WO2024025396A1 - Nouveau médicament précurseur d'auristatine - Google Patents

Nouveau médicament précurseur d'auristatine Download PDF

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WO2024025396A1
WO2024025396A1 PCT/KR2023/011084 KR2023011084W WO2024025396A1 WO 2024025396 A1 WO2024025396 A1 WO 2024025396A1 KR 2023011084 W KR2023011084 W KR 2023011084W WO 2024025396 A1 WO2024025396 A1 WO 2024025396A1
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cancer
compound
mmol
carcinoma
acid
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PCT/KR2023/011084
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Korean (ko)
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송호영
이건중
박창식
유병준
정철웅
김용주
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주식회사 레고켐 바이오사이언스
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Priority claimed from KR1020230099038A external-priority patent/KR20240016231A/ko
Publication of WO2024025396A1 publication Critical patent/WO2024025396A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/05—Dipeptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/02—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link

Definitions

  • the present invention relates to an auristatin prodrug, and more specifically, to an auristatin prodrug having a self-immolative group, a method for producing the same, a pharmaceutical composition containing the same, treatment of diseases and/ or its use for prophylaxis.
  • Auristatin is a major drug used in antibody drug conjugates (ADCs), the most well-known of which are monomethyl auristatin E (MMAE) and monomethyl auristatin F (monomethyl auristatin F). auristatin F, MMAF).
  • Auristatin acts by blocking the polymerization of tubulin, inhibiting mitosis, and interfering with cell proliferation.
  • auristatin has toxic side effects (e.g. neutropenia, Due to concerns about thrombocytopenia (platelet reduction, etc.), many attempts are being made to use antibody drug conjugates.
  • the linker is cleaved by enzymes such as cathepsin in tumor cells, and the drug acts.
  • the inventor of the present invention made efforts to develop an auristatin prodrug, and as a result, synthesized an auristatin prodrug that complements the shortcomings of existing drugs due to their high lipophilic properties and has excellent pharmacokinetic properties, cytotoxicity, etc. and confirmed its characteristics and activity to complete the present invention.
  • the present invention seeks to provide a novel auristatin prodrug with improved hydrophilicity, pharmacokinetic properties, etc.
  • the present invention provides a compound having a structure represented by the following general formula (I), an isomer thereof, or a pharmaceutically acceptable salt or solvate thereof.
  • MMAE is ego
  • E is an organic group derived from the substrate of a cancer cell-specific enzyme
  • R 1 and R 3 are independently in each case a divalent organic group
  • R 2 and R 4 are independently hydrogen or alkyl in each case
  • the N at the ** terminal of SIG2 can form an N-containing ring structure with the adjacent carbon atom in R 1 and R 2 ,
  • the N at the *** terminal of SIG1 can form an N-containing ring structure with the adjacent carbon atom in R 3 and R 4 ,
  • MMAE and SIG2 are connected at the * end;
  • SIG2 and SIG1 are connected at the ** end;
  • the bond between the *** end of SIG1 and E can be broken by the cancer cell-specific enzyme.
  • each connecting portion is located in the intracellular environment (e.g., lysosomes or endosomes or caveolae). It can be cut by cutting agents present in caveolea. For example, it may be cleaved by intracellular peptidase or protease enzymes, including but not limited to lysosomal or endosomal proteases.
  • Cleavage agents may include cathepsin B, cathepsin D, and plasmin, all of which are known to hydrolyze dipeptide drug derivatives, releasing the active drug within target cells (e.g., Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123).
  • it may contain structures that are cleavable by the thiol-dependent protease cathepsin-B, which is highly expressed in cancer tissues (e.g., Phe-Leu, Val-Cit, Phe-Lys, Val-Ala, etc.) .
  • structures that can be cleaved by cleavage agents include, for example, malonate linker (Johnson et al., 1995, Anticancer Res. 15:1387-93), maleimidobenzoyl linker (Lau et al., 1995, Bioorg-Med) -Chem. 3(10):1299-1304), 3'-N-amide analog (Lau et al., 1995, Bioorg-Med-Chem. 3(10):1305-12), beta-glucuronide ( ⁇ -glucuronide) linker (Jeffery et al., 2006, Bioconjug Chem. 17(3):832-40), or beta-galactosidase linker (Kolodych et al., 2017, Eur J Med Chem. Dec 15;142:376-382).
  • malonate linker Johnson et al., 1995, Anticancer Res. 15:1387-93
  • maleimidobenzoyl linker Liau et al., 1995, Bio
  • unsubstituted or substituted refers to a parent group that may be unsubstituted or substituted
  • substituted refers to a parent group having one or more substituents
  • a substituent refers to a parent group ( It refers to a chemical moiety covalently bonded to the parent group or fused to the parent group.
  • alkyl refers to a monovalent moiety obtained by removing a hydrogen atom from a carbon atom of an aliphatic or cycloaliphatic, saturated or unsaturated (unsaturated, fully unsaturated) hydrocarbon compound, generally 1 to 12, more specifically. It has 1 to 10 atoms.
  • saturated alkyl include methyl, ethyl, propyl, butyl, pentyl, hexyl, and heptyl
  • saturated straight-chain alkyl include methyl, ethyl, n-propyl, n-butyl, and n-. Pentyl (amyl), n-hexyl, n-heptyl, etc.
  • saturated branched alkyl examples include isopropyl, isobutyl, sec-butyl, tert-butyl, isopentyl, neopentyl, etc.
  • C 1 -C 5 alkyl herein refers to a straight or branched chain of 1 to 5 atoms, whether straight or branched, whether carbon atoms or heteroatoms.
  • alkyl includes “heteroalkyl” unless otherwise indicated.
  • heteroalkyl means alkyl in which one or more carbon atoms have been replaced by a heteroatom, such as a nitrogen atom, a sulfur atom, or an oxygen atom.
  • alkylene refers to a divalent moiety obtained by removing a hydrogen atom from a carbon atom of a straight-chain or branched aliphatic or alicyclic, saturated or unsaturated hydrocarbon compound.
  • lower alkylene, arylene, etc. generally refers to alkylene, arylene, etc. having 8 or less carbon atoms.
  • aryl refers to a monovalent moiety obtained by removing a hydrogen atom from an aromatic ring atom of an aromatic compound.
  • C 6 -C 10 aryl means a monovalent moiety obtained by removing a hydrogen atom from an aromatic ring atom of an aromatic compound, where the moiety has 6 to 10 ring atoms
  • C 5 -C 10 aryl means a monovalent moiety in which the moiety has 5 to 10 ring atoms and is obtained by removing a hydrogen atom from an aromatic ring atom of an aromatic compound.
  • the prefix refers to the number of ring atoms or a range of the number of ring atoms, regardless of whether they are carbon atoms or heteroatoms.
  • C 5 -C 6 aryl refers to an aryl group having 5 or 6 ring atoms.
  • the ring atoms may be all carbon atoms, as in a “carboaryl group”. Examples of carboaryl groups include, but are not limited to, those derived from benzene, naphthalene, azulene, anthracene, phenanthrene, naphthacene, and pyrene.
  • aryl groups containing fused rings include groups derived from indane, indene, isoindene, tetralin, acenaphthene, fluorene, phenalene, acephenanthrene, and aceanthrene, such as It is not limited. Additionally, “aryl” includes “heteroaryl” unless otherwise indicated, where heteroaryl refers to an aromatic ring atom in which the ring atom may contain one or more heteroatoms, examples of which include pyridinylene, It includes, but is not limited to, pyrimidinylene and thiophenylene.
  • arylene refers to a divalent moiety obtained by removing a minority atom from an aromatic ring atom of an aromatic compound.
  • alkaryl and “aralkyl” refer to alkylene-aryl and arylene-alkyl, where “alkarylene” and “aralkyl” refer to alkylene-arylene and arylene.
  • -Means alkylene, and each of alkyl, aryl, alkylene, and arylene is as defined above.
  • precursor or “prodrug” refers to the action of enzymes, etc. under physiological conditions in vivo (e.g., enzymatic oxidation, reduction, and/or hydrolysis, etc.). It refers to a compound that can be converted directly or indirectly into a drug.
  • the “pharmaceutically acceptable salt” may be an acid addition salt formed from a pharmaceutically acceptable free acid, and the free acid may be an organic acid or an inorganic acid.
  • the organic acids include, but are not limited to, citric acid, acetic acid, lactic acid, tartaric acid, maleic acid, fumaric acid, formic acid, propionic acid, oxalic acid, trifluoroacetic acid, benzoic acid, gluconic acid, metasulfonic acid, glycolic acid, succinic acid, 4-toluenesulfonic acid, Includes glutamic acid and aspartic acid.
  • the inorganic acids include, but are not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, and phosphoric acid.
  • a salt can be formed with the appropriate cation.
  • suitable inorganic cations include, but are not limited to, alkali metal ions such as Na + and K + , alkaline earth metal cations such as Ca 2+ and Mg 2+ and other cations such as Al 3+ .
  • suitable organic cations include, but are not limited to, ammonium ions (i.e., NH 4 + ) and substituted ammonium ions (e.g., NH 3 R + , NH 2 R 2 + , NHR 3 + , NR 4 + ).
  • Examples of some suitable substituted ammonium ions are those derived from: ethylamine, diethylamine, dicyclohexylamine, triethylamine, butylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, benzylamine. , phenylbenzylamine, choline, meglumine and tromethamine, as well as amino acids such as lysine and arginine.
  • An example of a common quaternary ammonium ion is N(CH 3 ) 4 + .
  • salts can be formed with the appropriate anion.
  • suitable inorganic anions include, but are not limited to, those derived from the following inorganic acids: hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, sulfurous acid, nitric acid, nitrous acid, phosphoric acid, and phosphorous acid.
  • Suitable organic anions include, but are not limited to, those derived from the following organic acids: 2-acetioxybenzoic acid, acetic acid, ascorbic acid, aspartic acid, benzoic acid, camphorsulfonic acid, cinnamic acid, citric acid, edetic acid, ethane.
  • Disulfonic acid ethanesulfonic acid, fumaric acid, gluteptonic acid, gluconic acid, glutamic acid, glycolic acid, hydroxymaleic acid, hydroxynaphthalene carboxylic acid, isethionic acid, lactic acid, lactobionic acid, lauric acid, maleic acid, Malic acid, methanesulfonic acid, mucilic acid, oleic acid, oxalic acid, palmitic acid, palmic acid, pantothenic acid, phenylacetic acid, phenylsulfonic acid, propionic acid, pyruvic acid, salicylic acid, stearic acid, succinic acid, sulfanilic acid, tartaric acid, toluenesulfonic acid and valeric acid.
  • suitable polymer organic anions include, but are not limited to, those derived from the following polymer acids: tannic acid, carboxymethyl cellulose, etc.
  • solvate refers to a molecular complex between the compound according to the present invention and solvent molecules.
  • solvates include water, isopropanol, ethanol, methanol, and dimethyl sulfoxide. It includes, but is not limited to, the compound according to the present invention combined with (dimethylsulfoxide), ethyl acetate, acetic acid, ethanolamine, or a mixed solvent thereof.
  • solvate is used herein in its conventional sense to refer to a complex of a solute (eg an active compound, a salt of an active compound) and a solvent.
  • a solute eg an active compound, a salt of an active compound
  • the solvent is water
  • the solvate may conveniently be referred to as a hydrate, such as monohydrate, dihydrate, trihydrate, etc.
  • SIG2, SIG1 and E are each independently a protease cleavable group, an acid-cleavable group, a disulfide, a self-immolative group, malonate, maleimidobenzoyl, 3' -Includes N-amide, beta-glucuronide, or beta-galactoside.
  • SIG2, SIG1, and E are each independently protease cleavage group, self-immolation group, beta-glucuronide, or beta-galactoside. am.
  • SIG 2 and SIG 1 are self-immolative groups and E is a protease cleavable group, beta-glucuronide, or beta-galactoside.
  • R 1 and R 3 are independently in each case substituted or unsubstituted alkylene, substituted or unsubstituted arylene, substituted or unsubstituted alkarylene, or substituted Or it is unsubstituted aralkylene.
  • R 1 is independently in each case substituted or unsubstituted lower alkylene, substituted or unsubstituted lower arylene, substituted or unsubstituted lower alkylene, or substituted or unsubstituted lower aralkylene.
  • R 1 is independently in each case substituted or unsubstituted C 1 -C 5 alkylene, substituted or unsubstituted C 6 -C 10 arylene, substituted or unsubstituted C 5 -C 10 alkarylene, or substituted or unsubstituted C 5 -C 10 aralkylene.
  • R 2 is hydrogen or lower alkyl.
  • R 2 is hydrogen or C 1 -C 3 alkyl.
  • each of R 1 and R 2 may be independently selected.
  • SIG2 is selected from the following group:
  • R 3 is independently substituted or unsubstituted arylene, substituted or unsubstituted alkarylene, or substituted or unsubstituted aralkylene.
  • R 3 is independently substituted or unsubstituted C 6 -C 14 arylene, substituted or unsubstituted C 6 -C 14 alkarylene, or substituted or unsubstituted C It is 6 -C 14 aralkylene.
  • R 3 is independently substituted or unsubstituted C 6 -C 10 arylene, substituted or unsubstituted C 6 -C 10 alkarylene, or substituted or unsubstituted C It is 6 -C 10 aralkylene.
  • R 4 is hydrogen or lower alkyl.
  • R 4 is hydrogen or C 1 -C 3 alkyl.
  • SIG1 is selected from the following group:
  • the cancer cell-specific enzyme is selected from the group consisting of ⁇ -glucuronidase, ⁇ -galactosidase, and cathepsin B. do.
  • the substrate of the cancer cell-specific enzyme is glucuronic acid, galactoside, or a derivative thereof, an amino acid or a derivative thereof, or a polypeptide or a derivative thereof.
  • the amino acids are alanine, ⁇ -alanine, ⁇ -aminobutyric acid, arginine, asparagine, aspartic acid, ⁇ -carboxyglutamic acid, citrulline, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, At least one selected from the group consisting of lysine, methionine, norleucine, norvaline, ornithine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine and derivatives thereof,
  • Polypeptides include alanine, ⁇ -alanine, ⁇ -aminobutyric acid, arginine, asparagine, aspartic acid, ⁇ -carboxyglutamic acid, citrulline, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, norleucine, norleucine. It is formed from two or more selected from the group consisting of valine, ornithine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine and derivatives thereof.
  • E is selected from the following group.
  • R 5 is -OH, or C 1-4 alkoxy
  • m is an integer of 1 to 12, and the meaning of *** is as described above.
  • E is selected from the following group:
  • the compound of general formula I is selected from the following group:
  • a ligand-drug conjugate comprising a compound having the structure of General Formula I and a ligand, or a pharmaceutically acceptable salt or solvate thereof is provided.
  • the term “conjugate” refers to a cell binding agent that is covalently linked to one or more molecules of a cytotoxic compound.
  • the “cell binding agent” is a molecule with affinity for a biological target, for example, a ligand, protein, antibody, specifically a monoclonal antibody, protein or antibody fragment, peptide, oligonucleotide, or oligosaccharide.
  • the binder functions to direct the biologically active compound to the biological target.
  • biological target in this specification refers to an antigen located on the surface of a tumor, cancer cell, or extracellular matrix.
  • conjugates can be designed to target tumor cells via cell surface antigens.
  • the antigen may be a cell surface antigen that is overexpressed or expressed on abnormal cell types.
  • the target antigen may be expressed only on proliferating cells (eg, tumor cells).
  • Target antigens can usually be selected based on differential expression between proliferative and normal tissues.
  • the ligand is coupled to a linker.
  • ligand refers to a molecule capable of forming a complex with a target biomolecule.
  • An example of a ligand is a molecule that transmits a signal by binding to a specific location on a target protein.
  • a ligand may be an antigen-binding moiety, an antibody, or an antigen-binding fragment, which can bind to a location on a target biomolecule (e.g., a protein) and transmit a signal.
  • the ligand-drug conjugate may refer to a cell binding agent in which a ligand and a drug (eg, a compound having the structure of Formula I) are covalently bound through a linker.
  • the ligand may be an antigen-binding moiety, an antibody, or an antigen-binding fragment that can bind to the biomolecule targeted by the cell binding agent, and the ligand-drug conjugate is an antibody-drug conjugate (ADC).
  • ADC antibody-drug conjugate
  • an “antibody” is an immunoglobulin molecule that can specifically bind to a target, such as carbohydrates, polynucleotides, lipids, polypeptides, etc., through at least one antigen recognition site located in the variable region of the immunoglobulin molecule.
  • antibody refers to an intact polyclonal or monoclonal antibody, as well as any antigen-binding portion of an intact antibody that retains the ability to specifically bind to a given antigen (e.g., “antigen-binding fragment”) or single chains thereof, fusion proteins comprising antibodies, and any other modified arrangement of immunoglobulin molecules comprising an antigen recognition site, including, but not limited to, Fab; Fab'; F(ab')2 Fd fragment; Fv fragment; single domain antibody (dAb) fragment; Isolated complementarity determining region (CDR); Single chain (scFv) and single domain antibodies (e.g., shark and camelid antibodies), maxibodies, minibodies, intrabodies, diabodies, triabodies, tetrabodies, v-NAR and bis-scFv ( See, for example, Hollinger and Hudson, 2005, Nature Biotechnology 23(9): 1126-1136.
  • Antibodies include antibodies of any class, such as IgG, IgA or IgM (or subclasses thereof), and the antibodies need not be of any particular class.
  • immunoglobulins can be assigned to different classes. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, several of which are further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. can be classified.
  • the heavy chain (HC) constant domains corresponding to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively.
  • the subunit structure and three-dimensional coordination of different classes of immunoglobulins are well known.
  • the antibody of the present invention can be produced using techniques well known in the related art, such as recombinant technology, phage display technology, synthetic technology, or a combination of the above techniques, or other techniques readily known in the related art.
  • isolated antibody refers to an antibody that is substantially free of other antibodies with different antigenic specificities and may be substantially free of other cellular material and/or chemicals.
  • Antibodies useful in the present invention include monoclonal antibodies, polyclonal antibodies, antibody fragments (e.g., Fab, Fab', F(ab')2, Fv, Fc, etc.), chimeric antibodies, bispecific antibodies, and heterologous antibodies.
  • Antibodies may be murine, rat, human or of any other origin (including chimeric or humanized antibodies).
  • the compound and the ligand having the structure of General Formula I may be linked through a linker.
  • the linker is any group or moiety that links, connects, or attaches a ligand described herein, specifically an antibody or antigen binding protein, to a therapeutic moiety, such as a compound having the structure of General Formula I above.
  • suitable binder linkers for the antibody conjugates described herein are those that are sufficiently stable to take advantage of the circulating half-life of the antibody, while simultaneously being capable of releasing the payload after antigen binding and/or antigen-mediated internalization of the conjugate.
  • the linker included in the ligand-drug conjugate disclosed herein may be cleavable or non-cleavable.
  • the linker is cleavable in vitro and in vivo.
  • Cleavable linkers may include chemically or enzymatically unstable or degradable linkages.
  • Cleavable linkers generally rely on processes inside the cell to liberate the drug, such as reduction in the cytoplasm, exposure to acidic conditions in lysosomes, or cleavage by specific proteases or other enzymes inside the cell.
  • Cleavable linkers generally contain one or more chemical bonds that are chemically or enzymatically cleavable, while the remainder of the linker is non-cleavable.
  • the linker contains chemically labile groups such as hydrazone and/or disulfide groups.
  • Linkers containing chemically labile groups take advantage of differential properties between plasma and some cytoplasmic compartments. Intracellular conditions to facilitate drug release for hydrazone-containing linkers are the acidic conditions of endosomes and lysosomes, while disulfide-containing linkers are reduced in the cytosol containing high thiol concentrations, such as glutathione.
  • the plasma stability of a linker comprising a chemically labile group can be increased by using substituents near the chemically labile group to induce steric hindrance.
  • Acid-labile groups such as hydrazone
  • hydrazone remain intact during systemic circulation in the neutral pH environment of blood (pH 7.3 to 7.5), and once ADC is transferred to intermediate acidic endosomes (pH 5.0 to 6.5) and lysosomes (pH 4.5 to 5.0) of cells. Once internalized into the compartment, it hydrolyzes and releases the drug. This pH-dependent release mechanism was associated with non-specific release of the drug.
  • the linker can be altered by chemical modifications, such as substitutions, to achieve more efficient release from the lysosome with minimal loss of circulation.
  • Cleavable linkers may also include disulfide groups.
  • Disulfides are thermodynamically stable at physiological pH and are designed to release drugs upon internalization inside cells, where the cytosol provides a significantly more reducing environment compared to the extracellular environment. Cleavage of the disulfide bond generally requires the presence of a cytosolic thiol cofactor, such as (reduced) glutathione (GSH), such that the disulfide-containing linker is adequately stable in the circulation, thereby selectively releasing the drug into the cytosol. do.
  • GSH reduced glutathione
  • Intracellular enzymes protein disulfide isomerase, or similar enzymes capable of cleaving disulfide bonds may also contribute to preferential cleavage of disulfide bonds inside cells.
  • GSH is reported to be present in cells in a concentration range of 0.5 to 10 mM compared to the significantly lower concentration of GSH or the most abundant low molecular weight thiol, cysteine, in circulation at approximately 5 ⁇ M.
  • the in vivo stability of disulfide-containing linkers can be improved by chemical modification of the linker, such as the use of steric hindrance adjacent to the disulfide bond.
  • linker that is specifically cleaved by enzymes.
  • Such linkers are typically peptidic or contain a peptide region that acts as a substrate for an enzyme.
  • Peptide-based linkers tend to be more stable in the cytoplasm and extracellular environment than chemically unstable linkers.
  • Peptide bonds generally have good serum stability because lysosomal proteases have very low activity in the blood due to endogenous inhibitors and due to the unfavorably high pH value of blood compared to lysosomes. Release of the drug from the antibody occurs specifically due to the action of lysosomal proteases, such as cathepsins and plasmin. These proteases may be present at elevated levels in certain tumor tissues.
  • the linker is cleavable by lysosomal enzymes. In certain embodiments, the linker is cleavable by a lysosomal enzyme, and the lysosomal enzyme is cathepsin B. In certain embodiments, the linker is cleavable by a lysosomal enzyme, and the lysosomal enzyme is ⁇ -glucuronidase or ⁇ -galactosidase.
  • Enzymatically cleavable linkers may include self-immolative spacers to spatially space the drug from the enzymatic cleavage site. Direct attachment of a drug to a peptide linker may result in proteolytic release of the drug's amino acid adducts, reducing its activity.
  • the use of a self-immolative spacer allows removal of a fully active, chemically unmodified drug upon amide bond hydrolysis.
  • One self-immolative spacer is a peptide via an amino group, while an amine-containing drug can be attached to the benzylic hydroxyl group of the linker via a carbamate functional group (giving p-amidobenzylcarbamate, PABC). It is a difunctional para-aminobenzyl alcohol group that is connected to, forming an amide bond.
  • the resulting prodrug is activated upon protease-mediated cleavage, resulting in a 1,6-elimination reaction that releases the remainder of the unmodified drug, carbon dioxide, and linker group.
  • the enzymatically cleavable linker is a ⁇ -galactoside based linker. While ⁇ -galactoside is abundantly present in lysosomes, its enzymatic activity outside the cell is low. Additionally, Bc1-xL inhibitors containing phenol groups can be covalently attached to the linker via the phenolic oxygen.
  • One such linker disclosed in US Patent Application Publication No. 2009/0318668, is a methodology in which diamino-ethane "SpaceLink" is used in conjugation with a traditional "PABO” based self-immolative group to deliver phenol. It depends.
  • a cleavable linker may comprise a non-cleavable moiety or segment and/or a cleavable segment or moiety may be included in an otherwise non-cleavable linker to render it cleavable.
  • polyethylene glycol (PEG) and related polymers may contain cleavable groups in the polymer backbone.
  • polyethylene glycol or polymer linkers may contain one or more cleavable groups such as disulfide, hydrazone, or dipeptide.
  • degradable linkages that may be included in the linker include ester linkages formed by the reaction between PEG carboxylic acids or activated PEG carboxylic acids and alcohol groups on the biologically active agent, wherein such ester groups are generally hydrolyzed under physiological conditions. Decomposes to release biologically active agents.
  • Hydrolytically degradable linkages include, but are not limited to, the ends of polymers and the 5' hydroxyl groups of oligonucleotides, including, but not limited to, carbonate linkages; An imine linkage resulting from the reaction of an amine and an aldehyde; a phosphate ester linkage formed by the reaction of an alcohol with a phosphate group; Acetal linkage, a reaction product of an aldehyde and an alcohol; Orthoester linkage, a reaction product of promate and alcohol; and oligonucleotide linkages formed by phosphoramidite groups.
  • linkers comprising the ADCs disclosed herein need not be cleavable.
  • drug release does not depend on differential properties between plasma and some cytoplasmic compartments. Release of the drug is assumed to occur after internalization of the ADC through antigen-mediated endocytosis and delivery to the lysosomal compartment where the antibody is degraded to the level of amino acids through intracellular proteolytic degradation. This process releases the drug derivative formed by the drug, the linker, and the amino acid residues to which the linker is covalently attached.
  • Amino acid drug metabolites from conjugates with non-cleavable linkers are more hydrophilic and generally less membrane permeable, resulting in less bystander effects and less non-specific toxicity compared to conjugates with cleaved linkers.
  • ADCs with non-cleavable linkers have greater stability in circulation than ADCs with cleaved linkers.
  • the non-cleavable linker may be an alkylene chain or may be polymeric in nature, for example based on polyalkylene glycol polymers, amide polymers, or of alkylene chains, polyalkylene glycols and/or amide polymers. Can contain segments.
  • the linker comprises polyethylene glycol having 1 to 6 ethylene glycol units.
  • the present invention provides a pharmaceutically acceptable compound or conjugate, wherein the compound of the general formula I or an isomer thereof, and/or a ligand-drug conjugate containing the same is used as a pharmaceutical.
  • a salt or solvate is provided.
  • the present invention provides a pharmaceutically acceptable compound or conjugate, wherein the compound of the general formula I, or an isomer thereof, and/or a ligand-drug conjugate containing the same is used as a pharmaceutical for treating cancer or proliferative diseases.
  • a salt or solvate is provided.
  • the present invention provides a pharmaceutical composition containing a compound of the above general formula I, or an isomer thereof, and/or a ligand-drug conjugate containing the same, or a pharmaceutically acceptable salt or solvate thereof as an active ingredient.
  • the present invention provides a drug for the treatment of cancer or proliferative disease comprising a compound of the above general formula I, or an isomer thereof, and/or a ligand-drug conjugate containing the same, or a pharmaceutically acceptable salt or solvate thereof as an active ingredient.
  • a drug for the treatment of cancer or proliferative disease comprising a compound of the above general formula I, or an isomer thereof, and/or a ligand-drug conjugate containing the same, or a pharmaceutically acceptable salt or solvate thereof as an active ingredient.
  • Pharmaceutical compositions are provided.
  • the present invention relates to a compound of the above general formula I, or an isomer thereof, and/or a ligand-drug conjugate containing the same, or a pharmaceutically acceptable salt or solvate thereof; and at least one pharmaceutically acceptable excipient.
  • proliferative disease refers to unwanted or uncontrolled cell proliferation of undesirable excessive or abnormal cells, such as neoplastic or hyperplastic growth, in vitro or in vivo.
  • the above proliferative diseases include carcinoma, lymphoma, leukemia, blastoma, sarcoma, liposarcoma, neuroendocrinoma, mesothelioma, schwannoma, meningioma, adenocarcinoma, melanoma, squamous cell carcinoma, squamous cell carcinoma, lung cancer, small cell lung cancer, and non-small cell lung cancer.
  • lung adenocarcinoma lung squamous carcinoma, peritonoma, hepatocellular carcinoma, gastric carcinoma, gastrointestinal tumor, pancreatic cancer, brain cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, liver cancer, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrium.
  • it may be selected from the group consisting of uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, liver carcinoma, anal carcinoma, penile carcinoma, testicular cancer, esophageal varices cancer, biliary tract cancer, colon cancer, and head and neck cancer. It is not limited to this.
  • a treatment method using an antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof is also provided.
  • the antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof is provided to a patient.
  • Antibody-drug conjugates, pharmaceutically acceptable salts thereof, or solvates thereof inhibit the metastasis of cancer cells by binding to targets expressed on the surface of cancer cells.
  • the antibody binds to a target expressed on the surface of cancer cells in a form bound to a cytotoxic agent, thereby specifically delivering the cytotoxic agent bound to the antibody to the cancer cells, thereby inducing death of the cancer cells.
  • the antibody binds to a target expressed on the surface of cancer cells in the form of an antibody specific for the same target or a different target, thereby increasing the specificity of the polyantibody to cancer cells or forming a link between cancer cells and other types of cells such as immune cells. induces the death of cancer cells.
  • a pharmaceutical composition comprising a therapeutically effective amount of an antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof, and a pharmaceutically acceptable diluent, carrier, solubilizer, emulsifier, preservative and/or adjuvant. do. Also included are methods of treating cancer patients, for example, by administering such pharmaceutical compositions.
  • patient includes human patients.
  • the pharmaceutical composition may include a pharmaceutically acceptable carrier.
  • the carrier is used to include an excipient, diluent, or auxiliary agent.
  • the carriers include, for example, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, polyvinyl chloride. It may be selected from the group consisting of rolidone, water, physiological saline, buffer solutions such as PBS, methyl hydroxy benzoate, propyl hydroxy benzoate, talc, magnesium stearate, and mineral oil.
  • the composition may include fillers, anti-coagulants, lubricants, wetting agents, flavoring agents, emulsifiers, preservatives, or combinations thereof.
  • the pharmaceutical composition can be prepared in any dosage form according to conventional methods.
  • the composition may be formulated, for example, as an oral dosage form (e.g., powder, tablet, capsule, syrup, pill, or granule), or a parenteral dosage form (e.g., injection). Additionally, the composition may be prepared as a systemic formulation or a topical formulation.
  • Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc. These solid preparations contain one or more compounds and at least one excipient, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. It is prepared by mixing. In addition to simple excipients, lubricants such as magnesium stearate, talc, etc. may also be used. Meanwhile, when administered orally, proteins or peptides are digested, so when formulating them in the form of oral compositions, it is necessary to coat the active ingredients or protect them from decomposition in the stomach.
  • excipient such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. It is prepared by mixing. In addition to simple excipients, lubricants such as magnesium stearate, talc, etc. may also be used.
  • Liquid preparations for oral administration include suspensions, oral solutions, emulsions, and syrups.
  • various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. there is.
  • Preparations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories.
  • Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable esters such as ethyl oleate.
  • injectable esters such as ethyl oleate.
  • As a base for suppositories witepsol, macrogol, tween 61, cacao, laurel, glycerogelatin, etc. can be used.
  • the pharmaceutical composition is selected from the group consisting of injections, tablets, pills, powders, granules, capsules, suspensions, oral solutions, emulsions, syrups, sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations and suppositories. It can have any one formulation.
  • the active ingredient may be in the form of an acceptable aqueous solution for parenteral administration that is pyrogen-free and has appropriate pH, isotonicity and stability.
  • aqueous solution for parenteral administration that is pyrogen-free and has appropriate pH, isotonicity and stability.
  • isotonic vehicles such as, for example, aqueous sodium chloride solution, Ringer's solution, lactated Ringer's solution, etc., and preservatives, stabilizers, buffers, antioxidants or other additives may be included as required.
  • Solid forms suitable for injection can also be prepared as emulsions or in the form of polypeptides encapsulated in liposomes.
  • the conjugate according to the invention may be administered by any device capable of transporting the active substance to the target cell.
  • the pharmaceutical composition may contain an effective amount of the antibody or antigen-binding fragment thereof, an anticancer agent, or a combination thereof.
  • the term “effective amount” refers to an amount sufficient to produce a prophylactic or therapeutic effect when administered to an individual in need of such prophylaxis or treatment.
  • the effective amount can be appropriately selected by a person skilled in the art depending on the cell or organism selected. Factors including the severity of the disease, the patient's age, weight, health, gender, the patient's sensitivity to the drug, time of administration, route of administration and excretion rate, duration of treatment, drugs combined or used simultaneously with the composition used, and other fields of medicine. It can be determined according to well-known factors.
  • the dosage of the pharmaceutical composition may be, for example, 10 ⁇ g/kg to about 30 mg/kg, optionally 0.1 mg/kg to about 30 mg/kg, or alternatively 0.3 mg/kg to about 20 mg/kg, for an adult. It may be in the range of mg/kg.
  • the administration may be administered once a day, multiple times a day, once a week to four weeks, or once a year to 12 times.
  • the auristatin prodrug according to the present invention has the advantage that by introducing a self-immolative group, the shortcomings of existing drugs due to high lipophilicity, pharmacokinetic properties, cytotoxicity, etc. are improved, thereby showing excellent drug efficacy. There is.
  • Figure 1 is a diagram showing the active drug release mechanism of a prodrug according to one embodiment.
  • Figure 2 is a diagram showing an example of synthesis of an antibody-drug conjugate (ADC) using a prodrug according to an embodiment.
  • ADC antibody-drug conjugate
  • Figure 3 is a graph showing the results of analyzing the hydrophobicity of an antibody-drug conjugate according to an example through HIC-HPLC.
  • N,N' -dimethylethylene-1,2-diamine (5 g, 56.72 mmol) was dissolved in tetrahydrofuran (200 mL) and di- t -butyl dicarbonate (4.3 mL, 18.9 mmol) was dissolved at 0°C. was added and the reaction solution was stirred at room temperature for 17 hours. The reaction solution was concentrated under reduced pressure and purified by column chromatography to obtain Compound 1 (1.7 g, 47%).
  • reaction solution was diluted with ethyl acetate (100 mL), washed with saturated aqueous sodium bicarbonate solution (50 mL), then distilled water (50 mL), and dried over anhydrous sodium sulfate. After filtration, concentration under reduced pressure and purification by column chromatography, compound 8 (715 mg, 81%) was obtained.
  • reaction solution was diluted with dichloromethane (100 mL), washed with saturated aqueous ammonium chloride solution (50 mL) and distilled water (50 mL), and dried over anhydrous sodium sulfate. After filtration, concentration under reduced pressure and purification by column chromatography, compound 12 (633 mg, 83%) was obtained.
  • N -( t -butoxycarbonyl)-L-valine (2.0 g, 9.3 mmol) was dissolved in dichloromethane (30 mL) and then N -hydroxysuccinimide (1.6 g, 13.9 mmol) at 0°C. and N -(3-dimethylaminopropyl)- N' -ethylcarbodiimide hydrochloride (EDC ⁇ HCl, 2.7 g, 13.9 mmol) were added, and the reaction solution was stirred at room temperature for 3 hours.
  • Triethylene glycol (30 g, 199.8 mmol) was dissolved in dichloromethane (20 mL), and then 4-toluenesulfonyl chloride (76 g, 399.6 mmol) and triethylamine (67 mL, 479.5 mmol) were dissolved in dichloromethane (20 mL) at 0°C. mmol) was added and the reaction solution was stirred at room temperature for 16 hours.
  • the reaction solution was diluted with dichloromethane (500 mL), washed with saturated aqueous ammonium chloride solution (300 mL) and brine (200 mL) in that order, and dried over anhydrous sodium sulfate.
  • 3-Aminopentanedioic acid hydrochloride (5.0 g, 27.2 mmol) was dissolved in distilled water/1,4-dioxane (6 mL/44 mL) and then mixed with aqueous sodium hydroxide solution (4 M, 20.4 mL, 81.6 mmol) and di- t . -Butyl dicarbonate (6.87mL, 29.9 mmol) was added. The reaction solution was stirred at room temperature for 15 hours, then adjusted to pH 2 with an aqueous potassium hydrogen sulfate solution and extracted with ethyl acetate (3 x 50 mL).
  • D-glutamic acid -Methyl ester (HD-Glu(OMe)-OH, 40 g, 249.8 mmol) was dissolved in 1,4-dioxane (80 mL) and distilled water (40 mL), and then di- t -butyl was dissolved in nitrogen atmosphere at 0°C.
  • Dicarbonate (71 g, 324.7 mmol) and sodium bicarbonate (63 g, 749.3 mmol) were added.
  • reaction solution was stirred at room temperature for 5 hours, adjusted to pH about 4 with 1 N aqueous hydrochloric acid, diluted with ethyl acetate (200 mL), washed with distilled water (100 mL), and dried over anhydrous sodium sulfate. After filtration, the mixture was concentrated under reduced pressure, dissolved in N,N -dimethylformamide (20 mL), and potassium carbonate (46 g, 328.4 mmol) and benzyl bromide (60 mL, 505.2 mmol) were added under a nitrogen atmosphere at 0°C.
  • reaction solution was diluted with ethyl acetate (500 mL), washed with saturated aqueous sodium bicarbonate solution (300 mL), then distilled water (300 mL), and dried over anhydrous sodium sulfate. After filtration, concentration under reduced pressure and purification by column chromatography, compound 60 (13 g, 41%) was obtained.
  • reaction solution was diluted with ethyl acetate (200 mL), washed with saturated aqueous ammonium chloride solution (100 mL), saturated aqueous sodium bicarbonate solution (100 mL), and distilled water (100 mL), and dried over anhydrous sodium sulfate. After filtration, concentration under reduced pressure and purification by column chromatography, compound 65 (3.2 g, 79%) was obtained.
  • Compound 79 (Compound 79 was prepared by the method described in Korean Patent Application No. 10-2023-0032106), 1.5 g, 0.67 mmol) was dissolved in dichloromethane (24 mL) and then dissolved in pyridine (0.22 mL, 2.68 mmol). Bis(pentafluorophenyl)carbonate (0.79 g, 2.01 mmol) was added at room temperature under a nitrogen atmosphere and stirred for 16 hours. The reaction solution was diluted with ethyl acetate (80 mL), washed with saturated aqueous sodium bicarbonate solution (50 mL x 2) and brine (50 mL), and dried over anhydrous sodium sulfate.
  • Compound 85 (754 mg, 1.12 mmol, Compound 85 was prepared by the method described in published patent WO2020222573A1) and Compound 64 (1.3 g, 2.70 mmol) were dissolved in N,N -dimethylformamide (15 mL) and then dissolved in 0 N,N,N',N' -tetramethyl- O- (1 H -benzotriazol-1-yl)uronium hexafluorophosphate (HBTU, 1.4 g, 3.72 mmol) and N under nitrogen atmosphere at °C. ,N' -Diisopropyl-ethylamine (1 mL, 5.63 mmol) was sequentially added and stirred at room temperature for 22 hours.
  • HBTU N,N,N',N' -Diisopropyl-ethylamine
  • reaction solution was diluted with ethyl acetate (200 mL), washed with saturated aqueous ammonium chloride solution (100 mL), saturated aqueous sodium bicarbonate solution (100 mL), and distilled water (100 mL), and dried over anhydrous sodium sulfate. After filtration, concentration under reduced pressure and purification by column chromatography, compound 65 (347 mg, 19%) was obtained. EI-MS m/z: 1602.55.
  • Compound 93 (892 mg, 0.93 mmol, Compound 93 was prepared by the method described in published patent WO2017089895A1) and Compound 64 (539 mg, 1.11 mmol) were dissolved in N,N -dimethylformamide (5 mL) and then dissolved in 0 °C, under nitrogen atmosphere 1-[bis(dimethylamino)methylene]-1 H -1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU, 458 mg, 1.21 mmol) and N,N' -diisopropylethylamine (0.48 mL, 2.78 mmol) were sequentially added and stirred at room temperature for 22 hours.
  • HATU 1-[bis(dimethylamino)methylene]-1 H -1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate
  • reaction solution was concentrated under reduced pressure and diluted with dichloromethane (100 mL) and methanol (10 mL), followed by saturated aqueous ammonium chloride solution (50 mL), saturated aqueous sodium bicarbonate solution (50 mL), and distilled water (50 mL). It was wiped and dried with anhydrous sodium sulfate. After filtration, concentration under reduced pressure and purification by column chromatography, compound 94 (885 mg, 66%) was obtained. EI-MS m/z: [M+H] + 1429.31, 1/2[M+H] + 715.05.
  • reaction solution was stirred at 60°C for 20 hours, diluted with ethyl acetate (100 mL), washed with saturated aqueous ammonium chloride solution (50 mL) and distilled water (2 After filtration and concentration under reduced pressure, the concentrated filtrate was dissolved in N,N -dimethylformamide (10 mL), and t -butyldimethylsilyl chloride (419 mg, 2.78 mmol) and imidazole (189) were added under nitrogen atmosphere at 0°C. mg, 2.78 mmol) was added.
  • reaction solution was stirred at room temperature for 1 hour, diluted with ethyl acetate (100 mL), washed with saturated aqueous ammonium chloride solution (50 mL) and distilled water (2 After filtration, concentration under reduced pressure and purification by column chromatography, compound 106 (818 mg, 77%) was obtained.
  • reaction solution was stirred at room temperature for 3 hours, diluted with ethyl acetate (100 mL), washed with 0.1 N aqueous hydrochloric acid (50 mL) and distilled water (2 After filtration, concentration under reduced pressure and purification by column chromatography, compound 107 (1.1 g, 94%) was obtained.
  • Comparative compound 113 was prepared by the method described in published patent WO2020180121A1.
  • the ADC was manufactured through the following two steps, and the commonly used LCB14-0606 was manufactured by the method described in Korean Application No. 10-2013-7032628. This document is incorporated herein by reference in its entirety.
  • the structural formula of LCB14-0606 is as follows:
  • a prenylation reaction mixture of the DLK1 antibody 18A5-CaaX described in published patent WO2020180121A1 was prepared and reacted at 30°C for 16 hours.
  • the reaction mixture contained a total of 1000 mg of 48 ⁇ M antibody, 400 nM FTase (Genscript), and 288 ⁇ M LCB14-0606 in buffer solution (50 mM Tris-HCl (pH 7.4), 5 mM MgCl 2 , 10 ⁇ M ZnCl 2 , 0.5 ⁇ M ZnCl 2 ). It consisted of (mM DTT).
  • the prenylated antibody was decontaminated through ultrafiltration (Vivaspin 20, Satorius) using a PBS buffer solution. As a result, a total of 906 mg of prenylated antibody was prepared.
  • Step 2 Drug-conjugation method
  • the oxime bond formation reaction between the prenylated antibody and the linker-drug was performed using a total of 50.0 mg of 100 ⁇ M Na-acetate buffer pH 5.2, 10% DMSO, 48 ⁇ M prenylated antibody and 6 equivalents of the linker-drug (in house, Example 29).
  • Compound No. 113 was mixed and stirred at 500 to 600 rpm at 30°C for 24 hours. After the reaction, excess low-molecular-weight compounds were removed through ultrafiltration (Vivaspin 20, Satorius) using a PBS buffer solution, and the protein fraction was collected and concentrated. As a result, a total of 34.4 mg of ADC1 was obtained.
  • the oxime bond formation reaction between the prenylated antibody and the linker-drug was performed using a total of 50.0 mg of 100 ⁇ M Na-acetate buffer pH 5.2, 10% DMSO, 48 ⁇ M prenylated antibody and 6 equivalents of the linker-drug (in house, Example 15).
  • Compound No. 69 was mixed and stirred at 500 to 600 rpm at 30°C for 24 hours. After the reaction, excess low-molecular-weight compounds were removed through ultrafiltration (Vivaspin 20, Satorius) using a PBS buffer solution, and the protein fraction was collected and concentrated. As a result, a total of 39.9 mg of ADC2 was obtained.
  • the oxime bond formation reaction mixture between the prenylated antibody and the linker-drug was 100 ⁇ M Na-acetate buffer pH 5.2, 10% DMSO, 48 ⁇ M a total of 20.1 mg of prenylated antibody and 6 equivalents of the linker-drug (in house, Example 10) Compound No. 45) was mixed and stirred at 500 to 600 rpm at 30°C for 24 hours. After the reaction, excess low-molecular-weight compounds were removed through ultrafiltration (Vivaspin 20, Satorius) using a PBS buffer solution, and the protein fraction was collected and concentrated. As a result, a total of 11.2 mg of ADC3 was obtained.
  • ADC1, ADC2, and ADC3 all have different hydrophobicities, and the hydrophobicity was confirmed to be greater in the order ADC2 > ADC1 > ADC3, and the purity in terms of DAR (drug-antibody ratio) was confirmed to be more than 96%. did.
  • ADC Antibody Linker-toxin ADC1 18A5-CaaX Compound 113 ADC2 Compound 69 ADC3 Compound 45
  • the cell proliferation inhibitory ability of the compounds listed in Table 2 below was measured in cancer cell lines and hematopoietic stem cells.
  • MIA-PaCa2 a human pancreatic cancer cell, was used as a cancer cell line, and commercially available CD34-positive hematopoietic stem cells (Stem cells, Cat No. 70002.2) were used as hematopoietic stem cells.
  • CD34-positive hematopoietic stem cells Stem cells, Cat No. 70002.2
  • cancer cell lines were treated with the compounds at a concentration of 0.256 pM ⁇ 100 nM (5-fold serial dilution), and hematopoietic stem cells were treated at a concentration of 12.8 pM ⁇ 5 ⁇ M (5-fold serial dilution).
  • SRB Steforhodamine B
  • CTG Cell titer glo, Promega
  • CC 50 in cancer cell lines was measured at MMAE of 2.70 nM, and the remaining three compounds could not be measured because they all showed cell viability of more than 95% even at the highest treatment concentration.
  • MMAE was measured at 1.01 nM, Compound 17 at 176.6 nM, Compound 22 at 38.11 nM, and Compound 31 at 189.7 nM.
  • MMAE showed the strongest cytotoxicity, and Compound 17 and Compound 31 were measured at 189.7 nM.
  • Compound 31 was more than 100 times less toxic, confirming its superior safety as an MMAE prodrug.
  • the cell proliferation inhibitory ability of the ADCs listed in Table 3 below was measured in cancer cell lines and hematopoietic stem cells.
  • MIA-PaCa2_DLK1+ a human pancreatic cancer cell that overexpresses DLK1
  • HaCaT was used as a normal skin cell
  • Fa2N4 was used as a normal liver cell
  • hPBMC was used as a peripheral blood mononuclear cell.
  • MIA-PaCa2_DLK1+ a human pancreatic cancer cell that overexpresses DLK1
  • HaCaT was used as a normal skin cell
  • Fa2N4 was used as a normal liver cell
  • hPBMC was used as a peripheral blood mononuclear cell.
  • 3,000 MIA-PaCa2_DLK1+, 1000 HaCaT, 15,000 Fa2N4, and 60,000 hPBMC were seeded per well.
  • the compounds were treated at a concentration of 0.256 pM ⁇ 100 nM (5-fold serial dilution) in cancer cell lines, 2.56 pM ⁇ 1000 nM (5-fold serial dilution) in HaCaT and Fa2N4, and 12.8 pM ⁇ 5-fold serial dilution in hPBMC. Treated at ⁇ M (5-fold serial dilution) concentration.
  • the number of viable cancer cells, HaCaT, and Fa2N4 were measured using SRB (Sulforhodamine B) assay, and hPBMC was measured using CTG (Cell titer glo, Promega) assay.
  • the CC50 in cancer cell lines was measured to be 0.18, 0.24, and 0.85 nM for ADC1, ADC2, and ADC3, respectively.
  • the CC50 in normal skin cells, HaCaT, for ADC1, ADC2, and ADC3 were measured to be 43.9, 258.7, and 501.8 nM, respectively.
  • CC50 in normal liver cells Fa2N4 could not be measured because ADC1, ADC2, and ADC3 all showed cell viability of more than 50% at the highest treatment concentration, but the viability was measured at 64.5, 99.3, and 99.1%, respectively.
  • the CC50 in hPBMC a peripheral blood mononuclear cell, was measured to be 57.8, 2568.0, and 1746.0 nM for ADC1, ADC2, and ADC3, respectively.
  • ADC2 and ADC3 introduced with MMAE prodrug were confirmed to have superior safety in normal cells compared to ADC1 introduced with MMAE.
  • the drug therapeutic indices for HaCaT and hPBMC are listed in Table 4 below.
  • the drug therapeutic index was calculated by dividing the CC50 in normal cells by the CC50 in cancer cells.
  • Test samples TI (drug therapeutic index, times) CC50 HaCaT / CC50 MIA-PaCa2_DLK1+ CC50hPBMC/ CC50 MIA-PaCa2_DLK1+ ADC1 247 325 ADC2 1087 10786 ADC3 590 2054
  • the pharmacotherapy index of ADC1 was calculated as 247 and 325, respectively, the pharmacotherapy index of ADC2 was calculated as 1087 and 10786, respectively, and the pharmacotherapy index of ADC3 was calculated as 590 and 2054, respectively.

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Abstract

La présente invention concerne un médicament précurseur d'uristatine et, plus particulièrement, un médicament précurseur d'auristatine portant un groupe auto-immolable, son procédé de préparation, une composition pharmaceutique le comprenant, et son utilisation pour la prophylaxie et/ou la prévention d'une maladie.
PCT/KR2023/011084 2022-07-28 2023-07-28 Nouveau médicament précurseur d'auristatine WO2024025396A1 (fr)

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