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
  • C07K7/50—Cyclic peptides containing at least one abnormal peptide link
  • C07K7/54—Cyclic peptides containing at least one abnormal peptide link with at least one abnormal peptide link in the ring
  • C07K7/56—Cyclic peptides containing at least one abnormal peptide link with at least one abnormal peptide link in the ring the cyclisation not occurring through 2,4-diamino-butanoic acid
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/04—Antibacterial agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

• the invention relates to cyclic Nonapeptidamide and process for their preparation and their use for the preparation of medicaments for the treatment and / or prophylaxis of diseases, in particular bacterial infectious diseases.
• the bacterial cell wall is synthesized by a number of enzymes (cell wall biosynthesis) and is essential for the survival or multiplication of microorganisms.
• the structure of this macromolecule, as well as the proteins involved in its synthesis, are highly conserved within the bacteria. Due to their essential nature and uniformity, cell wall biosynthesis is an ideal target for new antibiotics (D.W.Green, The bacterial cell wall as a source of antibacterial targets, Expert Opin. Ther. Targets, 2002, 6, 1-19).
• Vancomycin and penicillins are inhibitors of bacterial cell wall biosynthesis and are successful examples of the antibiotic potency of this principle. They have been used for several decades in the clinic for the treatment of bacterial infections, especially with Gram-positive pathogens. Due to the increasing incidence of resistant germs, e.g. Methicillin-resistant staphylococci, penicillin-resistant pneumococci, and vancomycin-resistant enterococci (F. Baquero, Gram-positive resistance: challenge for the development of new antibiotics, J.
• the present invention describes a new class of cell wall biosynthesis inhibitors without cross-resistance to known antibiotic classes.
• lysobactin and some derivatives are described as having antibacterial activity in US 4,754,018.
• the isolation and antibacterial activity of lysobactin is also described in EP-A-196 042 and JP 01132600.
• WO04 / 099239 describes derivatives of lysobactin having antibacterial activity.
• the antibacterial activity of lysobactin and katanosin A is further described in O'Sullivan, J. et al., J. Antibiot. 1988, 41, 1740-1744, Bonner, DP et al, J. Antibiot. 1988, 41, 1745-1751, Shoji, J. et al, J. Antibiot. 1988, 41, 713-718 and Tymiak, AA et al, J. Org. Chem. 1989, 54, 1149-1157.
• the stability of an active substance is an important parameter for its suitability as a medicinal product. The stability plays u. a role in the storage and presentation of drugs. Many natural products show inadequate stability for drugs.
• the antibacterial depsipeptide lysobactin hydrolyzes in aqueous neutral to basic medium (pH> 7) within days. This produces the “antibacterially inactive” "ope” lysobactin which is opened on the lactone. "Therefore, effective analogs of lysobactin with a higher ring stability are desirable.
• An object of the present invention is to provide alternative compounds to lysobactin having comparable or improved antibacterial activity, better compatibility, e.g. B. lower nephrotoxicity, and improved stability in an aqueous neutral to basic environment for the treatment of bacterial diseases in humans and animals to provide.
• lysobactinamides (cyclic nonapeptide amides) have an analogous antibacterial action as lysobactin and are stable to hydrolysis in an aqueous neutral to basic medium.
• Lysobactinamides are previously undescribed aza analogs of lysobactin in which the central lactone functionality is replaced by a lactam functionality.
• the invention relates to compounds of the formula
• R is hydrogen or methyl
• R 7 is a group of the formula
• R 1 is C 1 -C 6 -alkyl, C 2 -C 6 -alkenyl, C 3 -C 6 -cycloalkyl or C 6 -C 10 -aryl,
• alkyl, alkenyl, cycloalkyl and aryl may be substituted with 0, 1, 2 or 3 substituents independently selected from the group consisting of halogen, hydroxy, amino, cyano, trimethylsilyl, Ci-C 6 alkyl, C 1 -C 6 -alkoxy, benzyloxy, C 3 -C 6 cycloalkyl, C 6 -C 10 aryl, 5- to 7-membered heterocyclyl, 5- to 10-membered heteroaryl, Ci-C ö alkylamino, C 6 -C 10 - arylamino, C 1 -C 6 - alkylcarbonylamino, C 6 -C 10 aryl-carbonyl-arnmo, Ci-C ⁇ -alkylcarbonyl, Ci-C 6 - alkoxycarbonyl, C 6 -Cio-arylcarbonyl, and Be ⁇ zyloxy-carbonylamino,
• cycloalkyl, aryl, heterocyclyl and heteroaryl in which cycloalkyl, aryl, heterocyclyl and heteroaryl in turn may be substituted by 0, 1, 2 or 3 substituents independently aus ⁇ selected from the group consisting of halogen, hydroxy, amino, cyano, nitro, trifluoromethyl, C 1 -C 6 -AllCyI , C r C 6 alkoxy, phenyl and 5- to 7-membered heterocyclyl,
• R 2 is C r C 6 alkyl, C 2 -C 6 alkenyl, C 3 -C 6 -cycloalkyl or C 6 -C represents 0 aryl,
• alkyl, alkenyl, cycloalkyl and aryl may be substituted by 0, 1, 2 or 3 substituents independently of one another selected from the group consisting of halogen, hydroxyl, amino, cyano, trimethylsilyl, C 1 -C 6 -alkyl, C r C 6 - alkoxy, benzyloxy, C 3 -C 6 cycloalkyl, C 6 -Cio-aryl, 5- to 7-membered heterocyclyl, 5- to 10-membered heteroaryl, Ci-C 6 -alkylamino, C 6 -C 0 - arylamino, C r C 6 - alkylcarbonylamino, C ⁇ -Cio-arylcarbonyl-amino, C) -C 6 alkylcarbonyl, Ci-C 6 - Alkoxycarbonyl, Q-Cio-arylcarbonyl and benzyloxycarbonylamino,
• cycloalkyl, aryl, heterocyclyl and heteroaryl for their part may be substituted with 0, 1, 2 or 3 substituents independently aus ⁇ selected from the group consisting of halogen, hydroxy, amino, cyano, nitro, trifluoromethyl, C r C 6 alkyl, C r C 6 alkoxy, phenyl and 5- to 7-membered heterocyclyl,
• R 4 is C, -C 6 alkyl, C 2 -C 6 alkenyl, C 3 -C 6 -cycloalkyl or C 6 -C 1 0 aryl,
• alkyl, alkenyl, cycloalkyl and aryl may be substituted by 0, 1, 2 or 3 substituents independently of one another selected from the group consisting of halogen, hydroxyl, amino, cyano, trimethylsilyl, C 1 -C 6 -alkyl, C 1 -C 6 - alkoxy,
• cycloalkyl, aryl, heterocyclyl and heteroaryl for their part may be substituted with 0, 1, 2 or 3 substituents independently aus ⁇ selected from the group consisting of halogen, hydroxy, amino, cyano, nitro, trifluoromethyl, C r C 6 alkyl, C r C 6 alkoxy, phenyl and 5- to 7-membered heterocyclyl,
• R 5 is C 1 -C 6 -alkyl, C 2 -C 6 -alkenyl, C 3 -C 6 -cycloalkyl or C 6 -C 10 -aryl,
• alkyl, alkenyl, cycloalkyl and aryl may be substituted by 0, 1, 2 or 3 substituents independently of one another selected from the group consisting of halogen, hydroxyl, amino, cyano, trimethylsilyl, C 1 -C 6 -alkyl, C 1 -C 6 - -alkoxy, Ben ⁇ yloxy, C 3 -C 6 cycloalkyl, C 6 -C 0 aryl, 5- to 7-membered heterocyclyl, 5- to 10-membered heteroaryl, Ci-C 6 -alkylamino, C 6 -C 0 -Arylamino, C r C 6 -
• cycloalkyl, aryl, heterocyclyl and heteroaryl may in turn be substituted by 0, 1, 2 or 3 substituents independently of one another selected from the group consisting of halogen, hydroxyl, amino, cyano,
• Compounds of the invention are the compounds of the formula (I), (Ia) and (Ib) and their salts, solvates, solvates of the salts and prodrugs, the compounds of the formulas (I), (Ia) and (Ib) of the formulas mentioned below and their salts, solvates, solvates of the salts and prodrugs, and the compounds of formula (I), (Ia) and (Ib), hereinafter referred to as exemplary compounds and their salts, solvates, solvates of the salts and prodrugs, as far as they are of compounds (I), (Ia) and (Ib), compounds mentioned below are not already salts, solvates, solvates of the salts and prodrugs.
• the compounds of the invention may exist in stereoisomeric forms (enantiomers, diastereomers).
• the invention therefore relates to the enantiomers or diastereomers and their respective mixtures. From such mixtures of enantiomers and / or diastereomers, the stereoisomerically uniform components can be isolated in a known manner.
• the present invention encompasses all tautomeric forms.
• Salts in the context of the present invention are physiologically acceptable salts of the compounds according to the invention. But are also included salts that are not suitable for pharmaceutical applications themselves but can be used for example for the isolation or purification of the inventive compounds or mixed salts.
• mixed salt is understood as meaning an addition salt which contains two or more different acids or bases, such as, for example, B. a trifluoroacetate mesylate salt.
• 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 acid, hydrobromic acid, sulfuric acid, phosphoric acid, methanesulfonic acid, ethanesulfonic acid, toluenesulfonic acid, benzenesulfonic acid, naphthalenedisulfonic acid, acetic acid, trifluoroacetic acid, propionic acid, lactic acid, tartaric acid, malic acid, citric acid, fumaric acid, maleic acid and benzoic acid.
• Salts of Hydrochloric acid hydrobromic acid, sulfuric acid, phosphoric acid, methanesulfonic acid, ethanesulfonic acid, toluenesulfonic acid, benzenesulfonic acid, naphthalenedisulfonic acid, acetic acid, trifluoroacetic acid, propionic acid
• 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, as exemplified and preferably, ethylamine, diethylamine, triethylamine, ethyldiisopropylamine, monoethanolamine, diethanolamine, triethanolamine, dicyclohexylamine, dimethylaminoethanol, procaine, dibenzylamine, N-methyl morpholine, arginine, lysine, ethylenediamine and N-methylpiperidine.
• 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)
• 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.
• Alkyl per se and "Alk” and "alkyl” in alkoxy, alkylamino, alkylcarbonyl, alkoxycarbonyl and alkylcarbonylamino is a linear or branched alkyl radical having usually 1 to 6, preferably 1 to 4, particularly preferably 1 to 3 carbon atoms, by way of example and preferably methyl, ethyl, n-propyl, isopropyl, tert-butyl, 2,2-dimethylprop-1-yl, 2,2-dimethylbut-1-yl, n-pentyl and n-hexyl.
• Alkoxy is, by way of example and by way of preference, methoxy, ethoxy, n-propoxy, isopropoxy, tert-butoxy, n-pentoxy and n-hexoxy.
• Alkenyl represents a straight-chain or branched alkenyl radical having 2 to 6 carbon atoms. Preference is given to a straight-chain or branched alkenyl radical having 2 to 4, particularly preferably 2 to 3, carbon atoms. Examples which may be mentioned by way of example and by way of preference: vinyl, alumina, n-prop-1-en-1-yl, n-but-2-en-1-yl, 2-methylprop-1-en-1-yl and 2-methylpropyl 2-en-l-yl.
• Alkylamino is an alkylamino radical having one or two (independently selected) alkyl substituents, by way of example and preferably methylamino, ethylamino, n-
• C 1 -C 3 -alkylamino is, for example, a monoalkylamino radical having 1 to 3 carbon atoms or a dialkylamino radical having 1 to 3 carbon atoms each
• Arylamino is an aryl substituent bonded via an amino group, where the amino group may optionally have a further substituent, such as e.g. Aryl or alkyl, is exemplified and preferably phenylamino, ⁇ aphthylamino, phenylmethylamino or diphenylamino.
• Alkylcarbonyl is by way of example and preferably methylcarbonyl, ethylcarbonyl, n-propyl carbonyl, isopropylcarbonyl, tert-butylcarbonyl, n-pentylcarbonyl and n-hexylcarbonyl.
• Alkoxycarbonyl is exemplified and preferably methoxycarboiryl, ethoxycarbonyl, n-propoxycarbonyl, isopropoxycarbonyl, terf-butoxycarbonyl, n-pentoxycarborryl and n-hexoxycarbonyl.
• Arylcarbonyl is an aryl substituent bonded via a carbonyl group, by way of example and preferably phenylcarbonyl, naphthylcarbonyl and phenanthrertylcarbonyl.
• Alkylcarbonylamino is by way of example and preferably methylcarbonylamino, ethylcarbonylamino, n-propylcarbonylamino, isopropylcarbonylamino, tert-butylcarbonylamino, n-pentylcarbonylamino and n-hexylcarbonylamino.
• Arylcarbonylamino is by way of example and preferably phenylcarbonylamino, naphthylcarbonylamino and phenanthrenylcarbonylamino.
• Cycloalkyl represents a cycloalkyl group having usually 3 to 6 Kolilenstoffatomen, by way of example and preferably cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl.
• Aryl is a mono- to tricyclic aromatic, carbocyclic radical of usually 6 to 10 carbon atoms; by way of example and preferably for phenyl and naphthyl.
• Heterocyclyl is a mono- or polycyclic, preferably mono- or bicyclic, heterocyclic radical having usually 5 to 7 ring atoms and up to 3, preferably up to 2 heteroatoms and / or hetero groups from the series N, O, S, SO, SO 2 .
• the heterocyclyl radicals may be saturated or partially unsaturated.
• Heteroaryl is an aromatic, mono- or bicyclic radical having usually 5 to 10, preferably 5 to 6 ring atoms and up to 5, preferably up to 4 heteroatoms from the series S, O and N, by way of example and preferably for thienyl, furyl , Pyrrolyl, thiazolyl, oxazolyl, imidazolyl, pyridyl, pyrimidyl, pyridazinyl, indolyl, indazolyl, benzofuranyl, benzothiophenyl, quinolinyl and isoquinolinyl.
• Halogen is fluorine, chlorine, bromine and iodine, preferably fluorine and chlorine.
• Fig. 1 Single crystal X-ray structure of Example 12A, Ortep plot (50%).
• Fig. 2 1 H-NMR (500 MHz, dj-pyridine) of Example 21A
• R 6 is methyl
• R 7 is a group of the formula
• R 1 is 2-methylprop-1-yl, 2,2-dimethylprop-1-yl, 2,2-dimethylbut-1-yl,
• Trimethylsilylmethyl 1-hydroxy-2-methylprop-1-yl, 1-hydroxy-2,2-dimethylprop-1-yl, 1-hydroxy-2,2-dimethyl-but-1-yl, 1-hydroxy-2-yl ethyl-2-ethylbut-1-yl, 1-hydroxy-2,2-diethylbut-1-yl, phenylmethyl, 1-hydroxy-1-phenylmethyl, 2-pyridylmethyl or 3-pyridylmethane 1 means
• 2-pyridylmethyl or 3-pyridylmethyl can be substituted by 0, 1, 2 or 3 substituents independently of one another selected from the group consisting of hydroxyl, amino, trifluoromethyl, methyl, methoxy and morpholinyl,
• R 2 is 2-methylprop-1-yl, 2,2-dimethylprop-1-yl, 2 3 2-dimethylbut-1-yl,
• Trimethylsilylmethyl 1-hydroxy-2-methylprop-1-yl, 1-hydroxy; y-2,2-dimethylprop-1-yl, 1-hydroxy-2,2-dimethyl-but-1-yl, 1-hydroxy 2-ethyl-2-methylbut-1-yl, 1-hydroxy-2,2-diethylbut-1-yl, phenylmethyl, 1-hydroxy-1-phenylmethyl, 2-pyridylmethyl, 1 or 3 pyridine lmethy 1 means
• 2-pyridylmethyl or 3-pyridylmethyl can be substituted by 0, 1, 2 or 3 substituents independently of one another selected from the group consisting of hydroxyl, amino, trifluoromethyl, methyl, methoxy and morpholinyl
• R 4 is 2-methylprop-1-yl, 2,2-dimethylprop-1-yl, 2,2-dimethylbut-1-yl,
• 2-pyridylniethyl or 3-pyridylmethyl can be substituted by 0, 1, 2 or 3 substituents independently of one another selected from the group consisting of hydroxyl, amino, trifluoromethyl, methyl, methoxy and morpholinyl,
• R 5 is 2-methylprop-1-yl, 2,2-dimethylprop-1-yl, 2,2-dimethylbut-1-yl,
• Trimethylsilylmethyl 1-hydroxy-2-methylprop-1-yl, 1-hydroxy-2,2-dimethylprop-1-yl, 1-hydroxy-2,2-dimethylbut-1-yl, 1-hydroxy-2-yl ethyl-2-methylbut-1-yl, 1-hydroxy-2,2-diethylbut-1-yl, phenylmethyl, 1-hydroxy-1-phenylmethyl, 2-pyridylmethyl or 3-pyridylmethyl,
• R 6 is methyl
• R 7 is a group of the formula
• R 1 is 2-methylprop-1-yl, 2,2-dimethylprop-1-yl, trimethylsilylmethyl or 3-pyridylmethyl,
• 3-pyridylmethyl may be substituted by 0, 1 or 2 substituents independently of one another selected from the group consisting of hydroxyl, amino, trifluoromethyl, methyl, methoxy and morpholinyl,
• R 2 is 2-methylprop-1-yl, 2,2-dimethylprop-1-yl, trimethylsilylmethyl or 3-pyridylmethyl,
• 3-pyridylmethyl may be substituted by 0, 1 or 2 substituents independently of one another selected from the group consisting of hydroxyl, amino, trifluoromethyl, methyl, methoxy and morpholinyl,
• R 4 is 2-methylprop-1-yl, 2,2-dimethylprop-1-yl, trimethylsilylmethyl or 3-pyridylmethyl,
• 3-pyridylmethyl may be substituted by O, 1 or 2 substituents independently of one another selected from the group consisting of hydroxyl, amino, trifluoromethyl, methyl, methoxy and morpholinyl,
• R 5 is 2-methylprop-1-yl, 2,2-dimethylprop-1-yl, trimethylsilylmethyl or 3-pyridylmethyl,
• 3-pyridylmethyl may be substituted by O, 1 or 2 substituents independently of one another selected from the group consisting of hydroxyl, amino, trifluoromethyl, methyl, methox.y and morpholinyl,
• R 6 is methyl
• R 7 is a group of the formula
• R 1 and R 2 have the meaning given above.
• R 5 is methyl
• R 7 is a group of the formula
• R 4 and R 5 are as defined above.
• the invention also relates to compounds of the formula
• R 1 is C r C 6 alkyl, C 2 -C 6 alkenyl, C 3 -C 6 -cycloalkyl or C 6 -C, 0 aryl,
• alkyl, alkenyl, cycloalkyl and aryl may be substituted with 0, 1, 2 or 3 substituents independently selected from the group consisting of halogen,
• Cycloalkyl C ⁇ -Cio-aryl, 5- to 7-membered heterocyclyl, 5- to 10-membered heteroaryl,
• cycloalkyl, aryl, heterocyclyl and heteroaryl in which cycloalkyl, aryl, heterocyclyl and heteroaryl in turn may be substituted by 0, 1, 2 or 3 substituents independently of one another selected from the group consisting of halogen, hydroxyl, amixio, cyano, nitro, trifluoromethyl, C 1 -C 6 -alkyl, C 1 - Ce-AIkOXy, phenyl and 5- to 7-membered heterocyclyl,
• R 2 Ci-C 6 alkyl, C 2 -C 6 alkenyl, C 3 -C 6 -C 6 -cycloalkyl or represents C 0 aryl,
• alkyl, alkenyl, cycloalkyl and aryl may be substituted by 0, 1, 2 or 3 substituents independently of one another selected from the group consisting of halogen, hydroxyl, amino, cyano, trimethylsilyl, C 1 -C 6 -alkyl, C 1 -C 6 - Alkoxy; y, benzyloxy, C 3 -C 6 - Cycloalkyl, C 6 -C 10 aryl, 5- to 7-membered heterocyclyl, 5- to 10-membered heteroaryl, Ci-C ö -Alkylammo, C 6 -Cio-Arylammo, Ci-Ce-alkylcarbonylamino, C ⁇ -CIO Arylcarbonylamino, C 1 -C 6 -alkylcarbonyl, C 1 -C 6 -alkoxycarbonyl, C 6 -C 10 -arylcarbonyl and benzyloxycarbonylamino,
• cycloalkyl, aryl, heterocyclyl and heteroaryl in which cycloalkyl, aryl, heterocyclyl and heteroaryl in turn may be substituted by 0, 1, 2 or 3 substituents independently of one another selected from the group consisting of halogen, hydroxyl, amino, cyano, nitro, trifluoromethyl, C 1 -C 6 -alkyl, ci C 6 alkoxy, phenyl and 5- to 7-membered heterocyclyl,
• R 1 is 2-methylprop-1-yl, 2,2-dimethylprop-1-yl, 2,2-dimethylbut-1-yl, trimethylsilylmethyl, 1-hydroxy-2-methylprop-1-yl, 1-hydroxy-2 , 2-dimethylprop-1-yl, 1-hydroxy-2,2-dimethyl-but-1-yl, 1-hydroxy-2-ethyl-2-methylbut-1-yl, 1-hydroxy-2,2-diethylbutane -l-yl, phenylmethyl, 1-hydroxy-1-phenylmethyl, 2-pyridylmethyl or 3-pyridylmethyl,
• 2-pyridylmethyl or 3-pyridylmethyl may be substituted by 0, 1, 2 or 3 substituents independently of one another selected from the group consisting of hydroxyl, amino, trifluoromethyl, methyl, methoxy and morpholinyl,
• R 2 is 2-methylprop-1-yl, 2,2-dimethylprop-1-yl, 2,2-dimethylbut-1-yl, trimethylsilylmethyl, 1-hydroxy-2-methylprop-1-yl, 1-hydroxy -2,2-dimethylprop-1-yl, 1-hydroxy-2,2-dimethyl-1-but-1-yl, 1-hydroxy-2-ethyl-2-methylbut-1-yl, 1-hydroxy-2 , 2-diethylbut-1-yl, phenylmethyl, 1-hydroxy-1-phenylmethyl, 2-pyridylmethyl or 3-pyridylmethyl,
• 2-pyridylmethyl or 3-pyridylmethyl may be substituted by 0, 1, 2 or 3 substituents independently of one another selected from the group consisting of hydroxyl, amino, trifluoromethyl, methyl, methoxy and morpholinyl,
• R 1 is 2-methylprop-1-yl, 2,2-dimethylprop-1-yl, trimethylsilylmethyl or 3-pyridylmethyl means
• 3-pyridylmethyl may be substituted by 0, 1 or 2 substituents independently of one another selected from the group consisting of hydroxyl, amino, trifluoromethyl, methyl, methoxy and morpholinyl,
• R 2 is 2-methylprop-1-yl, 2,2-dimethylprop-1-yl, trimethylsilylmethyl or 3-pyridylmethyl,
• 3-pyridylmethyl can be substituted by 0, 1 or 2 substituents independently of one another selected from the group consisting of hydroxyl, amino, trifluoromethyl, methyl, methoxy and morpholinyl,
• the invention also relates to compounds of the formula
• R 1 Ci-C 6 alkyl, C 2 -C 6 alkenyl, C 3 -C 6 -cycloalkyl or C 6 -C 0 -aryl,
• alkyl, alkenyl, cycloalkyl and aryl may be substituted by 0, 1, 2 or 3 substituents independently of one another selected from the group consisting of halogen, hydroxyl, amino, cyano, trimethylsilyl, C r C 6 -alkyl, C r C 6 - Alkoxy, benzyloxy, C 3 -C 6 - Cycloalkyl, C 6 -C 0 aryl, 5- to 7-membered heterocyclyl, 5- to 10-membered heteroaryl, Ci-C 6 -alkylamino, C ⁇ -Cio-arylamino, Ci-C ö alkylcarbonylamino, C6-CIO Arylcarbonylamino, C 1 -C 6 -alkylcarbonyl, C 1 -C 6 -alkoxycarbonyl, C 6 -C 10 -arylcarbonyl and benzyloxycarbonylamino,
• cycloalkyl, aryl, heterocyclyl and heteroaryl in turn may be substituted by 0, 1, 2 or 3 substituents independently selected from the group consisting of halogen, hydroxy, amino, cyano, nitro, trifluoromethyl, Ci-C ⁇ -AJkyl, Ci-C ⁇ -alkoxy, phenyl and 5- to 7-membered heterocyclyl,
• Ci-Ce-alkyl, C 2 -C 6 alkenyl, C 3 -C 6 -cycloalkyl or C 6 -C represents 0 aryl
• alkyl, alkenyl, cycloalkyl and aryl may be substituted by 0, 1, 2 or 3 substituents independently of one another selected from the group consisting of halogen, hydroxyl, amino, cyano, trimethylsilyl, C 1 -C 6 -alkyl, C 1 -C 6 -alkoxy, benzyloxy, C3 -C6- cycloalkyl, C ö -Cio-aryl, 5- to 7-membered heterocyclyl, 5- to 10-membered heteroaryl, Ci-C ⁇ alkylamino, Coe-Cio-arylamino, Ci-C ⁇ -alkylcarbonylamino, C 6 -C 10 -arylcyconebonylamino, C 1 -C 6 -alkylcarbonyl, C 1 -C 6 -alkoxycarbonyl, C 6 -C 10 -arylcarbonyl and benzyloxycarbonylamino,
• cycloalkyl, aryl, heterocyclyl and heteroaryl in which cycloalkyl, aryl, heterocyclyl and heteroaryl in turn may be substituted by 0, 1, 2 or 3 substituents independently of one another selected from the group consisting of halogen, hydroxyl, amino, cyano, nitro,
• the invention further provides a process for the preparation of the compounds of the formulas (Ib), wherein
• R 6 has the meaning given above
• R 5 R 5 R and R have the abovementioned meaning
• R 3 is butoxy-butoxycarbonyl or benzyloxycarbonyl
• X 1 is halogen, preferably bromine, chlorine or fluorine, or hydroxy
• R 6 has the meaning given above
• R, R, R and R have the abovementioned meaning
• a fluoride reagent such as tetrabutylammonium fluoride
• reaction of the first stage is carried out in.
• inert solvents optionally in the presence of a base, preferably in a temperature range from -30 ° C to 50 0 C at atmospheric pressure.
• Inert solvents are, for example, tetrahydrofuran, methylene chloride, pyridine, dioxane, dimethylacetamide, N-methylpyrrolidine or dimethylformamide, preference is given to pyridine or dimethylformamide.
• bases are triethylamine, diisopropylethylamine or N-methylmorpholine, preference is given to diisopropylethylamine.
• the reaction of the first stage is generally carried out in inert solvents, in the presence of a dehydrating reagent, optionally in the presence of a base, preferably in a temperature range from -30 0 C to 5O 0 C at atmospheric pressure.
• Inert solvents are, for example, halogenated hydrocarbons, such as dichloromethane or trichloromethane, hydrocarbons, such as benzene, nitromethane, dioxane, dimethylformamide or acetonitrile. It is likewise possible to use mixtures of the solvents. Particularly preferred is dichloromethane or dimethylformamide.
• Suitable dehydrating here for example, carbodiimides such as N 1 N'-diethyl-, N 1 N 1 dipropyl, N, N'-diisopropyl-, N, N'-dicyclohexylcarbodiimide, N- (3-di ⁇ methylaminoisopropyty-N'suitable ethylcarbodiimide hydrochloride (EDC), N-cyclohexylcarbodiimide-N'-propyloxymethyl-polystyrene (PS-carbodiimide) or carbonyl compounds such as carbonyldiimidazole, or 1,2-oxazolium compounds such as 2-ethyl-5-phenyl-1,2-oxazolium 3-sulphate or 2-tert-butyl-5-methylisoxazolium perchlorate, or acylamino compounds such as 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline, or propanephospho
• bases are alkali metal carbonates, such as sodium or potassium carbonate, or hydrogencarbonates drogencarbonat, or organic bases such as trialkylamines, for example triethylamine, N 'methylmorpholine, N-methylpiperidine, 4-dimethylaminopyridine or diisopropylethylamine.
• the condensation is carried out with HATU or with EDC in the presence of HOBt.
• the reaction takes place with an acid in the second step of the process preferably in a temperature range from 0 ° C to 4O 0 C at atmospheric pressure.
• Suitable acids in this case are hydrogen chloride in dioxane, hydrogen bromide in acetic acid or trifluoroacetic acid in methylene chloride.
• the hydrogenolysis in the second stage of the process is generally carried out in a solvent in the presence of hydrogen and palladium on activated carbon, preferably in a temperature range from 0 ° C. to 40 ° C. under normal pressure.
• Solvents are, for example, alcohols such as methanol, ethanol, n-propanol or isopropanol, in a mixture with water and acetic acid or aqueous hydrochloric acid, vorz ⁇ ugt is a mixture of ethanol, water and acetic acid or a mixture of iso-propanol and aqueous hydrochloric acid.
• reaction of the compounds of the formula (IV) with compounds of the formula (V) or (VII) is generally carried out in inert solvents, in the presence of a dehydrating reagent, if appropriate in the presence of a base, preferably in a temperature range from -3O 0 C to 50 0 C at normal pressure.
• Inert solvents are, for example, halogenated hydrocarbons, such as dichloromethane or trichloromethane, hydrocarbons, such as benzene, nitromethane, dioxane, dimethylformamide or acetonitrile. It is likewise possible to use mixtures of the solvents. Particularly preferred is dichloromethane or dimethylformamide.
• Suitable dehydrating reagents here are, for example, carbodiimides, such as N, N'-diethyl, N, N-dipropyl, NN'-diisopropyl, N, N'-dicyclohexylcarbodiimide, N- (3-dimethylaminoisopropyl) -N ' ethylcarbodiimide hydrochloride (EDC), N-cyclohexylcarbodiimide-N'-propyloxymethyl-polystyrene (PS-carbodiimide) or carbonyl compounds such as carbonyldiimidazole, or 1,2-oxazolium compounds such as 2-ethyl-5-phenyl-1,2-oxazolium 3-sulphate or 2-tert-butyl-5-methylisoxazoline perchlorate, or acylamino compounds such as 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline, or propane
• Bases are, for example, alkali carbonates, e.g. Sodium or potassium carbonate, or hydrogen carbonate, or organic bases such as trialkylamines e.g. Triethylamine, N-methylmorpholine, N-methylpiperidine, 4-dimethylarninopyridine or diisopropylethylamine.
• alkali carbonates e.g. Sodium or potassium carbonate
• hydrogen carbonate or organic bases
• organic bases such as trialkylamines e.g. Triethylamine, N-methylmorpholine, N-methylpiperidine, 4-dimethylarninopyridine or diisopropylethylamine.
• the condensation is carried out with HATU or with a mixture of EDC and HOBt.
• the reaction with tetrabutyl ammonium fluoride in the first stage (a) of the further process is generally carried out in inert solvents, preferably in a temperature range from 0 ° C. to 40 ° C. under normal pressure.
• Inert solvents are, for example, halogenated hydrocarbons, such as dichloromethane or trichloromethane, or ethers, tetrahydrofuran or dioxane. Particularly preferred is tetrahydrofuran.
• the reaction with an acid in the second stage (b) of the process is preferably carried out in a temperature range from 0 0 C to 40 0 C at atmospheric pressure.
• Suitable acids in this case are hydrogen chloride in dioxane, hydrogen bromide in acetic acid or trifluoroacetic acid in methylene chloride.
• the reaction in the third stage (c) of the process is analogous to the reaction of compounds of the formula (IV) with compounds of the formula (V) or (VII).
• the hydrogenolysis in the fourth step (d) of the process is generally carried out in a solvent in the presence of hydrogen and palladium on activated carbon, preferably in a temperature range from 0 ° C. to 40 ° C. under normal pressure.
• Solvents are, for example, alcohols such as methanol, ethanol, n-propanol or isopropanol, in a mixture with water and acetic acid or aqueous hydrochloric acid, preferably a mixture of ethanol, water and acetic acid or a mixture of isopropanol and aqueous hydrochloric acid.
• the compound of formula (H) can be synthesized by hydrogenation, enzymatic cleavage and cyclization from lysobactin (Example IA) or katanosin A as described in the experimental section under Example 2A, 3A and 2OA to 26A.
• the compound of formula (FV) is an intermediate in this synthetic sequence.
• the present invention further provides a process for the preparation of the compound of the formula 2- (trimethylsilyl) ethyl (3i?) -3-amino-N 2 - [(benzyloxy) carbonyl] -L-phenylalaninate trifluoroacetate (Example 1 9A)
• Another object of the present invention is the compound of formula
• the compounds of the invention show an unpredictable, valuable spectrum of pharmacological activity. They show an antibacterial effect.
• the compounds of the invention are characterized by a lower nephrotoxicity to lysobactin.
• the compounds according to the invention are distinguished by improved stability in an aqueous neutral to basic medium. This property improves the storage of the compounds according to the invention and the administration as medicaments.
• the described nonadepsipeptides act as inhibitors of bacterial cell wall biosynthesis.
• the preparations according to the invention are particularly effective against bacteria and bacteria-like microorganisms. They are therefore particularly well suited for the prophylaxis and chemotherapy of local and systemic infections in human and veterinary medicine, which are caused by these pathogens.
• the preparations according to the invention can be used against all bacteria and bacteria-like microorganisms which are in the possession of a bacterial cell wall (murein sacculus) or the associated enzyme systems, for example by the following pathogens or by mixtures of the following pathogens:
• Gram-negative cocci Neisseria gonorrhoeae
• Gram-negative rods such as Enterobacteriaceae, e.g. Escherichia coli, Haemophilus influenzae, Pseudomonas, Klebsiella, Citrobacter (C. freundii, C. divernis), Salmonella and Shigella; also Enterobacter (E. aerogenes, E. agglomerans), Hafnia, Serratia (S. marcescens), Providencia, Yersinia, and the genus Acinetobacter, Branhamella and Chlamydia.
• the antibacterial spectrum includes strictly anaerobic bacteria such as e.g.
• Bacteroides fragilis members of the genus Peptococcus, Peptostreptococcus and the genus Clostridium; furthermore, mycobacteria, e.g. M. tuberculosus.
• the compounds according to the invention exhibit particularly pronounced activity against gram-positive cocci, e.g. Staphylococci (S. aureus, S. epidermidis, S. haemolyticus, S. carnosus), enterococci (E. faecalis, E. faecium) and streptococci (S. agalactiae, S. pneumoniae, S. pyogenes).
• Staphylococci S. aureus, S. epidermidis, S. haemolyticus, S. carnosus
• enterococci E. faecalis, E. faecium
• streptococci S. agalactiae, S. pneumonia
• pathogens are merely exemplary and not to be construed as limiting.
• diseases which are caused by the named pathogens or mixed infections and which can be prevented, ameliorated or cured by the preparations according to the invention are:
• Infectious diseases in humans such as uncomplicated and complicated urinary tract infections, uncomplicated skin and surface infections, complicated skin and soft tissue infections, hospital and community-acquired pneumonia, nosocomial pneumonia, acute exacerbations and secondary bacterial infections of chronic bronchitis, acute otitis media, acute sinusitis, streptococcal Pharyngitis, bacterial meningitis, uncomplicated gonococcal and non-gonococcal urethritis / cervicitis, acute prostatitis, endocarditis, uncomplicated and complicated intra-abdominal infections, gynecological infections, pelvic inflammatory disease, bacterial vaginosis, acute and chronic osteomyelitis, acute bacterial arthritis, empiric therapy in febrile neutropenic patients, furthermore Bacteria, MRSA infections, acute infectious diarrhea, Helicobacter pylori infections, postoperative infections, odontogenic infections, ophthalmic infections, postoperative infections (including periprocal
• bacterial infections can also be treated in other species. Examples include:
• Pig diarrhea, enterotoxemia, sepsis, dysentery, salmonellosis, metritis-mastitis-agalactiae syndrome, mastitis;
• Ruminants (cattle, sheep, goats): diarrhea, sepsis, bronchopneumonia, salmonellosis, pasteurellosis, genital infections;
• Horse bronchopneumonia, foal disease, puerperal and postpuerperal infections, salmonellosis;
• Dog and cat bronchopneumonia, diarrhea, dermatitis, otitis, urinary tract infections, prostatitis;
• Poultry (chicken, turkey, quail, pigeon, ornamental birds and others): E. co / z infections, chronic respiratory diseases, salmonellosis, pasteurellosis, psittacosis.
• bacterial diseases in the rearing and keeping of farmed and ornamental fish can be treated, wherein the antibacterial spectrum on the aforementioned pathogen addition to other pathogens such.
• Pasteurella Brucella, Campylobacter, Listeria, Erysipelothris, Corynebacteria, Borellia, Treponema, Nocardia, Rikettsia, Yersinia.
• Another object of the present invention is the use of the compounds of the invention for the treatment and / or prophylaxis of diseases, especially of bacterial infectious diseases.
• Another object of the present invention is the use of the compounds of the invention for the treatment and / or prophylaxis 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 prophylaxis of diseases, in particular the aforementioned diseases.
• the compounds according to the invention are preferably used for the preparation of medicaments which are suitable for the prophylaxis and / or treatment of bacterial diseases.
• Another object of the present invention is a method for the treatment and / or prophylaxis of diseases, in particular the aforementioned diseases, using an antibacterially effective amount of the inventive compounds.
• compositions containing at least one compound of the invention and at least one or more other active ingredients, in particular for the treatment and / or prophylaxis of the aforementioned diseases.
• Preferred combination active ingredients are antibacterial compounds which have a different spectrum of activity, in particular complementary spectrum of action, and / or are synergistic with the compounds according to the invention.
• the compounds according to the invention can act systemically and / or locally.
• they may be applied in a suitable manner, e.g. oral, parenteral, pulmonary, nasal, sublingual, lingual, buccal, rectal, dermal, transdermal, conjunctival, otic or as an implant or stent.
• the compounds according to the invention can be administered in suitable administration forms.
• Parenteral administration can be done bypassing a resorption step (eg intravenously, intraarterially, intracardially, intraspinally or intralumbarly) or with involvement of resorption (eg intramuscular, subcutaneous, intracutaneous, percutaneous or intraperitoneal).
• a resorption step eg intravenously, intraarterially, intracardially, intraspinally or intralumbarly
• involvement of resorption eg intramuscular, subcutaneous, intracutaneous, percutaneous or intraperitoneal.
• parenteral administration are suitable as application forms, including injection and infusion preparations in the form of solutions, suspensions, emulsions, lyophilisates or sterile powders.
• Inhalation medicines including powder inhalers, nebulizers
• nasal drops solutions, 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 (such as patches)
• milk Pastes, foams, scattering powders, implants or stents.
• the compounds according to the invention can be converted into the mentioned administration forms. This can be done in a conventional manner by mixing with inert, non-toxic, pharmaceutically suitable excipients.
• Carriers for example microcrystalline cellulose, lactose, mannitol
• solvents eg liquid polyethylene glycols
• emulsifiers and dispersing or wetting agents for example sodium dodecyl sulfate, polyoxysorbitanoleate
• binders for example polyvinylpyrrolidone
• synthetic and natural polymers for example albumin
• stabilizers eg antioxidants such as ascorbic acid
• dyes eg, inorganic pigments such as iron oxides
• flavor and / or odor remedies include, among others.
• Carriers for example microcrystalline cellulose, lactose, mannitol
• solvents eg liquid polyethylene glycols
• emulsifiers and dispersing or wetting agents for example sodium dodecyl sulfate
• Another object of the present invention are pharmaceutical compositions containing at least one erfmdungswashe connection, usually together with one or more inert, non-toxic, pharmaceutically suitable excipients, and their use for the purposes mentioned above.
• TOF-HR-MS TOF HR-MS-ESI + spectra are recorded using a Micromass LCT apparatus (capillary voltage: 3.2 KV, cone Spanmmg: 42 V Source Temperature: 120 0 C, Desolvations Temperature: 280 0 C ).
• a syringe pump Hard Apparatus
• the standard is leucine-enkephalin (Tyr-Gly-Gly-Phe-Leu).
• Method 2 (Analytical HPLC, Synergi): Device Type HPLC: HP 1100 Series; UV DAD; S-column: Phenomenex Synergi 2 ⁇ Hydro-RP Mercury 20 mm x 4 mm; Eluent A: 1 l of water + 1 ml of 50% formic acid, eluent B: 1 l of acetonitrile; Gradient: 0.0 min 90% A -> 2.5 min 30% A -E ⁇ 3.0 min 5% A -> 4.5 min 5% A; Flow: 0.0 min 1 ml / min, 2.5 min / 3.0 min / 4.5 min. 2 ml / min; Oven: 5O 0 C; UV detection: 210 nm.
• Method 3 Analytical HPLC, Synergi: Device Type HPLC: HP 1050 Series; UV DAD; Column: Phenomenex Synergi 2 ⁇ Max-RP Mercury 20mm x 4mm; Eluent A: 1 1 water + O.05% Trifluoroacetic acid, eluent B: 1 liter acetonitrile; Gradient: 0.0 min 90% A -> 2.5 min 30% A - »3.0 min 5% A -> 4.5 min 5% A; Flow: 0.0 min 1 ml / min, 2.5 min / 3.0 min / 4.5 min. 2 ml / min; Oven: 5O 0 C; UV detection: 210 nm.
• Method 4 (Analytical HPLC): Device Type HPLC: HP 1050 Series; UV DAD 1100 Series; Column: Kromasil C] 8 , 60 ⁇ 2 mm, 3.-5 ⁇ m; Eluent A: water / 0.5% perchloric acid, eluent B: acetonitrile; Gradient: 0-0.5 min 2% B, 0.5-4.5 min 2-90% B, 4.5-6.5 min 90% B, 6.5-6.7 min 90- 2% B, 6.7-7.5 min 2% B; Flow: 0.75 ml / min, Oven: 3O 0 C, detection UV 210 nm.
• Method 5 Device Type MS: Micromass LCT (ESI pos./neg.); Device type HPLC: HP 1100 Series; UV DAD 1100 Series; Column SymmetryPrep TM Ci 8 , Waters, 50 x 2.1 mm, 3.5 ⁇ m; Eluent A: water / 0.1% formic acid, eluent B: acetonitrile / 0.1% formic acid; Gradient: 0-1 min 0% B, 1-6 min 0-90% B, 6-8 min 90-100% B, 8-10 min 100% B, 10-10.1 min 100-0% B, 10.1- 12 min 0% B, then regeneration of the chromatography column. Oven: 4O 0 C, flow: 0.5 ml / min (at 10.1 min in the short term to 1 ml / min), UV detection: 210 nm.
• Method 6 (Preparative HPLC, Xterra, C 18 , 0.1% TFA): Device: Gilson Abimed HPLC; binary pump system; Column: Xterra, Waters, 5 ⁇ m; 150 x 10 mm; Eluent A: water / 0.1% trifluoroacetic acid, eluent B: acetonitrile; Gradient: 0-7 min 5% B, 7-9 min 5-40% B, 9-11 min 40% B, 11-18 min 40-90% B, then regeneration of the chromatography column; Flow: 8 ml / min; UV detector 210 nm.
• Method 7 Device Type MS: Micromass ZQ; Device type HPLC: Waters Alliance 2795; Column: Phenomenex Synergi 2 ⁇ Hydro-RP Mercury 20 mm x 4 mm; 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 90% A -> 2.5 min 30% A -> 3.0 min 5% A -> 4.5 min 5% A; Flow: 0.0 min 1 ml / min, 2.5 min / 3.0 min / 4.5 min 2 ml / min; Oven: 5O 0 C; UV detection: 210 nm.
• Method 8 Instrument: Micromass Quattro LCZ with HPLC Agilent Series 1100; Column: Phenomenex Synergi 2 ⁇ Hydro-RP Mercury 20 mm x 4 mm; 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 90% A -> 2.5 min 30% A ⁇ »3.0 min 5% A -> 4.5 min 5% A; Flow: 0.0 min 1 ml / min, 2.5 min / 3.0 min / 4.5 min 2 ml / min; Oven: 5O 0 C; UV detection: 208-400 nm.
• Method 9 (Amino Acid Analysis): The amino acid analyzes are carried out with an Eppendorf / Biotronik LC3000 amino acid analyzer. A slightly modified standard separation program from Eppendorf / Biotronik is used. The separation programs and the function of the analyzer are described in detail in the manual of the device (Handbook of the amino acid analyzer LC 3000, Scientific Devices GmbH Biotronik, Maintal, 1996).
• Method 10 (Preparative HPLC, Nucleodur, TF A): Apparatus: Gilson Abimed HPLC, binary pump system Column: Nucleodur Ci 8 Gravity, Macherey-Nagel, 5 ⁇ m, 250 ⁇ 21 mm, eluent A: water / 0.1% trifluoroacetic acid , Eluent B: acetonitrile; gradient: 0-8 min 5% B, 8-40 min 5-60% B, 40-60 min 60% B, 60-75 min 60-100% B, 75-80 min 100% B, followed by regeneration of the chromatography column, flow: 7-15 ml / min, UV Detektox 210 nm.
• Method 11 (Preparative HPLC, Nucleodur, Acetic Acid): Device: Gilson Abimed HPLC; binary pump system; Column: Nucleodur Ci 8 Gravity, Macherey-Nagel, 5 ⁇ m; 250 x 40 mm; Eluent A: water / 0.2% acetic acid, eluent B: acetonitrile / 0.2% acetic acid; Gradient: 0-10 min 10% B, 10-24 min 10-30% B, 24-28 min 30-50% B, 28-35 min 50% B, 35-45 min 50-60% B, 45- 53 min 60-70% B, 53-60 min 60-90% B, 60-70 min 100% B, then regeneration of the chromatography column; Flow: 15-45 ml / min; UV Detettor 210 nm.
• Method 12 (Preparative HPLC, Symmetry, Acetic Acid): Device: Gilson Abimed HPLC; binary pump system; Column: SymmetryPrep TM C ] 8 , Waters, 7 ⁇ m; 300 x 19 mm; Eluent A: water / 0.5-0.25% acetic acid, eluent B: acetonitrile; Gradient: 0-2 min 5% B, 2-60 min 5-90% B, 60-80 min 100% B, then regeneration of the chromatography column; Flow: 7 ml / min; UV detector 210 nm.
• Method 13 (Preparative HPLC, Kromasil, acetic acid): Device: Gilson Abimed HPLC; binary pump system; Column: Kromasil-IOOA Ci 8 , 5 microns; 250 x 30 mm; Eluent A: water / 0.25% acetic acid, eluent B: acetonitrile; Gradient: 0-3 min 5% B, 3-30 min 5-100% B, 30-38 min 100% B, then regeneration of the chromatography column; Flow: 25 ml / min; UV detector 210 nm.
• Method 14 gel chromatography on Sephadex LH-20: Gel chromatography is performed without pressure on Sephadex LH-20 (Pharmacia). It is fractionated after UV activity (UV detector for 254 nm, Knauer) (fraction collector ISCO Foxy 200). Column dimensions: 32 x 7 cm (1000-100 ⁇ mol scale); 30 x 4 cm (100-10 ⁇ mol scale); 25 x 2 cm (10-1 ⁇ mol scale). The eluent used is methanol or methanol / 0.2% acetic acid.
• Method 15 Chiral preparative HPLC: column (Sta ⁇ ht column, dimension 250x30 mm); stationary phase (chiral silica / polyamide composite KBD 5326, based on the selector poly (N-L-leucine methacryolyl dicyclopropylmethylamide); eluent ethyl acetate, isocratic; flow: 25 ml / min; temperature: 24 0 C; UV detection: 225 nm Sample: Repetitive injection of 2000 ⁇ l.
• Method 16 determination of enantiomeric purity: column (steel column, dimension 250 x 4.6 mm); stationary phase (chiral silica / polyamide composite KBD 5326, based on the selector poly (N-methacryolyl-L-leucine-dicyclopropylxnethylamide); ethyl acetate eluent, isocratic; Flow: 1 ml / min; Temperature: 25 ° C; UV detection: 220 nm; Sample: Inject 10 ⁇ l.
• Method 17 (Analytical HPLC): Device Type HPLC: HP 1100 Series; UV DAD column: Zorbax Eclipse XBD-C8 (Agilent), 150 mm x 4.6 mm, 5 ⁇ m; Eluent A: 5 ml HC1O 4/1 water, eluent B: acetonitrile; Gradient: 0-1 min 10% B, 1-4 min 10-90% B, 4-5 min 90% B; Flow: 2.0 ml / min; Oven: 3O 0 C; UV detection: 210 and 254 nm.
• Method 18 (Analytical HPLC): Column: Kromasil RP-18, 60 mm ⁇ 2 mm, 3.5 ⁇ m; Eluent A: 5 ml HCIO 4 / l water, eluent B: acetonitrile; Gradient: 0 min 2% B, 0.5 min 2% B, 4.5 min 90% B, 9 min 90% B; Flow: 0.75 ml / min; Oven: 30 ° C; UV detection: 210 nm.
• Method 19 (Analytical HPLC): Column: Kromasil RP-18, 250 mm ⁇ 4 mm, 5 ⁇ m; Eluent A: 5 ml HCIO 4 / l water, eluent B: acetonitrile; Gradient: 0 min 5% B, 10 min 95% B; Flow: 1 ml / min; Oven: 40 ° C; UV detection: 210 nm.
• Method 20 (Analytical HPLC): Column: Kromasil RP-18, 250 mm x 4 mm, 5 ⁇ m; Eluent A: 2 ml HClO 4 / l of water, eluent B: acetonitrile; Isocratic: 45% B, 55% A; Flow: 1 ml / min; Oven: 40 ° C; UV detection: 210 nm.
• Method 21 (Preparative HPLC, Nucleodur, 0.05-0.1% TF A): Device: Gilson Abimed HPLC; binary pump system; Column: Nucleodur Qg Gravity, Macherey-Nagel, 5 ⁇ m, 250 ⁇ 21 mm; Eluent A: water / 0.05-0.1% TFA, eluent B: acetonitrile; Gradient: 0-8 min 5% B, 8-40 min 5-60% B, 40-60 min 60% B, 60-75 min 60-100% B, 75-80 min 100% B, then regeneration of the chromatography column ; Flow: 7-15 ml / min; UV detection: 210 nm.
• Method 22 (Analytical HPLC): Device Type HPLC: HP 1050 Series; UV DAD 1100 Series; Column: Kromasil C 18 , 60 mm x 2 mm, 3.5 ⁇ m; Eluent A: water / 0.5% perchloric acid, eluent B: acetonitrile; Gradient: 0-0.5 min 2% B, 0.5-4.5 min 2-90% B, 4.5-9.0 min 90% B, 9.0-9.2 mia 90-2% B, 9.2-10.0 min 2% B; Flow: 0.75 ml / min, oven: 30 ° C., UV detection 210 nm.
• Method 23 (Analytical HPLC): Agilent 1100 with DAD (G1315B), Binary Pump (G1312A), Autosampler (G1313A), Degasser (G1379A) and Column Thermostat (G1316A); Column: Synergi 4 ⁇ . Hydro-RP 80A, 4.6 x 150 x 5 now; Eluent A: water + 0.05% 70% perchloric acid; Eluent Br acetonitrile; Gradient: 0-1 min 10% B, ramp, 4-5 min 90% B, ramp, 5.5 min 10% B; Flow z 2.00 ml / min; Oven temperature: 3 O 0 C.
• Method 24 (Analytical HPLC): Device: Agilent 1100 with DAD (G1315B), binary pump (GI 312A), autosampler (GI 313A), solvent degasser (GI 379A) and column thermostat: (G1316A); Column: Agilent Eclipse XDB-C8 4.6 x 150 x 5 mm; Eluent A: 0.05% 70% perchloric acid in water; Eluent B: methanol; Isocratic: 0-7 min 55% B; Flow: 2.00 ml / min; Column temperature: 4O 0 C.
• Method 25 (Analytical HPLC): Device Type HPLC: HP 1050 Series; LW BAD; Column: Zorbax 300 mSB-C18 3.5 ⁇ , 4.6 mm x 150 mm; Eluent A: 1 l water + 0.1% trifluoroacetic acid, eluent B: 400 ml acetonitrile / 600 ml water + 0.1% trifluoroacetic acid; Gradient: 0.0 RNIN 100% A, 1.3 min 10% B, 18.0 min 80% B, 20.0 min 80% B, 21.0 min 100% B, 25.0 min 100 «% B, 26.0 min 0% B, 30.0 min 0% B , Flow: 1 ml / min; Oven: 40 ° C; UV detection: 210 nm.
• Method 26 (Chiral HPLC): Gilson Abimed HPLC; Column: Daicel Chiralpak AD-H 5 um; 250 mm x 20 mm; Eluent A: / so-hexane, eluent B: 0.2% acetic acid / 1% water / 2-propanol; isocratic; Flow: 15 ml / min; UV detector 212 nm.
• Method 27 (determination of enantiomeric purity): Steel column: dimension 250 mm x 4.6 mm; stationary phase: Daicel Chirapak AD-H, 5 ⁇ m; Eluent: ethanol / water / acetic acid 1000: 10: 2, isocratic; Flow: 0.7 ml / min; Temperature: 45 ° C .; UV detection: 215 nm; Sample: Inject 5 ⁇ l.
• Method 28 determination of enantiomeric purity: column (steel column, dimension 250 mm x 4.6 mm); stationary phase (Chirapak IA); Eluent iso-hexane / iso-propanol 4: 1, isocratic; Flow: 1 ml / min; Temperature: 25 ° C; UV detection: 254 nm; Sample: Injection "of 10 ul.
• Method 29 UV detection: 210 nm.
• Device Type MS Micromass ZQ
• Device type HPLC Waters Alliance 2795; Column: Phenomenex Synergi 2 ⁇ Hydro-RP Mercury 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 90% A -> 2.5 min 30% A -> 3.0 min 5 ° / ⁇ A -> 4.5 min 5% A; Flow: 0.0 min 1 ml / min, 2.5 min / 3.0 min / 4.5 min 2 ml / min; Oven: 5O 0 C; UV detection: 210 nm.
• Method 30 Instrument: Micromass Platform LCZ with HPLC Agilent Series 1100; Column: Phenomenex Synergi 2 ⁇ Hydro-RP Mercury 20 mm x 4 mm; Eluent A .: 1 liter of water + 0.5 ml of 50% formic acid, eluent B: 1 liter of acetonitrile + 0.5 ml of 50% formic acid; Gradient: 0.0 min 90% A -> 2.5 min 30% A -> 3.0 min 5% A -> 4.5 min 5% A; 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 31 (MALDI-MS): The MALDI-MS / MS examinations are performed on a 4700 proteomics analyzer (Applied Biosystems, Framingham, MA) using
• the quasi-molecules are accelerated in the ion source with 8 kV, selected with an electric deflector (MSl), and encountered in a collision cell, which is located between MSl and MS2, with argon atoms.
• MSl electric deflector
• the resulting fragment ions are post-accelerated at 15 kV and characterized by the second time-of-flight mass analyzer (MS2).
• Method 32 (Chiral Preparative HPLC): Steel Column: Dimension 420 mm x 75 mm; stationary phase: silica gel phase with the sector poly (N-methacryloyl-D-leucine-dicyclopropylmethylamide); Eluent A: te / t-butyl methyl ether, eluent B: methanol; Gradient: 0-22.17 min 100% A, 22.18- 27.24 min 100% B, 27.25-40.30 min 100% A; Flow: 100 ml / min; Temperature: 24 ° C; UV detection: 260 nm; Sample: Repetitive injection of 50000 ⁇ l.
• Method 33 (Preparative HPLC, oducleodur, 0.05-0.1% TFA): Device: Gilson Abimed HPLC; binary pump system; Column: ⁇ ucleodur Qs Gravity, Macherey- ⁇ agel, 5 ⁇ m; 250 mm x 40 mm; Eluent A: water / 0.05-0.1% trifluoroacetic acid, eluent B: acetonitrile; Gradient: 0-10 min 10% B, 10-24 min 10-30% B, 24-28 min 30-50% B, 28-35 min 50 ° / oB, 35-45 min 50- 60% B, 45 -53 min 60-70% B, 53-60 min 60-90% B, 60-70 min 10O% B, then regeneration of the chromatography column; Flow: 15-45 ml / min; UV detector ZlO nm.
• Method 34 (Preparative HPLC Kromasil 0.05% TFA): Device: Gilson Abimed HPLC; UV detector 210 nm; binary pump system; Column: Kromasil C ] 8 , 5 ⁇ m, 100 ⁇ , 250 mm ⁇ 20 mm; Eluent A: 0.05% trifluoroacetic acid in water, eluent B: 0.05% trifluoroacetic acid in acetonitrile: flow rate: 20 ml / min; 0-3 min 10% B, ramp, 30-38 min 90% B, 38-45 min 1 0% B.
• Method 35 (Preparative HPLC, oducleodur C 18 Gravity, 0.05-0.1% TFA): Device: Gilson Abimed HPLC; binary pump system; Column: ⁇ ucleodur Ci 8 Gravity, 5 ⁇ m; 250 mm x 40 mm; Eluent A: water / 0.05-0.1% trifluoroacetic acid, eluent B: acetonitrile; 0-10 min 10% B, ramp, 10.01-55 min 100% B; Flow: 10-60 ml / min; UV detector 210 nm.
• Method 36 (Preparative HPLC, Kromasil, 0.05% TFA): Device: Gilson Abimed HPLC; binary pump system; Column: Kromasil-IOOA Ci 8 , 5 microns; 250 mm x. 30 mm; Eluent A: water / 0.05% trifluoroacetic acid, eluent B: acetonitrile; 0-10 min 10% B, ramp, 10.01-55 min 100% B; Flow: 30 ml / min; UV detector 210 nm.
• Method 37 (Preparative HPLC, Kromasil, 0.05% TFA): Device: Gilson Abimed HPLC; binary pump system; Column: Kromasil-100A C ] 8 , 5 ⁇ m; 250 mm JC 21 mm; Eluent A: water / 0.05% trifluoroacetic acid, eluent B: acetonitrile; 0-10 min 10% B, ramp, 10.01-55 min 100% B; Flow: 20 ml / min; UV detector 210 nm.
• Method 38 (Preparative HPLC, Kromasil, 0.05% TFA): Device: Gilson Abimed HPLC; binary pump system; Column: Kromasil-100A Ci 8 , 5 ⁇ m; 250 mm x 21 mm; Eluent A: Water / 0.05% trifluoroacetic acid, eluent B: acetonitrile / water 50/50; 0-10 min 10% B, ramp, 10.01-55 min 100% B; Flow: 20 ml / min; UV detector 210 um.
• Method 39 (Preparative HPLC, Kromasil, 0.05% TFA): Device: Gilson Abimed HPLC; binary pump system; Column: Kromasil 100A Ci 8 , 5 ⁇ m; 250 mm x 30 mm; Eluent A: water / 0.05% trifluoroacetic acid, eluent B: acetonitrile; 0-10 min 10% B, ramp, 10.01-55 min 100% B; Flow: 10-60 ml / min; UV detector 210 nm.
• Method 40 (Preparative HPLC): Device: Gilson Abimed HPLC; UV detector 210 nm; binary pump system; Column: Waters Symmetry-Prep TM Ci 8 , 7 ⁇ m, 300 mm x 19 mm; Eluent A: 0.05% trifluoroacetic acid in water, eluent B: 0.05% trifluoroacetic acid in acetonitrile: flow rate: 20 ml / min; 0-3 min 10% B, ramp, 30-38 min 90% B, 38-45 min 10% B.
• Method 41 (Preparative HPLC, Grom-Sil, 0.1% TFA): Device: Gilson Abimed HPLC; UV detector 210 nm; binary pump system; Column: Grom-Sil SNr.4051 120 ODS-4HE, 10 ⁇ m, 250 mm x 40 mm; Eluent A: 0.1% trifluoroacetic acid in water, eluent B: 0.1% trifluoroacetic acid in acetonitrile; Flow rate: 50 ml / min; 0-3 min 10% B, 3-27 min gradient ramp, 27-35 min 95% B, 35-40 min 10% B.
• Method 42 Chromatic Preparative HPLC: column (steel column, dimension 500 mm x 50 mm); stationary phase (Chirapak AD, 20 ⁇ m); Eluent A: iso-hexane, eluent B: iso-propanol; Gradient: 0- 11.74 min 15% B, 11.74-11.75 min 100% B, 15.74 min 1 00% B, 15.75 min 15% B, 21.25 min 15% B; Sample preparation: dissolve in 50 ml iso-propanol / 200 ml iso-hexane; Injection volume: 30 ml, temperature: 24 0 C; UV detection: 220 nm.
• Method 43 (Preparative HPLC): Device: Gilson Abimed HPLC; UV detector 254 nm; binary pump system; Column: Nucleosil RP-18, 7 ⁇ m; 250 x 5 O mm; Flow: 30 ml / min; Eluent A: water / 0.1% trifluoroacetic acid, eluent B: acetonitrile / O.1% trifluoroacetic acid; Gradient: 0-40 minutes 20-25% B, 40-60 minutes 25% B, 60-110 minutes 25-50% B, 110-120 minutes 50% B, 120-130 minutes 50-100% B, 130- 160 min 100% B, then regeneration of the chromatography column.
• Method 44 (Preparative HPLC): Device: Gilson Abimed HPLC; UV detector 254 nm; binary pump system; Column: Nucleosil RP-18, 7 ⁇ m; 250 x 50 mm; Flow: 40 ml / min; Eluent A: water / 0.05% trifluoroacetic acid, eluent B: acetonitrile / 0.05% trifluoroacetic acid; Gradient: 0-105min 20-25% B, 105-111min 25% B, 111-131min 25-27 ° / l> B, 131-157min 27-35% B, 157-192min 35-40 % B, 40-45% B, 192-207 min, then regeneration of the chromatography column.
• General working instructions 0-10min 20-25% B, 105-111min 25% B, 111-131min 25-27 ° / l> B, 131-157min 27-35% B, 157-192min 35-40 % B, 40-45% B,
• the Boc-protected compound is suspended in dichloromethane (1 / 5-1 / 10 of the Uss ⁇ solution) and then under argon inert gas atmosphere with trifluoroacetic acid in dichloro-5 methane (30%, about 1 ml / 10 mg starting material in the 100- 1 millimole scale, about 1 ml / 1 mg in 100-1 micromole scale) and stirred at room temperature until the HPLC chromatogram shows complete conversion (method 2). Then the solvent is distilled off in vacuo, the bath temperature should not exceed 3O 0 C. The crude product is suspended in toluene, concentrated again on a rotary evaporator and dried under high vacuum. 0 This procedure is repeated several times with toluene or with dichloromethane (two to five times).
• the peptidic benzyl ester or the peptidic N-Cbz-protected amine (1.2 mmol) is dissolved in methanol (60 ml) and treated under argon protective gas atmosphere with 1 Oproz. Palladium-carbon (100 mg). At RT and normal pressure, hydrogenation is carried out until analytical HPLC 5 (method 2) indicates complete conversion.
• the reaction mixture is filtered (eg., Via diatomaceous earth, Celite ®), concentrated in vacuo and dried under high vacuum.
• reaction after reaction with water and methane «! are added and then after purification via preparative RP-HPLC or gel chromatography, the product is obtained.
• YM Yeast Malt Agar: D-glucose (4 g / l), yeast extract (4 g / l), malt extract (10 g / l), 1 liter of Lewatit water. Before sterilization (at 121 ° C for 20 minutes) the pH is adjusted to 7.2.
• HPM mannitol (5.4 g / L), yeast extract (5 g / L), meat peptone (3 g / L).
• the lyophilized strain (ATCC 53042) is grown in 50 ml of YM medium.
• Piston Fermentation Inoculate 150 ml of YM medium or 100 ml of HPM medium in a 1 1 Erlenmeyer flask with 2 ml of the working fluid and grow on a shaker at 240 rpm for 30-48 hours at 28 ° C.
• 30 1 Fermentation 300 ml of the piston fermentation (HPM medium) are used to inoculate a sterile 30 L culture medium solution (1 ml Antifoam SAG 5693/1). This culture is wm for 21 hours at 28 0 C, 300 rpm and aeration with sterile air of 0.3 grown.
• YM sterile 200 L culture medium solution
• the culture broth of the main culture is separated at 17000 rpm in supernatant and sediment.
• the supernatant (183 1) is adjusted to pH 6.5-7 with concentrated trifluoroacetic acid or sodium hydroxide solution and applied to a Lewapol column (OC 1064, 60 l content). It is then eluted with pure water, water / methanol 1: 1 and then with pure methanol (with 0.1% trifluoroacetic acid). This organic phase is concentrated in vacuo to a residual aqueous residue of 11.5 1.
• the remaining aqueous phase is bound to silica gel Cis and separated (MPLC, Biotage Flash 75, 75 ⁇ 30 cm, KP-Cl 8-WP, 15-20 ⁇ m, flow: 30 ml, eluent: acetonitrile / water with 0.1% trifluoroacetic acid; Gradient: 10%, 15% and 40% acetonitrile).
• the 40% acetonitrile phase containing the majority of Example IA is concentrated in vacuo and then lyophilized (about 13 g).
• Example IA This process provides 2250 mg of Example IA.
• the solvent is concentrated in vacuo and the residue is freeze-dried.
• the resulting lyophilizate (89.9 g) is taken up in methanol, filtered off, concentrated and separated (Method 20).
• Example IA is then purified by gel filtration (Sephadex LH-20, 5 x 68 cm, water / acetonitrile 9: 1 (with 0.05% trifluoroacetic acid), flow: 2.7 ml / min, fraction size 13.5 ml) to give the pure substance.
• Lysobactin bistrifluoroacetate (Example IA, 500 mg, 0.33 mmol) is dissolved in isopropanol / water 2: 1 (30 ml). Under argon protective gas atmosphere, palladium on carbon (10%, 100 mg) is added. The reaction mixture is stirred (after degassing) in a pressure autoclave at 80-70 bar hydrogen pressure and RT for 48 h. Palladium on carbon (10%, 100 mg) is again added to the reaction. The reaction mixture is (after degassing) again stirred in a pressure autoclave at 80-70 bar hydrogen pressure and RT for 48 h. Now it is no longer possible to detect lysobactin by HPLC (method 2).
• reaction mixture is filtered through a glass frit (pore size 4) or diatomaceous earth, concentrated in vacuo, taken up in 1% ethanol / 0.2% acetic acid, filtered through a syringe filter (Biotage, PTFE), concentrated in vacuo and dried under high vacuum. This gives 496 mg (quant.) Product (80% »Dihydrolysobactin, 20% octahydrolysobactin).
• cleavage solution Approximately 800 ml of the cleavage solution are filtered through a filter (0.2 ⁇ m) and chromatographed in two portions of approximately 400 ml each on a Source 15RPC column (40 ml) with an acetonitrile / TTA gradient. Conditions: eluent A: 0.1% TFA, eluent B: 0.1% TFA / acetoritritil; Gradient: 0% B to 45% B in 40 minutes; Flow: 2 ml / min; UV detection 210 nm; Fraction size 1.5 ml.
• the crude product after the aqueous workup can be purified by crystallization from cyclohexane / ethyl acetate.
• the reaction mixture is filtered through kieselguhr and the eluate is concentrated.
• the crude product is chromatographed (silica gel, eluent: cyclohexane / ethyl acetate 4: 1). There are obtained 60 g (99% of theory) of the title compound.
• N-tert-butoxycarbonyl-3-tert-butyl-D-alanine methyl ester (Example 5A, 60 g, 231 mmol) is dissolved in tetrahydrofuran p.a. (463 ml) dissolved. At RT, a solution of lithium hydroxide monohydrate (19.4 g, 462.7 mmol) in water (463 ml) is slowly added dropwise. When the HPLC chromatogram (Method 2) shows complete conversion (about 20 h), the reaction mixture is cautiously adjusted to pH 3-4 with 1 ⁇ aqueous hydrochloric acid while cooling with ice.
• the title compound is prepared according to protocol 2 from N-tert-butoxycarbonyl-3- / tert-butyl-L-alanine methyl ester (28.0 g, 108 mmol) and 4N hydrochloric acid in dioxane (280 ml). Yield 22 g (quantitative). The product is further reacted without purification.
• the organic phase is diluted with ethyl acetate (5O0 ml) and then treated with water * and then carefully adjusted to pH 3-4 with 1 ⁇ aqueous hydrochloric acid.
• the combined organic phases are washed with saturated aqueous common salt solution, dried over sodium sulfate, filtered, evaporated in vacuo and dried under high vacuum. This gives 97.4 g (97% of theory) of the title compound.
• Dibenzylazadicarboxylate (34.2 g, 114.6 mmol, 1.6 equiv.) Is added in one portion to the reaction mixture. It is stirred for 3 h at -60 to -45 ° C. In order to stop the reaction is again cooled down to -78 ° C and (20.5 ml, 358 mmol, 5 equivalents) of acetic acid was added and then heated to RT and finally 0 0 C. The reaction mixture is evaporated in vacuo and taken up in ethyl acetate (1000 ml).
• the reaction mixture is evaporated in vacuo and the residue is recrystallized from ethyl acetate to give the title compound as a solid (38.8 g, 24.3% of theory, crystallizate T).
• the mother liquor is diluted with ethyl acetate and then washed with water (twice) and saturated aqueous sodium chloride solution (twice).
• the organic phase is dried over sodium sulfate, filtered, evaporated in vacuo and dried under high vacuum.
• the crude product is recrystallized from ethyl acetate-diethyl ether.
• the title compound is obtained as a solid (50.4 g, 28% of theory, crystallization t 3).
• the reaction mixture is allowed to thaw and stirred until the HPLC chromatogram (Method 2) indicates complete conversion (approximately 60% product) (approximately 12 h) to stop the reaction by adding acetic acid (pH 4-6).
• the reaction mixture is evaporated in vacuo at RT and taken up in ethyl acetate.
• the organic phase is washed with water (twice), 5% citric acid (twice), water (once), saturated aqueous sodium bicarbonate solution (twice), saturated aqueous sodium chloride solution (once), dried over sodium sulfate, filtered, evaporated in vacuo, under high vacuum dried.
• the reaction mixture is thawed and stirred until the HPLC chromatogram indicates complete conversion (about 65% product) (about 12 h) to stop the reaction by adding acetic acid (23 ml, pH 4-6).
• the organic phase is washed with water (twice), saturated aqueous sodium bicarbonate solution (twice), saturated aqueous sodium chloride solution (once).
• the aqueous phases are reextracted with dichloromethane.
• the United organic phases are dried over sodium sulfate, filtered, evaporated in vacuo, dried under high vacuum.
• the crude product is purified by flash chromatography (6 1 silica gel, cyclohexane / ethyl acetate 3: 1). This gives 13.7 g (62% of theory, based on the recovered starting material 69%) of the title compound and 3.1 g of the starting compound (10% of theory).
• the reaction mixture is filtered under argon protective gas atmosphere over a glass frit, the glass frit is washed several times with methanol / water / 0.2% acetic acid. The filtrate is evaporated in vacuo and dried under high vacuum. This gives a solid (about 3 g) which is then suspended in ethyl acetate in an ultrasonic bath. To the suspension is added a solution of EDTA (400 mg) in 7% aqueous sodium bicarbonate (400 ml). The aqueous phase is extracted with ethyl acetate (100 ml 3 three times). The combined organic phases are then treated with saturated aqueous sodium bicarbonate.
• the reaction mixture is mixed with Kaliumdihydrogenphospha-t (5 equivalents), concentrated in vacuo and purified by preparative HPLC (Method 21 or Method 11 followed by re-salting the chromatography product by addition of TFA (2000 .mu.mol, as a 0.05% solution in acetonitrile-water 1 : 1)) cleaned. 531 mg (70% of theory) of the title compound are obtained.
• reaction mixture warmed slowly (about 12 h) to room temperature and then shows complete conversion of the amine component (HPLC control).
• the reaction mixture is admixed with solid potassium dihydrogen phosphate (5 equivalents, 350 ⁇ mol) and then concentrated under high vacuum and purified by chromatography (Method 21 or MC method 13 followed by re-salting of the chromatography product by addition of TFA (200 ⁇ mol, as 0.05% solution in Acetonitrile-water 1: 1)). 73.5 mg (72% of theory) of product are obtained.
• the nonapeptide trimethylsilyl ester (Example 21A, 70 mg, 46 ⁇ mol) and molecular sieve 4 A (about 10 mg) are initially charged in dry THF (3 ml) under argon protective gas atmosphere.
• a 1 N TBAF-THF solution (320 ul, 7 equivalents) is added dropwise with vigorous stirring and stirred until the HPLC chromatogram (Method 2) shows complete conversion (about 1 h).
• the reaction mixture is then neutralized with acetic acid (50 ⁇ l, ca. 20 equivalents).
• reaction mixture is evaporated in vacuo and purified by preparative HPLC (Method 21 or Method 13 followed by re-salting the chromatography product by addition of TFA (200 .mu.mol, as a 0.05% solution in acetonitrile-water 1: 1)). 50.2 mg (77% of theory) of the title compound are obtained.
• the nonapeptide acid (Example 22A, 54.4 mg, 38 ⁇ mol) is placed under argon protective gas atmosphere in dichloromethane (1 ml). At -20 0 C successively pentafluorophenol are (71 mg, 380 .mu.mol, 10 equivalents), EDC (17 mg, 192 .mu.mol, 2.3 equivalents) and DMAP (mg 1, 8 .mu.mol, 0.2 equivalents) was added. It is allowed to thaw slowly (about 3 h) and stirred at RT (about 12 h) until the HPLC chromatogram indicates complete conversion (method 2). The crude product is reacted further without purification.
• the N- (tert-butoxycarbonyl) cyclopeptide (Example 23 A, 60 mg, 40 .mu.mol) is reacted under argon protective gas atmosphere. Following microphilic drying, 50 mg (96% of theory) of product are obtained, which is reacted directly without further purification.
• the Cbz-protected cyclopeptide (Example 25A, 4.3 mg, 3.3 ⁇ mol) is dissolved in methanol (4 ml) and treated under argon protective gas atmosphere with 10%. Palladium-carbon (100 mg) and aqueous IN hydrochloric acid (200 ⁇ l) were added. At RT and atmospheric pressure, hydrogenation is carried out (about 1 h) until analytical HPLC (method 2) indicates complete conversion. The reaction mixture is filtered (through kieselguhr, Celite ® or a syringe filter, Fa. Biotage, PTFE), concentrated in vacuo and dried under high vacuum. 4 mg (quant.) Of the title compound are obtained.
• reaction mixture is stirred (about 15 minutes) and treated again with N-methylmorpholine (5.0 equivalents, 50 .mu.mol).
• the reaction mixture warmed slowly (about 12 h) to room temperature and then shows complete conversion of Amine component (HPLC control, method 3).
• the reaction mixture is combined with solid potassium dihydrogen phosphate (10 equivalents, 500 ⁇ mol) and then concentrated by evaporation in a high vacuum and purified by chromatography (Method 21 or Method 13 followed by re-salting the chromatography product by addition of TFA (100 ⁇ mol, as a 0.05% solution in Acetonitrile-water 1: 1)). 10.5 mg (70% of theory) of product are obtained.
• Example 3OA Preparation analogous to Example 27A from the compound from Example 26A (900 ⁇ g, 0.8 ⁇ mol) and N- (benzyloxycarbonyl) -D-leucyl-L-leucine (Example 3OA, 1200 ⁇ g, 3.2 ⁇ mol, 4 equivalents).
• the Crude product is purified by gel chromatography (method 14) to give 500 ⁇ g (41% of theory) of product as a solid after freeze-drying.
• the reaction mixture is concentrated, taken up in ethyl acetate (about 200 ml) and then with saturated aqueous sodium bicarbonate solution (once), 5% aqueous citric acid (twice), saturated aqueous sodium bicarbonate solution (once) and saturated sodium chloride solution washed. It is dried over sodium sulfate and filtered. The mixture is evaporated to dryness in vacuo and subsequently dried in a high vacuum. 1483 mg (93% of theory) of the title compound are obtained, which are reacted without further purification.
• Method A (5 g scale): A 1 N solution of lithium hexamethyldisilazide (39.4 mmol, 39.4 ml, 2.2 equivalents) in THF in the reaction solvent THF (200 ml) is placed under an argon protective gas atmosphere. At -78 0 C a solution of (S) -3 is - zugetropfit slowly [(tert-butoxycarbonyl) amino] -3-phenylpropanklar (5.0 g, 17.9 mmol). The mixture is stirred for 10 min at -25 0 C and then cooled again to -78 0 C.
• Method B (50 g scale): A 1 N solution of lithium hexamethyldisilazide (452.9 mmol, 452.9 ml, 2.2 equivalents) in THF in the reaction solvent THF (1800 ml) is placed under an argon protective gas atmosphere. At -7O 0 C a solution of (S) -3 is - (ml 600) was slowly added dropwise [(tert-butoxycarbonyl) amino] -3-phenylpropanklad. The mixture is stirred for 10 min at -25 ° C and then cooled again to -7O 0 C down.
• the reaction mixture is thawed and stirred until the HPLC chromatogram (Method 2) indicates complete conversion (about 60% of product) (about 12 h) and the reaction is then carried out by addition of acetic acid (10.5 ml, pH 4-6). to stop.
• the organic phase is washed with water (twice), saturated aqueous sodium bicarbonate solution (twice), saturated aqueous sodium chloride solution (once).
• the aqueous phases are with Dichloromethane reextracted.
• the combined organic phases are dried over sodium sulfate, filtered, evaporated in vacuo, dried under high vacuum.
• the crude product is purified by flash chromatography (silica gel, toluene / ethyl acetate 4: 1). This gives 2.4 g (61% of theory, based on recovered starting material 68%) of the title compound and 490 mg of the starting compound (12% of theory).
• the aqueous phase is reextra ⁇ hiert with dichloromethane and then washed with 5% aqueous citric acid, saturated aqueous ⁇ a-trium- bicarbonate solution and saturated brine, dried over ⁇ atriurnsulfat, filtered and concentrated in vacuo.
• the mixture is then purified by means of flash chromatography (silica gel, toluene / ethyl acetate 4: 1). 144 mg (47% of theory) of the title compound are obtained 51
• the reaction mixture is thawed (about 12 h) and stirred at RT until the HPLC chromatogram (method 2) indicates complete conversion (about 12 h).
• the reaction mixture is filtered through kieselguhr and then evaporated at RT in vacuo.
• the crude product is pre-fractionated on a RPig cartridge (eluent water, then acetonitrile) and purified by preparative flash chromatography (1.5 kg silica gel, toluene / ethyl acetate 4: 1). 3.6 g (73% of theory) of the title compound are obtained.
• reaction mixture in a rotary evaporator. concentrated (water bath 35 ° C) with ethyl acetate (100 ml) and then with saturated sodium bicarbonate solution (twice 20 ml), 5 percent. Citric acid (twice 20 ml), and washed with saturated aqueous sodium chloride solution. The organic phase is dried over sodium sulfate, filtered and concentrated.
• the product obtained is a solid (2.88 g, 99% of theory) which can be finely purified by preparative HPLC (Method 13).
• the basic aqueous phase is washed with dichloromethane (six times). It is then adjusted to pH 2-3 with 1 N aqueous hydrochloric acid. The watery Phase is then extracted with ethyl acetate (three times). The combined organic phases are treated with conc. washed aqueous sodium chloride solution, dried over sodium sulfate, filtered and concentrated in vacuo. The resulting crude product is purified by preparative HDPLC (Method 35). This gives 6.86 g (64% of theory) of the title compound.
• Methyl L-leucinate hydrochloride (600.9 mg, 3.31 mmol, 1.1 equivalents) is initially charged under argon blanket gas atmosphere in dichloromethane (250 ml), the solution is treated with HOBt (1625.3 mg,
• reaction solution is concentrated on a rotary evaporator at 30 0 C bath temperature, the
• the title compound is prepared from the methyl ester (Example 48A, 1.1 g, 2.71 mmol) by hydrolysis according to procedure 4. After a reaction time of 4 h, the HPLC chromatogram (Method 2) indicates almost complete conversion. For fine purification, the crude product is chromatographed on the preparative RP-HPLC (Method 36) and then 980 mg (92% of theory) of product.
• Example Compound 51A (10.15 g, 26.69 mmol) is dissolved in methanol p.a. (100 ml) dissolved. With a cannula, argon is passed through for about 5 minutes, then (-f-) -1,2-bis - [(2) S ', 5> S) diethylphospholano] benzene (cyclooctadiene) rhodium (I) triflate (289 mg, 400 ⁇ mol, 0.015 equivalents).
• Example Compound 52A (9.90 g, 25.89 mmol) is dissolved in methanol (100 mL). With a cannula, argon is passed for about 5 minutes, then Pd is added via C (10%, 990 mg). It is hydrogenated for 12 h at 4 bar hydrogen pressure and RT. It is then filtered through kieselguhr, concentrated and dried under high vacuum. Yield: 5.8 g (90% of theory) of the title compound.
• example compound 53A (6.34 g, 25.54 mmol) and N- (tert-butoxycarbonyl) -3-tert-butyl-D-alanine (6.27 g, 25.54 mmol, 1.0 equiv.)
• dry DMF 240 mL
• HATU HATU
• IR v max (NaCl, cm -1 ): 2959, 1742, 1655, 1520, 1336, 1160, 1136, 1087, 1050, 1027.
• Methyl ⁇ [tert-butoxycarbonyl] amino ⁇ (dimethoxyphosphoryl) acetate (39.2 g, 132.00 mmol) and cyclopentanecarbaldehyde (38.9 g, 396.00 mmol, 3 equiv.) are initially charged in THF (320 mL) and N, NNN-tetramethylguanidine (24.8 mL, 22.81 g, 198.00 mmol, 1.5 equivalents) is added at -7O 0 C to the solution. Thaw the cold bath, the reaction is slowly brought to RT. Within 2 days full turnover is achieved.
• the reaction is combined with ethyl acetate (1600 ml) and water (800 ml) and the separated organic phase is dried over sodium sulfate.
• the desiccant is separated by filtration and the filtrate is concentrated on a rotary evaporator. From the residue, the product is isolated by column chromatography (silica gel 60: 1 kg, eluent: cyclohexane / ethyl acetate 4: 1) with 34.8 g (98% of theory) yield.
• the example compound 56A (13.0 g, 48.27 mmol) is dissolved in ethanol (220 ml), the solution is purged with argon for 10 min and placed in an ultrasonic bath for 5 min. After the addition of (+) - l, 2-bis - [(2S, 55) diethylphospholano] benzene (cyclooctadiene) rhodium (I) triflate (100.0 mg, 0.13 mmol) is rinsed again with argon for 30 min, in an ultrasonic bath for 5 min degassed and then hydrogenated for 3 days at RT at a hydrogen pressure of 3.5 bar.
• the exemplary compound 59A (5.0 g, 18.43 mmol) is reacted according to protocol 4 within 2 h to the iV " protected amino acid to give 5.11 g (quant.) Of the title compound.
• the peptidic amine (Example 58A, 0.9 g, 4.27 mmol, 1.1 equivalents) is initially charged in dichloromethane (250 ml) under argon protective gas atmosphere, the solution cooled to -10 0 C and then closing successively with HOBt (2.1 g, 15.54 mmol , 4 equivalents), ⁇ MM (1.3 ml, 1.2 g, 11.67 mmol, 3 equivalents), the peptidic acid (Example 6OA, 1.0 g, 3.89 mmol), EDC (1.5 g, 7.77 mmol, 2 equivalents) and again with ⁇ MM ( 0.8 ml, 0.8 g, 7.76 mmol, 2 equivalents).
• the reaction is slowly brought to RT in the cooling bath, completely reacted overnight.
• water is added, concentrated on a rotary evaporator (water bath 35 0 C), treated with ethyl acetate and saturated sodium bicarbonate solution and the separated aqueous phase extracted twice with ethyl acetate, the combined organic phases successively with saturated, aqueous 5% citric acid Solution, saturated sodium bicarbonate solution and saturated sodium chloride solution.
• the organic phase is dried over sodium sulfate, filtered and concentrated.
• the crude product is 1.64 g (quant.) Of the title compound.
• the dipeptide (Example 61 A, 1.5 g, 3.65 mmol) is reacted according to Working Method 4 within 1 h to dipeptidic carboxylic acid. After the fine purification (Method 13) of the freeze-dried crude product, the title compound is isolated with 923.4 mg (64% of theory) of iso.
• Example 64A (1.1 g, 1.98 mmol) in THF and water 3: 1 was added (48 ml) and cooled to -2O 0 C. Lithium hydroxide (119 mg, 2.5 mmol, 4.95 equivalents) is added and the mixture is stirred at RT for 12 h. The reaction is stopped by the addition of potassium dihydrogen phosphate (1.35 g) and concentrated in vacuo. After chromatographic purification (Method 14), 0.57 g (67% of theory) of the title compound are obtained as a solid.
• the peptidic amine (Example 77A, 4.0 g, 14.68 mmol, 1.1 equivalents) is initially charged under argon inert gas atmosphere in dichloromethane (365 ml), the solution is washed successively with HOBt (7.2 g, 53.23 mmol, 4 equivalents), NMM (4.3 ml , 39.92 mmol, 3 equivalents), N- (tert-butoxycarbonyl) -. D-leucine (3.1 g, 13.31 mmol, 1 equivalent) [T. Kato, N. Izumiya, Bull. Chem. Soc.
• Example compound 68A (400 mg, 1.07 mmol) is reacted according to protocol 4 within 1 h to the dipeptide carboxylic acid.
• the solution is combined with potassium dihydrogenphosphate (219.2 mg, 1.61 mmol, 1.5 equivalents), concentrated by evaporation in a cold rotary evaporator and finely purified by preparative HPLC (eg Method 35).
• the title compound is obtained as lyophilisate (282.0 mg, 65% of theory). (For analytical data see WO2004099239).
• Example IIA The compound from Example IIA. (300 mg) is dissolved in methanol (3 ml) and cooled to 0 ° C. Trimethylsilyl chloride (590 mg, 4.7 equivalents) is dissolved in 30 min. added dropwise and the mixture warmed to RT overnight. The volatile components are removed on a Rotavapor and then under high vacuum. The target compound (224 mg, 92% of theory) is obtained.
• Example 73A The compound of Example 73A (2.0 g) is initially charged in THF (40 ml) under argon at 0 ° C. Triethylamine (2.3 g, 2.4 equivalents) is added and then a solution of JV-benzyloxycarbonyloxysuccinimide (2.83 g, 1.2 equivalents) in THF (20 ml) is added dropwise. The mixture is then stirred at RT overnight, concentrated by half, diluted with ethyl acetate, washed twice with water and once with saturated sodium chloride solution. The organic phase is dried over sodium sulfate and concentrated by rotary evaporation. The residue is purified by preparative HPLC (Method 41). The target compound (2.32 g, 79% of theory) is obtained.
• Example 74A To a solution of Example 74A (2.32 g, 7.5 mmol) in THF (14 mL) was added lithium chloride (0.64 g, 15 mmol) and sodium borohydride (0.57 g, 15 mmol) then ethanol (22 mL). The mixture is stirred overnight at RT. For workup, the mixture is adjusted to pH 4 under cooling with ice-water and with citric acid solution (10% in water), then diluted with 200 ml of water and extracted three times with dichloromethane. The combined organic phases are dried over magnesium sulfate, filtered and concentrated in vacuo. The residue is separated by HPLC (Method 41) and the appropriate fraction is concentrated in vacuo. The title compound is obtained as an oil (1.24 g, 59% of theory).
• a solution of pyridine-SO 3 complex (2.04 g, 12.8 mmol) in anhydrous DMSO (12 mL) is added to a solution of Example 75A (0.36 g, 1.28 mmol) and N, N-diisopropylethylamine (1.65 g, 12.8 mmol) 7 ml of DMSO under argon at 10 0 C was added.
• the cooling bath is removed and the mixture is left for 20 min. stirred. 400 ml of ice-water are added and the solution is extracted three times with diethyl ether.
• Example 77A Under argon, the compound Example 77A are (4:33 g, 15.9 mmol), Example Compound 76A (7.2 g, 20.6 mmol) and ⁇ atriumacetat (1.3 g, 15.9 mmol) were introduced into DMEF (140 ml) at 0 0 C. Natriumcyanborohydrid (1.99 g, 31.7 mmol) is added and the mixture stirred at 0 0 C for 2 h.
• the mixture is diluted with 1 1 ethyl acetate and washed twice with a 5% solution of sodium bicarbonate. The aqueous phases are reextracted with ethyl acetate. The combined organic phases are washed with sat. Washed sodium chloride solution, dried over magnesium sulfate, filtered and concentrated. The residue is purified by preparative HPLC (Method 39). This gives 6.02 g (71% of theory) of the title compound as crystals.
• Example compound 78A (6.02 g, 1.2 mmol) is dissolved in methanol (225 ml) and treated at RT with a 1 ⁇ lithium hydroxide solution (101 ml) in water (9 equivalents). The reaction mixture is stirred at RT overnight (1 h), then acidified with 1 N hydrochloric acid and freed from methanol on a rotary evaporator. The remaining suspension is diluted with 200 ml of water and extracted three times with ethyl acetate. The combined organic phases are dried over magnesium sulfate, filtered and concentrated. The residue is dried under high vacuum. This gives 5.3 g (quantitative) of the title compound, which is reacted further without Reiniguxig.

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• 2004
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• 2005
  • 2005-10-26 JP JP2007539500A patent/JP4843615B2/ja not_active Expired - Fee Related
  • 2005-10-26 CN CNA2005800459350A patent/CN101107265A/zh active Pending
  • 2005-10-26 CA CA002602070A patent/CA2602070A1/en not_active Abandoned
  • 2005-10-26 EP EP05798236A patent/EP1809657A1/de not_active Withdrawn
  • 2005-10-26 WO PCT/EP2005/011451 patent/WO2006048156A1/de active Application Filing
• 2007
  • 2007-05-04 US US11/800,495 patent/US7718611B2/en not_active Expired - Fee Related

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