WO2017032487A1 - Inhibiteur de protéasome comprenant une fraction d'émission de signal - Google Patents

Inhibiteur de protéasome comprenant une fraction d'émission de signal Download PDF

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WO2017032487A1
WO2017032487A1 PCT/EP2016/064957 EP2016064957W WO2017032487A1 WO 2017032487 A1 WO2017032487 A1 WO 2017032487A1 EP 2016064957 W EP2016064957 W EP 2016064957W WO 2017032487 A1 WO2017032487 A1 WO 2017032487A1
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proteasome
group
proteasome inhibitor
acetyl
alkyl
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PCT/EP2016/064957
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Christian DUBIELLA
Michael Groll
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Technische Universität München
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/08—Tripeptides
    • C07K5/0802—Tripeptides with the first amino acid being neutral
    • C07K5/0812—Tripeptides with the first amino acid being neutral and aromatic or cycloaliphatic
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • C07K5/06008—Dipeptides with the first amino acid being neutral
    • C07K5/06017—Dipeptides with the first amino acid being neutral and aliphatic
    • C07K5/06026—Dipeptides with the first amino acid being neutral and aliphatic the side chain containing 0 or 1 carbon atom, i.e. Gly or Ala
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/08—Tripeptides
    • C07K5/0802—Tripeptides with the first amino acid being neutral
    • C07K5/0804—Tripeptides with the first amino acid being neutral and aliphatic
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • C07K5/1002—Tetrapeptides with the first amino acid being neutral
    • C07K5/1005—Tetrapeptides with the first amino acid being neutral and aliphatic
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • C07K5/1002—Tetrapeptides with the first amino acid being neutral
    • C07K5/1016—Tetrapeptides with the first amino acid being neutral and aromatic or cycloaliphatic
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • Proteasome inhibitor comprising a signal-emitting moiety
  • the present invention refers to proteasome inhibitors comprising a signal-emitting compound or precursor thereof, which is covalently bound to a proteasome inhibitor forming a sulfonic ester group.
  • the invention further relates to the use of the proteasome inhibitor comprising the signal-emitting compound in methods for quantifying proteasome inhibition and/or proteasomes and to kits comprising such inhibitors.
  • proteasomes In eukaryotes, protein degradation is predominately mediated through the ubiquitin pathway in which proteins targeted for destruction are ligated to ubiquitin. Once targeted, ubiquitinated proteins then serve as substrates e.g., for the 20S proteasome, a multicatalytic protease, which cleaves proteins into short peptides through the action of its three major proteolytic activities. The main function of the proteasome is to degrade unneeded or damaged proteins by proteolysis. Proteasomes are part of a major mechanism by which cells regulate the concentration of particular proteins and degrade misfolded proteins. Proteasomes are protein complexes inside all eukaryotes and archaea, and in some bacteria. In eukaryotes, they are located in the nucleus and the cytoplasm.
  • the proteasome is a cylindrical complex containing a "core" of four stacked rings forming a central pore. Each ring is composed of seven individual proteins.
  • the inner two rings are made of seven ⁇ subunits that contain three to seven protease active sites. These sites are located on the interior surface of the rings, so that the target protein must enter the central pore before it is degraded.
  • the outer two rings each contain seven a subunits whose function is to maintain a "gate” through which proteins enter the barrel. These a subunits are controlled by binding to "cap” structures or regulatory particles that recognize polyubiquitin tags attached to protein substrates and initiate the degradation process.
  • the overall system of ubiquitination and proteasomal degradation is known as the ubiquitin-proteasome system.
  • proteasome plays a straightforward but critical role in the function of the adaptive immune system.
  • Peptide antigens are displayed by the major histocompatibility complex class I (MHC) proteins on the surface of antigen-presenting cells. These peptides are products of proteasomal degradation of proteins including proteins originated by the invading pathogen.
  • MHC major histocompatibility complex class I
  • constitutively expressed proteasomes can participate in this process, a specialized complex composed of proteins, whose expression is induced by interferon gamma, are the primary producers of peptides which are optimal in size and composition for MHC binding.
  • proteins whose expression increases during the immune response include the 19S regulatory particle, whose main known biological role is regulating the production of MHC ligands, and specialized 6 subunits called 6 li, 6 2i, and 6 5i with altered substrate specificity.
  • the complex formed with the specialized 6 subunits is known as the immunoproteasome.
  • Another 6 5i variant subunit, 6 5t is expressed in the thymus, leading to a thymus-specific "thymoproteasome".
  • proteasomes there are two major types of proteasomes: the constitutive proteasome that is normally expressed in all cell types, and the immunoproteasome that can be induced upon exposure to inflammatory cytokines such as tumor necrosis factor alpha (TNF-a) or interferon gamma (IFN- ⁇ ).
  • TNF-a tumor necrosis factor alpha
  • IFN- ⁇ interferon gamma
  • proteasomal activity Due to its role in generating the activated form of NF- ⁇ , an anti-apoptotic and proinflammatory regulator of cytokine expression, proteasomal activity has been linked to inflammatory and autoimmune diseases. Increased levels of proteasome activity correlate with disease activity and have been implicated in autoimmune diseases such as systemic lupus erythematosus and rheumatoid arthritis. To this end, proteasome inhibitors emerged as a promising new class of drugs in this field of therapeutic application. Proteasome inhibitors are drugs that block the action of proteasomes, cellular complexes that break down proteins, like the p53 protein. There are several examples of
  • proteasome inhibitors such as bortezomib, carfilzomib, epoxomycin etc.
  • these compounds generally lack specificity, e.g., regarding the interaction with specific subunits of the proteasome, stability, and/or potency necessary to explore and exploit the roles of the proteasome at the cellular and molecular level.
  • Dubiella et al. describe for example peptidic sulfonyl fluorides specifically interacting, i.e., binding to the 65i-subunit of immune proteasomes. These inhibitors are based on known inhibitors such as epoxyketone containing compounds like epoxomicin, wherein a sulfonyl fluoride moiety is introduced resulting in a peptidyl sulfonyl compound.
  • US 7,691,852 and US 7,417,042 refer to proteasome inhibitors, in particular immune proteasome inhibitors based on epoxyketone compounds for example ONX-0914, bortezomib, and carfilzomib, which are for use in treating auto immune diseases or multiple myeloma.
  • the present invention provides proteasome inhibitors comprising a signal emitting compound or precursor thereof, wherein the signal emitting compound is covalently bound to the peptidic proteasome inhibitor forming a sulfonic ester group.
  • the inhibitor as well as the active site of the proteasome are inactivated, i.e., the active site is cross- linked, thereby releasing the activated signal emitting compound.
  • the present invention results in exact stoichiometric determination of the amount of a proteasome and a specific subunit, respectively.
  • the present invention allows - amongst others - direct detection of relative proteasomal activity and concentration in solution by a simple readout.
  • the lack of linkers, spacers, or the need for downstream reactions for signal amplification avoids possible side-reactions, rendering the system based on the present invention precise and sensitive.
  • peptidic proteasome inhibitors which are covalently bound to a signal -emitting compound or a precursor thereof specifically and highly sensitively bind to a proteasome subunit, in particular an immunoproteasome subunit.
  • the signal- emitting compound is released and forms the basis for stoichiometric determination of the amount of proteasomes.
  • signal emitting compounds having a pK a in the range of 3 - 5 or 4 - 6 or ⁇ 4, ⁇ 5 or ⁇ 6 show inhibition in the nanomolar range.
  • the peptidic proteasome inhibitor comprises or consists of at least two, three, four, five or six amino acids, wherein one or more of the amino acids are unsubstituted or substituted.
  • the Coc carbon for example comprises a morpholino acetyl, methyl indene acetyl, thiazole, oxazole, pyramine or pyrazolo group.
  • the proteasome inhibitors of the present invention interact for example with a proteasome subunit selected from the group consisting of 6 5i (LMP7), 6 li (LMP2), ⁇ lc, ⁇ 2i (MECL-1), 6 2c and 6 5c.
  • the signal emitting compound or a precursor thereof is a radiation emitting compound or a light emitting compound or a precursor thereof.
  • the light emitting compound or precursor thereof is for example selected from the group consisting of a xanthene, a cyanine, a naphthalene, a coumarin, an oxadiazole, an anthracene, a pyrene, an oxazine, an acridine, an arylmethine, a tetrapyrrole, a halogenated paranitrophenole, a luciferin, , a phenoxayine, a flourescein and a precursor of any of the light emitting compounds.
  • the coumarin is for example a coumarin of formula II or III, a 6,8-difluoro-7-hydroxycoumarin, a 6-fluoro-7- hydroxycoumarin, a 7-hydroxy-8-methoxycoumarin or a derivative thereof.
  • the proteasome inhibitor of the present invention is for example for use as a diagnostic agent (e.g. in an immunoproteasome, proteasome-associated and/or proteasome-related disease), for use in quantifying proteasome inhibition and optionally quantification of proteasome concentrations in solution and/or various biological samples in fluorescence assays (e.g., using fluorescent read-out methods), for use as a drug (e.g. in an immunoproteasome, proteasome-associated and/or proteasome-related disease), for use in quantifying proteasome inhibition and optionally quantification of proteasome concentrations in solution and/or various biological samples in fluorescence assays (e.g., using fluorescent read-out methods), for use as a drug (e.g
  • immunoproteasome, proteasome-associated and/or proteasome-related disease for use in the quantification of proteasome inhibition in the diagnosis or monitoring of the therapy and/or prevention of an autoimmune disease, cancer, an inflammatory disease, a viral infection or a neurodegenerative disease, or for use in identifying and/or
  • the proteasome inhibitor of the present invention forms part of a kit, further comprising a buffer solution of e.g., pH 6 to 9 comprising for example excipients selected from the group consisting of salts, stabilizers, and surfactants.
  • a buffer solution e.g., pH 6 to 9 comprising for example excipients selected from the group consisting of salts, stabilizers, and surfactants.
  • the present invention refers to a method of producing a proteasome inhibitor comprising the steps of:
  • residues optionally modify the peptide by attaching residues, wherein the residues are selected from the group consisting of H, alkyl, alkenyl, alkinyl, aryl, aycloalkyl, nitril, heterocycloalkyl, heteroaryl, or actyl;
  • Re is a protecting group selected from Cbz and carbamate; (iv) deprotecting the N-terminus and coupling of the peptidic backbone and the sulfonyl halogen headgroup to form a compound of general Formula V,
  • Figure 1 shows a scheme of reaction of inhibitors of the present invention and proteasomes.
  • Figure 2 shows an example of a peptidic protease inhibitor (compound A) comprising a sulfonyl fluoride, wherein the inhibitor binds to an active site of the proteasome such as an immunoproteasome; the fluoride leaving group is substituted by a fluorophore resulting in inhibitor (compound B) characterized by a sulfonate ester binding to the fluorophore.
  • compound A peptidic protease inhibitor
  • compound B sulfonate ester binding to the fluorophore
  • Figure 3 depicts the quantifiable inhibition of the proteasome such as an
  • the inhibitor and the proteasome are inactivated, and the light emitting compound is released.
  • Figure 4c shows experiments, where 1 ⁇ of compound (C) comprising a fluorophore (see Figure 1) was added to different concentrations (1.0 to 0.001 mg/ml) of a purified human constitutive proteasome in 100 mM Tris and 1 mM DTT at pH 7.5. The samples were incubated for 1 h and afterwards the fluorescence intensity was measured
  • a proteasome inhibitor of the present invention comprising a signal-emitting compound or a precursor thereof inhibits the activity of a proteasome, in particular a specific subunit of a proteasome, in that the active site of the proteasome interacting with the inhibitor is crosslinked, and the signal emitting compound is released. Further, as the inhibitor and the proteasome become inactive after their interaction, measuring the activity of the released light emitting compound results in exact stoichiometric quantification of the proteasome and its subunit, respectively.
  • the interaction time of the proteasome inhibitor and the proteasome is short, i.e., about 5, 10, 15, 20, 25 or 30 min. and optionally remains constant for, e.g., 1, 2, 3, 4 or 5 hours.
  • proteasome comprises immuno- and constitutive proteasomes.
  • the inhibitors of the present invention specifically interact for example with a proteasome subunit selected from the group consisting of 6 5i (LMP7), 6 li (LMP2), 6 lc, 6 2i (MECL-1), 6 2c and 6 5c, depending on the applied peptidic backbone.
  • a peptidic proteasome inhibitor of the present invention comprises or consists of a peptide, i.e., natural or artificial amino acids, which are linked by a peptide bond.
  • the peptide comprises for example 2 to 25, 3 to 20, or 4 to 15 amino acids.
  • the peptide comprises or consists of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids.
  • Peptidic backbones of the proteasome inhibitors are for shown in Example 1.
  • the peptide is unbranched or branched and each amino acid is unsubstituted or substituted.
  • substituted refers to moieties having substituents replacing a hydrogen on one or more carbons of the backbone.
  • substitution is in accordance with permitted valence of the substituted atom and the substituent, and that the substitution, results in a stable compound, e.g., which does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc.
  • substituted is contemplated to include all permissible substituents of organic compounds.
  • the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and non- aromatic substituents of organic compounds.
  • the permissible substituents can be one or more and the same or different for appropriate organic compounds.
  • Substituents can include, for example, a halogen, a hydroxyl, a carbonyl (such as a carboxyl, an alkoxy-carbonyl, a formyl, or an acyl), a thiocarbonyl (such as a thioester, a thioacetate, or a thioformate), an alkoxyl, a phosphoryl, a phosphate, a phosphonate, a phosphinate, anamino, an amido, an amidine, an imine, a cyano, a nitro, an azido, a sulfhydryl, an alkylthio, a sulfate, a sulfonate, a sulfamoyl, a sulfonamido, a sulfonyl, a heterocyclyl, an aralkyl, or an aromatic or heteroaromatic moiety. It will
  • Proteasome inhibitors of the present invention comprise or consist for example of the general Formula I
  • Ri is selected from the group consisting of H, alkyl, alkenyl, alkinyl, aryl, arenyl, aycloalkyl, nitril, neterocycloalkyl, heteroaryl, acetyl, alkyl acetyl, alkenyl acetyl, alkinyl acetyl, aryl acetyl, aycloalkyl acetyl, nitril acetyl, heteroaryl acetyl, arenyl acetyl or heterocycloalkyl acetyl;
  • R2 is selected from the group consisting of H, alkyl, alkenyl, alkinyl, aryl, aycloalkyl, nitril, heterocycloalkyl, heteroaryl, or actyl; and wherein R2 is in (R) and/or (S) configuration;
  • R3 is aryl, Ci-e aralkyl, heteroaryl, Ci-e heteroaralkyl, phenyl, isopropanyl, cycloalkyl or heterocycloalkyl
  • R4 is a fluorophore or a fluorophore precursor, wherein the fluorophore or the
  • fluorophore precursor is covalently bound by a sulfonic ester bond
  • n is an integer of between 2 or 4.
  • Ri is a morpholino acetyl, methyl indene acetyl, thiazole, oxazole, pyramine or pyrazolo group, and/or R3 is selected from Cl-6 aralkyl and Cl-6 heteroaralkyl, wherein the alkyl moiety may contain six, five, four, three, two, or one carbon atoms.
  • R3 is substituted with one or more substituents selected from hydroxy, halogen, amide, amine, carboxylic acid (or a salt thereof), ester (including Cl-6 alkyl ester, Cl-5 alkyl ester, and aryl ester), thiol, or thioether.
  • R3 is substituted with a substituent selected from alkyl, trihaloalkyl, alkoxy, hydroxy, or cyano.
  • R3 is selected from Cl-6 alkyl-phenyl and Cl-6 alkyl-indolyl, or R3 is selected from
  • D is selected from H, OMe, OBu 1 , OH, CN, CF3 or CH3, and R is any suitable protecting group.
  • pharmaceutically acceptable salt refers to the relatively non-toxic, inorganic and organic acid addition salts of the inhibitor(s) . These salts are for example prepared in situ during the final isolation and purification of the inhibitor(s), or by separately reacting a purified inhibitor(s) in its free base form with a suitable organic or inorganic acid, and isolating the salt thus formed.
  • Representative salts include the hydro-bromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate, stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succi-nate, tartrate, naphthylate, mesylate, glucoheptonate, lacto-bionate, laurylsulphonate salts, and amino acid salts, and the like.
  • sulfate bisulfate
  • phosphate nitrate
  • acetate valerate
  • oleate palmitate
  • stearate laurate
  • benzoate lactate
  • phosphate tosylate
  • citrate maleate
  • fumarate succi-nate
  • tartrate tartrate
  • naphthylate mesylate
  • mesylate glucoheptonate
  • the signal emitting compound or precursor thereof is selected from the group consisting of a xanthene, a cyanine, a naphthalene, a coumarin, an oxadiazole, an anthracene, a pyrene, an oxazine, an acridine, an arylmethine, a tetrapyrrole, a halogenated paranitrophenole, a luciferin, a phenoxayine, a flourescein and a precursor of any of the light emitting compounds.
  • the coumarin may comprise general Formula II
  • Ri is the peptide, to which the light emitting compound is bound by a sulfonic ester bond
  • R2 is selected from the group consisting of H, flour, chloride, bromide, iodine and nitro groups;
  • R31S selected from the group consisting of N, O, S, alkyl, alkenyl, alkinyl, heteroalkyl, heteroalkenyl, and heteroalkinyl;
  • R41S selected from the group consisting of N, O, S, alkyl, alkenyl, alkinyl, heteroalkyl, heteroalkenyl, and heteroalkinyl;
  • R 3 and R 4 taken together form a 5- or 6-membered heteroaryl.
  • the coumarin is for example 6,8-difluoro-7-hydroxycoumarin or a derivative thereof having formula III:
  • R4 is H, OH, CN, C1-C18 alkyl, C1-C18 perfluoroalkyl, sulfomethyl, biologically compatible salt of sulfomethyl, halomethyl, aryl, or -L-SC;
  • R5 is H or C1-C6 alkoxy
  • R7 is H, or a monovalent moiety derived by removal of a hydroxy group from a phosphate, a thiophosphate, a sulfate, or a biologically compatible salt thereof; or a monovalent moiety derived by removal of a hydroxy group from a carboxy group of an aliphatic or aromatic carboxylic acid; or a monovalent moiety derived by removal of a hydroxy group from an alcohol or from a mono- or polysaccharide; or R7 is a photo labile caging group; wherein
  • aryl is an aromatic substituent having 6 conjugated carbon atoms that is optionally and independently substituted by H, halogen, cyano, sulfo, biologically compatible salts of sulfo, carboxy, nitro, alkyl, perfluoroalkyl, alkoxy, alkylthio, amino, monoalkylamino, dialkylamino or alkylamido;
  • heteroaryl is a 5- or 6-membered aromatic heterocycle that is optionally fused to additional six-membered aromatic rings, or is fused to one 5- or 6-membered
  • heteroaromatic ring said heteroaromatic rings containing at least 1 and as many as 3 heteroatoms that are selected from the group consisting of O, N and S in any
  • each L is independently a single covalent bond, or L is a covalent linkage having 1-24 nonhydrogen atoms selected from the group consisting of C, N, 0 and S and is composed of any combination of single, double, triple or aromatic carbon-carbon bonds, carbon- nitrogen bonds, nitrogen-nitrogen bonds, carbon-oxygen bonds and carbon-sulfur bonds; SC is a conjugated substance.
  • the coumarin is for example a 6-fluoro-7- hydroxycoumarin, a 7-hydroxy-8-methoxycoumarin, or a derivative thereof.
  • a signal emitting compound or its precursor is for example further selected from the group consisting of rhodamine, Oregon green, eosin, Texas red, indocarbocyanine, oxacarbocyanine, thiacarbocyanine, merocyanine, dansyl and prodan derivatives, difluorhydroxymethylcoumarin, oxadiazole, pyridyloxazole, nitrobenzoxadiazole, benzoxadiazole, DRAQ5, DRAQ7, CyTRAK Orange, cascade blue, Nile red, Nile blue, cresyl violet, oxazine 170, proflavin, acridine orange, acridine yellow, auramine, crystal violet, malachite green, porphin, phthalocyanine, bilirubin, firefly luciferin, latia luciferin, bacterial luciferin, D-luciferin, D-aminoluciferin, OH-cyanobenzothiazole
  • the signal emitted by a signal emitting compound of the present invention is detected and quantified by methods and devices known in the field.
  • the signal- emitting compound is a compound emitting a signal by means of radioactivity.
  • signals are of particular importance for example in in vivo diagnosis if the tissues are to be examined are too thick to allow fluorescence signals to transpass, even if the most sensitive fluorescence detectors are employed.
  • a radioactive signal may transpass these tissues and give a true picture of the specific inhibition reaction performed by the present compounds.
  • radioactive signal emitting compounds are, for example, radionuclides used, for example, in Positron emission spectroscopy (PET).
  • PET Positron emission spectroscopy
  • the proteasome inhibitor of the present invention is represented by the following formulas:
  • the inhibitor is be coupled to a cell penetration enhancer in certain embodiments to better facilitate cell penetration and reaching of the cytosolic or nuclear proteasome which should be inhibited.
  • a smaller signal emitting precursor such as a fluorophore precursor
  • a second compound which reacts with the precursor at the desired site of action to form an active signal emitting compound, is separately administered to enhance cell access.
  • the proteasome inhibitors comprising a signal emitting compound of the present invention are produced in different ways. One of these methods comprises for example the following steps:
  • residues are selected from the group consisting of H, alkyl, alkenyl, alkinyl, aryl, aycloalkyl, nitril, heterocycloalkyl, heteroaryl, or actyl;
  • ⁇ is a protecting group selected from Cbz and carbamate
  • R4 is for example a fluorinated umbelliferon such as 6,8-difluoro-4-methylumbelliferon
  • the pKa of R4 is for example in the range of 3 - 5 or 4 - 6 or ⁇ 4, ⁇ 5 or ⁇ 6 to reach inhibition in the nanomolar range as the pK a values of R4 for example correlate with reactivity of the proteasome inhibitor.
  • the carbamate is for example methyl carbamate, ethyl carbamate, t-butyl carbamate (Bot), or 9-fluorenylmethyl carbamate (Fmoc).
  • proteasome inhibitors comprising a signal emitting compound of the present invention are for use in different fields, for example as a diagnostic agent, in quantifying proteasome inhibition in luminescence imaging, wherein the inhibitor comprising the light emitting compound precursor is administered together with a co-compound, such as D-cysteine, and a luciferase enzyme, as a drug, as a medicament, use in the
  • proteasome inhibitors of the present invention are used in a method for detecting metastases.
  • An autoimmune disease which is for example diagnosed or quantified by use of a proteasome inhibitor of the present invention is for example selected from the group consisting of acute disseminated encephalomyelitis (AD EM), Addison's disease, Diabetes type I, Erythema nodosum, Leucemia, Lupus erythematosus, Myositis, Neuromyelitis optica Neuromyotonia, Occular cicatricial pemphigoid, Opsoclonus myoclonus syndrome, Ord's thyroiditis, Palindromic rheumatism, PANDAS (pediatric autoimmune disease
  • Sydenham chorea Sympathetic ophthalmia, Systemic lupus erythematosus, Takayasu's arteritis, Temporal arteritis thrombocytopenia, Tolosa-Hunt syndrome, transverse myelitis, ulcerative colitis, undifferentiated connective tissue disease, undifferentiated spondyloarthropathy, urticarial vasculitis vasculitis vitiligo and Wegener's
  • the present invention is further directed to a kit comprising a proteasome inhibitor of the present invention and a buffer solution of pH 6 to 9 comprising excipients selected from the group consisting of salts, stabilizers, and surfactants.
  • the pH is in the range of 6.3 to 8.5, 6.5 to 8.0 or 6.8 to 7.6.
  • the kit comprises the proteasome inhibitor in form of a lyophilisate, a solution, a suspension, a transdermal composition, a dry powder inhalation composition or a metered dose inhalation composition.
  • the present invention comprises compositions comprising the proteasome inhibitors and optionally any conventional additive or excipient, such as a binder, a disintegrating agent, a lubricant, a coregent, a solubilizing agent, a suspension aid, an emulsifying agent, a coating agent, a cyclodextrin, and/or a buffer.
  • a binder a disintegrating agent, a lubricant, a coregent, a solubilizing agent, a suspension aid, an emulsifying agent, a coating agent, a cyclodextrin, and/or a buffer.
  • a proteasome inhibitor of the present invention or a composition comprising such inhibitor is administered in various forms for example orally formulated as tablets, capsules, granules, powders, or syrups; or for example parenterally formulated as injections (intravenous, intramuscular, intratumoral, intraarterial, intrathecal, intracapsu-lar, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrasternal injection or subcutaneous), infusion, or suppository.
  • injections intravenous, intramuscular, intratumoral, intraarterial, intrathecal, intracapsu-lar, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrasternal injection or subcutaneous
  • injections intravenous, intramuscular
  • the inhibitor(s) are administered by aerosol. This is accomplished by preparing an aqueous aerosol, liposomal preparation, or solid particles containing the composition. Alternatively, the proteasome inhibitor(s) are administered via a transdermal patch.
  • the proteasome inhibitor comprising a signal emitting compound further comprises a prodrug, i.e., a compound that, under physiological conditions, is converted into a therapeutically active agent, which is released from the inhibitor once it is interacting with the proteasome.
  • the prodrug is released together with the signal emitting compound or before or after the signal emitting compound.
  • a proteasome inhibitor of the invention is conjointly administered with a chemotherapeutic for example to control the preventive or therapeutic effect.
  • chemotherapeutics comprise for example, natural products such as vinca alkaloids (i.e. vinblastine, vincristine, and vinorelbine), paclitaxel, epidipodophyllotoxins (i.e.
  • etoposite teniposite
  • antibiotics dactinomycin (actinomycin D), daunorubicin, doxorubicin and idarubicin
  • anthracyclines mitoxantrone, bleomycins, plicamycin (mithramycin) and mitomycin
  • enzymes L-asparaginase which systemically metabolizes L-asparagine and deprives cells which do not have the capacity to synthesize their own asparagine
  • anti-platelet agents antiproliferative/antimitotic alkylating agents such as nitrogen mustards (mechlorethamine, cyclophospha-mide and analogs, melphalan, chlorambucil), ethylenimines and mefhylmelamines (hexamethylmelamine and thiotepa), alkyl sulfonates (busulfan), nitrosoureas (carmustine (BCNU) and analogs
  • antimetabolites such as folic acid analogs (methotrexate), pyrimidine analogs
  • hormones i.e. estrogen
  • hormone agonists such as leutinizing hormone releasing hormone (LHRH) agonists (goserelin, leuprolide and triptorelin).
  • LHRH leutinizing hormone releasing hormone
  • Other chemo therapeutic agents may include mechlorethamine, camptothecin, ifosfamide, tamoxifen, raloxifene, gemcitabine, navelbine, or any analog or derivative variant of the foregoing.
  • Each of the proteasome inhibitors of the present invention interact with the active site of a proteasome, particularly with a specific subunit of the proteasome, wherein the specificity of the inhibitor is defined by its peptide backbone, i.e., the amino acid sequence of the inhibitor.
  • the scheme of reactions shown in Fig. 1 presents the different steps of the interaction between proteasome inhibitor and proteasome resulting in the cross-linking of the proteasome based on the example of the interaction of an inhibitor with subunit 6 5 of the immunoproteasome.
  • the active site of the 6 5 subunit is Thr (Tl), wherein the sulfonylation of ThrO Y is conceivable via two different mechanisms: a direct nucleophilic attack of ThrO Y on the electrophilic sulfur (see Fig. 1, (1)) or via elimination and sulfene formation (see Fig. 1, (2)).
  • the sulfonylation (3) of (1) or (2) results in an adduct formation of the proteasome inhibitor and the proteasome, whereas the activated fluorophore is released. It follows an SN2- displacement (see Fig. 1, (4)) resulting in a (S, S)-aziridinze-Tl' followed by the crosslinking of the active site of the proteasome (see Fig.
  • proteasome inhibitors of the present invention are characterized in that the leaving group of a sulfonate, e.g., a fluoride, is substituted by a signal emitting compound, e.g., a fluorophore resulting in an electrophilic sulfonate ester ("warhead"; see Fig. 2).
  • the inhibitor is highly effective in the nanomolar range ( ⁇ 1 ⁇ ).
  • the compound is signal emitting, e.g., is fluorescent.
  • the signal emitting compound and the inhibitor are covalently bound with or without a linker.
  • the released signal emitting compound is not further influenced or degraded, and provides a stoichiometric quantifiable determination of the proteasome (see Fig. 3).
  • the applied buffers consisted of a gradient mixture of 0.1% (v/v) formic acid in H2O (buffer A) and 0.1% (v/v) formic acid in ACN:H 2 0 90:10 (v/v) (buffer B).
  • ESI-MS and LC-ESI-MS mass spectra were recorded with a Dionex UltiMate 3000 HPLC system coupled with a
  • Thermo LCQ fleet Reversed-phase HPLC purification was accomplished with a system consisting of a Waters 1525 binary HPLC pump, Macherey-Nagel Nucleodur ® Prep C18 column (5 pm, 10 x 250 mm), Waters 2998 PDA detector and Waters Fraction Collector III (Waters Corp.).
  • the applied buffers were H2O with 0.1% TFA (v/v) (buffer A) and ACN with 0.1% TFA (v/v) (buffer B). Lyophilization was performed on a Christ Alpha 2-4 LD plus.
  • Example 1 Synthesis of peptide backbones of peptidic proteasome inhibitor Peptidic backbones were prepared via solid-phase peptide synthesis using Fmoc- protected amino acids and a PS3 Peptide Synthesizer (Protein Technologies, Inc.). In applicable cases, preloaded L-Tyr(OMe)-2-Chlorotrityl-Cl resin (0.63 mmol/g loading) was used in a 0.2 mmol scale, otherwise the designated C-terminal Fmoc protected amino acid was loaded on Trityl-Cl resin (0.74 mmol/g loading) via incubation with 4.00 eq DIPEA.
  • Fmoc-protected amino acids (0.4 mmol, 2.00 eq) were deprotected with 20% (v/v) piperidine in DMF.
  • Activation of amino acids (0.4 mmol, 2.00 eq) for coupling was performed using HCTU (0.4 mmol, 2.00 eq) and 0.4 M DIPEA in DMF.
  • Cleavage from the resin was performed with 25% l,l,l,3,3,3-hexafluoro-2-propanol (v/v) in CH2CI2 following evaporation and.
  • the residual solid was dissolved in H2O and lyophilized to yield the peptidic backbone as white powdery free acid.
  • the backbones were used in the next coupling step without further purification.
  • the following peptides were synthesized: MorphAc-hPhe-Leu-Phe-OH TFA (I)
  • the sulfonyl fluoride headgroups and precursors were previously described in literature (e.g., Brouwer et al., Tetrahedron Lett., 2009, 50, 3391-3393; Brouwer et al., Bioorg. Med. Chem., 2001, 19, 2397-2406; FanHua et al., Sci. China Chem., 2012, 55, 2548- 2553).
  • the sulfonyl chloride headgroup precursors derived from L-phenylalaninol were synthesized according to Dubiella et al. (Angew Chem Int Ed Engl 2014, 53, 11969- 11973). The following headgroups were synthesized:
  • NEt 3 (1.76 mL, 1.21 g, 12.0 mmol) was added to a solution of 1 (2.91 g, 10.0 mmol) in CH2CI2 (30 mL). After cooling the mixture to 0 °C methane sulfonyl chloride (929 ⁇ , 1.37 g, 12 mmol) was added dropwise and the reaction mixture was stirred overnight at RT. Then H2O (10 mL) was added and the mixture was extracted with CH2CI2
  • thioacetic acid (885 ⁇ ⁇ , 955 mg, 12.6 mmol) was added to a suspension of CS2CO3 (2.13 g, 6.53 mmol) in DMF (35 mL). After stirring for 10 min at RT, a solution of mesylate 2 (3.71 g, 10.0 mmol) in DMF (15 mL) was added at once and the mixture was stirred in an aluminium foil-covered flask at 50 °C overnight. After evaporation of the solvent the crude product was purified via flash column
  • NCS (306 mg, 2.29 mmol) was dissolved in a mixture of HC1 (143 ⁇ _, 2 M, 286 mmol) and acetonitrile (985 ⁇ .) at 0 °C and stirred for 15 min. Following addition of thioacetate 3 (200 mg, 572 ⁇ ) to the mixture and stirring for 15 min at RT, the solution was diluted with CH2CI2 (4 mL), washed with brine (3 4 mL), dried over MgS04 and filtered. After evaporation of the solvent, sulfonyl chloride 4 (278 mg, 744 ⁇ , quant, crude) was obtained as an orange colored oil which was used without further purification in the next step.
  • Cbz-3-cyclohexyl-L-Ala-[CH 2 S02]-DFHMC (5)
  • HBr in acetic acid (33%, 1.64 niL, 9.37 mmol) was added dropwise to a solution of sulfonate 5 (206 mg, 375 ⁇ ) in CH2CI2 (4.0 niL) and stirred at RT for 45 min. After concentration in vacuo the residue was dissolved in H2O (4.0 niL) and DOWEX ® 1X8 (Cl- form, 300 mg) was added to solution. The mixture was stirred for 15 min, the solution was filtered and the residue was washed with H2O. After lyophilization hydrochloride 6 (123 mg, 272 ⁇ , 73%) was obtained as a white powder which was used without further purification in the next step.
  • HBr in acetic acid (33%, 1.69 niL, 9.66 mmol, 25.0 eq) was added dropwise to a solution of sulfonate 7 (210 mg, 386 ⁇ ) in CH2CI2 (4.0 niL) and stirred at RT for 45 min. After concentration in vacuo the residue was dissolved in H2O (4.0 niL) and DOWEX ® 1X8 (Cl- form, 300 mg) was added to solution. The mixture was stirred for 15 min, the solution was filtered and the residue was washed with H2O. After lyophilization hydrochloride 8 (126 mg, 282 ⁇ , 73%) was obtained as a white powder which was used without further purification in the next step.
  • HBr in acetic acid (33%, 0.7 mL, 4.27 mmol) was added dropwise to a solution of sulfonate 9 (87 mg, 0.171 mmol) in CH2CI2 (1.2 mL) and stirred at RT for 45 min. After concentration in vacuo the residue was dissolved in H2O (1.2 mL) and DOWEX ® 1X8 (Cl- form, 300 mg) was added to solution. The mixture was stirred for 15 min, the solution was filtered and the residue was washed with H2O. After lyophilization hydrochloride 10 (59 mg, 0.143 mmol, 84%) was obtained as a white powder which was used without further purification in the next step.
  • NEt3 (5.01 niL, 35.9 mmol) was added to a solution of 11 (8.54 g, 29.9 mmol) in CH2CI2 (100 niL). After cooling to 0 °C Ms-Cl (2.8 niL, 35.9 mmol) was added dropwise and the reaction mixture was stirred at RT overnight. After the addition of CH2CI2 (25 mL) the organic layer was washed with H2O (2 x 20 mL) and brine (2 x 20 mL). The organic layer was separated, dried over Na2S04 and filtered. Concentration under reduced pressure and purification by flash column chromatography (PE ⁇ 10% EA PE, v/v) yielded 12 (10.22 g, 28.1 mmol, 94%) as a white solid.
  • NCS (467 mg, 3.49 mmol) was dissolved in a cooled (10°C) 0 °C mixture of HC1 (2 M, 233 ⁇ , 0.466 mmol) and ACN (1.39 mL) and stirred for 15 min.
  • the thioacetate ester 13 300 mg, 0.874 mmol was added at once to the mixture. After stirring for 15 min at RT the reaction mixture was diluted with CH2CI2 (4 mL), washed with brine (3 x 2 mL), dried over MgS04 and concentrated to give 14 (341 mg, 0.926 mmol, 106% (crude product)) as a light yellow solid which was used without further purification in the next step.
  • HATU 27.8 mg, 73.0 ⁇
  • CH2CI2 390 ⁇ 3 ⁇ 4 at 0 °C.
  • HATU (17.14 mg, 0.045 mmol) was added to a solution of peptidic backbone I (25 mg, 0.045 mmol) in CH2CI2 (241 ⁇ 3 ⁇ 4 at 0 °C.
  • the reaction mixture was stirred for 20 min before adding sulfonate headgroup 8 (18.3 mg, 0.041 mmol). Afterwards, the reaction mixture was stirred for 10 min at 0 °C and DIPEA (20.8 yiL, 0.119 mmol) was added dropwise. The reaction was then allowed to reach RT and was stirred overnight. After evaporation of the solvent the residue was dissolved in DMF (250 ⁇ 3 ⁇ 4.
  • HATU (30.5 mg, 0.080 mmol) was added to a solution of peptidic backbone II (53.2 mg, 0.080 mmol) in CH2CI2 (430 ⁇ 3 ⁇ 4 at 0 °C.
  • the reaction mixture was stirred for 20 min before adding sulfonate headgroup 10 (30 mg, 0.073 mmol). Afterwards, the reaction mixture was stirred for 10 min at 0 °C and DIPEA (37 LL, 0.211 mmol) was added dropwise. The reaction was then allowed to reach RT and was stirred overnight. After evaporation of the solvent the residue was dissolved in DMF (250 ⁇ 3 ⁇ 4.
  • HATU (11.6 mg, 110 ⁇ , 1.10 eq) was added to a solution of peptidic backbone III (46.3 mg, 110 ⁇ , 1.10 eq) in CH2CI2 (590 ⁇ 3 ⁇ 4 at 0 °C and strirred for 20 min before adding sulfonate headgroup X (45.0 mg, 100 ⁇ ).
  • Example 5 Determination of proteasome concentrations with 1 ⁇
  • a proteasome inhibitor compound B, see Fig. 2
  • compound B see Fig. 2
  • the concentrations of the proteasome were 1.380, 0.690, 0.345, 0.173, 0.086, 0.043, 0.022, 0.011 and 0.005 mg/ml.
  • a proteasome inhibitor compound B, see Fig. 2
  • compound B see Fig. 2
  • the concentrations of the proteasome were 1.380, 0.690, 0.345, 0.173, 0.086, 0.043, 0.022, 0.011, 0.005 and 0.003 mg/ml.

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Abstract

La présente invention concerne des composés tels que des peptides qui agissent en tant qu'inhibiteurs de protéasome. Les composés sont liés par covalence à un composé d'émission de signal ou à un précurseur de ce dernier, par l'intermédiaire d'un ester tel qu'un groupe ester sulfonique permettant l'imagerie de l'inhibition du protéasome. Le composé est destiné à être utilisé dans la quantification de l'inhibition de protéasome, le diagnostic et/ou la prévention et/ou le traitement de maladies auto-immunes, du cancer, de maladies neurodégénératives, d'infections virales et/ou de maladies associées à une inflammation. L'invention concerne en outre des procédés de production de ces composés.
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