Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
  • C07K7/02—Linear peptides containing at least one abnormal peptide link
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
  • A61K38/08—Peptides having 5 to 11 amino acids
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
  • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
  • C07K5/02—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link
  • C07K5/0205—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link containing the structure -NH-(X)3-C(=0)-, e.g. statine or derivatives thereof
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• the present application relates to novel derivatives of monomethylauristatin F, to processes for the preparation of these derivatives, to the use of these derivatives for the treatment and / or prevention of diseases and to the use of these derivatives for the production of medicaments for the treatment and / or prevention of diseases, in particular hyperproliferative and / or angiogenic diseases such as cancer.
• Such treatments may be monotherapy or in combination with other medicines or other therapeutic measures.
• Cancers are the result of uncontrolled cell growth of various tissues. In many cases, the new cells invade existing tissues (invasive growth) or they metastasize to distant organs. Cancers occur in various organs and often have tissue-specific disease courses. Therefore, the term cancer as a generic term describes a large group of defined diseases of various organs, tissues and cell types. If necessary, early stage tumors may be removed by surgical and radiotherapeutic measures. As a rule, metastatic tumors can only be treated palliatively by chemotherapeutic agents. The goal here is to achieve the optimal combination of improving the quality of life and extending the lifetime.
• chemotherapeutic agents administered parenterally today are often not targeted to tumor tissue or tumor cells, but are nonspecifically distributed in the body by systemic administration, i. even in places where drug exposure is undesirable, such as in healthy cells, tissues, and organs. This can lead to unwanted side effects and even serious general toxic effects, which then often severely limit the therapeutically useful dose range of the drug or require a complete discontinuation of the medication.
• trastuzumab Herceptin
• rituximab Renitux
• cetuximab Erbitux
• bevacizumab Avastin
• ligands from the active substance region of small molecules which bind selectively to a specific target such as a receptor
• a specific target such as a receptor
• conjugates of cytotoxic drug and addressing ligand which have a defined cleavage site between ligand and drug to release the drug.
• a "target breakpoint" may be, for example, in a peptide chain which can be selectively cleaved at a particular site by a specific enzyme at the site of action [see, e.g. R.A. Firestone and L.A. Telan, US Patent Application US 2002/0147138].
• Auristatin E (AE) and monomethylauristatin E (MMAE) are synthetic analogues of the dolastatins, a special group of linear pseudopeptides originally isolated from marine sources, some of which have potent cytotoxic activity against tumor cells [for review see, eg, GR Pettit , Prog. Chem. Org. Nat. Prod. 70, 1-79 (1997); GR Pettit et al, Anti-Cancer Drug Design 10, 529-544 (1995); GR Pettit et al, Anti-Cancer Drug Design 13, 243-277 (1998)].
• MMAE Monomethylauristatin E
• MMAE has the disadvantage of a comparatively high systemic toxicity.
• this compound when used in the form of antibody-drug conjugates (immunoconjugates), this compound is not compatible with linking units (linkers) between antibody and active substance which do not have an enzymatically cleavable desired breakpoint [S. Doronina, et al., Bioconjugate Chem. 17, 1 14-124 (2006)].
• Monomethylauristatin F is an auristatin derivative having a C-terminal phenylalanine moiety which has only moderate anti-proliferative activity compared to MMAE. This is most likely due to the free carboxyl group which, due to its polarity and charge, adversely affects the cellularity of this compound.
• MMAF-OMe methyl ester of MMAF
• MMAF-OMe methyl ester of MMAF
• MMAF-OMe has been described as a neutrally charged, prodrug cell-permeable derivative that exhibits several orders of magnitude increased in vitro cytotoxicity to various carcinoma cell lines compared to MMAF [S. Doronina, et al., Bioconjugate Chem. 17, 114-124 (2006)]. It is believed that this effect is caused by MMAF itself, which is rapidly released by intracellular ester hydrolysis after uptake of the prodrug into the cells.
• active compound compounds based on simple ester derivatives are generally subject to the risk of chemical instability due to an unspecific ester hydrolysis independent of the intended site of action, for example by esterases present in the blood plasma; this can significantly limit the applicability of such compounds in therapy. It was therefore an object of the present invention, starting from the only moderately effective monomethylauristatin F (MMAF), to identify new compounds and to provide them for the treatment, in particular, of cancers which, on the one hand, have markedly greater cytotoxic activity in whole cell assays and, on the other hand, increased plasma stability compared to simple ester derivatives such as MMAF-OMe.
• Such substances could be particularly suitable as toxophores for linking to proteins, such as antibodies, or even with low molecular weight ligands to anti-proliferative acting (immuno) conjugates th.
• MMAF Monomethylauristatin F
• auristatin analogues with a C-terminal, amidically substituted phenylalanine unit are described in WO 01/18032-A2.
• WO 02/088172-A2 and WO 2007/008603-A1 claim MMAF analogs which relate to side-chain modifications of phenylalanine, and in WO 2007/008848-A2 those in which the carboxyl group of the phenylalanine is modified.
• Auristatin conjugates linked via the C-terminus have recently been described in WO 2009/117531 -AI [see also S.O. Doronina et al., Bioconjugate Chem. 19, 1960-1963 (2008)].
• R 1 is hydrogen, (C 1 -C 6 ) -alkyl or a group of the formula Q'-L 1 - * or Q 2 -L 2 - *, in which * denotes the point of attachment to the nitrogen atom,
• Q 1 denotes hydroxycarbonyl, (C 1 -C 4 -alkoxycarbonyl or benzyloxycarbonyl,
• L 1 is straight-chain (Ci-C 2) alkanediyl is meant, which can be up to tetrasubstituted by methyl and in which (a) two carbon atoms in 1, 2-, 1, 3- or 1, 4-Rela- tion to each other at Inclusion of any carbon atoms between them may be bridged to a (C 3 -C 6) -cycloalkyl ring or a phenyl ring or (b) up to three mutually non-adjacent CH 2 groups may be substituted by -O-,
• Q 2 is hydroxy, amino or mono- (C 1 -C 4) -alkylamino
• L 2 is straight-chain (C 2 -C 2) represents alkanediyl which may be up to tetrasubstituted by methyl and in which (a) two carbon atoms in 1, 2-, 1, 3- or 1, 4-Rela- tion to each other can be bridged to a (C 3 -C 6) -cycloalkyl ring or a phenyl ring, including the carbon atoms which may be present between them, or (b) up to three mutually non-adjacent CH 2 groups can be exchanged for -O-,
• R 2 is methyl or hydroxy
• R 3 is hydrogen or methyl
• R 4 isopropyl, isobutyl, ec-butyl, tert. Butyl, phenyl, benzyl, 4-hydroxybenzyl, 4-hydroxy-3-nitrobenzyl, 1-phenylethyl, diphenylmethyl, 1H-imidazol-4-ylmethyl or 1H-indol-3-ylmethyl, or
• R 3 and R 4 together with the carbon atom to which they are both bonded, a 2-phenyl-cyclopropane-l, l -diyl group of the formula
• R 5 and R 6 are each hydrogen, hydroxy, (C 1 -C 4 -alkoxy or benzyloxy, and
• R 7 is hydrogen, fluorine, chlorine, cyano, methyl or trifluoromethyl, and their salts, solvates and solvates of the salts.
• Compounds according to the invention are the compounds of the formula (I) and their salts, solvates and solvates of the salts comprising the compounds of the formulas below and their salts, solvates and solvates of the salts and of the formula (I) encompassed by formula (I), hereinafter referred to as exemplary compounds and their salts, solvates and solvates of the salts, as far as the compounds of formula (I), the compounds mentioned below are not already salts, solvates and solvates of the salts.
• the compounds according to the invention may exist in different stereoisomeric forms, ie in the form of configurational isomers or optionally also as conformational isomers (enantiomers and / or diastereomers, including rather with atropisomers).
• the present invention therefore includes the enantiomers and diastereoisomers and their respective mixtures. From such mixtures of enantiomers and / or diastereomers, the stereoisomerically uniform constituents can be isolated in a known manner; Preferably, chromatographic methods are used for this, in particular HPLC chromatography on achiral or chiral phase.
• the present invention encompasses all tautomeric forms.
• Salts used in the context of the present invention are physiologically acceptable salts of the compounds according to the invention. Also included are salts which are themselves unsuitable for pharmaceutical applications but can be used, for example, for the isolation or purification of the compounds of the invention.
• Physiologically acceptable salts of the compounds of the invention include acid addition salts of mineral acids, carboxylic acids and sulfonic acids, e.g. Salts of hydrochloric, hydrobromic, sulfuric, phosphoric, methanesulfonic, ethanesulfonic, benzenesulfonic, toluenesulfonic, naphthalenedisulfonic, acetic, trifluoroacetic, propionic, lactic, tartaric, malic, citric, fumaric, maleic and benzoic acids.
• Salts of hydrochloric, hydrobromic, sulfuric, phosphoric, methanesulfonic, ethanesulfonic, benzenesulfonic, toluenesulfonic, naphthalenedisulfonic acetic, trifluoroacetic, propionic, lactic, tartaric, malic, citric, fumaric, maleic and benzoic acids.
• Physiologically acceptable salts of the compounds according to the invention also include salts of customary bases, such as, by way of example and by way of preference, alkali metal salts (for example sodium and potassium salts), alkaline earth salts (for example calcium and magnesium salts) and ammonium salts derived from ammonia or organic amines having from 1 to 16 carbon atoms.
• alkali metal salts for example sodium and potassium salts
• alkaline earth salts for example calcium and magnesium salts
• ammonium salts derived from ammonia or organic amines having from 1 to 16 carbon atoms such as, by way of example and by way of preference, alkali metal salts (for example sodium and potassium salts), alkaline earth salts (for example calcium and magnesium salts) and ammonium salts derived from ammonia or organic amines having from 1 to 16 carbon atoms.
• Atoms such as, by way of example and by way of preference, ethylamine, diethylamine, triethylamine, ethyldiisopropylamine, monoethanolamine, diethanolamine, triethanolamine, dicyclohexylamine, dimethylaminoethanol, procaine, dibenzylamine, N-methylpiperidine, N-methylmorpholine, arginine, lysine and 1,2-ethylenediamine.
• Solvates in the context of the invention are those forms of the compounds according to the invention which form a complex in the solid or liquid state by coordination with solvent molecules. Hydrates are a special form of solvates that coordinate with water. As solvates, hydrates are preferred in the context of the present invention.
• the present invention also includes prodrugs of the compounds of the invention.
• prodrugs refers to compounds which are themselves biologically active or inactive However, during their residence time in the body to be converted into compounds of the invention (for example, metabolically or hydrolytically).
• substituents have the following meaning: (C 1 -C 6) -alkyl and (C 1 -C 4 -alkyl in the context of the invention represent a straight-chain or branched alkyl radical having 1 to 6 or 1 to 4 carbon atoms a straight-chain or branched alkyl radical having 1 to 4 carbon atoms, by way of example and by preference: methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, 2-pentyl , 3-Pentyl, neopentyl, n-hexyl, 2-hexyl and 3-hexyl.CrCnVAlkandivj (CrC ⁇ -Alkandivi (CVCnVlkandiyl and (CyC ⁇ alkanediyl in
• C 1 -C 6 -cycloalkyl stands for a monocyclic, saturated cycloalkyl group having 3 to 6 carbon atoms, by way of example and by preference: cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl
• C 1 -C 4 -alkoxy represents a straight-chain or branched alkoxy radical having 1 The following may be mentioned by way of example and preferably: methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, ec-butoxy and tert-butoxy.
• (C 1 -C 4 -alkoxycarbonyl in the context of the invention represents a straight-chain or branched alkoxy radical having 1 to 4 carbon atoms which is linked via a carbonyl group [-C (0O) -] by way of example and by preference: methoxycarbonyl, ethoxycarbonyl, n-propoxycarbonyl, isopropoxycarbonyl, n-butoxycarbonyl and tert-butoxycarbonyl.
• Mono-fCrQValkylamino in the context of the invention represents an amino group having a straight-chain or branched alkyl substituent which has 1 to 4 carbon atoms.
• the meaning is independent of each other for all radicals which occur repeatedly. If radicals are substituted in the compounds according to the invention, the radicals can, unless otherwise specified, be monosubstituted or polysubstituted. Substitution with one or two identical or different substituents is preferred. Particularly preferred is the substitution with a substituent.
• R 1 is hydrogen, (C 1 -C 4 ) -alkyl or a group of the formula Q'-L 1 - * or Q 2 -L 2 - *, in which
• Q 1 denotes hydroxycarbonyl or (C 1 -C 4 -alkoxycarbonyl
• L 1 is straight-chain (Ci-Ci 2) -alkyl radical in which (A) two carbon atoms in 1, 3- or 1, 4-relationship to one another with the inclusion of one or of the two lying between them carbon atoms may be bridged to form a phenyl ring or (b) up to three mutually non-adjacent CH 2 groups can be exchanged for -O-,
• Q 2 is hydroxy, amino or methylamino
• L 2 is straight-chain (C 2 -C 2) -alkyl means, which may be mono- or disubstituted by methyl and in which up to three mutually non-adjacent CH 2 - groups by -O- may be replaced,
• R 2 is methyl or hydroxy
• R 3 is hydrogen
• R 4 is benzyl, 4-hydroxybenzyl, 1-phenylethyl or 1H-indol-3-ylmethyl, or
• R 3 and R 4 together with the carbon atom to which they are both bonded, a 2-phenyl-cyclopropane-l, l -diyl group of the formula form, in which
• R 1 represents hydrogen, methyl or a group of the formula Q'-L 1 - * or Q 2 -L 2 - *, in which the point of attachment to the nitrogen atom denotes
• L 1 denotes straight-chain (C 1 -C 6) -alkanediyl in which two carbon atoms in 1, 4 relation to each other can be bridged to form a phenyl ring, including the two carbon atoms lying between them,
• Q 2 is hydroxy or amino
• L 2 is straight-chain (C 2 -C 6) -alkanediyl, R 2 is methyl, R 3 is hydrogen,
• R 4 is benzyl, 1-phenylethyl or 1H-indol-3-ylmethyl, or
• R 3 and R 4 together with the carbon atom to which they are both bonded, are a (1S, 2R) -2-phenylcyclopropane-1,1-diyl group of the formula
• # 1 is the point of attachment to the adjacent nitrogen atom
• # 2 denote the site of attachment with the carbonyl group, and the ring A with the NO group contained therein for a mono- or bicyclic, optionally substituted heterocycle of the formula
• R 5 is hydrogen, hydroxy or benzyloxy, and their salts, solvates and solvates of the salts.
• R 1 , R 2 , R 3 , R 4 and the ring A have the meanings defined above and the C x carbon atom carrying the radicals R 3 and R 4 has the depicted S configuration, and their salts, solvates and solvates salts.
• the invention further provides a process for the preparation of the compounds of the formula (I) according to the invention, which comprises reacting a compound of the formula (II)
• PG is an amino-protecting group such as (9H-fluoren-9-ylmethoxy) carbonyl, tert-butoxycarbonyl or benzyloxycarbonyl, in an inert solvent with activation of the carboxyl function in (II) either
• R 1A has the abovementioned meaning of R 1 , but does not stand for hydrogen
• X is a leaving group such as, for example, chloride, bromide, iodide, mesylate, triflate or tosylate, in a compound of the formula (IC) according to the invention CH 3 O
• R has the meaning given above of R, but shortened in the alkyl chain length by a CH 2 unit, in the presence of a suitable reducing agent in a compound of the formula (ID) according to the invention
• Inert solvents for the coupling reactions are, for example, ethers such as Diethyl ether, diisopropyl ether, tert.
• activating / condensing agents for these couplings are carbodiimides, such as NN'-diethyl, NN'-dipropyl, NN'-diisopropyl, NN'-dicyclohexylcarbodiimide (DCC) or N- (3-dimethylarninoisopropyl) -N '-ethylcarbodiimide hydrochloride (EDC), phosgene derivatives such as NN'-carbonyldiimidazole (CDI) or isobutyl chloroformate, 1, 2-oxazolium compounds such as 2-ethyl-5-phenyl-l, 2-oxazolium-3-sulfate or 2 tert-butyl-5-methylisoxazolium perchlorate, acylamino compounds such as 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline, phosphorus compounds such as propanephosphonic anhydride, die
• the activating / condensing agent for such coupling reactions is preferably N- (3-dimethylanilinoisopropyl) -N'-ethylcarbodiimide hydrochloride (EDC) in combination with 1-hydroxybenzotriazole (HOBt) and N, N-diisopropylethylamine, or O- ( 7-azabenzotriazol-1-yl) -N, N, N ', N'-tetramethyluronium hexafluorophosphate (HATU) also used in conjunction with N, N-diisopropylethylamine.
• EDC 1-hydroxybenzotriazole
• HABt 1-hydroxybenzotriazole
• HATU 7-azabenzotriazol-1-yl
• HATU hexafluorophosphate
• the coupling reactions (II) + (III) -> (IV), (IV) + (V) -> (VI) and (II) + (VII) -> (VI) are usually in a temperature range of -20 ° C to + 60 ° C, preferably carried out at 0 ° C to + 40 ° C.
• the reactions can be carried out at normal, at elevated or at reduced pressure (for example from 0.5 to 5 bar); generally one works at normal pressure.
• Suitable inert solvents for the reaction (IB) + (VIII) -> ⁇ (IC) are suitable, for example, ethers such as diethyl ether, diisopropyl ether, methyl tert-butyl ether, tetrahydrofuran, 1,4-dioxane, 1,2-dimethoxyethane or bis - (2-methoxyethyl) ethers, hydrocarbons such as benzene, toluene, xylene, pentane, hexane, heptane, cyclohexane or petroleum fractions, or dipolar aprotic solvents such as acetone, methyl ethyl ketone, acetonitrile, dimethyl sulfoxide (DMSO), N, N-dimethylformamide (DMF) , N, N-dimethylacetamide (DMA), N, N'-dimethylpropyleneurea (DMPU), N-methyl
• Suitable bases for this alkylation reaction are in particular alkali metal hydroxides such as, for example, lithium, sodium or potassium hydroxide, alkali metal or alkaline earth metal carbonates such as lithium, sodium, potassium, calcium or cesium carbonate, or customary organic amines such as triethylamine, N-methylmorpholine, N-methylpiperidine, N, N-diisopropylethylamine, pyridine or 4-N, N-dimethylaminopyridine.
• potassium or cesium carbonate is used.
• an alkylation catalyst such as lithium bromide or iodide, sodium or potassium iodide, tetra-n-butylammonium bromide or iodide or Benzyltriethyl- ammonium bromide.
• the reaction (IB) + (VIII) -> (IC) is generally carried out in a temperature range of -20 ° C to + 60 ° C, preferably at 0 ° C to + 40 ° C.
• the reaction can be carried out at normal, at elevated or at reduced pressure (for example from 0.5 to 5 bar); usually one works at normal pressure.
• the reaction (IB) + (IX) -> (ID) takes place in the usual for a reductive amination, under the reaction conditions inert solvents, optionally in the presence of an acid and / or a dehydrating agent as a catalyst.
• Such solvents include, for example, Wise alcohols such as methanol, ethanol, n-propanol, isopropanol, n-butanol or tert. Butanol, ethers such as tetrahydrofuran, 1, 4-dioxane, 1, 2-dimethoxyethane or bis (2-methoxyethyl) ether, or other solvents such as dichloromethane, 1, 2-dichloroethane, N, N-dimethylformamide or water. It is also possible to use mixtures of these solvents.
• the solvent used is preferably a 1,4-dioxane / water mixture with addition of acetic acid or dilute hydrochloric acid as catalyst.
• reaction (I-B) + (IX) -> (I-D) is generally carried out in a temperature range from 0 ° C to + 120 ° C, preferably at + 50 ° C to + 100 ° C.
• the reaction may be carried out at normal, elevated or reduced pressure (e.g., from 0.5 to 5 bar); usually one works at normal pressure.
• R 1 , R 1A , R 1B , R 2 , R 4 , R 5 and / or R 6 optionally present functional groups - such as in particular amino, hydroxyl and carboxyl groups - can in the previously described process steps, if appropriate or required, also available in temporarily protected form.
• the introduction and removal of such protective groups is carried out here by customary methods known from peptide chemistry [see, for example, TW Greene and PGM Wuts, Protective Groups in Organic Synthesis, Wiley, New York, 1999; M. Bodanszky and A. Bodanszky, The Practice of Peptide Synthesis, Springer-Verlag, Berlin, 1984]. If several protected groups are present, their release may optionally be carried out simultaneously in a one-pot reaction or else in separate reaction steps.
• amino-protecting group is preferably tert.
• Butoxycarbonyl (Boc), benzyloxycarbonyl (Z) or (9H-fluoren-9-ylmethoxy) carbonyl (Fmoc); for a hydroxy or carboxyl function is preferably tert.
• Butoxycarbonyl group is usually carried out by treatment with a strong acid such as hydrogen chloride, hydrogen bromide or trifluoroacetic acid, in an inert solvent such as diethyl ether, 1,4-dioxane, dichloromethane or acetic acid; if appropriate, this reaction can also be carried out without addition of an inert solvent.
• a strong acid such as hydrogen chloride, hydrogen bromide or trifluoroacetic acid
• an inert solvent such as diethyl ether, 1,4-dioxane, dichloromethane or acetic acid
• this reaction can also be carried out without addition of an inert solvent.
• benzyl or benzyloxycarbonyl as a protective group these are preferably removed by hydrogenolysis in the presence of a suitable palladium catalyst, such as palladium on activated carbon.
• the (9H-fluoren-9-ylmethoxy) carbonyl group is generally cleaved off with the aid of a secondary amine base such as diethylamine or piperidine.
• a secondary amine base such as diethylamine or piperidine.
• the compounds of the formula (II) can be prepared by customary methods of peptide chemistry, for example by initially preparing N- (benzyloxycarbonyl) -L-valine of the formula (X)
• PG is an amino-protecting group such as (9H-fluoren-9-ylmethoxy) carbonyl, tert-butoxycarbonyl or benzyloxycarbonyl, to give a compound of formula (XIV)
• the hydrolysis of the ester group -C (O) OT in process step (XIV) -> (II) is carried out by conventional methods by treating the ester in an inert solvent with an acid or a base, in the latter variant the initially formed carboxylate salt is converted by subsequent addition of an acid in the free carboxylic acid.
• the cleavage is preferably carried out by means of an acid.
• the alkyl radical T in compound (XI) is chosen here so that the conditions of its cleavage are compatible with the protecting group PG used in each case from compound (XIII).
• the usual inorganic bases are suitable. These include in particular alkali metal or alkaline earth metal hydroxides such as lithium, sodium, potassium or barium hydroxide, or alkali metal or alkaline earth metal carbonates such as sodium, potassium or calcium carbonate. Preference is given to lithium, sodium or potassium hydroxide.
• Suitable acids for the ester cleavage are generally sulfuric acid, hydrochloric acid / hydrochloric acid, hydrobromic / hydrobromic acid, phosphoric acid, acetic acid, trifluoroacetic acid, toluenesulfonic acid, methanesulfonic acid or trifluoromethanesulfonic acid or mixtures thereof, optionally with the addition of water. Hydrogen chloride or trifluoroacetic acid are preferred in the case of a tert. Butyl ester and hydrochloric acid in a methyl ester. Suitable inert solvents for these reactions are water or the organic solvents customary for ester cleavage.
• These include preferably lower alcohols such as methanol, ethanol, n-propanol or isopropanol, ethers such as diethyl ether, tetrahydrofuran, 1,4-dioxane or 1, 2-dimethoxyethane, or other solvents such as dichloromethane, acetone, methyl ethyl ketone, N, N-dimethylformamide or dimethyl sulfoxide. It is also possible to use mixtures of these solvents. In the case of basic ester hydrolysis, preference is given to using mixtures of water with 1,4-dioxane, tetrahydrofuran, methanol, ethanol and / or dimethylformamide.
• lower alcohols such as methanol, ethanol, n-propanol or isopropanol
• ethers such as diethyl ether, tetrahydrofuran, 1,4-dioxane or 1, 2-dimethoxyethane
• the ester cleavage is generally carried out in a temperature range from -20 ° C to + 100 ° C, preferably at 0 ° C to + 50 ° C.
• the compounds of the formula (V) can be prepared in analogy to known processes, for example by reacting a protected amino acid of the formula (XV)
• the bases for the cycloalkylation in process step (XVIII) + (XIX) -> (XVII) are preferably alkali metal hydroxides, such as lithium, sodium or potassium hydroxide, or alkali metal or alkaline earth metal carbonates, such as lithium, sodium, potassium , Calcium or cesium carbonate used.
• Suitable inert solvents for this reaction are in particular alcohols such as methanol, ethanol, n-propanol, isopropanol, n-butanol or tert. Butanol, or dipolar aprotic solvents such as acetone, methyl ethyl ketone, N, N-dimethylformamide or dimethyl sulfoxide. Preferably, acetone is used.
• the reaction (XVIII) + (XIX) -> (XVII) is generally carried out in a temperature range from + 20 ° C to + 120 ° C, preferably at + 50 ° C to + 80 ° C.
• the reaction may be carried out at normal, elevated or reduced pressure (e.g., from 0.5 to 5 bar); usually one works at normal pressure.
• the coupling reaction (V) + (XX) - > (XXI) is in turn carried out under analogous conditions as described above in the coupling steps illustrated in processes [A] and [B].
• the compounds of formulas (III), (VIII), (IX), (X), (XI), (XIII), (XV), (XVI) and (XIX) including, where appropriate, chiral or diastereomeric forms thereof commercially available or as such described in the literature, or they can be prepared in a manner which is obvious to the person skilled in the art in analogy to methods published in the literature. Numerous detailed instructions and Bibliographical references for the preparation of the starting materials are also in the Experimental Section in the section on the preparation of the starting compounds and intermediates.
• separation of the compounds according to the invention into the corresponding enantiomers and / or diastereomers can advantageously also be carried out already at the stage of the compounds (V), (VI), (VII), (XV), (XVI ), (XVII), (XVIII) or (XXI), which are then further reacted in separated form according to the reaction steps described above.
• Such a separation of the stereoisomers can be carried out by customary methods known to the person skilled in the art. Prefers chromatographic methods are applied to achiral or chiral separation phases; Alternatively, in the case of free carboxylic acids as intermediates, it is also possible to separate them via diastereomeric salts with the aid of chiral bases.
• the compounds according to the invention have valuable pharmacological properties and can be used for the prevention and treatment of diseases in humans and animals.
• the compounds according to the invention Compared to monomethylauristatin F (MMAF), the compounds according to the invention have markedly greater cytotoxic activity and, moreover, have a significantly increased stability in plasma compared to known ester derivatives of MMAF, such as MMAF-OMe.
• the compounds of the invention are therefore due to this property profile in particular for the treatment of hyperproliferative diseases in humans and mammals in general.
• the compounds can inhibit, block, reduce or lower cell proliferation and cell division and, on the other hand, increase apoptosis.
• the hyperproliferative diseases for the treatment of which the compounds according to the invention can be used are, in particular, the group of cancer and tumor diseases.
• these include, but are not limited to, the following diseases: breast carcinomas and breast tumors (ductal and lobular forms, also in situ), respiratory tumors (small cell and non-small cell carcinoma, bronchial carcinoma), brain tumors (eg the brainstem and hypothalamic astrocytoma, medulloblastoma, ependymoma, as well as neuro-ectodermal and pineal tumors), tumors of the digestive organs (esophagus, stomach, gall bladder, small intestine, colon, rectum), liver tumors (including hepatocellular carcinoma, cholangiocarcinoma and mixed hepatocellular cholangiocarcinoma) , Tumors of the head and neck area (larynx, hypopharynx, nasopharynx, oropharyn
• intraocular melanoma and retinoblastoma tumors of the endocrine and exocrine glands (eg thyroid and parathyroid glands, pancreas and salivary gland), tumors of the urinary tract (bladder, penis, kidney, renal pelvis and ureteral tumors) and tumors of the reproductive organs (endometrium).
• endocrine and exocrine glands eg thyroid and parathyroid glands, pancreas and salivary gland
• tumors of the urinary tract bladedder, penis, kidney, renal pelvis and ureteral tumors
• tumors of the reproductive organs endometrium
• Cervical, ovarian, vaginal, vulvar and uterine carcinomas of the female as well as prostate and testicular carcinomas of the male include solid proliferative blood diseases and circulating blood cells such as lymphomas, leukemias and myeloproliferative disorders, e.g.
• lymphoma acute myeloid, acute lymphoblastic, chronic lymphocytic, chronic myelogenous and hairy cell leukemia; and AIDS-related lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, cutaneous T-cell lymphoma, Burkitt's lymphoma and central nervous system lymphoma.
• treatment or “treating” is used conventionally within the context of this invention and means the care, care and supervision of a patient with the aim of combating, reducing, alleviating or alleviating a disease or health deviation and improving living conditions that are affected by this disease, such as cancer.
• Another object of the present invention is thus the use of the compounds of the invention for the treatment and / or prevention of diseases, in particular the aforementioned diseases.
• Another object of the present invention is the use of the compounds of the invention for the manufacture of a medicament for the treatment and / or prevention of diseases, in particular the aforementioned diseases.
• Another object of the present invention is the use of the compounds of the invention in a method for the treatment and / or prevention of diseases, in particular the aforementioned diseases.
• Another object of the present invention is a method for the treatment and / or prevention of diseases, in particular the aforementioned diseases, using an effective amount of at least one of the compounds of the invention.
• the compounds according to the invention can be used alone or as needed in combination with one or more other pharmacologically active substances, as long as this combination does not lead to undesired and unacceptable side effects.
• Another object of the present invention are therefore pharmaceutical compositions containing at least one of the compounds of the invention and one or more other active ingredients, in particular for the treatment and / or prevention of the aforementioned diseases.
• the compounds of the present invention can be combined with known anti-hyperproliferative, cytostatic or cytotoxic substances for the treatment of cancers.
• suitable combination active ingredients are:
• the compounds of the invention may also be combined with biological therapeutics such as antibodies (e.g., Avastin, Rituxan, Erbitux, Herceptin).
• biological therapeutics such as antibodies (e.g., Avastin, Rituxan, Erbitux, Herceptin).
• the compounds according to the invention can also achieve positive effects in combination with anti-angiogenic therapies, for example with avastin, axitinib, recentin, regorafenib, sorafenib or sunitinib.
• Combinations with proteasome and mTOR inhibitors as well as combinations with antihormones and steroidal metabolic enzyme inhibitors are also particularly suitable because of their favorable side effect profile.
• the combination of compounds of the present invention with other cytostatic or cytotoxic agents can achieve the following objectives: improved efficacy in slowing down the growth of a tumor, reducing its size, or even its total elimination relative to one Single agent treatment;
• the compounds according to the invention can also be used in combination with a radiation therapy and / or a surgical intervention.
• compositions containing at least one compound of the invention are pharmaceutical compositions containing at least one compound of the invention, usually together with one or more inert, non-toxic, pharmaceutically suitable excipients, and their use for the purposes mentioned above.
• the compounds 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 compounds according to the invention can be administered in suitable administration forms.
• the compounds of the invention rapidly and / or modified donating application forms containing the compounds of the invention in crystalline and / or amorphized and / or dissolved form, such as tablets (uncoated or coated Tablets, for example with enteric or delayed-dissolving or insoluble coatings, which control the release of the compound of the invention), tablets or films / wafers, films / lyophilisates, capsules (for example hard or soft gelatine capsules), dragees, granules, rapidly disintegrating in the oral cavity Pellets, powders, emulsions, suspensions, aerosols or solutions.
• parenteral administration can be done bypassing a resorption step (eg, intravenous, intraarterial, intracardiac, intraspinal, or intralumbar) or with involvement of resorption (eg, intramuscular, subcutaneous, intracutaneous, percutaneous, or intraperitoneal).
• a resorption step eg, intravenous, intraarterial, intracardiac, intraspinal, or intralumbar
• suitable application forms include 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 to be applied films / wafers or capsules, suppositories, ear or eye preparations, vaginal capsules, aqueous suspensions ( Lotions, shake mixtures), lipophilic suspensions, ointments, creams, transdermal therapeutic systems (eg plasters), milk, pastes, foams, scattering powders, implants or stents.
• the compounds 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 adjuvants include, among others. Carriers (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 (for example albumin), stabilizers ( For example, antioxidants such as ascorbic acid), dyes (eg, inorganic pigments such as iron oxides) and flavor and / or odoriferous.
• Carriers for example microcrystalline cellulose, lactose, mannitol
• solvents for example liquid polyethylene glycols
• emulsifiers and dispersants or wetting agents for example sodium dodecy
• the dosage is about 0.01 to 100 mg / kg, preferably about 0.01 to 20 mg / kg and most preferably 0.1 to 10 mg / kg of body weight.
• Device type MS Micromass ZQ
• Device type HPLC HP 1100 Series
• UV DAD Column: Phenomenex Gemini 3 ⁇ 30 mm x 3.00 mm
• Eluent A 1 l of water + 0.5 ml of 50% strength formic acid
• eluent B 1 l of acetonitrile + 0.5 ml of 50% strength formic acid
• Flow 0.0 min 1 ml / min -> 2.5 min / 3.0 min / 4.5 min 2 ml / min
• Oven 50 ° C
• UV detection 210 nm.
• Method 5 Device: HP 1090 Series II; Column: Merck Chromolith SpeedROD RP-18e, 50 mm x 4.6 mm; Guard column: Merck Chromolith Guard Cartridge Kit RP-18e, 5 mm x 4.6 mm; Injection volume: 5 ⁇ ; Eluent A: 70% HC10 4 in water (4 ml / liter), eluent B: acetonitrile; Gradient: 0.00 min 20% B -> 0.50 min 20% B -> 3.00 min 90% B -> 3.50 min 90% B -> 3.51 min 20% B -> 4.00 min 20% B; Flow: 5 ml / min; Column temperature: 40 ° C.
• Method 6 HPLQ:
• Device Type MS Waters ZQ; Device type HPLC: Agilent 1 100 Series; UV DAD; Column: Thermo Hypersil GOLD 3 ⁇ 20mm x 4mm; Eluent A: 1 l of water + 0.5 ml of 50% strength formic acid, eluent B: 1 l of acetonitrile + 0.5 ml of 50% strength formic acid; Gradient: 0.0 min 100% A -> ⁇ 3.0 min 10% A -> ⁇ 4.0 min 10% A -> ⁇ 4.1 min 100% A (flow 2.5 ml / min); Oven: 55 ° C; Flow: 2 ml / min; UV detection: 210 nm.
• Device Type MS Waters ZQ; Device type HPLC: Agilent 1 100 Series; UV DAD; Column: Thermo Hypersil GOLD 3 ⁇ 20mm x 4mm; Eluent A: 1 l of water + 0.5 ml of 50% formic acid, eluent B: 1 l of acetonitrile + 0.5 ml of 50% formic acid; Gradient: 0.0 min 100% A -> ⁇ 2.0 min 60% A -> ⁇ 2.3 min 40% A -> 3.0 min 20% A -> 4.0 min 10% A -> 4.2 min 100% A (flow 2.5 ml / min ); Oven: 55 ° C; Flow: 2 ml / min; UV detection: 210 nm.
• the title compound can be prepared by literature procedures in several ways, see e.g. Pettit et al., Synthesis 1996, 719; Shioiri et al., Tetrahedron Lett. 1991, 32, 931; Shioiri et al., Tetrahedron 1993, 49, 1913; Koga et al., Tetrahedron Lett. 1991, 32, 2395; Vidal et al., Tetrahedron 2004, 60, 9715; Poncet et al., Tetrahedron 1994, 50, 5345. It was prepared here either as the free acid (as shown) or in the form of the corresponding dicyclohexylamine salt.
• the title compound can be prepared by literature procedures in several ways, see e.g. Pettit et al, J. Org. Chem. 1994, 59, 1796; Koga et al, Tetrahedron Lett. 1991, 32, 2395; Shioiri et al., Tetrahedron Lett. 1991, 32, 931; Shioiri et al., Tetrahedron 1993, 49, 1913.
• the title compound can be prepared according to literature procedures, see e.g. H. King, J. Chem. Soc. 1942, 432; it is also commercially available.
• the title compound can be prepared according to literature procedures, see e.g. H. King, J. Chem. Soc. 1942, 432.
• the title compound can be prepared in Boc-protected form according to literature procedure (see, e.g., C. Johnson et al., Tetrahedron Lett., 1998, 39, 2059); the deprotection was carried out in the usual way by treatment with trifluoroacetic acid and subsequent neutralization. Yield: 149 mg (89% of t.l.).
• the title compound can be prepared according to literature procedures, see e.g. N. Amlaiky, Synthesis 1982, 5, 426.
• the title compound was prepared according to a literature procedure (A. Ritter et al., J. Org. Chem. 1994, 59, 4602) starting from commercially available (IS, 2R) -l - [(tert-butoxycarbonyl) amino] -2 -phenylcyclopropanecarboxylic acid (C. Cativiela et al., Chirality 1999, 11, 583).
• the filter residue was taken up in 5 ml of dioxane / water (1: 1) and the solution was adjusted to pH 11 with 1 N sodium hydroxide solution. Under ultrasound treatment, a total of 349 mg (1.6 mmol) of di-tert-butyl dicarbonate were added in several portions, the pH of the solution being kept at 11. After completion of the reaction, the dioxane was evaporated and the aqueous solution was adjusted to a pH of 2-3 with citric acid. It was extracted twice with 50 ml of ethyl acetate. The organic phases were combined, dried over magnesium sulfate and concentrated in vacuo. The residue was taken up in diethyl ether and the product precipitated with pentane. Decantation separated the solvent. The residue was digested several times with pentane and finally dried under high vacuum. This gave 31 mg (93% of theory) of the title compound.
• the title compound was prepared in three steps analogously to the synthesis of intermediates 5 and 6 by coupling commercially available N- (tert -butoxycarbonyl) - ⁇ -phenyl-L-phenylalanine with 1,2- Oxazinan hydrochloride (starting compound 5), subsequent deprotection with trifluoroacetic acid and re-coupling with starting compound 1 produced.
• the final product was purified by preparative HPLC.
• Diastereomer 2 Yield: 86 mg (16% of theory)
• N - [(benzyloxy) carbonyl] -N-methyl-L-threonine was liberated from 237 mg (0.887 mmol) of its dicyclohexylamine salt by taking up in ethyl acetate and shaking with 5% aqueous sulfuric acid. The organic phase was dried over magnesium sulfate, filtered and concentrated.
• Example 1 Exemplary embodiments: Example 1
• the mixture was stirred at RT for 3 h.
• the batch was then poured into a mixture of half-saturated aqueous ammonium chloride solution and ethyl acetate.
• the organic phase was separated, washed successively with saturated sodium bicarbonate solution and saturated sodium chloride solution, dried over magnesium sulfate, filtered and concentrated.
• the mixture was stirred at RT overnight.
• the batch was then poured into a mixture of half-saturated aqueous ammonium chloride solution and ethyl acetate.
• the organic phase was separated, washed successively with saturated sodium bicarbonate solution and saturated sodium chloride solution, dried over magnesium sulfate, filtered and concentrated.
• the mixture was stirred at RT overnight.
• the batch was then poured into a mixture of half-saturated aqueous ammonium chloride solution and ethyl acetate.
• the organic phase was separated, washed successively with saturated sodium bicarbonate solution and saturated sodium chloride solution, dried over magnesium sulfate, filtered and concentrated.
• Example 8 The title compound was prepared in analogy to the synthesis of Example 8 by coupling starting compound 1 with intermediate 28, then deprotecting with trifluoroacetic acid, then coupling with intermediate 4 and finally deprotecting with piperidine. The final product was purified by preparative HPLC (eluent: acetonitrile / 0.1% aq. TFA). There were obtained 16.4 mg of the title compound.
• Example 8 The title compound was prepared in analogy to the synthesis of Example 8 by coupling starting compound 1 with intermediate 29, then deprotecting with trifluoroacetic acid, then coupling with intermediate 4 and finally deprotecting with piperidine. The final product was purified by preparative HPLC (eluent: acetonitrile / 0.1% aq. TFA). There were obtained 28 mg of the title compound.
• the title compound was prepared via three steps starting from intermediate 21, first by deprotection with trifluoroacetic acid, followed by coupling with intermediate 4, and finally deprotection with piperidine.
• the final product was purified by preparative HPLC (eluent: acetonitrile + 0.01% TFA / water + 0.01% TFA). 2.5 mg of the title compound were obtained.
• the title compound was prepared via three steps starting from intermediate 22, first by deprotection with trifluoroacetic acid, followed by coupling with intermediate 4, and finally deprotection with piperidine. Purification of the final product was by preparative HPLC (eluent: acetonitrile + 0.01% TFA / water + 0.01% TFA). There was obtained 20 mg of the title compound.
• the title compound was prepared via three steps starting from intermediate 23, first by deprotection with trifluoroacetic acid, followed by coupling with intermediate 4, and finally deprotection with piperidine.
• the final product was purified by preparative HPLC (eluent: acetonitrile / 0.1% aq. TFA). 9 mg of the title compound were obtained.
• the batch was then poured into a mixture of half-saturated aqueous ammonium chloride solution and ethyl acetate.
• the organic phase was separated, washed successively with saturated sodium bicarbonate solution and saturated sodium chloride solution, dried over sodium sulfate, filtered and concentrated.
• the residue was purified by preparative HPLC.
• Example 2 The title compound was prepared in analogy to the synthesis of Example 23 by reacting 50 mg of N-methyl-L-valyl-N- [(3R, 4S, 55) -3-methoxy-1 - ⁇ (25) -2- [(1R , 2R) - 1-methoxy-2-methyl-3 - ⁇ [(25) - 1- (1,2-oxazinan-2-yl) -1-oxo-3-phenylpropan-2-yl] amino ⁇ -3 -oxopropyl] pyrrolidin-1-yl ⁇ -5-methyl-1-oxoheptan-4-yl] -N-methyl-L-valine amide trifluoroacetic acid salt (Example 1) with 4-oxobutanoic acid.
• Example 17 The title compound was prepared in analogy to the synthesis of Example 23 by reacting 15 mg of N-methyl-L-valyl-N- [(3R, 4S, 5S) -3-methoxy-1 - ⁇ (25) -2 - [(1R , 2R) -1-methoxy-2-methyl-3 - ⁇ [(1S, 2R) -1- (1,2-oxazinan-2-ylcarbonyl) -2-phenylcyclopropyl] amino ⁇ -3-oxopropyl] pyrrolidine l -yl ⁇ -5-methyl-1-oxoheptan-4-yl] -N-methyl-L-valinamide trifluoroacetic acid salt (Example 17) with 4-formylbenzoic acid.
• Example 2 The title compound was prepared in analogy to the synthesis of Example 2 by reacting 68 mg of N-methyl-L-valyl-N- [(3R, 4S, 55) -3-methoxy-1 - ⁇ (25) -2- [( ⁇ R, 2R) - 1-methoxy-2-methyl-3 - ⁇ [(25) - 1 - (1, 2-oxazinan-2-yl) -1-oxo-3-phenylpropan-2-yl] amino ⁇ - 3 -oxopropyl] pyrrolidin-1-yl ⁇ -5-methyl-1-oxo-heptan-4-yl] -N-methyl-L-valine amide trifluoroacetic acid salt (Example 1) with fert. Butyl (2-oxoethyl) carbamate and subsequent cleavage of the Boc protective group with trifluoroacetic acid. Yield: 49 mg (62% of theory over two stages)
• Example 28 N- (3-Aminopropyl) -N-methyl-L-valyl-N- [(3R, 4S, 5S) -3-methoxy-1 - ⁇ (25) -2 - [(1R, 2R) -1 -methoxy-2-methyl-3 - ⁇ [(1S, 2R) -1- (1,2-oxazinan-2-ylcarbonyl) -2-phenylcyclopropyl] amino ⁇ -3-oxopropyl] pyrrolidin-1-yl ⁇ 5-methyl-1-oxoheptan-4-yl] -N-methyl-L-valinamide
• Example 1 The title compound was prepared analogously to the synthesis of Example 27 by reacting 25 mg (0.027 mmol) of N-methyl-L-valyl-N - [(3R, 4S, 5S) -3-methoxy-1 - ⁇ (2S) -2 - [(1R, 2R) -1-methoxy-2-methyl-3 - ⁇ [(1S, 2R) -1- (1,2-oxazinan-2-ylcarbonyl) -2-phenylcyclopropyl] amino ⁇ -3 - oxopropyl] pyrrolidin-1-yl ⁇ -5-methyl-1-oxoheptan-4-yl] -N-methyl-L-valine amide trifluoroacetic acid salt (Example 1 7) with benzyl (3-oxopropyl) carbamate and subsequent hydrogenolytic cleavage Z-protecting group (with 10% palladium on carbon as catalyst, in ethanol as solvent). Yield: 11 mg
• Example 2 The title compound was prepared in analogy to the synthesis of Example 23 by reacting 9.5 mg of N-methyl-L-valyl-N- [(3R, 4S, 55) -3-methoxy-1 - ⁇ (25) -2- [(1R , 2R) - 1-methoxy-2-methyl-3 - ⁇ [(25) - 1- (1,2-oxazinan-2-yl) -1-oxo-3-phenylpropan-2-yl] amino ⁇ -3 -oxopropyl] pyrrolidin-1-yl ⁇ -5-methyl-1-oxoheptan-4-yl] -N-methyl-L-valine amide trifluoroacetic acid salt (Example 1) with methyl 4-o-butanoate.
• Example 1 The title compound was prepared in analogy to the synthesis of Example 28 by reacting 20 mg (16 ⁇ ) of N-methyl-L-valyl-N- [(3R, 4S, 5S 3 -methoxy-1 - ⁇ (25) -2 - [ (1R, 2R) -1-methoxy-2-methyl-3 - ⁇ [(25) - 1 - (1,2-oxazinan-2-yl) -1-oxo-3-phenylpropan-2-yl] amino ⁇ 3 -oxopropyl] pyrrolidin-1-yl ⁇ -5-methyl-1-oxoheptan-4-yl] -N-methyl-L-valine amide trifluoroacetic acid salt (Example 1) with benzyl (6-oxohexyl) carbamate and subsequent hydrogenolytic cleavage of the Z-protecting group (with 10% palladium on carbon as catalyst, in methanol as solvent).
• Example 1 The title compound was prepared in analogy to the synthesis of Example 28 by reacting 200 mg (0.108 mmol) of N-methyl-L-valyl-N - [(3R, 4S, 5S) -3-methoxy-l- ⁇ (2S) -2 - [(1R, 2R) -1-methoxy-2-methyl-3 - ⁇ [(1S, 2R) -1- (1,2-oxazinan-2-ylcarbonyl) -2-phenylcyclopropyl] amino ⁇ -3 - oxopropyl] pyrrolidin-1-yl ⁇ -5-methyl-1-oxoheptan-4-yl] -N-methyl-L-valine amide trifluoroacetic acid salt (Example 1 7) with benzyl (6-oxohexyl) carbamate and subsequent hydrogenolytic cleavage Z-protecting group (with 5% palladium on carbon as catalyst, in methanol as solvent).
• the biological activity of the compounds according to the invention can be detected by in vitro and in vivo studies, as are known to the person skilled in the art.
• the pharmacological and pharmacokinetic properties of the compounds according to the invention can be determined by means of the assays described below:
• a defined cell number of the human renal cancer cell line 786-0 was seeded in a 96-well microtiter plate in complete medium (2500 or 7000 cells / well) and incubated overnight at 37 ° C / 5% CO 2 . After 18 hours, the seed medium was replaced with serum-free medium or medium containing 2% FCS. The treatment started with addition of the respective test substance in varying concentrations (10 -5 M to 10 -14 M) and incubation times of 48 h to 96 h were selected Proliferation was determined by means of the MTT assay (ATCC, Manassas, Virginia). USA, Catalog No.
• the MTT reagent was incubated with the cells for 4 h before lysis of the cells was performed overnight by addition of the detergent 570 nm
• the proliferation not with test substance but otherwise identically treated cells was defined as 100% value
• the data obtained from this test represent triplicate determinations and at least two independent experiments were performed.
• MMAF monomethylauristatin F
• HT29wt cell line A defined cell number of the human colon carcinoma cell line HT29wt (wild type) was seeded in a 96-well microtiter plate in complete medium (10% FCS-RPMI) (2500 cells / well) and overnight at 37 ° C / 5%> CO 2 incubated. After 18 hours, the seed medium was replaced with fresh medium with 10% FCS. The treatment started with the addition of the respective test substance. Of the substances to be tested dose-response curves were in a concentration range of 10 "5 M to 10" 14 M (l: 10 dilution series) was determined. Incubation times of 48 h to 96 h were chosen.
• Detection of proliferation was accomplished by the MTT assay (ATCC, Manassas, Virginia, USA, Catalog No. 30-1010K). At the end of the chosen incubation period, the MTT reagent was incubated with the cells for 4 h before lysis of the cells occurred overnight by addition of the detergent. The detection of the dye formed took place at 570 nm. Proliferation not with test substance but otherwise identically treated cells was defined as 100%. The data obtained from this test represent triplicate determinations and at least two independent experiments were performed.
• Cancer cells are degenerated cells that often lead to tumor formation through increased cell division.
• Microtubules form the spindle fibers of the spindle apparatus and are an essential part of the cell cycle.
• the controlled assembly and disassembly of microtubules allows the exact distribution of the chromosomes on the daughter cells and represents a continuously dynamic process. A disturbance of this dynamics leads to a defective cell division and ultimately to cell death.
• the increased cell division of cancer cells also makes them particularly susceptible to spindle fiber toxins, which are an integral part of chemotherapy.
• GTP is added to unpolymerized tubulin, allowing polymerization to occur spontaneously.
• the assay is based on the binding of the fluorophore 4 ', 6-diamidino-2-phenylindole (DAPI) to tubulin. Free and bound DAPI can be differentiated due to different emission spectra. Since DAPI shows a significantly higher affinity for polymerized compared to unpolymerized tubulin, tubule polymerization can be monitored by the increase in fluorescence of bound DAPI fluorophores.
• test substances dissolved in DMSO were diluted from their starting concentration of 10 mM to 1 ⁇ M in water.
• the assay also included polymerization-increasing paclitaxel and, on the other hand, polymerization-inhibiting vinblastine.
• Half-bottom 96-well perforated plates were used for the measurement.
• the kinetics of tubule polymerization were followed for 1 h at 37 ° C in a fluorimeter.
• the excitation wavelength was 355 nm, the emission was monitored at 460 nm.
• the fluorescence change per minute (AF / min) was calculated, which represents the rate of polymerization of the microtubules.
• the potency of the test substances was quantified by the respective reduction of the polymerization rate.
• Instrument Agilent 1200 with DAD, binary pump, autosampler, column oven and thermostat; Column: Kromasil 100 C18, 250 mm ⁇ 4 mm, 5 ⁇ m; Column temperature: 45 ° C; Eluent A: 5 ml of Chloric acid / L water, eluent B: acetonitrile; Gradient: 0-8 min 98% A, 2% B; 8-15 minutes 56% A, 44% B; 15-20 min 10% A, 90% B; 20-21 min 10% A, 90% B; 21-23 min 98% A, 2% B; 23-25 minutes 98% A, 2% B; Flow rate: 2 ml / min; UV detection: 220 nm.
• test substance was incubated in rat or human plasma at 37 ° C over a period of 5 h with gentle stirring. At various times (0, 2, 5, 10, 20, 30, 60, 120, 180, and 300 minutes), a 100 ⁇ aliquot was taken. After addition of internal standard (10 ⁇ ), the proteins were precipitated by adding 200 ⁇ acetonitrile and the mixture was centrifuged in an Eppendorf centrifuge for 5 minutes. After addition of 150 ⁇ l of ammonium acetate buffer pH 3 to 150 ⁇ l of the supernatant, the content of unaltered test substance was analyzed by means of LC / MSMS.
• Table 3 shows the half-lives (ti / 2 ) of representative exemplary embodiments in rat plasma determined from these data: TABLE 3
• MMAF-OMe monomethylauristatin F
• exemplary degradation of the compounds according to the invention in Examples 1, 17 and 21 was not observed after 24 h, whereas the methyl ester of monomethylauristatin F (MMAF-OMe) was degraded by about 20% during this period.
• MMAF-OMe monomethylauristatin F
• the cell permeability of a substance can be assayed by in vitro testing in a flux assay using Caco-2 cells [MD Troutman and DR Thakker, Pharm. Res. 20 (8), 1210-1224 (2003)] ,
• the cells were cultured on 24-hole filter plates for 15-16 days.
• the respective test substance in a HEPES buffer either apical (A) or basal (B) was added to the cells and incubated for 2 h. After 0 h and after 2 h, samples were taken from the cis and transcompartments. The samples were separated by HPLC (Agilent 1200, Boeblingen, Germany) using reverse phase columns.
• the HPLC system was coupled via a Turbo Ion spray interface to an API 4000 triple quadrupole mass spectrometer (Applied Biosystems Applera, Darmstadt, Germany).
• the permeability was evaluated on the basis of a P app value which was determined by means of the methods described by Schwab et al. calculated formula [D. Schwab et al., J. Med. Chem. 46, 1716-1725 (2003)].
• a substance was classified as actively transported when the ratio of P app (BA) to P app (AB) was> 2 or ⁇ 0.5.
• the respective test substance was added to the cells alone or in the presence of an inhibitor (such as ivermectin or verapamil) in a HEPES buffer either apically (A) or basal (B) and incubated for 2 h. After 0 h and after 2 h, samples were taken from the cis and transcompartments taken. The samples were separated by HPLC using reverse phase columns. The HPLC system was coupled to a triple quadrupole mass spectrometer API 3000 (Applied Biosystems Applera, Darmstadt, Germany) via a Turbo Ion Spray Interface. The permeability was evaluated on the basis of a P app value which was determined by means of the methods described by Schwab et al. calculated formula [D. Schwab et al., J. Med. Chem. 46, 1716-1725 (2003)]. A substance was classified as a P-gp substrate if the efflux ratio was P app (BA) to P app (AB)> 2.
• an inhibitor such as ivermectin or
• the efflux ratios in L-MDR1 and LLC-PK1 cells or the efflux ratio in the presence or absence of an inhibitor can be compared. If these values differ by more than a factor of 2, the substance in question is a P-gp substrate.
• the compounds according to the invention can be converted into pharmaceutical preparations as follows: Tablet:
• the mixture of compound of the invention, lactose and starch is granulated with a 5% solution (m / m) of the PVP in water.
• the granules are mixed after drying with the magnesium stearate for 5 minutes.
• This mixture is compressed with a conventional tablet press (for the tablet format see above).
• a pressing force of 15 kN is used as a guideline for the compression.
• Composition 1000 mg of the compound of the invention, 1000 mg of ethanol (96%), 400 mg of Rhodigel ® (xanthan gum of the firm FMC, Pennsylvania, USA) and 99 g of water.
• a single dose of 100 mg of the compound of the invention corresponds to 10 ml of oral suspension.
• the rhodigel is suspended in ethanol, the compound according to the invention is added to the suspension. While stirring, the addition of water. Stirring is continued for about 6 h until the swelling of the rhodule is complete.
• the compound of the invention is suspended in the mixture of polyethylene glycol and polysorbate with stirring. The stirring is continued until complete dissolution of the compound according to the invention. iv -Solution:
• the compound of the invention is dissolved at a concentration below saturation solubility in a physiologically acceptable solvent (e.g., isotonic saline, 5% glucose solution, and / or 30% PEG 400 solution).
• a physiologically acceptable solvent e.g., isotonic saline, 5% glucose solution, and / or 30% PEG 400 solution.
• the solution is sterile filtered and filled into sterile and pyrogen-free injection containers.

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