EP4330382A2 - Médicaments in vitro dérivés de promédicaments et leur utilisation - Google Patents

Médicaments in vitro dérivés de promédicaments et leur utilisation

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Publication number
EP4330382A2
EP4330382A2 EP22725220.2A EP22725220A EP4330382A2 EP 4330382 A2 EP4330382 A2 EP 4330382A2 EP 22725220 A EP22725220 A EP 22725220A EP 4330382 A2 EP4330382 A2 EP 4330382A2
Authority
EP
European Patent Office
Prior art keywords
prodrug
hydrogen
enzymes
activated
iupac
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP22725220.2A
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German (de)
English (en)
Inventor
Friedrich Jung
Jan-Heiner KÜPPER
Katrin Scheibner
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Brandenburgische Technische Universitaet Cottbus
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Brandenburgische Technische Universitaet Cottbus
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Application filed by Brandenburgische Technische Universitaet Cottbus filed Critical Brandenburgische Technische Universitaet Cottbus
Publication of EP4330382A2 publication Critical patent/EP4330382A2/fr
Pending legal-status Critical Current

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Classifications

    • 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.)
    • C12N9/0065—Oxidoreductases (1.) acting on hydrogen peroxide as acceptor (1.11)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/44—Oxidoreductases (1)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/13—Amines
    • A61K31/135—Amines having aromatic rings, e.g. ketamine, nortriptyline
    • A61K31/138—Aryloxyalkylamines, e.g. propranolol, tamoxifen, phenoxybenzamine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/4353—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/4365—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system having sulfur as a ring hetero atom, e.g. ticlopidine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/445—Non condensed piperidines, e.g. piperocaine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y111/00—Oxidoreductases acting on a peroxide as acceptor (1.11)
    • C12Y111/02—Oxidoreductases acting on a peroxide as acceptor (1.11) with H2O2 as acceptor, one oxygen atom of which is incorporated into the product (1.11.2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y111/00—Oxidoreductases acting on a peroxide as acceptor (1.11)
    • C12Y111/02—Oxidoreductases acting on a peroxide as acceptor (1.11) with H2O2 as acceptor, one oxygen atom of which is incorporated into the product (1.11.2)
    • C12Y111/02001—Unspecific peroxygenase (1.11.2.1)

Definitions

  • the subject matter of the invention is a process for the production of a medicament, in which a prodrug, ie such a prodrug with bioactivation by cytochrome P450 enzymes, is mixed with at least one peroxygenase and the product obtained is isolated.
  • a prodrug ie such a prodrug with bioactivation by cytochrome P450 enzymes
  • the invention furthermore relates to selected prodrugs for this purpose, in particular those such as thienopyridines and the products obtained according to the invention and their use.
  • liver is a central detoxification organ of the metabolism.
  • Liver cells hepatocytes represent 70-80% of all cells in the liver and are responsible for the important physiological liver functions.
  • the liver uses biotransformation or metabolism in order to be able to excrete or detoxify ingested substances (e.g. drugs, toxins, natural products).
  • ingested substances e.g. drugs, toxins, natural products.
  • the phase I enzymes of the cytochrome P-450 (CYP450) system are particularly important for biotransformation.
  • the CYP450 enzymes are oxidoreductases that cause an oxidative degradation or metabolization of numerous substances such as drugs.
  • CYP450 isoenzymes with different substrate specificities that exist in humans only the isoenzymes CYP1A2, -2C9, -2C19, -2D6, - 2E1 and -3A4 responsible for about 90% of all oxidative metabolism of drugs. In many cases, a large number of drugs only achieve their curative effectiveness through these biochemical changes or even cause toxic metabolites with undesirable drug effects.
  • Such curative liver metabolites are to be produced in sufficient quantities; in particular, these liver metabolites mostly have a high regioselective and stereoselective specificity as stereoisomers. Such regio- and stereoselective modifications are characteristic of biotransformative liver enzymes.
  • Phase I enzymes have been described for biotransformation or metabolism in liver cells, in particular the cytochrome P-450 (CYP450) system, so-called oxidoreductases.
  • CYP450 cytochrome P-450
  • biotransformational enzymes can also occur in other organisms, such as fungi and bacteria, due to evolutionary factors.
  • WO 2008/119780 A2 describes a method for the enzymatic hydroxylation of non-activated hydrocarbons, in particular aromatic rings of non-activated hydrocarbon molecules (for example the selective conversion of naphthalene to 1-naphthol) by using fungal peroxidases from basidiomycetes of the Bolbitiaceae family (e.g. Agrocybe sp. ) for the production of pharmaceuticals, terpenes, steroids or fatty acids.
  • fungal peroxidases from basidiomycetes of the Bolbitiaceae family (e.g. Agrocybe sp. ) for the production of pharmaceuticals, terpenes, steroids or fatty acids.
  • DE 102008034829 A1 discloses a one-step, enzymatic process for the regioselective hydroxylation of 2-phenoxypropionic acid to 2-(4-hydroxyphenoxy)propionic acid.
  • the enantioselective and regioselective monohydroxylation of 2-phenoxypropionic acid to 2-(4-hydroxyphenoxy)propionic acid by isolated biocatalysts (in vitro) can also be highly regioselectively converted to 2-phenoxypropionic acid by a stable extracellular fungal enzyme, Agrocybe aegerita peroxygenase (AaP). (4-Hydroxyphenoxy)propionic acid and are preferably converted to its (R)-enantiomer.
  • WO 2015071264 A1 describes the production of biogenic substances, but using human liver cells.
  • liver-specific metabolites from a prodrug and its use as a drug is not described in the prior art.
  • liver-specific human metabolites cannot be produced in sufficient yield and variety.
  • precursor molecules are mostly synthesized in the prior art.
  • Semi-synthetic processes are often additionally required, so that the necessary further regio- and stereoselective modifications have to be introduced into the substance(s) in a complex manner, which are decisive for the pharmacological effectiveness of the metabolites from the liver.
  • the object of the invention is to provide suitable metabolites from a prodrug in vitro and in this way to produce and provide a suitable drug.
  • These medicaments can be provided particularly advantageously for patients whose P450 enzyme set is impaired by mutations or polymorphism.
  • the liver-specific prodrug clopidogrel a thienopyridine
  • clopidogrel a thienopyridine
  • the invention therefore relates to the biotechnical production of metabolites from a prodrug, which are produced using a new method.
  • the task is solved by at least one patent claim from the technical teaching taught.
  • the invention is therefore a method for
  • a prodrug with bioactivation by cytochrome P450 enzymes is treated with at least one peroxygenase and the product obtained is isolated.
  • metabolites in particular liver-specific human metabolites, can be produced continuously in high yields at low cost, and it is also possible to obtain advantageously stereoselective compounds of such metabolites, although, for example, non-human peroxygenases are used.
  • the invention therefore relates to a new method for the regioselective, enzymatic oxyfunctionalization of a liver-specific prodrug or a prodrug with bioactivation by cytochrome P450 enzymes.
  • the inventive method causes a regioselective oxyfunctionalization of the prodrug in the presence of a
  • Oxidizing agent preferably in an aqueous medium.
  • the prodrug is bioactivated by cytochrome P450 enzymes isolated substance or pure substance freely before and at least not in a liver cell.
  • prodrug with bioactivation by cytochrome P450 enzymes means that a prodrug is present, the prodrug being activated mostly in the liver by cytochrome P450 enzymes, particularly those such as CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19 , CYP2D6, CYP2E1, CYP3A4, CYP3A5,
  • Cytochrome P450 enzymes are heme proteins with enzymatic activity (oxidoreductases) and mostly act as monooxygenases. According to the invention, preference is given to those prodrugs which are metabolized in the liver to form a medicament.
  • the invention therefore includes in particular such prodrugs with bioactivation by cytochrome P450 enzymes, such as:
  • Amiodarone (metabolized by CYP3A4 and CYP2C8 to desethylamiodarone), Latini R, Tognoni G, Rates RE: Clinical pharmacokinetics of amiodarone. Clin Pharmacokinet. 1984 Mar-Apr;9(2):136-56. doi: 10.2165/00003088-198409020-00002. [PubMed:6370540],
  • Aripiprazole lauroxil activation by CYP3A4 and CYP2D6: https://go .drugbank.com/drugs/DB01238,
  • Carbamazepine (activated by CYP3A4): https://go .drugbank.com/drugs/DB00564,
  • Carisoprodol (activated by CYP2C19): https://go .drugbank.com/drugs/DB00395,
  • Clomifene (activated by CYP enzymes): YAKUGAKU ZASSHI130(10) 1325—1337 (2010) The Pharmaceutical Society of Japan,
  • Clopidogrel (activated by CYP2C19, CYP1A2 and CYP2B6): https://go .drugbank.com/drugs/DB00758,
  • Cyclophosphamides (activated by CYP3A4, CYP2B6): Preissner et al., Advances in Pharmacology, Volume 74 Copyright # 2015 Elsevier Inc. ISSN 1054-3589, http://dx.doi.org/10.1016/bs. apha.2015.03.004,
  • Decarbazines (activated by CYP1A2): Preissner et al., Advances in Pharmacology, Volume 74 Copyright # 2015 Elsevier Inc. ISSN 1054-3589, http://dx.doi.org/10.1016/bs. apha.2015.03.004,
  • N-desmethyltamoxifen (activated by CYP2D6 to form endoxifen) : https://go.drugbank.com/drugs/DB0067,
  • Desogestrel (activated by CYP2C9 and CYP2C19): Br J Clin Pharmacol 60:169-75, 2005; DOI:10.1111/j.1365-2125.2005.02382.x,
  • N,N-Didesmethyltamoxifen (activated by CYP2D6 and forms endoxifen) : https://go.drugbank.com/drugs/DB00675,
  • Enalapril (activated by CYP3A4): https://proteopedia.org/wiki/index.php/Enalapril,
  • Fludrocortisone (activated by CYP3A family): https://go .drugbank.com/drugs/DBOO687,
  • Flutamide (activated by CYP1A2): https://go.drugbank.com/drugs/DB00499, Future Med Chem. 2013 February; 5(2): 213-228. doi:10.4155/fmc.12.197,
  • Ifosfamide (activated by CYP3A4, -2B6): Preissner et al., Advances in Pharmacology, Volume 74 Copyright # 2015 Elsevier Inc. ISSN 1054-3589, http://dx.doi.org/10.1016/bs. apha.2015.03.004,
  • Irinotecan (activated by CYP3A4, CYP3A5): Preissner et al., Advances in Pharmacology, Volume 74 Copyright # 2015 Elsevier Inc. ISSN 1054-3589, http://dx.doi.org/10.1016/bs. apha.2015.03.004,
  • Lansoprazole (activated by CYP3A4 to lansoprazole sulfone): https://go .drugbank.com/drugs/DB00448,
  • Leflunomide (activate by CYP1A2): https://go.drugbank.com/drugs/DBOl097,
  • Loratadine (activated by CYP3A4 and CYP2D6): Future Med Chem. 2013 February; 5(2): 213-228. doi:10.4155/fmc.12.197,
  • Lynestrenol (metabolized to norethisterone by CYP2C9, CYP2C19, and CYP3A4): Korhonen T, Turpeinen M, Tolonen A, Laine K, Pelkonen 0 (2008). "Identification of the human cytochrome P450 enzymes involved in the in vitro biotransformation of lynestrenol and norethindrone". J Future Med Chem 2013 February; 5(2): 213-228. doi:10.4155/fmc.12.197, Steroid Biochem. Mol. Biol. 110 (1-2):
  • Pantoprazole (activated by CYP3A4 to desmethylpantoprazole): https://go.drugbank.com/drugs/DB00213,
  • Parecoxib (metabolized by CYP3A4 & CYP2C9 to valdecoxib): https://go .drugbank.com/drugs/DB08439,
  • Prasugrel (activated by CYP3A4, CYP2C19, CYP2B6 and CYP2C9 to R-138727): Future Med Chem. 2013 February; 5(2): 213-228. doi:10.4155/fmc.12.197, Schror K, Siller-Matula JM, Huber K (2012) Pharmacokinetic basis of the antiplatelet action of prasugrel. Fundam Clin Pharmacol 26: 39-46,
  • Proguanil (activated by CYP2C19): https://www .pharmazeutician-zeitung.de/ausgäbe-282012/potente- metaboliten/,
  • Sibutramine (activated by CYP3A4 to desmethylsibutramine (Ml; BTS-54354) and didesmethylsibutramine (M2; BTS-54505): https://go .drugbank.com/drugs/DBOl105,
  • T Tafenoquine (activated by CYP2D6): Molecules 2020, 25, 884; doi:10.3390/molecules25040884,
  • Tamoxifen (activation to endoxifen via CYP2D6, CYP3A4 and CYP3A5): Desta Z, Ward BA, Soukhova NV, Flockhart DA. Comprehensive evaluation of tamoxifen essential biotransformation by the human cytochrome P450 system in vitro: prominent roles for CYP3A4 and CYP2D6. J. Pharmacol.
  • Tegafur (activated by CYP1A2, CYP2A6 and CYP2C8): Preissner et al., Advances in Pharmacology, Volume 74 Copyright # 2015 Elsevier Inc. ISSN 1054-3589. http://dx.doi.org/10.1016/bs.apha.2015.03.004, Future Med Chem. 2013 February; 5(2): 213-228. doi:10.4155/fmc.12.197,
  • Temozolomide (activated by CYP3A4): Preissner et al., Advances in Pharmacology, Volume 74 Copyright # 2015 Elsevier Inc. ISSN 1054-3589, http://dx.doi.org/10.1016/bs. apha.2015.03.004,
  • Treosulfan (activated by CYP1A2 & CYP3A4): Preissner et al., Advances in Pharmacology, Volume 74 Copyright # 2015 Elsevier Inc. ISSN 1054-3589, http://dx.doi.org/10.1016/bs. apha.2015.03.004,
  • Trofosamides (activated by CYP3A4 & CYP2B6): Preissner et al., Advances in Pharmacology, Volume 74 Copyright # 2015 Elsevier Inc. ISSN 1054-3589, http://dx.doi.org/10.1016/bs.apha .2015.03.004,
  • Valbenazine (activated by CYP3A4/5 to NBI-136110): https://go .drugbank.com/drugs/DBl1915,
  • the products obtained according to the invention contain at least one chemical substance, a mixture of substances, but at least one drug or active substance.
  • the chemical substances are preferably an organic molecule which, in addition to carbon (C) and hydrogen (H), can contain heteroatoms such as oxygen (O), nitrogen (N), sulfur (S) or phosphorus (P).
  • the chemical substances can have linear and/or ring-shaped carbon chains including heteroatoms.
  • Organic molecules with less than 1000 g/mol, in particular less than 750 g/mol, less than 500 g/mol, less than 250 g/mol are preferred.
  • at least one chemical substance contains at least one chiral carbon atom and is a stereoisomer.
  • a prodrug according to the invention or the products obtained according to the invention can be adequately analyzed, for example, by means of combined analytical methods such as GC/LC-MS, IR, NMR and MS and, if necessary, one be subjected to structure elucidation.
  • a “peroxygenase” within the meaning of this invention is an enzyme which can catalyze one or more biochemical reactions.
  • a peroxygenase according to the invention is capable of a prodrug (educts)—a substrate—a first
  • the peroxygenases selected according to the invention are preferably of fungal origin, in particular non-specific peroxygenases (“UPOs”, EC 1.11.2.1 (see enzyme.expasy.org)) Peroxygenases from the fungal kingdom, particularly from the subkingdom of the Dykarya, in particular from the Divisions of the Basidiomycota and Ascomycota.
  • UPOs non-specific peroxygenases
  • Peroxygenases from the fungal kingdom, particularly from the subkingdom of the Dykarya, in particular from the Divisions of the Basidiomycota and Ascomycota.
  • Preferred non-specific peroxygenases are derived from fungi of the order Agaricales, preferably from the families
  • Psathyrellaceae, Marasmiaceae and Strophariaceae particularly of the genus Agrocybe, such species as Agrocybe aegerita and Agrocybe parasitica, of the genus Psathyrella, such species as Psathyrella aberdarensis, and of the genus Marasmius, such species as Marasmius rotula and Marasmius wettsteinii.
  • peroxygenases catalyze reactions like the P450 monooxygenases, wherein only an oxidizing agent such as hydroperoxide (R-OOH) in particular hydrogen peroxide (H2O2) is required as a co-substrate for the oxyfunctionalization of a substrate (A):
  • R-OOH hydroperoxide
  • H2O2 hydrogen peroxide
  • organic hydroperoxides e.g. tert-butyl hydroperoxide
  • peroxycarboxylic acids R-C0-00H, e.g. meta-chloroperbenzoic acid
  • hydrogen peroxide adducts e.g. Carbamide peroxide
  • UPOs conducting the reaction using the UPOs preferred according to the invention has advantages in comparison to P450 enzymes in vitro, in particular high water solubility due to the strong glycosylation of the proteins.
  • UPOs have a high stability compared to intracellular mono- or dioxygenases (freely dissolved or membrane-bound).
  • UPOs can be used to advantage under non-sterile conditions.
  • a peroxygenase can preferably be obtained from non-human organisms such as fungi, yeasts, algae or bacteria.
  • the peroxygenases can be isolated from the organisms and purified by known methods. Furthermore, such peroxygenases can be produced recombinantly in a host.
  • the two-step oxyfunctionalization of thienopyridine/clopidogrel provides selectively the desired thiol metabolites (H1-H4, Fig. 1), such as H3 and H4 in a high yield of 20%.
  • the peroxygenases in particular UPOs, in a low concentration of 0.1 U me 1 to 10 U me 1 , 1 to 3 U me 1 for the oxyfunctionalization of
  • Prodrugs in particular thienopyridines (1 unit converts 1 pmol of veratryl alcohol per minute) are used.
  • the concentration of a prodrug according to the invention is 0.1 to 10 mM, preferably 0.2 to 5 mM, particularly preferably between 0.5 to 2 mM, especially 0.8 to 1.5 mM.
  • the prodrug is at least one thienopyridine.
  • thienopyridines are ticlopidine (IUPAC: (5-(2-chlorobenzyl)-4,5,6,7-tetrahydrothieno[3,2-c]pyridine), clopidogrel (IUPAC:
  • thienopyridines are active ingredients from the group of platelet aggregation inhibitors and are used for the treatment and prophylaxis of both atheorothrombotic events such.
  • the thienopyridines are “prodrugs with bioactivation by cytochrome P450 enzymes” or a prodrug, with their anticoagulant effect only occurring after hepatic bioactivation by cytochrome P450 enzymes.
  • the active metabolites of thienopyridines mediate the potent antithrombotic activity through a highly specific, irreversible inhibition of the platelet P2Y12 receptor.
  • the C2-carbon is first hydroxylated by P450 enzymes and then rearranged to the 2-oxo metabolite.
  • This intermediate is generated in vivo by rapid deesterification for prasugrel and vicagrel.
  • the 2-oxo metabolite is converted to the active thiol metabolite by ring opening (Formula III below), in each case oxidative conversion by cytochrome P450 enzymes occurs.
  • the free thiol group of the active metabolites form disulfide bonds with the extracellular cysteine residues (Cysl7 and Cys270) on the P2Y12 receptor, thereby inactivating the receptor.
  • the binding of the intrinsic substrate ADP is blocked and the ADP-induced thrombocyte aggregation is suppressed.
  • Clopidogrel (CPG, 1) is a prodrug which requires bioactivation via a two-step cytochrome P450-dependent oxidation to a pharmacologically active human metabolite and is shown in FIG. 1 and FIG.
  • Clopidogrel 2-oxo-clopidogrel clopidogrel-active metabolite Hl-HM CPG (1, Fig. 1) is first oxidized to 2-oxo-clopidogrel (2-oxo-CPG, 2, Fig. 1) (see formula II below), which exists in two configurations by epimerization. Further oxidation of the thiolactone leads to the formation of a sulfenic acid intermediate which is very unstable and is reduced directly by ascorbate to the active thiol or indirectly by glutathione (GSH) via a disulfide conjugate and subsequent reduction by another GSH molecule. Due to the stereochemistry, the thiol metabolite can exist in four isomeric structures (H1-H4) (Drug Metaboi. Dispos., 2012, 43(10), 1632-1641).
  • the antithrombotic activity is exclusively due to H4 (Tuffal, Gilles et al., An improved method for specific and quantitative determination of the Clopidogrel active metabolite isomers in human plasma, Thromb. Haemost., 2011, 105(4), 696-705) .
  • the invention further relates to such prodrugs Bioactivation by cytochrome P450 enzymes according to formula I: with independently
  • R 2 hydrogen, halogen, Ci, F, CN;
  • R 3 Hydrogen, OH, OCOCH 3 in a method according to the invention. According to formula I is
  • the invention relates to those products which can be obtained from formula I according to the invention, of formula II with independently Ri: hydrogen, COOCH3 , COC3 H5;
  • R 2 hydrogen, halogen, Cl, F, CN; or of the formula III, so-called 4-mercapto-3-piperidinylideneacetic acid derivatives with independently
  • R2 hydrogen, halogen, CI, F, CN.
  • the one-pot device can be equipped with a stirrer and other customary aids.
  • the method according to the invention can be carried out particularly preferably in aqueous, in particular buffered, solutions with a buffer concentration of 1 to 100 mM, preferably 10 to 20 mM.
  • the reaction process is carried out at pH values of 3 to 10, preferably 5 to 8, in particular at pH 7.
  • the reaction can be carried out without a solvent or in the presence of a water-miscible solvent, for example alcohols, acetone and acetonitrile, or in a two-phase system for example dichloromethane, but preferably with acetone or acetonitrile in the ranges 1-90% by volume, particularly preferably 2-50% by volume, in particular 5-30% by volume.
  • electron transport proteins and regulatory proteins can be dispensed with in the method according to the invention.
  • the process is carried out in the presence of free-radical scavengers.
  • Ascorbic acid or glutathione for example, can be used as radical scavengers.
  • clopidogrel for example, when ascorbic acid is used, H3 and H4 can be formed selectively and in equal parts.
  • glutathione H1-H4 are formed in equal parts.
  • the concentration can be 0.01 mM to about 100 mM, in particular 5 to 10 mM.
  • the process is carried out at atmospheric pressure and temperatures of 4-40.degree. C., preferably at 15-35.degree. C. and very particularly preferably at 20-25.degree.
  • the products or intermediates obtained can be isolated by means of customary purification, e.g. from the supernatant, for example by extraction, filtration, distillation, rectification, chromatography, treatment with ion exchangers, adsorbents or crystallization.
  • the products are preferably isolated by means of liquid-liquid extraction and purified chromatographically.
  • the products or chemical compounds obtained can be identified, for example, by means of LC/MS.
  • the metabolite is available as a powder (approx. 4 mg) in a dark container and is stored in a freezer at at least -80 °C until the solution is prepared.
  • the active substance is dissolved in 500 ml of a suitable sterile solvent (here: phosphate-buffered saline, PBS). Powder and solvent are rigorously mixed. The complete dissolution of the powder must be checked visually. The mixture is stored on ice or at about 0 °C until use.
  • the aggregation of human thrombocytes (blood platelets) in whole blood is determined using impedance aggregometry (Paniccia, R.; Antonucci, E.; Maggini, N.; Romano, E.; Gori, AM; Marcucci , R., Prisco, D., Abbate, R. Assessment of Platelet Function on Whole Blood by Multiple Electrode Aggregometry in High-Risk Patients with Coronary Artery Disease Receiving Antiplatelet Therapy.American journal of clinical pathology 2009, 131, 834-42, doi:10.1309/AJCPTE3K1SGAPOIZ) .
  • a sample container provided for this purpose (e.g. Sarstedt Monovette, Sarstedt, Nümbrecht, Germany). This contains, for example, tri-sodium citrate in a standard concentration of 0.106 mol -Ll.
  • adenosine diphosphate (ADP, 10 mM final concentration) induced thrombocyte aggregation is tested according to the instructions of the manufacturer of the Multiplate® Analyzer (Roche Diagnostics, Mannheim, Germany).
  • ADP activated whole blood sample without drug The volume of drug is replaced with a phosphate buffered saline solution.
  • FIG. 2 representatively shows the reduction in ADP-induced platelet aggregation after a two-minute pre-incubation of the metabolite in the blood of an apparently healthy test person.
  • the theoretical starting concentration of the metabolite in this experiment was 9.40 mg per 1 mL PBS solvent (4.70 mg metabolite with approx. 73% purity in 500 pL PBS solution).
  • the final concentration of the metabolite in the blood tested was 0.31 mg per 1 mL (20 ⁇ l of the metabolite solution in a 600 ⁇ L blood sample).
  • N-desmethyl-OH-tamoxifen (retention time 3.67) should be the demethylated product of hydroxytamoxifen (retention time 3.77), which occurs very dominantly with CglUPO.
  • the N-desmethyl-OH-tamoxifen (4.18) as the demethylation product of hydroxytamoxifen (4.22) at M576UPO.

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  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Organic Chemistry (AREA)
  • Veterinary Medicine (AREA)
  • Pharmacology & Pharmacy (AREA)
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  • Proteomics, Peptides & Aminoacids (AREA)
  • Microbiology (AREA)
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  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Heterocyclic Carbon Compounds Containing A Hetero Ring Having Oxygen Or Sulfur (AREA)

Abstract

L'invention concerne en outre un procédé de fabrication d'un médicament, selon lequel un promédicament à activation biologique par des enzymes cytochromes p450 est mélangé à au moins une peroxygénase, et le produit obtenu est isolé. L'invention concerne en outre des promédicaments sélectionnés à cet effet, tels que des thiénopyridines, et les produits obtenus selon l'invention ainsi que leur utilisation.
EP22725220.2A 2021-04-25 2022-04-25 Médicaments in vitro dérivés de promédicaments et leur utilisation Pending EP4330382A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE102021204094.2A DE102021204094A1 (de) 2021-04-25 2021-04-25 In-vitro Arzneimittel aus Prodrugs und deren Verwendung
PCT/EP2022/060814 WO2022229050A2 (fr) 2021-04-25 2022-04-25 Médicaments in vitro dérivés de promédicaments et leur utilisation

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EP4330382A2 true EP4330382A2 (fr) 2024-03-06

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EP22725220.2A Pending EP4330382A2 (fr) 2021-04-25 2022-04-25 Médicaments in vitro dérivés de promédicaments et leur utilisation

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US (1) US20240207369A1 (fr)
EP (1) EP4330382A2 (fr)
DE (1) DE102021204094A1 (fr)
WO (1) WO2022229050A2 (fr)

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE102007016139A1 (de) 2007-03-30 2008-10-02 Jenabios Gmbh Verfahren zur regioselektiven Oxygenierung von N-Heterozyklen
DE102008034829A1 (de) 2008-07-23 2010-02-04 Jenabios Gmbh Verfahren zur Herstellung von 2-(4-Hydroxyphenoxy)propionsäure
EP2871233A1 (fr) 2013-11-12 2015-05-13 Brandenburgische Technische Universität Cottbus-Senftenberg Procédé de fabrication de substances biogènes
EP3994252A1 (fr) * 2019-07-05 2022-05-11 bisy GmbH Oxygénases hème-thiolate de recombinaison

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US20240207369A1 (en) 2024-06-27
DE102021204094A1 (de) 2022-10-27
WO2022229050A3 (fr) 2022-12-22
WO2022229050A2 (fr) 2022-11-03

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