [l4]) as well as sensors of energy metabolism. [15].
[0004] 20G-abhängige Dioxygenasen katalysieren Oxidationsreaktionen, indem sie ein einzelnes Sauerstoffatom in ihre Substrate einbauen. Dies ist immer verbunden mit der Oxidation des Cosubstrats 20G zu Succinat und Kohlendioxid [16] . [0004] 20G-dependent dioxygenases catalyze oxidation reactions by incorporating a single oxygen atom into their substrates. This is always associated with the oxidation of cosubstrate 20G to succinate and carbon dioxide [16].
[0005] Die katalytische Aktivität vieler 20G-abhängiger Dioxygenasen soll abhängig sein von Reduktionsmitteln, die in der Lage sind den Cofaktor Eisen in seiner zweiwertigen Form zu halten bzw. zu dieser Oxidationsstufe zu reduzieren [1] [17-20] Dieser Mechanismus wird auch für Vitamin C (Ascorbat) diskutiert. Jedoch nicht alle Funktionen können darüber erklärt werden [21] The catalytic activity of many 20G-dependent dioxygenases is said to depend on reducing agents which are able to keep the cofactor iron in its divalent form or to reduce to this oxidation level [1] [17-20] This mechanism is also discussed for vitamin C (ascorbate). However, not all functions can be explained [21]
[0006] 20G-abhängige Dioxygenasen sind durch einen gemeinsamen katalytischen Mechanismus gekennzeichnet. Im ersten Schritt erfolgt die Bindung von 20G und Substrat in die aktive Bindungsstelle [22-24] 20G ist direkt koordiniert mit Eisen (II) (Ni II; Mn II) im Aktivitätszentrum, während das Substrat in unmittelbarer Nähe, aber nicht direkt koordinierend, am Metall bindet. Der zweite Schritt ist die Bindung von molekularem Sauerstoff, der eine dritte Stelle am Fe(II) (Nill; MnII)-Zentrum einnimmt. Diese ermöglichen eine oxidative Decarboxylierungsreaktion unter Bildung von Succinat, Kohlendioxid und einem reaktiven Metall(IV)-oxo-Zwischenprodukt, das anschließend das Substrat oxidiert [25-31] Der Ersatz und die Rolle von Eisen war Gegenstand von Untersuchungen [59] [0006] 20G-dependent dioxygenases are characterized by a common catalytic mechanism. In the first step, 20G and substrate bind to the active binding site [22-24] 20G is directly coordinated with iron (II) (Ni II; Mn II) in the activity center, while the substrate is in close proximity, but not directly coordinating, binds to the metal. The second step is the binding of molecular oxygen, which occupies a third position at the Fe (II) (Nill; MnII) center. These enable an oxidative decarboxylation reaction with the formation of succinate, carbon dioxide and a reactive metal (IV) oxo intermediate, which subsequently oxidizes the substrate [25-31] The replacement and role of iron has been the subject of studies [59]
[0007] Ein alternativer Mechanismus wurde 2004 für eine bakterielle 20G-abhängige Dioxygenase, Deacetoxycephalosporin-C-Synthase (DAOCS), diskutiert. Der vorgeschlagene "Ping-Pong"- Mechanismus unterscheidet sich vom Konsensmechanismus (s.o.) dadurch, dass 20G und Sauerstoff zuerst an das Fe(II) -Zentrum in der Enzym- Aktivstelle in Abwesenheit des Substrats gebunden werden. Die entkoppelte Oxidation von 20G erfolgt dann zur Erzeugung einer reaktiven Fe(IV)-Oxo-Spezies, gefolgt von der Freisetzung von Succinat und Kohlendioxid und der Bindung des Substrats, welches oxidiert wird. Spätere Studien im Jahr 2014 zeigten jedoch, dass auch die DAOCS mit hoher Wahrscheinlichkeit dem allgemeinen Konsensmechanismus der 20G-abhängigen Oxygenasen folgt An alternative mechanism was discussed in 2004 for a bacterial 20G-dependent dioxygenase, deacetoxycephalosporin C synthase (DAOCS). The proposed "ping-pong" mechanism differs from the consensus mechanism (see above) in that 20G and oxygen are first bound to the Fe (II) center in the enzyme active site in the absence of the substrate. The decoupled oxidation of 20G then takes place to produce a reactive Fe (IV) -oxo species, followed by the release of succinate and carbon dioxide and the binding of the substrate, which is oxidized. However, later studies in 2014 showed that DAOCS is also very likely to follow the general consensus mechanism of 20G-dependent oxygenases
[32] [32]
[0008] Alle 20G-abhängigen Dioxygenasen enthalten eine konservierte doppelsträngige ß-Helix (DSBH), die mit zwei ß-Blättem eine Spalte bildet [33, 34] Die aktive Seite enthält ein hochkonserviertes 2-His-l-Carboxylat (HXD/E...H)-Aminosäureresttriad-Motiv, in dem das katalytisch essentielle Metall von zwei Histidinresten und einem Asparaginsäure- oder Glutaminsäurerest fixiert wird [35] Jedoch unterscheiden sie sich in der Aminosäureanordnung im aktiven Zentrum. All 20G-dependent dioxygenases contain a conserved double-stranded ß-helix (DSBH), which forms a column with two ß-leaves [33, 34] The active side contains a highly conserved 2-His-l-carboxylate (HXD / E ... H) amino acid residue triad motif in which the catalytically essential metal is fixed by two histidine residues and an aspartic acid or glutamic acid residue [35] However, they differ in the amino acid arrangement in the active center.
[0009] Erkenntnisse aus der Röntgenkristallographie, von Molekulardynamikberechnung (MD) und der NMR-Spektroskopie zeigen das einige 20G-abhängige Dioxygenasen ihr Substrat über einen induzierten Anpassungsmechanismus binden. Beispielsweise wurden Proteinstrukturveränderungen bei der Substratbindung für die humane Prolyl-Hydroxylase Isoform 2 (PHD2) [36, 37], [38] eine 20G- abhängige Dioxygenase, die an der Sauerstoffhomöostase beteiligt ist [39] und die Isopenicillin-N- Synthase (IPNS), eine mikrobielle 20G-abhängige Dioxygenase, beobachtet [40] [0010] Bei vielen Erkrankungen ist die Aktivität von DIOs verändert, häufig auch vermindert. Angesichts der wichtigen biologischen Rolle, die die 20G-abhängige Dioxygenase spielt, sind sie ein wichtiges Target für die Therapie von Krankheiten. Findings from X-ray crystallography, molecular dynamics (MD) and NMR spectroscopy show that some 20G-dependent dioxygenases bind their substrate via an induced adaptation mechanism. For example, protein structure changes in substrate binding for the human prolyl hydroxylase isoform 2 (PHD2) [36, 37], [38] were a 20G-dependent dioxygenase that is involved in oxygen homeostasis [39] and isopenicillin-N synthase (IPNS ), a microbial 20G-dependent dioxygenase, observed [40] In many diseases, the activity of DIOs is changed, often also reduced. Given the important biological role played by the 20G-dependent dioxygenase, they are an important target for the treatment of diseases.
[0011] Der Gesichtspunkt des Einsatzes kleiner Moleküle als fünktioneller Ersatz von 20G als Cosubstrat war bisher in dieser Funktion unbekannt und ist ein völlig neuer pharmakologischer Ansatz in der Einflussnahme auf DIOs zur Behandlung von Krankheiten. Somit werden Krankheitsbilder die durch Mangel an 2-OG bzw. durch kompetitive Hemmung durch Oncometaboliten (z.B. 2HG) im Enzym entstehen, therapierbar. The aspect of the use of small molecules as a functional replacement of 20G as a cosubstrate was previously unknown in this function and is a completely new pharmacological approach in influencing DIOs for the treatment of diseases. This makes it possible to treat diseases caused by a lack of 2-OG or by competitive inhibition by oncometabolites (e.g. 2HG) in the enzyme.
[0012] a-Ketoglutarsäure (20G)-abhängige Oxygenasen (DIOs) katalysieren eine bemerkenswert breite Palette von oxidativen Reaktionen. Bei Menschen und Tieren sind dieses Hydroxylierungen und N-Demethylierungen, die über Hydroxylierungsreaktionen ablaufen; bei Pflanzen und Mikroben katalysieren sie Reaktionen wie Ringformationen, Umlagerungen, Desaturationen und Halogenierungen. [0012] a-Ketoglutaric acid (20G) -dependent oxygenases (DIOs) catalyze a remarkably wide range of oxidative reactions. In humans and animals, these are hydroxylations and N-demethylations, which take place via hydroxylation reactions; in plants and microbes they catalyze reactions such as ring formations, rearrangements, desaturations and halogenations.
[0013] In Ihrer biologischen Funktion spiegelt sich die katalytische Flexibilität der DIOs wieder. Nachdem die Rolle von DIOs in der Kollagenbiosynthese identifiziert wurde, konnte gezeigt werden, dass sie auch bei der Entwicklung von Pflanzen und Tieren, der Transkriptionsregulation, der Modifikation/Reparatur von Nukleinsäuren (DNS, RNS), dem Fettsäurestoffwechsel der Bildung und Stabilisierung von Stammzellen (PS iPS) und der Biosynthese von Sekundärmetaboliten, einschließlich medizinisch wichtiger Antibiotika, eine Rolle spielen. [0013] The biological function of the DIOs reflects the catalytic flexibility. After the role of DIOs in collagen biosynthesis was identified, it was shown that they also play a role in the development of plants and animals, the regulation of transcription, the modification / repair of nucleic acids (DNA, RNA), the fatty acid metabolism in the formation and stabilization of stem cells PS iPS) and the biosynthesis of secondary metabolites, including medically important antibiotics, play a role.
Aufgabe der Erfindung Object of the invention
[0014] Die vorliegende Erfindung soll Verbindungen zur Funktionsübemahme des Cosubstrates 20G in 20G-abhängigen Oxygenasen zur Verfügung stellen, sowie deren Verwendung bei der Behandlung von Erkrankungen die im Zusammenhang mit den 20G- abhängigen Oxygenasen stehen (DIOs).  The present invention is intended to provide compounds for performing the function of the cosubstrate 20G in 20G-dependent oxygenases, and their use in the treatment of diseases which are associated with the 20G-dependent oxygenases (DIOs).
Zusammenfassung der Erfindung Summary of the invention
[0015] Die vorliegende Erfindung stellt Verbindungen zur Verfügung, wobei die Verbindungen ausgewählt sind aus den Verbindungen gemäß Formel (I) oder Formel (II) The present invention provides compounds, the compounds being selected from the compounds of the formula (I) or formula (II)
wobei  in which
As die Aminosäuren aus der Bindungstasche des Enzyms darstellen;  As represent the amino acids from the binding pocket of the enzyme;
Me ein Metall aus dem katalytischen Zentrum;  Me a metal from the catalytic center;
Rl und R2 Sauerstoff (Hydroxyl) oder Carboxylgruppen, Halogene, insbesondere Fluor, Chor, oder Iod, eine einfach bis mehrfach halogenierte Methylgruppe, insbesondere CH2F bis zu CF3 sind; und  Rl and R2 are oxygen (hydroxyl) or carboxyl groups, halogens, in particular fluorine, chlorine, or iodine, a single to multiple halogenated methyl group, in particular CH2F up to CF3; and
Cn ein C-Atom, ein Heteroatom oder die Brücke zu einen Heterocyclus repräsentiert. die dadurch charakterisiert sind, dass sie einen reaktiven Abstand zum katalytischen Zentrum einnehmen können und dadurch für die notwendige Funktion als Cosubstrat die entsprechenden Übergangszustände (TS) bilden können.  Cn represents a C atom, a hetero atom or the bridge to a heterocycle. which are characterized by the fact that they can take a reactive distance from the catalytic center and thus can form the corresponding transition states (TS) for the necessary function as cosubstrate.
[0016] Weiterhin ist bei der Verbindung vorgesehen, bei der Rl Wasserstoff oder eine CH2R3 -Gruppe ist, wobei R3 Wasserstoff oder Sauerstoff (Hydroxyl, Carbonyl) oder eine kürzere C-Kette (Cl bis C4) ist. It is also provided in the compound in which Rl is hydrogen or a CH2R3 group, where R3 is hydrogen or oxygen (hydroxyl, carbonyl) or a shorter carbon chain (C1 to C4).
[0017] In einem weiteren Aspekt der Verbindung kann dieser Teil eines Ringsystems sein, wobei die Ringgröße zwischen 3 bis 5 Atomen liegt mit zumindest einem Heteroatom. [0018] In einer weiteren Ausführungsform der Verbindung kann C7 aus einer Kohlenstoffkette mit bis zu 5 Atomen bestehen und Doppelbindungen enthalten. In a further aspect of the compound, this can be part of a ring system, the ring size being between 3 to 5 atoms with at least one heteroatom. In a further embodiment of the compound, C7 can consist of a carbon chain with up to 5 atoms and contain double bonds.
[0019] Die Verbindung kann weiterhin R2 als Carbonsäure umfassen. The compound may further comprise R2 as a carboxylic acid.
[0020] Für eine Verbindung nach Formel (11) kann R2 ein Wasserstoffatom, eine Methylgruppe, eine Alkylgruppe mit bis zu 6 C-Atomen repräsentieren, die verzweigt gesättigt oder ungesättigt sein können oder selbst auch ein Heteroatom enthalten. For a compound of formula (11) R2 can represent a hydrogen atom, a methyl group, an alkyl group with up to 6 carbon atoms, which may be branched saturated or unsaturated or even contain a hetero atom.
[0021] Weiterhin ist für eine Verbindung nach Formel (11) vorgesehen, dass zwischen Rl und R2 eine brückenbildende zyklische Struktur angeordnet ist, mit Einfach- oder Doppelbindungen, und Heteroatome enthält. Furthermore, it is provided for a compound of formula (11) that a bridge-forming cyclic structure is arranged between Rl and R2, with single or double bonds, and contains heteroatoms.
[0022] Erfindungsgemäß ist auch Mischung der Verbindungen nach Formel (I) und Formel (II) vorgesehen. According to the invention, a mixture of the compounds of the formula (I) and formula (II) is also provided.
[0023] In einem weiteren Aspekt werden pharmazeutisch akzeptable Salze und Tautomere der jeweiligen Verbindung allein oder in Kombination in zuvor definierten Mischungsverhältnissen verwendet. In a further aspect, pharmaceutically acceptable salts and tautomers of the respective compound are used alone or in combination in previously defined mixing ratios.
[0024] Ein weiteres Objekt der vorliegenden Erfindung ist die Verwendung einer der zuvor beschrieben Verbindungen als Medikament. Another object of the present invention is the use of one of the compounds described above as a medicament.
[0025] Weiterhin ist ein Objekt der Erfindung die Verwendung einer Verbindung wie zuvor beschrieben als fünktionelles Cosubstrat mit anderen Wirkstoffen in einem Medikament, wobei die anderen Wirkstoffe ausgewählt sein können, aber nicht darauf beschränkt sind, aus der Gruppe umfassen Chemotherapeutika, Zytostatika (Alkylantien, Antimetabolite, Topoisomerase Hemmer, Mitosehemmstoffe, Antibiotika, Antikörper, Kinaseinhibitoren, Proteosominhibitoren und supportive Arzneistoffe der Tumortherapie wie Interferone, Zytokine, Tumornekrosefaktor und IDH-Hemmem) Furthermore, an object of the invention is the use of a compound as described above as a functional cosubstrate with other active ingredients in a medicament, wherein the other active ingredients can be selected, but are not limited to, from the group comprising chemotherapeutic agents, cytostatic agents (alkylating agents, Antimetabolites, topoisomerase inhibitors, mitotic inhibitors, antibiotics, antibodies, kinase inhibitors, proteosome inhibitors and supportive drugs for tumor therapy such as interferons, cytokines, tumor necrosis factor and IDH inhibitors)
[0026] Die Erfindung umfasst weiterhin eine Verwendung einer Verbindung, wie zuvor beschrieben, als Medikament zur Prävention, Behandlung oder Nachsorge von Krebserkrankungen, Neurodegenerativen Erkrankungen sowie von angeborenen oder erworbenen metabolischen Störungen. The invention further comprises the use of a compound as described above as a medicament for the prevention, treatment or aftercare of cancer, neurodegenerative diseases and of congenital or acquired metabolic disorders.
[0027] Ein weiterer Gegenstand der vorliegenden Erfindung ist die Verwendung einer Verbindung nach Formel (I) oder Formel (II) die den TS im Enzym zeigen zur Herstellung eines Medikaments für die Prävention, Behandlung oder Nachsorge von Krebserkrankungen, Neurodegenerativen Erkrankungen sowie von angeborenen oder erworbenen metabolischen Störungen. [0028] Letztlich umfasst die vorliegende Erfindung auch ein Medikament umfassend eine Verbindung nach Formel (I) oder Formel (II). Another object of the present invention is the use of a compound of formula (I) or formula (II) which show the TS in the enzyme for the manufacture of a medicament for the prevention, treatment or aftercare of cancer, neurodegenerative diseases and congenital or acquired metabolic disorders. Ultimately, the present invention also includes a medicament comprising a compound of formula (I) or formula (II).
Zusammenfassung der Figuren Summary of the figures
[0029] Die vorliegende Erfindung wird anhand von Figuren und Ausführungsbeispielen beschrieben und dargestellt. Für den zuständigen Fachmann ist es offensichtlich, dass die Erfindung nicht auf den Inhalt der Figuren und Ausführungsbeispiele beschränkt ist. Es zeigt: The present invention is described and illustrated with reference to figures and exemplary embodiments. It is obvious to the person skilled in the art that the invention is not restricted to the content of the figures and exemplary embodiments. It shows:
FIG. 1 Für die Funktion als Cosubstrat notwendigen Bindungsverhältnisse FIG. 1 Binding relationships necessary for the function as cosubstrate
im Enzym  in the enzyme
FIG. 2 TET2 Enzyms, Bindung des TET-Hemmers NGA (N-Oxalylglycin)  FIG. 2 TET2 enzyme, binding of the TET inhibitor NGA (N-oxalylglycine)
FIG. 3, 4 Bindungsverhältnisse von 20G im TET Enzym FIG. 3, 4 binding ratios of 20G in the TET enzyme
FIG. 5A, 5B Funktionsverlust TET Enzym durch Kompetition 2HG und 20G FIG. 5A, 5B loss of function of TET enzyme by competition 2HG and 20G
FIG. 6 Funktionelle Ersatz des Cosubstrates 20G durch DKA FIG. 6 Functional replacement of the 20G cosubstrate by DKA
FIG. 7 Bekannte Therapien sowie das neue pharmakologisches Konzept FIG. 7 Known therapies and the new pharmacological concept
gemäß der vorliegenden Erfindung  according to the present invention
FIG. 8 Toxizität DKA  FIG. 8 DKA toxicity
FIG. 9 Toxizität 20G FIG. 9 Toxicity 20G
FIG. 10 Kombination von IDH Hemmern mit Cosubstraten gemäß der FIG. 10 Combination of IDH inhibitors with cosubstrates according to the
vorliegenden Erfindung  present invention
FIG. 11 Korrelation 2-Hydroxyglutarat Level mit 5-hmdC levels in IDH1-MT  FIG. 11 Correlation of 2-hydroxyglutarate level with 5-hmdC levels in IDH1-MT
HCT116  HCT116
FIG. 12 Wiederherstellung Funktion TET Enzymes durch DKA in Gegenwart  FIG. 12 Restoration of TET enzyme function by DKA in the presence
des kompetitiven Inhibitors 2HG  of the competitive inhibitor 2HG
FIG. 13-18 Ausgewählte chemische Verbindungen die als fünktioneller Ersatz  FIG. 13-18 Selected chemical compounds as a functional replacement
des Cosubtrates 20G an TET und HIF dienen können  of the 20G co-substrate can serve at TET and HIF
FIG. 19-27 Ausgewählte chemische Verbindungen als fünktioneller Ersatz für  FIG. 19-27 Selected chemical compounds as a functional replacement for
das HF Enzym  the HF enzyme
FIG. 28 Wiederherstellung der Funktionalität mittels DKA  FIG. 28 Restoration of functionality using DKA
FIG. 29 MTT Cytotoxizitätsassays FIG. 29 MTT cytotoxicity assays
FIG. 30, 31 Apoptose Assays mit 2,3 -Diketogulonic acid FIG. 30, 31 apoptosis assays with 2,3-diketogulonic acid
FIG. 32, 33 hmdC-Messungen mit 2,3 -Diketogulonic acid FIG. 32, 33 hmdC measurements with 2,3-diketogulonic acid
FIG. 34 Western-Blot mit HCT116 Zellen FIG. 34 Western blot with HCT116 cells
Ausführliche Beschreibung der Erfindung [0030] Die vorliegende Erfindung betrifft ein alternatives Cosubstrate an Ketoglutarsäure abhängigen Dioxygenasen ([1] [6, 7] Tab.1-2; Tab. 4) zu deren Funktionsherstellung und Reglung mit dem Ziel therapeutische Effekte gegen Krebs-, Neurodegenerative-, und altersbedingten Erkrankungen zu erzielen. Epigenetisch bedingte Erkrankungen bedingt durch Dysregulation insbesondere auch durch metabolische Entgleisungen im Zitronensäurezyklus sind ebenfalls im Fokus dieser Therapie. Detailed description of the invention The present invention relates to an alternative cosubstrate of ketoglutaric acid-dependent dioxygenases ([1] [6, 7] Tab. 1-2; Tab. 4) for their function production and regulation with the aim of therapeutic effects against cancer, neurodegenerative, and age-related diseases. Epigenetic diseases caused by dysregulation, in particular also by metabolic derailments in the citric acid cycle, are also the focus of this therapy.
[0031] Der Begriff Cosubstrat bezeichnet in der vorliegenden Beschreibung der Erfindung niedermolekulare chemische Verbindungen, die bei einer enzymatischen Reaktion benötigt werden, um die Umsetzung des eigentlichen Substrats zu ermöglichen. Damit dienen Cosubstrat als eine Art "Hilfsmoleküle", die zusammen mit dem Substrat umgesetzt werden, und besitzen aber keine eigene katalytische Wirkung. In the present description of the invention, the term cosubstrate denotes low-molecular chemical compounds which are required in an enzymatic reaction in order to enable the actual substrate to be converted. Thus cosubstrate serve as a kind of "auxiliary molecules" that are reacted together with the substrate, but do not have their own catalytic effect.
[0032] Allgemein werden in Therapien von Erkrankungen Wirkstoffrezeptoren durch Hemmung der Zielstrukturen (kompetitiv, nichtkompetitiv, allosterisch, kovalent) beeinflusst. Auch wird bei den DIOs dieses Ziel verfolgt wie durch z.B. Hemmung der IDHs oder a-HIF mit teilweisen widersprüchlichen Ergebnissen und toxischen Events [8-13] Die vorliegende Erfindung betrifft einen neuen Weg der Enzymbeinflussung für Krankheitstherapien, den funktionellen Ersatz des nativen Cosubstrates durch eine unserer Substanzen. In general, in therapies for diseases, drug receptors are influenced by inhibiting the target structures (competitive, non-competitive, allosteric, covalent). This goal is also pursued at the DIOs, e.g. by Inhibition of IDHs or a-HIF with partially conflicting results and toxic events [8-13] The present invention relates to a new way of influencing the enzyme for disease therapies, the functional replacement of the native cosubstrate by one of our substances.
[0033] Eine Regulierung der DlOs durch entsprechenden Cosubstratersatz (Modulatoren) kann dieses optimieren, wobei dieses so weit geht, dass gezielt Einfluss auf epigenetische Regulierungen, Wiederherstellung und Regulierung des Zellstoffwechsels und der damit verbundenen weiteren genetischen Regulierungen genommen werden kann. A regulation of the D10 by appropriate cosubstrate replacement (modulators) can optimize this, this goes so far that a targeted influence on epigenetic regulation, restoration and regulation of the cell metabolism and the associated further genetic regulation can be exerted.
[0034] Weiterhin behandelbar sind DlO abhängige Orphan Erkrankungen wie 2-Hydroxy- Glutarazidurie. DlO-dependent orphan diseases such as 2-hydroxyglutaric aciduria can also be treated.
[0035] Eine Nachbehandlung von konventionell behandelten Tumoren stellt ebenfalls eine Anwendungsoption dar. Post-treatment of conventionally treated tumors is also an application option.
[0036] Die vorliegende Erfindung stellt Verbindungen zum Ersatz und damit zur Modulation oder Regulierung von a-Ketoglutarsäure (20G)-abhängige Oxygenasen zur Verfügung. Die erfindungsgemäßen Verbindungen können in der Behandlung von Erkrankungen verwendet werden, die abhängig sind von der Funktion (Aktivität) der a-Ketoglutarsäure an Dioxygenasen (DlOs). Diese Krankheiten umfassen insbesondere Krebs, Alzheimer, Morbus Parkinson, alters bedingte Erkrankungen. The present invention provides compounds for the replacement and thus for the modulation or regulation of a-ketoglutaric acid (20G) -dependent oxygenases. The compounds according to the invention can be used in the treatment of diseases which are dependent on the function (activity) of the a-ketoglutaric acid on dioxygenases (DlOs). This Diseases include in particular cancer, Alzheimer's, Parkinson's disease, age-related diseases.
[0037] Eine Variation der durch die Erfindung zur Verfügung gestellten Verbindungen, welche im Zusammenhang mit der Beschreibung der Erfindung auch als Cosubstrate bezeichnet werden, ermöglicht eine Regulierung und Anpassung an die jeweiligen Zielstrukturen bei einer Indikation. A variation of the compounds provided by the invention, which are also referred to as cosubstrates in connection with the description of the invention, enables regulation and adaptation to the respective target structures in the case of an indication.
[0038] Die Erfindung stellt die folgenden Verbindungen der Formel (I) und Formel (II) zur Verfügung: Die Verbindung gemäß Formel (I) kann wie folgt ausgestaltet sein: The invention provides the following compounds of the formula (I) and formula (II): The compound of the formula (I) can be configured as follows:
wobei in which
As die Aminosäuren aus der Bindungstasche des Enzyms darstellen sowie Me ein Metall aus dem katalytischen Zentrum; As represents the amino acids from the binding pocket of the enzyme and Me represents a metal from the catalytic center;
Rl und R2 können Sauerstoff (Hydroxyl) oder Carboxylgruppen, Halogene, insbesondere Fluor, Chor, oder Iod ,eine einfach bis mehrfach halogenierte Methylgruppe, insbesondere CH2F bis zu CF3 sein; R1 and R2 can be oxygen (hydroxyl) or carboxyl groups, halogens, in particular fluorine, choir, or iodine, a mono- to poly-halogenated methyl group, in particular CH2F up to CF3;
Rl kann auch einfach Wasserstoff oder eine CH2R3 -Gruppe sein, wobei R3 Wasserstoff oder Rl can also simply be hydrogen or a CH 2 R 3 group, where R 3 is hydrogen or
Sauerstoff (Hydroxyl, Carbonyl) oder eine kürzere C-Kette (Ci bis C4 ) ist. Is oxygen (hydroxyl, carbonyl) or a shorter C chain (Ci to C 4 ).
[0039] Ein Beispiel einer erfindungsgemäßen Verbindung ist im Folgenden gezeigt: An example of a compound according to the invention is shown below:
[0040] Die Verbindung gemäß Formel (I) kann Teil eines Ringsystems sein, wobei die Ringgröße 3 bis 5 Atome mit einem oder mehreren Heteroatomen enthält. C7 kann aus einer Kohlenstoffkette bis 5 Atomen bestehen und Doppelbindungen wie im folgenden Beispiel gezeigt enthalten: The compound of formula (I) can be part of a ring system, the ring size containing 3 to 5 atoms with one or more heteroatoms. C7 can consist of a carbon chain of up to 5 atoms and contain double bonds as shown in the following example:
2,3-Diketogulonic acid DKA 2-Ketogulonic acid 2-OKG (2KG)  2,3-diketogulonic acid DKA 2-ketogulonic acid 2-OKG (2KG)
[0041] Diese Kette kann auch wie folgt in einem Ringsystem eingebunden sein [0041] This chain can also be integrated in a ring system as follows
[0042] Cn stellt in Formel (I) zumindest ein C-Atom, ein Heteroatom oder die Brücke zu einen Heterocyclus dar, der ein oder mehrere Heteronome enthält.  In formula (I), Cn represents at least one carbon atom, a hetero atom or the bridge to a heterocycle which contains one or more heteronomes.
[0043] R2 repräsentiert eine Carbonsäure, Cn kann ein, zwei oder mehre C-Atome repräsentieren wie in 2-Oxoadipinsäure: R2 represents a carboxylic acid, Cn can represent one, two or more C atoms as in 2-oxoadipic acid:
oder Oxobutanedioat or oxobutane dioate
R2 und Cn können einen ungesättigten oder gesättigten Ring mit oder ohne Heteroatomen darstellen. R2 and Cn can represent an unsaturated or saturated ring with or without heteroatoms.
[0044] Die Verbindung gemäß Formel (II) kann wie folgt ausgestaltet sein: The compound of formula (II) can be configured as follows:
wobei in which
As die Aminosäuren aus der Bindungstasche des Enzyms darstellen sowie Me ein Metall aus dem katalytischen Zentrum; As represents the amino acids from the binding pocket of the enzyme and Me represents a metal from the catalytic center;
Rl und R2 können Sauerstoff (Hydroxyl) oder Carboxylgruppen, Halogene, insbesondere Fluor, Chor, oder Iod ,eine einfach bis mehrfach halogenierte Methylgruppe, insbesondere CH2F bis zu CF3 sein; R1 and R2 can be oxygen (hydroxyl) or carboxyl groups, halogens, in particular fluorine, choir, or iodine, a mono- to poly-halogenated methyl group, in particular CH2F up to CF3;
Rl kann einfach gebundener Wasserstoff oder eine CH2R3 -Gruppe sein, wobei R3 Wasserstoff oder Sauerstoff (Hydroxyl, Carbonyl) eine kürzere C-Kette (Ci bis C4 ) sein kann. Rl can be simply bonded hydrogen or a CH 2 R 3 group, where R 3 can be hydrogen or oxygen (hydroxyl, carbonyl) a shorter C chain (Ci to C 4 ).
[0045] Die Verbindung gemäß Formel (II) kann Teil eines Ringsystems sein, wobei die Ringgröße 3 bis 5 Atome mit einem oder mehreren Heteroatomen enthält. C7 kann aus einer Kohlenstoffkette bis 5 Atomen bestehen und Doppelbindungen, wie folgt beispielhaft gezeigt, enthalten: The compound of formula (II) can be part of a ring system, the ring size containing 3 to 5 atoms with one or more heteroatoms. C7 can consist of a carbon chain of up to 5 atoms and contain double bonds, as shown by way of example:
5-Oxohex-2-enedioic acid 5-oxohex-2-enedioic acid
[0046] Diese Kette kann auch in einem Ringsystem eingebunden sein:  [0046] This chain can also be integrated in a ring system:
[0047] Cn stellt in Formel (II) ein C-Atom oder ein Heteroatom dar. In formula (II), Cn represents a carbon atom or a heteroatom.
[0048] R2 repräsentiert eine Carbonsäure, Cn kann ein, zwei oder mehre C-Atome repräsentieren, wie beispielsweise in 2-Oxoadipinsäure (s.o.) oder Oxobutanedioat (s.o.). R2 represents a carboxylic acid, Cn can represent one, two or more C atoms, such as in 2-oxoadipic acid (see above) or oxobutane dioate (see above).
[0049] Cn kann Teil oder Brücke zu einem Heterocyclus sein, der ein oder mehrere Heteronome enthält, wie z.B. in: Cn can be part or bridge to a heterocycle containing one or more heteronomes, e.g. in:
[0050] In Formel (II) kann Rl ein Wasserstoffatom, eine Methylgruppe oder eine Alkylgruppe bis zu 5 C-Atomen sein die verzweigt gesättigt oder ungesättigt sein können oder auch Heteroatome enthalten. In formula (II) Rl can be a hydrogen atom, a methyl group or an alkyl group up to 5 carbon atoms which can be branched saturated or unsaturated or also contain heteroatoms.
[0051] R2 kann ebenso ein Wasserstoffatom, eine Methylgruppe, eine Alkylgruppe mit bis zu 6 C- Atomen sein, die verzweigt gesättigt oder ungesättigt sein können oder auch ein Heteroatom enthalten kann, wie in den folgenden Beispielen angegeben. R2 can also be a hydrogen atom, a methyl group, an alkyl group with up to 6 C atoms, which can be branched saturated or unsaturated or can also contain a hetero atom, as indicated in the following examples.
[0052] Zwischen Rl und R2 kann in Formel (II) eine brückenbildende zyklische Struktur angeordnet sein, die ungesättigt oder gesättigt oder Heteroatome enthalten kann. Der gebildete Zyklus kann ein 3- Rring, 4-Ring ein 5-Ring oder auch ein 6-Ring sein. Der Ring kann eine oder mehrere Doppelbindungen enthalten, ebenso dazu oder allein ein oder mehrere Heteroatome der gleichen Art oder auch gemischt, wie in den folgenden Beispielen angegeben. Between Rl and R2, a bridge-forming cyclic structure can be arranged in formula (II), which can contain unsaturated or saturated or heteroatoms. The cycle formed can be a 3-ring, a 4-ring, a 5-ring or a 6-ring. The ring can contain one or more double bonds, as well as this alone or one or more heteroatoms of the same type or mixed, as indicated in the following examples.
eine Doppelbindung, auch in Verbindung mit Heteroatomen a double bond, also in connection with heteroatoms
eine oder mehrere Doppelbindung, auch in Verbindung mit Heteroatomen one or more double bonds, also in connection with heteroatoms
eine oder mehrere Doppelbindung, auch in Verbindung mit Heteroatomen one or more double bonds, also in connection with heteroatoms
eine oder mehrere Doppelbindung, auch in Verbindung mit Heteroatomen one or more double bonds, also in connection with heteroatoms
eine oder mehrere Doppelbindung, auch in Verbindung mit Heteroatomen [0053] Die Kette der C-Atome kann verlängert werden und die Substituenten wie in den folgenden Beispielen gezeigt, Verwendung finden. one or more double bonds, also in connection with heteroatoms The chain of carbon atoms can be extended and the substituents can be used as shown in the following examples.
[0054] Die Verbindungen können sowohl als Substanz, als Salz und in gepufferter Form zur Anwendung kommen. Die Carbonsäuren können ebenfalls verestert zur Anwendung gelangen, wobei die Veresterung auch mit höherkettigen Alkoholen (bis C12 Atomen) möglich ist. The compounds can be used both as a substance, as a salt and in a buffered form. The carboxylic acids can also be used esterified, the esterification also being possible with higher-chain alcohols (up to C12 atoms).
[0055] Die Verarbeitung erfolgt auf pharmazeutisch-technischen Niveau. Zubereitungen wie z.B. Cremes, Salben, Gele, Nanoformulierungen, Infüsionslösungen, Tabletten, Kapseln. The processing takes place on a pharmaceutical-technical level. Preparations such as Creams, ointments, gels, nanoformulations, infusion solutions, tablets, capsules.
[0056] Als klassisches Prodrug kann Vitamin C fungieren welches in Körper zu Verbindungen gemäß Formel (I) oder nach Formel (II) sowie zu der gemäß der im Folgenden gezeigten Struktur nach Formel (II) metabolisiert werden kann. Veresterung der Carboxylgruppe führt ebenfalls zu Prodrugs mit verbesserter Resorption und Zellaufnahme. Weitere Optionen für eine Prodruggestaltung sind in [48] aufgeführt. Vitamin C can act as a classic prodrug which can be metabolized in the body to form compounds according to formula (I) or according to formula (II) and to the structure according to formula (II) shown below. Esterification of the carboxyl group also leads to prodrugs with improved absorption and cell uptake. Further options for prodrug design are listed in [48].
[0057] Die Kombination von Prodrug und Ersatz Cosubstrat (Modulator) sind möglich. Die Verabreichung kann, oral, lokal, oder durch Infüsion erfolgen. The combination of prodrug and replacement cosubstrate (modulator) are possible. Administration can be oral, local, or by infusion.
[0058] Die Mischung bzw. kombinierte Verabreichung von klassischen Tumortherapeutikum und Modulator zur Effizienzsteigerung der Therapie sind möglich und stellen eine Therapieoptimierung dar. The mixture or combined administration of classic tumor therapeutic and modulator to increase the efficiency of the therapy are possible and represent therapy optimization.
[0059] Für die beanspruchten Strukturelemente stehen eine Vielzahl von bekannten Strukturen (CAS) zur Auswahl, die je nach DIOs als Modulator zur Verfügung stehen können und die DIOs damit beeinflusst werden. A large number of known structures (CAS) are available for the claimed structural elements, which, depending on the DIOs, can be available as modulators and thus influence the DIOs.
[0060] Ebenso stehen Strukturen zur Auswahl die als Prodrug fünktionieren. Das einfachste Beispiel ist das Vitamin C und deren Derivate wie 2-O-a-D-Glucopyranosyl-l-ascorbinsäure die zu 2,3- Diketoglulonic acid (DKA)und auch zu 3,4,5-Trihydroxy-2-oxopentanoic acid (III; 2KGL) There are also structures to choose from that function as a prodrug. The simplest example is vitamin C and its derivatives such as 2-OaD-glucopyranosyl-l-ascorbic acid, which are used for 2,3-diketoglulonic acid (DKA) and also for 3,4,5-trihydroxy-2-oxopentanoic acid (III; 2KGL )
metabolisiert werden können und als untoxischer funktioneller Ersatz des Cosubstrat in den DIOs funktionieren. Der Wirkmechanismus von Vitamin C an den verschiedenen DlOs und den damit verbunden Prozessen war bisher ungeklärt und kann durch die Funktion als Prodrug für DKG und 2KGL erklärt werden. Damit ergibt sich auch die recht breite Aktivität von Vitamin C an unterschiedlichsten Zielstrukturen abseits von Redoxeffekten.[l7, 49-61]  can be metabolized and function as a non-toxic functional replacement of the cosubstrate in the DIOs. The mechanism of action of vitamin C on the various DlOs and the associated processes has so far not been clarified and can be explained by the function as a prodrug for DKG and 2KGL. This also results in the quite broad activity of vitamin C on a wide variety of target structures apart from redox effects. [L7, 49-61]
[0061] Durch den Einsatz der Modulatoren erfolgt eine Reaktivierung der DlOs und die kompetitive Verdrängung von Oncometaboliten (Hydroxyglutarate HG) aus dem aktiven Zentrum wird ermöglicht. Die Funktion wird wiederhergestellt und angepasst. By using the modulators, the D10s are reactivated and the competitive displacement of oncometabolites (hydroxyglutarate HG) from the active center is made possible. The function is restored and adjusted.
[0062] Beispiele für den Einfluss von HGs und Zielen der Cosubstrat Regulierung sind in den Tabellen 1 und 2 zusammengefasst: Examples of the influence of HGs and goals of cosubstrate regulation are summarized in Tables 1 and 2:
Tabelle 1 Table 1
Tabelle 2 [0063] Die Verwendung der erfindungsgemäßen Verbindungen ist auch zusammen mit pharmazeutisch anwendbaren Salzen, Tautomeren und Stereoisomeren der jeweiligen Verbindung einschließlich von Mischungen davon vorgesehen. Table 2 The use of the compounds according to the invention is also intended together with pharmaceutically acceptable salts, tautomers and stereoisomers of the respective compound, including mixtures thereof.
[0064] Die Verbindungen stellen eine Basis für die weitere Entwicklung und Modifikation der Grundformeln dar. Im Rahmen der vorliegenden Erfindung sind pharmazeutisch verträgliche oder anwendbare Salze, Prodrugs, Enantiomere, Diastereomere, racemische Gemische, kristalline Formen, nicht-kristalline Formen, amorphe Formen, unsolvatisierte Formen und Solvate der allgemeinen Formeln (I) wie offenbart. The compounds form a basis for the further development and modification of the basic formulas. Within the scope of the present invention are pharmaceutically acceptable or applicable salts, prodrugs, enantiomers, diastereomers, racemic mixtures, crystalline forms, non-crystalline forms, amorphous forms, unsolvated forms and solvates of general formulas (I) as disclosed.
[0065] Der Ausdruck "pharmazeutisch anwendbare Salze", wie er hier verwendet wird, schließt Salze der Verbindung nach den allgemeinen Formeln (I) und (II) ein, die mit relativ nicht-toxischen (dh. pharmazeutisch annehmbaren) Säuren oder Basen in Abhängigkeit von den speziellen gefundenen Substituenten hergestellt werden auf den Verbindungen der vorliegenden Erfindung. Wenn beispielsweise die Verbindungen der vorliegenden Erfindung saure Funktionalitäten enthalten, können Basenadditionssalze durch Inkontaktbringen der neutralen Form solcher Verbindungen mit einer ausreichenden Menge der gewünschten Base, entweder in reiner Form oder in einem geeigneten inerten Lösungsmittel, erhalten werden. Nicht einschränkende Beispiele für pharmazeutisch annehmbare Basenadditionssalze umfassen Natrium-, Kalium-, Calcium-, Ammonium-, organisches Amino- oder Magnesiumsalz oder ein ähnliches Salz. Wenn Verbindungen der vorliegenden Erfindung basische Funktionalitäten enthalten, können Säureadditionssalze erhalten werden, indem man die neutrale Form solcher Verbindungen mit einer ausreichenden Menge der gewünschten Säure entweder in reiner Form oder in einem geeigneten inerten Lösungsmittel in Kontakt bringt. Nicht einschränkende Beispiele für pharmazeutisch verträgliche Säureadditionssalze umfassen solche, die von anorganischen Säuren, wie Salzsäure, Bromwasserstoffsäure, Salpetersäure, Kohlensäure, Phosphorsäure, teilweise neutralisierte Phosphorsäuren, Schwefelsäure, teilweise neutralisierte sulfürische, lodwasserstoff- oder phosphorige Säure und dergleichen abgeleitet sind ebenso wie die Salze, die von relativ nichttoxischen organischen Säuren wie Essigsäure, Propionsäure, Isobuttersäure, Maleinsäure abgeleitet sind. Malonsäure-, Benzoe-, Bernstein-, Su-Ber-, Fumar-, Mandel-, Phthal-, Benzolsulfon-, p-Tolylsulfon-, Zitronen-, Wein-, Methansulfonsäure und dergleichen. Ebenfalls eingeschlossen sind Salze von Aminosäuren, wie Arginat und dergleichen, und Salze von organischen Säuren, wie Glucuron- oder Galactussäuren und dergleichen. Bestimmte spezifische Verbindungen der vorliegenden Erfindung können sowohl basische als auch saure Funktionalitäten enthalten, die es ermöglichen, dass die Verbindungen entweder in Basen oder Säureadditionssalze umgewandelt werden. Das Inkontaktbringen des Salzes mit einer Base kann die neutralen Formen der Verbindungen der vorliegenden Erfindung oder Säure regenerieren und die Stammverbindung auf herkömmliche Weise isolieren. Die Ausgangsform der Verbindung unterscheidet sich von den verschiedenen Salzformen in bestimmten physikalischen Eigenschaften, wie der Löslichkeit in polaren Lösungsmitteln, jedoch sind die Salze im übrigen für die Zwecke der vorliegenden Erfindung der Ausgangsform der Verbindung äquivalent. Die Verbindungen der vorliegenden Erfindung können chirale oder asymmetrische Kohlenstoff- Atome (optische Zentren) und / oder Doppelbindungen besitzen. Die Racemate, Diastereomere, geometrischen lsomere und individuellen optischen lsomere sind von der vorliegenden Erfindung umfasst. Die Verbindungen der vorliegenden Erfindung können sowohl in unsolvatisierten Formen als auch in solvatisierten Formen, einschließlich hyperierten Formen, vorliegen lm Allgemeinen sind die solvatisierten Formen äquivalent zu unsolvatisierten Formen und werden auch von der vorliegenden Erfindung umfasst. Die Verbindungen der vorliegenden Erfindung können weiterhin in multiplen kristallinen oder amorphen Formen existieren. The term "pharmaceutically acceptable salts" as used herein includes salts of the compound of general formulas (I) and (II), which are in relatively non-toxic (ie. Pharmaceutically acceptable) acids or bases Dependence on the particular substituents found are made on the compounds of the present invention. For example, when the compounds of the present invention contain acidic functionality, base addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired base, either in pure form or in a suitable inert solvent. Nonlimiting examples of pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amino or magnesium salt or a similar salt. When compounds of the present invention contain basic functionalities, acid addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired acid, either in pure form or in a suitable inert solvent. Nonlimiting examples of pharmaceutically acceptable acid addition salts include those derived from inorganic acids such as hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, phosphoric acid, partially neutralized phosphoric acids, sulfuric acid, partially neutralized sulfuric, hydroiodic or phosphoric acid and the like as well as the salts, which are derived from relatively non-toxic organic acids such as acetic acid, propionic acid, isobutyric acid, maleic acid. Malonic, benzoic, amber, Su-Ber, fumaric, almond, phthalic, benzenesulfonic, p-tolylsulfonic, citric, wine, methanesulfonic acid and the like. Also included are salts of amino acids such as arginate and the like, and salts of organic acids such as glucuronic or galactic acids and the like. Certain specific compounds of the present invention may contain both basic and acid functionalities that allow the compounds to be converted to either bases or acid addition salts. Contacting the salt with a base can regenerate the neutral forms of the compounds of the present invention or acid and isolate the parent compound in a conventional manner. The starting form of the compound differs from the different salt forms in certain physical properties, such as that Solubility in polar solvents, however, the salts are otherwise equivalent to the starting form of the compound for the purposes of the present invention. The compounds of the present invention can have chiral or asymmetrical carbon atoms (optical centers) and / or double bonds. The racemates, diastereomers, geometric isomers and individual optical isomers are encompassed by the present invention. The compounds of the present invention can exist in unsolvated forms as well as in solvated forms, including hyperated forms. Generally, the solvated forms are equivalent to unsolvated forms and are also encompassed by the present invention. The compounds of the present invention may further exist in multiple crystalline or amorphous forms.
[0066] Die Verbindungen der vorliegenden Erfindung können weiterhin in einer sogenannten Prodrugform vorliegen. Prodrugs der Verbindungen der Erfindung sind jene Verbindungen, die leicht unter physiologischen Bedingungen chemische Veränderungen eingehen, um die Verbindungen der vorliegenden Erfindung bereitzustellen. Zusätzlich können Prodrugs in den Verbindungen der vorliegenden Erfindung durch chemische oder biochemische Verfahren in einer Ex-vivo-Umgebung umgewandelt werden. Zum Beispiel können Prodrugs langsam in die Verbindungen der vorliegenden Erfindung umgewandelt werden, wenn sie zum Beispiel in einem transdermalen Pflasterreservoir mit einem geeigneten Enzym oder chemischen Reagenz platziert werden. The compounds of the present invention may also be in a so-called prodrug form. Prodrugs of the compounds of the invention are those compounds that readily undergo chemical changes under physiological conditions to provide the compounds of the present invention. In addition, prodrugs in the compounds of the present invention can be converted by chemical or biochemical methods in an ex vivo environment. For example, prodrugs can be slowly converted to the compounds of the present invention when placed, for example, in a transdermal patch reservoir with an appropriate enzyme or chemical reagent.
[0067] Die hierin beschriebenen Verbindungen der Erfindung kann Säugetieren, wie Mensch oder Haustier in einer geeigneten Dosis verabreicht werden. Nicht einschränkende Beispiele für Haustiere sind Schweine, Kühe, Büffel, Schafe, Ziegen, Kaninchen, Pferde, Esel, Hühner, Enten, Katzen, Hunde, echte Schweine oder Hamster. Am meisten bevorzugt wird es an Menschen verabreicht. Die bevorzugte Art der Verabreichung hängt von der Form der Verbindung der Erfindung (mit der allgemeinen Formel (1)) ab. Wie oben beschrieben, kann die Verbindung mit der allgemeinen Formel (1) in Form von pharmazeutisch verträglichen Salzen, Prodrugs, Enantiomeren, Diastereomeren, racemischen Mischungen, kristallinen Formen, nicht-kristallinen Formen, amorphen Formen, nichtsolvatisierte Formen oder Solvate. Die Verbindung der Erfindung kann oral, parenteral, wie subkutan, intraventral, intramuskulär, intraperitoneal, intrathekal, intraokular, transdermal, transmucosally, subdural, lokal oder topisch über eine lontophorese, sublingual, durch lnhalationsspray verabreicht werden. Aerosol oder rektal und dergleichen in Dosierungseinheitsformulierungen, die gegebenenfalls weiterhin herkömmliche pharmazeutisch akzeptable Exzipienten umfassen. Die Verbindung der Erfindung zur Verwendung in Übereinstimmung mit der vorliegenden Erfindung kann als eine pharmazeutische Zusammensetzung unter Verwendung von einem oder mehreren physiologischen Trägem oder Exzipienten formuliert werden. [0068] Für die orale Verabreichung kann die pharmazeutische Zusammensetzung der Erfindung beispielsweise die Form von Tabletten oder Kapseln annehmen, die auf herkömmliche Weise mit pharmazeutisch annehmbaren Hilfsstoffen wie Bindemitteln (z. B. vorgelatinierte Maisstärke, Polyvinylpyrrolidon, Hydroxypropylmethylcellulose) hergestellt werden ), Füllstoffe (z. B. Lactose, mikrokristalline Cellulose, Calciumhydrogenphosphat), Gleitmittel (z. B. Magnesiumstearat, Talk, Siliciumdioxid), Sprengmittel (z. B. Pota- Stärke, Natriumstärkeglycolat) oder Benetzungsmittel (z. B. Natriumlauryl) Sulfat). Die pharmazeutische Zusammensetzung kann einem Patienten mit einem physiologisch verträglichen Träger verabreicht werden. In einer spezifischen Ausführungsform bedeutet der Ausdruck "pharmazeutisch verträglich", dass er von einer Regulierungsbehörde oder einem anderen allgemein anerkannten Arzneibuch zur Verwendung in Tieren und insbesondere in Menschen zugelassen ist. Der Ausdruck "Träger" bezieht sich auf ein Verdünnungsmittel, Adjuvans, Exzipient oder Vehikel, mit dem das Therapeutikum verabreicht wird. Solche pharmazeutischen Träger können sterile Flüssigkeiten sein, wie Wasser und Öle, einschließlich solchen aus Erdöl, tierischem, pflanzlichem oder synthetischem Ursprung, wie Erdnussöl, Sojabohnenöl, Mineralöl, Sesamöl und dergleichen. Wasser ist ein bevorzugter Träger, wenn die pharmazeutische Zusammensetzung intravenös verabreicht wird. Salzlösungen und wässrige Dextrose- und Glycerinlösungen können auch als flüssige Träger, insbesondere für injizierbare Lösungen, verwendet werden. Geeignete pharmazeutische Hilfsstoffe umfassen Stärke, Glucose, Lactose, Saccharose, Gelatine, Malz, Reis, Mehl, Kreide, Silicagel, Natriumstearat, Glycerinmonostearat, Talk, Natriumion, getrocknete Magermilch, Glycerin, Propylen, Glycol, Wasser, Ethanol und dergleichen. Die Zusammensetzung kann gewünschtenfalls auch geringe Mengen an Netz- oder Emulgiermitteln oder pH-Puffermittel enthalten. Diese Zusammensetzungen können in Form von Salben, Lösungen, Suspensionen, Emulsionen, Tabletten, Pillen, Kapseln, Pulvern, Formulierungen mit verzögerter Freisetzung und dergleichen vorliegen. Eine bevorzugte Form ist eine Salbe. Die Zusammensetzung kann als Suppositorium mit traditionellen Bindemitteln und Trägem wie Triglyceriden formuliert werden. Die orale Formulierung kann Standardträger enthalten, wie Mannitol, Lactose, Stärke, Magnesiumstearat, Natriumsaccharin, Cellulose, Magnesiumcarbonat usw. von pharmazeutischer Qualität. E. W. Martin beschreibt Beispiele von geeigneten pharmazeutischen Trägem in "Remington's Pharmaceutical Sciences". Solche Zusammensetzungen enthalten eine therapeutisch wirksame Menge der vorstehend erwähnten Verbindungen, vorzugsweise in gereinigter Form, zusammen mit einer geeigneten Menge an Träger, um so die Form für die richtige Verabreichung an den Patienten bereitzustellen. Die Formulierung sollte der Art der Verabreichung entsprechen. Flüssige Zubereitungen für die orale Verabreichung können beispielsweise in Form von Lösungen, Sirupen oder Suspensionen vorliegen oder können als trockenes Produkt zur Verwendung mit Wasser oder einem anderen geeigneten Vehikel vor der Verwendung präsentiert werden. Eine solche flüssige Zubereitung kann auf herkömmliche Weise mit pharmazeutisch verträglichen Zusätzen wie Suspendiermitteln (z. B. Sorbitol, Sirup, Cellulosederivaten, hydrierten genießbaren Feten), Emulgatoren (z. B. Lecithin, Akaziengummi), nichtwäßrigen Vehikeln (z. B. Mandelöl) hergestellt werden Öl, ölige Ester, Ethylalkohol, fraktionierte Pflanzenöle), Konservierungsmitel (zB Methyl- oder Propyl-p-hydroxycarbonate, Sorosäuren). Die Zubereitungen können gegebenenfalls auch Puffersalze, Geschmacks-, Färbe- und Süßungsmitel enthalten. Zubereitungen für die orale Verabreichung können geeignet formuliert werden, um eine kontrollierte Freisetzung der pharmazeutischen Zusammensetzung der Erfindung zu ergeben. The compounds of the invention described herein can be administered in a suitable dose to mammals such as humans or pets. Non-limiting examples of pets are pigs, cows, buffalos, sheep, goats, rabbits, horses, donkeys, chickens, ducks, cats, dogs, real pigs or hamsters. Most preferably it is administered to humans. The preferred mode of administration depends on the form of the compound of the invention (having the general formula (1)). As described above, the compound represented by the general formula (1) can take the form of pharmaceutically acceptable salts, prodrugs, enantiomers, diastereomers, racemic mixtures, crystalline forms, non-crystalline forms, amorphous forms, unsolvated forms or solvates. The compound of the invention can be administered orally, parenterally, such as subcutaneously, intraventrally, intramuscularly, intraperitoneally, intrathecally, intraocularly, transdermally, transmucosally, subdurally, locally or topically via iontophoresis, sublingually, by inhalation spray. Aerosol or rectally and the like in unit dosage formulations, which may further comprise conventional pharmaceutically acceptable excipients. The compound of the invention for use in accordance with the present invention can be formulated as a pharmaceutical composition using one or more physiological carriers or excipients. For oral administration, the pharmaceutical composition of the invention can take the form of tablets or capsules, for example, which are prepared in a conventional manner with pharmaceutically acceptable excipients such as binders (e.g. pregelatinized corn starch, polyvinylpyrrolidone, hydroxypropylmethyl cellulose), fillers ( e.g. lactose, microcrystalline cellulose, calcium hydrogen phosphate), lubricants (e.g. magnesium stearate, talc, silicon dioxide), disintegrants (e.g. pota starch, sodium starch glycolate) or wetting agents (e.g. sodium lauryl) sulfate). The pharmaceutical composition can be administered to a patient with a physiologically acceptable carrier. In a specific embodiment, the term "pharmaceutically acceptable" means that it is approved by a regulatory or other generally recognized pharmacopoeia for use in animals, and particularly in humans. The term "carrier" refers to a diluent, adjuvant, excipient or vehicle with which the therapeutic agent is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be used as liquid carriers, especially for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium ion, dried skim milk, glycerin, propylene, glycol, water, ethanol and the like. If desired, the composition can also contain small amounts of wetting or emulsifying agents or pH buffering agents. These compositions may be in the form of ointments, solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations and the like. A preferred form is an ointment. The composition can be formulated as a suppository with traditional binders and carriers such as triglycerides. The oral formulation may contain standard carriers such as mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc. of pharmaceutical quality. EW Martin describes examples of suitable pharmaceutical carriers in "Remington's Pharmaceutical Sciences". Such compositions contain a therapeutically effective amount of the above-mentioned compounds, preferably in purified form, together with an appropriate amount of carrier so as to provide the form for proper administration to the patient. The formulation should correspond to the route of administration. Liquid preparations for oral administration may, for example, be in the form of solutions, syrups or suspensions, or may be presented as a dry product for use with water or other suitable vehicle before use. Such a liquid preparation can be hydrogenated in a conventional manner with pharmaceutically acceptable additives such as suspending agents (e.g. sorbitol, syrup, cellulose derivatives edible fetuses), emulsifiers (e.g. lecithin, acacia gum), non-aqueous vehicles (e.g. almond oil), oil, oily esters, ethyl alcohol, fractionated vegetable oils), preservatives (e.g. methyl or propyl p-hydroxycarbonates, soro acids) ). The preparations can optionally also contain buffer salts, flavoring, coloring and sweetening agents. Preparations for oral administration can be suitably formulated to provide controlled release of the pharmaceutical composition of the invention.
[0069] Zur Verabreichung durch lnhalation wird die pharmazeutische Zusammensetzung der Erfindung in Form einer Aerosolspray-Präsentation aus einer Druckpackung oder einem Vernebler unter Verwendung eines geeigneten Treibmittels (z. B. Dichlordifluormethan, Trichlorfluormethan) bequem abgegeben B. Dichlortetrafluorethan, Kohlendioxid oder ein anderes geeignetes Gas) lm Falle eines vorexispergierten Aerosols kann die Dosierungseinheit bestimmt werden, indem ein Ventil zur Abgabe einer abgemessenen Menge bereitgestellt wird. Kapseln und Patronen aus beispielsweise Gelatine zur Verwendung in einem lnhalator oder lnsufflator können formuliert werden, die eine Pulvermischung der pharmazeutischen Zusammensetzung der Erfindung und eine geeignete Pulverbase, wie Lactose oder Stärke, enthalten. For administration by inhalation, the pharmaceutical composition of the invention is conveniently delivered in the form of an aerosol spray presentation from a pressure pack or nebulizer using a suitable propellant (e.g. dichlorodifluoromethane, trichlorofluoromethane) B. dichlorotetrafluoroethane, carbon dioxide or other suitable Gas) In the case of a pre-dispersed aerosol, the dosage unit can be determined by providing a valve for dispensing a measured amount. Capsules and cartridges of, for example, gelatin for use in an inhaler or insufflator can be formulated which contain a powder mixture of the pharmaceutical composition of the invention and a suitable powder base such as lactose or starch.
[0070] Die pharmazeutische Zusammensetzung der Erfindung kann zur parenteralen Verabreichung durch lnjektion, zum Beispiel durch Bolusinjektion oder kontinuierliche Infusion formuliert werden. Die Stelle der lnjektionen ist intravensal, intraperitoneal oder subkutan. Formulierungen zur lnjektion können in Einheitsdosierungsform (z. B. in Ampullen, in Mehrfachdosisbehältem) und mit einem zusätzlichen Konservierungsmitel vorgelegt werden. Die pharmazeutische Zusammensetzung der Erfindung kann solche Formen wie Suspensionen, Lösungen oder Emulsionen in öligen oder wßrigen Vehikeln annehmen und kann formulierende Mitel enthalten, wie beispielsweise suspendierende, stabilisierende oder dispergierende Mitel. Alternativ dazu kann das Mitel in Pulverform zur Konstitution mit einem geeigneten Vehikel (z. B. steriles pyrogenfreies Wasser) vor der Verwendung vorliegen. Typischerweise sind die Zusammensetzungen für die intravenöse Verabreichung Lösungen in sterilen isotonischen wässrigen Puffern. Falls erforderlich, kann die Zusammensetzung auch ein Solubilisierungsmitel und ein Lokalanästhetikum, wie Lignocain, enthalten, um den Schmerz an der lnjektionsstelle zu lindem lm allgemeinen werden die Bestandteile entweder getrennt oder in Einheitsdosierungsform zusammengemischt, beispielsweise als trockenes lyophilisiertes Pulver oder wasserfreies Konzentrat in einem hermetisch verschlossenen Behälter, wie einer Ampulle oder einem Beutel, der die Menge an aktivem Mitel anzeigt. Wenn die Zusammensetzung durch lnfusion verabreicht werden soll, kann auf eine Infusionsflasche verzichtet werden, die steriles Wasser oder Kochsalz von pharmazeutischer Qualität enthält. Wenn die Zusammensetzung durch lnjektion verabreicht wird, kann eine Ampulle mit sterilem Wasser zur Injektion oder Kochsalzlösung bereitgestellt werden, so dass die Bestandteile vor der Verabreichung gemischt werden können. [0071] Es ist für einen Durchschnittsfachmann auf dem Gebiet offensichtlich, dass die vorliegende Erfindung auch Dosierungsformen mit verzögerter Freisetzung umfasst, die so gestaltet sind, dass sie ein Arzneimittel mit einer vorbestimmten Rate freisetzen, um eine konstante Arzneimittelkonzentration für ein Arzneimittel aufrechtzuerhalten bestimmter Zeitraum mit minimalen Nebenwirkungen. Dies kann durch eine Vielzahl von Formulierungen oder Vorrichtungen erreicht werden, einschließlich Mikrokügelchen, Nanopartikeln, Liposomen und anderen Polymermatrices, wie Arzneimittel-Polymer- Konjugate wie Hydrogele oder biologisch abbaubare Stoffe wie Poly (milchsäure-co-glykolsäure) (PLGA), die den Wirkstoff einkapseln. Es ist bevorzugt, die Freisetzung an die spezifischen Bedürfnisse zur Behandlung von bestimmten Krankheiten anzupassen, z. wie anhaltende Freisetzung von lnjektionen bei der Behandlung von Diabetes. Die Definition der verzögerten Freisetzung ähnelt mehr einer "kontrollierten Freisetzung" oder "Depot-Medikation" als einer "anhaltenden". [0070] The pharmaceutical composition of the invention can be formulated for parenteral administration by injection, for example by bolus injection or continuous infusion. The location of the injections is intravensal, intraperitoneal, or subcutaneous. Formulations for injection can be presented in unit dosage form (e.g. in ampoules, in multiple dose containers) and with an additional preservative. The pharmaceutical composition of the invention can take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles and can contain formulating agents such as suspending, stabilizing or dispersing agents. Alternatively, the agent may be in powder form for constitution with a suitable vehicle (e.g., sterile pyrogen-free water) before use. Typically, the compositions for intravenous administration are solutions in sterile isotonic aqueous buffers. If necessary, the composition may also include a solubilizing agent and a local anesthetic, such as lignocaine, to alleviate the pain at the injection site. Generally, the ingredients are either separated or mixed together in unit dosage form, for example as a dry lyophilized powder or anhydrous concentrate in a hermetically sealed Container, such as an ampoule or pouch, that shows the amount of active agent. If the composition is to be administered by infusion, an infusion bottle containing sterile water or pharmaceutical grade saline can be omitted. When the composition is administered by injection, an ampoule can be provided with sterile water for injection or saline so that the ingredients can be mixed prior to administration. It will be apparent to one of ordinary skill in the art that the present invention also includes sustained release dosage forms designed to release a drug at a predetermined rate to maintain a constant drug concentration for a drug over a period of time minimal side effects. This can be achieved by a variety of formulations or devices, including microspheres, nanoparticles, liposomes and other polymer matrices such as drug-polymer conjugates such as hydrogels or biodegradable substances such as poly (lactic acid-co-glycolic acid) (PLGA), which are the active ingredient encapsulate. It is preferred to adapt the release to the specific needs for the treatment of certain diseases, e.g. such as sustained release of injections in the treatment of diabetes. The definition of delayed release is more like "controlled release" or "depot medication" than "sustained".
[0072] Die pharmazeutische Zusammensetzung der Erfindung kann, falls gewünscht, auch in einer Packung oder einem Spender bereitgestellt werden, die eine oder mehrere Einheitsdosierungsformen enthalten können, die das Mittel enthalten. Die Packung kann beispielsweise Metall- oder Kunststofffolie, wie Blisterpackung, umfassen. Das Pack- oder Dispensiergerät kann mit Anweisungen für die Verabreichung begleitet sein. The pharmaceutical composition of the invention may also be provided in a package or dispenser, if desired, which may contain one or more unit dosage forms containing the agent. The pack can include, for example, metal or plastic film, such as a blister pack. The pack or dispenser may be accompanied with instructions for administration.
[0073] Die pharmazeutische Zusammensetzung der Erfindung kann als alleiniger Wirkstoff verabreicht werden oder kann in Kombination mit anderen Wirkstoffen verabreicht werden. Solche zusätzlichen aktiven Mittel sollten primär aus Wirkstoffen ausgewählt werden, die mit der Behandlung der gleichen Krankheit in Verbindung stehen. Für den Fall, dass Fettleibigkeit behandelt werden soll, sollte ein zusätzlicher Wirkstoff aus der Gruppe der Medikamente gegen Fettleibigkeit ausgewählt werden ln Analogie können Antidiabetika und auch Anti-NAFLD / NASH- sowie Antidyslipidämiemedikamente als weitere Wirkstoffe verwendet werden. Darüber hinaus sollte ein solcher zusätzlicher Wirkstoff aus Wirkstoffen ausgewählt werden, die mit Nebenwirkungen wie Körpergewichtszunahme wie antipsychotische Behandlungen in Verbindung stehen. The pharmaceutical composition of the invention can be administered as the sole active ingredient or can be administered in combination with other active ingredients. Such additional active agents should primarily be selected from agents that are related to the treatment of the same disease. In the event that obesity is to be treated, an additional active ingredient should be selected from the group of anti-obesity drugs. Analogously, anti-diabetic agents and also anti-NAFLD / NASH and anti-dyslipidemic drugs can be used as further active ingredients. In addition, such an additional active ingredient should be selected from active ingredients that are associated with side effects such as body weight gain and antipsychotic treatments.
[0074] Die erfindungsgemäßen Verbindungen bzw. die erfindungsgemäßen Zusammensetzungen können als Cosubstrat zur Prävention, Behandlung und Nachbehandlung von Tumorerkrankungen verwendet werden. Vorzugsweise ist die Tumorerkrankung eine Erkrankung ausgewählt aus der Gruppe umfassend Tumoren der Hals-Nasen-Ohren-Region, umfassend Tumoren der inneren Nase, Nasennebenhöhlen, Nasopharynx, Lippen, Mundhöhle, Oropharynx, Larynx, Hypopharynx, Ohr, Speicheldrüsen, und Paragangliome, Lungengeschwulste, umfassend nicht-parvicelläre Bronchialkarzinome, parvicelluläre Bronchialkarzinome, Tumoren des Mediastinums, Tumoren des Gastrointestinaltraktes, umfassend Tumoren der Speiseröhre, des Magens, der Bauchspeicheldrüse, der Leber, der Gallenblase und der Gallenwege, Dünndarm-, Darm- und Darmkarzinome und Analkarzinome, Urogenitaltumoren, umfassend Tumoren der Nieren, Harnleiter, Blase, Prostatadrüse, Harnröhre, Penis und Hoden, gynäkologische Tumoren, die Tumore der Zervix, Vagina, Vulva, Gebärmutterkrebs, maligne umfassen Trophoblast-Krankheit, Ovarialkarzinom, Tuben des Uterusrohrs (TubaFaloppii), Tumore der Bauchhöhle, Mammakarzinome, Tumoren der Endokrinorgane, umfassend Tumoren der Schilddrüse, Nebenschilddrüse, Nebennierenrinde, endokrine Pankreastumoren, Karzinoide und Karzinoidsyndrom, multiple endokrine Neoplasien, Knochen- und Weichteilsarkome, Mesotheliome, Hauttumoren, Melanome aus kutanen und intraokularen Melanomen, Tumore der Zentralnervensystem, Tumoren im Säuglingsalter, umfassend Retinoblastom, Wilms-Tumor, Neurofibromatose, Neuroblastom, Ewing-Sarkom-Tumorfamilie, Rhabdomyosarkom, Lymphome, die Non-Hodgkin-Lymphome umfassen, kutane T-Zell-Lymphome, primäre Lymphome des zentralen Nervensystems, Morbus Hodg-kin, Leukämien, die akute Leukämien, chronische myeloische und lymphatische Leukämien, Plasmazellneoplasmen, Myelodysplasie-Syndrome, paraneoplastische Syndrome, Metastasen mit unbekanntem Primärtumor (CUP-Syndrom), metastasierende Tumore, die Himmetastasen umfassen, Lungenmetastasen, Lebermetastasen, Knochenmetastasen, Pleura und Perikardmetastasen und malignen Aszites, Peritonealkarzinose, Immunsup pressionsbedingte Malignität, die AIDS-assoziierte Malignität umfasst, wie Kaposi-Sarkom, AIDS-assoziierte Lymphome, AIDS-assoziierte Lymphome des zentralen Nervensystems, AIDS-assoziiertes Morbus Hodgkin und AIDS-assoziierte anogenitale Tumoren, Transplantations-bezogene Malignität. The compounds according to the invention or the compositions according to the invention can be used as a cosubstrate for the prevention, treatment and aftertreatment of tumor diseases. The tumor disease is preferably a disease selected from the group comprising tumors of the ear, nose and throat region, including tumors of the inner nose, paranasal sinuses, nasopharynx, lips, oral cavity, oropharynx, larynx, hypopharynx, ear, salivary glands, and paragangliomas, lung tumors, comprising non-parvicellar bronchial carcinomas, parvicellular bronchial carcinomas, tumors of the mediastinum, tumors of the gastrointestinal tract, including tumors of the esophagus, stomach, pancreas, the Liver, gallbladder and biliary tract, small bowel, colon and intestinal carcinoma and anal carcinoma, urogenital tumors, including tumors of the kidneys, ureters, bladder, prostate gland, urethra, penis and testicles, gynecological tumors, tumors of the cervix, vagina, vulva, uterine cancer , malignant include trophoblast disease, ovarian carcinoma, tubes of the uterine tube (TubaFaloppii), tumors of the abdominal cavity, breast carcinomas, tumors of the endocrine organs, including tumors of the thyroid gland, adrenal cortex, endocrine pancreatic tumors, carcinoma endocrine and carcinoma of the neoplasms , Mesotheliomas, skin tumors, melanomas from cutaneous and intraocular melanomas, tumors of the central nervous system, tumors in infancy, including retinoblastoma, Wilms tumor, neurofibromatosis, neuroblastoma, Ewing's sarcoma tumor family, rhabdomyosarcoma, lymphomas, which include non-Homeg T-cell lymphomas, primary lymphomas of the central nervous system, Hodg-kin disease, leukaemias, acute leukaemias, chronic myeloid and lymphatic leukaemias, plasma cell neoplasms, myelodysplasia syndromes, paraneoplastic syndromes, metastases with unknown primary tumor (CUP syndrome), metastatic tumors, live metastases, which include metastases, Bone metastases, pleura and pericardial metastases and malignant ascites, peritoneal carcinosis, immunosuppression-related malignancy, which includes AIDS-associated malignancy, such as Kaposi's sarcoma, AIDS-associated lymphoma, AIDS-associated lymphoma of the central nervous system, AIDS-associated disease, Hodgkinitale's disease and AIDS Tumors, transplant-related malignancy.
[0075] Im Zusammenhang mit der Behandlung von Tumor erkrankungen werden die erfindungsgemäßen Verbindungen mit Chemotherapeutika kombiniert, welche ausgewählt sein können aus der gruppe umfassend Antikörper, Alkylierungsmittel, Platin-Analoga, Interkalationsmittel, Antibiotika, Mitose-Suppressoren, Taxane, Topoisomerase-Suppressoren, Antimetaboliten und / oder L-Asparaginase, Hydroxycarbamid, Mitotane und / oder Amanitine.  In connection with the treatment of tumor diseases, the compounds according to the invention are combined with chemotherapeutic agents, which can be selected from the group comprising antibodies, alkylating agents, platinum analogues, intercalation agents, antibiotics, mitosis suppressors, taxanes, topoisomerase suppressors, antimetabolites and / or L-asparaginase, hydroxycarbamide, mitotane and / or amanitine.
[0076] Weitere Aspekte, Merkmale und Vorteile der vorliegenden Erfindung ergeben sich ohne weiteres aus der folgenden detaillierten Beschreibung, in der einfach bevorzugte Ausführungsformen und Implementierungen dargestellt sind. Die vorliegende Erfindung kann auch in anderen und unterschiedlichen Ausführungsformen verwirklicht werden und ihre verschiedenen Details können in verschiedenen, offensichtlichen Aspekten modifiziert werden, ohne Lehre und Umfang der vorliegenden Erfindung zu verlassen. Dementsprechend sind die Zeichnungen und Beschreibungen als veranschaulichend und nicht als einschränkend anzusehen. Zusätzliche Aufgaben und Vorteile der Erfindung werden teilweise in der folgenden Beschreibung dargelegt und werden teilweise aus der Beschreibung offensichtlich oder können der Ausführung der Erfindung entnommen werden [0077] FIG. 1 zeigt die für die Funktion als Cosubstrat notwendigen Bindungsverhältnisse im Enzym. Dazu befindet sich ein zweiwertiges Eisenatom 1 , welches nicht kovalent mit dem Cosubstrat interagiert im Reaktionszentrum des Enzyms. Der notwendige Abstand Cosubstrat Eisen liegt dabei im Bereich von 2, 1-2,4 Ä (Pfeil 2). Das Wassermolekül hat einen Abstand von 2,3Ä (Pfeil 3) und spielt für die ablaufende Reaktion ebenfalls eine wichtige Rolle. Zusammen wird hier der ÜbergangzustandFurther aspects, features and advantages of the present invention are readily apparent from the following detailed description, in which simply preferred embodiments and implementations are shown. The present invention can be embodied in other and different embodiments and its various details can be modified in various obvious aspects without departing from the teachings and scope of the present invention. Accordingly, the drawings and descriptions are to be regarded in an illustrative rather than a restrictive sense. Additional objects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned from the practice of the invention [0077] FIG. 1 shows the binding relationships in the enzyme which are necessary for the function as cosubstrate. For this there is a divalent iron atom 1, which does not interact covalently with the cosubstrate in the reaction center of the enzyme. The necessary distance cosubstrate iron is in the range of 2, 1-2.4 Ä (arrow 2). The water molecule is 2.3Ä apart (arrow 3) and also plays an important role in the reaction taking place. Together the transition state becomes here
(Transistion state; TS) für die Reaktion im Enzym des Cosubstrat bzw. Cosubstrate Funktionsersatz dargestellt. Weiterhin zeigt F1G. 1 das Umfeld im aktiven Zentrum von DlOs und die Einbettung und die nichtkovalente Fixierung des Cosubstrat/Cofaktor Komplexes 4 (vgl. auch faciler 2-His-l- Carboxylate-Triade [41] [42]). Es ist weiterhin ein Kohlenstoffatom mit gleichen elektronischen Eigenschaften 5 dargestellt. (Transistion state; TS) for the reaction in the enzyme of the cosubstrate or cosubstrate function replacement. F1G also shows. 1 the environment in the active center of DlOs and the embedding and noncovalent fixation of the cosubstrate / cofactor complex 4 (cf. also facile 2-His-l-carboxylate triad [41] [42]). A carbon atom with the same electronic properties 5 is also shown.
[0078] Die Verbindungen der vorliegenden Erfindung gemäß Formel (1) und (11) können über Transporter (z.B. DKA, 2-OKG) oder nach entsprechender Derivatisierung aktiv oder passiv in die Zielzelle gelangen. The compounds of the present invention according to formulas (1) and (11) can reach the target cell actively or passively via transporters (e.g. DKA, 2-OKG) or after corresponding derivatization.
wobei in which
As die Aminosäuren aus der Bindungstasche des Enzyms darstellen; Me ein Metall aus dem katalytischen Zentrum; As represent the amino acids from the binding pocket of the enzyme; Me a metal from the catalytic center;
Rl und R2 können Sauerstoff (Hydroxyl) oder Carboxylgruppen, Halogene, insbesondere Fluor, Chor, oder Iod, eine einfach bis mehrfach halogenierte Methylgruppe, insbesondere CH2F bis zu CF3 sein; und  R1 and R2 can be oxygen (hydroxyl) or carboxyl groups, halogens, in particular fluorine, choir, or iodine, a mono- to poly-halogenated methyl group, in particular CH2F up to CF3; and
Cn ein C-Atom, ein Heteroatom oder die Brücke zu einem Heterocyclus repräsentiert.  Cn represents a C atom, a hetero atom or the bridge to a heterocycle.
[0079] Diese Herangehensweise Funktionsäquivalente des physiologischen Cosubstrates 20G in DIOs einzusetzen ist bisher unbekannt und stellt insgesamt einen Paradigmenwechsel in der Funktionsbeeinflussung und -Wiederherstellung von humanen metabolischen Enzymen zur Behandlung von Krankheiten dar. Es grenzt sich daher von anderen Optionen ab, die darauf zielen, das katalytische Metall (Cofaktor) des Enzyms zu beeinflussen oder die Bildung von Oncometaboliten wie 2HG zu reduzieren (sogenannte IDH Inhibitoren). Damit wird dieser Paradigmenwechsel ausdrücklich deutlich. Es werden durch diese neue Methode und der gezeigten Effektivität dieser, bisher offene Fragen der Funktionsweise von DIOs wie den Ten-eleven Translocation (TET) Enzymen und HIF Prolinhydroxylasen erklärbar [21] This approach of using functional equivalents of the physiological cosubstrate 20G in DIOs is hitherto unknown and overall represents a paradigm shift in the function influencing and restoration of human metabolic enzymes for the treatment of diseases. It therefore differs from other options which aim to to influence the catalytic metal (cofactor) of the enzyme or to reduce the formation of oncometabolites such as 2HG (so-called IDH inhibitors). This makes this paradigm shift explicit. This new method and the demonstrated effectiveness of these previously unanswered questions about the functioning of DIOs such as the ten-eleven translocation (TET) enzymes and HIF proline hydroxylases can be explained [21]
[0080] Die vorliegende Erfindung basiert auf der Entdeckung von Verbindungen, welche die Funktion von DIOs auch in Gegenwart von Oncometaboliten (HG) wiederherstellen und damit weitreichende Behandlungsmöglichkeiten von Krankheiten verfügbar werden. Bisher sind keine Verbindungen beschrieben die die physiologische Funktion des 20G als Cosubstrat nachahmen können. The present invention is based on the discovery of compounds which restore the function of DIOs even in the presence of oncometabolites (HG) and thus far-reaching treatment options for diseases become available. So far, no compounds have been described that can mimic the physiological function of the 20G as a cosubstrate.
[0081] Zu einem ist es ein völlig neues Herangehen an die Beeinflussung von Enzymen in lebenden Zellen und zum anderen ermöglicht es die vorliegende Erfindung umfänglich und erstmalig offene Fragen zu den DIOs zu beantworten und dieses in neue Therapieoptionen für DIO abhängige Erkrankungen überführen. On the one hand, it is a completely new approach to influencing enzymes in living cells and, on the other hand, the present invention makes it possible to answer extensive and for the first time open questions about the DIOs and to convert them into new therapy options for DIO-dependent diseases.
[0082] Die vorliegende Erfindung basiert weiterhin auf Dockingexperimente und molekulardynamische Untersuchungen mit Verfahren und Algorithmen nach Homann, wodurch es möglich war die tatsächlichen Verhältnisse in der aktiven Site (Bindungs- oder lnteraktionsstelle) des DlO Enzyms zu zeigen und durch entsprechende Experimente in Zellfreien und Zellsystemen belegen zu können. The present invention is further based on docking experiments and molecular dynamic investigations with methods and algorithms according to Homann, which made it possible to show the actual conditions in the active site (binding or interaction site) of the D10 enzyme and by corresponding experiments in cell-free and cell systems to be able to prove.
[0083] So ist aus Röntgenstrukturuntersuchungen am TET2 Enzyms die Bindung des TET-Hemmers NGA (N-Oxalylglycin) bekannt (vgl. F1G. 2). [0084] Die Bindungsverhältnisse von 20G im TET Enzym sind bisher nicht bekannt und wurden analog Fig. 2 angenommen. In FIG. 3 und Fig. 4 werden diese Bindungsverhältnisse (im TS) erstmals gezeigt. In Anlehnung an Fig. 1. und Fig. 2 kann man sehen, dass die für die Enzymreaktion notwendigen Bindungsabstände eingehalten werden. Das Wasser ist wie in Figl schon erläutert eine notwendige Komponente im Enzymkomplex und wird hier mit dargestellt. Thus, the binding of the TET inhibitor NGA (N-oxalylglycine) is known from X-ray structure studies on the TET2 enzyme (cf. F1G. 2). The binding ratios of 20G in the TET enzyme are hitherto unknown and were assumed to be analogous to FIG. 2. In FIG. 3 and Fig. 4, these binding relationships (in the TS) are shown for the first time. Based on FIGS. 1 and 2, it can be seen that the binding distances necessary for the enzyme reaction are observed. As already explained in Figl, the water is a necessary component in the enzyme complex and is also shown here.
[0085] Um weitegehend den Funktionsverlust von DIOs (hier am Bsp. von TET) durch den Oncometaboliten 2HG erklären zu können, musste auch dessen räumliche Struktur im Enzym geklärt werden (vgl. FIG. 4). Hier können wir zeigen wie 2HG im TET und HIF Enzym interagiert und durch die Wechselwirkung der Hydroxygruppe mit dem katalytischen Metall (Fe2+) die Reaktion als Cosubstrat nicht eingehen kann. Zugleich wird durch seine stärkere Affinität des 2HG im Enzym 20G kompetitiv verdrängt. Damit wird das Enzym funktionslos (Fig. 5A und Fig. 5B). In order to be able to largely explain the loss of function of DIOs (here in the example of TET) by the oncometabolite 2HG, its spatial structure in the enzyme also had to be clarified (cf. FIG. 4). Here we can show how 2HG interacts in the TET and HIF enzyme and because of the interaction of the hydroxy group with the catalytic metal (Fe2 + ) the reaction as a cosubstrate cannot take place. At the same time, its stronger affinity for 2HG in the 20G enzyme means that it is competitively displaced. The enzyme thus becomes inoperative (FIGS. 5A and 5B).
[0086] Auf Basis der zuvor geschilderten Untersuchungen und Ergebnissen ergab sich die Erkenntnis, dass eine chemische Verbindung, die die Funktionalität des 20G wahmehmen soll, in gleicher Art sich im Enzym unter ähnlichen elektronischen Verhältnissen befinden muss (Fig. 1). On the basis of the investigations and results described above, it was found that a chemical compound that is supposed to perceive the functionality of the 20G must be in the same way in the enzyme under similar electronic conditions (FIG. 1).
[0087] Aus diesen ersten Ergebnissen ergab sich, dass zum funktionellen Ersatz des 20G und zur Wiederherstellung der DIO Funktion nur Verbindungen in Frage kommen, die stärker als 20G im DIO- Enzym binden, aber zugleich die Funktionalität als Cosubstrat haben und übernehmen können. Eine reine stärkere Bindung allein würde zu einer Hemmung der DIOs führen, die dann ähnlich zu denen des 2HGs wäre und wie 2HG zu einer kompetitiven Hemmung führt [43] Dieses ist kontraproduktiv zur angestrebten Funktion als Funktionsäquivalent. From these first results, it was found that only compounds which bind more than 20G in the DIO enzyme, but at the same time have and can take over the functionality as cosubstrate, are suitable for the functional replacement of the 20G and for restoring the DIO function. A stronger bond alone would lead to an inhibition of the DIOs, which would then be similar to that of the 2HG and, like 2HG, would lead to a competitive inhibition [43]. This is counterproductive to the desired function as a function equivalent.
[0088] Geht jedoch die stärkere Bindung im DIO-Enzym einher als Funktionsäquivalent, welches die entsprechenden Reaktionen eingeht, liegen Verbindungen vor, die 2HG verdrängen können und durch die Cosubstratfünktion die DIOs (z.B. TET Funktion der Demethylierung von 5-Methylcytosin 5mC) wiederherstellen. Diese Verbindungen allein sind in der Lage das 20G zu ersetzen. However, if the stronger binding in the DIO enzyme goes hand in hand as a functional equivalent, which enters into the corresponding reactions, there are compounds which can displace 2HG and restore the DIOs (e.g. TET function of demethylation of 5-methylcytosine 5mC) through the cosubstrate function. These connections alone are able to replace the 20G.
[0089] Der fünktionelle Ersatz des Cosubstrates 20G durch DKA ist in FIG. 6 dargestellt, welche die Ergebnisse eines zellfreien Assays zur Funktion von DKA im TET Enzym als fünktioneller Cofaktor zeigen. Dazu erfolgten die Untersuchungen im zellfreien TET-Assay (in 50 mM HEPES puffer mit 50mM NaCl 8mM Fe(NH4)2(SO i)2 5mM ATP, 3mM DTT, 0,25pg TET-Enzym und Herings-Sperma DNS. Nach einer 8 ständigen Inkubation erfolgte die Auswertung mittel Massenspektrometrie Das native 20G wurde ersetzt durch das fünktionelle Coenzyme DKA) Hierbei zeigte sich, dass DKA die Funktion des nativen 20G übernehmen kann und als Ersatz des nativen Cosubstrats dienen kann. [0090] Mutationen in den Genen, die die Isocitratdehydrogenase (IDH) kodieren, fordern die Reduktion von 20G zu den Onkometaboliten D-2-Hydroxyglutarat (2HG), was zu einer Hemmung der Demethylierung von 5-Methylcytosin(5mC) in der DNS zu 5-Hydroxymethylcytosin(5hmC) durch (TET), sowie zu methylierte Histonlysin (HK)-Reste (HKme) durch Jumonji C-Domänendemethylasen (JMJC) und N6-Methyladenosin (m6A) durch FTO führt [44, 45] The functional replacement of the cosubstrate 20G by DKA is shown in FIG. 6, which show the results of a cell-free assay for the function of DKA in the TET enzyme as a functional cofactor. For this purpose, the investigations were carried out in a cell-free TET assay (in 50 mM HEPES buffer with 50mM NaCl 8mM Fe (NH4) 2 (SO i) 2 5mM ATP, 3mM DTT, 0.25pg TET enzyme and herring sperm DNA. After an 8 Constant incubation was carried out using mass spectrometry. The native 20G was replaced by the functional coenzyme DKA) It was shown that DKA can take over the function of the native 20G and can serve as a replacement for the native cosubstrate. Mutations in the genes encoding isocitrate dehydrogenase (IDH) require the reduction of 20G to the oncometabolites D-2-hydroxyglutarate (2HG), which leads to an inhibition of the demethylation of 5-methylcytosine (5mC) in the DNA 5-hydroxymethylcytosine (5hmC) by (TET), as well as methylated histone lysine (HK) residues (HKme) by Jumonji C domain demethylases (JMJC) and N6-methyladenosine (m6A) by FTO [44, 45]
[0091] 2HG ist ein Onkometabolit, der zahlreiche Demethylasen hemmt, was zu Veränderungen in genomischen und transkriptomischen Methylierungsprofilen sowie zu Veränderungen in der Genexpression und Genomtopologie führt [46] Daraus resultieren entsprechende Krankheitsbilder u.a. Krebs und bei Betrachtung der Gesamtmethylierung des Genomes Alterungsprozesse. [0091] 2HG is an oncometabolite that inhibits numerous demethylases, which leads to changes in genomic and transcriptomic methylation profiles as well as to changes in gene expression and genome topology [46]. Cancer and considering the total methylation of the genome aging processes.
[0092] Der Eckpfeiler der Krebstherapie, einschließlich des späteren Erfolgs epigenetischer Therapien, ist der Einsatz effektiver und rationeller Medikamentenkombinationen. Im Mittelpunkt steht die Kombination epigenetische wirksame Medikamente (auch unsere 20G funktionellen Ersatzcosubstrate könnten dazu gezählt werden) mit anderen Therapien und die Optimierung dieser. The cornerstone of cancer therapy, including the later success of epigenetic therapies, is the use of effective and rational drug combinations. The focus is on the combination of epigenetically effective drugs (our 20G functional replacement cosubstrates could also be counted) with other therapies and the optimization of these.
[0093] Die TET Enzyme stellen insofern durch ihre zentrale epigenetische Rolle ein besonderes Ziel für den Ersatz des nativen Coenzyms 20G dar. In this respect, the TET enzymes, because of their central epigenetic role, represent a particular target for the replacement of the native 20G coenzyme.
[0094] Die Verwendung der Verbindungen gemäß der vorliegenden Erfindung wird im folgendem am TET Enzym dargestellt. Die bisherige bekannte wissenschaftliche Literatur geht, wenn TET-Enzyme als Target für Therapien betrachtet werden von den in FIG. 7 dargestellten Zusammenhängen ausfolgenden aus. The use of the compounds according to the present invention is shown below on the TET enzyme. The previous known scientific literature goes when TET enzymes are considered as a target for therapies of the in FIG. 7 shown relationships from the following.
[0095] In FIG. 7 werden bekannte Therapien dargestellt sowie das neue pharmakologisches Konzept gemäß der vorliegenden Erfindung ergänzt. Dabei haben die Abkürzungen folgende Bedeutungen: HDAC Histondeacetylasen; EZH2 Enhancer of zeste homolog 2 ist ein Histone-Lysin N- Methyltransferase Enzym; DOT1L (Disruptor of telomeric silencing l-like; BET Bromodomain and extra-terminal motif. In FIG. 7, known therapies are shown and the new pharmacological concept according to the present invention is supplemented. The abbreviations have the following meanings: HDAC histone deacetylases; EZH2 Enhancer of zeste homolog 2 is a histone-lysine N-methyltransferase enzyme; DOT1L (Disruptor of telomeric silencing l-like; BET Bromodomain and extra-terminal motif.
[0096] Ein fünktioneller Ersatz des Cosubstrates und die Funktionsübemahme durch eine extern zugeführte Verbindung ist im Satnd der Technik bisher nie in Erwägung gezogen worden und stellt somit einen Paradigmenwechsel dar. Die Verbindungen gemäß der vorliegenden Erfindung, insbesondere das DKA und das 2-Keto-Gulonicacid (2 OG) können durch ihre nicht toxische Wirkung, auch in den entsprechenden pharmakologischen Konzentrationen eingesetzt werden (höchste eingesetzte Konzentration liegt bei lmM), wie aus FIG. 8 und FIG. 9 hervorgeht, welche die Ergebnisse von Untersuchungen zur Toxizität zeigen. [0097] In klinischen Studien werden derzeit verschiedene Kombinationsstrategien verfolgt, darunter die Kombination von epigenetischen Therapien mit Chemotherapie, sowie zielgerichteten Therapien und mit Immuntherapien. Funktionsäquivalente des 20G in TETs konnten bisher in Kombinationstherapien nicht berücksichtigt worden, da sie bis dato unbekannt waren und erst mit der vorliegenden Erfindung in Betracht kommen. A functional replacement of the cosubstrate and the functional takeover by an externally supplied connection has never been considered in the prior art and thus represents a paradigm shift. The compounds according to the present invention, in particular the DKA and the 2-keto Due to their non-toxic effect, gulonicacid (2nd floor) can also be used in the corresponding pharmacological concentrations (the highest concentration used is 1 mm), as shown in FIG. 8 and FIG. 9 shows the results of toxicity studies. Various combination strategies are currently being pursued in clinical studies, including the combination of epigenetic therapies with chemotherapy, as well as targeted therapies and with immunotherapies. Functional equivalents of 20G in TETs have so far not been taken into account in combination therapies, since they were previously unknown and can only be considered with the present invention.
[0098] Der Nachweis, dass Krebszellen durch transkriptioneile Anpassung dem selektiven Druck entkommen können, liefert eine molekulare Begründung für die Verwendung der epigenetischen Therapie zur Blockade oder Umkehrung der Resistenz. In IDH Hemmern, die sich zurzeit in ersten klinischen Studien befinden, zeigt sich dieses ebenfalls [47] Durch die Kombination von IDH Hemmern mit Cosubstraten gemäß der vorliegenden Erfindung kann auf Zellebene eine Potenzierung der Wirksamkeit der IDH -Hemmer gezeigt werden (vgl. FIG. 10), wodurch das Potential der neuen Verbindungen gemäß der vorliegenden Erfindung im Umfeld einer neuen Enzymbeeinflussung eindrucksvoll gezeigt wird. The proof that cancer cells can escape the selective pressure by transcriptional adaptation provides a molecular reason for the use of epigenetic therapy to block or reverse the resistance. This is also shown in IDH inhibitors, which are currently in the first clinical studies [47]. The combination of IDH inhibitors with cosubstrates according to the present invention can potentiate the effectiveness of the IDH inhibitors at the cell level (cf. FIG. 10), which impressively shows the potential of the new compounds according to the present invention in the context of a new enzyme influencing.
[0099] Zugleich tritt eine Förderung der sekundären Apoptose ein, was auf die Wiederherstellung der Zellfunktion auf molekularer Ebene verweist, (vgl. FIG. 11). Das native 2 OG geht wieder in den normalen Zellmetabolismus ein und hemmt damit wieder die maligne Entgleisung der Zelle (sekundärer Weg der Bildung von 20G aus Glutaminsäure fällt weg). Die Inkubation mit dem starken IDH-Hemmer ML309, der den 2-HG-Spiegel signifikant reduzierte, aktiviert die gehemmten TET-Enzyme in IDH1R132H/+ Zellen nicht. Durch den Zusatz des funktionellen Cosubstrates DKA erfolgte eine Aktivierung des TET Enzymes. At the same time, secondary apoptosis is promoted, which points to the restoration of cell function at the molecular level (cf. FIG. 11). The native 2 OG goes back to normal cell metabolism and thus inhibits the malignant derailment of the cell (secondary way of the formation of 20G from glutamic acid is eliminated). Incubation with the powerful IDH inhibitor ML309, which significantly reduced the 2-HG level, did not activate the inhibited TET enzymes in IDH1R132H / + cells. The TET enzyme was activated by adding the functional cosubstrate DKA.
[00l00]Ein weiterer Fakt, der die Vorteile von Verbindungen gemäß der vorliegenden Erfindung belegt, die das native Cosubstrat 20G ersetzen, ist, dass dadurch neue Verbindungen entwickelt werden können, die auch an mutierten DIOs aktiv sind und sich in der Kinetik und Bindungsaffinität vom natürlichen Cosubstrat unterscheiden. Another fact that demonstrates the benefits of compounds in accordance with the present invention that replace the native 20G cosubstrate is that it can be used to develop new compounds that are also active on mutant DIOs and that differ in kinetics and binding affinity distinguish natural cosubstrate.
[00l0l]Es ist bekannt, dass IDH 1 -Mutationen mit einer veränderten IDH 1 -Enzymfunktion verbunden sind, die die Überproduktion von neomorphem Metaboliten 2-Hydroxyglutarat induziert. Um die erhöhte Häufigkeit von 2-HG in HCT116 IDH1R132H/+ Zellen im Vergleich zu HCT116 IDH1+/+ Zellen zu validieren, wurde der 2-HG Gehalt mittels LC-MS/MS Analyse analysiert (Abbildung 1). Intrazelluläres 2-HG in IDH1R132H/+ Zellen war 56 mal höher als in IDH1 Wildtyp-Zellen (8,5 nmoPmg Protein bzw. 0,15 nmol/mg Protein; 56 mal höher; p < 0,0001). Darüber hinaus führte die Behandlung mit dem IDH 1 -Inhibitor ML309 (10 mM) zu einem signifikanten Rückgang des Onkometaboliten 2-HG im HCT1 16 IDH1R132H/+ Zellen auf (0,3 nmol/mg Protein) und zu einem geringeren Grad an IDH1+/+ (0,04 nmol/mg Protein) (Abb. ). Im Gegensatz dazu produzierte DKA nur minimale Veränderungen der 2-HG-Konzentrationen in den mutierten Zellen (5,9 nmol/mg Protein). Interessanterweise führte die kombinatorische Behandlung mit 10 mM ML309 und 1 mM DKA zu dem stärksten Rückgang von 2-HG bei IDH1R132H/+ und erreichte ähnliche Konzentration wie bei den IDH1 -Wildtypzellen ( 0,12 nmol/mg Protein), vgl. FIG. 10. It is known that IDH 1 mutations are associated with an altered IDH 1 enzyme function, which induces the overproduction of neomorphic metabolite 2-hydroxyglutarate. In order to validate the increased frequency of 2-HG in HCT116 IDH1R132H / + cells compared to HCT116 IDH1 + / + cells, the 2-HG content was analyzed using LC-MS / MS analysis (Figure 1). Intracellular 2-HG in IDH1R132H / + cells was 56 times higher than in IDH1 wild-type cells (8.5 nmoPmg protein or 0.15 nmol / mg protein; 56 times higher; p <0.0001). In addition, treatment with the IDH 1 inhibitor ML309 (10 mM) led to a significant decrease in the oncometabolite 2-HG in the HCT1 16 IDH1R132H / + cells (0.3 nmol / mg protein) and to one lower level of IDH1 + / + (0.04 nmol / mg protein) (Fig.). In contrast, DKA produced only minimal changes in the 2-HG concentrations in the mutated cells (5.9 nmol / mg protein). Interestingly, the combinatorial treatment with 10 mM ML309 and 1 mM DKA led to the greatest decrease in 2-HG in IDH1R132H / + and reached a concentration similar to that of IDH1 wild-type cells (0.12 nmol / mg protein), cf. FIG. 10th
[00102] FIG. 11 zeigt, dass 2-Hydroxyglutarat Level mit 5-hmdC levels in IDH1-MT HCT116 korrelieren. [00102] FIG. Figure 11 shows that 2-hydroxyglutarate levels correlate with 5-hmdC levels in IDH1-MT HCT116.
[00103] FIG. 12 zeigt, dass durch die Verwendung von DKA allein als fünktionelles Cosubstrat die Funktion des TET Enzymes auch in Gegenwart des kompetitiven Inhibitors 2HG wiederhergestellt werden kann. [00103] FIG. 12 shows that by using DKA alone as a functional cosubstrate, the function of the TET enzyme can be restored even in the presence of the competitive inhibitor 2HG.
[00104] In den Figuren FIG. 13-18 sind weitere ausgewählte chemische Verbindungen die als fünktioneller Ersatz des Cosubtrates 20G an TET und HIF agieren können in ihrer Konformation im Enzym aufgeführt. Dazu werden weiter unten entsprechende und Zellexperiment aufgeführt. In the figures FIG. 13-18 are other selected chemical compounds that can act as functional substitutes for cosubtrate 20G on TET and HIF in their conformation in the enzyme. Corresponding and cell experiments are listed below.
[00105] FIG. 13 zeigt für das TET Enzym DKA und gibt in FIG. 13 an welche Atome Aminosäuren AS koordinieren. In FIG. 114 ist DKA räumlich dargestellt und in FIG. 15 die räumliche Koordination von DKA im Enzym. Der Pfeil in FIG. 15 weist auf den Abstand des Fe 2+ vom DKA mit 2,2 Ä hin. Es wird gezeigt, dass DKA im TS im Enzym liegt und die vorgegebenen Bedingungen (Fig. 1) auch hier erreicht werden, so dass ein fünktioneller Ersatz des Cosubstrates 20G ermöglicht wird. [00105] FIG. 13 shows DKA for the TET enzyme and in FIG. 13 to which atoms coordinate amino acids AS. In FIG. 114 DKA is shown spatially and in FIG. 15 the spatial coordination of DKA in the enzyme. The arrow in FIG. 15 indicates the distance of the Fe 2 + from the DKA with 2.2 Ä. It is shown that DKA is in the TS in the enzyme and that the specified conditions (FIG. 1) are also achieved here, so that a functional replacement of the cosubstrate 20G is made possible.
[00106] FIG. 16A zeigt 2-Keto-Gulonicacid (2-OKG) und zeigt, wie das 2-OKG im TS im Enzym liegt und die vorgegebenen Bedingungen (Fig. 1) auch hier erreicht werden, so das ein fünktioneller Ersatz des Cosubstrates 20G ermöglicht wird. In FIG. 16B wird im Assay gezeigt, dass 2-OGK einen Effekt abhängig von der Dosis hat. [00106] FIG. 16A shows 2-keto-gulonicacid (2-OKG) and shows how the 2-OKG in the TS lies in the enzyme and the specified conditions (FIG. 1) are also achieved here, so that a functional replacement of the cosubstrate 20G is made possible. In FIG. 16B, the assay shows that 2-OGK has an effect depending on the dose.
[00107] FIG. 17 zeigt 4-Methyl-5-oxohex-2-enedioic acid mit Fe 2+ unter Angabe der Aminosäuren, die interagieren. [00107] FIG. 17 shows 4-methyl-5-oxohex-2-enedioic acid with Fe 2 + , indicating the amino acids that interact.
[00108] In Figur 18A ist AOF (2-(Furan-2-yl)-2-oxoacetate mit Fe 2+ und Aminosäuren gezeigt, in FIG. 18B der Effekt und in FIG. 18C die Interaktion zwischen Enzym und AOF. FIG. 18A shows AOF (2- (furan-2-yl) -2-oxoacetate with Fe 2 + and amino acids, the effect in FIG. 18B and the interaction between enzyme and AOF in FIG. 18C.
[00109] Figur 19-27 zeigt Verbindungen für das HF Enzym. In FIG. 19A wird die Struktur gezeigt und in FIG. 19B ist in 2G19 ist 2-((hydroxy(4-hydroxy-8-iodoisoquinolin-2-ium-3- yl)methylene)amino)acetateals in Hypoxie-induzierten Faktor (Hypoxia-Inducible Factor Prolyl Hydroxylase ,PHD2) als Antagonist gebunden. Figure 19-27 shows compounds for the HF enzyme. In FIG. 19A, the structure is shown and shown in FIG. 19B is in 2G19 is 2 - ((hydroxy (4-hydroxy-8-iodoisoquinolin-2-ium-3- yl) methylene) amino) acetates as an antagonist in hypoxia-induced factor (hypoxia-inducible factor prolyl hydroxylase, PHD2).
[00110] In FIG. 20 ist 20G im Enzym PHD2 (nach Methode Homann) gezeigt. Die Lage im Enzym entspricht dem notwendigen Prinzip für die Funktion als Cosubstrat. In FIG. 20 is shown 20G in the enzyme PHD2 (according to the Homann method). The position in the enzyme corresponds to the principle necessary for the function as a cosubstrate.
[00111] In FIG. 21 ist NGA als Antagonist im Enzym dargestellt. [00111] In FIG. 21 NGA is shown as an antagonist in the enzyme.
[00112] FIG. 22 zeigt den kompetitiven Antagonisten 2 HG im Enzym, der im Enzym kompetitiv mit 20G interagiert und zum Funktionsverlust des Enzyms führt. [00112] FIG. 22 shows the competitive antagonist 2 HG in the enzyme, which competitively interacts with 20G in the enzyme and leads to loss of function of the enzyme.
[00113] FIG. 23 zeigt 2-OKG im Enzym als fünktionelles Coenzym. Die notwendigen Prinzipien für diese Funktion werden eingehalten. [00113] FIG. 23 shows 2-OKG in the enzyme as a functional coenzyme. The necessary principles for this function are observed.
[001 l4]FIG. 24 zeigt 3-Bromo-2-oxopentanoate im Enzym als fünktionelles Coenzym. [001 l4] FIG. 24 shows 3-bromo-2-oxopentanoate in the enzyme as a functional coenzyme.
[00115] FIG. 25 zeigt (E)-5-oxohex-2-enedioic acid im Enzym als fünktionelles Coenzym. [00115] FIG. 25 shows (E) -5-oxohex-2-enedioic acid in the enzyme as a functional coenzyme.
[00116] FIG. 26 zeigt 4-(S)-Methyl-5-oxohex-2-enedioic acid im Enzym als funktionelles Coenzym. [00117] FIG. 27 zeigt DKA im Enzym als funktionelles Coenzym. [00116] FIG. 26 shows 4- (S) -methyl-5-oxohex-2-enedioic acid in the enzyme as a functional coenzyme. [00117] FIG. 27 shows DKA in the enzyme as a functional coenzyme.
Ausführungsbeispiele embodiments
Tet Enzyme (Ten-eleven Translocation (TET) Enzym) Tet Enzyme (Ten-eleven Translocation (TET) enzyme)
[00118] In Beispiel 1 (FIG. 28) konnte die Wiederherstellung der Funktionalität gezeigt werden und epigenetische Veränderungen, die zur Krebsentstehung führen rückgängig gemacht werden. In example 1 (FIG. 28), the restoration of the functionality could be shown and epigenetic changes which lead to the development of cancer could be reversed.
[00119] Außerdem ist die Regenerierung des normalen Metabolismus der Zelle möglich und der Krebs Metabolismus (Cancer Metabolismus) wird unterbrochen. Dies führt in Verbindung mit anderen Chemotherapien (Kombinationstherapie) oder allein zu einer Normalisierung der Zellfunktion und der Möglichkeit der Apoptose, die in entarteten Zellen unterbunden ist. Die Reaktivierung von Tumorsuppressor Genen (CDKN1A (p2l)) erfolgt ebenfalls durch die wieder fünktionsfähigen TETs. In addition, the regeneration of the normal metabolism of the cell is possible and the cancer metabolism (cancer metabolism) is interrupted. In conjunction with other chemotherapy (combination therapy) or alone, this leads to normalization of cell function and the possibility of apoptosis, which is prevented in degenerated cells. The reactivation of tumor suppressor genes (CDKN1A (p2l)) is also carried out by the again functional TETs.
[00l20]FIG. 28 zeigt DKA - 2,3-Diketogulonic acid aus Beispiel 1 und 20G (2 KG - 2-Keto-L-gulonic acid: [00l20] FIG. 28 shows DKA - 2,3-diketogulonic acid from Examples 1 and 20G (2 KG - 2-keto-L-gulonic acid:
Zellkultur und Behandlung Cell culture and treatment
[00121JHCT116 (ATCC-Nr. CCL-247), eine humane Kolorektalkarzinom-Zelllinie, wurde von der American Type Culture Collection (ATCC; https://www.atcc.org/) erworben. Humane Kolonkarzinomzellen HCT116 IDH1+/+ und HCT116 IDH1R132H/+ stammen von Horizon Discovery und wurden uns für Testzwecke überlassen.Die Zellen wurden in Dulbecco's Modified Eagle's Medium (DMEM) mit 2 mM L-Glutamin, ergänzt mit 10% fötalem Rinderserum (FBS), 45 lU/ml Penicillin und 45 lU/ml Streptomycin kultiviert. Die Zelllinien wurden innerhalb von sechs Monaten vor der Anwendung negativ auf Mykoplasmeninfektionen getestet. [00121JHCT116 (ATCC No. CCL-247), a human colorectal cancer cell line, was purchased from the American Type Culture Collection (ATCC; https://www.atcc.org/). Human colon carcinoma cells HCT116 IDH1 + / + and HCT116 IDH1R132H / + are from Horizon Discovery and were given to us for testing purposes. The cells were in Dulbecco's Modified Eagle's Medium (DMEM) with 2 mM L-glutamine, supplemented with 10% fetal bovine serum (FBS), 45 lU / ml penicillin and 45 lU / ml streptomycin cultured. The cell lines were tested negative for mycoplasma infections within six months prior to use.
Zelllebensfähigkeitstest Cell viability test
[00122] Die Untersuchung der möglichen zytotoxischen Effekte der getesteten Substanzen wurde mit dem MTT-Reduktionsassay durchgeführt, wie bereits beschrieben. HCT1 l6-Zellen wurden in 96-Well- Platten (TPP, Trasadingen, Schweiz) eingesetzt. Nach 24 h wurden die angegebenen Konzentrationen für 24, 48 und 72 h addiert. Anschließend wurden die Zellen mit 100 pL MTT-Lösung (0,5 mg/ml in PBS) für 4 h inkubiert. Nach Entfernung der Überstände wurden 50 pL Dimethylsulfoxid hinzugefügt, um das Formazonsalz aufzulösen, und die optische Dichte (OD) wurde mit einem Mikroplattenleser (Tecan, Crailsheim, Deutschland) gemessen.Die Anregung wurde auf 540 nm einstellte. Die Positivkontrollen wurden mit 0,002% SDS behandelt. Eine Zelllebensfähigkeit <75% sagt zytotoxische Effekte voraus. The investigation of the possible cytotoxic effects of the tested substances was carried out with the MTT reduction assay, as already described. HCT1 16 cells were used in 96-well plates (TPP, Trasadingen, Switzerland). After 24 h, the indicated concentrations for 24, 48 and 72 h were added. The cells were then incubated with 100 pL MTT solution (0.5 mg / ml in PBS) for 4 h. After the supernatants were removed, 50 pL of dimethyl sulfoxide was added to dissolve the formazone salt and the optical density (OD) was measured with a microplate reader (Tecan, Crailsheim, Germany). The excitation was set to 540 nm. The positive controls were treated with 0.002% SDS. Cell viability <75% predicts cytotoxic effects.
Apoptose-Assay Apoptosis Assay
[00123] Das Niveau apoptotischer und toter Zellen wurde mittels Durchflusszytometrie unter Verwendung von eBioscience™ Annexin V Apoptosis Detection Kit APC (Thermo Fisher, Darmstadt, Deutschland) bestimmt.Die Zellen wurden 2 x 105 HCT116 Zellen/Wells in 6-Well-Platten (TPP, Trasadingen, Schweiz) ausgesät. Nach 24 h wurden die Zellen mit den Substanzen in den angegebenen Konzentrationen für 72 h inkubiert. Anschließend wurden die Zellen gewaschen und mit Annexin V und Propidiumjodid nach den Anweisungen des Herstellers gefärbt. Die Zellen wurden auf einem FACSCanto 11 (BD Biosciences, Heidelberg, Deutschland) analysiert. Für die Datenanalyse wurde die Software FlowJo (Treestar, Ashland, USA) eingesetzt. RT-PCR The level of apoptotic and dead cells was determined by flow cytometry using eBioscience ™ Annexin V Apoptosis Detection Kit APC (Thermo Fisher, Darmstadt, Germany). The cells were 2 x 105 HCT116 cells / wells in 6-well plates ( TPP, Trasadingen, Switzerland). After 24 hours, the cells were incubated with the substances in the stated concentrations for 72 hours. The cells were then washed and stained with Annexin V and propidium iodide according to the manufacturer's instructions. The cells were analyzed on a FACSCanto 11 (BD Biosciences, Heidelberg, Germany). The FlowJo software (Treestar, Ashland, USA) was used for the data analysis. RT-PCR
[00124] Die RNA wurde gemäß den Anweisungen des RNA High Pure RNA Kit (Roche, Mannheim, Deutschland) extrahiert und 0,5-5 pg (idealerweise 3 mg) der RNA wurde mit Hilfe der RevertAid Reverse Transkriptase (Thermo Fisher, Darmstadt, Deutschland) gemäß dem Protokoll reverse transkribiert. Die qRT-PCR wurde mit dem Maxima SYBR Green qPCR Mix (ThermoFisher, Darmstadt, Deutschland) auf einem Lightcycler 480 II Real-Time PCR System (Roche, Mannheim, Deutschland) durchgeführt. Die Quantifizierung wurde mit der Methode DD Ct durchgeführt, und der GAPDH-Ausdruck wurde als interne Referenz verwendet. Die Schmelzkurvenanalyse bestätigte, dass alle qRT-PCR-Produkte in Form von doppelsträngiger DNA generiert wurden. Die verwendeten Primer sind in Tabelle 3 aufgeführt. The RNA was extracted according to the instructions of the RNA High Pure RNA Kit (Roche, Mannheim, Germany) and 0.5-5 pg (ideally 3 mg) of the RNA was extracted using the RevertAid Reverse Transcriptase (Thermo Fisher, Darmstadt, Germany) reverse transcribed according to the protocol. The qRT-PCR was carried out with the Maxima SYBR Green qPCR Mix (ThermoFisher, Darmstadt, Germany) on a Lightcycler 480 II Real-Time PCR System (Roche, Mannheim, Germany). The quantification was performed using the DD Ct method and the GAPDH expression was used as an internal reference. The melting curve analysis confirmed that all qRT-PCR products were generated in the form of double-stranded DNA. The primers used are listed in Table 3.
Tabelle 3: Bestimmung der genomweiten DNA-Methylierung und Hydroxymethylierung mittels Table 3: Determination of genome-wide DNA methylation and hydroxymethylation using
Isotopenverdünnung, Flüssigchromatographie, Tandem-Massenspektrometrie (LC-MS/MS) Isotope dilution, liquid chromatography, tandem mass spectrometry (LC-MS / MS)
[00125] Proben genomischer DNA (20 pg) wurden mit mikrokokkaler Nuklease aus Staphylococcus aureus, boviner Milz-Phosphodiesterase und kalbs intestinaler alkalischer Phosphatase (alle von Sigma- Aldrich, Taufkirchen, Deutschland) zu 2'-Deoxynukleosiden hydrolysiert, wie beschrieben [62]mit Anwendungsmodifikationen. Es wurden 10 pL von 50 nM 5-hmdC-d3 (Toronto Research Chemicals, Toronto, Kanada) als interner Standard dem DNA- Aufschlussgemisch zugesetzt und die Inkubationszeit der zweistufigen Hydrolyse betrug jeweils 1 h. Anschließend wurden DNA-Hydrolysate zentrifugiert (5 min, 16.000 x g) und 10 pL der Überstände wurden zur Quantifizierung von dC und 5-mdC stabile markierten Referenzen verwendet. [00125] Genomic DNA (20 pg) samples were hydrolyzed to 2'-deoxynucleosides with 2'-deoxynucleosides as described with micrococal nuclease from Staphylococcus aureus, bovine spleen phosphodiesterase and calf's intestinal alkaline phosphatase (all from Sigma-Aldrich, Taufkirchen, Germany), as described [62] with application modifications. 10 pL of 50 nM 5-hmdC-d3 (Toronto Research Chemicals, Toronto, Canada) were added as an internal standard to the DNA digestion mixture and the incubation time of the two-stage hydrolysis was 1 hour each. DNA hydrolyzates were then centrifuged (5 min, 16,000 x g) and 10 pL of the supernatants were used to quantify dC and 5-mdC stable labeled references.
[00l26]Verbindungen[l5N2,l3Cl]dC und 5-mdC-d3 (beide von Toronto Research Chemicals, Toronto, Kanada). Die Reste der DNA-Hydrolysate (~ 310 pL) wurden mit einem Savant SpeedVac Koncentrator (Thermo Fisher Scientific, Dreieich, Deutschland) unter reduziertem Druck bis zur Trockenheit verdampft. Nach Zugabe von 100 pL Methanol zu den getrockneten Rückständen und kurzem Vortexen wurden die Proben über Nacht bei -20 °C gelagert. Am nächsten Tag wurden die Proben 10 Minuten lang gründlich vortexed (1.400 U/min) und anschließend 10 Minuten lang bei 16.000 x g zentrifugiert. Überstände wurden in neue Probenröhrchen überführt. Die Extraktion der Proteinpellets wurde durch Zugabe von weiteren 100 pL Methanol und Zentrifugation (1.400 U/min) für 5 min wiederholt. Nach der Zentrifugation bei 16.000 x g für 10 min wurden beide methanolischen Fraktionen zusammengegeben und unter vermindertem Druck zur Trockenheit verdampft. Die getrockneten Rückstände wurden in 50 pL Wasser mit einem Gehalt von 0,0075 % Ameisensäure rekonstituiert, das für 10 min ultra-sonifiziert wurde, gefolgt von 5 min Zentrifugation (1.400 U/min) und Zentrifugation für 5 min bei 16.000 x g. Die LC-MS/MS-Analysen der Überstände wurden mit einem Agilent 1260 Infmity LC-System in Verbindung mit einem Agilent 6490 Triple Quadrupol- Massenspektrometer (beide aus Waldbronn, Deutschland) durchgeführt, das mit einer Elektrospray- Ionenquelle im Positiv-Ionen-Modus (ESI+) verbunden ist. Chromatographische Bedingungen und Einstellungen der ESI-Quelle wie für die Quantifizierung von dC und 5-mdC beschrieben[62]. Als Trennsäule wurde eineAgilent Poroshell 120 EC-C18 (2.7 pm, 3.0 x 150 mm) verwendet, das Injektionsvolumen betrug 5 pL. Die Quantifizierung von 5-hmdC in Bezug auf den stabilen isotopenmarkierten Standard, die beide bei 4,9 min von der LC-Säule eluiert wurden (die Retentionszeiten von dC und 5-mdC betrugen 4,7 bzw. 6,0 min), mit Hilfe des Multiplen Reaction Monitoring (MRM)-Ansatzes wurde die Quantifizierung durchgeführt. Dabei wurden folgende Massenübergänge (Verlust von 2'-Desoxyribose) als Quantifizierer (optimierte Kollisionsenergien in Klammern) verwendet: 5-HmdC: m/z 258,1 > 142,0 (8 eV) und 5-HmdC-d3: m/z 261,1 > 145,0 (8 eV). Zur eindeutigen Identifizierung wurden zusätzliche Massenübergänge aufgezeichnet. Die Verweilzeit für jeden der vier analysierten Massenübergänge betrug 50 ms. Compounds [15N2, 13Cl] dC and 5-mdC-d3 (both from Toronto Research Chemicals, Toronto, Canada). The residues of the DNA hydrolysates (~ 310 pL) were evaporated to dryness with a Savant SpeedVac Concentrator (Thermo Fisher Scientific, Dreieich, Germany) under reduced pressure. After adding 100 pL of methanol to the dried residues and briefly vortexing, the samples were stored at -20 ° C. overnight. The next day, the samples were thoroughly vortexed (1400 rpm) for 10 minutes and then centrifuged at 16,000 xg for 10 minutes. Supernatants were transferred to new sample tubes. The extraction of the protein pellets was repeated by adding a further 100 pL of methanol and centrifuging (1,400 rpm) for 5 min. After centrifugation at 16,000 xg for 10 min, both methanolic fractions were combined and evaporated to dryness under reduced pressure. The dried residues were reconstituted in 50 pL of water containing 0.0075% formic acid, which was ultrasonified for 10 min, followed by centrifugation for 5 min (1400 rpm) and centrifugation for 5 min at 16,000 x g. The LC-MS / MS analyzes of the supernatants were carried out using an Agilent 1260 Infmity LC system in conjunction with an Agilent 6490 triple quadrupole mass spectrometer (both from Waldbronn, Germany) using an electrospray ion source in positive ion mode (ESI +) is connected. Chromatographic conditions and settings of the ESI source as described for the quantification of dC and 5-mdC [62]. An Agilent Poroshell 120 EC-C18 (2.7 pm, 3.0 x 150 mm) was used as the separation column, the injection volume was 5 pL. The quantification of 5-hmdC with respect to the stable isotope-labeled standard, both eluted from the LC column at 4.9 min (the retention times of dC and 5-mdC were 4.7 and 6.0 min, respectively) with The quantification was carried out using the multiple reaction monitoring (MRM) approach. The following mass transitions (loss of 2'-deoxyribose) were used as quantifiers (optimized collision energies in brackets): 5-HmdC: m / z 258.1> 142.0 (8 eV) and 5-HmdC-d3: m / z 261.1> 145.0 (8 eV). Additional mass transitions were recorded for clear identification. The dwell time for each of the four mass transitions analyzed was 50 ms.
Versuche an Human IDH1 (R132H/+) HCT116 Cell Line Trials on Human IDH1 (R132H / +) HCT116 Cell Line
[00127]FIG. 29 zeigt die Ergebnisse eines MTT Cytotoxizitätsassays, der wie folgt durchgeführt wurde: • Inkubation mit DKA in steigenden Konzentrationen (100 mM - 10 mM) für 24h, 48h und 72h [00127] FIG. 29 shows the results of an MTT cytotoxicity assay, which was carried out as follows: Incubation with DKA in increasing concentrations (100 mM-10 mM) for 24 h, 48 h and 72 h
Kolonkarzinomzelllinie HCT116 mit heterozygoter Mutation IDH1-(R132H/+) - MT und HCT116 - IDH1-WT Colon carcinoma cell line HCT116 with heterozygous mutation IDH1- (R132H / +) - MT and HCT116 - IDH1-WT
[00l28]FIG. 30 zeigt die Ergebnisse eines Apoptose Assays, der wie folgt durchgeführt wurde: [00l28] FIG. 30 shows the results of an apoptosis assay performed as follows:
• 72h Inkubation mit DKA in steigenden Konzentrationen (100 mM - 10 mM)  72h incubation with DKA in increasing concentrations (100mM - 10mM)
• Kolonkarzinomzelllinie HCT116 mit heterozygoter Mutation IDH1 -(Rl 32H/+) - MT und HCT116 - IDH1-WT • Colon carcinoma cell line HCT116 with heterozygous mutation IDH1 - (Rl 32H / +) - MT and HCT116 - IDH1-WT
Statistik: Two-way ANOVA mit Dunnetts Korrektur- ****= p < 0.0001 Statistics: Two-way ANOVA with Dunnetts correction- **** = p <0.0001
[00129]FIG. 31 zeigt die Ergebnisse eines weiteren Apoptose Assays, der wie folgt durchgeführt wurde:[00129] FIG. 31 shows the results of another apoptosis assay, which was carried out as follows:
• 48h Inkubation DKA mit aufsteigenden Konzentrationen (100 mM - 10 mM) + 24h TNFa(10 ng/mL)/CHX (1 pG/mL) 48h incubation DKA with increasing concentrations (100mM - 10mM) + 24h TNFa (10ng / mL) / CHX (1 pG / mL)
• Kolonkarzinomzelllinie HCT116 mit heterozygoter Mutation IDH1 -(Rl 32H/+) - MT und HCT116 - IDH1-WT • Colon carcinoma cell line HCT116 with heterozygous mutation IDH1 - (Rl 32H / +) - MT and HCT116 - IDH1-WT
Statistik: Two-way ANOVA mit Dunnetts Korrektur- ****= p<0.0001 ; ***= p<0.001 Statistics: Two-way ANOVA with Dunnetts correction- **** = p <0.0001; *** = p <0.001
[00130]FIG. 32 zeigt die Ergebnisse von hmdC-Messungen (verschiedene Darstellungen), die wie folgt durchgeführt wurden: [00130] FIG. 32 shows the results of hmdC measurements (various representations), which were carried out as follows:
• 72h Inkubation mit DKA in steigenden Konzentrationen (100 mM - 10 mM)  72h incubation with DKA in increasing concentrations (100mM - 10mM)
• Kolonkarzinomzelllinie HCT116 mit heterozygoter Mutation IDH1 -(Rl 32H/+) - MT und HCT116 - IDH1-WT • Colon carcinoma cell line HCT116 with heterozygous mutation IDH1 - (Rl 32H / +) - MT and HCT116 - IDH1-WT
Statistik: Two-way ANOVA mit Dunnetts Korrektur- ****= p < 0.0001 ; *= p<0.05 [00l3l]FIG. 33 zeigt die Ergebnisse von hmdC-Messungen (verschiedene Darstellungen), die wie folgt durchgeführt wurden: Statistics: Two-way ANOVA with Dunnetts correction- **** = p <0.0001; * = p <0.05 [00l3l] FIG. 33 shows the results of hmdC measurements (various representations) which were carried out as follows:
hmdC-Messung (andere Darstellung - in % relativ zur unbehandelten Kontrolle als 100 %) hmdC measurement (different representation - in% relative to the untreated control than 100%)
• 72h Inkubation mit DKA in steigenden Konzentrationen (10 mM - 1 mM) 72h incubation with DKA in increasing concentrations (10mM - 1mM)
• Kolonkarzinomzelllinie HCT116 mit heterozygoter Mutation IDH1 -(Rl 32H/+) - MT und HCT116 - IDH1-WT • Colon carcinoma cell line HCT116 with heterozygous mutation IDH1 - (Rl 32H / +) - MT and HCT116 - IDH1-WT
Statistik: Two-way ANOVA mit Dunnetts Korrektur- ****= p < 0.0001; *= p<0.05 Statistics: Two-way ANOVA with Dunnetts correction- **** = p <0.0001; * = p <0.05
[00132] FIG. 34 zeigt einen Western Blot mit HCT116 Zellen, der wie folgt durchgeführt wurde:[00132] FIG. 34 shows a Western blot with HCT116 cells, which was carried out as follows:
• 72h Inkubation mit DKA in steigenden Konzentrationen (100 mM - 1 mM) 72h incubation with DKA in increasing concentrations (100mM - 1mM)
• Kolonkarzinomzelllinie HCT116 mit heterozygoter Mutation IDH1 -(Rl 32H/+) - MT und HCT116 - IDH1-WT • Colon carcinoma cell line HCT116 with heterozygous mutation IDH1 - (Rl 32H / +) - MT and HCT116 - IDH1-WT
[00133]An den Beispielen konnte gezeigte werden, dass der Anspruch eines Cosubstratersatzes und Modulation mit einer atoxischen Beispielsubstanz wie DKG erfolgreich ist. Die Effekte wie sekundäre Apoptose weisen auf eine Rekonstruktion des Zellstoffwechsels hin. The examples showed that the claim of a cosubstrate replacement and modulation with an atoxic example substance such as DKG is successful. The effects such as secondary apoptosis indicate a reconstruction of the cell metabolism.
[00134] Durch den erhöhten bzw. veränderten Energiebedarf von Tumorzellen sind diese nicht mehr in der Lage diesen aus normalen Stoffwechselkreisläufen zu generieren und kommen so durch die Behandlung in eine sekundäre Apoptose. Die Aktivitätsherstellung der TETs führt zu einer Demethylierung des Cytosins und Rückgängigmachung von epigenetischen Veränderungen verbunden mit der Regenerierung von Tumorsuppressorgenen. Due to the increased or changed energy requirements of tumor cells, they are no longer able to generate them from normal metabolic cycles and thus come into secondary apoptosis due to the treatment. The production of activity of the TETs leads to demethylation of the cytosine and reversal of epigenetic changes associated with the regeneration of tumor suppressor genes.
[00135]Dieses alles zeigt die Funktionsfähigkeit von Cosubstrat Ersatz (Modulatoren) zur Einflussnahme auf DIOs und den damit verbundenen verschiedenen pathologischen Zuständen. All of this shows the functionality of cosubstrate substitutes (modulators) for influencing DIOs and the various pathological conditions associated therewith.
[00136] Menschliche DIOs, insbesondere solche, welche die Transkription regulieren, sind Gegenstand aktueller Forschungsansätze für therapeutische Angriffspunkte bei verschiedenen Anämien und Krebserkrankungen. [63-66] Die DIOs haben Einfluss und regulieren einer Vielzahl von Proteinen, was aus der Anfangs aufgezeigten Vielzahl von Reaktionen hergeleitet werden kann. [67] Human DIOs, especially those that regulate transcription, are the subject of current research approaches for therapeutic targets for various anemias and cancer. [63-66] The DIOs influence and regulate a large number of proteins, which can be derived from the large number of reactions shown at the beginning. [67]
[00137] Bekannte und putative 20G-abhängige Dioxygenasen in der GenBank DNA-Datenbank sind in Tabelle 4 dargestellt. Diese kommen gemäß der vorliegenden Erfindung zur Modulation in Betracht: [00137] Known and putative 20G-dependent dioxygenases in the GenBank DNA database are shown in Table 4. According to the present invention, these are suitable for modulation:
Referenzen credentials
1. Flashman, E. and C.J. Schofield, The most versatile of all reactive intermediates? Nature 1. Flashman, E. and C.J. Schofield, The most versatile of all reactive intermediates? Nature
Chemical Biology, 2007. 3: p. 86.  Chemical Biology, 2007. 3: p. 86th
2. Hausinger, R.P., Fe(ll)/a-Ketoglutarate-Dependent Hydroxylases and Related Enzymes. Critical Reviews in Biochemistry and Molecular Biology, 2004. 39(1): p. 21-68.  2. Hausinger, R.P., Fe (ll) / a-Ketoglutarate-Dependent Hydroxylases and Related Enzymes. Critical Reviews in Biochemistry and Molecular Biology, 2004. 39 (1): p. 21-68.
3. Perry, C., et al., Rieske non-heme iron-dependent oxygenases catalyse diverse reactions in natural product biosynthesis. Natural Product Reports, 2018. 35(7): p. 622-632.  3. Perry, C., et al., Rieske non-heme iron-dependent oxygenases catalyze diverse reactions in natural product biosynthesis. Natural Product Reports, 2018. 35 (7): p. 622-632.
4. Loenarz, C. and C.J. Schofield, Physiological and biochemical aspects of hydroxylations and demethylations catalyzed by human 2-oxoglutarate oxygenases. Trends in Biochemical Sciences, 2011. 36(1): p. 7-18.  4. Loenarz, C. and C.J. Schofield, Physiological and biochemical aspects of hydroxylations and demethylations catalyzed by human 2-oxoglutarate oxygenases. Trends in Biochemical Sciences, 2011. 36 (1): p. 7-18.
5. Prescott, A.G. and M.D. Lloyd, The iron(ll) and 2-oxoacid-dependent dioxygenases and their rote in metabolism. Natural Product Reports, 2000. 17(4): p. 367-383.  5. Prescott, A.G. and M.D. Lloyd, The iron (ll) and 2-oxoacid-dependent dioxygenases and their red in metabolism. Natural Product Reports, 2000. 17 (4): p. 367-383.
6. Scotti, J.S., et al., Human oxygen sensing may have origins in prokaryotic elongation factor Tu prolyl-hydroxylation. Proceedings of the National Academy of Sciences, 2014. 111(37): p. 13331-13336.  6. Scotti, J.S., et al., Human oxygen sensing may have origins in prokaryotic elongation factor Tu prolyl-hydroxylation. Proceedings of the National Academy of Sciences, 2014. 111 (37): p. 13,331 to 13,336.
7. Clifton, I.J., et al., Crystal Structure of Carbapenem Synthase (CarC). Journal of Biological Chemistry, 2003. 278(23): p. 20843-20850.  7. Clifton, I.J., et al., Crystal Structure of Carbapenem Synthase (CarC). Journal of Biological Chemistry, 2003. 278 (23): p. 20843-20850.
8. Farrow, S.C. and P.J. Facchini, Functional diversity of 2-oxoglutarate/Fe(ll)-dependent  8. Farrow, S.C. and P.J. Facchini, Functional diversity of 2-oxoglutarate / Fe (ll) -dependent
dioxygenases in plant metabolism. Frontiers in Plant Science, 2014. 5: p. 524.  dioxygenases in plant metabolism. Frontiers in Plant Science, 2014. 5: p. 524th
9. Cheng, A.X., et al., The function and catalysis of 2-oxoglutarate-dependent oxygenases  9. Cheng, A.X., et al., The function and catalysis of 2-oxoglutarate-dependent oxygenases
involved in plant flavonoid biosynthesis. Int J Mol Sei, 2014. 15(1): p. 1080-95.  involved in plant flavonoid biosynthesis. Int J Mol Sei, 2014. 15 (1): p. 1080-95.
10. Zhang, Z., et al., Crystal Structure and Mechanistic Implications of 1-Aminocyclopropane-l- Carboxylic Acid Oxidase— The Ethylene-Forming Enzyme. Chemistry & Biology, 2004. 11(10): p. 1383-1394.  10. Zhang, Z., et al., Crystal Structure and Mechanistic Implications of 1-Aminocyclopropane-l-Carboxylic Acid Oxidase - The Ethylene-Forming Enzyme. Chemistry & Biology, 2004. 11 (10): p. 1383-1394.
11. Myllyharju, J., Prolyl 4-hydroxyiases, the key enzymes of Collagen biosynthesis. Matrix  11. Myllyharju, J., Prolyl 4-hydroxyiases, the key enzymes of collagen biosynthesis. matrix
Biology, 2003. 22(1): p. 15-24.  Biology, 2003. 22 (1): p. 15-24.
12. Leung, I.K.H., et al., Structural and Mechanistic Studies on y-Butyrobetaine Hydroxylase.  12. Leung, I.K.H., et al., Structural and Mechanistic Studies on y-Butyrobetaine Hydroxylase.
Chemistry & Biology, 2010. 17(12): p. 1316-1324.  Chemistry & Biology, 2010. 17 (12): p. From 1316 to 1324.
13. Markolovic, S., S.E. Wilkins, and C.J. Schofield, Protein Hydroxylation Catalyzed by 2- Oxoglutarate-dependent Oxygenases. Journal of Biological Chemistry, 2015. 290(34): p. 20712-20722.  13. Markolovic, S., S.E. Wilkins, and C.J. Schofield, Protein Hydroxylation Catalyzed by 2-Oxoglutarate-dependent Oxygenases. Journal of Biological Chemistry, 2015. 290 (34): p. 20,712 to 20,722.
14. Walport, L.J., R.J. Hopkinson, and C.J. Schofield, Mechanisms of human histone and nucleic acid demethylases. Current Opinion in Chemical Biology, 2012. 16(5): p. 525-534.  14. Walport, L.J., R.J. Hopkinson, and C.J. Schofield, Mechanisms of human histone and nucleic acid demethylases. Current Opinion in Chemical Biology, 2012. 16 (5): p. 525-534.
15. Salminen, A., A. Kauppinen, and K. Kaarniranta, 2-Oxoglutarate-dependent dioxygenases are sensors of energy metabolism , oxygen availability , and iron homeostasis: potential role in the regulation of aging process. Cellular and Molecular Life Sciences, 2015. 72(20): p. 3897-3914. 15. Salminen, A., A. Kauppinen, and K. Kaarniranta, 2-Oxoglutarate-dependent dioxygenases are sensors of energy metabolism, oxygen availability, and iron homeostasis: potential role in the regulation of aging process. Cellular and Molecular Life Sciences, 2015. 72 (20): p. 3897-3914.
16. Martinez, S. and R.P. Hausinger, Catalytic Mechanisms of Fe(ll)- and 2-Oxoglutarate- dependent Oxygenases. Journal of Biological Chemistry, 2015. 290(34): p. 20702-20711.16. Martinez, S. and R.P. Hausinger, Catalytic Mechanisms of Fe (ll) - and 2-Oxoglutarate-dependent Oxygenases. Journal of Biological Chemistry, 2015. 290 (34): p. 20,702 to 20,711.
17. Myllylä, R., et al., Ascorbate is consumed stoichiometrically in the uncoupled reactions 17. Myllyla, R., et al., Ascorbate is consumed stoichiometrically in the uncoupled reactions
catalyzed by prolyl 4-hydroxylase and lysyl hydroxylase. Journal of Biological Chemistry, 1984. 259(9): p. 5403-5405.  catalyzed by prolyl 4-hydroxylase and lysyl hydroxylase. Journal of Biological Chemistry, 1984. 259 (9): p. From 5403 to 5405.
18. Flashman, E., et al., Investigating the dependence of the hypoxia-inducible factor  18. Flashman, E., et al., Investigating the dependence of the hypoxia-inducible factor
hydroxylases (factor inhibiting HIF and prolyl hydroxylase domain 2) on ascorbate and other reducing agents. Biochem J, 2010. 427(1): p. 135-42.  hydroxylases (factor inhibiting HIF and prolyl hydroxylase domain 2) on ascorbate and other reducing agents. Biochem J, 2010. 427 (1): p. 135-42.
19. Lappin, T. and N. Masson, Two antioxidants are better than one. Blood, 2011. 117(20): p.  19. Lappin, T. and N. Masson, Two antioxidants are better than one. Blood, 2011. 117 (20): p.
5276.  5276th
20. Mastrangelo, D., et al., Mechanisms of anti -cancer effects of ascorbate: Cytotoxic activity and epigenetic modulation. Blood Cells, Molecules, and Diseases, 2018. 69: p. 57-64. Shenoy, N., et al., Ascorbic Acid in Cancer Treatment: Let the Phoenix Fly. Cancer Cell, 2018. Solomon, E.I., A. Decker, and N. Lehnert, Non-heme iron enzymes: Contrasts to heme catalysis. Proceedings of the National Academy of Sciences, 2003. 100(7): p. 3589. 20. Mastrangelo, D., et al., Mechanisms of anti-cancer effects of ascorbate: Cytotoxic activity and epigenetic modulation. Blood Cells, Molecules, and Diseases, 2018. 69: p. 57-64. Shenoy, N., et al., Ascorbic Acid in Cancer Treatment: Let the Phoenix Fly. Cancer Cell, 2018. Solomon, EI, A. Decker, and N. Lehnert, Non-heme iron enzymes: Contrasts to heme catalysis. Proceedings of the National Academy of Sciences, 2003. 100 (7): p. 3589th
Huang, C.W., et al., The different catalytic roles of the metal-binding ligands in human 4- hydroxyphenylpyruvate dioxygenase. Biochem J, 2016. 473(9): p. 1179-89. Huang, C.W., et al., The different catalytic roles of the metal-binding ligands in human 4-hydroxyphenylpyruvate dioxygenase. Biochem J, 2016. 473 (9): p. 1179-89.
Hewitson, K.S., et al., Oxidation by 2-oxoglutarate oxygenases: non-haem iron Systems in catalysis and signalling. Philosophical Transactions of the Royal Society A: Mathematical, Physical and Engineering Sciences, 2005. 363(1829): p. 807. Hewitson, K.S., et al., Oxidation by 2-oxoglutarate oxygenases: non-haem iron systems in catalysis and signaling. Philosophical Transactions of the Royal Society A: Mathematical, Physical and Engineering Sciences, 2005. 363 (1829): p. 807th
Raili, M., T. Leena, and K.K. I., Mechanism of the Prolyl Hydroxylase Reaction. European Journal of Biochemistry, 1977. 80(2): p. 349-357. Raili, M., T. Leena, and K.K. I., Mechanism of the Prolyl Hydroxylase Reaction. European Journal of Biochemistry, 1977. 80 (2): p. 349-357.
Price, J.C., et al., The First Direct Characterization of a High-Valent Iron Intermediate in the Reaction of an a-Ketoglutarate-Dependent Dioxygenase: A High-Spin Fe(IV) Complex in Taurine/a-Ketoglutarate Dioxygenase (TauD)from Escherichia coli. Biochemistry, 2003. 42(24): p. 7497-7508. Price, JC, et al., The First Direct Characterization of a High-Valent Iron Intermediate in the Reaction of an a-Ketoglutarate-Dependent Dioxygenase: A High-Spin Fe (IV) Complex in Taurine / a-Ketoglutarate Dioxygenase (TauD) from Escherichia coli. Biochemistry, 2003. 42 (24): p. 7497 to 7508.
Proshlyakov, D.A., et al., Direct Detection of Oxygen Intermediates in the Non-Heme Fe Enzyme Taurine/a-Ketoglutarate Dioxygenase. Journal of the American Chemical Society, 2004. 126(4): p. 1022-1023. Proshlyakov, D.A., et al., Direct Detection of Oxygen Intermediates in the Non-Heme Fe Enzyme Taurine / a-Ketoglutarate Dioxygenase. Journal of the American Chemical Society, 2004. 126 (4): p. 1022 to 1023.
Welford, R.W., et al., Incorporation of oxygen into the succinate co-product ofiron(ll) and 2- oxoglutarate dependent oxygenases from bacteria, plants and humans. FEBS Lett, 2005. 579(23): p. 5170-4. Welford, R.W., et al., Incorporation of oxygen into the succinate co-product ofiron (ll) and 2-oxoglutarate dependent oxygenases from bacteria, plants and humans. FEBS Lett, 2005. 579 (23): p. 5170-4.
Grzyska, P. K., et al., Insight into the mechanism of an iron dioxygenase by resolution of Steps following the FelV=HO species. Proc Natl Acad Sei U S A, 2010. 107(9): p. 3982-7. Grzyska, P.K., et al., Insight into the mechanism of an iron dioxygenase by resolution of Steps following the FelV = HO species. Proc Natl Acad Sei U S A, 2010. 107 (9): p. 3982-7.
Valegärd, K., et al., The structural basis of Cephalosporin formation in a mononuclear ferrous enzyme. Nature Structural &Amp; Molecular Biology, 2003. 11: p. 95. Valegard, K., et al., The structural basis of cephalosporin formation in a mononuclear ferrous enzyme. Nature Structural & Molecular Biology, 2003. 11: p. 95th
Wiek, C.R., et al., Structural Insight into the Prolyl Hydroxylase PHD2: A Molecular Dynamics and DFT Study. European Journal of Inorganic Chemistry, 2012. 2012(31): p. 4973-4985. Tarhonskaya, H., et al., Studies on Deacetoxycephalosporin C Synthase Support a Consensus Mechanism for 2-Oxoglutarate Dependent Oxygenases. Biochemistry, 2014. 53(15): p. 2483- 2493. Wiek, C.R., et al., Structural Insight into the Prolyl Hydroxylase PHD2: A Molecular Dynamics and DFT Study. European Journal of Inorganic Chemistry, 2012. 2012 (31): p. 4973 to 4985. Tarhonskaya, H., et al., Studies on Deacetoxycephalosporin C Synthase Support a Consensus Mechanism for 2-Oxoglutarate Dependent Oxygenases. Biochemistry, 2014. 53 (15): p. 2483-2493.
McDonough, M.A., et al., Structural studies on human 2-oxoglutarate dependent oxygenases. Current Opinion in Structural Biology, 2010. 20(6): p. 659-672. McDonough, M.A., et al., Structural studies on human 2-oxoglutarate dependent oxygenases. Current Opinion in Structural Biology, 2010. 20 (6): p. 659-672.
Clifton, I.J., et al., Structural studies on 2-oxoglutarate oxygenases and related double- stranded beta-helix fold proteins. J Inorg Biochem, 2006. 100(4): p. 644-69. Clifton, I.J., et al., Structural studies on 2-oxoglutarate oxygenases and related double-stranded beta-helix fold proteins. J Inorg Biochem, 2006. 100 (4): p. 644-69.
L, H.E. and J.L. Que, The 2-His-l-Carboxylate Facial Triad— An Emerging Structural Motifin Mononuclear Non-Heme Iron(ll) Enzymes. European Journal of Biochemistry, 1997. 250(3): p. 625-629. L, H.E. and J.L. Que, The 2-His-l-Carboxylate Facial Triad - An Emerging Structural Motifin Mononuclear Non-Heme Iron (II) Enzymes. European Journal of Biochemistry, 1997. 250 (3): p. 625-629.
Chowdhury, R., et al., Structural basis for binding of hypoxia-inducible factor to the oxygen- sensing prolyl hydroxylases. Structure, 2009. 17(7): p. 981-9. Chowdhury, R., et al., Structural basis for binding of hypoxia-inducible factor to the oxygen-sensing prolyl hydroxylases. Structure, 2009. 17 (7): p. 981-9.
Deng, G., et al., Novel complex crystal structure of prolyl hydroxylase domain-containing protein 2 (PHD2): 2,8-Diazaspiro[4.5]decan-l-ones as potent, orally bioavailable PHD2 Inhibitors. Bioorg Med Chem, 2013. 21(21): p. 6349-58. Deng, G., et al., Novel complex crystal structure of prolyl hydroxylase domain-containing protein 2 (PHD2): 2,8-diazaspiro [4.5] decan-l-ones as potent, orally bioavailable PHD2 inhibitors. Bioorg Med Chem, 2013. 21 (21): p. 6349-58.
Chowdhury, R., et al., Structural basis for oxygen degradation domain selectivity of the HIF prolyl hydroxylases. Nature Communications, 2016. 7: p. 12673. Chowdhury, R., et al., Structural basis for oxygen degradation domain selectivity of the HIF prolyl hydroxylases. Nature Communications, 2016. 7: p. 12,673th
Willam, C., et al., The prolyl hydroxylase enzymes that act as oxygen sensors regulating destruction of hypoxia-inducible factor alpha. Adv Enzyme Regul, 2004. 44: p. 75-92. Willam, C., et al., The prolyl hydroxylase enzymes that act as oxygen sensors regulating destruction of hypoxia-inducible factor alpha. Adv Enzyme Regul, 2004. 44: p. 75-92.
Rabe, P., et al., Roles of 2-oxoglutarate oxygenases and isopenicillin N synthase in 6-lactam biosynthesis. Natural Product Reports, 2018. Rabe, P., et al., Roles of 2-oxoglutarate oxygenases and isopenicillin N synthase in 6-lactam biosynthesis. Natural Product Reports, 2018.
Hegg, E.L. and L.Q. Jr, The 2-His-l-Carboxylate Facial Triad— An Emerging Structural Motif in Mononuclear Non-Heme Iron(ll) Enzymes. European Journal of Biochemistry, 1997. 250(3): p. 625-629. Kal, S. and L. Que, Dioxygen activation by nonheme iron enzymes with the 2-His-l-carboxylate facial triad that generate high-valent oxoiron oxidants. JBIC Journal of Biological Inorganic Chemistry, 2017. 22(2): p. 339-365. Hegg, EL and LQ Jr, The 2-His-l-Carboxylate Facial Triad - An Emerging Structural Motif in Mononuclear Non-Heme Iron (ll) Enzymes. European Journal of Biochemistry, 1997. 250 (3): p. 625-629. Kal, S. and L. Que, Dioxygen activation by nonheme iron enzymes with the 2-His-l-carboxylate facial triad that generate high-valent oxoiron oxidants. JBIC Journal of Biological Inorganic Chemistry, 2017. 22 (2): p. 339-365.
Xu, W., et al., Oncometabolite 2-Hydroxyglutarate Is a Competitive Inhibitor of a- Ketoglutarate-Dependent Dioxygenases. Cancer Cell, 2011. 19(1): p. 17-30. Xu, W., et al., Oncometabolite 2-Hydroxyglutarate Is a Competitive Inhibitor of a-Ketoglutarate-Dependent Dioxygenases. Cancer Cell, 2011. 19 (1): p. 17-30.
Xu, W., et al., Oncometabolite 2-hydroxygiutarate is a competitive inhibitor of alpha- ketoglutarate-dependent dioxygenases. Cancer Cell, 2011. 19(1): p. 17-30. Xu, W., et al., Oncometabolite 2-hydroxygiutarate is a competitive inhibitor of alphaketoglutarate-dependent dioxygenases. Cancer Cell, 2011. 19 (1): p. 17-30.
Elkashef, S.M., et al., IDH Mutation, Competitive Inhibition of FTO, and RNA Methylation. Cancer Cell, 2017. 31(5): p. 619-620. Elkashef, S.M., et al., IDH Mutation, Competitive Inhibition of FTO, and RNA Methylation. Cancer Cell, 2017. 31 (5): p. 619-620.
Flavahan, W.A., et al., Insulator dysfunction and oncogene activation in IDH mutant gliomas. Nature, 2016. 529(7584): p. 110-4. Flavahan, W.A., et al., Insulator dysfunction and oncogene activation in IDH mutant gliomas. Nature, 2016. 529 (7584): p. 110-4.
Buege, M.J., A.J. DiPippo, and C.D. DiNardo, Evolving Treatment Strategies for Elderly Leukemia Patients with IDH Mutations. Cancers (Basel), 2018. 10(6). Buege, M.J., A.J. DiPippo, and C.D. DiNardo, Evolving Treatment Strategies for Elderly Leukemia Patients with IDH Mutations. Cancers (Basel), 2018. 10 (6).
Rautio, J., et al., The expanding role of prodrugs in Contemporary drug design and development. Nat Rev Drug Discov, 2018. 17(8): p. 559-587. Rautio, J., et al., The expanding role of prodrugs in Contemporary drug design and development. Nat Rev Drug Discov, 2018. 17 (8): p. 559-587.
van Gorkom, G., et al., Influence of Vitamin C on Lymphocytes: An OverView. Antioxidants, 2018. 7(3): p. 41. van Gorkom, G., et al., Influence of Vitamin C on Lymphocytes: An OverView. Antioxidants, 2018. 7 (3): p. 41st
Cimmino, L, B.G. Neel, and I. Aifantis, Vitamin C in Stern Cell Reprogramming and Cancer. Trends Cell Biol, 2018. Cimmino, L, B.G. Neel, and I. Aifantis, Vitamin C in Stern Cell Reprogramming and Cancer. Trends Cell Biol, 2018.
Li, X., et al., Ten-eleven translocation 2 demethylates the MMP9 promoter, and its down- regulation in preeclampsia impairs trophoblast migration and invasion. J Biol Chem, 2018. Sant, D.W., et al., Vitamin C promotes apoptosis in breast cancer cells by increasing TRAIL expression. Scientific Reports, 2018. 8(1): p. 5306. Li, X., et al., Ten-eleven translocation 2 demethylates the MMP9 promoter, and its down-regulation in preeclampsia impairs trophoblast migration and invasion. J Biol Chem, 2018. Sant, D.W., et al., Vitamin C promotes apoptosis in breast cancer cells by increasing TRAIL expression. Scientific Reports, 2018. 8 (1): p. 5306th
Park, S., et al., Vitamin C in Cancer: A Metabolomics Perspective. Frontiers in Physiology, 2018. 9(762). Park, S., et al., Vitamin C in Cancer: A Metabolomics Perspective. Frontiers in Physiology, 2018. 9 (762).
Monacelli, F., et al., Vitamin C, Aging and Alzheimer's Disease. Nutrients, 2017. 9(7). Monacelli, F., et al., Vitamin C, Aging and Alzheimer's Disease. Nutrients, 2017. 9 (7).
Ebata, K.T., et al., Vitamin C induces specific demethylation of H3K9me2 in mouse embryonic stem cells via Kdm3a/b. Epigenetics & Chromatin, 2017. 10(1): p. 36. Ebata, K.T., et al., Vitamin C induces specific demethylation of H3K9me2 in mouse embryonic stem cells via Kdm3a / b. Epigenetics & Chromatin, 2017. 10 (1): p. 36th
D'Aniello, C., et al., Vitamin C and I-Proline Antagonistic Effects Capture Alternative States in the Pluripotency Continuum. Stem Cell Reports, 2017. 8(1): p. 1-10. D'Aniello, C., et al., Vitamin C and I-Proline Antagonistic Effects Capture Alternative States in the Pluripotency Continuum. Stem Cell Reports, 2017. 8 (1): p. 1-10.
Schonberger, K. and N. Cabezas-Wallscheid, Vitamin C: C-ing a New Way to Fight Leukemia. Cell Stem Cell, 2017. 21(5): p. 561-563. Schonberger, K. and N. Cabezas-Wallscheid, Vitamin C: C-ing a New Way to Fight Leukemia. Cell Stem Cell, 2017. 21 (5): p. 561-563.
Unlu, A., et al., High-dose vitamin C and cancer. Journal of Oncological Science, 2016. 1: p. 10-12. Unlu, A., et al., High-dose vitamin C and cancer. Journal of Oncological Science, 2016. 1: p. 10-12.
Li, R., Vitamin C, a Multi-Tasking Molecule, Finds a Molecular Target in Killing Cancer Cells. React Oxyg Species (Apex), 2016. 1(2): p. 141-156. Li, R., Vitamin C, a Multi-Tasking Molecule, Finds a Molecular Target in Killing Cancer Cells. React Oxyg Species (Apex), 2016. 1 (2): p. 141-156.
Levine, M. and P.-C. Violet, Data Triumph at C. Cancer Cell, 2017. 31(4): p. 467-469. Levine, M. and P.-C. Violet, Data Triumph at C. Cancer Cell, 2017. 31 (4): p. 467-469.
Monfort, A. and A. Wutz, Breathing-in epigenetic change with vitamin C. EMBO Rep, 2013. 14(4): p. 337-46. Monfort, A. and A. Wutz, Breathing-in epigenetic change with vitamin C. EMBO Rep, 2013. 14 (4): p. 337-46.
Schumacher, F., et al., Optimized enzymatic hydrolysis of DNA for LC-MS/MS analyses of adducts of l-methoxy-3-indolylmethyl glucosinolate and methyleugenol. Anal Biochem, 2013. 434(1): p. 4-11. Schumacher, F., et al., Optimized enzymatic hydrolysis of DNA for LC-MS / MS analyzes of adducts of l-methoxy-3-indolylmethyl glucosinolate and methyleugenol. Anal Biochem, 2013. 434 (1): p. 4-11.
Islam, M.S., et al., 2-Oxoglutarate-Dependent Oxygenases. Annual Review of Biochemistry, 2018. 87(1): p. 585-620. Islam, M.S., et al., 2-Oxoglutarate-Dependent Oxygenases. Annual Review of Biochemistry, 2018. 87 (1): p. 585-620.
Lv, X., et al., The role of hypoxia-inducible factors in tumor angiogenesis and cell metabolism. Genes & Diseases, 2017. 4(1): p. 19-24. Lv, X., et al., The role of hypoxia-inducible factors in tumor angiogenesis and cell metabolism. Genes & Diseases, 2017. 4 (1): p. 19-24.
Wigerup, C., S. Pahlman, and D. Bexell, Therapeutic targeting of hypoxia and hypoxia- inducible factors in cancer. Pharmacol Ther, 2016. 164: p. 152-69. Wigerup, C., S. Pahlman, and D. Bexell, Therapeutic targeting of hypoxia and hypoxia-inducible factors in cancer. Pharmacol Ther, 2016. 164: p. 152-69.
Xia, Y., H.K. Choi, and K. Lee, Recent advances in hypoxia-inducible factor (HIF)-l inhibitors.Xia, Y., H.K. Choi, and K. Lee, Recent advances in hypoxia-inducible factor (HIF) -l inhibitors.
Eur J Med Chem, 2012. 49: p. 24-40. 67. Semenza, G.L., A compendium of proteins that interact with HIF-lalpha. Exp Cell Res, 2017. 356(2): p. 128-135. Eur J Med Chem, 2012. 49: p. 24-40. 67. Semenza, GL, A compendium of proteins that interact with HIF-lalpha. Exp Cell Res, 2017. 356 (2): p. 128-135.

Claims

ANSPRÜCHE EXPECTATIONS
1. Eine Verbindung ausgewählt aus den Verbindungen gemäß Formel (I) oder Formel (II) 1. A compound selected from the compounds of the formula (I) or formula (II)
wobei  in which
As die Aminosäuren aus der Bindungstasche des Enzyms darstellen;  As represent the amino acids from the binding pocket of the enzyme;
Me ein Metall aus dem katalytischen Zentrum;  Me a metal from the catalytic center;
Rl und R2 Sauerstoff (Hydroxyl) oder Carboxylgruppen, Halogene, insbesondere Fluor, Chor, oder Iod, eine einfach bis mehrfach halogenierte Methylgruppe, insbesondere CH2F bis zu CF3 sind; und  Rl and R2 are oxygen (hydroxyl) or carboxyl groups, halogens, in particular fluorine, chlorine, or iodine, a single to multiple halogenated methyl group, in particular CH2F up to CF3; and
Cn ein C-Atom, ein Heteroatom oder die Brücke zu einen Heterocyclus repräsentiert.  Cn represents a C atom, a hetero atom or the bridge to a heterocycle.
2. Die Verbindung nach Anspruch 1, wobei Rl Wasserstoff oder eine CH2R3 -Gruppe ist, wobei R3 Wasserstoff oder Sauerstoff (Hydroxyl, Carbonyl) oder eine kürzere C-Kette (Ci bis C4) ist. 2. The compound of claim 1, wherein Rl is hydrogen or a CH 2 R 3 group, wherein R 3 is hydrogen or oxygen (hydroxyl, carbonyl) or a shorter C chain (Ci to C 4 ).
3. Die Verbindung nach Anspruch 1 oder 2, wobei die Verbindung Teil eines Ringsystems ist. 3. The compound of claim 1 or 2, wherein the compound is part of a ring system.
4. Die Verbindung nach Anspruch 3, wobei die Ringgröße zwischen 3 bis 5 Atomen liegt mit zumindest einem Heteroatom. 4. The compound of claim 3, wherein the ring size is between 3 to 5 atoms with at least one hetero atom.
5. Die Verbindung nach einem der Ansprüche 1 bis 4, wobei C7 aus einer Kohlenstoffkette mit bis zu 5 Atomen besteht und Doppelbindungen enthält. 5. The compound according to any one of claims 1 to 4, wherein C7 consists of a carbon chain with up to 5 atoms and contains double bonds.
6. Die Verbindung nach einem der Ansprüche 1 bis 5, wobei R2 eine Carbonsäure repräsentiert. 6. The compound according to any one of claims 1 to 5, wherein R2 represents a carboxylic acid.
7. Die Verbindung nach einem der Ansprüche 1 bis 6, wobei in einer Verbindung nach Formel (II) R2 ein Wasserstoffatom, eine Methylgruppe, eine Alkylgruppe mit bis zu 6 C-Atomen repräsentiert, die verzweigt gesättigt oder ungesättigt sein können oder selbst auch ein Heteroatom enthalten. 7. The compound according to any one of claims 1 to 6, wherein in a compound of formula (II) R2 represents a hydrogen atom, a methyl group, an alkyl group having up to 6 carbon atoms, which can be branched saturated or unsaturated or even one Heteroatom included.
8. Die Verbindung nach einem der Ansprüche 1 bis 7, wobei in einer Verbindung nach Formel (II) zwischen Rl und R2 eine brückenbildende zyklische Struktur angeordnet ist, die ungesättigte oder gesättigte Heteroatome enthält. 8. The compound according to any one of claims 1 to 7, wherein in a compound of formula (II) between Rl and R2 a bridge-forming cyclic structure is arranged, which contains unsaturated or saturated heteroatoms.
9. Die Verbindung nach einem der Ansprüche 1 bis 8, wobei Mischungen der die Verbindungen nach Formel (I) und Formel (II) verwendet werden. 9. The compound according to any one of claims 1 to 8, wherein mixtures of the compounds of formula (I) and formula (II) are used.
10. Die Verbindung nach einem der Ansprüche 1 bis 9, wobei pharmazeutisch akzeptable Salze und Tautomere der jeweiligen Verbindung allein oder in Kombination in zuvor definierten Mischungsverhältnissen verwendet werden. 10. The compound according to any one of claims 1 to 9, wherein pharmaceutically acceptable salts and tautomers of the respective compound are used alone or in combination in previously defined mixing ratios.
11. Eine V erwendung einer V erbindung nach einem der Ansprüche 1 bis 10 als Medikament. 11. A use of a compound according to any one of claims 1 to 10 as a medicament.
12. Eine Verwendung einer Verbindung nach einem der Ansprüche 1 bis 10 als Cosubstrat mit anderen Wirkstoffen in einem Medikament. 12. A use of a compound according to any one of claims 1 to 10 as a cosubstrate with other active ingredients in a medicament.
13. Die Verwendung nach Anspruch 12, wobei die anderen Wirkstoffe ausgewählt sind aus der Gruppe umfassend Chemotherapeutika, Zytostatika, wie Alkylantien, Antimetabolite, Topoisomerase Hemmer, Mitosehemmstoffe, Antibiotika, Antikörper, Kinaseinhibitoren, Proteosominhibitoren und supportive Arzneistoffe der Tumortherapie wie insbesondere Interferone, Zytokine, Tumornekrosefaktor und IDH-Hemmer. 13. The use according to claim 12, wherein the other active ingredients are selected from the group comprising chemotherapeutics, cytostatics, such as alkylating agents, antimetabolites, topoisomerase inhibitors, mitotic inhibitors, antibiotics, antibodies, kinase inhibitors, proteosome inhibitors and supportive drugs for tumor therapy, such as in particular interferons, cytokines, Tumor necrosis factor and IDH inhibitor.
14. Eine Verwendung einer Verbindung nach einem der Ansprüche 1 bis 10 als Medikament zur Prävention, Behandlung oder Nachsorge von Krebserkrankungen, Neurodegenerativen Erkrankungen sowie von angeborenen oder erworbenen metabolischen Störungen. 14. A use of a compound according to any one of claims 1 to 10 as a medicament for the prevention, treatment or aftercare of cancer, neurodegenerative diseases and of congenital or acquired metabolic disorders.
15. Eine Verwendung einer Verbindung nach einem der Ansprüche 1 bis 10 zur Herstellung eines Medikaments für die Prävention, Behandlung oder Nachsorge von Krebserkrankungen, Neurodegenerativen Erkrankungen sowie von angeborenen oder erworbenen metabolischen Störungen. 15. A use of a compound according to any one of claims 1 to 10 for the manufacture of a medicament for the prevention, treatment or aftercare of cancer, Neurodegenerative diseases and congenital or acquired metabolic disorders.
16. Ein Medikament umfassend eine Verbindung nach einem der Ansprüche 1 bis 10.  16. A medicament comprising a compound according to any one of claims 1 to 10.
EP19750128.1A 2018-08-10 2019-08-12 Compounds for modulation and as functional replacement of alpha-ketoglutaric acid (2og)-dependent oxygenases Pending EP3762038A1 (en)

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LU100900A LU100900B1 (en) 2018-08-10 2018-08-10 COMPOUNDS FOR MODULATING A-KETOGLUTARIC ACID (2KG) -DEPENDENT OXYGENASES
PCT/EP2019/071620 WO2020030826A1 (en) 2018-08-10 2019-08-12 Compounds for modulation and as functional replacement of alpha-ketoglutaric acid (2og)-dependent oxygenases

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