EP3140321A1 - Method for targeted conjugation of peptides and proteins by paired c2 bridging of cysteine amino acids - Google Patents

Method for targeted conjugation of peptides and proteins by paired c2 bridging of cysteine amino acids

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Publication number
EP3140321A1
EP3140321A1 EP15719245.1A EP15719245A EP3140321A1 EP 3140321 A1 EP3140321 A1 EP 3140321A1 EP 15719245 A EP15719245 A EP 15719245A EP 3140321 A1 EP3140321 A1 EP 3140321A1
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EP
European Patent Office
Prior art keywords
amino
alkoxy
peptide
alkoxycarbonyl
alkyl
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Application number
EP15719245.1A
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German (de)
French (fr)
Inventor
Nils Griebenow
Stefan BRÄSE
Alicia DILMAC
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Bayer Pharma AG
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Bayer Pharma AG
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Publication of EP3140321A1 publication Critical patent/EP3140321A1/en
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Classifications

    • 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
    • A61K47/56—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 macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—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 macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—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 macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • 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
    • 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
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/64—Cyclic peptides containing only normal peptide links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/55—Fab or Fab'

Definitions

  • the present application relates to a novel method for the targeted conjugation of peptides and proteins, which is characterized by the pairwise C2-bridging of cysteine amino acids via their thiol groups, and also to the conjugates of peptides and proteins obtainable by such a process, and the use of such conjugates for the diagnosis and / or treatment of diseases.
  • peptides and proteins are altered or modulated by conjugation.
  • Therapeutically relevant peptides and proteins may, for example, be conjugated to biocompatible polymers to increase the half-life of the subject peptide or protein in the plasma circulation and thus its duration of action or to counteract possible immunogenicity [Veronese and Maro, 2008; Roberts et al., 2002].
  • peptides and proteins can be conjugated with biochemical markers, dyes or reactive groups, which then provide information on the binding process in certain organs or cell regions after administration [Miller and Cornish, 2005].
  • Another widely researched field are peptide and protein conjugates with active ingredients that target these drugs to specific cell or organ regions for their intended site of action (so-called drug targeting).
  • ADCs Antibody Drug Conjugates
  • peptides and proteins can be provided with reactive groups by different methods, which in turn serve as a point of attachment for drugs or diagnostics [Ramil and Lin, 2013].
  • peptides and proteins can be conjugated via the functionality of their amino acids [Widdison et al., 2006].
  • the challenge here is to provide a Method that allows a selective conversion of the desired functionality in the peptide or protein in the presence of other, usually unprotected (free) amino acid groups.
  • Another possibility is to generate reactive groups by biochemical transformations, which are accessible to subsequent conjugation.
  • a prominent example is the reduction of one or more cysteine-formed disulfide bonds in a peptide or protein to then conjugate the thus liberated thiol groups.
  • This can be done by conjugation of a single thiol, preferably with maleimides [Ghosh et al., 1990], or by bridging both thiol groups of the former disulfide bond.
  • maleimides preferably with maleimides [Ghosh et al., 1990]
  • bridging both thiol groups of the former disulfide bond For example, for such a bridging of thiols, double Michael acceptors are described which lead to a Cl- or C3-bridging [Liberatore et al., 1990; Godwin et al., Int. Pat. Appl. WO 2005/007197-A2, WO 2010/100430-A1], as well as bifunctional
  • Scheme 1 Bis-reactive conjugation reagents: a) equilibrium transfer alkylating cross-link (ETAC) reagent for disulfide C3 bridging; b) functional maleimides for disulfide C2 bridging.
  • ADCs antibody-drug conjugates
  • interchain-ol bonds of the antibody in question are reduced to free thiol groups and subsequently conjugated in a bridging manner.
  • a method has now been found which opens up new access to C2-bridged peptide and protein conjugates. In this method, after reduction of the cysteine-formed disulfide bridge, the two thiols are selectively reacted with alkynes by a so-called thiol-yn reaction: Scheme 2:
  • Thiol-yne reactions (thiol-yne reaction, thiol-yne coupling (TYC)) on peptides and proteins are known per se. However, they have not been used for the C2-bridging conjugation of cysteines with free thiol groups, but each thiol group has been conjugated separately and non-bridging [Lo Conte et al., 2011; Lo Conte et al., 2010; Massi and Nanni, 2012; Minozzi et al., 2011; Krannig et al., 2013; Aimetti et al., 2010].
  • thiol-yn reactions to peptides which serve the synthesis of cyclopeptides by substituting a linear precursor peptide at an appropriate position with an alkyne group which then reacts with a free cysteine of the same peptide under cyclization [Aimetti et al. , Int. Pat. Appl. WO 2011/156686-A2].
  • the vinyl sulfide formed in this case can be reacted in a subsequent reaction step with another (other) thiol.
  • the invention provides a method which allows a C2-conjugating bridging of cysteines with free thiol groups in peptides and proteins by means of a selective thiol-yn reaction with alkyne derivatives.
  • Corresponding free thiols can be generated for example by the reduction of disulfide bonds.
  • the alkyne derivatives may be suitably provided with substituents, functional groups and / or linker moieties, allowing for a corresponding modulation of the molecular properties of the target conjugates.
  • the present invention is a process for the preparation of homogeneous peptide and protein conjugates, which is characterized in that a peptide or protein of the formula (II)
  • S 1 and S 2 represent cysteine-sulfur atoms of this peptide or protein bound in a disulfide bridge, under reducing conditions into a peptide or protein of the formula ( ⁇ )
  • R 1 and R 2 independently of one another represent hydrogen, alkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, hydroxyl, alkoxy, amino, alkylamino, dialkylamino, hydroxycarbonyl, alkoxycarbonyl, alkylcarbonylamino or alkoxycarbonylamino, where alkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, Alkoxy, alkylamino, dialkylamino, alkoxycarbonyl, alkylcarbonylamino and alkoxycarbonylamino, in turn, one or more times, identically or differently, with halogen, hydroxyl, alkoxy, amino, Alkylamino, dialkylamino, hydroxycarbonyl, alkoxycarbonyl, alkylcarbonylamino and alkoxycarbonylamino, for a bond or a hydrocarbon chain having 1 to 100 carbon atoms of alkylene,
  • Cycloalkyl ring may be monosubstituted or polysubstituted, identically or differently, with fluorine, alkyl, hydroxy, hydroxyalkyl and alkoxy, is a bond or a linker, n-fold may be present and a drug molecule, polymer, alkaloid, peptide, Protein, carbohydrate, nucleotide, nucleoside, steroid, terpene, porphyrin, chlorin, corrin, eicosanoid, pheromone, vitamin, biotin, a dye molecule or a cryptand or represents hydrogen, hydroxyl, alkoxy, amino, alkylamino, dialkylamino, hydroxycarbonyl, Alk
  • reaction steps a) and b) depicted in Scheme 3 can be carried out either in separate form, with intermediate isolation of the intermediate (III), or successively in the same reaction. are carried out. Preferably, the reactions are carried out in the last-mentioned "one-pot process”.
  • the reduction of the disulfide ( ⁇ ) to the free dithiol (III) is preferably carried out with tris (2-carboxyethyl) phosphine (TCEP).
  • the thiol-yn reaction of the dithiol ( ⁇ ) with the alkyne derivative of the formula (IV) to give the conjugate of the formula (I) can be achieved by photochemical radical initiators or by oxidatively generated radicals, such as, for example, triethylborane with small amounts of oxygen, mediated.
  • photochemical free radical initiators such as bis are preferred (2,4,6-trimefhyl- benzoyl) phenylphosphine oxide (Irgacure ® 819) or lithium-phenyl-2,4,6-trimethylbenzoylphos- phinat (LAP) [Gong et al., 2013 ], used.
  • UV light of a wavelength between 350 nm and 400 nm is preferably used, the wavelengths of 365 nm and 385 nm are particularly preferred.
  • Suitable inert solvents for reaction steps a) and b) are preferably water, aqueous buffer solutions or mixtures of water a water-soluble organic solvent such as methanol or ethanol.
  • the reactions are generally carried out in a temperature range from 0 ° C to 40 ° C; preference is given to the reaction procedure at room temperature.
  • the inventive method in this case relates to the "direct" reaction of the dithiol ( ⁇ ) with the alkyne derivative (IV) to the conjugate of formula (I) under the conditions previously described for reaction step b).
  • the method according to the invention also comprises the preparation of multiple conjugates of a peptide or protein of the formula (II) with the alkyne derivative (IV) according to the above-described reaction sequence in cases where in the peptide or protein of the formula (II) more of such Conjugation accessible disulfide bridges are present.
  • Another object of the present invention are peptide and protein conjugates of the general formula (I)
  • S 1 and S 2 are previously in a disulfide bridge bound cysteine sulfur atoms of a peptide or protein
  • R 1 and R 2 independently of one another represent hydrogen, alkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, hydroxyl, alkoxy, amino, alkylamino, dialkylamino, hydroxycarbonyl, alkoxycarbonyl, alkylcarbonylamino or alkoxycarbonylamino, where alkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, Alkoxy, alkylamino, dialkylamino, alkoxycarbonyl, alkylcarbonylamino and alkoxycarbonylamino in turn may be mono- or polysubstituted, identically or differently, by halogen, hydroxyl, alkoxy, amino, alkylamino, dialkylamino, hydroxycarbonyl, alkoxycarbonyl, alkylcarbonylamino and alkoxycarbonylamino,
  • R 2 and A are linked together and, together with the carbon atoms between them, form an 8-membered carbocycle which may be fused to a 3- to 6-membered cycloalkyl ring, the 8-membered carbocycle and optionally the fused cycloalkyl ring may be monosubstituted or polysubstituted, identically or differently, by fluorine, alkyl, hydroxy, hydroxyalkyl and alkoxy,
  • L is a bond or a linker
  • X can be present n times and a drug molecule, polymer, alkaloid, peptide, protein, carbohydrate, nucleotide, nucleoside, steroid, terpene, porphyrin, chlorin, corrin, eicosanoid, pheromone, vitamin, biotin, a dye molecule or a cryptand represents or represents hydrogen, hydroxy, alkoxy, amino, alkylamino, dialkylamino, hydroxy carbonyl, alkoxycarbonyl, alkylcarbonylamino, alkoxycarbonylamino, alkyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl, where alkyl itself is mono- or polysubstituted, identically or differently, by halogen, hydroxyl, alkoxy, amino, alkylamino, dialkylamino, hydroxycarbonyl, alkoxycarbonyl, alkylcarbonylamino and Alkoxycarbonyla
  • the present invention also encompasses corresponding multiple conjugates of such a peptide or protein, ie conjugates, in which a pairwise C2-bridging in the sense of formula (I) at several positions of the respective precursor peptide, or peptides or proteins with several disulfide bridges accessible by the conjugation method according to the invention protein is done.
  • the substituents and radicals unless specified otherwise, have the following meaning:
  • Alkyl in the context of the invention is a straight-chain or branched alkyl radical having 1 to 10, preferably 1 to 8, particularly preferably 1 to 6 carbon atoms.
  • Alkylene in the context of the invention is a straight-chain or branched, divalent alkyl radical (alkanediyl radical) having 1 to 10, preferably 1 to 8, particularly preferably 1 to 6 carbon atoms.
  • alkanediyl radical alkanediyl radical
  • Alkanediyl radical a straight-chain or branched, divalent alkyl radical (alkanediyl radical) having 1 to 10, preferably 1 to 8, particularly preferably 1 to 6 carbon atoms.
  • hydroxyalkyl is a straight-chain or branched alkyl radical having 1 to 6, preferably 1 to 4, carbon atoms which carries a hydroxy group as substituent within the chain or terminally.
  • Examples which may be mentioned by way of example are: hydroxymethyl, 1-hydroxyethyl, 2-hydroxyethyl, 1-hydroxy-1-methylethyl, 1,1-dimethyl-2-hydroxyethyl, 1-hydroxypropyl, 2-hydroxypropyl, 3-hydroxypropyl, 1 Hydroxy-2-methylpropyl, 2-hydroxy-1-methylpropyl, 2-hydroxy-2-methylpropyl, 1-hydroxybutyl, 2-hydroxybutyl, 3-hydroxybutyl, 4-hydroxybutyl, 5-hydroxypentyl and 6-hydroxyhexyl.
  • Alkoxy in the context of the invention is a straight-chain or branched alkoxy radical having 1 to 6, preferably 1 to 4, carbon atoms. Examples which may be mentioned by way of example and with preference are: methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, where-butoxy, sec-butoxy, ferric. Butoxy, pentoxy, isopentoxy, 1-ethylpropoxy, 1-methylbutoxy, 2-methylbutoxy, 3-methylbutoxy and n-hexoxy.
  • alkoxycarbonyl is a straight-chain or branched alkoxy radical having 1 to 6, preferably 1 to 4, carbon atoms which is linked to the rest of the molecule via a carbonyl group [-C (0O) -] bound to the oxygen atom is.
  • Alkylamino in the context of the invention represents an amino group having a straight-chain or branched alkyl substituent which has 1 to 6, preferably 1 to 4, carbon atoms.
  • Dialkylamino is in the context of the invention an amino group having two identical or different straight-chain or branched alkyl substituents, each having 1 to 6, preferably 1 to 4 carbon atoms.
  • Examples which may be mentioned by way of example and by way of preference are: -dimethylamino, -dibylamino, -ethylmethylamino, -methyl-n-propylamino, / V-isopropylmethylamino, -sopropyl-n-propylamino, / V, / V- Diisopropylamino, n-butyl-methyl-amino and N-ethyl-butyl-N-methylamino.
  • Alkoxycarbonylamino in the context of the invention represents an amino group having a straight-chain or branched alkoxycarbonyl substituent which has 1 to 6, preferably 1 to 4, carbon atoms in the alkoxy radical and is linked via the carbonyl group to the nitrogen atom.
  • Examples which may be mentioned by preference include: methoxycarbonylamino, ethoxycarbonylamino, -propoxycarbonylamino, isopropoxycarbonylamino, n-butoxycarbonylamino and ieri.-butoxycarbonylamino.
  • Examples which may be mentioned are: acetyl, propionyl, n-butyryl, where-butyryl, n-pentanoyl and pivaloyl.
  • Alkylcarbonylamino in the context of the invention is an amino group having a straight-chain or branched alkylcarbonyl substituent which has 1 to 6, preferably 1 to 4 carbon atoms in the alkyl radical and is linked via the carbonyl group to the nitrogen atom. Examples which may be mentioned are: acetylamino, propionylamino, n-butyrylamino, isobutyrylamino, n-pentanoylamino and pivaloylamino.
  • Cycloalkyl in the context of the invention is a monocyclic, saturated carbocycle having 3 to 10, preferably 3 to 8, particularly preferably 3 to 6 ring carbon atoms. Examples which may be mentioned by way of example include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl and cyclodecyl.
  • Cycloalkylene in the context of the invention is a monocyclic, saturated, divalent cycloalkyl radical (cycloalkanediyl radical) having 3 to 10, preferably 3 to 8, particularly preferably 3 to 6 ring carbon atoms.
  • cycloalkanediyl radical having 3 to 10, preferably 3 to 8, particularly preferably 3 to 6 ring carbon atoms. Examples which may be mentioned by way of example and are preferably: cyclopropan-1, 1-diyl, cyclopropan-1, 2-diyl, cyclobutane-1,1-diyl, cyclobutane-1,2-diyl, cyclobutane-1,3-diyl, cyclopentane l, l-diyl, cyclopentane-l, 2-diyl, cyclopentane, 3-diyl, cyclohexane-l, l-diyl, cyclohexane-1,
  • azetidinyl oxetanyl, thietanyl, pyrrolidinyl, pyrrolinyl, pyrazolidinyl, dihydropyrazolyl, tetrahydrofuranyl, thiolanyl, 1,1-diaminothiolanyl, 1,3-oxazolidinyl, 1,3-thiazolidinyl, piperidinyl, tetrahydropyridyl, piperazinyl, tetrahydropyranyl, Dihydropyranyl, tetrahydrothiopyranyl, 1,3-dioxanyl, 1,4-dioxanyl, morpholinyl, thiomorpholinyl, 1,1-dioxideothiomorpholinyl, hexahydroazepinyl, hexahydro-1,4-dihydroxypropyl azepinyl, oct
  • Examples which may be mentioned are: azetidinyl, oxetanyl, thietanyl, pyrrolidinyl, pyrazolidinyl, tetrahydrofuranyl, thiolanyl, 1,3-oxazolidinyl, piperidinyl, piperazinyl, tetrahydropyranyl, tetrahydrothiopyranyl, 1,4-dioxanyl, morpholinyl and thiomorpholinyl.
  • Aryl in the context of the invention is an aromatic carbocycle having 6 or 10 ring carbon atoms, such as phenyl and naphthyl.
  • Arylene in the context of the invention is a divalent aryl radical, for example 1,2-phenylene, 1,3-phenylene, 1,4-phenylene, naphthalene-2,3-diyl, naphthalene-1,4-diyl, naphthalene-1, 5-diyl, naphthalene-2,6-diyl and naphthalene-1,8-diyl.
  • Heteroaryl is in the context of the invention for a 5- to 10-membered, mono- or optionally bicyclic aromatic heterocycle (heteroaromatic) having a total of 5 to 10 ring atoms containing up to four ring heteroatoms from the series N, O and / or S. and linked via a ring carbon atom or optionally a ring nitrogen atom.
  • heterocycle mono- or optionally bicyclic aromatic heterocycle
  • Examples which may be mentioned are: furyl, pyrrolyl, thienyl, pyrazolyl, imidazolyl, thiazolyl, oxazolyl, isoxazolyl, isothiazolyl, triazolyl, oxadiazolyl, thiadiazolyl, tetrazolyl, pyridyl, pyrimidinyl, pyridazinyl, pyrazinyl, triazinyl, benzofuranyl, benzothienyl, benzimidazolyl, benzoxazolyl, benzothiazolyl, Benzotriazolyl, indolyl, indazolyl, quinolinyl, isoquinolinyl, naphthyridinyl, quinazolinyl, quinoxalinyl, phthalazinyl and pyrazolo [3,4-b] pyridinyl.
  • Examples which may be mentioned are: furyl, thienyl, thiazolyl, oxazolyl, isothiazolyl, isoxazolyl, pyrazolyl, imidazolyl, triazolyl, oxadiazolyl, thiadiazolyl, pyridyl, pyrimidinyl, pyridazinyl, pyrazinyl and triazinyl.
  • Halogen in the context of the invention includes fluorine, chlorine, bromine and iodine. Preference is given to chlorine, fluorine or bromine, more preferably fluorine or chlorine.
  • radicals in the compounds according to the invention are present Unless otherwise specified, the radicals may be monosubstituted or polysubstituted. Substitution with one or two or three identical or different substituents is preferred. Particularly preferred is the substitution with one or two identical or different substituents. Very particular preference is given to the substitution with a substituent.
  • in vivo cleavable group can be subdivided into groups which are cleavable in vivo chemically (eg by acid hydrolysis or redox processes) and those which are enzymatically isolated, ie under the influence of an endogenous enzyme, are cleavable in vivo.
  • Both types of cleavable groups should initially be stable in the circulation and only at or in the target cell by the altered there chemical or enzymatic environment (eg lower pH, increased glutathione concentration, presence of lysosomal enzymes such as cathepsin or plasmin) are cleaved.
  • Suitable structural fragments which can be cleaved chemically in vivo are in particular disulfide, hydrazone, acetal and aminal groups for this purpose.
  • Enzymatic cleavable structural elements in particular are oligopeptide units of 2 to 8 amino acids and especially dipeptide groups. Such designated peptide cleavage sites are described in numerous forms in the literature.
  • Prominent examples are the dipeptide units valine-alanine, valine-lysine, valine-citrulline, alanine-lysine and phenylalanine-lysine [see eg JJ Petersen and CF Meares, Bioconjugate Chem. 9, 618-626 (1998); GM Dubowchik and RA Firestone, Bioorg. Med. Chem. Lett. 8, 3341-3346 (1998); GM Dubowchik et al, Bioconjugate Chem. 13, 855-869 (2002)].
  • the linker described above may also contain an in vivo transient stable group.
  • transiently stable groups are cleaved under the influence of the chemical or enzymatic environment, for example in the bloodstream over a relatively long period of time (of hours or days) [see, eg, Flamme et al., Int. Pat. Appl. WO 2013/064455-A1].
  • active ingredient molecules in the above definition of the group X in particular pharmaceutical active ingredients for cancer therapy, such as cytotoxins and cytostatics into consideration. In detail, these agents can damage the cancer cell, initiate programmed cell death, and / or inhibit cell growth and cell proliferation.
  • agents for cancer treatment are: antimetabolites such as methotrexate, cladribine, fludarabine, mercaptopurine, tioguanine, pentostatin, cytarabine, fluorouracil, capecitabine and gemcitabine, alkylating substances such as cyclophosphamide, ifosfamide, mitomycin, trofosfamide, thiotepa , Busulfan, treosulfan, carmustine, lomustine, nimustine, procarbazine, dacarbazine and the platinum compounds cisplatin, carboplatin and oxaliplatin, topoisomerase inhibitors such as topotecan, irinotecan, etoposide and teniposide, kinase inhibitors such as sorafenib, regorafinib, sunitinib, afatinib, Erlo - tinib and
  • Group X include cytotoxic compounds and toxins, as experimentally and clinically tested as "toxophore” in antibody-drug conjugates (ADCs), especially for cancer therapy.
  • ADCs antibody-drug conjugates
  • examples include, in particular, substances such as maytansine and maytansinoids (DM-1, DM-4), dolastatins, auristatins (MMAE, MMAF), calicheamicins, duocarmycins, camptothecins (topotecan, exatecan, irinotecan, SN-38), doxorubicin , Amatoxins (amanitin), pyrrolobenzodiazepines (PBDs) and kinesin spindle protein (KSP) inhibitors.
  • DM-1, DM-4 dolastatins
  • MMAE, MMAF auristatins
  • calicheamicins duocarmycins
  • camptothecins topotecan, exatecan, ir
  • the peptides and proteins of formula (II) contain at least two cysteine amino acids which form or are capable of disulfide bonding.
  • Such peptides include, for example, peptide hormones such as insulin, somatostatin, oxitocin, terli- pressin, adrenomedullin, calcitonin or vasopressin.
  • proteins of this type are antibodies, cytokines such as interleukins and albumins.
  • an immunoglobulin molecule preferably represents a molecule having four polypeptide chains, consisting of two heavy chains (H chains, HC) and two light chains (L chains, LC), which are typically linked together by disulfide bridges (so-called intercam-disulfide bridges) are linked.
  • Each heavy chain comprises a variable domain (abbreviated VH) and a constant domain (CH).
  • the heavy chain constant domain in turn may have three (CHI, CH2, CH3) or four subdomains.
  • Each light chain includes also a variable domain (VL) and a constant domain (CL) -
  • VH and VL domains can be further subdivided into regions of hypervariability, also called complementarity determining regions (CDRs), and in regions with lower sequence variability ("framework region", FR).
  • Each VH and VL region is typically composed of three CDRs and up to four FRs, for example, from the amino to the carboxy terminus in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • An antibody can be obtained from any suitable species, eg monkey, pig, rabbit, mouse or rat. In a particular embodiment, the antibody is of human or murine origin. Such an antibody may be, for example, human, humanized or chimeric.
  • Another object of the present invention is the use of the conjugates of the formula (I) for the diagnosis and / or treatment of diseases, in particular for the diagnosis and / or treatment of cancer and tumor diseases.
  • Another object of the present invention is the use of the conjugates of the formula (I) in a method for the diagnosis and / or treatment of diseases, in particular cancer and tumor diseases.
  • Another object of the present invention is a method for the diagnosis and / or treatment of diseases, in particular cancer and tumor diseases, using one or more conjugates of the formula (I).
  • the term “treatment” or “treating” includes inhibiting, delaying, arresting, alleviating, reducing, restricting, reducing, suppressing, restraining or curing a disease, a disease, a disease, an injury or a health disorder , the unfolding, the course or progression of such conditions and / or the symptoms of such conditions.
  • the term “therapy” is understood to be synonymous with the term “treatment”.
  • diagnosis in the context of the present invention in the usual sense understood as (discriminating) detection, detection, determination, assessment, classification and naming of a disease, a disease, a disease, a disease symptom, an injury or a health disorder.
  • Another object of the present invention are pharmaceutical compositions containing at least one of the conjugates of formula (I), usually together with one or more inert, non-toxic, pharmaceutically suitable excipients, and their use for the purposes mentioned above.
  • the conjugates of the formula (I) according to the invention can act systemically and / or locally. For this purpose, they may be applied in a suitable manner, such as, for example, orally, parenterally, pulmonarily, nasally, sublingually, lingually, buccally, rectally, dermally, transdermally, conjunctivally, otically or as an implant or stent.
  • the conjugates according to the invention can be administered in suitable administration forms.
  • the prior art uses rapidly and / or modified delivery forms which contain the conjugates according to the invention in crystalline and / or amorphized and / or dissolved form, e.g. Tablets (uncoated or coated tablets, for example with enteric or delayed-release or insoluble coatings controlling the release of the conjugates of the invention), tablets or films / wafers rapidly breaking down in the oral cavity, films / lyophilisates, capsules (e.g. Soft gelatin capsules), dragees, granules, pellets, powders, emulsions, suspensions, aerosols or solutions.
  • Tablets uncoated or coated tablets, for example with enteric or delayed-release or insoluble coatings controlling the release of the conjugates of the invention
  • tablets or films / wafers rapidly breaking down in the oral cavity
  • films / lyophilisates e.g. Soft gelatin capsules
  • dragees granules, pellets, powders, emulsions, suspensions, aerosols or solutions
  • Parenteral administration can be accomplished by bypassing a resorption step (e.g., intravenously, intraarterially, intracardially, intraspinal, or intralumbar) or by resorting to absorption (e.g., intramuscularly, subcutaneously, intracutaneously, percutaneously, or intraperitoneally).
  • a resorption step e.g., intravenously, intraarterially, intracardially, intraspinal, or intralumbar
  • absorption e.g., intramuscularly, subcutaneously, intracutaneously, percutaneously, or intraperitoneally.
  • parenteral administration are suitable as application forms u.a. Injection and infusion preparations in the form of solutions, suspensions, emulsions, lyophilisates or sterile powders.
  • Inhalation medicaments including powder inhalers, nebulizers
  • nasal drops solutions or sprays
  • lingual, sublingual or buccal tablets films / wafers or capsules
  • suppositories ear or ophthalmic preparations
  • vaginal capsules aqueous suspensions (lotions, shake mixtures), lipophilic suspensions
  • Ointments creams, transdermal therapeutic systems (eg patches), milk, pastes, foams, powdered powders, implants or stents.
  • the conjugates according to the invention can be converted into the stated administration forms. This can be done in a conventional manner by mixing with inert, non-toxic, pharmaceutically suitable excipients.
  • excipients include, among others, excipients (for example microcrystalline cellulose, lactose, mannitol), solvents (for example liquid polyethylene glycols), emulsifiers and dispersants or wetting agents (for example sodium dodecyl sulfate, polyoxysorbitanoleate), binders (for example polyvinylpyrrolidone), synthetic and natural polymers (eg albumin), stabilizers (eg antioxidants such as Ascorbic acid), dyes (eg, inorganic pigments such as iron oxides) and flavor and / or odoriferous.
  • excipients for example microcrystalline cellulose, lactose, mannitol
  • solvents for example liquid polyethylene glycols
  • emulsifiers and dispersants or wetting agents for example sodium do
  • Device Type MS Waters Synapt G2S
  • Device type UPLC Waters Acquity I-Class
  • Eluent A 1: 1 water + 0.01% formic acid
  • eluent B 1: 1 acetonitrile + 0.01% formic acid
  • Oven 50 ° C
  • Flow 1.20 ml / min
  • UV detection 210 nm.
  • Device Type MS Thermo Fisher Scientific LTQ-Orbitrap-XL
  • Device type HPLC Agilent 1200SL
  • Column Agilent Poroshell 120 SB-C18 2.7 ⁇ , 3 x 150 mm
  • Eluent A 1: 1 water + 0.1% trifluoroacetic acid
  • eluent B 1: 1 acetonitrile + 0.1% trifluoroacetic acid
  • Gradient 0.0 min 2% B-> 1.5 min 2% B -> 15.5 min 95% B -> 18.0 min 95% B
  • Oven 40 ° C
  • Flow 0.75 ml / min
  • UV detection 210 nm.
  • Example 1 Exemplary embodiments: Example 1
  • reaction mixture was then purified by separation of the isomeric products by preparative HPLC (column: Waters X-Bridge BEH130 Prep C18 10 ⁇ OBD, 19 ⁇ 250 mm, eluent A: water with 0.05% TFA, eluent B: acetonitrile with 0.05% TFA; Gradient: 0.0 min 5% B -> 40 min 40% B).
  • An LED UV-pen (OmniCure LX400, diameter 12 mm, igb-tech GmbH, Germany) was introduced through a cut of the two-necked flask (distance to the reaction mixture about 40 mm), and the reaction mixture was UV for 365 h at 365 nm Light irradiated. The addition of 50 ⁇ L of the LAP solution and the subsequent irradiation for 1 h with 365 nm UV light were repeated twice more.
  • reaction mixture was then fractionated by preparative HPLC (column: Waters X -Bridge BEH130 Prep C18 10 ⁇ OB IX 19 ⁇ 250 mm, eluent A: water with 0.1% TFA, eluent B: acetonitrile with 0.1% TFA, gradient: 0.0 min 5% B -> 3 min 5% B -> 43 min 40% B 44.30 min 95% B 49.30 min 95% B).
  • terlipresin acetate (Bachem, Switzerland, sequence: H-Gly-Gly-Gly-Cys-Tyr-Phe-Gln-Asn-Cys-) were dissolved in an argon atmosphere in a two-necked flask.
  • the reaction mixture was stirred for 2 h at RT and then treated with a solution of 4.9 mg (15.84 ⁇ ) 4- (hex-5-yn-l-yl) -4-methylmorpholin-4-iumiodid in 500 ⁇ DPBS buffer. Subsequently, 4.70 mg (15.98 ⁇ ) of LAP [prepared by literature-known method (Gong et al, 2013)] were dissolved in 500 ⁇ DPBS buffer and 50 ⁇ of this LAP solution was added to the reaction mixture.
  • An LED UV-pen (OmniCure LX400, diameter 12 mm, igb-tech GmbH, Germany) was introduced through a cut of the two-necked flask (distance to the reaction mixture about 40 mm), and the reaction mixture was UV for 365 h at 365 nm Light irradiated. The addition of 50 ⁇ L of the LAP solution and the subsequent irradiation for 1 h with 365 nm UV light were repeated twice more.
  • reaction mixture was then fractionated by preparative HPLC (column: Phenomenex Kinetex Prep 5 ⁇ C18 100 ⁇ AXIA Packed LC Column, 21.2 ⁇ 100 mm, eluent A: water with 0.1% TFA, eluent B: acetonitrile with 0.08% TFA; 0.0 min 5% B -> 3 min 5% B -> 63 min 40% B - »64.30 min 95% B 69.30 min 95% B).
  • Product fraction 1 Product fraction 1:
  • the reaction mixture was stirred at RT for 1.5 h and then treated with 200 ⁇ DPBS buffer and a solution of 1.6 mg (7.54 ⁇ ) / V-Boc-L-propargylglycine in 100 ⁇ DPBS buffer. Subsequently 3.20 mg (10.87 ⁇ mol) of LAP [prepared by the method known from the literature (Gong et al., 2013)] were dissolved in 500 ⁇ l of DPBS buffer and 50 ⁇ l of this LAP solution were added to the reaction mixture.
  • An LED UV pencil (OmniCure LX400, diameter 12 mm, igb-tech GmbH, Germany) was introduced through a cut of the two-necked flask (distance to the reaction mixture about 40 mm), and the reaction mixture was allowed to stand for 1 h nm irradiated UV light. The addition of 50 ⁇ L of the LAP solution and the subsequent irradiation for 1 h with 365 nm UV light were repeated twice more.
  • reaction mixture was then fractionated by preparative HPLC (column: Phenomenex Kinetex Prep 5 ⁇ L C18 100 ⁇ AXIA Packed LC Column, 21 ⁇ 2 ⁇ 100 mm, eluant A: water with 0.1% IIA, eluent B: acetonitrile with 0.08% I TA; Gradient: 0.0 min 'AB - + 3 min 5% B -> 63 min 40% B 65.3 min 95% B 70 min 95% B).
  • the reaction mixture was stirred for 2.5 h at RT and then treated with 200 .mu.l 0.1% aqueous acetic acid, 200 ⁇ ⁇ acetonitrile and a solution of 2.5 mg (19.82 ⁇ ⁇ ) hept-6-acid in 100 .mu.l 0.1% aqueous acetic acid , 5.8 mg (19.72 ⁇ mol) of LAP [prepared by the method known from the literature (Gong et al., 2013)] were then dissolved in 500 ⁇ l of 0.1% strength aqueous acetic acid, and 50 ⁇ l of this LAP solution were added to the reaction mixture.
  • reaction mixture was then fractionated by preparative HPLC (column: Waters X-Bridge BEI II 30 Prep C18 10 ⁇ OBD, 19 ⁇ 250 mm, eluant A: water with 0.1% TFA, eluent B: acetonitrile with 0.08% I LA; Gradient: 0.0 min 5% B 3 min 5% B -> 33 min 40% B).
  • reaction mixture was then fractionated by preparative HPLC (column: Phenomenex Kinetex Prep 5 ⁇ C18 100 ⁇ AXIA Packed LC Column, 21.2 ⁇ 100 mm, eluent A: water with 0.1% TFA, eluent B: acetonitrile with 0.08% TFA, gradient: 0.0 min 5 ' ⁇ B-> 3 min 5% B ⁇ 63 min 40% B 64.30 min 95% B 69.30 min 95% B).
  • the quantification of the covalent FAB fragment was carried out by RP chromatography of the reduced and denatured FAB fragment. Guanidinium hydrochloride (GuHCl) (28.6 mg) and a solution of DL-dithiothreitol (DTT) (500 mM, 3 ⁇ ) were added to the sample solution (1 mg / ml, 50 ⁇ ). The mixture was incubated for one hour at 55 ° C and then analyzed by HPLC.
  • GuHCl Guanidinium hydrochloride
  • DTT DL-dithiothreitol
  • HPLC analysis was performed on an Agilent 1260 HPLC system with detection at 220 nm.
  • a Polymer Laboratories PLRP-S Polymerized Reversed Phase column (2.1 mm x 150 mm, 8 ⁇ m particle size, 1000 ⁇ ; Catalog no. PL1912-3802) at a flow rate of 1 ml / min using the following eluent system: eluent A: 0.05% trifluoroacetic acid in water, eluent B: 0.05% trifluoroacetic acid in acetonitrile; Gradient: 0 min 25% B, 3 min 25% B, 28 min 50% B.
  • the signal detected exclusively in the conjugated sample was assigned to the covalent, non-reducible linked FAB.
  • the percentage of covalently linked FAB was calculated from the signal areas determined by integration. For this purpose, the quotient of the signal area of the FAB fragment to the total area of all signals was formed and multiplied by 100. The resulting chromatograms and the calculated percentages are shown in Figure 1 1 and 12.
  • the denatured and reduced sample was mass spectrometrically after online desalting over a Grom-Sil 300 Butyl lSt column (particle size 5 ⁇ , column dimension 5 mm x 500 ⁇ ) by HPLC-ESI-TOF (Impact HD, Bruker Daltonik).
  • the flow rate was 5 ⁇ / min with the following eluent system: Eluent A: 0.1% formic acid in water, eluent B: 0.1% formic acid in 80% isopropanol, 10% acetonitrile and 10% water; Gradient: 0 minutes 22% B, 8 minutes 22% B, 10 minutes 24% B, 12 minutes 80% B, 18 minutes 95% B, 27 minutes 95% B, 30 minutes 22% B.
  • MINOZZI M., MONESI, A., NANNI, D., SPAGNOLO, P., MARCHETTI, N. & MASSI, A., 2011.
  • WIDDISON, W.C, WILHELM, SD CAVANAGH, EE, WHITEMAN, KR, LEECE, BA, KOVTUN, Y., GOLDMACHER, VS, XIE, H., STEEVES, RM, LUTZ, RJ, ZHAO, R., WANG , L., BLATTLER, WA & CHARI, RV, 2006.

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Abstract

The present application concerns a new method for targeted conjugation of peptides and proteins which is characterized by the C2 bridging of cysteine amino acids in pairs via their thiol groups, the conjugates of peptides and proteins that are obtainable by such a method, and the use of such conjugates in the diagnosis and/or treatment of disorders.

Description

Verfahren zur gezielten Konjugation von Peptiden und Proteinen mittels paarweiser C2- Verbrückung von Cystein-Aminosäuren  Method for targeted conjugation of peptides and proteins by pairwise C2 bridging of cysteine amino acids
Die vorliegende Anmeldung betrifft ein neues Verfahren zur gezielten Konjugation von Peptiden und Proteinen, das durch die paarweise C2-Verbrückung von Cystein-Aminosäuren über deren Thiol-Gruppen gekennzeichnet ist, ferner die Konjugate von Peptiden und Proteinen, die nach einem solchen Verfahren erhältlich sind, sowie die Verwendung solcher Konjugate zur Diagnose und/oder Behandlung von Erkrankungen. The present application relates to a novel method for the targeted conjugation of peptides and proteins, which is characterized by the pairwise C2-bridging of cysteine amino acids via their thiol groups, and also to the conjugates of peptides and proteins obtainable by such a process, and the use of such conjugates for the diagnosis and / or treatment of diseases.
Die Peptid- und Proteinkonjugation hat in den letzten Jahren enorme Bedeutung in der Bereitstellung von therapeutisch wichtigen Wirkstoffen, in der Diagnostik von Krankheitsgeschehen sowie bei der Aufklärung biochemischer Mechanismen erlangt [Flygare et al., 2013; Jones et al., 2013] . Peptide and protein conjugation has gained tremendous importance in recent years in the delivery of therapeutically important drugs, in the diagnosis of disease and in the elucidation of biochemical mechanisms [Flygare et al., 2013; Jones et al., 2013].
Im Wesentlichen werden durch Konjugation die Eigenschaften von Peptiden und Proteinen verändert oder moduliert. Therapeutisch relevante Peptide und Proteine können beispielsweise mit biokompatiblen Polymeren konjugiert werden, um die Halbwertszeit des betreffenden Peptids oder Proteins in der Plasmazirkulation und damit seine Wirkdauer zu verlängern oder um einer möglichen Immunogenizität entgegenzuwirken [Veronese und Maro, 2008; Roberts et al., 2002]. Weiterhin können Peptide und Proteine mit biochemischen Markern, Farbstoffen oder Reaktivgruppen konjugiert werden, die dann nach Applikation Aufschluss über das Bindungsgeschehen in gewissen Organen oder Zellregionen geben [Miller und Cornish, 2005]. Eine weiteres, breit beforschtes Feld sind Peptid- und Proteinkonjugate mit Wirkstoffen, die diese Wirkstoffe zielgerichtet in bestimmte Zell- oder Organregionen zum beabsichtigten Wirkort führen (sogenanntes Drug Targeting). Prominentes Beispiel hierfür sind Antikörper -Wirkstoff-Konjugate (sogenannte Antibody Drug Conjugates, ADCs), die gezielt an bestimmte Domänen/ Antigene binden und nach Internalisierung in die Zelle sowie anschließender Prozessierung (beispielsweise in den Lysosomen) den Wirkstoff im Kompartiment des Wirkorts freisetzen [Garnett, 2001 ; Wu und Senter, 2005; Sapra et al., 2011; Chari et al., 2014; Klinguer-Hamour et al., 2014; Tian et al., 2014]. Essentially, the properties of peptides and proteins are altered or modulated by conjugation. Therapeutically relevant peptides and proteins may, for example, be conjugated to biocompatible polymers to increase the half-life of the subject peptide or protein in the plasma circulation and thus its duration of action or to counteract possible immunogenicity [Veronese and Maro, 2008; Roberts et al., 2002]. Furthermore, peptides and proteins can be conjugated with biochemical markers, dyes or reactive groups, which then provide information on the binding process in certain organs or cell regions after administration [Miller and Cornish, 2005]. Another widely researched field are peptide and protein conjugates with active ingredients that target these drugs to specific cell or organ regions for their intended site of action (so-called drug targeting). A prominent example of this are antibody-drug conjugates (so-called Antibody Drug Conjugates, ADCs) that bind specifically to specific domains / antigens and after internalization in the cell and subsequent processing (for example, in the lysosomes) release the drug in the compartment of the site of action [Garnett , 2001; Wu and Senter, 2005; Sapra et al., 2011; Chari et al., 2014; Klinguer-Hamour et al., 2014; Tian et al., 2014].
Die Methoden zur Konjugation von Peptiden oder Proteinen sind vielfältig. So können Peptide und Proteine nach unterschiedlichen Verfahren mit reaktiven Gruppen versehen werden, die dann ihrerseits als Anknüpfungsstelle für Wirkstoffe oder Diagnostika dienen [Ramil und Lin, 2013] . Darüber hinaus können Peptide und Proteine über die Funktionalitäten ihrer Aminosäuren konjugiert werden [Widdison et al., 2006] . Die Herausforderung hierbei ist die Bereitstellung einer Methode, die eine selektive Umsetzung der gewünschten Funktionalität im Peptid oder Protein in Gegenwart der weiteren, in der Regel ungeschützten (freien) Aminosäuregruppen erlaubt. The methods for conjugating peptides or proteins are manifold. Thus, peptides and proteins can be provided with reactive groups by different methods, which in turn serve as a point of attachment for drugs or diagnostics [Ramil and Lin, 2013]. In addition, peptides and proteins can be conjugated via the functionality of their amino acids [Widdison et al., 2006]. The challenge here is to provide a Method that allows a selective conversion of the desired functionality in the peptide or protein in the presence of other, usually unprotected (free) amino acid groups.
Eine weitere Möglichkeit besteht darin, durch biochemische Transformationen reaktive Gruppen zu generieren, die einer nachfolgenden Konjugation zugänglich sind. Prominentes Beispiel ist die Reduktion einer oder mehrerer über Cysteine ausgebildeter Disulfid-Bindungen in einem Peptid oder Protein, um die so frei werdenden Thiol-Gruppen dann zu konjugieren. Dies kann durch Konjugation eines einzelnen Thiols geschehen, vorzugsweise mit Maleinimiden [Ghosh et al., 1990], oder durch die Überbrückung beider Thiol-Gruppen der ehemaligen Disulfid-Bindung. Für eine solche Überbrückung von Thiolen sind beispielsweise doppelte Michael-Akzeptoren beschrieben, die zu einer Cl- bzw. C3-Verbrückung führen [Liberatore et al., 1990; Godwin et al., Int. Pat. Appl. WO 2005/007197-A2, WO 2010/100430-A1], sowie bifunktionelle elektrophile Maleinimi- de, die eine C2-Verbrückung ermöglichen [Schumacher et al., 2013] : Another possibility is to generate reactive groups by biochemical transformations, which are accessible to subsequent conjugation. A prominent example is the reduction of one or more cysteine-formed disulfide bonds in a peptide or protein to then conjugate the thus liberated thiol groups. This can be done by conjugation of a single thiol, preferably with maleimides [Ghosh et al., 1990], or by bridging both thiol groups of the former disulfide bond. For example, for such a bridging of thiols, double Michael acceptors are described which lead to a Cl- or C3-bridging [Liberatore et al., 1990; Godwin et al., Int. Pat. Appl. WO 2005/007197-A2, WO 2010/100430-A1], as well as bifunctional electrophilic maleimides which enable C2 bridging [Schumacher et al., 2013]:
Schema la: Scheme la:
Schema lb: Scheme Ib:
Schema 1: Bis-reaktive Konjugationsreagenzien: a) "equilibrium transfer alkylating cross-link" (ETAC)-Reagenz für die Disulfid-C3-Verbrückung; b) funktionelle Maleinimide zur Disulfid-C2- Verbrückung. Die in Schema la und lb skizzierten Methoden haben bei der Bereitstellung von Antikörper -Wirk- stoff-Konjugaten (ADCs) erfolgreich Anwendung gefunden. Hierbei werden interchain-Ois lüd- brücken des betreffenden Antikörpers zu freien Thiol-Gruppen reduziert und anschließend verbrückend konjugiert. Im Rahmen der vorliegenden Erfindung wurde nun eine Methode gefunden, die einen neuen Zugang zu C2-verbrückten Peptid- und Proteinkonjugaten eröffnet. Bei dieser Methode werden die beiden Thiole nach Reduktion der über Cysteine ausgebildeten Disulfid-Brücke mittels einer sogenannten Thiol-yn-Reaktion selektiv mit Alkinen umgesetzt: Schema 2: Scheme 1: Bis-reactive conjugation reagents: a) equilibrium transfer alkylating cross-link (ETAC) reagent for disulfide C3 bridging; b) functional maleimides for disulfide C2 bridging. The methods outlined in Schemes 1a and 1b have been successfully applied to the delivery of antibody-drug conjugates (ADCs). In this case, interchain-ol bonds of the antibody in question are reduced to free thiol groups and subsequently conjugated in a bridging manner. In the context of the present invention, a method has now been found which opens up new access to C2-bridged peptide and protein conjugates. In this method, after reduction of the cysteine-formed disulfide bridge, the two thiols are selectively reacted with alkynes by a so-called thiol-yn reaction: Scheme 2:
Schema 2: Thiol-yn-Reaktion zur C2-Verbrückung von reduzierten Disulfid-Bindungen in Peptiden und Proteinen. Scheme 2: Thiol-yn reaction for C2 bridging of reduced disulfide bonds in peptides and proteins.
Thiol-yn-Reaktionen [engl, thiol-yne reaction, thiol-yne coupling (TYC)] an Peptiden und Pro- teinen sind an sich bekannt. Sie wurden bisher jedoch nicht für die C2-verbrückende Konjugation an Cysteine mit freien Thiol-Gruppen genutzt, vielmehr wurde jede Thiol-Gruppe separat und nicht-verbrückend konjugiert [Lo Conte et al., 2011 ; Lo Conte et al., 2010; Massi und Nanni, 2012; Minozzi et al., 2011 ; Krannig et al., 2013; Aimetti et al., 2010]. Weiterhin sind Thiol-yn- Reaktionen an Peptiden bekannt, die der Synthese von Cyclopeptiden dienen, indem ein lineares Vorläuferpeptid an einer geeigneten Position mit einer Alkin-Gruppe substituiert wird, welche dann mit einem freien Cystein desselben Peptids unter Cyclisierung reagiert [Aimetti et al., Int. Pat. Appl. WO 2011/156686-A2] . Das hierbei gebildete Vinylsulfid kann in einem nachfolgenden Reaktionsschritt mit einem weiteren (anderen) Thiol umgesetzt werden. Im Gegensatz zur hier beschriebenen erfindungsgemäßen Methode werden dabei jedoch nicht zwei freie Cysteine eines Peptids oder Proteins miteinander verbrückt, sondern es wird jeweils ein peptid- oder proteingebundenes Alkin mit einem Cystein dieses Peptids oder Proteins zur Reaktion gebracht. Ferner ist in der Literatur eine verbrückende Thiol-yn-Reaktion von zwei verbundenen Mercaptoessigsäure- estern mit freien Thiol-Gruppen zur Synthese von makrocyclischen Kronenether- und Rotaxan- Strukturen beschrieben [Zhou et al., 2010]. Weiterhin sind Thiol-yn-Reaktionen mit Peptiden und Proteinen beschrieben, welche dem Aufbau dreidimensionaler Netzwerke, wie zum Beispiel Hydrogelen, dienen [Anseth et al., Int. Pat. Appl. WO 2012/103445-A2; Kazantsev et al., US Pat. Appl. US 2014/0273153-A1]. Im Gegensatz zur hier beschriebenen erfindungsgemäßen Methode resultieren dabei nicht homogene Konjugate, sondern heterogene polymere Netzwerke. Wesentliches Merkmal der oben beschriebenen Cystein-Verbrückung ist, dass dabei die Stabilität bzw. Konformation des Peptids oder Proteins, welche vormals durch die entsprechende Disulfid- Brücke vermittelt wurde, weitgehend erhalten bleibt. Ferner wird durch eine C2-Brücke die Funktionalität, d.h. die Affinität zum biologischen Target des Peptids oder Proteins vielfach bewahrt [Jones et al., 2012; Gerona-Navarro et al., 2011]. Thiol-yne reactions (thiol-yne reaction, thiol-yne coupling (TYC)) on peptides and proteins are known per se. However, they have not been used for the C2-bridging conjugation of cysteines with free thiol groups, but each thiol group has been conjugated separately and non-bridging [Lo Conte et al., 2011; Lo Conte et al., 2010; Massi and Nanni, 2012; Minozzi et al., 2011; Krannig et al., 2013; Aimetti et al., 2010]. Furthermore, thiol-yn reactions to peptides are known which serve the synthesis of cyclopeptides by substituting a linear precursor peptide at an appropriate position with an alkyne group which then reacts with a free cysteine of the same peptide under cyclization [Aimetti et al. , Int. Pat. Appl. WO 2011/156686-A2]. The vinyl sulfide formed in this case can be reacted in a subsequent reaction step with another (other) thiol. In contrast to the method according to the invention described here, however, not two free cysteines of a peptide or protein are bridged with each other, but in each case a peptide- or protein-bound alkyne is reacted with a cysteine of this peptide or protein. Furthermore, the literature describes a bridging thiol-yn reaction of two linked mercaptoacetic acid esters with free thiol groups for the synthesis of macrocyclic crown ether and rotaxane structures [Zhou et al., 2010]. Furthermore, thiol-yn reactions with peptides and proteins are described, which serve the construction of three-dimensional networks, such as hydrogels [Anseth et al., Int. Pat. Appl. WO 2012/103445-A2; Kazantsev et al., US Pat. Appl. US 2014/0273153-A1]. In contrast to the method according to the invention described here, this results in non-homogeneous conjugates but heterogeneous polymeric networks. An essential feature of the cysteine bridge described above is that the stability or conformation of the peptide or protein, which was previously mediated by the corresponding disulfide bridge, is largely retained. Furthermore, functionality, ie affinity for the biological target of the peptide or protein, is often preserved by a C2 bridge [Jones et al., 2012; Gerona-Navarro et al., 2011].
Von Michael-Addukten, wie im Falle der oben beschriebenen Cl- bzw. C3 -verbrückten Konjugate (siehe Schema la), ist andererseits bekannt, dass diese unter physiologischen Bedingungen Retro- und Austauschreaktionen mit anderen Thiol- Verbindungen, wie beispielsweise Glutathion oder Serumalbumin, eingehen können und somit einem gewissen Abbau in der Plasmazirkulation unter - liegen [Baldwin und Kiick, 2011; Toda et al., 2013; Shen et al., 2012]. Um diese oftmals unerwünschte Eigenschaft zu unterdrücken, sind nachfolgende chemische Transformationen nötig. Im Falle der über Maleinimid C2 -verbrückten Konjugate (siehe Schema lb) ist eine Öffnung des Maleinimids oder im gesättigten Fall des Succinimids erforderlich, um eine gegenüber Thiolen stabile Konjugation zu erreichen [Castaneda et al., 2013]. Bei den nach dem erfindungsgemäßen Verfahren über Thiol-yn-Reaktion erhältlichen Verbindungen handelt es sich nicht um Konjugate von Michael-Akzeptoren, so dass derartige Retro- und Austauschreaktionen hier nicht zu erwarten sind. On the other hand, Michael adducts, as in the case of the above-described Cl- or C3-bridged conjugates (see scheme Ia), are known to undergo retro and exchange reactions with other thiol compounds, such as glutathione or serum albumin, under physiological conditions. and thus a certain degradation in the plasma circulation lie under [Baldwin and Kiick, 2011; Toda et al., 2013; Shen et al., 2012]. To suppress this often undesirable property, subsequent chemical transformations are necessary. In the case of maleinimide C2-bridged conjugates (see Scheme Ib), an opening of the maleimide or in the saturated case of the succinimide is required to achieve thiol-stable conjugation [Castaneda et al., 2013]. The compounds obtainable by the process according to the invention via thiol-yn reaction are not conjugates of Michael acceptors, so that such retro- and exchange reactions are not to be expected here.
Vor dem geschilderten Hintergrund war es Aufgabe der vorliegenden Erfindung, eine neue Methode bereitzustellen, nach der Peptide und Proteine selektiv über ihre Cystein-Gruppen verbrückend und in stabiler Form konjugiert werden können und die so für die Herstellung verschiedenartiger, definierter Peptid- und Proteinkonjugate von Nutzen ist. Against the background described, it was an object of the present invention to provide a novel method in which peptides and proteins can be selectively bridged and stably conjugated via their cysteine groups and thus useful for the preparation of various, defined peptide and protein conjugates is.
Zur Lösung dieser Aufgabe stellt die Erfindung ein Verfahren bereit, das eine konjugierende C2- Verbrückung von Cysteinen mit freien Thiol-Gruppen in Peptiden und Proteinen auf dem Wege einer selektiven Thiol-yn-Reaktion mit Alkin-Derivaten ermöglicht. Entsprechende freie Thiole können beispielsweise durch die Reduktion von Disulfid-Bindungen generiert werden. Weiterhin können die Alkin-Derivate in geeigneter Weise mit Substituenten, funktionellen Gruppen und/oder Linker-Einheiten versehen werden, was eine entsprechende Modulation der molekularen Eigenschaften der Ziel-Konjugate erlaubt. To achieve this object, the invention provides a method which allows a C2-conjugating bridging of cysteines with free thiol groups in peptides and proteins by means of a selective thiol-yn reaction with alkyne derivatives. Corresponding free thiols can be generated for example by the reduction of disulfide bonds. Furthermore, the alkyne derivatives may be suitably provided with substituents, functional groups and / or linker moieties, allowing for a corresponding modulation of the molecular properties of the target conjugates.
Gegenstand der vorliegenden Erfindung ist ein Verfahren zur Herstellung von homogenen Peptid- und Proteinkonjugaten, welches dadurch gekennzeichnet ist, dass man ein Peptid oder Protein der Formel (II) The present invention is a process for the preparation of homogeneous peptide and protein conjugates, which is characterized in that a peptide or protein of the formula (II)
in welcher S1 und S2 in einer Disulfid-Brücke gebundene Cystein-Schwefelatome dieses Peptids oder Proteins darstellen, unter reduzierenden Bedingungen in ein Peptid oder Protein der Formel (ΙΠ) in which S 1 and S 2 represent cysteine-sulfur atoms of this peptide or protein bound in a disulfide bridge, under reducing conditions into a peptide or protein of the formula (ΙΠ)
überführt und dieses dann unter radikalischen Reaktionsbedingungen mit einem Alkin-Derivat der Formel (IV) and this then under radical reaction conditions with an alkyne derivative of the formula (IV)
(IV) in welcher  (IV) in which
R1 und R2 unabhängig voneinander für Wasserstoff, Alkyl, Cycloalkyl, Heterocycloalkyl, Aryl, Heteroaryl, Hydroxy, Alkoxy, Amino, Alkylamino, Dialkylamino, Hydroxycarbonyl, Alkoxycarbonyl, Alkylcarbonylamino oder Alkoxycarbonylamino stehen, wobei Alkyl, Cycloalkyl, Heterocycloalkyl, Aryl, Heteroaryl, Alkoxy, Alkylamino, Dialkylamino, Alkoxycarbonyl, Alkylcarbonylamino und Alkoxycarbonylamino ihrerseits ein- oder mehrfach, gleich oder verschieden, mit Halogen, Hydroxy, Alkoxy, Amino, Alkylamino, Dialkylamino, Hydroxycarbonyl, Alkoxycarbonyl, Alkylcarbonylamino und Alkoxycarbonylamino substituiert sein können, für eine Bindung oder eine Kohlenwasserstoffkette mit 1 bis 100 Kohlenstoffatomen aus Alkylen-, Cycloalkylen- und/oder Arylengruppen steht, die ein- oder mehrfach, gleich oder verschieden, durch eine Gruppe ausgewählt aus -O-, -S-, -S(=0)-, -S(=0)2-, -NH-, -N(CH3)-, -C(=0)-, -NH-C(=0)-, -C(=0)-NH-, -0-C(=0)-, -C(=0)-0-, -S02-NH-, -NH-SO2-, -NH-NH-, -SO2-NH-NH-, -NH-NH-SO2-, -C(=0)-NH-NH-, -NH-NH-C(=0)-, -NH-C(=0)-NH-, -0-C(=0)-NH-, -NH-C(=0)-0- und einem 4- bis 10-gliedrigen, aromatischen oder nicht-aromatischen Heterocyclus mit bis zu 4 Heteroatomen aus der Reihe N, O, S, S(=0) und/oder S(=0)2 unterbrochen sein kann, R 1 and R 2 independently of one another represent hydrogen, alkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, hydroxyl, alkoxy, amino, alkylamino, dialkylamino, hydroxycarbonyl, alkoxycarbonyl, alkylcarbonylamino or alkoxycarbonylamino, where alkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, Alkoxy, alkylamino, dialkylamino, alkoxycarbonyl, alkylcarbonylamino and alkoxycarbonylamino, in turn, one or more times, identically or differently, with halogen, hydroxyl, alkoxy, amino, Alkylamino, dialkylamino, hydroxycarbonyl, alkoxycarbonyl, alkylcarbonylamino and alkoxycarbonylamino, for a bond or a hydrocarbon chain having 1 to 100 carbon atoms of alkylene, cycloalkylene and / or arylene groups which are mono- or polysubstituted, identical or different, by a Group selected from -O-, -S-, -S (= O) -, -S (= O) 2-, -NH-, -N (CH 3 ) -, -C (= O) -, -NH -C (= O) -, -C (= O) -NH-, -O-C (= O) -, -C (= O) -O-, -SO 2 -NH-, -NH-SO 2 - , -NH-NH-, -SO 2 -NH-NH-, -NH-NH-SO 2 -, -C (= O) -NH-NH-, -NH-NH-C (= O) -, -NH- C (= O) -NH-, -O-C (= O) -NH-, -NH-C (= O) -O- and a 4- to 10-membered, aromatic or non-aromatic heterocycle with up to 4 heteroatoms from the series N, O, S, S (= 0) and / or S (= 0) 2 may be interrupted,
und A miteinander verknüpft sind und zusammen mit den Kohlenstoffatomen, die sich zwischen ihnen befinden, einen 8-gliedrigen Carbocyclus bilden, welcher mit einem 3- bis 6-gliedrigen Cycloalkyl-Ring anelliert sein kann, wobei der 8-gliedrige Carbocyclus und gegebenenfalls der anellierte Cycloalkyl-Ring ein- oder mehrfach, gleich oder verschieden, mit Fluor, Alkyl, Hydroxy, Hydroxyalkyl und Alkoxy substituiert sein können, für eine Bindung oder einen Linker steht, n-fach vorhanden sein kann und ein Wirkstoffmolekül, Polymer, Alkaloid, Peptid, Protein, Kohlenhydrat, Nucleotid, Nucleosid, Steroid, Terpen, Porphyrin, Chlorin, Corrin, Eico- sanoid, Pheromon, Vitamin, Biotin, ein Farbstoffmolekül oder einen Kryptanden darstellt oder für Wasserstoff, Hydroxy, Alkoxy, Amino, Alkylamino, Dialkylamino, Hydroxycarbonyl, Alkoxycarbonyl, Alkylcarbonylamino, Alkoxycarbonylamino, Alkyl, Cycloalkyl, Heterocycloalkyl, Aryl oder Heteroaryl steht, wobei Alkyl seinerseits ein- oder mehrfach, gleich oder verschieden, mit Halogen, Hydroxy, Alkoxy, Amino, Alkylamino, Dialkylamino, Hydroxycarbonyl, Alkoxycarbonyl, Alkylcarbonylamino und Alkoxycarbonylamino substituiert sein kann und wobei Cycloalkyl, Heterocycloalkyl, Aryl und Heteroaryl ihrerseits ein- oder mehrfach, gleich oder verschieden, mit Halogen, Alkyl, Hydroxy, Alkoxy, Amino, Alkylamino, Dialkylamino, Hydroxycarbonyl, Alkoxycarbonyl, Alkylcarbonylamino und Alkoxycarbo- nylamino substituiert sein können, und n für eine ganze Zahl im Bereich von 1 bis einschließlich 10 steht, wobei im Fall, dass die Gruppe X mehrfach vorhanden ist, ihre individuellen Bedeutungen gleich oder verschieden sein können, zu einem Peptid- bzw. Protein-Konjugat der Formel (I) and A are linked together and, together with the carbon atoms located between them, form an 8-membered carbocycle which may be fused to a 3- to 6-membered cycloalkyl ring, the 8-membered carbocycle and optionally the fused one Cycloalkyl ring may be monosubstituted or polysubstituted, identically or differently, with fluorine, alkyl, hydroxy, hydroxyalkyl and alkoxy, is a bond or a linker, n-fold may be present and a drug molecule, polymer, alkaloid, peptide, Protein, carbohydrate, nucleotide, nucleoside, steroid, terpene, porphyrin, chlorin, corrin, eicosanoid, pheromone, vitamin, biotin, a dye molecule or a cryptand or represents hydrogen, hydroxyl, alkoxy, amino, alkylamino, dialkylamino, hydroxycarbonyl, Alkoxycarbonyl, alkylcarbonylamino, alkoxycarbonylamino, alkyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl, wherein alkyl in turn one or more times, the same or differently, may be substituted by halogen, hydroxyl, alkoxy, amino, alkylamino, dialkylamino, hydroxycarbonyl, alkoxycarbonyl, alkylcarbonylamino and alkoxycarbonylamino and where cycloalkyl, heterocycloalkyl, aryl and heteroaryl in turn one or more times, identically or differently, with halogen, alkyl, Hydroxy, alkoxy, amino, alkylamino, Dialkylamino, hydroxycarbonyl, alkoxycarbonyl, alkylcarbonylamino and alkoxycarbonylamino, and n is an integer in the range of 1 to 10, inclusive, wherein in the event that the group X is present more than once, their individual meanings may be the same or different , to a peptide or protein conjugate of the formula (I)
in welcher R1, R2, A, L, X und n die oben angegebenen Bedeutungen haben, umsetzt. in which R 1 , R 2 , A, L, X and n have the meanings given above, is reacted.
Das erfindungsgemäße Verfahren ist in dem nachfolgenden Reaktionsschema zusammenfassend dargestellt: The process according to the invention is summarized in the following reaction scheme:
Schema 3 Scheme 3
(I I) (III )  (I) (III)
[a): Reduktion; b): Thiol-yn-Reaktion]. [a): reduction; b): thiol-yn reaction].
Die in Schema 3 abgebildeten Reaktionsschritte a) und b) können entweder in separater Form, unter zwischenzeitlicher Isolierung des Intermediats (III), oder nacheinander im gleichen Reak- tionsgefäß durchgeführt werden. Vorzugsweise werden die Umsetzungen im zuletzt genannten "Eintopfverfahren" durchgeführt. Die Reduktion des Disulfids (Π) zum freien Dithiol (III) erfolgt bevorzugt mit Tris(2-carboxyethyl)phosphin (TCEP). Die über einen Radikalmechanismus verlaufende Thiol-yn-Reaktion des Dithiols (ΙΠ) mit dem Alkin-Derivat der Formel (IV) zum Konjugat der Formel (I) kann durch photochemische Radikalinitiatoren oder durch oxidativ erzeugte Radikale, wie beispielsweise Triethylboran mit geringen Mengen Sauerstoff, vermittelt werden. Bevorzugt werden bekannte photochemische Radikalinitiatoren, wie beispielsweise Bis(2,4,6-trimefhyl- benzoyl)phenylphosphinoxid (Irgacure® 819) oder Lithium-phenyl-2,4,6-trimethylbenzoylphos- phinat (LAP) [Gong et al., 2013], verwendet. Zur photochemischen Initiierung in Verbindung mit dem Radikalstarter eignet sich UV-Licht. Bevorzugt wird UV-Licht einer Wellenlänge zwischen 350 nm und 400 nm eingesetzt, besonders bevorzugt sind die Wellenlängen von 365 nm und 385 nm. Als inerte Lösungsmittel für die Reaktionsschritte a) und b) eignen sich vorzugsweise Wasser, wässrige Pufferlösungen oder Gemische von Wasser mit einem wasserlöslichen organischen Lösungsmittel wie Methanol oder Ethanol. Die Umsetzungen erfolgen im Allgemeinen in einem Temperaturbereich von 0°C bis 40°C; bevorzugt ist die Reaktionsführung bei Raumtemperatur. The reaction steps a) and b) depicted in Scheme 3 can be carried out either in separate form, with intermediate isolation of the intermediate (III), or successively in the same reaction. are carried out. Preferably, the reactions are carried out in the last-mentioned "one-pot process". The reduction of the disulfide (Π) to the free dithiol (III) is preferably carried out with tris (2-carboxyethyl) phosphine (TCEP). The thiol-yn reaction of the dithiol (ΙΠ) with the alkyne derivative of the formula (IV) to give the conjugate of the formula (I) can be achieved by photochemical radical initiators or by oxidatively generated radicals, such as, for example, triethylborane with small amounts of oxygen, mediated. Known photochemical free radical initiators, such as bis are preferred (2,4,6-trimefhyl- benzoyl) phenylphosphine oxide (Irgacure ® 819) or lithium-phenyl-2,4,6-trimethylbenzoylphos- phinat (LAP) [Gong et al., 2013 ], used. For photochemical initiation in conjunction with the radical starter UV light is suitable. UV light of a wavelength between 350 nm and 400 nm is preferably used, the wavelengths of 365 nm and 385 nm are particularly preferred. Suitable inert solvents for reaction steps a) and b) are preferably water, aqueous buffer solutions or mixtures of water a water-soluble organic solvent such as methanol or ethanol. The reactions are generally carried out in a temperature range from 0 ° C to 40 ° C; preference is given to the reaction procedure at room temperature.
Wenn das erfindungsgemäß zu konjugierende Peptid oder Protein bereits in der Dithiol-Form (ΙΠ) vorliegt, entfällt der oben aufgeführte Reduktionsschritt a); das erfindungsgemäße Verfahren betrifft in diesem Fall die "direkte" Umsetzung des Dithiols (ΙΠ) mit dem Alkin-Derivat (IV) zum Konjugat der Formel (I) unter den zuvor für Reaktionsschritt b) beschriebenen Bedingungen. If the peptide or protein to be conjugated according to the invention is already present in the dithiol form (ΙΠ), the abovementioned reduction step a); the inventive method in this case relates to the "direct" reaction of the dithiol (ΙΠ) with the alkyne derivative (IV) to the conjugate of formula (I) under the conditions previously described for reaction step b).
Das erfindungsgemäße Verfahren umfasst auch die Herstellung von Mehrfach-Konjugaten eines Peptids oder Proteins der Formel (II) mit dem Alkin-Derivat (IV) gemäß der oben beschriebenen Reaktionssequenz in Fällen, wo in dem Peptid oder Protein der Formel (II) mehrere einer solchen Konjugation zugängliche Disulfidbrücken vorhanden sind. Weiterer Gegenstand der vorliegenden Erfindung sind Peptid- und Protein-Konjugate der allgemeinen Formel (I) The method according to the invention also comprises the preparation of multiple conjugates of a peptide or protein of the formula (II) with the alkyne derivative (IV) according to the above-described reaction sequence in cases where in the peptide or protein of the formula (II) more of such Conjugation accessible disulfide bridges are present. Another object of the present invention are peptide and protein conjugates of the general formula (I)
in welcher in which
S1 und S2 vormals in einer Disulfid-Brücke gebundene Cystein-Schwefelatome eines Peptids oder Proteins darstellen, S 1 and S 2 are previously in a disulfide bridge bound cysteine sulfur atoms of a peptide or protein,
R1 und R2 unabhängig voneinander für Wasserstoff, Alkyl, Cycloalkyl, Heterocycloalkyl, Aryl, Heteroaryl, Hydroxy, Alkoxy, Amino, Alkylamino, Dialkylamino, Hydroxycarbonyl, Alkoxycarbonyl, Alkylcarbonylamino oder Alkoxycarbonylamino stehen, wobei Alkyl, Cycloalkyl, Heterocycloalkyl, Aryl, Heteroaryl, Alkoxy, Alkylamino, Dialkylamino, Alkoxycarbonyl, Alkylcarbonylamino und Alkoxycarbonylamino ihrerseits ein- oder mehrfach, gleich oder verschieden, mit Halogen, Hydroxy, Alkoxy, Amino, Alkylamino, Dialkylamino, Hydroxycarbonyl, Alkoxycarbonyl, Alkylcarbonylamino und Alkoxycarbonylamino substituiert sein können, R 1 and R 2 independently of one another represent hydrogen, alkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, hydroxyl, alkoxy, amino, alkylamino, dialkylamino, hydroxycarbonyl, alkoxycarbonyl, alkylcarbonylamino or alkoxycarbonylamino, where alkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, Alkoxy, alkylamino, dialkylamino, alkoxycarbonyl, alkylcarbonylamino and alkoxycarbonylamino in turn may be mono- or polysubstituted, identically or differently, by halogen, hydroxyl, alkoxy, amino, alkylamino, dialkylamino, hydroxycarbonyl, alkoxycarbonyl, alkylcarbonylamino and alkoxycarbonylamino,
A für eine Bindung oder eine Kohlenwasserstoffkette mit 1 bis 100 Kohlenstoffatomen aus Alkylen-, Cycloalkylen- und/oder Arylengruppen steht, die ein- oder mehrfach, gleich oder verschieden, durch eine Gruppe ausgewählt aus -O-, -S-, -S(=0)-, -S(=0)2-, -NH-, -N(CH3)-, -C(=0)-, -NH-C(=0)-, -C(=0)-NH-, -0-C(=0)-, -C(=0)-0-, -S02-NH-, -NH-SO2-, -NH-NH-, -SO2-NH-NH-, -NH-NH-SO2-, -C(=0)-NH-NH-, -NH-NH-C(=0)-, -NH-C(=0)-NH-, -0-C(=0)-NH-, -NH-C(=0)-0- und einem 4- bis 10-gliedrigen, aromatischen oder nicht-aromatischen Heterocyclus mit bis zu 4 Heteroatomen aus der Reihe N, O, S, S(=0) und/oder S(=0)2 unterbrochen sein kann, oder A is a bond or a hydrocarbon chain having 1 to 100 carbon atoms of alkylene, cycloalkylene and / or arylene groups which are mono- or polysubstituted, identically or differently, by a group selected from -O-, -S-, -S ( = 0) -, -S (= 0) 2-, -NH-, -N (CH 3 ) -, -C (= 0) -, -NH-C (= 0) -, -C (= 0) -NH-, -O-C (= O) -, -C (= O) -O-, -SO 2 -NH-, -NH-SO 2 -, -NH-NH-, -SO 2 -NH-NH- , -NH-NH-SO 2, -C (= O) -NH-NH-, -NH-NH-C (= O) -, -NH-C (= O) -NH-, -O-C ( = 0) -NH-, -NH-C (= O) -O- and a 4- to 10-membered, aromatic or non-aromatic heterocycle having up to 4 heteroatoms from the series N, O, S, S (= 0) and / or S (= 0) 2 may be interrupted, or
R2 und A miteinander verknüpft sind und zusammen mit den Kohlenstoffatomen, die sich zwischen ihnen befinden, einen 8-gliedrigen Carbocyclus bilden, welcher mit einem 3- bis 6-gliedrigen Cycloalkyl-Ring anelliert sein kann, wobei der 8-gliedrige Carbocyclus und gegebenenfalls der anellierte Cycloalkyl-Ring ein- oder mehrfach, gleich oder verschieden, mit Fluor, Alkyl, Hydroxy, Hydroxyalkyl und Alkoxy substituiert sein können, R 2 and A are linked together and, together with the carbon atoms between them, form an 8-membered carbocycle which may be fused to a 3- to 6-membered cycloalkyl ring, the 8-membered carbocycle and optionally the fused cycloalkyl ring may be monosubstituted or polysubstituted, identically or differently, by fluorine, alkyl, hydroxy, hydroxyalkyl and alkoxy,
L für eine Bindung oder einen Linker steht, L is a bond or a linker,
X n-fach vorhanden sein kann und ein Wirkstoffmolekül, Polymer, Alkaloid, Peptid, Protein, Kohlenhydrat, Nucleotid, Nucleosid, Steroid, Terpen, Porphyrin, Chlorin, Corrin, Eico- sanoid, Pheromon, Vitamin, Biotin, ein Farbstoffmolekül oder einen Kryptanden darstellt oder für Wasserstoff, Hydroxy, Alkoxy, Amino, Alkylamino, Dialkylamino, Hydroxy- carbonyl, Alkoxycarbonyl, Alkylcarbonylamino, Alkoxycarbonylamino, Alkyl, Cycloalkyl, Heterocycloalkyl, Aryl oder Heteroaryl steht, wobei Alkyl seinerseits ein- oder mehrfach, gleich oder verschieden, mit Halogen, Hydroxy, Alkoxy, Amino, Alkylamino, Dialkylamino, Hydroxycarbonyl, Alkoxycarbonyl, Alkylcarbonylamino und Alkoxycarbonylamino substituiert sein kann und wobei Cycloalkyl, Heterocycloalkyl, Aryl und Heteroaryl ihrerseits ein- oder mehrfach, gleich oder verschieden, mit Halogen, Alkyl, Hydroxy, Alkoxy, Amino, Alkylamino, Dialkylamino, Hydroxycarbonyl, Alkoxycarbonyl, Alkylcarbonylamino und Alkoxycarbo- nylamino substituiert sein können, und n für eine ganze Zahl im Bereich von 1 bis einschließlich 10 steht, wobei im Fall, dass die Gruppe X mehrfach vorhanden ist, ihre individuellen Bedeutungen gleich oder verschieden sein können. Die vorliegende Erfindung umfasst bei Peptiden oder Proteinen mit mehreren der erfindungsgemäßen Konjugationsmethode zugänglichen Disulfidbrücken auch entsprechende Mehrfach- Konjugate eines solchen Peptids oder Proteins, d.h. Konjugate, bei denen eine paarweise C2- Verbrückung im Sinne der Formel (I) an mehreren Positionen des betreffenden Vorläuferpeptids oder -proteins erfolgt ist. Im Rahmen der vorliegenden Erfindung haben die Substituenten und Reste, soweit nicht anders spezifiziert, die folgende Bedeutung: X can be present n times and a drug molecule, polymer, alkaloid, peptide, protein, carbohydrate, nucleotide, nucleoside, steroid, terpene, porphyrin, chlorin, corrin, eicosanoid, pheromone, vitamin, biotin, a dye molecule or a cryptand represents or represents hydrogen, hydroxy, alkoxy, amino, alkylamino, dialkylamino, hydroxy carbonyl, alkoxycarbonyl, alkylcarbonylamino, alkoxycarbonylamino, alkyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl, where alkyl itself is mono- or polysubstituted, identically or differently, by halogen, hydroxyl, alkoxy, amino, alkylamino, dialkylamino, hydroxycarbonyl, alkoxycarbonyl, alkylcarbonylamino and Alkoxycarbonylamino can be substituted and wherein cycloalkyl, heterocycloalkyl, aryl and heteroaryl in turn mono- or polysubstituted, identically or differently, by halogen, alkyl, hydroxy, alkoxy, amino, alkylamino, dialkylamino, hydroxycarbonyl, alkoxycarbonyl, alkylcarbonylamino and alkoxycarbonyl-nylamino , and n is an integer in the range of 1 to 10, inclusive, and in case that the group X is multiple, their individual meanings may be the same or different. The present invention also encompasses corresponding multiple conjugates of such a peptide or protein, ie conjugates, in which a pairwise C2-bridging in the sense of formula (I) at several positions of the respective precursor peptide, or peptides or proteins with several disulfide bridges accessible by the conjugation method according to the invention protein is done. In the context of the present invention, the substituents and radicals, unless specified otherwise, have the following meaning:
Alkyl steht im Rahmen der Erfindung für einen geradkettigen oder verzweigten Alkylrest mit 1 bis 10, bevorzugt 1 bis 8, besonders bevorzugt 1 bis 6 Kohlenstoffatomen. Beispielhaft und vorzugsweise seien genannt: Methyl, Ethyl, n-Propyl, Isopropyl, n-Butyl, wo-Butyl, sec.-Butyl, ieri.-Butyl, n-Pentyl, Isopentyl, 1-Ethylpropyl, 1-Methylbutyl, 2-Methylbutyl, 3-Methylbutyl, Neopentyl, n-Hexyl, 1-Methylpentyl, 2-Methylpentyl, 3-Methylpentyl, 4-Methylpentyl, 3,3-Dimethylbutyl, 1-Ethylbutyl, 2-Ethylbutyl, n-Heptyl, n-Octyl, n-Nonyl und n-Decyl. Alkyl in the context of the invention is a straight-chain or branched alkyl radical having 1 to 10, preferably 1 to 8, particularly preferably 1 to 6 carbon atoms. Examples which may be mentioned by way of example and are preferably: methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, 1-ethylpropyl, 1-methylbutyl, 2 Methylbutyl, 3-methylbutyl, neopentyl, n-hexyl, 1-methylpentyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 3,3-dimethylbutyl, 1-ethylbutyl, 2-ethylbutyl, n-heptyl, n-octyl, n-nonyl and n-decyl.
Alkylen steht im Rahmen der Erfindung für einen geradkettigen oder verzweigten, divalenten Alkylrest (Alkandiyl-Rest) mit 1 bis 10, bevorzugt 1 bis 8, besonders bevorzugt 1 bis 6 Kohlen- Stoffatomen. Beispielhaft und vorzugsweise seien genannt: Methylen, Ethan-l,l-diyl, Ethan-1,2- diyl (1,2-Ethylen), Propan-l,l-diyl, Propan-l,2-diyl, Propan-2,2-diyl, Propan-l,3-diyl (1,3-Pro- pylen), Butan- 1,2-diyl, Butan-l,3-diyl, Butan-2,3-diyl, Butan- 1,4-diyl (1,4-Butylen), Pentan-1,5- diyl (1,5-Pentylen), Hexan- 1,6-diyl (1,6-Hexylen), Heptan-l,7-diyl (1,7-Hexylen), Octan- 1,8 -diyl (1,8-Octylen), Nonan-l,9-diyl (1,9-Nonylen) und Decan-l,10-diyl (1,10-Decylen). Hydroxyalkyl steht im Rahmen der Erfindung für einen geradkettigen oder verzweigten Alkylrest mit 1 bis 6, bevorzugt 1 bis 4 Kohlenstoffatomen, der innerhalb der Kette oder endständig eine Hydroxy-Gruppe als Substituenten trägt. Beispielhaft und vorzugsweise seien genannt: Hydroxy- methyl, 1-Hydroxyethyl, 2-Hydroxyethyl, 1 -Hydro xy-l-methylethyl, l,l-Dimethyl-2-hydroxyethyl, 1-Hydroxypropyl, 2-Hydroxypropyl, 3-Hydroxypropyl, l-Hydroxy-2-methylpropyl, 2-Hydroxy- 1-methylpropyl, 2-Hydroxy-2-methylpropyl, 1-Hydroxybutyl, 2-Hydroxybutyl, 3-Hydroxybutyl, 4- Hydroxybutyl, 5-Hydroxypentyl und 6-Hydroxyhexyl. Alkylene in the context of the invention is a straight-chain or branched, divalent alkyl radical (alkanediyl radical) having 1 to 10, preferably 1 to 8, particularly preferably 1 to 6 carbon atoms. By way of example and preferably mention may be made of: methylene, ethane-1, 1-diyl, ethane-1,2- diyl (1,2-ethylene), propan-1, 1-diyl, propan-1, 2-diyl, propan-2,2-diyl, propan-1,3-diyl (1,3-propylene), Butane-1,2-diyl, butane-l, 3-diyl, butane-2,3-diyl, butane-1,4-diyl (1,4-butylene), pentane-1,5-diyl (1.5 Pentylene), hexane-1,6-diyl (1,6-hexylene), heptane-1,17-diyl (1,7-hexylene), octane-1,8-diyl (1,8-octylene), nonane -l, 9-diyl (1,9-nonylene) and decane-1, 10-diyl (1,10-decylene). In the context of the invention, hydroxyalkyl is a straight-chain or branched alkyl radical having 1 to 6, preferably 1 to 4, carbon atoms which carries a hydroxy group as substituent within the chain or terminally. Examples which may be mentioned by way of example are: hydroxymethyl, 1-hydroxyethyl, 2-hydroxyethyl, 1-hydroxy-1-methylethyl, 1,1-dimethyl-2-hydroxyethyl, 1-hydroxypropyl, 2-hydroxypropyl, 3-hydroxypropyl, 1 Hydroxy-2-methylpropyl, 2-hydroxy-1-methylpropyl, 2-hydroxy-2-methylpropyl, 1-hydroxybutyl, 2-hydroxybutyl, 3-hydroxybutyl, 4-hydroxybutyl, 5-hydroxypentyl and 6-hydroxyhexyl.
Alkoxy steht im Rahmen der Erfindung für einen geradkettigen oder verzweigten Alkoxyrest mit 1 bis 6, bevorzugt 1 bis 4 Kohlenstoffatomen. Beispielhaft und vorzugsweise seien genannt: Meth- oxy, Ethoxy, n-Propoxy, Isopropoxy, n-Butoxy, wo-Butoxy, sec.-Butoxy, feri.-Butoxy, «-Pentoxy, Isopentoxy, 1-Ethylpropoxy, 1-Methylbutoxy, 2-Methylbutoxy, 3-Methylbutoxy und n-Hexoxy. Alkoxy in the context of the invention is a straight-chain or branched alkoxy radical having 1 to 6, preferably 1 to 4, carbon atoms. Examples which may be mentioned by way of example and with preference are: methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, where-butoxy, sec-butoxy, ferric. Butoxy, pentoxy, isopentoxy, 1-ethylpropoxy, 1-methylbutoxy, 2-methylbutoxy, 3-methylbutoxy and n-hexoxy.
Alkoxycarbonyl steht im Rahmen der Erfindung für einen geradkettigen oder verzweigten Alkoxyrest mit 1 bis 6, bevorzugt 1 bis 4 Kohlenstoffatomen, der über eine an das O-Atom gebundene Carbonyl-Gruppe [-C(=0)-] mit dem Rest des Moleküls verknüpft ist. Beispielhaft und vorzugsweise seien genannt: Methoxycarbonyl, Ethoxycarbonyl, n-Propoxycarbonyl, Isopropoxycarbonyl, n-Butoxycarbonyl und ieri.-Butoxycarbonyl. In the context of the invention, alkoxycarbonyl is a straight-chain or branched alkoxy radical having 1 to 6, preferably 1 to 4, carbon atoms which is linked to the rest of the molecule via a carbonyl group [-C (0O) -] bound to the oxygen atom is. By way of example and preferably mention may be made of: methoxycarbonyl, ethoxycarbonyl, n-propoxycarbonyl, isopropoxycarbonyl, n-butoxycarbonyl and ieri.-butoxycarbonyl.
Alkylamino steht im Rahmen der Erfindung für eine Amino-Gruppe mit einem geradkettigen oder verzweigten Alkylsubstituenten, der 1 bis 6, bevorzugt 1 bis 4 Kohlenstoffatome aufweist. Beispielhaft und vorzugsweise seien genannt: Methylamino, Ethylamino, n-Propylamino, Isopropyl- amino, n-Butylamino und ieri.-Butylamino. Dialkylamino steht im Rahmen der Erfindung für eine Amino-Gruppe mit zwei gleichen oder verschiedenen geradkettigen oder verzweigten Alkylsubstituenten, die jeweils 1 bis 6, bevorzugt 1 bis 4 Kohlenstoffatome aufweisen. Beispielhaft und vorzugsweise seien genannt: -Dimefhylamino, -Diefhylamino, -Ethyl- -mefhylamino, -Methyl- -n-propylamino, /V-Isopropyl- -methyl- amino, -rsopropyl- -n-propylamino, /V,/V-Diisopropylamino, -n-Butyl- -mefhylamino und N-ieri.-Butyl-N-methylamino. Alkylamino in the context of the invention represents an amino group having a straight-chain or branched alkyl substituent which has 1 to 6, preferably 1 to 4, carbon atoms. By way of example and by way of preference: methylamino, ethylamino, n-propylamino, isopropylamino, n-butylamino and ieri.-butylamino. Dialkylamino is in the context of the invention an amino group having two identical or different straight-chain or branched alkyl substituents, each having 1 to 6, preferably 1 to 4 carbon atoms. Examples which may be mentioned by way of example and by way of preference are: -dimethylamino, -dibylamino, -ethylmethylamino, -methyl-n-propylamino, / V-isopropylmethylamino, -sopropyl-n-propylamino, / V, / V- Diisopropylamino, n-butyl-methyl-amino and N-ethyl-butyl-N-methylamino.
Alkoxycarbonylamino steht im Rahmen der Erfindung für eine Amino-Gruppe mit einem geradkettigen oder verzweigten Alkoxycarbonyl-Substituenten, der 1 bis 6, bevorzugt 1 bis 4 Kohlenstoffatome im Alkoxyrest aufweist und über die Carbonylgruppe mit dem N-Atom verknüpft ist. Beispielhaft und vorzugsweise seien genannt: Methoxycarbonylamino, Ethoxycarbonylamino, «-Propoxycarbonylamino, Isopropoxycarbonylamino, n-Butoxycarbonylamino und ieri.-Butoxy- carbonylamino. Alkoxycarbonylamino in the context of the invention represents an amino group having a straight-chain or branched alkoxycarbonyl substituent which has 1 to 6, preferably 1 to 4, carbon atoms in the alkoxy radical and is linked via the carbonyl group to the nitrogen atom. Examples which may be mentioned by preference include: methoxycarbonylamino, ethoxycarbonylamino, -propoxycarbonylamino, isopropoxycarbonylamino, n-butoxycarbonylamino and ieri.-butoxycarbonylamino.
Alkylcarbonyl steht im Rahmen der Erfindung für einen geradkettigen oder verzweigten Alkylrest mit 1 bis 6, bevorzugt 1 bis 4 Kohlenstoffatomen, der über eine Carbonyl-Gruppe [-C(=0)-] mit dem Rest des Moleküls verknüpft ist. Beispielhaft und vorzugsweise seien genannt: Acetyl, Propionyl, n-Butyryl, wo-Butyryl, n-Pentanoyl und Pivaloyl. In the context of the invention, alkylcarbonyl is a straight-chain or branched alkyl radical having 1 to 6, preferably 1 to 4, carbon atoms which is linked to the rest of the molecule via a carbonyl group [-C (= O) -]. Examples which may be mentioned are: acetyl, propionyl, n-butyryl, where-butyryl, n-pentanoyl and pivaloyl.
Alkylcarbonylamino steht im Rahmen der Erfindung für eine Amino-Gruppe mit einem geradkettigen oder verzweigten Alkylcarbonyl-Substituenten, der 1 bis 6, bevorzugt 1 bis 4 Kohlenstoff- atome im Alkylrest aufweist und über die Carbonylgruppe mit dem N-Atom verknüpft ist. Beispielhaft und vorzugsweise seien genannt: Acetylamino, Propionylamino, n-Butyrylamino, iso- Butyrylamino, n-Pentanoylamino und Pivaloylamino. Alkylcarbonylamino in the context of the invention is an amino group having a straight-chain or branched alkylcarbonyl substituent which has 1 to 6, preferably 1 to 4 carbon atoms in the alkyl radical and is linked via the carbonyl group to the nitrogen atom. Examples which may be mentioned are: acetylamino, propionylamino, n-butyrylamino, isobutyrylamino, n-pentanoylamino and pivaloylamino.
Cycloalkyl steht im Rahmen der Erfindung für einen monocyclischen, gesättigten Carbocyclus mit 3 bis 10, bevorzugt 3 bis 8, besonders bevorzugt 3 bis 6 Ring-Kohlenstoffatomen. Beispielhaft und vorzugsweise seien genannt: Cyclopropyl, Cyclobutyl, Cyclopentyl, Cyclohexyl, Cycloheptyl, Cyclooctyl, Cyclononyl und Cyclodecyl. Cycloalkyl in the context of the invention is a monocyclic, saturated carbocycle having 3 to 10, preferably 3 to 8, particularly preferably 3 to 6 ring carbon atoms. Examples which may be mentioned by way of example include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl and cyclodecyl.
Cycloalkylen steht im Rahmen der Erfindung für einen monocyclischen, gesättigten, divalenten Cycloalkyl-Rest (Cycloalkandiyl-Rest) mit 3 bis 10, bevorzugt 3 bis 8, besonders bevorzugt 3 bis 6 Ring-Kohlenstoffatomen. Beispielhaft und vorzugsweise seien genannt: Cyclopropan-l,l-diyl, Cyclopropan-l,2-diyl, Cyclobutan-l,l-diyl, Cyclobutan-l,2-diyl, Cyclobutan-l,3-diyl, Cyclo- pentan-l,l-diyl, Cyclopentan-l,2-diyl, Cyclopentan- l,3-diyl, Cyclohexan-l,l-diyl, Cyclohexan- 1,2-diyl, Cyclohexan-l,3-diyl, Cyclohexan-l,4-diyl, Cycloheptan-l,l-diyl, Cycloheptan-l,2-diyl, Cycloheptan-l,4-diyl, Cyclooctan-l,2-diyl, Cyclooctan-l,5-diyl, Cyclononan-l,2-diyl, Cyclo- nonan-l,5-diyl, Cyclodecan-l,2-diyl und Cyclodecan-l,6-diyl. Heterocycloalkyl steht im Rahmen der Erfindung für einen 4- bis 10-gliedrigen, mono- oder gegebenenfalls bicyclischen, gesättigten oder eine Doppelbindung enthaltenden, nicht-aromatischen Heterocyclus mit insgesamt 4 bis 10 Ringatomen, der bis zu vier Ring-Heteroatome aus der Reihe N, O, S, S(=0) und/oder S(=0)2 enthält und über ein Ring-Kohlenstoffatom oder gegebenenfalls ein Ring-Stickstoffatom verknüpft ist. Beispielhaft seien genannt: Azetidinyl, Oxetanyl, Thietanyl, Pyrrolidinyl, Pyrrolinyl, Pyrazolidinyl, Dihydropyrazolyl, Tetrahydrofuranyl, Thiolanyl, 1,1-Di- oxidothiolanyl, 1,3-Oxazolidinyl, 1,3-Thiazolidinyl, Piperidinyl, Tetrahydropyridyl, Piperazinyl, Tetrahydropyranyl, Dihydropyranyl, Tetrahydrothiopyranyl, 1,3-Dioxanyl, 1,4-Dioxanyl, Morpho- linyl, Thiomorpholinyl, 1,1-Dioxidothiomorpholinyl, Hexahydroazepinyl, Hexahydro-l,4-di- azepinyl, Octahydroazocinyl, Octahydropyrrolo[3,4-b]pyrrolyl, Octahydroisoindolyl, Octahydro- pyrrolo[3,4-b]pyridyl, Hexahydropyrrolo[3,4-c]pyridyl, Octahydropyrrolo[l,2-a]pyrazinyl, Deca- hydroisochinolinyl, Octahydropyrido[l,2-a]pyrazinyl, 7-Azabicyclo[2.2.1]heptanyl, 3-Azabicyclo- [3.2.0]heptanyl, 3-Azabicyclo[3.2.1]octanyl, 8-Azabicyclo[3.2.1]octanyl und 8-Oxa-3-azabicyclo- [3.2.1]octanyl. Bevorzugt ist ein 4- bis 6-gliedriger, monocyclischer, gesättigter Heterocyclus mit insgesamt 4 bis 6 Ringatomen, der ein oder zwei Ring-Heteroatome aus der Reihe N, O und/oder S enthält und über ein Ring-Kohlenstoffatom oder gegebenenfalls ein Ring-Stickstoffatom verknüpft ist. Beispielhaft seien genannt: Azetidinyl, Oxetanyl, Thietanyl, Pyrrolidinyl, Pyrazolidinyl, Tetra- hydrofuranyl, Thiolanyl, 1,3-Oxazolidinyl, Piperidinyl, Piperazinyl, Tetrahydropyranyl, Tetra- hydrothiopyranyl, 1,4-Dioxanyl, Morpholinyl und Thiomorpholinyl. Cycloalkylene in the context of the invention is a monocyclic, saturated, divalent cycloalkyl radical (cycloalkanediyl radical) having 3 to 10, preferably 3 to 8, particularly preferably 3 to 6 ring carbon atoms. Examples which may be mentioned by way of example and are preferably: cyclopropan-1, 1-diyl, cyclopropan-1, 2-diyl, cyclobutane-1,1-diyl, cyclobutane-1,2-diyl, cyclobutane-1,3-diyl, cyclopentane l, l-diyl, cyclopentane-l, 2-diyl, cyclopentane, 3-diyl, cyclohexane-l, l-diyl, cyclohexane-1,2-diyl, cyclohexane-l, 3-diyl, cyclohexane-l, 4-diyl, cycloheptan-1, 1-diyl, cycloheptan-1, 2-diyl, cycloheptan-1, 4-diyl, cyclooctan-1, 2-diyl, cyclooctan-1, 5-diyl, cyclononan-1, 2 diyl, cyclo-nonane-l, 5-diyl, cyclodecane-l, 2-diyl and cyclodecane-l, 6-diyl. Heterocycloalkyl is in the context of the invention for a 4- to 10-membered, mono- or optionally bicyclic, saturated or a double bond-containing, non-aromatic heterocycle having a total of 4 to 10 ring atoms, up to four ring heteroatoms from the series N, O, S, S (= 0) and / or S (= 0) 2 and is linked via a ring carbon atom or optionally a ring nitrogen atom. Examples which may be mentioned are: azetidinyl, oxetanyl, thietanyl, pyrrolidinyl, pyrrolinyl, pyrazolidinyl, dihydropyrazolyl, tetrahydrofuranyl, thiolanyl, 1,1-diaminothiolanyl, 1,3-oxazolidinyl, 1,3-thiazolidinyl, piperidinyl, tetrahydropyridyl, piperazinyl, tetrahydropyranyl, Dihydropyranyl, tetrahydrothiopyranyl, 1,3-dioxanyl, 1,4-dioxanyl, morpholinyl, thiomorpholinyl, 1,1-dioxideothiomorpholinyl, hexahydroazepinyl, hexahydro-1,4-dihydroxypropyl azepinyl, octahydroazocinyl, octahydropyrrolo [3,4-b] pyrrolyl, octahydroisoindolyl, octahydro-pyrrolo [3,4-b] pyridyl, hexahydropyrrolo [3,4-c] pyridyl, octahydropyrrolo [1,2-a] pyrazinyl, deca- hydroisoquinolinyl, octahydropyrido [1,2-a] pyrazinyl, 7-azabicyclo [2.2.1] heptanyl, 3-azabicyclo [3.2.0] heptanyl, 3-azabicyclo [3.2.1] octanyl, 8-azabicyclo [3.2.1 ] octanyl and 8-oxa-3-azabicyclo [3.2.1] octanyl. Preference is given to a 4- to 6-membered, monocyclic, saturated heterocycle having a total of 4 to 6 ring atoms which contains one or two ring heteroatoms from the series N, O and / or S and via a ring carbon atom or optionally a ring Nitrogen atom is linked. Examples which may be mentioned are: azetidinyl, oxetanyl, thietanyl, pyrrolidinyl, pyrazolidinyl, tetrahydrofuranyl, thiolanyl, 1,3-oxazolidinyl, piperidinyl, piperazinyl, tetrahydropyranyl, tetrahydrothiopyranyl, 1,4-dioxanyl, morpholinyl and thiomorpholinyl.
Aryl steht im Rahmen der Erfindung für einen aromatischen Carbocyclus mit 6 oder 10 Ring- Kohlenstoffatomen wie Phenyl und Naphthyl. Aryl in the context of the invention is an aromatic carbocycle having 6 or 10 ring carbon atoms, such as phenyl and naphthyl.
Arylen steht im Rahmen der Erfindung für einen divalenten Arylrest wie beispielsweise 1,2- Phenylen, 1,3-Phenylen, 1,4-Phenylen, Naphthalin-2,3-diyl, Naphthalin- 1,4-diyl, Naphthalin- 1,5- diyl, Naphthalin-2,6-diyl und Naphthalin-l,8-diyl. Arylene in the context of the invention is a divalent aryl radical, for example 1,2-phenylene, 1,3-phenylene, 1,4-phenylene, naphthalene-2,3-diyl, naphthalene-1,4-diyl, naphthalene-1, 5-diyl, naphthalene-2,6-diyl and naphthalene-1,8-diyl.
Heteroaryl steht im Rahmen der Erfindung für einen 5- bis 10-gliedrigen, mono- oder gegebenenfalls bicyclischen aromatischen Heterocyclus (Heteroaromaten) mit insgesamt 5 bis 10 Ringatomen, der bis zu vier Ring-Heteroatome aus der Reihe N, O und/oder S enthält und über ein Ring-Kohlenstoffatom oder gegebenenfalls ein Ring-Stickstoffatom verknüpft ist. Beispielhaft seien genannt: Furyl, Pyrrolyl, Thienyl, Pyrazolyl, Imidazolyl, Thiazolyl, Oxazolyl, Isoxazolyl, Isothiazolyl, Triazolyl, Oxadiazolyl, Thiadiazolyl, Tetrazolyl, Pyridyl, Pyrimidinyl, Pyridazinyl, Pyrazinyl, Triazinyl, Benzofuranyl, Benzothienyl, Benzimidazolyl, Benzoxazolyl, Benzothiazolyl, Benzotriazolyl, Indolyl, Indazolyl, Chinolinyl, Isochinolinyl, Naphthyridinyl, Chinazolinyl, Chinoxalinyl, Phthalazinyl und Pyrazolo[3,4-b]pyridinyl. Bevorzugt ist ein 5- oder 6-gliedriger, monocyclischer Heteroaryl-Rest mit insgesamt 5 oder 6 Ringatomen, der bis zu drei Ring-Heteroatome aus der Reihe N, O und/oder S enthält und über ein Ring-Kohlenstoffatom oder gegebenenfalls ein Ring-Stickstoffatom verknüpft ist. Beispielhaft seien genannt: Furyl, Thienyl, Thiazolyl, Oxazolyl, Isothiazolyl, Isoxazolyl, Pyrazolyl, Imidazolyl, Triazolyl, Oxadiazolyl, Thiadiazolyl, Pyridyl, Pyrimidinyl, Pyridazinyl, Pyrazinyl und Triazinyl. Halogen schließt im Rahmen der Erfindung Fluor, Chlor, Brom und Iod ein. Bevorzugt sind Chlor, Fluor oder Brom, besonders bevorzugt Fluor oder Chlor. Heteroaryl is in the context of the invention for a 5- to 10-membered, mono- or optionally bicyclic aromatic heterocycle (heteroaromatic) having a total of 5 to 10 ring atoms containing up to four ring heteroatoms from the series N, O and / or S. and linked via a ring carbon atom or optionally a ring nitrogen atom. Examples which may be mentioned are: furyl, pyrrolyl, thienyl, pyrazolyl, imidazolyl, thiazolyl, oxazolyl, isoxazolyl, isothiazolyl, triazolyl, oxadiazolyl, thiadiazolyl, tetrazolyl, pyridyl, pyrimidinyl, pyridazinyl, pyrazinyl, triazinyl, benzofuranyl, benzothienyl, benzimidazolyl, benzoxazolyl, benzothiazolyl, Benzotriazolyl, indolyl, indazolyl, quinolinyl, isoquinolinyl, naphthyridinyl, quinazolinyl, quinoxalinyl, phthalazinyl and pyrazolo [3,4-b] pyridinyl. Preference is given to a 5- or 6-membered, monocyclic heteroaryl radical having a total of 5 or 6 ring atoms and containing up to three ring heteroatoms from the series N, O and / or S and being bonded via a ring carbon atom or optionally a ring atom. Nitrogen atom is linked. Examples which may be mentioned are: furyl, thienyl, thiazolyl, oxazolyl, isothiazolyl, isoxazolyl, pyrazolyl, imidazolyl, triazolyl, oxadiazolyl, thiadiazolyl, pyridyl, pyrimidinyl, pyridazinyl, pyrazinyl and triazinyl. Halogen in the context of the invention includes fluorine, chlorine, bromine and iodine. Preference is given to chlorine, fluorine or bromine, more preferably fluorine or chlorine.
Im Rahmen der vorliegenden Erfindung gilt, dass für alle Reste, die mehrfach auftreten, deren Bedeutung unabhängig voneinander ist. Wenn Reste in den erfindungsgemäßen Verbindungen sub- stituiert sind, können die Reste, soweit nicht anders spezifiziert, ein- oder mehrfach substituiert sein. Eine Substitution mit einem oder mit zwei oder drei gleichen oder verschiedenen Substitu- enten ist bevorzugt. Besonders bevorzugt ist die Substitution mit einem oder mit zwei gleichen oder verschiedenen Substituenten. Ganz besonders bevorzugt ist die Substitution mit einem Substi- tuenten. In the context of the present invention, the meaning is independent of each other for all radicals which occur repeatedly. When radicals in the compounds according to the invention are present Unless otherwise specified, the radicals may be monosubstituted or polysubstituted. Substitution with one or two or three identical or different substituents is preferred. Particularly preferred is the substitution with one or two identical or different substituents. Very particular preference is given to the substitution with a substituent.
Ein "Linker" stellt im Sinne der vorliegenden Erfindung eine Kohlenwasserstoffkette mit 1 bis 100 Kohlenstoffatomen aus Alkylen-, Cycloalkylen- und/oder Arylengruppen dar, die ein- oder mehrfach, gleich oder verschieden, durch eine Gruppe ausgewählt aus -O-, -S-, -S(=0)-, -S(=0)2-, -NH-, -N(CH3)-, -C(=0)-, -NH-C(=0)-, -C(=0)-NH-, -0-C(=0)-, -C(=0)-0-, -S02-NH-, -NH-S02-, -NH-NH-, -SO2-NH-NH-, -NH-NH-SO2-, -C(=0)-NH-NH-, -NH-NH-C(=0)-, -NH-C(=0)-NH-, -0-C(=0)-NH-, -NH-C(=0)-0- und einem 4- bis 10-gliedrigen, aromatischen oder nicht-aromatischen Heterocyclus mit bis zu 4 Heteroatomen aus der Reihe N, O, S, S(=0) und/oder S(=0)2 sowie durch eine in vivo spaltbare Gruppe oder durch eine in vivo transient stabile Gruppe unterbrochen sein kann. Der Begriff "in vivo spaltbare Gruppe" kann unterteilt werden in Gruppen, die auf chemischem Wege in vivo spaltbar sind (z.B. durch saure Hydrolyse oder Redox-Prozesse), und in solche, die auf enzymatische Weise, d.h. unter dem Einfluss eines körpereigenen Enzyms, in vivo spaltbar sind. Beide Arten von spaltbaren Gruppen sollten im Blutkreislauf zunächst stabil sein und erst an oder in der Zielzelle durch die dort veränderte chemische bzw. enzymatische Umgebung (z.B. niedrigerer pH-Wert, erhöhte Glutathion-Konzentration, Anwesenheit lysosomaler Enzyme wie Cathepsin oder Plasmin) gespalten werden. Als chemisch in vivo spaltbare Strukturfragmente eignen sich für diesen Zweck insbesondere Disulfid-, Hydrazon-, Acetal- und Aminal-Gruppierungen. Enzymatisch in vivo spaltbare Strukturelemente sind insbesondere Oligopeptid-Einheiten aus 2 bis 8 Aminosäuren und hier vor allem Dipeptid-Gruppierungen. Solche designierten Peptid-Spaltstel- len sind in zahlreicher Form in der Literatur beschrieben. Prominente Beispiele sind die Dipeptid- Einheiten Valin-Alanin, Valin-Lysin, Valin-Citrullin, Alanin-Lysin und Phenylalanin-Lysin [siehe z.B. J. J. Petersen und C. F. Meares, Bioconjugate Chem. 9, 618-626 (1998); G. M. Dubowchik und R. A. Firestone, Bioorg. Med. Chem. Lett. 8, 3341-3346 (1998); G. M. Dubowchik et al, Bioconjugate Chem. 13, 855-869 (2002)] . Der oben beschriebene Linker kann auch eine in vivo transient stabile Gruppe enthalten. Solche transient stabilen Gruppen werden unter dem Einfluss der chemischen bzw. enzymatischen Umgebung beispielsweise im Blutkreislauf über einen längeren Zeitraum (von Stunden oder Tagen) hinweg gespalten [siehe z.B. Flamme et al., Int. Pat. Appl. WO 2013/064455-A1]. Als Wirkstoffmoleküle in der obigen Definition der Gruppe X kommen insbesondere Arzneimittel- Wirkstoffe zur Krebstherapie, wie Zytotoxine und Zytostatika, in Betracht. Im Detail vermögen diese Wirkstoffe die Krebszelle zu schädigen, den programmierten Zelltod einzuleiten und/oder das Zellwachstum und die Zellvermehrung zu unterbinden. Als solche Wirkstoffe zur Krebsthera- pie sind beispielsweise zu nennen: Antimetabolite wie Methotrexat, Cladribin, Fludarabin, Mer- captopurin, Tioguanin, Pentostatin, Cytarabin, Fluorouracil, Capecitabin und Gemcitabin, alkylie- rende Stoffe wie Cyclophosphamid, Ifosfamid, Mitomycin, Trofosfamid, Thiotepa, Busulfan, Treosulfan, Carmustin, Lomustin, Nimustin, Procarbazin, Dacarbazin und die Platin- Verbindungen Cisplatin, Carboplatin und Oxaliplatin, Topoisomerase-Hemmstoffe wie Topotecan, Irinotecan, Etoposid und Teniposid, Kinase-Inhibitoren wie Sorafenib, Regorafinib, Sunitinib, Afatinib, Erlo- tinib und Gefitinib, Mitose-Hemmstoffe wie Vinblastin, Vincristin, Vinorelbin, Paclitaxol und Docetaxel sowie Antibiotika wie Dactinomycin, Daunorubicin, Doxorubicin, Idarubicin, Epiru- bicin, Bleomycin, Mitoxantron und Amsacrin. A "linker" in the context of the present invention represents a hydrocarbon chain having 1 to 100 carbon atoms from alkylene, cycloalkylene and / or arylene groups which are mono- or polysubstituted, identically or differently, by a group selected from -O-, -S -, -S (= O) -, -S (= O) 2-, -NH-, -N (CH 3 ) -, -C (= O) -, -NH-C (= O) -, - C (= O) -NH-, -O-C (= O) -, -C (= O) -O-, -SO 2 -NH-, -NH-SO 2 -, -NH-NH-, - SO2-NH-NH-, -NH-NH-SO2-, -C (= O) -NH-NH-, -NH-NH-C (= O) -, -NH-C (= O) -NH- , -O-C (= O) -NH-, -NH-C (= O) -O- and a 4- to 10-membered, aromatic or non-aromatic heterocycle having up to 4 heteroatoms from the series N, O. , S, S (= 0) and / or S (= 0) 2 and may be interrupted by an in vivo cleavable group or by an in vivo transient stable group. The term "in vivo cleavable group" can be subdivided into groups which are cleavable in vivo chemically (eg by acid hydrolysis or redox processes) and those which are enzymatically isolated, ie under the influence of an endogenous enzyme, are cleavable in vivo. Both types of cleavable groups should initially be stable in the circulation and only at or in the target cell by the altered there chemical or enzymatic environment (eg lower pH, increased glutathione concentration, presence of lysosomal enzymes such as cathepsin or plasmin) are cleaved. Suitable structural fragments which can be cleaved chemically in vivo are in particular disulfide, hydrazone, acetal and aminal groups for this purpose. Enzymatic cleavable structural elements in particular are oligopeptide units of 2 to 8 amino acids and especially dipeptide groups. Such designated peptide cleavage sites are described in numerous forms in the literature. Prominent examples are the dipeptide units valine-alanine, valine-lysine, valine-citrulline, alanine-lysine and phenylalanine-lysine [see eg JJ Petersen and CF Meares, Bioconjugate Chem. 9, 618-626 (1998); GM Dubowchik and RA Firestone, Bioorg. Med. Chem. Lett. 8, 3341-3346 (1998); GM Dubowchik et al, Bioconjugate Chem. 13, 855-869 (2002)]. The linker described above may also contain an in vivo transient stable group. Such transiently stable groups are cleaved under the influence of the chemical or enzymatic environment, for example in the bloodstream over a relatively long period of time (of hours or days) [see, eg, Flamme et al., Int. Pat. Appl. WO 2013/064455-A1]. As active ingredient molecules in the above definition of the group X, in particular pharmaceutical active ingredients for cancer therapy, such as cytotoxins and cytostatics into consideration. In detail, these agents can damage the cancer cell, initiate programmed cell death, and / or inhibit cell growth and cell proliferation. Examples of such agents for cancer treatment are: antimetabolites such as methotrexate, cladribine, fludarabine, mercaptopurine, tioguanine, pentostatin, cytarabine, fluorouracil, capecitabine and gemcitabine, alkylating substances such as cyclophosphamide, ifosfamide, mitomycin, trofosfamide, thiotepa , Busulfan, treosulfan, carmustine, lomustine, nimustine, procarbazine, dacarbazine and the platinum compounds cisplatin, carboplatin and oxaliplatin, topoisomerase inhibitors such as topotecan, irinotecan, etoposide and teniposide, kinase inhibitors such as sorafenib, regorafinib, sunitinib, afatinib, Erlo - tinib and gefitinib, mitotic inhibitors such as vinblastine, vincristine, vinorelbine, paclitaxol and docetaxel, as well as antibiotics such as dactinomycin, daunorubicin, doxorubicin, idarubicin, epirubicin, bleomycin, mitoxantrone and amsacrine.
Weitere vom Bedeutungsumfang der Gruppe X erfasste Wirkstoffe stellen zelltoxische Verbindun- gen und Toxine dar, wie sie experimentell und klinisch als "Toxophore" in Antikörper -Wirkstoff- Konjugaten (ADCs) insbesondere für die Krebstherapie untersucht werden. Beispielhaft zu nennen sind hier insbesondere Substanzen wie Maytansin und Maytansinoide (DM-1, DM -4), Dolastatine, Auristatine (MMAE, MMAF), Calicheamicine, Duocarmycine, Camptothecine (Topotecan, Exa- tecan, Irinotecan, SN-38), Doxorubicin, Amatoxine (Amanitin), Pyrrolobenzodiazepine (PBDs) und Kinesin-Spindel-Protein (KSP)-Inhibitoren. Other compounds covered by Group X include cytotoxic compounds and toxins, as experimentally and clinically tested as "toxophore" in antibody-drug conjugates (ADCs), especially for cancer therapy. Examples include, in particular, substances such as maytansine and maytansinoids (DM-1, DM-4), dolastatins, auristatins (MMAE, MMAF), calicheamicins, duocarmycins, camptothecins (topotecan, exatecan, irinotecan, SN-38), doxorubicin , Amatoxins (amanitin), pyrrolobenzodiazepines (PBDs) and kinesin spindle protein (KSP) inhibitors.
Gemäß der vorliegenden Erfindung enthalten die Peptide und Proteine der Formel (II) mindestens zwei Cystein-Aminosäuren, die eine Disulfid-Bindung ausbilden oder dazu befähigt sind. Zu solchen Peptiden zählen beispielsweise Peptidhormone wie Insulin, Somatostatin, Oxitocin, Terli- pressin, Adrenomedullin, Calcitonin oder Vasopressin. Als Proteine dieser Art seien beispielhaft Antikörper, Cytokine wie Interleukine sowie Albumine genannt. According to the present invention, the peptides and proteins of formula (II) contain at least two cysteine amino acids which form or are capable of disulfide bonding. Such peptides include, for example, peptide hormones such as insulin, somatostatin, oxitocin, terli- pressin, adrenomedullin, calcitonin or vasopressin. Examples of proteins of this type are antibodies, cytokines such as interleukins and albumins.
Der Begriff "Antikörper" wird gemäß der vorliegenden Erfindung in seinem breitesten Sinne verstanden und bezeichnet Immunglobulinmoleküle, beispielsweise intakte oder modifizierte monoklonale Antikörper, polyklonale Antikörper oder multispezifische Antikörper (z.B. bispezifische Antikörper), sowie Fragmente hiervon. Ein Immunglobulinmolekül stellt bevorzugt ein Molekül mit vier Polypeptidketten dar, bestehend aus zwei schweren Ketten (H-Ketten, HC) und zwei leichten Ketten (L-Ketten, LC), welche typischerweise durch Disulfidbrücken (sogenannte inter- c^am-Disulfidbrücken) miteinander verknüpft sind. Jede schwere Kette umfasst eine variable Domäne (abgekürzt VH) und eine konstante Domäne (CH). Die konstante Domäne der schweren Kette ihrerseits kann drei (CHI, CH2, CH3) oder vier Unter-Domänen besitzen. Jede leichte Kette umfasst ebenfalls eine variable Domäne (VL) und eine konstante Domäne (CL)- Die VH- und VL-Domänen können weiter unterteilt werden in Regionen mit Hypervariabilität, auch komplementaritäts- bestimmende Regionen genannt ("complementarity determining region", CDR), und in Regionen mit geringerer Sequenzvariabilität ("framework region", FR). Jede VH- und VL-Region setzt sich typischerweise aus drei CDRs und bis zu vier FRs zusammen, beispielsweise vom Amino- zum Carboxy-Terminus in der Reihenfolge FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Ein Antikörper kann aus jeder dafür geeigneten Spezies erhalten werden, z.B. Affe, Schwein, Kaninchen, Maus oder Ratte. In einer besonderen Ausführungsform ist der Antikörper humanen oder murinen Ursprungs. Ein solcher Antikörper kann z.B. human, humanisiert oder chimär sein. Weiterer Gegenstand der vorliegenden Erfindung ist die Verwendung der Konjugate der Formel (I) zur Diagnose und/oder Behandlung von Erkrankungen, insbesondere zur Diagnose und/oder Behandlung von Krebs- und Tumorerkrankungen. The term "antibody" is understood in its broadest sense according to the present invention and refers to immunoglobulin molecules, for example intact or modified monoclonal antibodies, polyclonal antibodies or multispecific antibodies (eg bispecific antibodies), as well as fragments thereof. An immunoglobulin molecule preferably represents a molecule having four polypeptide chains, consisting of two heavy chains (H chains, HC) and two light chains (L chains, LC), which are typically linked together by disulfide bridges (so-called intercam-disulfide bridges) are linked. Each heavy chain comprises a variable domain (abbreviated VH) and a constant domain (CH). The heavy chain constant domain in turn may have three (CHI, CH2, CH3) or four subdomains. Each light chain includes also a variable domain (VL) and a constant domain (CL) - The VH and VL domains can be further subdivided into regions of hypervariability, also called complementarity determining regions (CDRs), and in regions with lower sequence variability ("framework region", FR). Each VH and VL region is typically composed of three CDRs and up to four FRs, for example, from the amino to the carboxy terminus in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. An antibody can be obtained from any suitable species, eg monkey, pig, rabbit, mouse or rat. In a particular embodiment, the antibody is of human or murine origin. Such an antibody may be, for example, human, humanized or chimeric. Another object of the present invention is the use of the conjugates of the formula (I) for the diagnosis and / or treatment of diseases, in particular for the diagnosis and / or treatment of cancer and tumor diseases.
Weiterer Gegenstand der vorliegenden Erfindung ist die Verwendung der Konjugate der Formel (I) in einem Verfahren zur Diagnose und/oder Behandlung von Erkrankungen, insbesondere von Krebs- und Tumorerkrankungen. Another object of the present invention is the use of the conjugates of the formula (I) in a method for the diagnosis and / or treatment of diseases, in particular cancer and tumor diseases.
Weiterer Gegenstand der vorliegenden Erfindung ist ein Verfahren zur Diagnose und/oder Behandlung von Erkrankungen, insbesondere von Krebs- und Tumorerkrankungen, unter Verwendung eines oder mehrerer Konjugate der Formel (I). Another object of the present invention is a method for the diagnosis and / or treatment of diseases, in particular cancer and tumor diseases, using one or more conjugates of the formula (I).
Der Begriff "Behandlung" oder "behandeln" umfasst im Sinne der vorliegenden Erfindung ein Hemmen, Verzögern, Aufhalten, Lindern, Abschwächen, Einschränken, Verringern, Unterdrücken, Zurückdrängen oder Heilen einer Krankheit, eines Leidens, einer Erkrankung, einer Verletzung oder einer gesundheitlichen Störung, der Entfaltung, des Verlaufs oder des Fortschreitens solcher Zustände und/oder der Symptome solcher Zustände. Der Begriff "Therapie" wird hierbei als synonym mit dem Begriff "Behandlung" verstanden. Der Begriff "Diagnose" wird im Rahmen der vorliegenden Erfindung im üblichen Sinne verstanden als (unterscheidende) Erkennung, Feststellung, Bestimmung, Beurteilung, Zuordnung und Benennung einer Krankheit, eines Leidens, einer Erkrankung, eines Krankheitssymptoms, einer Verletzung oder einer gesundheitlichen Störung. For the purposes of the present invention, the term "treatment" or "treating" includes inhibiting, delaying, arresting, alleviating, reducing, restricting, reducing, suppressing, restraining or curing a disease, a disease, a disease, an injury or a health disorder , the unfolding, the course or progression of such conditions and / or the symptoms of such conditions. The term "therapy" is understood to be synonymous with the term "treatment". The term "diagnosis" in the context of the present invention in the usual sense understood as (discriminating) detection, detection, determination, assessment, classification and naming of a disease, a disease, a disease, a disease symptom, an injury or a health disorder.
Weiterer Gegenstand der vorliegenden Erfindung sind pharmazeutische Zusammensetzungen, die mindestens eines der Konjugate der Formel (I), üblicherweise zusammen mit einem oder mehreren inerten, nicht-toxischen, pharmazeutisch geeigneten Hilfsstoffen, enthalten, sowie deren Verwendung zu den zuvor genannten Zwecken. Die erfindungsgemäßen Konjugate der Formel (I) können systemisch und/oder lokal wirken. Zu diesem Zweck können sie auf geeignete Weise appliziert werden, wie z.B. oral, parenteral, pulmonal, nasal, sublingual, lingual, buccal, rectal, dermal, transdermal, conjunctival, otisch oder als Implantat oder Stent. Für diese Applikationswege können die erfindungsgemäßen Konjugate in geeigneten Applikationsformen verabreicht werden. Another object of the present invention are pharmaceutical compositions containing at least one of the conjugates of formula (I), usually together with one or more inert, non-toxic, pharmaceutically suitable excipients, and their use for the purposes mentioned above. The conjugates of the formula (I) according to the invention can act systemically and / or locally. For this purpose, they may be applied in a suitable manner, such as, for example, orally, parenterally, pulmonarily, nasally, sublingually, lingually, buccally, rectally, dermally, transdermally, conjunctivally, otically or as an implant or stent. For these administration routes, the conjugates according to the invention can be administered in suitable administration forms.
Für die orale Applikation eignen sich nach dem Stand der Technik funktionierende, die erfindungsgemäßen Konjugate schnell und/oder modifiziert abgebende Applikationsformen, die die erfindungsgemäßen Konjugate in kristalliner und/oder amorphisierter und/oder gelöster Form enthal- ten, wie z.B. Tabletten (nicht-überzogene oder überzogene Tabletten, beispielsweise mit magensaftresistenten oder sich verzögert auflösenden oder unlöslichen Überzügen, die die Freisetzung der erfindungsgemäßen Konjugate kontrollieren), in der Mundhöhle schnell zerfallende Tabletten oder Filme/Oblaten, Filme/Lyophylisate, Kapseln (beispielsweise Hart- oder Weichgelatinekapseln), Dragees, Granulate, Pellets, Pulver, Emulsionen, Suspensionen, Aerosole oder Lösungen. Die parenterale Applikation kann unter Umgehung eines Resorptionsschrittes geschehen (z.B. intravenös, intraarteriell, intrakardial, intraspinal oder intralumbal) oder unter Einschaltung einer Resorption (z.B. intramuskulär, subcutan, intracutan, percutan oder intraperitoneal). Für die parenterale Applikation eignen sich als Applikationsformen u.a. Injektions- und Infusionszubereitungen in Form von Lösungen, Suspensionen, Emulsionen, Lyophilisaten oder sterilen Pulvern. Für die sonstigen Applikationswege eignen sich z.B. Inhalationsarzneiformen (u.a. Pulverinhalatoren, Nebulizer), Nasentropfen, -lösungen oder -sprays, lingual, sublingual oder buccal zu applizierende Tabletten, Filme/Oblaten oder Kapseln, Suppositorien, Ohren- oder Augenpräparationen, Vaginalkapseln, wässrige Suspensionen (Lotionen, Schüttelmixturen), lipophile Suspensionen, Salben, Cremes, transdermale therapeutische Systeme (z.B. Pflaster), Milch, Pasten, Schäume, Streupuder, Implantate oder Stents. For oral administration, the prior art uses rapidly and / or modified delivery forms which contain the conjugates according to the invention in crystalline and / or amorphized and / or dissolved form, e.g. Tablets (uncoated or coated tablets, for example with enteric or delayed-release or insoluble coatings controlling the release of the conjugates of the invention), tablets or films / wafers rapidly breaking down in the oral cavity, films / lyophilisates, capsules (e.g. Soft gelatin capsules), dragees, granules, pellets, powders, emulsions, suspensions, aerosols or solutions. Parenteral administration can be accomplished by bypassing a resorption step (e.g., intravenously, intraarterially, intracardially, intraspinal, or intralumbar) or by resorting to absorption (e.g., intramuscularly, subcutaneously, intracutaneously, percutaneously, or intraperitoneally). For parenteral administration are suitable as application forms u.a. Injection and infusion preparations in the form of solutions, suspensions, emulsions, lyophilisates or sterile powders. For the other routes of administration are suitable, for example Inhalation medicaments (including powder inhalers, nebulizers), nasal drops, solutions or sprays, lingual, sublingual or buccal tablets, films / wafers or capsules, suppositories, ear or ophthalmic preparations, vaginal capsules, aqueous suspensions (lotions, shake mixtures), lipophilic suspensions , Ointments, creams, transdermal therapeutic systems (eg patches), milk, pastes, foams, powdered powders, implants or stents.
Bevorzugt ist die parenterale Applikation, insbesondere die intravenöse Applikation. Preference is given to parenteral administration, in particular intravenous administration.
Die erfindungsgemäßen Konjugate können in die angeführten Applikationsformen überführt werden. Dies kann in an sich bekannter Weise durch Mischen mit inerten, nichttoxischen, pharmazeutisch geeigneten Hilfsstoffen geschehen. Zu diesen Hilfsstoffen zählen u.a. Trägerstoffe (bei- spielsweise mikrokristalline Cellulose, Lactose, Mannitol), Lösungsmittel (z.B. flüssige Poly- ethylenglycole), Emulgatoren und Dispergier- oder Netzmittel (beispielsweise Natriumdodecyl- sulfat, Polyoxysorbitanoleat), Bindemittel (beispielsweise Polyvinylpyrrolidon), synthetische und natürliche Polymere (beispielsweise Albumin), Stabilisatoren (z.B. Antioxidantien wie beispiels- weise Ascorbinsäure), Farbstoffe (z.B. anorganische Pigmente wie beispielsweise Eisenoxide) und Geschmacks- und/oder Geruchskorrigentien. The conjugates according to the invention can be converted into the stated administration forms. This can be done in a conventional manner by mixing with inert, non-toxic, pharmaceutically suitable excipients. These excipients include, among others, excipients (for example microcrystalline cellulose, lactose, mannitol), solvents (for example liquid polyethylene glycols), emulsifiers and dispersants or wetting agents (for example sodium dodecyl sulfate, polyoxysorbitanoleate), binders (for example polyvinylpyrrolidone), synthetic and natural polymers (eg albumin), stabilizers (eg antioxidants such as Ascorbic acid), dyes (eg, inorganic pigments such as iron oxides) and flavor and / or odoriferous.
Die nachfolgenden Ausführungsbeispiele erläutern die Erfindung. Die Erfindung ist nicht auf die Beispiele beschränkt. The following embodiments illustrate the invention. The invention is not limited to the examples.
A. Beispiele A. Examples
Abkürzungen und Akronyme: abs. absolut, von absoluter Reinheit  Abbreviations and acronyms: abs. absolutely, of absolute purity
aq. wässrig, wässrige Lösung aq. aqueous, aqueous solution
Boc ieri.-Butoxycarbonyl  Boc ieri-butoxycarbonyl
c Konzentration c concentration
ca. circa, ungefähr about circa, about
DMF N, N-Dimethylformamid  DMF N, N-dimethylformamide
DMPA 2,2-Dimethoxy-2-phenylacetophenon  DMPA 2,2-dimethoxy-2-phenylacetophenone
DMSO Dimethylsulfoxid  DMSO dimethyl sulfoxide
DPBS engl. Dulbecco's phosphate buffered saline  DPBS engl. Dulbecco's phosphate buffered saline
d. Th. der Theorie (bei chemischer Ausbeute) d. Th. Of theory (in chemical yield)
DTT Dithiothreitol  DTT dithiothreitol
ELSD Lichtstreudetektor (engl, evaporative light scattering detector)  ELSD light scattering detector (evaporative light scattering detector)
eq. Äquivalent(e) eq. Equivalent (s)
ES oder ESI Elektrospray-Ionisation (bei MS)  ES or ESI electrospray ionization (in MS)
FAB engl, fragment antigen-binding  FAB engl, fragment antigen-binding
h Stunde(n) h hour (s)
HPLC Hochdruck-, Hochleistungsflüssigchromatographie  HPLC high pressure, high performance liquid chromatography
HRMS hochaufgelöste Massenspektrometrie  HRMS high-resolution mass spectrometry
Irgacure® 819 Bis(2,4,6-trimethylbenzoyl)phenylphosphinoxid Irgacure ® 819 bis (2,4,6-trimethylbenzoyl) phenylphosphine
konz. konzentriert (bei Lösung) conc. concentrated (at solution)
LAP Lithium-phenyl-2,4,6-trimethylbenzoylphosphinat  LAP lithium phenyl-2,4,6-trimethylbenzoylphosphinate
LC/MS Flüssigchromatographie-gekoppelte Massenspektrometrie  LC / MS liquid chromatography-coupled mass spectrometry
LED Licht-emittierende Diode  LED light-emitting diode
Lit. Literatur(stelle)  Literature (position)
m Multiplett (bei NMR) m multiplet (by NMR)
m/z Masse zu Ladung- Verhältnis (bei MS) min Minute(n) m / z mass to charge ratio (in MS) min minute (s)
MPLC Mitteldruckflüssigchromatographie (über Kieselgel; auch "flash- MPLC medium pressure liquid chromatography (over silica gel; also "flash"
Chromatographie" genannt) Called chromatography ")
MS Massenspektrometrie  MS mass spectrometry
NMR Kernresonanzspektrometrie  NMR nuclear magnetic resonance spectrometry
PBS engl, phosphate buffered saline  PBS English, phosphate buffered saline
RP reverse phase (Umkehrphase, bei HPLC)  RP reverse phase (reversed phase, for HPLC)
RT Raumtemperatur  RT room temperature
Rt Retentionszeit (bei HPLC, LC/MS) R t retention time (for HPLC, LC / MS)
TCEP-HC1 Tris(2-carboxyethyl)phosphin-Hydrochlorid  TCEP-HC1 tris (2-carboxyethyl) phosphine hydrochloride
tert. tertiär tert. tertiary
TFA Trifluoressigsäure  TFA trifluoroacetic acid
THF Tetrahydrofuran  THF tetrahydrofuran
TOF engl, time-of-flight (Flugzeit)-Massenspektrometrie  TOF English time-of-flight mass spectrometry
UV Ultraviolett(-Spektrometrie)  UV ultraviolet (spectrometry)
v/v Volumen zu Volumen- Verhältnis (einer Lösung) v / v volume to volume ratio (of a solution)
LC/MS-Methoden: LC / MS methods:
Methode 1 : Method 1:
Instrument: Waters Acquity SQD UPLC System; Säule: Waters Acquity UPLC HSS T3 1.8 μιη, 50 x 1 mm; Eluent A: 1 1 Wasser + 0.25 ml 99%-ige Ameisensäure, Eluent B: 1 1 Acetonitril + 0.25 ml 99%-ige Ameisensäure; Gradient: 0.0 min 95% A -> 6.0 min 5% A -> 7.5 min 5% A; Ofen: 50°C; Fluss: 0.35 ml/min; UV-Detektion: 210-400 nm.  Instrument: Waters Acquity SQD UPLC System; Column: Waters Acquity UPLC HSS T3 1.8 μιη, 50 x 1 mm; Eluent A: 1 l of water + 0.25 ml of 99% formic acid, eluent B: 1 l of acetonitrile + 0.25 ml of 99% formic acid; Gradient: 0.0 min 95% A -> 6.0 min 5% A -> 7.5 min 5% A; Oven: 50 ° C; Flow: 0.35 ml / min; UV detection: 210-400 nm.
Methode 2: Method 2:
Gerätetyp MS: Waters Synapt G2S; Gerätetyp UPLC: Waters Acquity I-Class; Säule: Waters HSS T3, 2.1 x 50 mm, C18 1.8 μιη; Eluent A: 1 1 Wasser + 0.01 % Ameisensäure, Eluent B: 1 1 Acetonitril + 0.01 % Ameisensäure; Gradient: 0.0 min 2% B -> 2.0 min 2% B -> 13.0 min 90% B -> 15.0 min 90% B; Ofen: 50°C; Fluss: 1.20 ml/min; UV-Detektion: 210 nm.  Device Type MS: Waters Synapt G2S; Device type UPLC: Waters Acquity I-Class; Column: Waters HSS T3, 2.1 x 50 mm, C18 1.8 μιη; Eluent A: 1: 1 water + 0.01% formic acid, eluent B: 1: 1 acetonitrile + 0.01% formic acid; Gradient: 0.0 min 2% B -> 2.0 min 2% B -> 13.0 min 90% B -> 15.0 min 90% B; Oven: 50 ° C; Flow: 1.20 ml / min; UV detection: 210 nm.
Methode 3: Method 3:
Gerätetyp MS: Thermo Fisher Scientific LTQ-Orbitrap-XL; Gerätetyp HPLC: Agilent 1200SL; Säule: Agilent Poroshell 120 SB-C18 2.7 μιη, 3 x 150 mm; Eluent A: 1 1 Wasser + 0.1% Trifluoressigsäure, Eluent B: 1 1 Acetonitril + 0.1% Trifluoressigsäure; Gradient: 0.0 min 2% B— > 1.5 min 2% B -> 15.5 min 95% B -> 18.0 min 95% B; Ofen: 40°C; Fluss: 0.75 ml/min; UV-Detektion: 210 nm. Device Type MS: Thermo Fisher Scientific LTQ-Orbitrap-XL; Device type HPLC: Agilent 1200SL; Column: Agilent Poroshell 120 SB-C18 2.7 μιη, 3 x 150 mm; Eluent A: 1: 1 water + 0.1% trifluoroacetic acid, eluent B: 1: 1 acetonitrile + 0.1% trifluoroacetic acid; Gradient: 0.0 min 2% B-> 1.5 min 2% B -> 15.5 min 95% B -> 18.0 min 95% B; Oven: 40 ° C; Flow: 0.75 ml / min; UV detection: 210 nm.
Ausgangsverbindungen : Verbindung A Starting compounds: Compound A
N-(2,5,8,l l,14,17,20,23-Octaoxapentacosan-25-yl)hex-5-inamid N- (2,5,8,11,14,17,20,23-octaoxapentacosan-25-yl) hex-5-ynamide
27.8 μΐ (0.25 mmol) Hex-5-insäure und 48.3 mg (0.25 mmol) Ι,Γ-Carbonyldiimidazol wurden unter einer Argonatmosphäre in 1 ml absolutem DMF vorgelegt. Das Gemisch wurde 2 h bei RT gerührt. Anschließend wurden 100.0 mg (0.25 mmol) 2,5,8,1 l,14,17,20,23-Octaoxapentacosan-25- amin, gelöst in 0.5 ml abs. DMF, hinzugefügt. Das Gemisch wurde über Nacht bei RT gerührt und dann bei reduziertem Druck eingeengt. Der Rückstand wurde mittels präparativer MPLC gereinigt. Die produkthaltigen Fraktionen wurden eingeengt, der Rückstand wurde mit 10 ml Wasser versetzt und dann dreimal mit jeweils 10 ml Essigsäureethylester extrahiert. Die vereinigten organischen Phasen wurden über Magnesiumsulfat getrocknet, im Vakuum eingeengt und der Rückstand im Hochvakuum getrocknet (Ausbeute an Produkt: 22 mg). Die zuvor erhaltene wässrige Phase wurde lyophilisiert und das Lyophilisat durch Kieselgel-Chromatographie gereinigt (Eluent: zunächst Di- chlormethan/Methanol 100:5, dann Dichlormethan/Methanol 9: 1). Es wurden 37.8 mg eines transparenten Öls erhalten. Gesamtausbeute: 59.8 mg (51 % d. Th.)  27.8 μΐ (0.25 mmol) of hex-5-acetic acid and 48.3 mg (0.25 mmol) of Ι, Γ-carbonyldiimidazole were placed under an argon atmosphere in 1 ml of absolute DMF. The mixture was stirred for 2 h at RT. Subsequently, 100.0 mg (0.25 mmol) of 2,5,8,11,14,17,20,23-octaoxapentacosan-25-amine dissolved in 0.5 ml of abs. DMF, added. The mixture was stirred at RT overnight and then concentrated under reduced pressure. The residue was purified by preparative MPLC. The product-containing fractions were concentrated, the residue was admixed with 10 ml of water and then extracted three times with 10 ml of ethyl acetate each time. The combined organic phases were dried over magnesium sulfate, concentrated in vacuo and the residue was dried under high vacuum (yield of product: 22 mg). The aqueous phase previously obtained was lyophilized and the lyophilizate was purified by silica gel chromatography (eluent: first dichloromethane / methanol 100: 5, then dichloromethane / methanol 9: 1). There were obtained 37.8 mg of a transparent oil. Total yield: 59.8 mg (51% of theory)
Ή-NMR (400 MHz, CDC13): δ [ppm] = 1.87 (quin, J = 7.1 Hz, 2H), 1.99 (t, J = 2.5 Hz, 1H), 2.26 (td, J = 6.9 und 2.7 Hz, 2H), 2.32 (t, J = 7.4 Hz, 2H), 3.38 (s, 3H), 3.45 (q, J = 5.4 Hz, 2H), 3.55 (m, 4H), 3.60-3.71 (m, 26H), 6.18 (br. s, 1H). Ή NMR (400 MHz, CDC1 3 ): δ [ppm] = 1.87 (quin, J = 7.1 Hz, 2H), 1.99 (t, J = 2.5 Hz, 1H), 2.26 (td, J = 6.9 and 2.7 Hz , 2H), 2.32 (t, J = 7.4 Hz, 2H), 3.38 (s, 3H), 3.45 (q, J = 5.4 Hz, 2H), 3.55 (m, 4H), 3.60-3.71 (m, 26H) , 6.18 (brs s, 1H).
13C-NMR (125.78 MHz, CDC13): δ [ppm] = 172.21, 83.61, 71.93, 70.60, 70.56, 70.53, 70.51, 70.25, 69.87, 69.12, 59.03, 39.18, 35.00, 24.18, 17.88. Verbindung B 13 C-NMR (125.78 MHz, CDC1 3): δ [ppm] = 172.21, 83.61, 71.93, 70.60, 70.56, 70.53, 70.51, 70.25, 69.87, 69.12, 59.03, 39.18, 35.00, 24.18, 17.88. Compound B
4-(Hex-5-in-l-yl)-4-methylmorpholiri-4-iumiodid 4- (Hex-5-yne-l-yl) -4-4-ium iodide methylmorpholiri-
3.0 ml (22.7 mmol) 6-Iodhex-l-in wurden zu 2.5 ml (22.7 mmol) 4-Methylmorpholin getropft. Das Gemisch wurde 5 Minuten bei RT und anschließend 10 Minuten bei 54°C gerührt. Danach wurde auf RT abgekühlt, 16 h nachgerührt und dann mit geringen Mengen Petrolether, Diethylether und zuletzt Essigsäureethylester versetzt. Der Niederschlag wurde abfiltriert und das Filtrat bei vermindertem Druck aufkonzentriert. Der daraus resultierende Niederschlag wurde abfiltriert und mit Essigsäureethylester gewaschen. Das weisse bis beigefarbene Pulver wurde im Hochvakuum ge- trocknet. Ausbeute: 593.6 mg (9% d. Th.). 3.0 ml (22.7 mmol) of 6-iodohex-1-yl were added dropwise to 2.5 ml (22.7 mmol) of 4-methylmorpholine. The mixture was stirred at RT for 5 minutes and then at 54 ° C. for 10 minutes. It was then cooled to RT, stirred for 16 h and then treated with small amounts of petroleum ether, diethyl ether and finally ethyl acetate. The precipitate was filtered off and the filtrate concentrated at reduced pressure. The resulting precipitate was filtered off and washed with ethyl acetate. The white to beige powder was dried in a high vacuum. Yield: 593.6 mg (9% of theory).
Ή-NMR (400 MHz, D20): δ [ppm] = 1.62 (quin, J = 7.3 Hz, 2H), 1.95 (m, 2H), 2.33 (td, / = 7 und 2.6 Hz, 2H), 2.41 (t, J = 2.6 Hz, 1H), 3.21 (s, 3H), 3.46-3.60 (m, 6H), 4.06 (br. s, 4H). Ή NMR (400 MHz, D 2 O): δ [ppm] = 1.62 (quin, J = 7.3 Hz, 2H), 1.95 (m, 2H), 2.33 (td, / = 7 and 2.6 Hz, 2H), 2.41 (t, J = 2.6 Hz, 1H), 3.21 (s, 3H), 3.46-3.60 (m, 6H), 4.06 (br, s, 4H).
13C-NMR (125.78 MHz, D20): δ [ppm] = 84.64, 70.01, 66.59, 60.42, 59.64, 59.58, 24.23, 20.13, 17.11. 13 C-NMR (125.78 MHz, D 2 O): δ [ppm] = 84.64, 70.01, 66.59, 60.42, 59.64, 59.58, 24.23, 20.13, 17.11.
MS (ESpos): m/z MS (ESpos): m / z
Ausführungsbeispiele : Beispiel 1 Exemplary embodiments: Example 1
(5R, 12R)- 12-{ [(Benzyloxy)carbonyl] amino } -5-carboxy-8-(8-carboxyoctyl)-3-oxo-l -phenyl-2-oxa- 7,10-dithia-4-azatridecan-13-säure (5R, 12R) - 12- {[(benzyloxy) carbonyl] amino} -5-carboxy-8- (8-carboxyctyl) -3-oxo-1-phenyl-2-oxa-7,10-dithia-4- azatridecane-13-oic acid
In einem Zweihalsrundkolben wurden unter einer Argonatmosphäre 100.00 mg (0.19 mmol) Ν,Ν'- Bis[(benzyloxy)carbonyl]-L-cystin und 79.47 mg (277.25 μιηοΐ) TCEP-HC1 in 1 ml Wasser/ Methanol (1 : 1 v/v) vorgelegt. Das Gemisch wurde 2.5 h bei RT gerührt. Anschließend wurden 33.69 mg (0.19 mmol) Undec-10-insäure sowie 3.87 mg (9.24 μιηοΐ) Irgacure® 819 hinzugefügt. Durch einen Schliff des Zweihalskolbens wurde ein LED-UV-Stift (OmniCure LX400, Durchmesser 12 mm; igb-tech GmbH, Deutschland) eingeführt (Abstand zum Reaktionsgemisch ca. 40 mm), und das Reaktionsgemisch wurde für 1 h mit 365 nm UV -Licht bestrahlt. Der Umsatz gemäß HPLC (Eluent: Gradient Acetonitril/Wasser + 0.1% TFA; ELSD) war quantitativ. Die Reaktions- lösung wurde danach mit Methanol verdünnt und mittels präparativer HPLC gereinigt (Eluent: Gradient Acetonitril/Wasser + 0.1 % TFA). Es wurden 39 mg (27% d. Th., LC/MS -Reinheit 88%) der Zielverbindung erhalten. 100.00 mg (0.19 mmol) of Ν, Ν'-bis [(benzyloxy) carbonyl] -L-cystine and 79.47 mg (277.25 μmol) of TCEP-HC1 in 1 ml of water / methanol (1: 1 v / v) were used in a two-necked round-bottomed flask under an argon atmosphere / v). The mixture was stirred at RT for 2.5 h. Then 33.69 mg (0:19 mmol) of undec-10-ynoic acid and 3.87 mg (9.24 μιηοΐ) Irgacure ® 819 added. By grinding the two-necked flask, an LED UV-stick (OmniCure LX400, diameter 12 mm, igb-tech GmbH, Germany) was introduced (distance to the reaction mixture about 40 mm), and the reaction mixture was exposed to 365 nm UV for 1 h. Light irradiated. The conversion by HPLC (eluent: gradient acetonitrile / water + 0.1% TFA, ELSD) was quantitative. The reaction solution was then diluted with methanol and purified by preparative HPLC (eluent: gradient acetonitrile / water + 0.1% TFA). 39 mg (27% of theory, LC / MS purity 88%) of the target compound were obtained.
LC/MS (Methode 1): Rt = 3.45 min; MS (ESIpos): m/z = 693 [M+H]+ LC / MS (Method 1): R t = 3.45 min; MS (ESIpos): m / z = 693 [M + H] +
Ή-NMR (400 MHz, DMSO-d6): δ [ppm] = 1.24-1.50 (m, 13H), 1.55-1.60 (m, 2H), 1.72-1.79 (m, 1H), 2.66-3.18 (m, 7H), 4.34-4.40 (m, 2H), 5.07-5.14 (m, 4H), 7.26-7.38 (m, 10H). Ή NMR (400 MHz, DMSO-d 6 ): δ [ppm] = 1.24-1.50 (m, 13H), 1.55-1.60 (m, 2H), 1.72-1.79 (m, 1H), 2.66-3.18 (m , 7H), 4.34-4.40 (m, 2H), 5.07-5.14 (m, 4H), 7.26-7.38 (m, 10H).
Beispiel 2A und 2B Example 2A and 2B
2A: 5-[(6R,9S, 125, 155, 185,2 lR)-21 - { [( { [( Aminoacetyl)amino] acetyl } amino)acetyl] amino } -6- { [(25)-2-( { (25)-6-amino- 1 - [(2-amino-2-oxoethyl)amino] - 1 -oxohexan-2-yl } carbamoyl)pyrrolidin- l-yl]carbonyl}-9-(2-amino-2-oxoethyl)-12-(3-amino-3-oxopropyl)-15-benzyl-18-(4-hydroxy- benzyl)-8,l l,14,17,20-pentaoxo-l,4-dithia-7,10,13,16,19-pentaazacyclodocosan-3-yl]pentansäure 2A: 5 - [(6R, 9S, 125, 155, 185.2 IR) -21 - {[({[(aminoacetyl) amino] acetyl} amino) acetyl] amino} -6- {[(25) -2 - ({(25) -6-amino-1 - [(2-amino-2-oxo-ethyl) -amino] -1-oxo-hexan-2-yl} -carbamoyl) -pyrrolidin-1-yl] -carbonyl} -9- (2- amino-2-oxoethyl) -12- (3-amino-3-oxopropyl) -15-benzyl-18- (4-hydroxybenzyl) -8,11,14,17,20-pentaoxo-1,4-dithia -7,10,13,16,19-pentaazacyclodocosan-3-yl] pentanoic acid
2B : 5-[(6R,9S,l2S, 155, 185,2 lR)-21 - { [( { [( Aminoacetyl)amino] acetyl } amino)acetyl] amino } -6- { [(25)-2-( { (25)-6-amino- 1 - [(2-amino-2-oxoethyl)amino] - 1 -oxohexan-2-yl } carbamoyl)pyrrolidin- l-yl]carbonyl}-9-(2-amino-2-oxoethyl)-12-(3-amino-3-oxopropyl)-15-benzyl-18-(4-hydroxy- benzyl)-8,l l,14,17,20-pentaoxo-l,4-dithia-7,10,13,16,19-pentaazacyclodocosan-2-yl]pentansäure 2B: 5 - [(6R, 9S, 12S, 155, 185.2 IR) -21 - {[({[(aminoacetyl) amino] acetyl} amino) acetyl] amino} -6- {[(25) -2 - ({(25) -6-amino-1 - [(2-amino-2-oxoethyl) amino] -1-oxohexan-2-yl} carbamoyl) pyrrolidine 1-yl] carbonyl} -9- (2-amino-2-oxoethyl) -12- (3-amino-3-oxopropyl) -15-benzyl-18- (4-hydroxybenzyl) -8,11,14 , 17,20-pentaoxo-l, 4-dithia-7,10,13,16,19-pentaazacyclodocosan-2-yl] pentanoic acid
2A: 2A:
2B: 2 B:
Unter einer Argonatmosphäre wurden in einem Zweihalskolben 50.17 mg (37.23 μιηοΐ) Terlipres- sin-Acetat (Fa. Bachem, Schweiz; Sequenz: H-Gly-Gly-Gly-Cys-Tyr-Phe-Gln-Asn-Cys-Pro-Lys- Gly-NEb., als cyclisches Cys-Disulfid) in 1.1 ml Wasser vorgelegt und mit 16.01 mg (55.85 μιηοΐ) TCEP-HCl versetzt. Das Reaktionsgemisch wurde 2 h bei RT gerührt und dann mit einer Lösung von 4.70 mg (37.23 mmol) Hept-6-insäure in 100 μΐ Methanol versetzt. Anschließend wurden 0.55 mg (1.86 μιηοΐ) LAP [hergestellt nach literaturbekanntem Verfahren (Gong et al., 2013)] in 150 μΐ Wasser hinzugefügt. Es wurde ein LED-UV-Stift (OmniCure LX400, Durchmesser 12 mm; igb-tech GmbH, Deutschland) durch einen Schliff des Zweihalskolbens eingeführt (Abstand zum Reaktionsgemisch ca. 40 mm), und das Reaktionsgemisch wurde für 1 h mit 365 nm UV-Licht bestrahlt. Danach wurden nochmals 0.55 mg (1.86 μιηοΐ) LAP hinzugefügt, und das Reaktionsge- misch wurde erneut für 1 h mit 365 nm UV-Licht bestrahlt. Das Reaktionsgemisch wurde dann unter Auftrennung der isomeren Produkte mittels präparativer HPLC gereinigt (Säule: Waters X-Bridge BEH130 Prep C18 10 μηι OBD, 19 x 250 mm; Eluent A: Wasser mit 0.05% TFA, Eluent B: Acetonitril mit 0.05% TFA; Gradient: 0.0 min 5% B -> 40 min 40% B). 50.17 mg (37.23 μιηοΐ) of terlipresin acetate (Bachem, Switzerland, sequence: H-Gly-Gly-Gly-Cys-Tyr-Phe-Gln-Asn-Cys-Pro-Lys.) Were dissolved in an argon atmosphere in a two-necked flask - Gly-NEb., As cyclic Cys disulfide) in 1.1 ml of water and treated with 16.01 mg (55.85 μιηοΐ) TCEP-HCl. The reaction mixture was stirred for 2 h at RT and then treated with a solution of 4.70 mg (37.23 mmol) of hept-6-acetic acid in 100 μΐ methanol. Subsequently, 0.55 mg (1.86 μιηοΐ) LAP [prepared by literature method (Gong et al., 2013)] in 150 μΐ of water added. An LED UV-pen (OmniCure LX400, diameter 12 mm, igb-tech GmbH, Germany) was introduced through a cut of the two-necked flask (distance to the reaction mixture about 40 mm), and the reaction mixture was UV for 365 h at 365 nm Light irradiated. Thereafter, another 0.55 mg (1.86 μmol) of LAP was added, and the reaction mixture was irradiated once again with 365 nm UV light for 1 h. The reaction mixture was then purified by separation of the isomeric products by preparative HPLC (column: Waters X-Bridge BEH130 Prep C18 10 μηι OBD, 19 × 250 mm, eluent A: water with 0.05% TFA, eluent B: acetonitrile with 0.05% TFA; Gradient: 0.0 min 5% B -> 40 min 40% B).
Isomer 1 : Isomer 1:
LC/MS (Methode 2): Rt = 3.53 min; MS (ESpos): m/z = 1355 [M+H]+. Isomer 2: LC / MS (Method 2): R t = 3.53 min; MS (ESpos): m / z = 1355 [M + H] + . Isomer 2:
Ausbeute: 1.3 mg (2.6% d. Th.)  Yield: 1.3 mg (2.6% of theory)
LC/MS (Methode 2): Rt = 3.57 min; MS (ESpos): m/z = 1355 [M+H]+ LC / MS (method 2): R t = 3.57 min; MS (ESpos): m / z = 1355 [M + H] +
13C-NMR (125.78 MHz, D20, δ (1,4-Dioxan) = 67.4 ppm): δ [ppm] = 183.1 (S), 182.1 (S), 178.5 (S), 175.2 (3S), 174.7 (S), 174.0 (S), 173.9 (S), 172.7 (S), 172.5 (S), 171.5 (S), 170.9, 168.7, 155.2 (S), 137.0 (S), 131.4 (S), 130.0 (S), 129.7 (S), 128.8 (S), 128.2 (S), 116.3 (S), 61.7 (D), 57.1 (D), 55.6 (D), 55.3 (D), 54.5 (D), 53.4 (D), 52.6 (D), 50.7 (D), 48.8 (T), 45.7 (D), 43.2 (T), 43.0 (T), 42.9 (T), 41.3 (T), 40.1 (T), 38.8 (T), 37.0 (T), 36.6 (T), 36.4 (2T), 34.0 (T), 33.6 (T), 31.9 (2T), 30.9 (T), 30.1 (T), 27.0 (T), 26.9 (T), 26.8 (T), 25.6 (T), 25.5 (T), 22.9 (T) [entspricht einer ring- geschlossenen Struktur, da keine olefinischen Kohlenstoff atome zu detektieren sind]. 13 C-NMR (125.78 MHz, D 2 0, δ (1,4-dioxane) = 67.4 ppm): δ [ppm] = 183.1 (S), 182.1 (S), 178.5 (S), 175.2 (3S), 174.7 (S), 174.0 (S), 173.9 (S), 172.7 (S), 172.5 (S), 171.5 (S), 170.9, 168.7, 155.2 (S), 137.0 (S), 131.4 (S), 130.0 (S), 129.7 (S), 128.8 (S), 128.2 (S), 116.3 (S), 61.7 (D), 57.1 (D), 55.6 (D), 55.3 (D), 54.5 (D), 53.4 (D), 52.6 (D), 50.7 (D), 48.8 (T), 45.7 (D), 43.2 (T), 43.0 (T), 42.9 (T), 41.3 (T), 40.1 (T), 38.8 (T), 37.0 (T), 36.6 (T), 36.4 (2T), 34.0 (T), 33.6 (T), 31.9 (2T), 30.9 (T), 30.1 (T), 27.0 (T), 26.9 (T), 26.8 (T), 25.6 (T), 25.5 (T), 22.9 (T) [corresponds to a closed-ring structure, since no olefinic carbon atoms are to be detected].
Das ^-NMR-Spektrum dieses Isomers ist in Abbildung 1 wiedergegeben. The ^ -NMR spectrum of this isomer is shown in Figure 1.
Beispiel 3A und 3B Example 3A and 3B
3A: (25)- 1 - { [(6R,95, 125, 155, 185,2 lR)-21 - { [( { [(Aminoacetyl)amino] acetyl } amino)acetyl] - amino}-9-(2-amino-2-oxoethyl)-12-(3-amino-3-oxopropyl)-15-benzyl-18-(4-hydroxybenzyl)- 8,l l,14,17,20-pentaoxo-3-(27-oxo-2,5,8,l l,14,17,20,23-octaoxa-26-azatriacontan-30-yl)-l,4-di- thia-7, 10, 13, 16, 19-pentaazacyclodocosan-6-yl]carbonyl } -N- { (25)-6-amino- 1 - [(2-amino-2-oxo- ethyl)amino] -1 -oxohexan-2-yl }pyrrolidin-2-carboxamid 3A: (25) - 1 - {[(6R, 95, 125, 155, 185.2 IR) -21 - {[({[(aminoacetyl) amino] acetyl} amino) acetyl] -amino} -9- ( 2-amino-2-oxoethyl) -12- (3-amino-3-oxopropyl) -15-benzyl-18- (4-hydroxybenzyl) -8,11,14,17,20-pentaoxo-3- (27- oxo-2,5,8,11,14,17,20,23-octaoxa-26-azatriacontan-30-yl) -1,4-di-thia-7,10,13,16,19-pentaazacyclodocosan-6 -yl] carbonyl} -N- {(25) -6-amino-1 - [(2-amino-2-oxo-ethyl) -amino] -1-oxo-hexan-2-yl} -pyrrolidine-2-carboxamide
3B : (25)- 1 - { [(6R,95, 125, 155, 185,2 lR)-21 - { [( { [(Aminoacetyl)amino] acetyl } amino)acetyl] - amino}-9-(2-amino-2-oxoethyl)-12-(3-amino-3-oxopropyl)-15-benzyl-18-(4-hydroxybenzyl)- 8,l l,14,17,20-pentaoxo-2-(27-oxo-2,5,8,l l,14,17,20,23-octaoxa-26-azatriacontan-30-yl)-l,4-di- thia-7, 10, 13, 16, 19-pentaazacyclodocosan-6-yl]carbonyl } -N- { (2S)-6-amino- 1 - [(2-amino-2-oxo- ethyl)amino] -1 -oxohexan-2-yl }pyrrolidin-2-carboxamid 3B: (25) - 1 - {[(6R, 95, 125, 155, 185.2 IR) -21 - {[({[(aminoacetyl) amino] acetyl} amino) acetyl] -amino} -9- ( 2-amino-2-oxoethyl) -12- (3-amino-3-oxopropyl) -15-benzyl-18- (4-hydroxybenzyl) -8,11,14,17,20-pentaoxo-2- (27- oxo-2,5,8, ll, 14,17,20,23-octaoxa-26-azatriacontan-30-yl) -l, 4-di- thia-7, 10, 13, 16, 19-pentaazacyclodocosan-6-yl] carbonyl} - N - {(2S) -6-amino-1 - [(2-amino-2-oxo-ethyl) -amino] -1 -oxohexan-2-yl} pyrrolidine-2-carboxamide
3A: 3A:
3B: 3B:
Unter einer Argonatmosphäre wurden in einem Zweihalskolben 20.64 mg (15.32 μιηοΐ) Terlipres- sin-Acetat (Fa. Bachem, Schweiz; Sequenz: H-Gly-Gly-Gly-Cys-Tyr-Phe-Gln-Asn-Cys-Pro-Lys- Gly-N I I;, als cyclisches Cys-Disulfid) in 500 μΐ DPBS-Puffer vorgelegt und mit 13.30 mg (46.39 μ ιηοΐ ) TCEP-HCl versetzt. Das Reaktionsgemisch wurde 2 h bei RT gerührt und dann mit einer Lösung von 7.20 mg (15.15 μιηοΐ) 7V-(2,5,8,l l,14,17,20,23-Octaoxapentacosan-25-yl)hex-5-inamid in 150 μ Ι DPBS-Puffer versetzt. Anschließend wurden 4.4 mg (14.95 μιηοΐ) LAP [hergestellt nach literaturbekanntem Verfahren (Gong et al., 2013)] in 500 μΐ DPBS-Puffer gelöst und 50 μΐ dieser LAP-Lösung zum Reaktionsgemisch hinzugefügt. Es wurde ein LED-UV-Stift (OmniCure LX400, Durchmesser 12 mm; igb-tech GmbH, Deutschland) durch einen Schliff des Zweihalskolbens eingeführt (Abstand zum Reaktionsgemisch ca. 40 mm), und das Reaktionsgemisch wurde für 1 h mit 365 nm UV-Licht bestrahlt. Die Zugabe von 50 μΐ der LAP-Lösung und die nachfolgende Bestrahlung für 1 h mit 365 nm UV -Licht wurden noch zweimal wiederholt. Das Reaktionsgemisch wurde dann mittels präparativer HPLC fraktioniert (Säule: Waters X -Bridge BEH130 Prep C18 10 μ ιη OB IX 19 x 250 mm; Eluent A: Wasser mit 0.1 % TFA, Eluent B: Acetonitril mit 0.1 % TFA; Gradient: 0.0 min 5% B -> 3 min 5% B -> 43 min 40% B 44.30 min 95% B 49.30 min 95% B). 20.64 mg (15.32 μιηοΐ) terlipresin acetate (Bachem, Switzerland; sequence: H-Gly-Gly-Gly-Cys-Tyr-Phe-Gln-Asn-Cys-Pro-Lys.) Were dissolved in an argon atmosphere in a two-necked flask Gly-N II; as cyclic Cys disulfide) in 500 μΐ DPBS buffer and with 13.30 mg (46.39 μ ιηοΐ) TCEP-HCl. The reaction mixture was stirred for 2 h at RT and then with a solution of 7.20 mg (15.15 μιηοΐ) 7V- (2,5,8, ll, 14,17,20,23-Octaoxapentacosan-25-yl) hex-5-ynamide added in 150 μ Ι DPBS buffer. Subsequently, 4.4 mg (14.95 μιηοΐ) LAP [prepared by the method known from the literature (Gong et al., 2013)] were dissolved in 500 μΐ DPBS buffer and 50 μΐ of this LAP solution was added to the reaction mixture. An LED UV-pen (OmniCure LX400, diameter 12 mm, igb-tech GmbH, Germany) was introduced through a cut of the two-necked flask (distance to the reaction mixture about 40 mm), and the reaction mixture was UV for 365 h at 365 nm Light irradiated. The addition of 50 μL of the LAP solution and the subsequent irradiation for 1 h with 365 nm UV light were repeated twice more. The reaction mixture was then fractionated by preparative HPLC (column: Waters X -Bridge BEH130 Prep C18 10 μηι OB IX 19 × 250 mm, eluent A: water with 0.1% TFA, eluent B: acetonitrile with 0.1% TFA, gradient: 0.0 min 5% B -> 3 min 5% B -> 43 min 40% B 44.30 min 95% B 49.30 min 95% B).
Produktfraktion 1 : Product fraction 1:
Ausbeute: 6.20 mg (23% d. Th.); Reinheit nach LC7MS (Methode 3): 99% Yield: 6.20 mg (23% of theory); Purity to LC7MS (Method 3): 99%
LC/MS (Methode 3 ): K, = 7.4 1 min; MS (ESpos): m/z = 853.9101 [M+2H]2+ LC / MS (Method 3): K, = 7.4 1 min; MS (ESpos): m / z = 853.9101 [M + 2H] 2+
1 IKMS: für C75H121O24N17S2 [M+2H]2+ berechnet: 853.9100, gemessen: 853.9100. 1 IKMS: Calculated for C75H121O24N17S2 [M + 2H] 2+ : 853.9100, measured: 853.9100.
Die ' I I- und "C-N K-Spektren dieser Produktfraktion sind in Abbildung 2 und 3 wiedergegeben. The 'I I and C-N K spectra of this product fraction are shown in Figures 2 and 3.
Produktfraktion 2: Product fraction 2:
Ausbeute: 1.70 mg (7% d. Th.) Yield: 1.70 mg (7% of theory)
LC/MS (Methode 2): R, = 4.09 min; MS (ESpos): m/z = 853.9149 [M+2H]2+ LC / MS (Method 2): R, = 4.09 min; MS (ESpos): m / z = 853.9149 [M + 2H] 2+
I IRMS: für C75H121O24N17S2 [M+2H]2+ berechnet: 853.9100, gemessen: 853.9095. IRMS: calculated for C75H121O24N17S2 [M + 2H] 2+ : 853.9100, measured: 853.9095.
Beispiel 4A und 4B Example 4A and 4B
4Λ: 4-{ 4-[(6R,9S,l2S, 15S, 18S,21R)-21 - { [( { [(Aminoacetyl)amino] acetyl } amino)acetyl] amino } - 6-{ [(2lSr)-2-({(2lS')-6-amino-l-[(2-amino-2-oxoethyl)amino]-l-oxohexan-2-yl }carbamoyl)pyrrolidin- l-yl]carbonyl}-9-(2-amino-2-oxoethyl)-12-(3-amino-3-oxopropyl)-15-benzyl-18-(4-hydroxy- benzyl)-8, 11,14, 17,20-pentaoxo-l,4-dithia-7, 10, 13,16, 19-pentaazacyclodocosan-3-yl]butyl }-4- methylmorpholin-4-ium-Trifluoracetat 4B : 4- { 4-[(6R,95,125, 155, 185,21R)-21 - { [( { [(Aminoacetyl)amino] acetyl } amino)acetyl] amino } - 6-j | ( 2.S')-2-( j ( 2.S')-6-ainiiH)- l -| (2-ainiiH)-2-()x()cthyl )aniiiH) |- l -()x()liexan-2-yl IcarbanioyDpyrrolidin- l-yl]carbonyl}-9-(2-amino-2-oxoethyl)-12-(3-amino-3-oxopropyl)-15-benzyl-18-(4-hydroxy- benzyl)-8, 1 1 , 14, 17,20-pentaoxo-l ,4-dithia-7, 10, 13, 16, 19-pentaazacyclodocosan-2-yl]butyl }-4- methylmorpholin-4-ium-Trifluoracetat 4Λ: 4- {4 - [(6R, 9S, 12S, 15S, 18S, 21R) -21 - {[({[(aminoacetyl) amino] acetyl} amino) acetyl] amino} - 6- {[(2 l S r ) -2 - ({(2 L S ') - 6-amino-1 - [(2-amino-2-oxoethyl) amino] -1-oxohexan-2-yl} carbamoyl) pyrrolidin-1-yl] carbonyl} -9- (2-amino-2-oxo-ethyl) -12- (3-amino-3-oxo-propyl) -15-benzyl-18- (4-hydroxybenzyl) -8,11,14,17,20 -pentaoxo-l, 4-dithia-7,10,13,16,19-pentaazacyclodocosan-3-yl] butyl} -4-methylmorpholine-4-ium trifluoroacetate 4B: 4- {4 - [(6R, 95,125, 155, 185,21R) -21 - {[({[(Aminoacetyl) amino] acetyl} amino) acetyl] amino} -6-j (2.S ' ) -2- (j (2.S ' ) -6-ainiiH) - l - | (2-ainiiH) -2 - () x () cyl) aniiiH) | - l - () x () liexan-2-ylcarbanioxy-pyrrolidin-1-yl] carbonyl} -9- (2-amino-2-oxoethyl ) -12- (3-amino-3-oxopropyl) -15-benzyl-18- (4-hydroxybenzyl) -8,1,1,1,4,17,20-pentaoxo-1,4-dithia-7,10 , 13, 16, 19-pentaazacyclodocosan-2-yl] butyl} -4-methylmorpholine-4-ium trifluoroacetate
4A: 4A:
Unter einer Argonatmosphäre wurden in einem Zweihalskolben 2 1 .54 mg (15.98 μιηοΐ) Terlipres- sin-Acetat (Fa. Bachem, Schweiz; Sequenz: H-Gly-Gly-Gly-Cys-Tyr-Phe-Gln-Asn-Cys-Pro-Lys- Gly-N ! L. als cyclisches Cys-Disulfid) in 800 μΐ DPBS-Puffer vorgelegt und mit 6.50 mg (22.68 μιηοΐ) TCEP-HCl versetzt. Das Reaktionsgemisch wurde 2 h bei RT gerührt und dann mit einer Lösung von 4.9 mg (15.84 μιηοΐ) 4-(Hex-5-in-l-yl)-4-methylmorpholin-4-iumiodid in 500 μΐ DPBS-Puffer versetzt. Anschließend wurden 4.70 mg (15.98 μιηοΐ) LAP [hergestellt nach literatur- bekanntem Verfahren (Gong et al, 2013)] in 500 μΐ DPBS-Puffer gelöst und 50 μΐ dieser LAP- Lösung zum Reaktionsgemisch hinzugefügt. Es wurde ein LED-UV-Stift (OmniCure LX400, Durchmesser 12 mm; igb-tech GmbH, Deutschland) durch einen Schliff des Zweihalskolbens eingeführt (Abstand zum Reaktionsgemisch ca. 40 mm), und das Reaktionsgemisch wurde für 1 h mit 365 nm UV-Licht bestrahlt. Die Zugabe von 50 μ Ι der LAP-Lösung und die nachfolgende Bestrah- lung für 1 h mit 365 nm UV -Licht wurden noch zweimal wiederholt. Das Reaktionsgemisch wurde dann mittels präparativer HPLC fraktioniert (Säule: Phenomenex Kinetex Prep 5 μ ιη C18 100 Ä AXIA Packed LC Column, 21.2 x 100 mm; Eluent A: Wasser mit 0.1 % TFA, Eluent B: Acetonitril mit 0.08% TFA; Gradient: 0.0 min 5% B -> 3 min 5% B -> 63 min 40% B -» 64.30 min 95% B 69.30 min 95% B). Produktfraktion 1 : 2 1.54 mg (15.98 μmol) terlipresin acetate (Bachem, Switzerland, sequence: H-Gly-Gly-Gly-Cys-Tyr-Phe-Gln-Asn-Cys-) were dissolved in an argon atmosphere in a two-necked flask. Pro-Lys Gly-N! L. as cyclic Cys disulfide) in 800 .mu.ΐ DPBS buffer and treated with 6.50 mg (22.68 μιηοΐ) TCEP-HCl. The reaction mixture was stirred for 2 h at RT and then treated with a solution of 4.9 mg (15.84 μιηοΐ) 4- (hex-5-yn-l-yl) -4-methylmorpholin-4-iumiodid in 500 μΐ DPBS buffer. Subsequently, 4.70 mg (15.98 μιηοΐ) of LAP [prepared by literature-known method (Gong et al, 2013)] were dissolved in 500 μΐ DPBS buffer and 50 μΐ of this LAP solution was added to the reaction mixture. An LED UV-pen (OmniCure LX400, diameter 12 mm, igb-tech GmbH, Germany) was introduced through a cut of the two-necked flask (distance to the reaction mixture about 40 mm), and the reaction mixture was UV for 365 h at 365 nm Light irradiated. The addition of 50 μL of the LAP solution and the subsequent irradiation for 1 h with 365 nm UV light were repeated twice more. The reaction mixture was then fractionated by preparative HPLC (column: Phenomenex Kinetex Prep 5 μηι C18 100 Å AXIA Packed LC Column, 21.2 × 100 mm, eluent A: water with 0.1% TFA, eluent B: acetonitrile with 0.08% TFA; 0.0 min 5% B -> 3 min 5% B -> 63 min 40% B - »64.30 min 95% B 69.30 min 95% B). Product fraction 1:
Ausbeute: 1.5 mg (4% d. Th.); Reinheit nach LC/MS (Methode 3): 65%  Yield: 1.5 mg (4% of theory); Purity to LC / MS (Method 3): 65%
LC/MS (Methode 3): R, = 6.49 min; MS (ESpos): m/z = 705.8397 [M+H]2+ LC / MS (Method 3): R, = 6.49 min; MS (ESpos): m / z = 705.8397 [M + H] 2+
1 IRMS: für C63H97O16N17S2 [M+H]2+ berechnet: 705.8365, gemessen: 705.8361. 1 IRMS: Calculated for C63H97O16N17S2 [M + H] 2+ : 705.8365, measured: 705.8361.
Das 11 l-NM R-Spektrum dieser Produktfraktion ist in Abbildung 4 wiedergegeben. Produktfraktion 2: The 11 L NMR spectrum of this product fraction is shown in Figure 4. Product fraction 2:
Ausbeute: 2.2 mg (8% d. Th.); Reinheit nach LC/MS (Methode 3): 87%  Yield: 2.2 mg (8% of theory); Purity to LC / MS (Method 3): 87%
LC/MS (Methode 3): R, = 6.48 min; S (ESpos): m/z = 705.8401 [M+H]2+ LC / MS (Method 3): R, = 6.48 min; S (ESpos): m / z = 705.8401 [M + H] 2+
1 IRMS: für C63H97O16N17S2 [M+H]2+ berechnet: 705.8365, gemessen: 705.8377. 1 IRMS: Calculated for C63H97O16N17S2 [M + H] 2+ : 705.8365, measured: 705.8377.
Das Ί I-NM R-Speklrum dieser Produktfraktion ist in Abbildung 5 wiedergegeben. Beispiel 5A und 5B The Ί I-NM R spectrum of this product fraction is shown in Figure 5. Example 5A and 5B
5A: (2S)-3-[(6R,9S,l2S, 155, 18S,21R)-21 - { [( { [(Aminoacetyl)amino] acetyl } amino)acetyl] - amino } -6- { [(2S)-2-( { (2S)-6-amino- 1 -[(2-amino-2-oxoethyl)amino]- l-oxohexan-2-yl Jcarbamoyl)- pyrrolidin-l-yl]carbonyl}-9-(2-amino-2-oxoethyl)-12-(3-amino-3-oxopropyl)-15-benzyl-18-(4- hydroxybenzyl)-8, 11,14, 17,20-pentaoxo- 1 ,4-dithia-7, 10, 13, 16, 19-pentaazacyclodocosan-3-yl] -2- [(ierf.-butoxycarbonyl)amino]propansäure 5A: (2S) -3 - [(6R, 9S, 12S, 155, 18S, 21R) -21 - {[({[(aminoacetyl) amino] acetyl} amino) acetyl] -amino} -6- {[( 2S) -2- ({(2S) -6-amino-1 - [(2-amino-2-oxoethyl) amino] -1-oxohexan-2-ylcarbamoyl) -pyrrolidin-1-yl] carbonyl} -9 - (2-amino-2-oxoethyl) -12- (3-amino-3-oxopropyl) -15-benzyl-18- (4- hydroxybenzyl) -8,11,14,17,20-penta-oxo-1,4-dithia-7,10,13,16,19-pentaazacyclodocosan-3-yl] -2- [(ε-butoxycarbonyl) amino] propanoic acid
5B : (2S)-3-[(6R,9S,l2S, 155,18S,21R)-21 - { [( { [(Aminoacetyl)amino] acetyl } amino)acetyl] - amino } -6- { [(2S)-2-( { (2^-6-amino- 1 -[(2-amino-2-oxoethyl)amino]- l-oxohexan-2-yl Jcarbamoyl)- Pyrrolidin- l-yl]carbonyl}-9-(2-amino-2-oxoethyl)-12-(3-amino-3-oxopropyl)-15-benzyl-18-(4- hydroxybenzyl)-8, 1 1.14.17,20-pentaoxo- 1 ,4-dithia-7, 10,13,16,19-pentaazacyclodocosan-2-yl] -2- [(ierf.-butoxycarbonyl)amino]propansäure 5B: (2S) -3 - [(6R, 9S, 12S, 155, 18S, 21R) -21 - {[({[(aminoacetyl) amino] acetyl} amino) acetyl] -amino} -6- {[( 2S) -2- ({(2 ^ -6-amino-1 - [(2-amino-2-oxoethyl) amino] -1-oxohexan-2-ylcarbamoyl) -pyrrolidin-1-yl] carbonyl} -9 (2-amino-2-oxo-ethyl) -12- (3-amino-3-oxo-propyl) -15-benzyl-18- (4-hydroxy-benzyl) -8, 1, 1.14, 17, 20-penta-oxo-1, 4- dithia-7, 10,13,16,19-pentaazacyclodocosan-2-yl] -2- [(ε-butoxycarbonyl) amino] propanoic acid
5A: 5A:
5B: 5B:
Unter einer Argonatmosphäre wurden in einem Zweihalskolben 10.16 mg (7.54 μιηοΐ) Terlipres- sin-Acetat (Fa. Bachem, Schweiz; Sequenz: H-Gly-Gly-Gly-Cys-Tyr-Phe-Gln-Asn-Cys-Pro-Lys- Gly-N I I;. als cyclisches Cys-Disulfid) in 300 μΐ DPBS-Puffer vorgelegt und mit 3.20 mg (11.17 μιηοΐ) TCEP-HCl versetzt. Das Reaktionsgemisch wurde 1.5 h bei RT gerührt und dann mit 200 μΐ DPBS-Puffer sowie einer Lösung von 1.6 mg (7.54 μ ιηοΐ ) /V-Boc-L-Propargylglycin in 100 μΐ DPBS-Puffer versetzt. Anschließend wurden 3.20 mg (10.87 μ mol ) LAP [hergestellt nach literaturbekanntem Verfahren (Gong et al, 2013)] in 500 μΐ DPBS-Puffer gelöst und 50 μΐ dieser LAP- Lösung zum Reaktionsgemisch hinzugefügt. Es wurde ein LED-UV-Stift (OmniCure LX400, Durchmesser 12 mm; igb-tech GmbH, Deutschland) durch einen Schliff des Zweihalskolbens ein- geführt (Abstand zum Reaktionsgemisch ca. 40 mm), und das Reaktionsgemisch wurde für 1 h mit 365 nm UV-Licht bestrahlt. Die Zugabe von 50 μΐ der LAP-Lösung und die nachfolgende Bestrahlung für 1 h mit 365 nm UV -Licht wurden noch zweimal wiederholt. Das Reaktionsgemisch wurde dann mittels präparativer HPLC fraktioniert (Säule: Phenomenex Kinetex Prep 5 μ ιη C18 100 Ä AXIA Packed LC Column, 21 .2 x 100 mm; Eluent A: Wasser mit 0.1 % 'I I A, Eluent B: Acetonitril mit 0.08% 'I TA; Gradient: 0.0 min 'A B -+ 3 min 5% B -> 63 min 40% B 65.3 min 95% B 70 min 95% B). 10.16 mg (7.54 μιηοΐ) of terlipresin acetate (Bachem, Switzerland, sequence: H-Gly-Gly-Gly-Cys-Tyr-Phe-Gln-Asn-Cys-Pro-Lys.) Were dissolved in an argon atmosphere in a two-necked flask - Gly-N II; as a cyclic Cys disulfide) in 300 .mu.ΐ DPBS buffer and 3.20 mg (11.17 μιηοΐ) TCEP-HCl. The reaction mixture was stirred at RT for 1.5 h and then treated with 200 μΐ DPBS buffer and a solution of 1.6 mg (7.54 μιηοΐ) / V-Boc-L-propargylglycine in 100 μΐ DPBS buffer. Subsequently 3.20 mg (10.87 μmol) of LAP [prepared by the method known from the literature (Gong et al., 2013)] were dissolved in 500 μl of DPBS buffer and 50 μl of this LAP solution were added to the reaction mixture. An LED UV pencil (OmniCure LX400, diameter 12 mm, igb-tech GmbH, Germany) was introduced through a cut of the two-necked flask (distance to the reaction mixture about 40 mm), and the reaction mixture was allowed to stand for 1 h nm irradiated UV light. The addition of 50 μL of the LAP solution and the subsequent irradiation for 1 h with 365 nm UV light were repeated twice more. The reaction mixture was then fractionated by preparative HPLC (column: Phenomenex Kinetex Prep 5 μL C18 100 Å AXIA Packed LC Column, 21 × 2 × 100 mm, eluant A: water with 0.1% IIA, eluent B: acetonitrile with 0.08% I TA; Gradient: 0.0 min 'AB - + 3 min 5% B -> 63 min 40% B 65.3 min 95% B 70 min 95% B).
Produktfraktion 1 : Product fraction 1:
Ausbeute: 0.4 mg (4% cl. Th.) Yield: 0.4 mg (4% Cl. Th.)
LC/MS (Methode 3): K, = 7. 1 1 min; MS (ESpos): m/z = 721.8130 [M+2H]2+ IKMS: für C62H93O19N17S2 [M+2H]2+ berechnet: 721.8132, gemessen: 721.8130. Das Ί I-NM K-Spektrum dieser Produktfraktion ist in Abbildung 6 wiedergegeben. Produktfraktion 2: LC / MS (Method 3): K, = 7. 1 1 min; MS (ESpos): m / z = 721.8130 [M + 2H] 2+ IKMS: Calculated for C62H93O19N17S2 [M + 2H] 2+ : 721.8132, measured: 721.8130. The Ί I-NM K spectrum of this product fraction is shown in Figure 6. Product fraction 2:
Ausbeute: 1.6 mg (15% d. Th.); Reinheit nach LC/MS (Methode 3): 94% LC/MS (Methode 3): R, = 7.36 min; MS (ESpos): m z = 1442. 1 4 [M+H]+ I I KMS: für C62H93O19N17S2 [M+2H]2+ berechnet: 721.8132, gemessen: 721.8132. Das T I-NM K-Spektrum dieser Produktfraktion ist in Abbildung 7 wiedergegeben. Produktfraktion 3: Yield: 1.6 mg (15% of theory); Purity to LC / MS (Method 3): 94% LC / MS (Method 3): R, = 7.36 min; MS (ESpos): mz = 1442. 1 4 [M + H] + II KMS: Calculated for C62H93O19N17S2 [M + 2H] 2+ : 721.8132, measured: 721.8132. The T I-NM K spectrum of this product fraction is shown in Figure 7. Product fraction 3:
Ausbeute: 0.3 mg (3% d. Th.); Reinheit nach LC/MS (Methode 3): 89%  Yield: 0.3 mg (3% of theory); Purity to LC / MS (Method 3): 89%
LC/MS (Methode 3): K, = 7.34 min; MS (ESpos): m/z = 721.8122 [M+2H]2+ HRMS: für C62H92O19N17S2 [M+H]+ berechnet: 1442.6191, gemessen: 1442.6200. Beispiel 6A und 6B LC / MS (Method 3): K, = 7.34 min; MS (ESpos): m / z = 721.8122 [M + 2H] 2+ HRMS: for C6 2 H9 2 O 1 9N 17 S 2 [M + H] + calcd: 1442.6191, measured: 1442.6200. Example 6A and 6B
6Λ: 6Λ:
15-benzyl-3-(4-carboxybutyl)- 18-(4-hydroxybenzyl)-8, 1 1 , 14.1 7 ,20-pentaoxo- 1.4-dithia- 7, 10, 13,16, 19-pentaazacyclodocosan-6-carbonsäure 15-benzyl-3- (4-carboxybutyl) -18- (4-hydroxybenzyl) -8, 11, 14.1 7, 20-pentaoxo-1,4-dithia-7, 10, 13, 16, 19-pentaazacyclodocosan-6 carboxylic acid
6B: 6B:
15-benzyl-2-(4-carboxybutyl)- 1 8-(4-hydroxybenzyl)-8, 1 1.14.17,20-pentaoxo- 1 ,4-dithia- 7,10,13,16,19-pentaazacyclodocosan-6-carbonsäure 15-benzyl-2- (4-carboxybutyl) -18- (4-hydroxybenzyl) -8,1,1,1,17,20-pentaoxo-1,4-dithia-7,10,13,16,19-pentaazacyclodocosane 6-carboxylic acid
6A: 6A:
Unter einer Argonatmosphäre wurden in einem Zweihalskolben 15.33 mg (19.78 μιηοΐ) Pressinoic Acid (Fa. Bachem, Schweiz; Sequenz: H-Cys-Tyr-Phe-Gln-Asn-Cys-OH, als cyclisches Cys-Disul- fid) in 500 μΐ 0.1 %-iger wässriger Essigsäure und 500 μΐ Acetonitril vorgelegt und mit 8.70 mg (30.35 μ mol ) TCEP-HC1 versetzt. Das Reaktionsgemisch wurde 2.5 h bei RT gerührt und dann mit 200 μΐ 0.1%-iger wässriger Essigsäure, 200 μ ΐ Acetonitril sowie einer Lösung von 2.5 mg (19.82 μ ιηοΐ ) Hept-6-insäure in 100 μΐ 0.1 %-iger wässriger Essigsäure versetzt. Anschließend wurden 5.8 mg (19.72 μιηοΐ) LAP [hergestellt nach literaturbekanntem Verfahren (Gong et al., 2013)] in 500 μΐ 0.1 %-iger wässriger Essigsäure gelöst und 50 μΐ dieser LAP-Lösung zum Reaktionsgemisch hinzugefügt. Es wurde ein LED-UV-Stift (Omni Cure LX400, Durchmesser 12 mm; igb-tech GmbH, Deutschland) durch einen Schliff des Zweihalskolbens eingeführt (Abstand zum Reaktionsgemisch ca. 40 mm), und das Reaktionsgemisch wurde für 1 h mit 365 nm UV -Licht bestrahlt. Die Zugabe von 50 μΐ der LAP-Lösung und die nachfolgende Bestrahlung für 1 h mit 365 nm UV- Licht wurden noch zweimal wiederholt. Das Reaktionsgemisch wurde dann mittels präparativer HPLC fraktioniert (Säule: Waters X-Bridge BEI I I 30 Prep C18 10 μ ιη OBD, 19 x 250 mm; Eluent A: Wasser mit 0.1 % TFA, Eluent B: Acetonitril mit 0.08% 'I LA; Gradient: 0.0 min 5% B 3 min 5% B -> 33 min 40% B). 15.33 mg (19.78 μmol) of Pressinoic Acid (Bachem, Switzerland, sequence: H-Cys-Tyr-Phe-Gln-Asn-Cys-OH, as a cyclic Cys disulphide) in 500 ml were used in an argon atmosphere in a two-necked flask Submitted μΐ 0.1% aqueous acetic acid and 500 μΐ acetonitrile and treated with 8.70 mg (30.35 μ mol) TCEP-HC1. The reaction mixture was stirred for 2.5 h at RT and then treated with 200 .mu.l 0.1% aqueous acetic acid, 200 μ ΐ acetonitrile and a solution of 2.5 mg (19.82 μ ιηοΐ) hept-6-acid in 100 .mu.l 0.1% aqueous acetic acid , 5.8 mg (19.72 μmol) of LAP [prepared by the method known from the literature (Gong et al., 2013)] were then dissolved in 500 μl of 0.1% strength aqueous acetic acid, and 50 μl of this LAP solution were added to the reaction mixture. An LED UV pencil (Omni Cure LX400, diameter 12 mm, igb-tech GmbH, Germany) was introduced through a cut of the two-necked flask (distance to the reaction mixture about 40 mm), and the reaction mixture was at 365 nm for 1 h UV light irradiated. The addition of 50 μL of the LAP solution and the subsequent irradiation for 1 h with 365 nm UV light were repeated twice more. The reaction mixture was then fractionated by preparative HPLC (column: Waters X-Bridge BEI II 30 Prep C18 10 μηι OBD, 19 × 250 mm, eluant A: water with 0.1% TFA, eluent B: acetonitrile with 0.08% I LA; Gradient: 0.0 min 5% B 3 min 5% B -> 33 min 40% B).
Produktfraktion 1 : Product fraction 1:
Ausbeute: 1 .4 mg (8% d. Th.) Yield: 1 .4 mg (8% of theory)
LC/MS (Methode 1): R, = 1 .25 min; MS (ESpos): m/z = 903.4 [M+H]+. Das Ί I-NM K-Spektrum dieser Produktfraktion ist in Abbildung 8 wiedergegeben. LC / MS (Method 1): R, = 1 .25 min; MS (ESpos): m / z = 903.4 [M + H] + . The Ί I-NM K spectrum of this product fraction is shown in Figure 8.
Produktfraktion 2: Product fraction 2:
Ausbeute: 0.6 mg (3% d. Th.) Yield: 0.6 mg (3% of theory)
LC/MS (Methode 1 ): K, = 1 .25 min; MS (ESpos): m/z = 903.4 [M+H]+ und R, = 1.27 min; MS (ESpos): m/z = 903.3 [M+H]+. IKMS: für C40H55O12N8S2 [M+H]+ berechnet: 903.3375 gemessen: 903.3371. LC / MS (Method 1): K, = 1 .25 min; MS (ESpos): m / z = 903.4 [M + H] + and R, = 1.27 min; MS (ESpos): m / z = 903.3 [M + H] + . IKMS: Calculated for C40H55O12N8S2 [M + H] + : 903.3375 measured: 903.3371.
Produktfraktion 3: Product fraction 3:
Ausbeute: 0.7 mg (4% d. Th.) Yield: 0.7 mg (4% of theory)
LC/MS (Methode 1): R, = 1.27 min; MS (ESpos): m/z = 903.3 [M+H]+. LC / MS (method 1): R, = 1.27 min; MS (ESpos): m / z = 903.3 [M + H] + .
Das 11 1-N.M K-Spektrum dieser Produktfraktion ist in Abbildung 9 wiedergegeben. Beispiel 7A und 7B The 1 1 1-NM K spectrum of this product fraction is shown in Figure 9. Example 7A and 7B
7A: (2S)-l-{ [(3R,65,95, 125, 155, 18R,22aR,235,23a5)- 18- { [( { [( Aminoacetyl)amino] acetyl } - amino)acetyl]amino}-6-(2-amino-2-oxoethyl)-9-(3-amino-3-oxopropyl)-12-benzyl-15-(4-hydroxy- benzyl)-23-(hydroxymethyl)-5,8,l l,14,17-pentaoxohexacosahydro-20aii-cyclopropa[5,6]cyclo- octa[ 1 ,2-b] [ 1 ,4,7, 10, 13, 16, 19]dithiapentaazacyclodocosin-3-yl]carbonyl } -N- { (25)-6-amino- 1 -[(2- amino-2-oxoethyl)amino] - l-oxohexan-2-yl }pyrrolidin-2-carboxamid 7A: (2S) - 1 - {[(3R, 65.95, 125, 155, 18R, 22aR, 235, 23a5) - 18 - {[({[(Aminoacetyl) amino] acetyl} - amino) acetyl] amino } -6- (2-amino-2-oxoethyl) -9- (3-amino-3-oxopropyl) -12-benzyl-15- (4-hydroxybenzyl) -23- (hydroxymethyl) -5.8, II, 14, 17-pentaoxohexacosahydro-20aii-cyclopropa [5,6] cycloocta [1, 2-b] [1, 4, 7, 10, 13, 16, 19] dithiapentaazacyclodocosin-3-yl] carbonyl} - N- {(25) -6-amino-1 - [(2-amino-2-oxoethyl) amino] -1-oxohexan-2-yl} pyrrolidine-2-carboxamide
7B : (25)- 1 - { [(3R,65,95, 125, 155, 18R,22a5,23R,23aR)- 18- { [( { [( Aminoacetyl)amino] acetyl } - amino)acetyl]amino}-6-(2-amino-2-oxoethyl)-9-(3-amino-3-oxopropyl)-12-benzyl-15-(4-hydroxy- benzyl)-23-(hydroxymethyl)-5,8,l l,14,17-pentaoxohexacosahydro-20aii-cyclopropa[5,6]cyclo- octa[ 1 ,2-b] [ 1 ,4,7, 10, 13, 16, 19]dithiapentaazacyclodocosin-3-yl]carbonyl } -N- { (25)-6-amino- 1 -[(2- amino-2-oxoethyl)amino] - l-oxohexan-2-yl }pyrrolidin-2-carboxamid 7B: (25) - 1 - {[(3R, 65.95, 125, 155, 18R, 22a5, 23R, 23aR) - 18 - {[({[(aminoacetyl) amino] acetyl} - amino) acetyl] amino } -6- (2-amino-2-oxoethyl) -9- (3-amino-3-oxopropyl) -12-benzyl-15- (4-hydroxybenzyl) -23- (hydroxymethyl) -5.8, II, 14, 17-pentaoxohexacosahydro-20aii-cyclopropa [5,6] cycloocta [1, 2-b] [1, 4, 7, 10, 13, 16, 19] dithiapentaazacyclodocosin-3-yl] carbonyl} - N- {(25) -6-amino-1 - [(2-amino-2-oxoethyl) amino] -1-oxohexan-2-yl} pyrrolidine-2-carboxamide
7A: 7A:
7B: 7B:
Unter einer Argonatmosphäre wurden in einem Zweihalskolben 26.24 mg (19.47 μιηοΐ) Terlipres- sin-Acetat (Fa. Bachem, Schweiz; Sequenz: H-Gly-Gly-Gly-Cys-Tyr-Phe-Gln-Asn-Cys-Pro-Lys- Gly-N i l;. als cyclisches Cys-Disulfid) und 8.4 mg (29.31 μιηοΐ) TCEP-HCl in 1 .5 ml DPBS-Puffer vorgelegt. Das Reaktionsgemisch wurde 2.5 h bei KT gerührt und dann mit einer Lösung von 2.9 mg (19.32 μιηοΐ) in 200 μΐ Methanol versetzt. Anschließend wurden 5.70 mg (19.38 μιηοΐ) LAP [hergestellt nach literaturbekanntem Verfahren (Gong et al, 2013)] in 500 μ ΐ DPBS-Puffer gelöst und 50 μΐ dieser LAP-Lösung zum Reak- tionsgemisch hinzugefügt. Es wurde ein LED-UV-Stift (OmniCure LX400, Durchmesser 12 mm; igb-tech GmbH, Deutschland) durch einen Schliff des Zweihalskolbens eingeführt (Abstand zum Reaktionsgemisch ca. 40 mm), und das Reaktionsgemisch wurde für 1 h mit 365 nm UV-Licht bestrahlt. Die Zugabe von 50 μΐ der LAP-Lösung und die nachfolgende Bestrahlung für 1 h mit 365 nm UV -Licht wurden noch zweimal wiederholt. Das Reaktionsgemisch wurde dann mittels präparativer HPLC fraktioniert (Säule: Phenomenex Kinetex Prep 5 μιη C18 100 Ä AXIA Packed LC Column, 21.2 x 100 mm; Eluent A: Wasser mit 0.1% TFA, Eluent B: Acetonitril mit 0.08% TFA; Gradient: 0.0 min 5' < B— > 3 min 5% B ^ 63 min 40% B 64.30 min 95% B 69.30 min 95% B). In a two-necked flask, 26.24 mg (19.47 μmol) of terlipresin acetate (Bachem, Switzerland, sequence: H-Gly-Gly-Gly-Cys-Tyr-Phe-Gln-Asn-Cys-Pro-Lys - Gly-N il ;. As a cyclic Cys disulfide) and 8.4 mg (29.31 μιηοΐ) TCEP-HCl in 1 .5 ml DPBS buffer submitted. The reaction mixture was stirred for 2.5 h at KT and then with a solution of 2.9 mg (19.32 μιηοΐ) in 200 μΐ methanol. Subsequently, 5.70 mg (19.38 μmol) of LAP [prepared by the method known from the literature (Gong et al, 2013)] were dissolved in 500 μl of DPBS buffer and 50 μl of this LAP solution were added to the reaction mixture. An LED UV-pen (OmniCure LX400, diameter 12 mm, igb-tech GmbH, Germany) was introduced through a cut of the two-necked flask (distance to the reaction mixture about 40 mm), and the reaction mixture was UV for 365 h at 365 nm Light irradiated. The addition of 50 μL of the LAP solution and the subsequent irradiation for 1 h with 365 nm UV light were repeated twice more. The reaction mixture was then fractionated by preparative HPLC (column: Phenomenex Kinetex Prep 5 μιη C18 100 Å AXIA Packed LC Column, 21.2 × 100 mm, eluent A: water with 0.1% TFA, eluent B: acetonitrile with 0.08% TFA, gradient: 0.0 min 5 '<B-> 3 min 5% B ^ 63 min 40% B 64.30 min 95% B 69.30 min 95% B).
Produktfraktion 1 : Product fraction 1:
Ausbeute: 3.1 mg (7.5% d. Th.); Reinheit nach LC/MS (Methode 3): 65% LC/MS (Methode 3): R, = 6.92 min; MS (ESpos): m/z = 903.3 [M+H]+ HRMS: für C62H91O16N16S2 [M+H]+ berechnet: 1379.6235, gemessen: 1379.6244. Das Ί I-N R-Spektrum dieser Produktfraktion ist in Abbildung 10 wiedergegeben. Beispiel 8 Yield: 3.1 mg (7.5% of theory); Purity to LC / MS (Method 3): 65% LC / MS (Method 3): R, = 6.92 min; MS (ESpos): m / z = 903.3 [M + H] + HRMS: Calculated for C62H91O16N16S2 [M + H] + : 1379.6235, measured: 1379.6244. The Ί IN R spectrum of this product fraction is shown in Figure 10. Example 8
C2-verbrückende Thiol-yn-Reaktion mit einem Antikörper-F AB -Fragment: C2-bridging thiol-yn reaction with an antibody F AB fragment:
Unter einer Argonatmosphäre wurden in einem Zweihalskolben 108.4 μΐ einer Lösung des FAB- Fragments M14-G07 [beschrieben in Dittmer et al, US Pat. Appl. US 2014/0050743-A1, Seite 13, Abschnitte 0105, 0106 und 0113ff.] in PBS-Puffer vorgelegt (c = 46.1 mg/ml, 0.107 μιηοΐ). 4.60 mg TCEP-HC1 wurden in 400 μΐ DPBS-Puffer gelöst und 4 μΐ dieser Lösung zur Lösung des FAB-Fragments hinzugefügt. Das Reaktionsgemisch wurde 1 h bei RT gerührt und dann mit 4 μΐ einer Lösung von 1.36 μΐ (10.75 μ ιηοΐ ) Hept-6-insäure in 398 μΐ Methanol versetzt. Anschließend wurden 3.10 mg (10.53 μπιοΐ) LAP [hergestellt nach literaturbekanntem Verfahren (Gong et al., 2013)] in 500 μ ΐ DPBS-Puffer gelöst und 1 μΐ dieser LAP-Lösung zum Reaktionsgemisch hinzugefügt. Der Reaktionskolben wurde mittels eines Eisbads gekühlt (5°C < T < 10°C). Es wurde ein LED-UV-Stift (Omni Cure LX400, Durchmesser 12 mm; igb-tech GmbH, Deutschland) durch einen Schliff des Zweihalskolbens eingeführt (Abstand zum Reaktionsgemisch ca. 40 mm), und das Reaktionsgemisch wurde für 1 h mit 365 nm UV-Licht bestrahlt. In drei aufeinanderfolgenden Intervallen wurde erneut jeweils 1 μΐ der LAP-Lösung hinzugefügt und das Reaktionsgemisch nachfolgend für jeweils 1 h mit 365 nm UV-Licht bestrahlt. Anschließend wurde das Reaktionsgemisch mit 2.384 ml DPBS-Puffer verdünnt und über eine Sephadex® G-25M PD-10-Säule (GE Healthcare), welche zuvor fünfmal mit jeweils 5 ml DPBS-Puffer konditioniert worden war, frak- tioniert. Die daraus resultierenden Fraktionen wurde 5 Minuten lang zentrifugiert (10°C, 4000 U/ min). Danach wurde die Lösung in Zentrifugenfiltergefäße (Ultracel® 30 K - Amicon® Ultra-4) pipettiert und 15 Minuten lang erneut zentrifugiert. Das resultierende Konzentrat wurde mehrmals mit insgesamt 2.5 ml DPBS-Puffer verdünnt. 108.4 μΐ of a solution of FAB fragment M14-G07 [described in Dittmer et al., US Pat. Appl. Appl. No. 2014/0050743-A1, page 13, sections 0105, 0106 and 0113ff.] In PBS buffer (c = 46.1 mg / ml, 0.107 μιηοΐ). 4.60 mg TCEP-HC1 were dissolved in 400 μΐ DPBS buffer and 4 μΐ of this solution added to the solution of the FAB fragment. The reaction mixture was stirred for 1 h at RT and then treated with 4 μΐ a solution of 1.36 μΐ (10.75 μ ιηοΐ) hept-6-acid in 398 μΐ methanol. Subsequently, 3.10 mg (10.53 μπιοΐ) of LAP [prepared by the method known from the literature (Gong et al., 2013)] were dissolved in 500 μΐ DPBS buffer and 1 μΐ of this LAP solution was added to the reaction mixture. The reaction flask was cooled by means of an ice bath (5 ° C <T <10 ° C). An LED UV pencil (Omni Cure LX400, diameter 12 mm, igb-tech GmbH, Germany) was introduced through a cut of the two-necked flask (distance to the reaction mixture about 40 mm), and the reaction mixture was at 365 nm for 1 h UV light irradiated. 1 μΐ of the LAP solution was added again at three successive intervals and the reaction mixture was subsequently irradiated with 365 nm UV light for 1 h each. Then the reaction mixture was diluted with 2,384 ml DPBS buffer and passed over a Sephadex ® G-25M PD-10 column (GE Healthcare), which had been previously conditioned five times with 5 ml of DPBS buffer, parliamentary groups tioniert. The resulting fractions were centrifuged for 5 minutes (10 ° C, 4000 rpm). Thereafter, the solution in centrifugal filter vessels (Ultracel ® 30 K - Amicon ® Ultra-4) was pipetted and centrifuged for 15 minutes again. The resulting concentrate was diluted several times with a total of 2.5 ml of DPBS buffer.
Bestätigung und Quantifizierung des kovalent verknüpften FAB-Fragments: Confirmation and quantification of the covalently linked FAB fragment:
Aus den erhaltenen Proben in DPBS-Puffer wurde die kovalente Verknüpfung des FAB-Fragments wie folgt quantifiziert und identifiziert: From the samples obtained in DPBS buffer, the covalent linkage of the FAB fragment was quantified and identified as follows:
Die Quantifizierung des kovalenten FAB-Fragments erfolgte mittels RP-Chromatographie des reduzierten und denaturierten FAB-Fragments. Zur Probenlösung (1 mg/ml, 50 μΐ) wurde Guani- dinium-Hydrochlorid (GuHCl) (28.6 mg) und eine Lösung von DL-Dithiothreitol (DTT) (500 mM, 3 μΐ) gegeben. Die Mischung wurde für eine Stunde bei 55°C inkubiert und dann über HPLC analysiert. The quantification of the covalent FAB fragment was carried out by RP chromatography of the reduced and denatured FAB fragment. Guanidinium hydrochloride (GuHCl) (28.6 mg) and a solution of DL-dithiothreitol (DTT) (500 mM, 3 μΐ) were added to the sample solution (1 mg / ml, 50 μΐ). The mixture was incubated for one hour at 55 ° C and then analyzed by HPLC.
Die HPLC- Analyse wurde auf einem Agilent 1260 HPLC-System mit Detektion bei 220 nm durchgeführt. Es wurde eine Polymer Laboratories PLRP-S Polymerie Reversed Phase-Säule (2.1 mm x 150 mm, 8 μηι Partikelgröße, 1000 Ä; Katalog-Nr. PL1912-3802) bei einer Flussrate von 1 ml/min mit folgendem Eluentensystem verwendet: Eluent A: 0.05% Trifluoressigsäure in Wasser, Eluent B: 0.05% Trifluoressigsäure in Acetonitril; Gradient: 0 min 25% B, 3 min 25% B, 28 min 50% B. HPLC analysis was performed on an Agilent 1260 HPLC system with detection at 220 nm. A Polymer Laboratories PLRP-S Polymerized Reversed Phase column (2.1 mm x 150 mm, 8 μm particle size, 1000 Å; Catalog no. PL1912-3802) at a flow rate of 1 ml / min using the following eluent system: eluent A: 0.05% trifluoroacetic acid in water, eluent B: 0.05% trifluoroacetic acid in acetonitrile; Gradient: 0 min 25% B, 3 min 25% B, 28 min 50% B.
Die detektierten Peaks wurden durch Retentionszeitvergleich mit der leichten Kette (L0) und der schweren Kette (VH-CH1 = HO) des nicht-konjugierten FAB-Fragments zugeordnet. Das Signal, welches ausschließlich in der konjugierten Probe detektiert wurde, wurde dem kovalenten, nicht- reduzierbar verknüpften FAB zugeordnet. Der prozentuale Anteil an kovalent verknüpftem FAB wurde aus den durch Integration bestimmten Signalflächen berechnet. Hierzu wurde der Quotient der Signalfläche des FAB-Fragments zur Gesamtfläche aller Signale gebildet und mit 100 multipli- ziert. Die resultierenden Chromatogramme und die berechneten prozentualen Anteile sind in Abbildung 1 1 und 12 wiedergegeben. The detected peaks were assigned by retention time comparison with the light chain (L0) and the heavy chain (VH-CH1 = HO) of the unconjugated FAB fragment. The signal detected exclusively in the conjugated sample was assigned to the covalent, non-reducible linked FAB. The percentage of covalently linked FAB was calculated from the signal areas determined by integration. For this purpose, the quotient of the signal area of the FAB fragment to the total area of all signals was formed and multiplied by 100. The resulting chromatograms and the calculated percentages are shown in Figure 1 1 and 12.
Zur Bestätigung des kovalent gebundenen FAB-Fragments wurde die denaturierte und reduzierte Probe massenspektrometrisch nach Online -Entsalzung über eine Grom-Sil 300 Butyl-lSt-Säule (Partikelgröße 5 μιη, Säulendimension 5 mm x 500 μιη) mittels HPLC-ESI-TOF (Impact HD, Bru- ker Daltonik) analysiert. Die Flussrate betrug 5 μΐ/min mit folgendem Eluentensystem: Eluent A: 0.1 % Ameisensäure in Wasser, Eluent B: 0.1 % Ameisensäure in 80% Isopropanol, 10% Acetonitril und 10% Wasser; Gradient: 0 min 22% B, 8 min 22% B, 10 min 24% B, 12 min 80% B, 18 min 95% B, 27 min 95% B, 30 min 22% B. To confirm the covalently bound FAB fragment, the denatured and reduced sample was mass spectrometrically after online desalting over a Grom-Sil 300 Butyl lSt column (particle size 5 μιη, column dimension 5 mm x 500 μιη) by HPLC-ESI-TOF (Impact HD, Bruker Daltonik). The flow rate was 5 μΐ / min with the following eluent system: Eluent A: 0.1% formic acid in water, eluent B: 0.1% formic acid in 80% isopropanol, 10% acetonitrile and 10% water; Gradient: 0 minutes 22% B, 8 minutes 22% B, 10 minutes 24% B, 12 minutes 80% B, 18 minutes 95% B, 27 minutes 95% B, 30 minutes 22% B.
Die über das TIC (total ion chromatogram)-Signal erhaltenen Spektren wurden addiert und das Molekulargewicht der verschiedenen Spezies auf Basis von MaxEnt-Dekonvolution kalkuliert. Durch Vergleich der erhaltenen Massen mit den theoretischen Massen von leichter Kette und schwerer Kette (VH-CH1) sowie des kovalent verknüpften FAB-Fragments konnte die erwünschte kovalente Verknüpfung des FAB-Fragments eindeutig bestätigt werden. Das resultierende Spektrum ist in Abbildung 13 wiedergegeben. The spectra obtained via the TIC (total ion chromatogram) signal were added together and the molecular weight of the different species calculated on the basis of MaxEnt deconvolution. By comparison of the obtained masses with the theoretical masses of light chain and heavy chain (VH-CH1) as well as the covalently linked FAB fragment, the desired covalent linkage of the FAB fragment could be clearly confirmed. The resulting spectrum is shown in Figure 13.
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Claims

Patentansprüche claims
1. Verfahren zur Herstellung von paarweise über Cystein- Aminosäuren C2-verbrückten Peptid- oder Proteinkonjugaten, dadurch gekennzeichnet, dass man ein Peptid oder Protein der Formel (II) 1. Process for the preparation of pairs of cysteine-amino acids C2-bridged peptide or protein conjugates, characterized in that a peptide or protein of the formula (II)
O l)  O l)
in welcher S1 und S2 in einer Disulfid-Brücke gebundene Cystein-Schwefelatome dieses Peptids oder Proteins darstellen, unter reduzierenden Bedingungen in ein Peptid oder Protein der Formel (III) in which S 1 and S 2 are cysteine-sulfur atoms of this peptide or protein bonded in a disulfide bridge, under reducing conditions into a peptide or protein of the formula (III)
(I II ) überführt und dieses dann unter radikalischen Reaktionsbedingungen mit einem Alkin- Derivat der Formel (IV)  (I II) and this then under radical reaction conditions with an alkyne derivative of the formula (IV)
(IV) in welcher  (IV) in which
R1 und R2 unabhängig voneinander für Wasserstoff, Alkyl, Cycloalkyl, Heterocycloalkyl, Aryl, Heteroaryl, Hydroxy, Alkoxy, Amino, Alkylamino, Dialkylamino, Hydroxy- carbonyl, Alkoxycarbonyl, Alkylcarbonylamino oder Alkoxycarbonylamino stehen, wobei Alkyl, Cycloalkyl, Heterocycloalkyl, Aryl, Heteroaryl, Alkoxy, Alkylamino, Dialkylamino, Alkoxycarbonyl, Alkylcarbonylamino und Alkoxycarbonylamino ihrerseits ein- oder mehrfach, gleich oder verschieden, mit Halogen, Hydroxy, Alkoxy, Amino, Alkylamino, Dialkylamino, Hydroxycarbonyl, Alkoxycarbonyl, Alkylcarbonylamino und Alkoxycarbonylamino substituiert sein können, R 1 and R 2 are independently hydrogen, alkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, hydroxy, alkoxy, amino, alkylamino, dialkylamino, hydroxy carbonyl, alkoxycarbonyl, alkylcarbonylamino or alkoxycarbonylamino, where alkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, alkoxy, alkylamino, dialkylamino, alkoxycarbonyl, alkylcarbonylamino and alkoxycarbonylamino themselves in turn one or more times, identically or differently, with halogen, hydroxyl, alkoxy, amino, Alkylamino, dialkylamino, hydroxycarbonyl, alkoxycarbonyl, alkylcarbonylamino and alkoxycarbonylamino may be substituted,
A für eine Bindung oder eine Kohlenwasserstoffkette mit 1 bis 100 Kohlenstoffatomen aus Alkylen-, Cycloalkylen- und/oder Arylengruppen steht, die ein- oder mehrfach, gleich oder verschieden, durch eine Gruppe ausgewählt aus -O-, -S-, -S(=0)-, -S(=0)2-, -NH-, -N(CH3)-, -C(=0)-, -NH-C(=0)-, -C(=0)-NH-, -0-C(=0)-, -C(=0)-0-, -SO2-NH-, -NH-SO2-, -NH-NH-, -SO2-NH-NH-, -NH-NH-SO2-, -C(=0)-NH-NH-, -NH-NH-C(=0)-, -NH-C(=0)-NH-, -0-C(=0)-NH-, -NH-C(=0)-0- und einem 4- bis 10-gliedrigen, aromatischen oder nicht-aromatischen Heterocyclus mit bis zu 4 Heteroatomen aus der Reihe N, O, S, S(=0) und/oder S(=0)2 unterbrochen sein kann, oder A is a bond or a hydrocarbon chain having 1 to 100 carbon atoms of alkylene, cycloalkylene and / or arylene groups which are mono- or polysubstituted, identically or differently, by a group selected from -O-, -S-, -S ( = 0) -, -S (= O) 2 -, -NH-, -N (CH 3 ) -, -C (= O) -, -NH-C (= O) -, -C (= O) -NH-, -O-C (= O) -, -C (= O) -O-, -SO 2 -NH-, -NH-SO 2 -, -NH-NH-, -SO 2 -NH-NH-, -NH-NH-SO2-, -C (= O) -NH-NH-, -NH-NH-C (= O) -, -NH-C (= O) -NH-, -O-C (= 0) -NH-, -NH-C (= O) -O- and a 4- to 10-membered, aromatic or non-aromatic heterocycle having up to 4 heteroatoms from the series N, O, S, S (= 0 ) and / or S (= 0) 2 may be interrupted, or
R2 und A miteinander verknüpft sind und zusammen mit den Kohlenstoffatomen, die sich zwischen ihnen befinden, einen 8-gliedrigen Carbocyclus bilden, welcher mit einem 3- bis 6-gliedrigen Cycloalkyl-Ring anelliert sein kann, wobei der 8-gliedrige Carbocyclus und gegebenenfalls der anellierte Cycloalkyl- Ring ein- oder mehrfach, gleich oder verschieden, mit Fluor, Alkyl, Hydroxy, Hydroxyalkyl und Alkoxy substituiert sein können, R 2 and A are linked together and together with the carbon atoms between them form an 8-membered carbocycle which may be fused to a 3- to 6-membered cycloalkyl ring, the 8-membered carbocycle and optionally the fused cycloalkyl ring may be monosubstituted or polysubstituted, identically or differently, by fluorine, alkyl, hydroxy, hydroxyalkyl and alkoxy,
L für eine Bindung oder einen Linker steht, L is a bond or a linker,
X n-fach vorhanden sein kann und ein Wirkstoffmolekül, Polymer, Alkaloid, Peptid, Protein, Kohlenhydrat, Nucleotid, Nucleosid, Steroid, Terpen, Porphyrin, Chlorin, Corrin, Eicosanoid, Pheromon, Vitamin, Biotin, ein Farbstoffmolekül oder einen Kryptanden darstellt oder für Wasserstoff, Hydroxy, Alkoxy, Amino, Alkylamino, Dialkylamino, Hydroxycarbonyl, Alkoxycarbonyl, Alkylcarbonylamino, Alkoxycarbonylamino, Alkyl, Cycloalkyl, Heterocycloalkyl, Aryl oder Heteroaryl steht, wobei Alkyl seinerseits ein- oder mehrfach, gleich oder verschieden, mit Halogen, Hydroxy, Alkoxy, Amino, Alkylamino, Dialkylamino, Hydroxycarbonyl, Alkoxy- carbonyl, Alkylcarbonylamino und Alkoxycarbonylamino substituiert sein kann und wobei Cycloalkyl, Heterocycloalkyl, Aryl und Heteroaryl ihrerseits ein- oder mehrfach, gleich oder verschieden, mit Halogen, Alkyl, Hydroxy, Alkoxy, Amino, Alkylamino, Dialkylamino, Hydroxycarbonyl, Alkoxycarbonyl, Alkylcarbonylamino und Alkoxycarbonylamino substituiert sein können, und n für eine ganze Zahl im Bereich von 1 bis einschließlich 10 steht, wobei im Fall, dass die Gruppe X mehrfach vorhanden ist, ihre individuellen Bedeutungen gleich oder verschieden sein können, n Konjugat der Formel (I) X can be present n times and represents a drug molecule, polymer, alkaloid, peptide, protein, carbohydrate, nucleotide, nucleoside, steroid, terpene, porphyrin, chlorin, corrin, eicosanoid, pheromone, vitamin, biotin, a dye molecule or a cryptand or represents hydrogen, hydroxyl, alkoxy, amino, alkylamino, dialkylamino, hydroxycarbonyl, alkoxycarbonyl, alkylcarbonylamino, alkoxycarbonylamino, alkyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl, in which alkyl may in turn be monosubstituted or polysubstituted, identically or differently, by halogen, hydroxyl, alkoxy, amino, alkylamino, dialkylamino, hydroxycarbonyl, alkoxycarbonyl, alkylcarbonylamino and alkoxycarbonylamino, and cycloalkyl, heterocycloalkyl, aryl and heteroaryl in turn may be the same or different, may be substituted by halogen, alkyl, hydroxy, alkoxy, amino, alkylamino, dialkylamino, hydroxycarbonyl, alkoxycarbonyl, alkylcarbonylamino and alkoxycarbonylamino, and n is an integer in the range of 1 to 10 inclusive, in which case in that the group X is present more than once, their individual meanings may be the same or different, n conjugate of the formula (I)
in welcher R1, R2, A, L, X und n die oben angegebenen Bedeutungen haben, umsetzt. in which R 1 , R 2 , A, L, X and n have the meanings given above, is reacted.
Peptid- oder Protein-Konjugat der allgemeinen Formel (I) Peptide or protein conjugate of the general formula (I)
in welcher in which
S1 und S2 vormals in einer Disulfid-Brücke gebundene Cystein-Schwefelatome eines Peptids oder Proteins darstellen, S 1 and S 2 are previously in a disulfide bridge bound cysteine sulfur atoms of a peptide or protein,
R1 und R2 unabhängig voneinander für Wasserstoff, Alkyl, Cycloalkyl, Heterocycloalkyl, Aryl, Heteroaryl, Hydroxy, Alkoxy, Amino, Alkylamino, Dialkylamino, Hydroxy- carbonyl, Alkoxycarbonyl, Alkylcarbonylamino oder Alkoxycarbonylamino stehen, wobei Alkyl, Cycloalkyl, Heterocycloalkyl, Aryl, Heteroaryl, Alkoxy, Alkylamino, Dialkylamino, Alkoxycarbonyl, Alkylcarbonylamino und Alkoxycarbonylamino ihrerseits ein- oder mehrfach, gleich oder verschieden, mit Halogen, Hydroxy,R 1 and R 2 independently of one another are hydrogen, alkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, hydroxyl, alkoxy, amino, alkylamino, dialkylamino, hydroxycarbonyl, alkoxycarbonyl, alkylcarbonylamino or alkoxycarbonylamino, where alkyl, cycloalkyl, heterocycloalkyl, aryl, Heteroaryl, alkoxy, alkylamino, dialkylamino, alkoxycarbonyl, alkylcarbonylamino and alkoxycarbonylamino, in turn, one or more times, identically or differently, with halogen, hydroxyl,
Alkoxy, Amino, Alkylamino, Dialkylamino, Hydroxycarbonyl, Alkoxycarbonyl, Alkylcarbonylamino und Alkoxycarbonylamino substituiert sein können, Alkoxy, amino, alkylamino, dialkylamino, hydroxycarbonyl, alkoxycarbonyl, alkylcarbonylamino and alkoxycarbonylamino may be substituted,
A für eine Bindung oder eine Kohlenwasserstoffkette mit 1 bis 100 Kohlenstoffatomen aus Alkylen-, Cycloalkylen- und/oder Arylengruppen steht, die ein- oder mehrfach, gleich oder verschieden, durch eine Gruppe ausgewählt aus -O-, -S-,A represents a bond or a hydrocarbon chain having 1 to 100 carbon atoms from alkylene, cycloalkylene and / or arylene groups which are mono- or polysubstituted, identically or differently, by a group selected from -O-, -S-,
-S(=0)-, -S(=0)2-, -NH-, -N(CH3)-, -C(=0)-, -NH-C(=0)-, -C(=0)-NH-, -0-C(=0)-, -C(=0)-0-, -SO2-NH-, -NH-SO2-, -NH-NH-, -SO2-NH-NH-, -NH-NH-SO2-, -C(=0)-NH-NH-, -NH-NH-C(=0)-, -NH-C(=0)-NH-, -0-C(=0)-NH-, -NH-C(=0)-0- und einem 4- bis 10-gliedrigen, aromatischen oder nicht-aromatischen Heterocyclus mit bis zu 4 Heteroatomen aus der Reihe N, O, S,-S (= O) -, -S (= O) 2 -, -NH-, -N (CH 3 ) -, -C (= O) -, -NH-C (= O) -, -C ( = 0) -NH-, -O-C (= O) -, -C (= O) -O-, -SO 2 -NH-, -NH-SO 2 -, -NH-NH-, -SO 2 -NH- NH, -NH-NH-SO 2, -C (= O) -NH-NH, -NH-NH-C (= O), -NH-C (= O) -NH-, -O- C (= O) -NH-, -NH-C (= O) -O- and a 4- to 10-membered, aromatic or non-aromatic heterocycle having up to 4 heteroatoms from the series N, O, S,
S(=0) und/oder S(=0)2 unterbrochen sein kann, oder S (= 0) and / or S (= 0) 2 may be interrupted, or
R2 und A miteinander verknüpft sind und zusammen mit den Kohlenstoffatomen, die sich zwischen ihnen befinden, einen 8-gliedrigen Carbocyclus bilden, welcher mit einem 3- bis 6-gliedrigen Cycloalkyl-Ring anelliert sein kann, wobei der 8-gliedrige Carbocyclus und gegebenenfalls der anellierte Cycloalkyl- Ring ein- oder mehrfach, gleich oder verschieden, mit Fluor, Alkyl, Hydroxy, Hydroxyalkyl und Alkoxy substituiert sein können, R 2 and A are linked together and, together with the carbon atoms between them, form an 8-membered carbocycle which may be fused to a 3- to 6-membered cycloalkyl ring, the 8-membered carbocycle and optionally the fused cycloalkyl ring may be monosubstituted or polysubstituted, identically or differently, with fluorine, alkyl, hydroxy, hydroxyalkyl and alkoxy,
L für eine Bindung oder einen Linker steht, X n-fach vorhanden sein kann und ein Wirkstoffmolekül, Polymer, Alkaloid, Peptid,L is a bond or a linker, X may be present n times and a drug molecule, polymer, alkaloid, peptide,
Protein, Kohlenhydrat, Nucleotid, Nucleosid, Steroid, Terpen, Porphyrin, Chlorin, Corrin, Eicosanoid, Pheromon, Vitamin, Biotin, ein Farbstoffmolekül oder einen Kryptanden darstellt oder für Wasserstoff, Hydroxy, Alkoxy, Amino, Alkylamino, Dialkylamino, Hydroxycarbonyl, Alkoxycarbonyl, Alkylcarbonylamino, Alkoxy- carbonylamino, Alkyl, Cycloalkyl, Heterocycloalkyl, Aryl oder Heteroaryl steht, wobei Alkyl seinerseits ein- oder mehrfach, gleich oder verschieden, mit Halogen, Hydroxy, Alkoxy, Amino, Alkylamino, Dialkylamino, Hydroxycarbonyl, Alkoxycarbonyl, Alkylcarbonylamino und Alkoxycarbonylamino substituiert sein kann und wobei Cycloalkyl, Heterocycloalkyl, Aryl und Heteroaryl ihrerseits ein- oder mehrfach, gleich oder verschieden, mit Halogen, Alkyl, Hydroxy, Alkoxy, Amino, Alkylamino, Dialkylamino, Hydroxycarbonyl, Alkoxycarbonyl, Alkylcarbonylamino und Alkoxycarbonylamino substituiert sein können, Protein, carbohydrate, nucleotide, nucleoside, steroid, terpene, porphyrin, chlorin, Corrin, eicosanoid, pheromone, vitamin, biotin, a dye molecule or a cryptand, or is hydrogen, hydroxy, alkoxy, amino, alkylamino, dialkylamino, hydroxycarbonyl, alkoxycarbonyl, alkylcarbonylamino, alkoxycarbonylamino, alkyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl where alkyl may in turn be monosubstituted or polysubstituted, identically or differently, by halogen, hydroxyl, alkoxy, amino, alkylamino, dialkylamino, hydroxycarbonyl, alkoxycarbonyl, alkylcarbonylamino and alkoxycarbonylamino, and where cycloalkyl, heterocycloalkyl, aryl and heteroaryl are themselves mono- or polysubstituted , identical or different, may be substituted by halogen, alkyl, hydroxy, alkoxy, amino, alkylamino, dialkylamino, hydroxycarbonyl, alkoxycarbonyl, alkylcarbonylamino and alkoxycarbonylamino,
für eine ganze Zahl im Bereich von 1 bis einschließlich 10 steht, wobei im Fall, dass die Gruppe X mehrfach vorhanden ist, ihre individuellen Bedeutungen gleich oder verschieden sein können. is an integer in the range of 1 to 10 inclusive, and in the case that the group X is multiple, their individual meanings may be the same or different.
Peptid- oder Protein-Konjugat, wie in Anspruch 2 definiert, zur Diagnose und/oder Behandlung von Erkrankungen. A peptide or protein conjugate as defined in claim 2 for the diagnosis and / or treatment of diseases.
Peptid- oder Protein-Konjugat, wie in Anspruch 2 definiert, zur Verwendung in einem Verfahren zur Diagnose und/oder Behandlung von Krebs- und Tumorerkrankungen. A peptide or protein conjugate as defined in claim 2 for use in a method for the diagnosis and / or treatment of cancer and tumor diseases.
Pharmazeutische Zusammensetzung enthaltend ein Peptid- oder Protein-Konjugat, wie in Anspruch 2 definiert, in Kombination mit einem oder mehreren inerten, nicht-toxischen, pharmazeutisch geeigneten Hilfsstoffen. A pharmaceutical composition containing a peptide or protein conjugate as defined in claim 2, in combination with one or more inert non-toxic pharmaceutically acceptable excipients.
Pharmazeutische Zusammensetzung nach Anspruch 5 zur Diagnose und/oder Behandlung von Krebs- und Tumorerkrankungen. Pharmaceutical composition according to claim 5 for the diagnosis and / or treatment of cancer and tumor diseases.
7. Verfahren zur Diagnose und/oder Behandlung von Krebs- und Tumorerkrankungen unter Verwendung eines Peptid- oder Protein-Konjugats, wie in Anspruch 2 definiert, oder einer pharmazeutischen Zusammensetzung, wie in Anspruch 5 definiert. A method of diagnosing and / or treating cancer and tumor diseases using a peptide or protein conjugate as defined in claim 2 or a pharmaceutical composition as defined in claim 5.
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