WO2023042643A1 - リンカー化合物及びそれを含む複合体化合物 - Google Patents

リンカー化合物及びそれを含む複合体化合物 Download PDF

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WO2023042643A1
WO2023042643A1 PCT/JP2022/032220 JP2022032220W WO2023042643A1 WO 2023042643 A1 WO2023042643 A1 WO 2023042643A1 JP 2022032220 W JP2022032220 W JP 2022032220W WO 2023042643 A1 WO2023042643 A1 WO 2023042643A1
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compound
linker
drug
formula
group
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French (fr)
Japanese (ja)
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健彦 和田
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Tohoku University NUC
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Tohoku University NUC
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Priority to EP22869790.0A priority Critical patent/EP4403549A1/en
Priority to JP2023548386A priority patent/JPWO2023042643A1/ja
Priority to US18/691,722 priority patent/US20240374750A1/en
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C321/00—Thiols, sulfides, hydropolysulfides or polysulfides
    • C07C321/24—Thiols, sulfides, hydropolysulfides, or polysulfides having thio groups bound to carbon atoms of six-membered aromatic rings
    • C07C321/28—Sulfides, hydropolysulfides, or polysulfides having thio groups bound to carbon atoms of six-membered aromatic rings
    • 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
    • A61K47/68—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 antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6889—Conjugates wherein the antibody being the modifying agent and wherein the linker, binder or spacer confers particular properties to the conjugates, e.g. peptidic enzyme-labile linkers or acid-labile linkers, providing for an acid-labile immuno conjugate wherein the drug may be released from its antibody conjugated part in an acidic, e.g. tumoural or environment
    • 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
    • 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
    • A61K47/645—Polycationic or polyanionic oligopeptides, polypeptides or polyamino acids, e.g. polylysine, polyarginine, polyglutamic acid or peptide TAT
    • 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
    • A61K47/68—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 antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/6807—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug or compound being a sugar, nucleoside, nucleotide, nucleic acid, e.g. RNA antisense
    • 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
    • A61K47/68—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 antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
    • 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
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C323/00—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups
    • C07C323/01—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and halogen atoms, or nitro or nitroso groups bound to the same carbon skeleton
    • C07C323/09—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and halogen atoms, or nitro or nitroso groups bound to the same carbon skeleton having sulfur atoms of thio groups bound to carbon atoms of six-membered aromatic rings of the carbon skeleton
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C323/00—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups
    • C07C323/10—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and singly-bound oxygen atoms bound to the same carbon skeleton
    • C07C323/18—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and singly-bound oxygen atoms bound to the same carbon skeleton having the sulfur atom of at least one of the thio groups bound to a carbon atom of a six-membered aromatic ring of the carbon skeleton
    • C07C323/20—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and singly-bound oxygen atoms bound to the same carbon skeleton having the sulfur atom of at least one of the thio groups bound to a carbon atom of a six-membered aromatic ring of the carbon skeleton with singly-bound oxygen atoms bound to carbon atoms of the same non-condensed six-membered aromatic ring
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C323/00—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups
    • C07C323/50—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and carboxyl groups bound to the same carbon skeleton
    • C07C323/62—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and carboxyl groups bound to the same carbon skeleton having the sulfur atom of at least one of the thio groups bound to a carbon atom of a six-membered aromatic ring of the carbon skeleton

Definitions

  • the present invention relates to a conjugated compound in which a drug compound used for treatment is conjugated with another compound, a linker compound used to form the conjugated compound, and a conjugated compound using the same.
  • Cancer is a disease that ranks high in both morbidity and mortality in Japan and the world, and the establishment of its treatment is one of the most important issues.
  • drugs for cancer relatively high-molecular-weight biopharmaceuticals produced by using gene recombination techniques have been developed from low-molecular-weight drugs in recent years.
  • antibody drugs using the distinguishing property of antibodies have been developed.
  • Antibody drugs have the advantages of high target specificity and fewer side effects than low-molecular-weight drugs. For this reason, antibody drugs are expected to be applied to cancer treatments by specifically acting on cancer cells.
  • Antibody drugs can be developed in a short period of time using existing drugs and antibodies, and are expected to be developed with a drug development strategy that has relatively low development costs and a high probability of success. It is also expected in terms of possibility.
  • antibody drugs generally have many problems such as complicated manufacturing processes, high manufacturing and development costs, high molecular weight drugs, and antigenicity.
  • due to its large molecular weight, etc. its cell membrane permeability is extremely low, and its target is sometimes limited to extracellular proteins such as cell surface layers.
  • an antibody-drug conjugate (antibody-drug conjugate) is created by combining an antibody and a drug with a linker compound, and by giving the linker compound a dissociable function, the drug exerts its function independently of the antibody. Technologies to enable this are also being developed.
  • Patent Document 1 discloses a drug-antibody conjugate compound in which an antibody and a drug are linked via a linker compound.
  • This drug-antibody conjugate compound uses a linker compound having a sulfide group to form a disulfide bond with a cysteine in the Fc portion of the antibody to form a conjugate of the pyrrolobenzodiazepine prodrug monomer (drug), the antibody and the linker compound. It is what we are trying to form.
  • Non-Patent Document 1 discloses an alkane-type disulfide linker as a linker that binds an antibody and a drug. This linker cleaves the disulfide linker in a glutathione concentration-dependent manner, and the cleavage is to be evaluated using FRET.
  • Non-Patent Document 2 discloses a dithiobenzylurethane linker as a linker that binds an antibody and a drug. This linker attempts to cleave the disulfide linker in a glutathione concentration-dependent manner.
  • the disulfide bond of the linker compound binds the antibody and the drug-composing compound, and the antibody and the drug-composing compound are cleaved according to the glutathione concentration, and the drug is treated. function can be exhibited.
  • the concentration of glutathione is said to be 5 to 25 ⁇ M in blood and 0.4 mM to less than 10 mM in normal cells.
  • Patent Document 1 identifies a range of glutathione concentrations greater than three digits (mM order) as the intracellular concentration. When the antibody-drug conjugate is administered into the blood, the glutathione concentration is low ( ⁇ M order), so the conjugated state is maintained.
  • the glutathione concentration becomes high (mM order)
  • the disulfide bond of the linker compound is cleaved
  • the conjugate of the antibody and the compound that constitutes the drug is dissociated, and the drug is released. It can exert its medicinal effect in cells.
  • the linker compound can only dissociate the complex of the antibody and the compound constituting the drug at a certain concentration according to the structure of each compound.
  • the conjugate dissociates when the glutathione concentration reaches the mM order. exert its medicinal effect. Therefore, since the drug dissociates even in normal cells, the drug may act on unwanted cells and cause side effects.
  • cancer cells are known to have a glutathione concentration of 10 to 100 times higher than that of normal cells, averaging about 10 to 200 mM.
  • the present inventors considered whether it would be possible to distinguish between cancer cells and normal cells based on the difference in glutathione concentration between cancer cells and normal cells.
  • the linker compound disclosed in Patent Document 1 can distinguish between ⁇ M-order and mM-order glutathione concentrations due to the structure of the linker compound.
  • the linker using a disulfide bond used in Non-Patent Documents 1 and 2 is an alkane-type linker based on a thiol group such as cysteine.
  • the alkane-type linker can adjust the easiness of dissociation and binding by adjusting the steric hindrance of the S atom, but it is difficult to give a high degree of selectivity according to various glutathione concentrations.
  • glutathione concentration in cancer cells varies depending on the cell type and cancer state.
  • linker compounds that dissociate at a given concentration of glutathione may be insufficient for therapeutic use in a variety of cancer cells and cancer conditions.
  • the present inventors have found that by using a multifunctional linker compound with a higher degree of freedom in designing functions, various cancer cells can be identified, target cells can be accurately selected, and effective It was considered that it can be used for cancer treatment. Specifically, if the glutathione concentration at which disulfide bonds dissociate can be adjusted by molecular design, it is possible to obtain a multifunctional linker compound that can distinguish various cancer cells and cancer states depending on the glutathione concentration. Further research was carried out on linker compounds.
  • the present invention has been made in view of the above circumstances, and the concentration of glutathione at which disulfide bonds dissociate can be designed by molecular design, cancer cells can be accurately identified, and the glutathione is stable and targeted outside the target cells.
  • An object of the present invention is to provide a linker compound and a conjugate compound containing the linker compound that can effectively release a drug compound in cells.
  • a linker compound used in a conjugate compound containing a drug compound the linker compound having a structure represented by the following formula (1).
  • R 1 and R 2 are each a compound containing hydrogen, carbon, oxygen, nitrogen, or a halogen element, or a derivative thereof.
  • the linker compound, wherein the conjugate compound is an antibody drug conjugate compound in which the drug compound and the drug compound transit peptide are conjugated via the linker compound.
  • the conjugate compound, which is an antibody-drug conjugate compound in which the drug compound and an antibody are conjugated via the linker compound.
  • the complex compound, wherein the drug compound is an apoptosis fusion peptide.
  • the complex compound, wherein the drug compound is a nucleic acid.
  • the concentration of glutathione at which disulfide bonds dissociate can be designed by molecular design, cancer cells can be accurately identified, and a drug compound can be released stably outside target cells and effective in target cells.
  • a linker compound and a conjugate compound containing the same are obtained.
  • FIG. 2 is a graph showing the NMR spectrum of the compound used in Test Example 1.
  • FIG. FIG. 2 is a graphical representation comparing the NMR spectra of the compounds of FIG. 1 overlaid;
  • 4 is a graph showing the NMR spectrum of the compound used in Comparative Example 1.
  • FIG. 4 is a graph showing the NMR spectrum of the compound used in Test Example 2.
  • FIG. 5 is a partially enlarged view of FIG. 4;
  • FIG. FIG. 5 is a graph showing the result of plotting changes in absorption intensity at 242 nm in FIG. 4 with respect to time;
  • 3 is a graph showing NMR spectra of some compounds used in Test Example 3.
  • FIG. 3 is a graph showing NMR spectra of some compounds used in Test Example 3.
  • FIG. 1 is a graph showing NMR spectra of some compounds used in Test Example 3.
  • FIG. 3 is a graph showing NMR spectra of some compounds used in Test Example 3.
  • FIG. 3 is a graph showing NMR spectra of some compounds used in Test Example 3.
  • FIG. 4 is a graph showing the NMR spectrum of the compound used in Test Example 4.
  • FIG. 3 is a graph showing an NMR spectrum of another compound used in Test Example 3.
  • FIG. 13 is a partially enlarged view of FIG. 12;
  • FIG. 13 is another partially enlarged view of FIG. 12;
  • FIG. 10 is a confocal microscopic view of fluorescent coloring of cells used in Test Example 9.
  • the linker compound of this embodiment is a linker compound used for a complex compound containing a drug compound, and has a structure represented by the following formula (1).
  • R 1 and R 2 are each a compound containing hydrogen, carbon, oxygen, or a halogen element, or a derivative thereof.
  • a linker compound is a compound that is used to join other compounds and form a conjugate compound with those other compounds. Primarily through substitution on either the benzene ring of formula (1) or on R 1 , R 2 , it binds to other compounds to form complex compounds.
  • a linker compound is used for a conjugate compound containing a drug compound. That is, at least one of the plurality of compounds directly or indirectly bound to the linker compound to form the complex compound is a drug compound.
  • Pharmaceutical compounds broadly refer to compounds that act as drugs and compounds that exert therapeutic effects.
  • the pharmaceutical compound is used in the treatment of cancer.
  • the complex compound is preferably used against cancer cells.
  • the linker-binding portion dissociates according to the concentration of glutathione. Therefore, when introduced into cancer cells with a high concentration of glutathione, the drug compound separates from the complex compound and is suitable for acting as a drug. ing.
  • R 1 and R 2 are substituents in the aromatic ring in the formula.
  • R 1 and R 2 may be substituted at any position in the aromatic ring.
  • R 1 is substituted on one aromatic ring
  • R 2 is substituted on the other aromatic ring. It may be a group, and may be substituted at multiple positions on the aromatic ring.
  • R 1 , R 2 , and a plurality of substituents that can be contained therein may be the same substituents or different substituents.
  • the substituent may be substituted at any position ortho, meta or para with respect to the position to which S is attached on the aromatic ring.
  • the present inventors found that the conditions under which the S--S bond dissociates, specifically reducing conditions such as glutathione concentration, can be adjusted by adjusting the S atom of the S--S bond.
  • the conditions under which the S--S bond dissociates specifically reducing conditions such as glutathione concentration
  • it might be possible by adjusting the electron density of Mutual thiol-disulfide exchange reactions between S atoms and S—S bonds are controlled by the acidity (pKa) of the thiol, the electronic environment of the S atom, steric hindrance, entropy, and the like. Therefore, to control the reactivity of the disulfide linker with GSH, it is necessary to change the above factors.
  • linker candidate compound As a linker candidate compound, by adjusting the electronic environment in the aromatic ring with the substituent (-R) of the aromatic ring bonded to or adjacent to the S atom, the environment of the S atom is controlled and the reactivity with GSH is controlled. I expected. Therefore, at first, attention was paid to the use of benzylthiol derivatives (Formulas (10) and (11) described later) that can be synthesized based on commercially available isophthalic acid. However, with benzylthiol derivatives, it was not possible to obtain a difference in electron density by proton NMR depending on the presence or absence of substituents, and it was considered difficult to design them as linker compounds.
  • R 1 and R 2 are each preferably a halogen element, a hydrocarbon group, an oxide group, or a nitride group.
  • each of R 1 and R 2 is preferably a halogen element, methyl group, t-butyl group, methoxy group, nitro group, carboxyl group, cyano group or dimethylamino group.
  • R 1 and R 2 are each preferably a nitro group, a fluorine group, or a carboxyl group.
  • R 1 and R 2 are each preferably a nitro group, a fluorine group, or a carboxyl group.
  • substituents or compounds by selecting R 1 and R 2 from these elements, substituents or compounds, the electron density of the S atom in the above formula (1) is designed, and the glutathione concentration in cancer cells is selected. It can be a structure suitable for dissociating effectively. Specifically, by setting R 1 and R 2 to ortho-coordinated carboxyl groups, 80% or more of normal cells do not dissociate one day after administration, and a cancer cell model with a glutathione concentration of 20 mM It can be a structure in which nearly 100% dissociate one day after administration.
  • both R 1 and R 2 are ortho-coordinated carboxyl groups and meta-coordinated fluorine elements
  • 70% or more at 100 minutes after administration to a cancer cell model with a glutathione concentration of 20 mM can be a structure that dissociates.
  • 70% or more at 100 minutes after administration to a cancer cell model with a glutathione concentration of 20 mM can be a structure that dissociates.
  • R 1 and R 2 are meta-coordinated carboxyl groups and para-coordinated nitro group elements, 80% after administration to a cancer cell model with a glutathione concentration of 20 mM at 100 minutes after administration A structure in which the above dissociates can be obtained.
  • the S—S bond is cleaved at a glutathione concentration of 10 to 200 mM.
  • the S—S bond can be cleaved at glutathione concentrations in various cancer cells.
  • the scale (order) of glutathione concentration in tissues is considered to be approximately 10 -6 to 10 -5 M in blood, approximately 1000 to 100000 ⁇ M in normal cells, and approximately 10000 to 100000 ⁇ M in cancer cells in terms of molar concentration. It is In particular, on average, it ranges from 5 to 25 ⁇ M in blood, from 0.4 mM to less than 10 mM in normal cells, and from 10 mM to 200 mM in cancer cells.
  • the linker compound and the linker portion of the conjugate compound containing the linker compound are contained at a glutathione concentration of 10 to 200 mM in cancer cells under specific conditions. can be designed to cleave at a glutathione concentration of 10 to 200 mM.
  • the structure of the compound of formula (1) more specifically, the structure of R 1 and R 2 , and the substitution positions on the aromatic ring can be designed to increase the electron density of the S atom.
  • the linker compounds of the present embodiments can be widely used to form conjugate compounds with drug compounds and other compounds. Specifically, as described later, it can be used for an antibody-drug conjugate compound in which the drug compound and the antibody are conjugated via the linker compound.
  • the complex compound of this embodiment contains the linker compound. That is, the conjugate compound includes the drug compound and the linker compound. In particular, it includes compounds in which drug compounds and other compounds are conjugated via said linker compounds.
  • the conjugate compound is preferably an antibody-drug conjugate compound in which the drug compound and the drug compound transit peptide are conjugated via the linker compound.
  • the drug compound transit peptide broadly refers to a peptide that has the function of complexing with a drug compound and guiding the drug compound to a target.
  • the term "peptide" as used herein refers broadly to a conjugate of a plurality of amino acids, ranging from a few to large polypeptides (so-called proteins).
  • a target refers to, for example, a particular cell or a particular location in a cell.
  • a specific cell is a cell having a specific property such as a cancer cell, and a specific position in a cell refers to the inside of the cell, the surface of the cell, or the like.
  • the drug compound transit peptide is selected from the group consisting of antibodies and cell membrane penetrating peptides.
  • These effects include, for example, antibodies that deliver drug compounds to target cancer cells, and cell membrane-penetrating peptides such as linker peptides that deliver drug compounds into cells. It has the function of leading to a place.
  • the cell membrane penetrating peptide is also preferably a linker peptide (peptide linker) that binds the above linker compound to another compound.
  • a linker peptide for example, an arginine linker having an oligoarginine sequence in which multiple arginines are bound can be used.
  • Peptides having an oligoarginine sequence or arginine-rich peptides facilitate intracellular uptake. Therefore, these peptides can be used as arginine linkers to bind to other compounds, allowing compounds to enter the cytoplasm from the extracellular space. can be taken into.
  • the conjugated compound is also preferably an antibody-drug conjugated compound in which the drug compound and the antibody are conjugated via the linker compound.
  • An antibody-drug conjugate compound can be used as a kind of antibody drug. That is, the antibody-drug conjugate compound has highly selective selectivity due to specific binding of the antibody to the target object (antigen).
  • the linker compound is cleaved according to the intracellular environment, for example, the reducing environment (glutathione concentration), and the drug compound is released from the antibody-drug conjugate compound. Particularly high selectivity can be exhibited.
  • the drug compound is also preferably a so-called drug, an amino acid, an oligopeptide, a protein drug, or a nucleic acid drug.
  • the oligopeptide of the drug compound is also preferably an apoptosis-inducing peptide.
  • Apoptosis-inducing peptides include, for example, PAD/kla(KLAKLAK) 2 and the like.
  • Apoptosis fusion peptides induce apoptosis in cells and can be used to kill and treat cancer cells. Since the linker compound of the present embodiment and the complex compound containing it can specifically target cancer cells and cancer cells under specific conditions, they are used for treatment to kill specific cancer cells. be able to.
  • the drug compound of this embodiment is a nucleic acid.
  • the nucleic acid of the drug compound can be selected from those used as nucleic acid drugs.
  • Nucleic acid medicines are therapeutic drugs that target chromosomal DNA, various functional RNAs, and proteins, and exhibit efficacy through functional control using nucleic acid derivatives.
  • the antisense method which targets functional RNA in particular for target diseases, enables the design of various nucleic acid medicines based on the sequence, expression timing, expression site, etc., and has many potential applications.
  • the linker compound of the present embodiment can be applied as a linker with polyethylene glycol (PEG), which is frequently used as a general-purpose DDS system. It can also be used as a linker compound that binds a molecule and a drug. It can also be used as a prodrug and as a binding linker to a deactivating molecule.
  • PEG polyethylene glycol
  • the linker compound is not limited to the above-mentioned compounds as the drug to be bound/loaded, and can be applied to various drugs.
  • the complex compound of the present embodiment can appropriately select target cells by reducing conditions, particularly glutathione concentration conditions.
  • it is preferably used against cancer cells.
  • Cancer cells have a specific range of glutathione concentration, and the complex compound of this embodiment can specifically select a target.
  • the action of the linker compound of the present embodiment includes the structure represented by formula (1), and the disulfide bond (S—S bond) in the structure of formula (1) is dissociated under reducing conditions. For example, it dissociates when glutathione (GSH) concentration increases.
  • GSH glutathione
  • the reductive conditions for dissociation such as glutathione concentration, vary depending on the structure represented by formula (1).
  • the redox potential of the disulfide bond differs depending on the coordination position and structure of the substituent R on the aromatic ring of formula (1). In other words, the design of the substituent R on the aromatic ring can set the conditions for the dissociation of the disulfide bond.
  • the linker compound of the present embodiment distinguishes between mM glutathione concentrations, specifically glutathione concentrations within the range of 10 to 200 mM, and dissociates when placed under specific concentration conditions. Therefore, the linker compound does not dissociate under specific concentration conditions, for example, when introduced into blood or normal cells with a glutathione concentration in the ⁇ M level. On the other hand, the linker compound can be dissociated when introduced into target disease cells having a glutathione concentration above the specific concentration condition, or having a certain intracellular glutathione concentration.
  • the linker compound of the present embodiment has the above-described actions, it can specifically identify cancer cells. Specifically, according to the difference in glutathione concentration between normal cells and cancer cells, and also according to the difference in glutathione concentration according to the type of cancer cell and the state of cancer, the disulfide bonds are dissociated and the linker Conditions can be set for the compound to dissociate. Therefore, it is possible to accurately identify cancer cells and their types and states, dissociate the linker compound, and release the drug compound in the cells. This results in a linker compound that is stable outside the target cell and capable of effective release of the drug compound in the target cell. That is, it can be applied as a multifunctional linker that has designability and selectivity and exhibits advanced functions.
  • linker compounds that can be dissociated by other glutathione concentrations or reducing conditions, and dissociate under conditions that can be designed in various ways. It can be widely applied as
  • the composite compound of this embodiment has a linker compound having the above-described actions and effects.
  • the conjugated compounds of the present embodiments can be used for drug delivery in a variety of situations where discrimination of reduction states by linker compounds can be applied, such as drug delivery in the treatment of various diseases. It can be widely used as a complex compound that can dissociate and release drug compounds depending on the glutathione concentration, which can be designed in various ways, in various situations, not limited to distinguishing between normal cells and cancer cells, or whether intracellular dissociation exists or not. .
  • a compound with specific selectivity such as an antibody
  • a linker compound to form a complex compound stable and efficient cell transduction other than the target is possible. Effective drug release in the targeted cytoplasm is possible.
  • the complex compound of the present embodiment can be suitably used particularly for cancer treatment.
  • cancer treatment it is possible to selectively release drugs in the vicinity of cancer cells, effectively escape drugs from endosomes, and selectively release drugs in the cell environment characteristic of cancer cells.
  • a selective drug delivery system (DDS) is needed.
  • the conjugated compound of the present embodiment is a conjugated compound obtained by binding a compound having specific selectivity such as an antibody with a linker compound to a drug compound. Since the linker compounds of the embodiments enable cancer cell selection, effective drug escape from endosomes, and selective drug release in cell environments characteristic of cancer cells, they can be widely applied to the DDS.
  • FIG. 1 shows a comparison of spectral results of proton NMR in CDCl3 for the compounds of formulas (2) and (4).
  • FIG. 2 shows a graph in which the spectra in FIG. 1 are superimposed for comparison.
  • THF used was Wako Pure Chemical Industries, Ltd., product code 207-17765, ultra-dehydrated, stabilizer-free, and a cell of 10 mm ⁇ 10 mm was used.
  • a reduction reaction of the oxidized form (disulfide) of the unsubstituted model compound of formula (2) was carried out in the cell.
  • the reaction was carried out using an excess amount (11 equivalents) of LiBH 4 with respect to the oxidized form of the compound of formula (2) (45 ⁇ Min THF).
  • LiBH 4 was prepared by diluting a commercially available THF 2M solution and used in the reaction.
  • FIG. 4 shows changes in the UV spectrum as the reaction progresses.
  • FIG. 5 shows an enlarged graph of absorption 1.0 to 1.5 on the vertical axis and wavelength 216 to 228 nm on the horizontal axis in FIG.
  • NMR NMR was used to investigate the stability of disulfide bonds in a mixed system under various concentration conditions. From Test Example 2, it is possible to track the reaction by UV, but NMR measurement is used below because NMR can track a complex system more.
  • the compounds of formula (6), formula (7) and formula (8) having the substituent (--COOH) are represented as ArSSrA(H), ArSSrA(F) and ArSSrA(NO 2 ), respectively.
  • a comparison of the NMR spectra in FIGS. 7 and 8 shows that there is a clear difference between GSH and GSSG.
  • a particularly striking difference is the chemical shift of the number 7 proton at the root of the S atom.
  • the proton of number 7 of GSH is located at 2.144 ppm, while the proton of number 7 of GSSG is divided into two peaks of 3.292 and 2.956 ppm.
  • the difference in chemical shift is due to the difference in electron density depending on the state of the S atom being SH or SS. It is thought that it is because it stopped rotating because it was crowded. Since it was found that there was a difference in the peaks of GSH and GSSG, it was thought that the respective compound ratios in the mixed system of GSH and GSSG could be calculated from the integrated values.
  • FIG. 9 shows the NMR spectrum of reduced ArSH (formula (15) in the figure) and
  • FIG. 10 shows the NMR spectrum of oxidized ArSSrA (formula (7)).
  • Equation (16) shows the parallel relationship between ArSSrA(H) and GSH (left side) and between ArSH(H) and GS-SG (right side).
  • An NMR tube was prepared by using a double tube (SHIGEMI model SP-405) and putting a TMS-containing CDCl3 solution (LOCK solvent) on the outside.
  • LOCK solvent TMS-containing CDCl3 solution
  • the abundance ratio of each compound can be derived from the comparison of the initial concentration of the input and the integral value of the obtained spectrum. was found to exist in the system as an intermediate.
  • ArSSrA(F) and ArSSrA(NO2) were tested in the same manner as in Test Example 6 to examine their respective cleavage reactions in normal cell models and cancer cell models.
  • the results of ArSSrA(F)ArSSrA(NO2) are shown in Tables 6 and 7, respectively.
  • Tables 6 and 7 both ArSSrA (F) and ArSSrA (NO2) were cleaved about 90% after 100 minutes, and all were cleaved after 1 day. It was found that it can be expected as a linker that cleaves in response to cancer cell GSH concentration.
  • Arg 8 acts as a cell membrane penetrating peptide (arginine linker) and has the effect of taking the bound compound into the cytoplasm.
  • FAM and Dbs each develop a color by fluorescence, but the compound to which FAM-Dbs binds hardly develops color.
  • FAM and DBS are separated and emit light, so that their intracellular distribution can be detected. Therefore, examining the fluorescence emission of the Arg 8 -Dbs-linker-FAM compound detects whether the compound was taken up into the cytoplasm, cleaved intracellularly, and the intracellular distribution of the cleaved compound. be able to.
  • FIG. 15 shows a diagram of fluorescence development observed with a confocal microscope after incubating various cells with an Arg 8 -Dbs-linker-FAM compound (formula (22)) (25 ⁇ M) for 30 minutes in the F-12(-) state. show.
  • the various cells are HeLa (human cervical epithelial cancer cells), HT-1080 (human fibroblastoma cells), HepG2 (human liver cancer cells), and CHO-K1 (Chinese hamster ovary-derived cells), respectively.
  • strong intracellular fluorescence was observed, indicating that the compound was incorporated into each cell via a cell membrane penetrating peptide (arginine linker).
  • the fact that fluorescence was strong indicates that Dbs and FAM were separated from the compound of formula (22), that is, the disulfide bond of the linker compound was cleaved. From this result, the compound bound by the linker compound of the present embodiment can be introduced into cells by the cell membrane penetrating peptide, and the linker compound can be cleaved only in cancer cells where the glutathione concentration is above a certain level. shown.
  • the glutathione concentration at which the disulfide bond dissociates can be designed by molecular design, cancer cells can be accurately identified, and stable and stable outside the target cell. can provide effective drug compound release. Therefore, it can be widely applied to antibody drugs using antibody-drug conjugates and drugs using other conjugates, and can be used for cancer treatment with few side effects.

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