EP1807443A1 - Acylated nonadepsipeptides used as lysobactin derivatives - Google Patents

Acylated nonadepsipeptides used as lysobactin derivatives

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Publication number
EP1807443A1
EP1807443A1 EP05798234A EP05798234A EP1807443A1 EP 1807443 A1 EP1807443 A1 EP 1807443A1 EP 05798234 A EP05798234 A EP 05798234A EP 05798234 A EP05798234 A EP 05798234A EP 1807443 A1 EP1807443 A1 EP 1807443A1
Authority
EP
European Patent Office
Prior art keywords
hplc
mmol
compound
water
salts
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP05798234A
Other languages
German (de)
French (fr)
Inventor
Franz Von Nussbaum
Nina Brunner
Rainer Endermann
Chantal FÜRSTNER
Elke Hartmann
Holger Paulsen
Jacques Ragot
Guido Schiffer
Joachim Schuhmacher
Niels Svenstrup
Joachim Telser
Sonja Anlauf
Michael-Alexander BRÜNING
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Aicuris GmbH and Co KG
Original Assignee
Aicuris GmbH and Co KG
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Aicuris GmbH and Co KG filed Critical Aicuris GmbH and Co KG
Publication of EP1807443A1 publication Critical patent/EP1807443A1/en
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K11/00—Depsipeptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K11/02—Depsipeptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof cyclic, e.g. valinomycins ; Derivatives thereof

Definitions

  • the invention relates to nonadepsipeptides and processes for their preparation and their use for the production 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 represent successful examples of the antibiotic potency of this active 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 anti-bacterial activity.
  • An object of the present invention is to provide alternative compounds having comparable or improved antibacterial activity, better compatibility, e.g. B. lower nephrotoxicity, and better distribution in the body, ie better pharmacokinetic properties, such. B. increase of the free fraction (f u ) to provide for the treatment of bacterial diseases in humans and animals.
  • the invention relates to compounds of the formula
  • R 1 is hydrogen
  • R 2 is 2,2-dimethylbut-1-yl, 2-ethyl-2-methylbut-1-yl, 2,2-diethyl-but-1-yl, 2,2-dimethylpent-1-yl or trimethylsilylmethyl,
  • R 1 is trifluoromethyl
  • R 2 2,2-dimethylprop-1-yl, 2,2-dimethylbut-1-yl, 2-ethyl-2-methylbut-1-yl, 2,2-diethyl-but-1-yl, 2,2- Dimethylpent-1-yl or trimethylsilylmethyl,
  • Compounds according to the invention are the compounds of the formula (I) and their salts, solvates, solvates of the salts and prodrugs, the compounds of the formulas below and their salts, solvates, solvates of the salts and prodrugs and of the formula (I) I), hereinafter referred to as exemplary compounds and their salts, solvates, solvates of the salts and prodrugs, as far as the compounds encompassed by formula (I) 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 also included are salts which are not suitable for pharmaceutical applications themselves but can be used for example for the isolation or purification of the compounds of the invention 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, hydrobromic, sulfuric, phosphoric, methanesulfonic, ethanesulfonic, toluenesulfonic, benzenesulfonic, naphthalenedisulfonic, acetic, trifluoroacetic, propionic, lactic, tartaric, malic, citric, fumaric, maleic and benzoic acids.
  • Salts of hydrochloric, hydrobromic, sulfuric, phosphoric, methanesulfonic, ethanesulfonic, toluenesulfonic, benzenesulfonic, 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, such as, by way of example and by way of illustration, ethylamine, diethylamine, triethylamine, ethyldiisopropylamine, monoethanolamine, diethanolamine, triethanolamine, dicyclohexylamine, dimethylaminoethanol, procaine, dibenzylamine, N-methylmorpholine, arginine, lysine, ethylenediamine and N-methylpiperidine.
  • solvates 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 solvox,
  • R 1 is hydrogen
  • R 2 is 2,2-dimethylbut-1-yl or trimethylsilylmethyl
  • R 1 is trifluoromethyl
  • R 2 is 2,2-dimethylprop-1-yl, 2,2-dimethylbut-1-yl or trimethylsilylmethyl,
  • R 1 is hydrogen
  • R 2 is 2,2-dimethylbut-1-yl, 2-ethyl-2-methylbut-1-yl, 2,2-diethyl-but-1-yl or trimethylsilyl-methyl,
  • R 1 is hydrogen
  • R 2 is 2,2-dimethylbut-1-yl, 2-ethyl-2-methylbut-1-yl, 2,2-diethyl-but-1-yl, 2,2-dimethylpent-1-yl or trimethylsilylmethyl.
  • the invention furthermore relates to a process for the preparation of the compounds of the formulas (I), where the compound of the formula
  • R 1 and R 2 have the meaning given above, and
  • X 1 is halogen, preferably bromine, chlorine or fluorine, or hydroxy
  • the reaction is generally carried out in inert solvents, if appropriate in the presence of a base, preferably in a temperature range from -3O 0 C to 50 0 C at atmospheric pressure.
  • Inert solvents are, for example, tetrahydrofuran, methylene chloride, pyridine, dioxane or dimethylformamide, preference is given to methylene chloride or dimethylformamide.
  • bases are triethylamine, diisopropylethylamine or N-methylmorpholine, preferably diisopropylethylamine.
  • the reaction 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 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, dipropyl, N, N'-diisopropyl-, N, N'-dicyclohexylcarbodiimide, N- (3-di- methylaminoisopropyl) are - N'-ethylcarbodiimide hydrochloride (EDC), N-cyclohexylcarbodiimide-N'-propyloxymethyl-polystyrene (PS-carbodiimide) or carbonyl compounds such as carbonyldiimida- ,
  • EDC N'-ethylcarbodiimide hydrochloride
  • PS-carbodiimide N-cyclohexylcarbodiimide-N'-propyloxymethyl-polystyrene
  • carbonyl compounds such as carbonyldiimida-
  • 1,2-oxazolium compounds such as 2-ethyl-5-phenyl-l, 2-oxazolium-3-sulfate or 2-tert-butyl-5-methyl-isoxazolium perchlorate, or acylamino compounds such as 2-ethoxy -l-ethoxycarbonyl-l, 2-dihydroquinoline, or propanephosphonic anhydride, or isobutylchlorofo ⁇ nat, or bis (2-oxo-3-oxazolidinyl) -phosphorylchlorid or Benzotriazolyloxy-tri (dimethylamino) - phosphoniumhexafluorophosphat, or O- (Benzot ⁇ iazol-l -yl) -N, N, N ', N'-tetra-methyluronium hexafluorophosphate (HBTU), 2- (2-oxo-l- (2H) -pyridyl) -1,3,
  • 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-dimethylaminopyridine 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-dimethylaminopyridine or diisopropylethylamine.
  • the condensation is carried out with HATU or with EDC in the presence of HOBt.
  • the compounds of the formula (HI) optionally carry protective groups, so that in these cases the reaction of the compound of the formula (II) with compounds of the formula (DI) involves cleavage of the protective groups with trifluoroacetic acid by methods known to the person skilled in the art.
  • the free base of the salts of the compounds of the formula (I) can be obtained, for example, by addition of a base and subsequent extraction, precipitation or chromatographic separation of the compound by methods known to the person skilled in the art.
  • polymer-bound bases such as, for example, polymer-bound bicarbonate.
  • the invention further provides a process for the preparation of the compounds of the formula (I) or their solvates according to claim 1, in which salts of the compounds or solvates of the salts of the compounds are converted into the compounds by addition of a base.
  • the compound of the formula (H) can be synthesized by double Edmann degradation from lysobactin (Example 1), as described in the experimental section under Example 2A.
  • the compounds of the formula (III) are known or can be synthesized by known processes from the corresponding starting materials.
  • the compounds of the invention show an unpredictable, valuable pharmacological and pharmacokinetic spectrum of activity. They show an antibacterial effect.
  • the compounds according to the invention are distinguished by a lower nephrotoxicity compared to lysobactin.
  • the compounds according to the invention are distinguished by a better pharmacokinetics than by lysobactin. They show a better distribution in the body with the same or improved pharmacological effect, which results in a lower therapeutic dose and a broader therapeutic window of treatment.
  • the compounds of the invention have a higher free fraction (f u ) in plasma than lyso ⁇ bactin.
  • 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 suitable 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 bacterium-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), Sahnonella and Shigella; also Enterobacter (E. aerogenes, E. agglomerans), Hafhia, Serratia (S. marcescens), Providencia, Yersinia, as well as the genera Acinetobacter, Branhamella and Chlamydia.
  • Enterobacteriaceae e.g. Escherichia coli, Haemophilus influenzae, Pseudomonas, Klebsiella, Citrobacter (C. freundii, C. divernis), Sahnonella and Shigella
  • Enterobacter E. aerogenes,
  • 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 show particularly pronounced activity against gram-positive cocci, e.g. Staphylococci (S. aureus, S. epidermidis, S. haemolyticus, S. carnosus), enterococci (E. faeca-Us, E. faecium) and streptococci (S. agalactiae, S. pneumoniae, S. pyogenes).
  • Staphylococci S. aureus, S. epidermidis, S. haemolyticus, S. carnosus
  • enterococci E. faeca-Us, E. f
  • pathogens are merely exemplary and by no means restrictive.
  • 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:
  • Human infectious diseases 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, empirical therapy in febrile neutropenic patients, further bacteremia, MRSA infections, acute infectious diarrhea, Helicobacter pylori infections, postoperative infections, odontogenic infections, ophthalmological In infections, postoperative infections (incl. peripro
  • 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, salmonella;
  • 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, with the antibacterial spectrum on the aforementioned pathogens out to further pathogens such.
  • Pasteurella Brucella, Campylobacter, Listeria, Erysipelothris, Corynebacteria, Borellia, Treponema, Nocardia, Rikettsia, Yersinia.
  • a further subject of the present invention is the use of the compounds according to the invention for the treatment and / or prophylaxis of diseases, in particular of bacterial infectious diseases.
  • Another object of the present invention is the use of Ver ⁇ compounds of the invention for the treatment and / or prophylaxis of diseases, in particular the aforementioned ge diseases.
  • Another object of the present invention is the use of Ver ⁇ compounds of the invention for the preparation of a medicament for the treatment and / or prophylaxis of Erkran- kungen, 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 Pro ⁇ phylaxis of diseases, in particular the aforementioned diseases, using an antibacterially effective amount of the compounds of the invention.
  • 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.
  • Tablets uncoated or coated tablets, for example, with enteric or delayed-dissolving or insoluble coatings containing the
  • Soft gelatin capsules Soft gelatin capsules
  • dragees granules, pellets, powders, emulsions, suspensions, aerosols or solutions.
  • Parenteral administration can be accomplished by bypassing a resorption step (e.g., intravenously, intraarterially, intracardially, intraspinal, or intralumbar) or by resorting to absorption (e.g., intramuscularly, subcutaneously, intracutaneously, percutaneously, or intraperitoneally).
  • a resorption step e.g., intravenously, intraarterially, intracardially, intraspinal, or intralumbar
  • absorption e.g., intramuscularly, subcutaneously, intracutaneously, percutaneously, or intraperitoneally.
  • injection and infusion preparations in the form of solutions, suspensions, emulsions, lyophilisates or sterile powders.
  • inhalation medicaments including powder inhalers, nebulizers
  • nasal drops solutions, sprays
  • lingual, sublingual or buccal tablets to be applied
  • films / wafers or capsules films / wafers or capsules, suppositories, ear or eye 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 are used as therapeutically active agents.
  • the compounds according to the invention can be converted into the stated administration forms. This can be done in a manner known per se by mixing with inert, non-toxic, pharmaceutically suitable auxiliaries.
  • 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 (eg antioxidants such as ascorbic acid), dyes (eg inorganic pigments such as iron oxides) and flavor and / or odoriferous agents.
  • Carriers for example microcrystalline cellulose, lactose, mannitol
  • solvents for example liquid polyethylene glycols
  • emulsifiers and dispersants or wetting agents for example sodium dode
  • the present invention further relates to medicaments which comprise at least one compound according to the invention, usually together with one or more inert, non-toxic, pharmaceutically suitable excipients, and to their use for the purposes mentioned above.
  • EDC 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide also EDCI
  • TOF-HR-MS-ESI + spectra are provided with a Micromass LCT apparatus (capillary voltage: 3.2 KV, cone voltage: 42 V, source temperature: 12O 0 C, desolvation temperature: 280 0 C).
  • a syringe pump Hard Apparatus
  • the standard is leucine-enkephalin (Tyr-Gly-Gly-Phe-Leu).
  • Method 2 (Preparative HPLC): Device: Gilson Abimed HPLC; UV detector 210 nm; binary pump system; Column: Waters Symmetry-Prep TM Ci 8 , 7 ⁇ m, 300 x 19 mm; Eluent A: 0.2% trifluoroacetic acid in water, eluent B: acetonitrile; Flow rate: 25 mL / min; Column temperature RT; 0 min 20% B, ramp 0-10 min 70% B, ramp 10-10.1 min 20% B, 15 min 20% B.
  • Method 3 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). At scales of 1 mmol to 11 mmol, a column of the dimension 80 ⁇ 30 cm is used. In this case, the fractions are collected manually and without upstream UV detector. The assignment of the fractions is carried out by HPLC (Method 9).
  • Method 4 (preparative HPLC, Kromasil, acetic acid): Device: Gilson Abimed HPLC; UV detector 210 nm; binary pump system; Column: Kromasil 100 A Ci 8 , 5 ⁇ m; 250 x 20 mm; Flow: 25 mL / min; Eluent A: water / 0.25-0.5% 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.
  • Method 5 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 water + 0.5 mL 50% formic acid, eluent B: 1 L acetonitrile + 0.5 mL 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 6 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 water + 0.5 mL 50% formic acid, eluent B: 1 L acetonitrile + 0.5 mL 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 7 Device Type MS: Micromass ZQ; Device type HPLC: HP 1100 Series; UV DAD; Column: Phenomenex Synergi 2 ⁇ Hydro-RP Mercury 20 mm x 4 mm; Eluent A: 1 L water + 0.5 mL 50% formic acid, eluent B: 1 L acetonitrile + 0.5 mL 50% formic acid; Gra ⁇ serves: 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 (Analytical HPLC): Device Type HPLC: HP 1050 Series; UV DAD 1100 Series; Column: Kromasil Ci 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, 4.5-9.0 min 90% B, 9.0-9.2 min 90-2% B, 9.2-10.0 min 2% B; Flow: 0.75 mL / min, oven: 30 ° C., UV detection 210 nm.
  • Method 9 (analytical HPLC, Agilent Zorbax C 8 ): Device: Agilent 1100 with DAD (GI 315B), binary pump (G1312A), autosampler (G1313A), solvent degasser (G1379A) and column thermostat (G1316A); Column: Agilent Zorbax Eclipse XDB-C8 4.6 x 150 x 5 mm; Eluent A: 0.05% 70% perchloric acid in water; Eluent B: acetonitrile; Gradient: 0-1 min 10% B, ramp, 4-5 min 90% B, ramp, 5.5 min 10% B; Flow: 2.00 mL / min; Column temperature: 3O 0 C.
  • Method 11 (Preparative HPLC Symmetry): Device: Gilson Abimed HPLC; binary pump system; Column: SymmetryPrep TM C 18 , Waters, 7 ⁇ m; 300mm x 19mm; Eluent A: water / 0.2% trifluoroacetic acid, eluent B: acetonitrile; Gradient: 0-10 min 15-65% B, then regeneration of the chromatography column; Flow: 25 mL / min; UV detection 210 nm.
  • Method 12 (Preparative HPLC Kromasil): Device: Gilson Abimed HPLC; binary pump system; Column: Kromasil Ci 8 , 5 ⁇ m, 100 A, 250 ⁇ 20 mm; Eluent A: 0.05% trifluoroacetic acid in water, eluent B: 0.05% trifluoroacetic acid in acetonitrile; Gradient: 0-3 min 10% B, ramp, 30-38 min 90% B, 38-45 min 10% B; Flow: 20 mL / min; UV detection 210 nm.
  • Method 13 (Preparative HPLC Waters Symmetry): Device: Gilson Abimed HPLC; binary pump system; Column: Waters Symmetry-Prep TM Ci 8 , 7 ⁇ m, 300 x 19 mm; Eluent A: 0.05% trifluoroacetic acid in water, eluent B: 0.05% trifluoroacetic acid in acetonitrile; Gradient: 0-3 min 10% B, ramp, 30-38 min 90% B, 38-45 min 10% B; Flow: 20 mL / min; UV detection 210 nm.
  • Method 14 (preparative HPLC): Device: Gilson Abimed HPLC; binary pump system; Column: Waters Symmetry-Prep TM Ci 8 , 7 ⁇ m, 300 x 19 mm; Eluent A: water / 0.2% trifluoroacetic acid, eluent B: acetonitrile; Gradient: 0-10 min 25-65% B, then regeneration of the chromatographic column; Flow: 25 mL / min; UV detection 210 nm.
  • Method 15 Chiral HPLC Daicel Chiralpak: Agilent 1100 HPLC; Column: Daicel Chiralpak AD-H 5 ⁇ m; 250 x 20 mm; isocratic: 75% hexane, 25% 2-propanol with 0.2% trifluoroacetic acid and 1% water; Flow: 1.0 mL / min; Oven: 25 ° C; UV detector 212 nm.
  • Method 16 (Preparative HPLC): Device: Gilson Abimed HPLC; binary pump system; Column: YMC ODS-AQ 5 ⁇ m, 250 x 30 mm; Eluent A: 0.05% trifluoroacetic acid in water, eluent B: 0.05% trifluoroacetic acid in acetonitrile; Gradient: 0-3 min 10% B, ramp, 30-38 min 90% B, 38- 45 min 10% B; Flow: 50 mL / min; UV detector 210 nm.
  • Method 17 Instrument: Micromass GCT, GC6890; Column: Restek RTX-35MS, 30 m ⁇ 250 ⁇ m ⁇ 0.25 ⁇ m; Gradient: 60 0 C (0.30 keep min), 50 ° C / min ⁇ 12O 0 C, 16 ° C / min ⁇ 25O 0 C, C / min ⁇ C (hold 1.7 min) 300 30 ° 0; constant flow with helium: 0.88 mL / min; Oven: 60 ° C; Let: 25O 0 C.
  • Method 19 (HPLC): Column: Kromasil RP-18, 60 mm ⁇ 2 mm, 3.5 ⁇ m; Eluent A: 5 mL HCIO 4 / I 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: 3O 0 C; UV detection: 210 nm.
  • Method 20 (HPLC): Column: Kromasil RP-18, 250 mm x 4 mm, 5 ⁇ m; Eluent A: 5 mL HClO 4 /! 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 21 (HPLC): Column: Kromasil RP-18, 250 mm x 4 mm, 5 ⁇ m; Eluent A: 2 mL HCIO 4 / l water, eluent B: acetonitrile; Isocratic: 45% B, 55% A; Flow: 1 mL / min; Oven: 4O 0 C; UV detection: 210 nm.
  • Method 22 Device Type MS: Micromass ZQ; Device type HPLC: HP 1100 Series; UV DAD; Column: Grom-Sil 120 ODS-4 HE 50 ⁇ 2 mm, 3.0 ⁇ m; Eluent A: water / 0.025% formic acid / l, eluent B: acetonitrile / 0.025% formic acid; Gradient: 0-2.9 min 0-70% B, 2.9-3.1 min 70-90% B, 3.1-4.5 min 70-90% B; Oven: 5O 0 C, flow: 0.8 mL / min, UV detection: 210 nm.
  • Method 23 Device Type HPLC: HP 1050 Series; UV DAD 1100 Series; Column Symmetry-Prep TM Ci 8 , Waters, 50 x 2.1 mm, 3.5 ⁇ m; Eluent A: water / 0.05% trifluoroacetic acid, eluent B: acetonitrile; Gradient: 0-9 min 0-100% B, 9-11 min 100% B, 11-12 min 100-0% B, subsequent regeneration of the chromatography column; Oven: 4O 0 C, flow: 0.4 mL / min, UV detection: 210 nm.
  • Method 24 Quantitative 19 F-NMR Spectroscopy: Approximately 10 mg of sample substance weighed in exactly and approximately 20 mg of 1,4-dibromotetrafluorobenzene weighed in exactly are dissolved in pyridine and measured by 19 F-NMR spectroscopy, ⁇ -74 (cf. TFA) and -132.0 (1,4-dibromotetrafluorobenzene) are integrated and compared. The TFA content is expressed in percent TFA of the mass of the sample substance.
  • Method 25 Ion Chromatography: Ion chromatography system with suppressor system and conductivity detector; Guard column: A SUPP 4/5 Guard, separation column: A SUPP 5 4.0 x 250 mm; Eluent: 3.2 mM sodium carbonate and 2.4 mM sodium bicarbonate in water; Flow: 0.7 mL / min.
  • the sample is dissolved in methanol (20% of the final sample volume), treated in an ultrasonic bath for 3 minutes and made up with water.
  • the sample is filtered through an ion-free cellulose acetate filter (Por 0.45 ⁇ m) and injected. Quantification against external standards (0.5 mg / L - 10 mg / L).
  • 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 (20 minutes at 121 0 C), 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 YM medium.
  • Piston Fermentation Inoculate 150 mL of YM medium or 100 mL of HPM medium in a 1 L Erlenmeyer flask with 2 mL of the working fluid and allow to grow for 30-48 hours at 28 0 C on a shaker at 240 rpm.
  • 30 L 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 media solution
  • the culture broth of the main culture is separated at 17000 rpm in supernatant and sediment.
  • the supernatant (183 L) 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). The mixture 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 remaining aqueous residue of 11.5 L.
  • aqueous phase is bound to silica gel Cig and separated (MPLC, Biotage Flash 75, 75 ⁇ 30 cm, KP-C18-WP, 15-20 ⁇ m, flow: 30 mL, eluent: acetonitrile / water with 0.1% trifluoroacetic acid, gradient : 10%, 15% and 40% acetonitrile).
  • MPLC Biotage Flash 75, 75 ⁇ 30 cm, KP-C18-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 lyophilizate obtained (89.9 g) is taken up in methanol, filtered off, concentrated and separated on silica gel (Method 21).
  • Example IA is then purified by gel filtration (Sephadex LH-20, 5 ⁇ 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 (60.0 g, 39.88 mmol) is dissolved under argon atmosphere in pyridine (840 WLL). Then phenylisothiocyanate (32.35 g, 239.28 mmol, 6 equivalents) is added ben and the reaction mixture is stirred at 37 ° C for 7 h. Subsequently, the solvent is distilled off on a rotary evaporator at 4O 0 C bath temperature. The residue is treated with methyl tert-butyl ether (1400 ml) and stirred vigorously for 30 min. Then it is filtered off with suction through a glass frit (pore width 3, 13 cm diameter). The intermediate product (Edman OJ degradation product) is isolated in a crude yield of 72 g and further reacted without workup.
  • the crude product is dissolved under argon atmosphere in trifluoroacetic acid (1026 mL) and stirred at RT for 30 min. Then the solution is concentrated on a rotary evaporator under vacuum at 2O 0 C bath temperature. The residue is taken up in methyl tert-butyl ether (1400 ml) and stirred vigorously until a powdery amorphous solid is formed. This is filtered off with a vacuum over a frit (pore width 3, 18 cm in diameter). The solid is then stirred with diethyl ether (1400 ml) and filtered off again. The same procedure is repeated with 2 portions of dichloromethane (900 mL each). The crude product is dried in vacuo. This gives 58 g of crude des (lD-leucyl) -lysobactin-bis-trifluoroacetate (Edman , o degradation product).
  • the intermediate product ( Edman® 5 -degradation product) is isolated in a crude yield of 65 g and, after drying in an oil pump vacuum, dissolved directly under argon atmosphere in trifluoroacetic acid (1240 mL) and stirred at RT for 30 min. Then, the solution is Lö ⁇ on a rotary evaporator under vacuum at 20 0 C. bath temperature concentrated. The residue is taken up in methyl tert-butyl ether (1400 ml) and stirred vigorously until a pulverulent amorphous solid is formed. This is filtered off with a vacuum over a frit (pore width 3, 18 cm in diameter).
  • X 1113x (qualitative) 220 nm (s), 255-270 (w).
  • 6-Trifluoromethylpyridine-3-carbaldehyde (4.85 g, 27.70 mmol) and methyl ⁇ [(benzyloxy) carbonyl] amino] (dimethoxyphosphoryl) acetate (9.17 g, 27.70 mmol, 1.0 equiv.) Are dissolved in THF (70 mL ) and cooled to -70 0 C.
  • N, N, N-tetramethylguanidine (6:38 g, 55.39 mmol, 6.95 mL, 2.0 equivalents) was added dropwise slowly and then stirred for 4 h then at -70 0 C for 12 h at RT.
  • Example 3A The compound of Example 3A (10.15 g, 26.69 mmol) is dissolved in methanol (10 mL). With a cannula, argon is passed through for about 5 minutes, then (+) - l, 2-bis - [(2> S r , 5 ) S) diethylphospholano] benzene (cyclooctadiene) rhodium (I) triflate (289 mg , 400 ⁇ mol 0.015 equivalent). It is hydrogenated for 12 h at 4 bar hydrogen pressure and RT. It is then filtered through Kie selgur (methanol) and the eluate is concentrated. The crude product is chromatographed (Kie selgel, eluent: toluene / ethyl acetate 5: 1). There are obtained 9.9 g (97% of theory) of the title compound.
  • Example 4A The compound from Example 4A (9.90 g, 25.89 mmol) is dissolved in methanol (100 mL). With a cannula, argon is passed for about 5 minutes, then Pd on charcoal (10%, 990 mg) is added. It is hydrogenated for 12 h at 4 bar hydrogen pressure and RT. It is then filtered through kieselguhr, concentrated and dried in an oil pump vacuum. Yield: 5.8 g (90% of theory) of the title compound.
  • IR v max (NaCl, cm -1 ): 2959, 1742, 1655, 1520, 1336, 1160, 1136, 1087, 1050, 1027.
  • Example 2A To a solution of the compound from Example 2A (7.00 g, 1.0 equivalents, 5.48 mmol) and Example 7A (3.03 g, 1.2 equivalents, 6.57 mmol) in dry DMF (119 mL) at -3O 0 C slowly N-methylmorpholine (2.77 g, 3.01 mL, 5 eq, 27.38 mmol) and HATU (4.37 g, 2.1 eq, 11.50 mmol). The reaction mixture warmed slowly to RT (about 1 h), with complete conversion being observed by means of HPLC / UV-Vis (Method 9). The reaction is quenched with potassium dihydrogen phosphate (7.45 g, 10.0 equivalents, 54.76 mmol).
  • reaction mixture is purified by gel chromatography (method 10, eluent methanol / acetone 4: 1) to give 12.63 g (quant.) Product.
  • HPLC / UV-Vis (Method 8): R 4 4.78 min.
  • Example I IA The compound from Example I IA (11.8 g, 42.09 mmol) is dissolved in trifluoroacetic acid in dichloromethane (160 mL, 30% solution) and 30 min. stirred at RT. Then you narrow in a vacuum. The residue is taken up in a little water and lyophilized. The lyophilizate is then treated with toluene and concentrated in vacuo. Finally, it is dried to constant weight in an oil pump vacuum. Yield: 17.15 g (quant).
  • Example 9A The compound from Example 9A (10.31 g, 39.4 mmol) and the compound from Example 12A (16.10 g, 39.4 mmol, 1 equivalent) are dissolved at 0 ° C. in DMF (186 ml). Then N-methylmorpholine (17.34 mL, 16.00 g, 4 equiv) and HATU (22.49 g, 59.16 mmol, 1.5 equiv) are added. The batch is stirred for two hours at RT. It is mixed with tert-butyl methyl ether and washed with saturated sodium carbonate solution.
  • aqueous phase is back-extracted once with tert-butyl methyl ether, then the combined organic phases are washed with 1 M aqueous citric acid and again with saturated sodium carbonate solution, dried over sodium sulfate, filtered and concentrated in vacuo. It is filtered through silica gel (cyclohexane / ethyl acetate 2: 1). Yield: 14.1 g (84% of theory).
  • Example 13A The compound from Example 13A (7.4 g, 17:56 mmol) in THF / water (6: 4) was added, cooled to 0 0 C, and (g 1:47, 35.13 mmol, 2 equivalents) of lithium hydroxide monohydrate is ver ⁇ . The mixture is stirred at 0 ° C. After one hour, another equivalent (0.74 g) of lithium hydroxide monohydrate is added and the mixture is stirred for a further hour. The majority of the THF is distilled off in vacuo, washed with two portions of methyl tert-butyl ether and then the aqueous phase is adjusted to pH 4 by addition of citric acid. A solid precipitates out.
  • Example 2A The compound from Example 2A (3.00 g, 2.35 mmol) and the compound from Example 14A (1.44 g, 3.52 mmol, 1.5 equivalents) are dissolved in DMF (50 mL) and cooled to 0 ° C. Then, firstly 4.7 mL (4.7 mmol, 2 equivalents) of a 1 M solution of 4-methylmorpholine in DMF are added. Subsequently, HATU (1.52 g, 3.99 mmol, 1.7 equivalents) is added immediately and stirred at 0 ° C. for 15 min. Then another 4.7 mL (4.7 mmol, 2 equivalents) of the 1 M solution of 4-methylmorpholine in DMF are added dropwise. The batch is then stirred for 2 h at RT. The crude product is gel-chromatographed (Method 3). The product is further reacted without fine cleaning. Yield: 3.6 g (82% of theory).
  • 2,2-Dimethyl-1-butanol (4.0 g, 39 mmol) is dissolved in dichloromethane (136 mL) and washed with alumina (7.98 g, 78 mmol, 2 equivalents) and with pyridinium chlorochromate (16.88 g, 78 mmol, 2 Equivalents). The batch is stirred at RT for 1 h and then filtered through a layer of silica gel. The filtrate is carefully concentrated and the residue is distilled at normal pressure (boiling point: 102 0 C (990 mbar)). Yield: 2.97 g (75% of theory).
  • Example 19A The compound from Example 19A (430 mg, 1.47 mmol) and the compound from Example 12A (809 mg, 1.47 mmol, 1 equivalent) are dissolved at 0 ° C. in DMF (5 mL), then 4- Methylmorpholine (644 .mu.l, 5.86 mmol, 4 equivalents) and HATU (836 mg, 2.20 mmol, 1.5 equivalents) was added. The batch is stirred for three hours at RT. It is mixed with ethyl acetate and washed with saturated sodium bicarbonate solution.
  • aqueous phase is extracted once with ethyl acetate, then the combined organic phases are washed with 1 M aqueous citric acid and again with saturated sodium bicarbonate solution, dried over sodium sulfate, filtered and concentrated in vacuo. The residue is chromatographed (method 16). Yield: 496 mg (74% of theory).
  • Example 2A The compound from Example 2A (0.28 g, 0.22 mmol) and the compound from Example 21A (148 mg, 0.33 mmol, 1.5 equivalents) are dissolved in DMF (4 mL) and cooled to 0 ° C. Then first 0.47 mL (0.44 mmol, 2 equivalents) of a 1 M solution of N-methylmorpholine in DMF is added. Then immediately HATU (141 mg, 0.37 mmol, 1.7 equivalents) was added and stirred at 0 0 C for 15 min. Then another 0.44 mL (0.47 mmol, 2 equivalents) of the 1 M solution of 4-methylmorpholine in DMF are added dropwise. The mixture is then stirred overnight at RT.
  • Example 15A The compound from Example 15A (9.12 g, 4.93 mmol, crude product) is taken up in trifluoroacetic acid in dichloromethane (65 mL, 30% solution). The batch is stirred at RT for 20 min. The solvent is distilled off. The residue is dried in an oil pump vacuum and then purified by chromatography (Method 14). Yield: 5.54 g (67% of theory).
  • the structure is confirmed by a single-crystal X-ray structure analysis.
  • Example 2 3- (trimethylsilyl) -D-alanyl-3- (pyridin-3-yl) -L-alanyl-des (ID-leucyl-2-L-leucyl) -lysobactin tris-trifluoroacetate (Example 2) dissolved in 90 mL of water and the solution was abandoned and slowly eluted. The column is refilled with approx. 20 mL of water. flushes. Product-containing eluates are combined, finely filtered (pore size 0.20 ⁇ m) and lyophilized. The column is conditioned again and reused. This gives 1.79 g (1.10 mmol, 92% of theory) of Example 3 from 2000 mg (1.19 mmol) of Example 2.
  • Example 22A The compound from Example 22A (149 mg, 0.09 mmol) is dissolved in methanol / containing 0.05% trifluoroacetic acid (10 mL). Palladium on activated carbon (10%, 20 mg) is added, then a total of 2.5 h is hydrogenated at RT and atmospheric pressure with hydrogen. The crude product is filtered off from the catalyst and the filtrate is concentrated. The residue is purified by chromatography (Method 13). Yield: 68 mg (46% of theory).
  • Example 2 2.00 g (1.19 mmol) of 3- (trimethylsilyl) -D-alanyl-3- (pyridin-3-yl) -L-alanyl-des (ID-leucyl-2-L-leucyl) -lysobactin tris-trifluoroacetate (Example 2) dissolved in 90 mL of water and the solution was abandoned and slowly eluted. The column is rinsed with approx. 20 mL of water. Product-containing eluates are combined, finely filtered (pore size 0.20 ⁇ m) and lyophilized. The column is conditioned again and reused. This gives 1.54 g (1.06 mmol, 89% of theory) of Example 6 from 2000 mg (1.19 mmol) of Example 2.
  • Example 7 is obtained from 2000 mg (1.19 mmol) of Example 2.
  • Example 2 100 mg (59 ⁇ mol) of Example 2 are then dissolved in 10 mL of water and applied to the column. Then it is washed in several portions of about 10 mL of water. The educt dissolves only moderately and remains partly on the column. Product-containing eluate fractions are combined and lyophilized. 57 mg (31 ⁇ mol, 52% of theory) of the product are obtained.
  • the integration of the 1 H-NMR spectrum indicates a stoichiometry 1: 3 for (trimethylsilyl) -D-alanyl-3- (pyridin-3-yl) -L-alanyl-des (ID-leucyl-2-L-leucyl) -lysobactin: D-tartaric acid.
  • Example 3 By recrystallization of Example 3 from TFA-doped water at RT, the title compound can be obtained as a crystalline substance (slow evaporation of the solution).
  • the MIC is determined in the liquid dilution test according to the NCCLS guidelines. Overnight cultures of Staphylococcus aureus 133, Entercococcus faecalis 27159, E. faecium 4147 and Streptococcus pneumoniae G9a are incubated with the described test substances in a 1: 2 dilution series. The MIC determination is carried out with a cell count of 10 5 germs per 1 ml in Iso-sensitest medium (Difco Co., Irvine / USA), with the exception of S. pneumoniae, which is in BHI broth (Difco, Irvine / USA) is tested with 10% bovine serum at a cell count of 10 6 germs per mL. The cultures are incubated at 37 ° C for 18-24 hours, S. pneumoniae in the presence of 10% CO 2 .
  • the lowest substance concentration at which no visible bacterial growth occurs is defined as MIC.
  • the MIC values are given in ⁇ g / mL.
  • S. aureus 133 cells are grown overnight in BHI broth (Oxoid, New York / USA). The overnight culture is diluted 1: 100 in fresh BHI broth and incubated for 3 hours. The cells, which are then in the logarithmic growth phase, are centrifuged off and washed twice with buffered physiological saline. Thereafter, a cell suspension in saline solution with an extinction of 50 units is adjusted by photometry. After a dilution step (1:15), this suspension is mixed 1: 1 with a 10% mucin solution. 0.25 ml / 20 g of mouse are administered intraperitoneally from this infectious solution (corresponding to 1 ⁇ 10 6 germs / mouse). Therapy is intraperitoneal or intravenous 30 minutes after infection. For the infection experiment, female CFW1 mice are used. The survival of the animals is recorded over 6 days.
  • Nephrotoxic side effects of nonadepsipeptides are analyzed by histopathological investigations of the kidneys in mice after repeated administration of a specific dose. 5-6 animals are treated daily either intravenously (iv) or intraperitoneally (ip) with substances which are dissolved in aqueous solution or with the addition of solutol. Kidney toxic effects are determined by light microscopic analysis of hematoxylin and eosin (H & E) stained paraffin sections of the kidneys. A Periodic Acid Schiff (PAS) reaction is optionally carried out to better display glycoproteins.
  • PAS Periodic Acid Schiff
  • Nephrotoxic side effects of the nonadepsipeptides are analyzed by histopathological investigations of the kidneys in rats after repeated administration of a specific dose. For this purpose, 5 animals are treated intravenously (i.v.) daily with substances which are dissolved in saline or Ringer's lactate solution. Kidney toxic effects are determined by light microscopic evaluation of hematoxylin and eosin (H & E) stained paraffin sections of the kidneys. A periodic acid Schiff (PAS) reaction is optionally carried out for the better presentation of glycoproteins.
  • PAS periodic acid Schiff
  • the average severity of tubular degeneration / regeneration and the incidence (number of affected animals) are calculated per animal group or derivative. Beyond kidney changes such as tubular dilatation and necrosis and accumulation of necrotic material are also listed.
  • the plasma is usually diluted with isotonic phosphate-buffer (pH 7.4) and then suspended with Transil ®.
  • the determination of f u '(free fraction in diluted plasma) in this diluted protein solution is carried out analogously to the determination of f u .
  • the free fraction in undiluted plasma is calculated from f u 'and the dilution factor. Comparison to this method also: Schuhmacher, Joachim; Kohlsdorfer, Christian; Buehner, Klaus; Brandenburger, Tim; Kruk, Renate, "High-throughput determination of the free fraction of drugs bound to plasma proteins.” Journal of Pharmaceutical Sciences 2004, 93, 816-830.
  • the mixtures are incubated for 30 min at room temperature, for example on a mini shaker at an angle of about 45 ° at about 400 rpm.
  • at least one aliquot of, for example, 100 .mu.l is taken, the remaining batch is centrifuged for about 10 min at about 1800 g. From each sample, at least 2 aliquots (eg, 100 ⁇ l) of the supernatant are taken for concentration determination.
  • Transil ® volume The total incubation and the added Transil ® volume will depend on the expected free fraction. In general, the total volume of 0.5 mL -1 is that Transil ® volume 10- 100 ul. In the case of very low free fractions of the plasma to be tested is Spe ⁇ zies with isotonic buffer solution, pH 7.4, for example, 10 -400fach diluted and then added with Tran ⁇ sil ®. The further procedure is as described above for the determination of the MAb Uffer values.
  • the plasma of the species to be examined is filtered through a semipermeable membrane.
  • the concentration of the substance in the filtrate is measured and from this the free fraction fu is calculated.
  • the Centrifree micropartition system from Millipore / Amicon is used.
  • the ultrafiltration membranes have an exclusion size of 30,000 Da.
  • the substance is added to 1 mL plasma at a concentration of approx. 1 ⁇ g / mL.
  • the solvent content should be ⁇ 2%.
  • the plasma is pipetted into the ultrafiltration system and centrifuged for 10 minutes at 1800 g.
  • the compounds according to the invention can be converted into pharmaceutical preparations as follows:
  • Example 1 100 mg of the compound of Example 1, 50 mg of lactose (monohydrate), 50 mg of corn starch (native), 10 mg of polyvinylpyrrolidone (PVP 25) (BASF, Ludwigshafen, Germany) and 2 mg of magnesium stearate.
  • the mixture of active ingredient, 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 min.
  • 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.
  • a single dose of 100 mg of the compound according to the invention corresponds to 10 ml of oral suspension.
  • the rhodigel is suspended in ethanol, the active ingredient is added to the suspension. While stirring, the addition of water. Until the swelling of the Rhodigels is complete, it is stirred for about 6 hours.
  • Intravenously administrable solution
  • Example 1 The compound of Example 1 is dissolved together with polyethylene glycol 400 in the water with stirring.
  • the solution is sterile-filtered (pore diameter 0.22 ⁇ m) and filled under aseptic conditions into heat-sterilized infusion bottles. These are closed with infusion stoppers and crimp caps.

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Abstract

The invention relates to nonadepsipeptides (lysobactin derivatives), methods for the production thereof, and the use thereof for producing medicaments used for treating and/or preventing diseases, especially bacterial infectious diseases. Said novel lysobactin derivatives distinguish themselves from the natural substance by the N-terminal peptide chain which comprises a 3-(3-pyridyl)-alanyl radical.

Description

. ,
ACYLIERTE NONADEPSΪPEPTIDE ALS LYSOBACTINDERIVATEACYLATED NONADESPΪPEPTIDES AS LYSOBACTIN DERIVATIVES
Die Erfindung betrifft Nonadepsipeptide und Verfahren zu ihrer Herstellung sowie ihre Ver¬ wendung zur Herstellung von Arzneimitteln zur Behandlung und/oder Prophylaxe von Krankhei¬ ten, insbesondere bakteriellen Infektionskrankheiten.The invention relates to nonadepsipeptides and processes for their preparation and their use for the production of medicaments for the treatment and / or prophylaxis of diseases, in particular bacterial infectious diseases.
Die bakterielle Zellwand wird durch eine Reihe von Enzymen synthetisiert (Zellwandbiosynthese) und ist essentiell für das Überleben bzw. die Vermehrung von Mikroorganismen. Die Struktur dieses Makromoleküls ebenso wie die an ihrer Synthese beteiligten Proteine sind innerhalb der Bakterien stark konserviert. Aufgrund ihrer essentiellen Natur und Einheitlichkeit ist die Zellwand¬ biosynthese ein idealer Angriffspunkt für neue Antibiotika (D.W.Green, The bacterial cell wall as a source of antibacterial targets, Expert Opin. Ther. Targets, 2002, 6, 1-19).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 und Penicilline sind Inhibitoren der bakteriellen Zellwandbiosynthese und stellen erfolgreiche Beispiele für die antibiotische Potenz dieses Wirkprinzips dar. Sie werden seit mehre¬ ren Jahrzehnten in der Klinik zur Behandlung von bakteriellen Infektionen, vor allem mit Gram¬ positiven Erregern eingesetzt. Durch das wachsende Auftreten von resistenten Keimen, z.B. Methicillin-resistenten Staphylokokken, Penicillin-resistenten Pneumokokken und Vancomycin- resistenten Enterokokken (F. Baquero, Gram-positive resistance: challenge for the development of new antibiotics, J. Antimicrob. Chemother., 1997, 39, Suppl A: 1-6; A.P. Johnson, D.M. Liver- more, G. S. Tillotson, Antimicrobial susceptibility of Gram-positive bacteria: what's current, what's anticipated ?, J. Hosp. Infect., 2001, (49), Suppl A: 3-11) sowie jüngst auch erstmals Vancomycin- resistenten Staphylokokken (B. Goldrick, First reported case of VRSA in the United States, Am. J. Nurs., 2002, 102, 17) verlieren diese Substanzen zunehmend ihre therapeutische Wirksamkeit.Vancomycin and penicillins are inhibitors of bacterial cell wall biosynthesis and represent successful examples of the antibiotic potency of this active 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. Antimicrob., Chemother., 1997, 39, Suppl A: 1-6; AP Johnson, DM Livermore, GS Tillotson, Antimicrobial Susceptibility of Gram-positive Bacteria: What's Current, What's Anticipated ?, J. Hosp. Infect., 2001, (49), Suppl A: 3-11) and most recently for the first time Vancomycin-resistant staphylococci (B.Goldrick, First reported case of VRSA in the United States, Am.J. Nurs., 2002, 102, 17) are increasingly losing their therapeutic efficacy.
Die vorliegende Erfindung beschreibt eine neue Klasse von Zellwandbiosynthese-Inhibitoren ohne Kreuzresistenzen zu bekannten Antibiotika-Klassen.The present invention describes a new class of cell wall biosynthesis inhibitors without cross-resistance to known antibiotic classes.
Der Naturstoff Lysobactin und einige Derivate sind als antibakteriell wirksam beschrieben in US 4,754,018. Die Isolierung und antibakterielle Wirksamkeit von Lysobactin ist auch in EP-A-196 042 und JP 01132600 beschrieben. WO04/099239 beschreibt Derivate des Lysobactins mit anti¬ bakterieller Wirksamkeit.The natural product 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 anti-bacterial activity.
Die antibakterielle Wirkung von Lysobactin und Katanosin A wird weiterhin beschrieben in O'Sullivan, J. et al, J. Antibiot. 1988, 41, 1740 - 1744, Bonner, D. P. et al, J. Antibiot. 1988, 41, 1745 - 1751, Shoji, J. et al, J. Antibiot. 1988, 41, 713 - 718 und Tymiak, A. A. et al, J. Org Chem. 1989, 54, 1149 - 1157. Eine Aufgabe der vorliegenden Erfindung ist es, alternative Verbindungen mit vergleichbarer oder verbesserter antibakterieller Wirkung, besserer Verträglichkeit, z. B. geringerer Nephrotoxizität, und besserer Verteilung im Körper, d. h. besseren pharmakokinetischen Eigenschaften, wie z. B. Erhöhung der freien Fraktion (fu), zur Behandlung von bakteriellen Erkrankungen bei Menschen und Tieren zur Verfügung zu stellen.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. An object of the present invention is to provide alternative compounds having comparable or improved antibacterial activity, better compatibility, e.g. B. lower nephrotoxicity, and better distribution in the body, ie better pharmacokinetic properties, such. B. increase of the free fraction (f u ) to provide for the treatment of bacterial diseases in humans and animals.
Gegenstand der Erfindung sind Verbindungen der FormelThe invention relates to compounds of the formula
in welcherin which
R1 Wasserstoff bedeutet undR 1 is hydrogen and
R2 2,2-Dimethylbut-l-yl, 2-Ethyl-2-methylbut-l-yl, 2,2-Diethyl-but-l-yl, 2,2-Dimethylpent-l- yl oder Trimethylsilylmethyl bedeutet,R 2 is 2,2-dimethylbut-1-yl, 2-ethyl-2-methylbut-1-yl, 2,2-diethyl-but-1-yl, 2,2-dimethylpent-1-yl or trimethylsilylmethyl,
oderor
R1 Trifluormethyl bedeutet undR 1 is trifluoromethyl and
R2 2,2-Dimethylprop-l-yl, 2,2-Dimethylbut-l-yl, 2-Ethyl-2-methylbut-l-yl, 2,2-Diethyl-but-l- yl, 2,2-Dimethylpent-l-yl oder Trimethylsilylmethyl bedeutet,R 2 2,2-dimethylprop-1-yl, 2,2-dimethylbut-1-yl, 2-ethyl-2-methylbut-1-yl, 2,2-diethyl-but-1-yl, 2,2- Dimethylpent-1-yl or trimethylsilylmethyl,
und ihre Salze, ihre Solvate und die Solvate ihrer Salze. Erfindungsgemäße Verbindungen sind die Verbindungen der Formel (I) und deren Salze, Solvate, Solvate der Salze und Prodrugs, die von Formel (I) umfassten Verbindungen der nachfolgend genannten Formeln und deren Salze, Solvate, Solvate der Salze und Prodrugs sowie die von Formel (I) umfassten, nachfolgend als Ausfuhrungsbeispiele genannten Verbindungen und deren Salze, Solvate, Solvate der Salze und Prodrugs, soweit es sich bei den von Formel (I) umfassten, nachfolgend genannten Verbindungen nicht bereits um Salze, Solvate, Solvate der Salze und Prodrugs handelt.and their salts, their solvates, and the solvates of their salts. Compounds according to the invention are the compounds of the formula (I) and their salts, solvates, solvates of the salts and prodrugs, the compounds of the formulas below and their salts, solvates, solvates of the salts and prodrugs and of the formula (I) I), hereinafter referred to as exemplary compounds and their salts, solvates, solvates of the salts and prodrugs, as far as the compounds encompassed by formula (I) below are not already salts, solvates, solvates of the salts and prodrugs.
Die erfindungsgemäßen Verbindungen können in Abhängigkeit von ihrer Struktur in stereoisomeren Formen (Enantiomere, Diastereomere) existieren. Die Erfindung betrifft deshalb die Enantiomeren oder Diastereomeren und ihre jeweiligen Mischungen. Aus solchen Mischungen von Enantiomeren und/oder Diastereomeren lassen sich die stereoisomer einheitlichen Bestandteile in bekannter Weise isolieren.Depending on their structure, 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.
Sofern die erfindungsgemäßen Verbindungen in tautomeren Formen vorkommen können, umfasst die vorliegenden Erfindung sämtliche tautomere Formen.If the compounds according to the invention can occur in tautomeric forms, the present invention encompasses all tautomeric forms.
Als Salze sind im Rahmen der vorliegenden Erfindung physiologisch unbedenkliche Salze der erfin¬ dungsgemäßen Verbindungen bevorzugt. Umfasst sind aber auch Salze, die für pharmazeutische Anwendungen selbst nicht geeignet sind aber beispielsweise für die Isolierung oder Reinigung der erfindungsgemäßen Verbindungen verwendet werden können oder Mischsalze. Unter Mischsalz wird im Rahmen der vorliegenden Erfindung ein Additionssalz verstanden, das zwei oder mehrere unterschiedliche Säuren bzw. Basen enthält, wie z. B. ein Trifluoracetat-Mesylat-Salz.Salts in the context of the present invention are physiologically acceptable salts of the compounds according to the invention. But also included are salts which are not suitable for pharmaceutical applications themselves but can be used for example for the isolation or purification of the compounds of the invention or mixed salts. For the purposes of the present invention, 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.
Physiologisch unbedenkliche Salze der erfindungsgemäßen Verbindungen umfassen Säure¬ additionssalze von Mineralsäuren, Carbonsäuren und Sulfonsäuren, z.B. Salze der Chlorwasserstoffsäure, Bromwasserstoffsäure, Schwefelsäure, Phosphorsäure, Methansulfonsäure, Ethansulfonsäure, Toluolsulfonsäure, Benzolsulfonsäure, Naphthalindisulfonsäure, Essigsäure, Trifluoressigsäure, Propionsäure, Milchsäure, Weinsäure, Äpfelsäure, Zitronensäure, Fumarsäure, Maleinsäure und Benzoesäure.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, toluenesulfonic, benzenesulfonic, naphthalenedisulfonic, acetic, trifluoroacetic, propionic, lactic, tartaric, malic, citric, fumaric, maleic and benzoic acids.
Physiologisch unbedenkliche Salze der erfindungsgemäßen Verbindungen umfassen auch Salze üblicher Basen, wie beispielhaft und vorzugsweise Alkalimetallsalze (z.B. Natrium- und Kaliumsalze), Erdalkalisalze (z.B. Calcium- und Magnesiumsalze) und Ammoniumsalze, abgeleitet von Ammoniak oder organischen Aminen mit 1 bis 16 C-Atomen, wie beispielhaft und vorzugsweise Ethylamin, Diethylamin, Triethylamin, Ethyldiisopropylamin, Monoethanolamin, Diethanolamin, Triethanolamin, Dicyclohexylamin, Dimethylaminoethanol, Prokain, Dibenzylamin, N- Methylmorpholin, Arginin, Lysin, Ethylendiamin und N-Methylpiperidin. AIs Solvate werden im Rahmen der Erfindung solche Formen der erfindungsgemäßen Verbindungen bezeichnet, welche in festem oder flüssigem Zustand durch Koordination mit Lösungsmittelmolekü¬ len einen Komplex bilden. Hydrate sind eine spezielle Form der Solvate, bei denen die Koordination mit Wasser erfolgt.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, such as, by way of example and by way of illustration, ethylamine, diethylamine, triethylamine, ethyldiisopropylamine, monoethanolamine, diethanolamine, triethanolamine, dicyclohexylamine, dimethylaminoethanol, procaine, dibenzylamine, N-methylmorpholine, arginine, lysine, ethylenediamine and N-methylpiperidine. In the context of the invention, solvates 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.
Bevorzugt sind Verbindungen der Formel (I), in welcherPreference is given to compounds of the formula (I) in which
R1 Wasserstoff bedeutet undR 1 is hydrogen and
R2 2,2-Dimethylbut- 1 -yl oder Trimethylsilylmethyl bedeutet,R 2 is 2,2-dimethylbut-1-yl or trimethylsilylmethyl,
oderor
R1 Trifluormethyl bedeutet undR 1 is trifluoromethyl and
R2 2,2-Dimethylprop- 1 -yl, 2,2-Dimethylbut- 1 -yl oder Trimethylsilylmethyl bedeutet,R 2 is 2,2-dimethylprop-1-yl, 2,2-dimethylbut-1-yl or trimethylsilylmethyl,
und ihre Salze, ihre Solvate und die Solvate ihrer Salze.and their salts, their solvates, and the solvates of their salts.
Bevorzugt sind auch Verbindungen der Formel (I), in welcherPreference is also given to compounds of the formula (I) in which
R1 Wasserstoff bedeutet undR 1 is hydrogen and
R2 2,2-Dimethylbut- 1-yl, 2-Ethyl-2-methylbut-l-yl, 2,2-Diethyl-but-l-yl oder Trimethylsilyl- methyl bedeutet,R 2 is 2,2-dimethylbut-1-yl, 2-ethyl-2-methylbut-1-yl, 2,2-diethyl-but-1-yl or trimethylsilyl-methyl,
und ihre Salze, ihre Solvate und die Solvate ihrer Salze.and their salts, their solvates, and the solvates of their salts.
Bevorzugt sind auch Verbindungen der Formel (I), in welcherPreference is also given to compounds of the formula (I) in which
R1 Wasserstoff bedeutet undR 1 is hydrogen and
R2 2,2-Dimethylbut-l-yl, 2-Ethyl-2-methylbut-l-yl, 2,2-Diethyl-but-l-yl, 2,2-Dimethylpent-l- yl oder Trimethylsilylmethyl bedeutet.R 2 is 2,2-dimethylbut-1-yl, 2-ethyl-2-methylbut-1-yl, 2,2-diethyl-but-1-yl, 2,2-dimethylpent-1-yl or trimethylsilylmethyl.
Insbesondere bevorzugt ist die Verbindung 3-(Trimethylsilyl)-D-alanyl-3-(pyridin-3-yl)-L-alanyl- des( 1 -D-leucy 1-2-L-leucy l)-lysobactin Especially preferred is the compound 3- (trimethylsilyl) -D-alanyl-3- (pyridin-3-yl) -L-alanyl-des (1-D-leucate 1-2-L-leucyl) -lysobactin
oder eines ihrer Salze, ihrer Solvate oder der Solvate ihrer Salze.or one of its salts, its solvates or the solvates of its salts.
Gegenstand der Erfindung ist weiterhin ein Verfahren zur Herstellung der Verbindungen der For¬ meln (I), wobei die Verbindung der FormelThe invention furthermore relates to a process for the preparation of the compounds of the formulas (I), where the compound of the formula
mit Verbindungen der Formel .with compounds of the formula ,
in welcherin which
R1 und R2 die oben angegebene Bedeutung haben, undR 1 and R 2 have the meaning given above, and
X1 Halogen, bevorzugt Brom, Chlor oder Fluor, oder Hydroxy bedeutet,X 1 is halogen, preferably bromine, chlorine or fluorine, or hydroxy,
umgesetzt werden.be implemented.
Falls X1 für Halogen steht, erfolgt die Umsetzung im Allgemeinen in inerten Lösungsmitteln, ge¬ gebenenfalls in Gegenwart einer Base, bevorzugt in einem Temperaturbereich von -3O0C bis 500C bei Normaldruck.If X 1 is halogen, the reaction is generally carried out in inert solvents, if appropriate in the presence of a base, preferably in a temperature range from -3O 0 C to 50 0 C at atmospheric pressure.
Inerte Lösungsmittel sind beispielsweise Tetrahydrofuran, Methylenchlorid, Pyridin, Dioxan oder Dimethylformamid, bevorzugt ist Methylenchlorid oder Dimethylformamid.Inert solvents are, for example, tetrahydrofuran, methylene chloride, pyridine, dioxane or dimethylformamide, preference is given to methylene chloride or dimethylformamide.
Basen sind beispielsweise Triethylamin, Diisopropylethylamin oder N-Methylmorpholin, bevor¬ zugt ist Diisopropylethylamin.Examples of bases are triethylamine, diisopropylethylamine or N-methylmorpholine, preferably diisopropylethylamine.
Falls X1 für Hydroxy steht, erfolgt die Umsetzung im Allgemeinen in inerten Lösungsmitteln, in Gegenwart eines Dehydratisierungsreagenzes, gegebenenfalls in Gegenwart einer Base, bevorzugt in einem Temperaturbereich von -3O0C bis 500C bei Normaldruck.If X 1 is hydroxy, the reaction 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 atmospheric pressure.
Inerte Lösungsmittel sind beispielsweise Halogenkohlenwasserstoffe wie Dichlormethan oder Trichlormethan, Kohlenwasserstoff wie Benzol, Nitromethan, Dioxan, Dimethylformamid oder Acetonitril. Ebenso ist es möglich, Gemische der Lösemittel einzusetzen. Besonders bevorzugt ist Dichlormethan oder Dimethylformamid.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.
Als Dehydratisierungsreagenzien eignen sich hierbei beispielsweise Carbodiimide wie z.B. N1N'- Diethyl-, N1N, -Dipropyl-, N,N'-Diisopropyl-, N.N'-Dicyclohexylcarbodiimid, N-(3-Di- methylaminoisopropyl)-N'-ethylcarbodiimid-hydrochlorid (EDC), N-Cyclohexylcarbodiimid-N '- propyloxymethyl-Polystyrol (PS-Carbodiimid) oder Carbonylverbindungen wie Carbonyldiimida- .Suitable dehydrating here, for example, carbodiimides such as N 1 N'-diethyl-, N 1 N, dipropyl, N, N'-diisopropyl-, N, N'-dicyclohexylcarbodiimide, N- (3-di- methylaminoisopropyl) are - N'-ethylcarbodiimide hydrochloride (EDC), N-cyclohexylcarbodiimide-N'-propyloxymethyl-polystyrene (PS-carbodiimide) or carbonyl compounds such as carbonyldiimida- ,
- 7 - zol, oder 1,2-Oxazoliumverbindungen wie 2-Ethyl-5-phenyl-l,2-oxazolium-3-sulfat oder 2-tert- Butyl-5-methyl-isoxazolium-perchlorat, oder Acylaminoverbindungen wie 2-Ethoxy-l-ethoxy- carbonyl-l,2-dihydrochinolin, oder Propanphosphonsäureanhydrid, oder Isobutylchlorofoπnat, oder Bis-(2-oxo-3-oxazolidinyl)-phosphorylchlorid oder Benzotriazolyloxy-tri(dimethylamino)- phosphoniumhexafluorophosphat, oder O-(Benzotτiazol-l-yl)-N,N,N',N'4etra-methyluronium- hexafluorophosphat (HBTU), 2-(2-Oxo-l-(2H)-pyridyl)-l,l,3,3-tetramethyluroniumtetrafluoro- borat (TPTU) oder 0-(7-Azabenzotriazol-l-yl)-N,N,N',N'-tetramethyl-uroniumhexafluorophosphat (HATU), oder 1-Hydroxybenzotriazol (HOBt), oder Benzotriazol-l-yloxytris(dimethylamino)- phosphoniumhexafluorophosphat (BOP), oder N-Hydroxysuccinimid, oder Mischungen aus die- sen, mit Basen.7 -ol, or 1,2-oxazolium compounds such as 2-ethyl-5-phenyl-l, 2-oxazolium-3-sulfate or 2-tert-butyl-5-methyl-isoxazolium perchlorate, or acylamino compounds such as 2-ethoxy -l-ethoxycarbonyl-l, 2-dihydroquinoline, or propanephosphonic anhydride, or isobutylchlorofoπnat, or bis (2-oxo-3-oxazolidinyl) -phosphorylchlorid or Benzotriazolyloxy-tri (dimethylamino) - phosphoniumhexafluorophosphat, or O- (Benzotτiazol-l -yl) -N, N, N ', N'-tetra-methyluronium hexafluorophosphate (HBTU), 2- (2-oxo-l- (2H) -pyridyl) -1,3,3-tetramethyluronium tetrafluoroborate ( TPTU) or 0- (7-azabenzotriazol-1-yl) -N, N, N ', N'-tetramethyl-uronium hexafluorophosphate (HATU), or 1-hydroxybenzotriazole (HOBt), or benzotriazol-1-yloxytris (dimethylamino) - phosphonium hexafluorophosphate (BOP), or N-hydroxysuccinimide, or mixtures thereof, with bases.
Basen sind beispielsweise Alkalicarbonate, wie z.B. Natrium- oder Kaliumcarbonat, oder -hy- drogencarbonat, oder organische Basen wie Trialkylamine z.B. Triethylamin, N-Methylmorpholin, N-Methylpiperidin, 4-Dimethylaminopyridin oder Diisopropylethylamin.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-dimethylaminopyridine or diisopropylethylamine.
Vorzugsweise wird die Kondensation mit HATU oder mit EDC in Gegenwart von HOBt durchge- führt.Preferably, the condensation is carried out with HATU or with EDC in the presence of HOBt.
Die Verbindungen der Formel (HI) tragen gegebenenfalls Schutzgruppen, so dass sich in diesen Fällen der Umsetzung der Verbindung der Formel (II) mit Verbindungen der Formel (DI) eine Ab¬ spaltung der Schutzgruppen mit Trifluoressigsäure nach dem Fachmann bekannten Methoden an¬ schließt.The compounds of the formula (HI) optionally carry protective groups, so that in these cases the reaction of the compound of the formula (II) with compounds of the formula (DI) involves cleavage of the protective groups with trifluoroacetic acid by methods known to the person skilled in the art.
Die freie Base der Salze der Verbindungen der Formel (I) kann zum Beispiel durch Zusatz einer Base und anschließender Extraktion, Präzipitation oder chromatographischer Trennung der Ver¬ bindung nach dem Fachmann bekannten Verfahren erhalten werden. Insbesondere durch Verwen¬ dung polymergebundener Basen, wie zum Beispiel polymergebundenem Hydrogencarbonat.The free base of the salts of the compounds of the formula (I) can be obtained, for example, by addition of a base and subsequent extraction, precipitation or chromatographic separation of the compound by methods known to the person skilled in the art. In particular by using polymer-bound bases, such as, for example, polymer-bound bicarbonate.
Weiterer Gegenstand der Erfindung ist ein Verfahren zur Herstellung der Verbindungen der For- mel (I) oder ihrer Solvate nach Anspruch 1, bei dem Salze der Verbindungen oder Solvate der Sal¬ ze der Verbindungen durch Zusatz einer Base in die Verbindungen überführt werden.The invention further provides a process for the preparation of the compounds of the formula (I) or their solvates according to claim 1, in which salts of the compounds or solvates of the salts of the compounds are converted into the compounds by addition of a base.
Die Verbindung der Formel (H) lässt sich durch doppelten Edmann-Abbau aus Lysobactin (Bei¬ spiel IA) synthetisieren, wie im experimentellen Teil unter Beispiel 2A beschrieben.The compound of the formula (H) can be synthesized by double Edmann degradation from lysobactin (Example 1), as described in the experimental section under Example 2A.
Die Verbindungen der Formel (IH) sind bekannt oder lassen sich nach bekannten Verfahren aus den entsprechenden Edukten synthetisieren.The compounds of the formula (III) are known or can be synthesized by known processes from the corresponding starting materials.
Die Herstellung der erfϊndungsgemäßen Verbindungen kann durch folgendes Syntheseschema ver- deutlicht werden.The preparation of the compounds according to the invention can be carried out by the following synthesis scheme: be clear.
Syntheseschema:Synthesis scheme:
Die erfindungsgemäßen Verbindungen zeigen ein nicht vorhersehbares, wertvolles pharmakologi¬ sches und pharmakokinetisches Wirkspektrum. Sie zeigen eine antibakterielle Wirkung.The compounds of the invention show an unpredictable, valuable pharmacological and pharmacokinetic spectrum of activity. They show an antibacterial effect.
Sie eignen sich daher zur Verwendung als Arzneimittel zur Behandlung und/oder Prophylaxe von Krankheiten bei Menschen und Tieren.They are therefore suitable for use as medicaments for the treatment and / or prophylaxis of diseases in humans and animals.
Die erfϊndungsgemäßen Verbindungen zeichnen sich durch eine geringere Nephrotoxizität gegen- über Lysobactin aus.The compounds according to the invention are distinguished by a lower nephrotoxicity compared to lysobactin.
Die erfindungsgemäßen Verbindungen zeichnen sich durch eine bessere Pharmakokinetik gegen¬ über Lysobactin aus. Sie zeigen bei gleicher oder verbesserter pharmakologischer Wirkung eine bessere Verteilung im Körper, was eine geringere therapeutische Dosis sowie ein breiteres thera¬ peutisches Behandlungsfenster zur Folge hat.The compounds according to the invention are distinguished by a better pharmacokinetics than by lysobactin. They show a better distribution in the body with the same or improved pharmacological effect, which results in a lower therapeutic dose and a broader therapeutic window of treatment.
Die erfindungsgemäßen Verbindungen haben eine höhere freie Fraktion (fu) im Plasma als Lyso¬ bactin.The compounds of the invention have a higher free fraction (f u ) in plasma than lyso¬ bactin.
Die beschriebenen Nonadepsipeptide wirken als Inhibitoren der bakteriellen Zellwandbiosynthese. Besonders wirksam sind die erfindungsgemäßen Zubereitungen gegen Bakterien und bakterienähn¬ liche Mikroorganismen. Sie sind daher besonders gut zur Prophylaxe und Chemotherapie von loka¬ len und systemischen Infektionen in der Human- und Tiermedizin geeignet, die durch diese Erreger hervorgerufen werden.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 suitable for the prophylaxis and chemotherapy of local and systemic infections in human and veterinary medicine, which are caused by these pathogens.
Grundsätzlich können die erfindungsgemäßen Zubereitungen gegen alle Bakterien und bakterien¬ ähnlichen Mikroorganismen verwendet werden, die im Besitz einer bakteriellen Zellwand (Murein sacculus) bzw. den dazugehörigen Enzymsystemen sind, beispielsweise durch die folgenden Erre¬ ger oder durch Mischungen der folgenden Erreger:In principle, the preparations according to the invention can be used against all bacteria and bacterium-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 Kokken (Neisseria gonorrhoeae) sowie Gram-negative Stäbchen wie Enterobakte- riaceen, z.B. Escherichia coli, Hämophilus influenzae, Pseudomonas, Klebsiella, Citrobacter (C. freundii, C. divernis), Sahnonella und Shigella; ferner Enterobacter (E. aerogenes, E. agglome- rans), Hafhia, Serratia (S. marcescens), Providencia, Yersinia, sowie die Gattung Acinetobacter, Branhamella und Chlamydia. Darüber hinaus umfasst das antibakterielle Spektrum strikt anaerobe Bakterien wie z.B. Bacteroides fragilis, Vertreter der Gattung Peptococcus, Peptostreptococcus sowie die Gattung Clostridium; ferner Mykobakterien, z.B. M. tuberculosus. Besonders ausgepräg¬ te Wirkung zeigen die erfindungsgemäßen Verbindungen gegen Gram-positive Kokken, z.B. Staphylokokken (S. aureus, S. epidermidis, S. haemolyticus, S. carnosus), Enterokokken (E. faeca- Us, E. faecium) und Streptokokken (S. agalactiae, S. pneumoniae, S. pyogenes).Gram-negative cocci (Neisseria gonorrhoeae) as well as Gram-negative rods such as Enterobacteriaceae, e.g. Escherichia coli, Haemophilus influenzae, Pseudomonas, Klebsiella, Citrobacter (C. freundii, C. divernis), Sahnonella and Shigella; also Enterobacter (E. aerogenes, E. agglomerans), Hafhia, Serratia (S. marcescens), Providencia, Yersinia, as well as the genera Acinetobacter, Branhamella and Chlamydia. In addition, 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 show particularly pronounced activity against gram-positive cocci, e.g. Staphylococci (S. aureus, S. epidermidis, S. haemolyticus, S. carnosus), enterococci (E. faeca-Us, E. faecium) and streptococci (S. agalactiae, S. pneumoniae, S. pyogenes).
Die obige Aufzählung von Erregern ist lediglich beispielhaft und keineswegs beschränkend aufzu- fassen. Als Krankheiten, die durch die genannten Erreger oder Mischinfektionen verursacht und durch die erfindungsgemäßen Zubereitungen verhindert, gebessert oder geheilt werden können, seien beispielsweise genannt:The above list of pathogens is merely exemplary and by no means restrictive. Examples of 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:
Infektionskrankheiten beim Menschen wie z.B. unkomplizierte und komplizierte Harnwegsinfekte, unkomplizierte Haut- und Oberflächeninfektionen, komplizierte Haut- und Weichteilinfektionen, im Hospital und ambulant erworbene Lungenentzündung, nosokomiale Pneumonien, akute Exa- zerbationen und sekundäre bakterielle Infektionen der chronischen Bronchitis, akute Otitis media, akute Sinusitis, streptokokkale Pharyngitis, bakterielle Meningitis, unkomplizierte gonokokkale und nicht-gonokokkale Urethritis/Cervizitis, akute Prostatitis, Endocarditis, unkomplizierte und komplizierte intra-abdominale Infektionen, gynäkologische Infektionen, pelvische Entzündungs- krankheit, bakterielle Vaginose, akute und chronische Osteomyelitis, akute bakterielle Arthritis, empirische Therapie in febrilen neutropenischen Patienten, desweiteren Bakterämien, MRSA In¬ fektionen, akute infektiöse Diarrhoe, Helicobacter pylori Infektionen, postoperative Infektionen, odontogene Infektionen, ophthalmologische Infektionen, postoperative Infektionen (inkl. periprok- taler Abszess, Wundinfektionen, Galleninfektionen, Mastitis und akute Appendizitis), zystische Fibrose und Bronchiectasis.Human infectious diseases 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, empirical therapy in febrile neutropenic patients, further bacteremia, MRSA infections, acute infectious diarrhea, Helicobacter pylori infections, postoperative infections, odontogenic infections, ophthalmological In infections, postoperative infections (incl. periprosthetic abscess, wound infections, bile infections, mastitis and acute appendicitis), cystic Fibrosis and bronchiectasis.
Außer beim Menschen können bakterielle Infektionen auch bei anderen Spezies behandelt werden. Beispielhaft seien genannt:Apart from humans, bacterial infections can also be treated in other species. Examples include:
Schwein: Diarrhoe, Enterotoxamie, Sepsis, Dysenterie, Salmonellose, Metritis-Mastitis- Agalaktiae-Syndrom, Mastitis;Pig: diarrhea, enterotoxemia, sepsis, dysentery, salmonellosis, metritis-mastitis-agalactiae syndrome, mastitis;
Wiederkäuer (Rind, Schaf, Ziege): Diarrhoe, Sepsis, Bronchopneumonie, Salmonellose, Pasteurel- lose, Genitalinfektionen;Ruminants (cattle, sheep, goats): diarrhea, sepsis, bronchopneumonia, salmonellosis, pasteurellosis, genital infections;
Pferd: Bronchopneumonien, Fohlenlähme, puerperale und postpuerperale Infektionen, Salmonello¬ se;Horse: bronchopneumonia, foal disease, puerperal and postpuerperal infections, salmonella;
Hund und Katze: Bronchopneumonie, Diarrhoe, Dermatitis, Otitis, Harnwegsinfekte, Prostatitis;Dog and cat: bronchopneumonia, diarrhea, dermatitis, otitis, urinary tract infections, prostatitis;
Geflügel (Huhn, Pute, Wachtel, Taube, Ziervögel und andere): E. co/z-Infektionen, chronische Luftwegserkrankungen, Salmonellose, Pasteurellose, Psittakose.Poultry (chicken, turkey, quail, pigeon, ornamental birds and others): E. co / z infections, chronic respiratory diseases, salmonellosis, pasteurellosis, psittacosis.
Ebenso können bakterielle Erkrankungen bei der Aufzucht und Haltung von Nutz- und Zierfischen behandelt werden, wobei sich das antibakterielle Spektrum über die vorher genannten Erreger hin- aus auf weitere Erreger wie z.B. Pasteurella, Brucella, Campylobacter, Listeria, Erysipelothris, Corynebakterien, Borellia, Treponema, Nocardia, Rikettsia, Yersinia, erweitert.Likewise, bacterial diseases in the rearing and keeping of farmed and ornamental fish can be treated, with the antibacterial spectrum on the aforementioned pathogens out to further pathogens such. Pasteurella, Brucella, Campylobacter, Listeria, Erysipelothris, Corynebacteria, Borellia, Treponema, Nocardia, Rikettsia, Yersinia.
Weiterer Gegenstand der vorliegenden Erfindung ist der Einsatz der erfindungsgemäßen Verbin¬ dungen zur Behandlung und/oder Prophylaxe von Erkrankungen, insbesondere von bakteriellen Infektionskrankheiten.A further subject of the present invention is the use of the compounds according to the invention for the treatment and / or prophylaxis of diseases, in particular of bacterial infectious diseases.
Weiterer Gegenstand der vorliegenden Erfindung ist die Verwendung der erfindungsgemäßen Ver¬ bindungen zur Behandlung und/oder Prophylaxe von Erkrankungen, insbesondere der zuvor ge¬ nannten Erkrankungen.Another object of the present invention is the use of Ver¬ compounds of the invention for the treatment and / or prophylaxis of diseases, in particular the aforementioned ge diseases.
Weiterer Gegenstand der vorliegenden Erfindung ist die Verwendung der erfindungsgemäßen Ver¬ bindungen zur Herstellung eines Arzneimittels zur Behandlung und/oder Prophylaxe von Erkran- kungen, insbesondere der zuvor genannten Erkrankungen.Another object of the present invention is the use of Ver¬ compounds of the invention for the preparation of a medicament for the treatment and / or prophylaxis of Erkran- kungen, in particular the aforementioned diseases.
Bevorzugt werden die erfindungsgemäßen Verbindungen zur Herstellung von Arzneimitteln ver¬ wendet, die zur Prophylaxe und/oder Behandlung von bakteriellen Erkrankungen geeignet sind.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.
Weiterer Gegenstand der vorliegenden Erfindung ist ein Verfahren zur Behandlung und/oder Pro¬ phylaxe von Erkrankungen, insbesondere der zuvor genannten Erkrankungen, unter Verwendung einer antibakteriell wirksamen Menge der erfindungsgemäßen Verbindungen.Another object of the present invention is a method for the treatment and / or Pro¬ phylaxis of diseases, in particular the aforementioned diseases, using an antibacterially effective amount of the compounds of the invention.
Weiterer Gegenstand der vorliegenden Erfindung sind Arzneimittel, enthaltend mindestens eine erfindungsgemäße Verbindung und mindestens einen oder mehrere weitere Wirkstoffe, insbeson¬ dere zur Behandlung und/oder Prophylaxe der zuvor genannten Erkrankungen. Bevorzugte Kom- binationswirkstoffe sind antibakteriell wirkende Verbindungen, die ein anderes Wirkspektrum, insbesondere ergänzendes Wirkspektrum, haben und/oder synergistisch zu den erfindungsgemäßen Verbindungen sind.Another object of the present invention are pharmaceutical 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.
Die erfindungsgemäßen Verbindungen können systemisch und/oder lokal wirken. Zu diesem Zweck können sie auf geeignete Weise appliziert werden, wie z.B. oral, parenteral, pulmonal, na- sal, sublingual, lingual, buccal, rectal, dermal, transdermal, conjunctival, otisch oder als Implantat bzw. Stent.The compounds according to the invention can act systemically and / or locally. For this purpose, 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.
Für diese Applikationswege können die erfindungsgemäßen Verbindungen in geeigneten Applika¬ tionsformen verabreicht werden.For these administration routes, the compounds according to the invention can be administered in suitable administration forms.
Für die orale Applikation eignen sich nach dem Stand der Technik funktionierende schnell und/oder modifiziert die erfindungsgemäßen Verbindungen abgebende Applikationsformen, die die erfindungsgemäßen Verbindungen in kristalliner und/ oder amorphisierter und/oder gelösterFor the oral administration are according to the prior art functioning rapidly and / or modified compounds of the invention donating application forms, the compounds of the invention in crystalline and / or amorphised and / or dissolved
Form enthalten, wie z.B. Tabletten (nichtüberzogene oder überzogene Tabletten, beispielsweise mit magensaftresistenten oder sich verzögert auflösenden oder unlöslichen Überzügen, die dieContain form such. Tablets (uncoated or coated tablets, for example, with enteric or delayed-dissolving or insoluble coatings containing the
Freisetzung der erfindungsgemäßen Verbindung kontrollieren), in der Mundhöhle schnell zerfal- lende Tabletten oder Filme/Oblaten, Filme/Lyophilisate, Kapseln (beispielsweise Hart- oderControl release of the compound according to the invention), rapidly disintegrating tablets or films / wafers, films / lyophilisates, capsules (for example hard or soft tissue) in the oral cavity
Weichgelatinekapseln), Dragees, Granulate, Pellets, Pulver, Emulsionen, Suspensionen, Aerosole oder Lösungen.Soft gelatin capsules), dragees, granules, pellets, powders, emulsions, suspensions, aerosols or solutions.
Die parenterale Applikation kann unter Umgehung eines Resorptionsschrittes geschehen (z.B. intravenös, intraarteriell, intrakardial, intraspinal oder intralumbal) oder unter Einschaltung einer Resorption (z.B. intramuskulär, subcutan, intracutan, percutan oder intraperitoneal). Für die paren¬ terale Applikation eignen sich als Applikationsformen u.a. Injektions- und Infusionszubereitungen in Form von Lösungen, Suspensionen, Emulsionen, Lyophilisaten oder sterilen Pulvern.Parenteral administration can be accomplished by bypassing a resorption step (e.g., intravenously, intraarterially, intracardially, intraspinal, or intralumbar) or by resorting to absorption (e.g., intramuscularly, subcutaneously, intracutaneously, percutaneously, or intraperitoneally). For the parenteral application are suitable as application forms u.a. Injection and infusion preparations in the form of solutions, suspensions, emulsions, lyophilisates or sterile powders.
Für die sonstigen Applikationswege eignen sich z.B. Inhalationsarzneiformen (u.a. Pulverinhalato¬ ren, Nebulizer), Nasentropfen, -lösungen, -sprays; lingual, sublingual oder buccal zu applizierende Tabletten, Filme/Oblaten oder Kapseln, Suppositorien, Ohren- oder Augen-präparationen, Vagi¬ nalkapseln, wäßrige Suspensionen (Lotionen, Schüttelmixturen), lipophile Suspensionen, Salben, Cremes, transdermale therapeutische Systeme (wie beispielsweise Pflaster), Milch, Pasten, .For other routes of administration are, for example, inhalation medicaments (including powder inhalers, nebulizers), nasal drops, solutions, 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 (such as patches ), Milk, pastes, ,
- 12 -- 12 -
Schäume, Streupuder, Implantate oder Stents.Foams, scattering powders, implants or stents.
Die erfindungsgemäßen Verbindungen können in die angeführten Applikationsformen überfuhrt werden. Dies kann in an sich bekannter Weise durch Mischen mit inerten, nichttoxischen, pharma¬ zeutisch geeigneten Hilfsstoffen geschehen. Zu diesen Hilfsstoffen zählen u.a. Trägerstoffe (bei- spielsweise mikrokristalline Cellulose, Laktose, Mannitol), Lösungsmittel (z.B. flüssige Polyethy- lenglycole), Emulgatoren und Dispergier- oder Netzmittel (beispielsweise Natriumdodecylsulfat, Polyoxysorbitanoleat), Bindemittel (beispielsweise Polyvinylpyrrolidon), synthetische und natürli¬ che Polymere (beispielsweise Albumin), Stabilisatoren (z.B. Antioxidantien wie beispielsweise Ascorbinsäure), Farbstoffe (z.B. anorganische Pigmente wie beispielsweise Eisenoxide) und Ge- schmacks- und / oder Geruchskorrigentien.The compounds according to the invention can be converted into the stated administration forms. This can be done in a manner known per se by mixing with inert, non-toxic, pharmaceutically suitable auxiliaries. 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 (eg antioxidants such as ascorbic acid), dyes (eg inorganic pigments such as iron oxides) and flavor and / or odoriferous agents.
Weiterer Gegenstand der vorliegenden Erfindung sind Arzneimittel, die mindestens eine erfin¬ dungsgemäße Verbindung, üblicherweise zusammen mit einem oder mehreren inerten, nichttoxi¬ schen, pharmazeutisch geeigneten Hilfsstoffen enthalten, sowie deren Verwendung zu den zuvor genannten Zwecken.The present invention further relates to medicaments which comprise at least one compound according to the invention, usually together with one or more inert, non-toxic, pharmaceutically suitable excipients, and to their use for the purposes mentioned above.
Im Allgemeinen hat es sich als vorteilhaft erwiesen, bei intravenöser Applikation Mengen von etwa 0.001 bis 100 mg/kg, vorzugsweise etwa 0.1 bis 10 mg/kg Körpergewicht zur Erzielung wirk¬ samer Ergebnisse zu verabreichen, und bei oraler Applikation beträgt die Dosierung etwa 0.01 bis 50 mg/kg, vorzugsweise 0.5 bis 10 mg/kg Körpergewicht.In general, it has proved to be advantageous to administer amounts of about 0.001 to 100 mg / kg, preferably about 0.1 to 10 mg / kg of body weight to achieve effective results when administered intravenously, and for oral administration the dosage is about 0.01 to 50 mg / kg, preferably 0.5 to 10 mg / kg of body weight.
Trotzdem kann es gegebenenfalls erforderlich sein, von den genannten Mengen abzuweichen, und zwar in Abhängigkeit von Körpergewicht, Applikationsweg, individuellem Verhalten gegenüber dem Wirkstoff, Art der Zubereitung und Zeitpunkt bzw. Intervall, zu welchem die Applikation erfolgt. So kann es in einigen Fällen ausreichend sein, mit weniger als der vorgenannten Mindest¬ menge auszukommen, während in anderen Fällen die genannte obere Grenze überschritten werden muss. Im Falle der Applikation größerer Mengen kann es empfehlenswert sein, diese in mehreren Einzelgaben über den Tag zu verteilen.Nevertheless, it may be necessary to deviate from the stated amounts, depending on body weight, route of administration, individual behavior towards the active ingredient, type of preparation and time or interval at which the application is carried out. Thus, in some cases it may be sufficient to make do with less than the aforementioned Mindest¬ amount, while in other cases, the said upper limit must be exceeded. In the case of the application of larger quantities, it may be advisable to distribute these in several single doses throughout the day.
Die Prozentangaben in den folgenden Tests und Beispielen sind, sofern nicht anders angegeben, Gewichtsprozente; Teile sind Gewichtsteile. Lösungsmittelverhältnisse, Verdünnungsverhältnisse und Konzentrationsangaben von flüssig/flüssig-Lösungen beziehen sich jeweils auf das Volumen. A1 BeispieleThe percentages in the following tests and examples are by weight unless otherwise indicated; Parts are parts by weight. Solvent ratios, dilution ratios and concentration data of liquid / liquid solutions are based on volume. A 1 examples
AbkürzungenAbbreviations
AHg. Allgemein(e)AHG. General (e)
Area (Peak)fläche ber. berechnetArea (peak) area calculated
BHI Brain Heart InfusionBHI Brain Heart Infusion
Boc tert-Butyloxycarbonyl br. breites Signal (bei NMR-Spektren)Boc tert-butyloxycarbonyl br. broad signal (in NMR spectra)
Bsp. Beispiel d Dublett (bei NMR-Spektren)Example d doublet (in NMR spectra)
DC DünnschichtchromatographieTLC thin layer chromatography
DCI direkte chemische Ionisation (bei MS)DCI direct chemical ionization (in MS)
DCM DichlormethanDCM dichloromethane
DIEA N,N-DiisopropylethylaminDIEA N, N-diisopropylethylamine
DMAP 4-N,N-DirnethylaminopyridinDMAP 4-N, N-dimethylaminopyridine
DMSO DimethylsulfoxidDMSO dimethyl sulfoxide
DMF N,N-Dimethylforrnamid d. Th. der TheorieDMF N, N-dimethylformamide d. Th. Of theory
EDC l-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (auch EDCI)EDC 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (also EDCI)
EDCxHCl 1 -Ethyl-3 -(3 -dimethy laminopropy l)carbodiimide-hydrochloridEDCxHCl 1 -ethyl-3 - (3-dimethylaminopropyl) carbodiimide hydrochloride
EE Ethylacetat (Essigsäureethylester)EE ethyl acetate (ethyl acetate)
EI Elektronenstoß-Ionisation (bei MS)EI electron impact ionization (in MS)
ESI Elektrospray-Ionisation (bei MS)ESI electrospray ionization (in MS)
ESIneg Anionenscan bei der ESI-MassenspektroskopieESIneg anion scan in ESI mass spectroscopy
ESIpos Kationenscan bei der ESI-MassenspektroskopieESIpos cation scan in ESI mass spectroscopy
Fp. Schmelzpunkt gef. gefunden ges. gesättigt h StundeMp melting point found ges. saturated h hour
HATU O-(7-Azabenzotriazol-l-yl)-NNN',N'-tetramethyluronium-hexa- fluorphosphatHATU O- (7-azabenzotriazol-1-yl) -NNN ', N'-tetramethyluronium hexafluorophosphate
HOBt 1 -HydroxybenzotriazolHOBt 1-hydroxybenzotriazole
HPLC Hochdruck-, HochleistungsflüssigchromatographieHPLC high pressure, high performance liquid chromatography
HR High Resolution (Hochauflösung) i. V. im Vakuum konz. KonzentriertHR High Resolution i. V. in a vacuum conc. Concentrated
LC-MS Flüssigchromatographie-gekoppelte MassenspektroskopieLC-MS liquid chromatography-coupled mass spectrometry
LDA Lithium-Diisopropylamid m middle (mittel) (bei UV- und IR-Spektren) m Multiple« (bei NMR-Spektren)LDA lithium diisopropylamide m middle (medium) (in UV and IR spectra) m multiple «(in NMR spectra)
MALDI Matrix-Assisted Laser Desorption/IonizationMALDI Matrix-Assisted Laser Desorption / Ionization
MHK Minimale Hemmkonzentration min Minute/MinutenMIC Minimum inhibitory concentration min minute / minutes
MRSA Methicillin-resistenter Staphylococcus aureusMRSA Methicillin-resistant Staphylococcus aureus
MS MassenspektroskopieMS mass spectroscopy
NCCLS National Committee for Clinical Laboratory Standards neg. negativNCCLS National Committee for Clinical Laboratory Standards neg. Negative
NMM N-MethylmorpholinNMM N-methylmorpholine
NMR Kernresonanzspektroskopie (nuclear magnetic resonance) p.a. pro analysiNMR Nuclear Magnetic Resonance (NMR) p.a. pro analysi
Pd PalladiumPd palladium
Pd-C Palladium auf Kohle pos. positiv proz. ProzentigPd-C palladium on carbon pos. positive percent. Percent
PTFE Polytetrafluorethylen quant. quantitativPTFE polytetrafluoroethylene quant. quantitatively
RP-HPLC Reverse Phase HPLCRP-HPLC reverse phase HPLC
RT RaumtemperaturRT room temperature
Rt Retentionszeit (bei HPLC)Retention time (on HPLC)
S strong (stark) (bei UV- und IR-Spektren)S strong (in UV and IR spectra)
S Singulett (bei ΝMR-Spektren)S singlet (in ΝMR spectra)
TBTU 0-(Benzotriazol-l-yl)-N,N,N',N'-tetramethyluronium-TBTU 0- (benzotriazol-1-yl) -N, N, N ', N'-tetramethyluronium
Tetrafluoroborattetrafluoroborate
TCTU O-( 1 H-6-Chlorbenzotriazol- 1 -yl)- 1,1,3,3 -tetramethyluronium-TCTU O- (1H-6-chlorobenzotriazol-1-yl) -1,1,3,3-tetramethyluronium
Tetrafluoroborattetrafluoroborate
TFA TrifluoressigsäureTFA trifluoroacetic acid
TFE 2,2,2-TrifluorethanolTFE 2,2,2-trifluoroethanol
THF TetrahydrofuranTHF tetrahydrofuran
TOF Flugzeit (time of flight)TOF time of flight
UV UltraviolettUV ultraviolet
Vis sichtbar (visible) VRSA Vancomycin-resistenter Stapylococcus aureusVisible (visible) VRSA vancomycin-resistant Stapylococcus aureus
W weak (schwach) (bei UV- und IR-Spektren) wässr. wässrige(r)W weak (in UV and IR spectra) aq. aqueous (r)
Z, Cbz BenzyloxycarbonylZ, Cbz benzyloxycarbonyl
Literaturliterature
Zur Nomenklatur der Peptide und Cyclodepsipeptide vergleiche:For the nomenclature of peptides and cyclodepsipeptides, see:
1. A Guide to IUPAC Nomenclature of Organic Compounds (Recommendations 1993), 1993, Blackwell Scientific publications.1. A Guide to IUPAC Nomenclature of Organic Compounds (Recommendations 1993), 1993, Blackwell Scientific publications.
2. Nomenclature and symbolism for amino acids and peptides. Recommendations 1983. IUPAC- IUB Joint Commission on Biochemical Nomenclature, UK. Biochemical Journal 1984, 219, 345-373. Sowie zitierte Literatur.2. Nomenclature and symbolism for amino acids and peptides. Recommendations 1983. IUPAC-IUB Joint Commission on Biochemical Nomenclature, UK. Biochemical Journal 1984, 219, 345-373. As well as cited literature.
Allgemeine Methoden GC-MS, LC-MS, HR-MS, HPLC und GelchromatographieGeneral methods GC-MS, LC-MS, HR-MS, HPLC and gel chromatography
Methode 1 (TOF-HR-MS): TOF-HR-MS-ESI+ Spektren werden mit einem Micromass LCT Ge¬ rät (Kapillarspannung: 3.2 KV, Cone-Spannung: 42 V, Quellentemperatur: 12O0C, Desolvations- Temperatur: 2800C) aufgenommen. Hierzu wird eine Spritzenpumpe (Fa. Harvard Apparatus) für die Probenzuführung verwendet. Als Standart dient Leucin-Enkephalin (Tyr-Gly-Gly-Phe-Leu).Method 1 (TOF-HR-MS): TOF-HR-MS-ESI + spectra are provided with a Micromass LCT apparatus (capillary voltage: 3.2 KV, cone voltage: 42 V, source temperature: 12O 0 C, desolvation temperature: 280 0 C). For this purpose, a syringe pump (Harvard Apparatus) is used for sample introduction. The standard is leucine-enkephalin (Tyr-Gly-Gly-Phe-Leu).
Methode 2 (präparative HPLC): Gerät: Gilson Abimed HPLC; UV-Detektor 210 nm; binäres Pumpensystem; Säule: Waters Symmetry-Prep™ Ci8, 7 μm, 300 x 19 mm; Eluent A: 0.2% Trifluo- ressigsäure in Wasser, Eluent B: Acetonitril; Flussrate: 25 mL/min; Säulentemperatur RT; 0 min 20% B, Rampe 0-10 min 70% B, Rampe 10-10.1 min 20% B, 15 min 20% B.Method 2 (Preparative HPLC): Device: Gilson Abimed HPLC; UV detector 210 nm; binary pump system; Column: Waters Symmetry-Prep ™ Ci 8 , 7 μm, 300 x 19 mm; Eluent A: 0.2% trifluoroacetic acid in water, eluent B: acetonitrile; Flow rate: 25 mL / min; Column temperature RT; 0 min 20% B, ramp 0-10 min 70% B, ramp 10-10.1 min 20% B, 15 min 20% B.
Methode 3 (Gelchromatographie an Sephadex LH-20): Gelchromatographie wird ohne Druck an Sephadex LH-20 (Fa. Pharmacia) durchgeführt. Es wird nach UV-Aktivität (UV Detektor für 254 nm, Fa. Knauer) fraktioniert (Fraktionssammler ISCO Foxy 200). Säulendimensionen: 32 x 7 cm (1000-100 μmol Maßstab); 30 x 4 cm (100-10 μmol Maßstab); 25 x 2 cm (10-1 μmol Maßstab). Bei Maßstäben 1 mmol bis 11 mmol wird eine Säule der Dimension 80 x 30 cm ver¬ wendet. In diesem Fall werden die Fraktionen manuell und ohne vorgeschalteten UV-Detektor gesammelt. Die Zuordnung der Fraktionen erfolgt per HPLC (Methode 9). Methode 4 (präparative HPLC; Kromasil, Essigsäure): Gerät: Gilson Abimed HPLC; UV De¬ tektor 210 nm; binäres Pumpensystem; Säule: Kromasil- 100A Ci8, 5 μm; 250 x 20 mm; Fluss: 25 mL/min; Eluent A: Wasser/0.25-0.5% Essigsäure, Eluent B: Acetonitril; Gradient: 0-3 min 5% B, 3-30 min 5-100% B, 30-38 min 100% B, anschließend Regeneration der Chromatographiesäule.Method 3 (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 μmol scale); 30 x 4 cm (100-10 μmol scale); 25 x 2 cm (10-1 μmol scale). At scales of 1 mmol to 11 mmol, a column of the dimension 80 × 30 cm is used. In this case, the fractions are collected manually and without upstream UV detector. The assignment of the fractions is carried out by HPLC (Method 9). Method 4 (preparative HPLC, Kromasil, acetic acid): Device: Gilson Abimed HPLC; UV detector 210 nm; binary pump system; Column: Kromasil 100 A Ci 8 , 5 μm; 250 x 20 mm; Flow: 25 mL / min; Eluent A: water / 0.25-0.5% 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.
Methode 5 (LC-MS): Instrument: Micromass Quattro LCZ mit HPLC Agilent Serie 1100; Säule: Phenomenex Synergi 2μ Hydro-RP Mercury 20 mm x 4 mm; Eluent A: 1 L Wasser + 0.5 mL 50%ige Ameisensäure, Eluent B: 1 L Acetonitril + 0.5 mL 50%ige Ameisensäure; Gradient: 0.0 min 90%A -> 2.5 min 30%A -> 3.0 min 5%A -> 4.5 min 5%A; Fluss: 0.0 min 1 mL/min, 2.5 min/3.0 min/4.5 min 2 mL/min; Ofen: 5O0C; UV-Detektion: 208- 400 nm.Method 5 (LC-MS): Instrument: Micromass Quattro LCZ with HPLC Agilent Series 1100; Column: Phenomenex Synergi 2μ Hydro-RP Mercury 20 mm x 4 mm; Eluent A: 1 L water + 0.5 mL 50% formic acid, eluent B: 1 L acetonitrile + 0.5 mL 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.
Methode 6 (LC-MS): Gerätetyp MS: Micromass ZQ; Gerätetyp HPLC: Waters Alliance 2795; Säule: Phenomenex Synergi 2μ Hydro-RP Mercury 20 mm x 4 mm; Eluent A: 1 L Wasser + 0.5 mL 50%ige Ameisensäure, Eluent B: 1 L Acetonitril + 0.5 mL 50%ige Ameisensäure; Gradient: 0.0 min 90%A ■* 2.5 min 30%A -> 3.0 min 5%A -> 4.5 min 5%A; Fluss: 0.0 min 1 mL/min, 2.5 min/3.0 min/4.5 min 2 mL/min; Ofen: 5O0C; UV-Detektion: 210 nm.Method 6 (LC-MS): Device Type MS: Micromass ZQ; Device type HPLC: Waters Alliance 2795; Column: Phenomenex Synergi 2μ Hydro-RP Mercury 20 mm x 4 mm; Eluent A: 1 L water + 0.5 mL 50% formic acid, eluent B: 1 L acetonitrile + 0.5 mL 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.
Methode 7 (LC-MS): Gerätetyp MS: Micromass ZQ; Gerätetyp HPLC: HP 1100 Series; UV DAD; Säule: Phenomenex Synergi 2μ Hydro-RP Mercury 20 mm x 4 mm; Eluent A: 1 L Wasser + 0.5 mL 50%ige Ameisensäure, Eluent B: 1 L Acetonitril + 0.5 mL 50%ige Ameisensäure; Gra¬ dient: 0.0 min 90%A -> 2.5 min 30%A -> 3.0 min 5%A -> 4.5 min 5%A; Fluss: 0.0 min 1 mL/min, 2.5 min/3.0 min/4.5 min. 2 mL/min; Ofen: 5O0C; UV-Detektion: 210 nm.Method 7 (LC-MS): Device Type MS: Micromass ZQ; Device type HPLC: HP 1100 Series; UV DAD; Column: Phenomenex Synergi 2μ Hydro-RP Mercury 20 mm x 4 mm; Eluent A: 1 L water + 0.5 mL 50% formic acid, eluent B: 1 L acetonitrile + 0.5 mL 50% formic acid; Gra¬ serves: 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.
Methode 8 (analytische HPLC): Gerätetyp HPLC: HP 1050 Series; UV DAD 1100 Series; Säule: Kromasil Ci8, 6O x 2 mm, 3.5 μm; Eluent A: Wasser/0.5% Perchlorsäure, Eluent B: Acetonitril; 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 min 90-2%B, 9.2- 10.0 min 2%B; Fluss: 0.75 mL/min, Ofen: 300C, UV-Detektion 210 nm.Method 8 (Analytical HPLC): Device Type HPLC: HP 1050 Series; UV DAD 1100 Series; Column: Kromasil Ci 8 , 60 × 2 mm, 3.5 μm; 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 min 90-2% B, 9.2-10.0 min 2% B; Flow: 0.75 mL / min, oven: 30 ° C., UV detection 210 nm.
Methode 9 (analytische HPLC, Agilent Zorbax C8): Gerät: Agilent 1100 mit DAD (Gl 315B), binärer Pumpe (G1312A), Autosampier (G1313A), Lösemittel-Entgaser (G1379A) und Säulen¬ thermostat (G1316A); Säule: Agilent Zorbax Eclipse XDB-C8 4.6 x 150 x 5 mm; Eluent A: 0.05% 70%ige Perchlorsäure in Wasser; Eluent B: Acetonitril; Gradient: 0—1 min 10%B, Rampe, 4—5 min 90%B, Rampe, 5.5 min 10%B; Fluss: 2.00 mL/min; Säulentemperatur: 3O0C.Method 9 (analytical HPLC, Agilent Zorbax C 8 ): Device: Agilent 1100 with DAD (GI 315B), binary pump (G1312A), autosampler (G1313A), solvent degasser (G1379A) and column thermostat (G1316A); Column: Agilent Zorbax Eclipse XDB-C8 4.6 x 150 x 5 mm; Eluent A: 0.05% 70% perchloric acid in water; Eluent B: acetonitrile; Gradient: 0-1 min 10% B, ramp, 4-5 min 90% B, ramp, 5.5 min 10% B; Flow: 2.00 mL / min; Column temperature: 3O 0 C.
Methode 10 (Gelchromatographie an Sephadex LH-20): Gelchromatographie wird ohne Druck an Sephadex LH-20 (Fa. Pharmacia) durchgeführt. Es wird nach UV-Aktivität (UV Detektor fürMethod 10 (gel chromatography on Sephadex LH-20): Gel chromatography is performed without pressure on Sephadex LH-20 (Pharmacia). It will after UV activity (UV detector for
254 nm, Fa. Knauer) fraktioniert (Fraktionssammler ISCO Foxy 200). Säulendimensionen:254 nm, Knauer) fractionated (fraction collector ISCO Foxy 200). Column dimensions:
32 x 7 cm (1000-100 μmol Maßstab); 30 x 4 cm (100-10 μmol Maßstab); 25 x 2 cm (10-1 μmol Maßstab).32 x 7 cm (1000-100 μmol scale); 30 x 4 cm (100-10 μmol scale); 25 x 2 cm (10-1 μmol Scale).
Methode 11 (präparative HPLC Symmetry): Gerät: Gilson Abimed HPLC; binäres Pumpensys¬ tem; Säule: SymmetryPrep™C18, Fa. Waters, 7 μm; 300 mm x 19 mm; Eluent A: Wasser/0.2% Trifluoressigsäure, Eluent B: Acetonitril; Gradient: 0-10 min 15-65%B, anschließend Regenera- tion der Chromatographiesäule; Fluss: 25 mL/min; UV-Detektion 210 nm.Method 11 (Preparative HPLC Symmetry): Device: Gilson Abimed HPLC; binary pump system; Column: SymmetryPrep ™ C 18 , Waters, 7 μm; 300mm x 19mm; Eluent A: water / 0.2% trifluoroacetic acid, eluent B: acetonitrile; Gradient: 0-10 min 15-65% B, then regeneration of the chromatography column; Flow: 25 mL / min; UV detection 210 nm.
Methode 12 (präparative HPLC Kromasil): Gerät: Gilson Abimed HPLC; binäres Pumpensys¬ tem; Säule: Kromasil Ci8, 5 μm, 100 A, 250 x 20 mm; Eluent A: 0.05% Trifluoressigsäure in Was¬ ser, Eluent B: 0.05% Trifluoressigsäure in Acetonitril; Gradient: 0-3 min 10%B, Rampe, 30- 38 min 90%B, 38-45 min 10%B; Fluss: 20 mL/min; UV-Detektion 210 nm.Method 12 (Preparative HPLC Kromasil): Device: Gilson Abimed HPLC; binary pump system; Column: Kromasil Ci 8 , 5 μm, 100 A, 250 × 20 mm; Eluent A: 0.05% trifluoroacetic acid in water, eluent B: 0.05% trifluoroacetic acid in acetonitrile; Gradient: 0-3 min 10% B, ramp, 30-38 min 90% B, 38-45 min 10% B; Flow: 20 mL / min; UV detection 210 nm.
Methode 13 (präparative HPLC Waters Symmetry): Gerät: Gilson Abimed HPLC; binäres Pumpensystem; Säule: Waters Symmetry-Prep™ Ci8, 7 μm, 300 x 19 mm; Eluent A: 0.05% Trifluoressigsäure in Wasser, Eluent B: 0.05% Trifluoressigsäure in Acetonitril; Gradient: 0-3 min 10%B, Rampe, 30-38 min 90%B, 38-45 min 10%B; Fluss: 20 mL/min; UV-Detektion 210 nm.Method 13 (Preparative HPLC Waters Symmetry): Device: Gilson Abimed HPLC; binary pump system; Column: Waters Symmetry-Prep ™ Ci 8 , 7 μm, 300 x 19 mm; Eluent A: 0.05% trifluoroacetic acid in water, eluent B: 0.05% trifluoroacetic acid in acetonitrile; Gradient: 0-3 min 10% B, ramp, 30-38 min 90% B, 38-45 min 10% B; Flow: 20 mL / min; UV detection 210 nm.
Methode 14 (präparative HPLC): Gerät: Gilson Abimed HPLC; binäres Pumpensystem; Säule: Waters Symmetry-Prep™ Ci8, 7 μm, 300 x 19 mm; Eluent A: Wasser/0.2% Trifluoressigsäure, Eluent B: Acetonitril; Gradient: 0-10 min 25-65%B, anschließend Regeneration der Chroma¬ tographiesäule; Fluss: 25 mL/min; UV-Detektion 210 nm.Method 14 (preparative HPLC): Device: Gilson Abimed HPLC; binary pump system; Column: Waters Symmetry-Prep ™ Ci 8 , 7 μm, 300 x 19 mm; Eluent A: water / 0.2% trifluoroacetic acid, eluent B: acetonitrile; Gradient: 0-10 min 25-65% B, then regeneration of the chromatographic column; Flow: 25 mL / min; UV detection 210 nm.
Methode 15 (Chirale HPLC Daicel Chiralpak): Agilent 1100 HPLC; Säule: Daicel Chiralpak AD-H 5 μm; 250 x 20 mm; isokratisch: 75% wo-Hexan, 25% 2-Propanol mit 0.2% Trifluoressig- säure und 1% Wasser; Fluss: 1.0 mL/min; Ofen: 250C; UV-Detektor 212 nm.Method 15 (Chiral HPLC Daicel Chiralpak): Agilent 1100 HPLC; Column: Daicel Chiralpak AD-H 5 μm; 250 x 20 mm; isocratic: 75% hexane, 25% 2-propanol with 0.2% trifluoroacetic acid and 1% water; Flow: 1.0 mL / min; Oven: 25 ° C; UV detector 212 nm.
Methode 16 (präparative HPLC): Gerät: Gilson Abimed HPLC; binäres Pumpensystem; Säule: YMC ODS-AQ 5 μm, 250 x 30 mm; Eluent A: 0.05% Trifluoressigsäure in Wasser, Eluent B: 0.05% Trifluoressigsäure in Acetonitril; Gradient: 0-3 min 10%B, Rampe, 30-38 min 90%B, 38- 45 min 10%B; Fluss: 50 mL/min; UV-Detektor 210 nm.Method 16 (Preparative HPLC): Device: Gilson Abimed HPLC; binary pump system; Column: YMC ODS-AQ 5 μm, 250 x 30 mm; Eluent A: 0.05% trifluoroacetic acid in water, eluent B: 0.05% trifluoroacetic acid in acetonitrile; Gradient: 0-3 min 10% B, ramp, 30-38 min 90% B, 38- 45 min 10% B; Flow: 50 mL / min; UV detector 210 nm.
Methode 17 (GC-MS): Instrument: Micromass GCT, GC6890; Säule: Restek RTX-35MS, 30 m x 250 μm x 0.25 μm; Gradient: 600C (0.30 min halten), 50°C/min → 12O0C, 16°C/min → 25O0C, 30°C/min → 3000C (1.7 min halten); konstanter Fluss mit Helium: 0.88 mL/min; Ofen: 600C; In- let: 25O0C.Method 17 (GC-MS): Instrument: Micromass GCT, GC6890; Column: Restek RTX-35MS, 30 m × 250 μm × 0.25 μm; Gradient: 60 0 C (0.30 keep min), 50 ° C / min → 12O 0 C, 16 ° C / min → 25O 0 C, C / min → C (hold 1.7 min) 300 30 ° 0; constant flow with helium: 0.88 mL / min; Oven: 60 ° C; Let: 25O 0 C.
Methode 18 (HPLC): Gerätetyp HPLC: HP 1100 Series; UV DAD Säule: Zorbax Eclipse XBD- C8 (Agilent), 150 mm x 4.6 mm, 5 μm; Eluent A: 5 mL HClO4/! Wasser, Eluent B: Acetonitril; .Method 18 (HPLC): Device Type HPLC: HP 1100 Series; UV DAD column: Zorbax Eclipse XBD-C8 (Agilent), 150 mm x 4.6 mm, 5 μm; Eluent A: 5 mL HClO 4 /! Water, eluent B: acetonitrile; ,
- 18 -- 18 -
Gradient: 0-1 min 10%B, 1-4 min 10-90%B, 4-5 min 90%B; Fluss: 2.0 mL/min; Ofen: 300C; UV- Detektion: 210 und 254 nm.Gradient: 0-1 min 10% B, 1-4 min 10-90% B, 4-5 min 90% B; Flow: 2.0 mL / min; Oven: 30 ° C; UV detection: 210 and 254 nm.
Methode 19 (HPLC): Säule: Kromasil RP-18, 60 mm x 2 mm, 3.5 μm; Eluent A: 5 mL HCIO4/I Wasser, Eluent B: Acetonitril; Gradient: 0 min 2%B, 0.5 min 2%B, 4.5 min 90%B, 9 min 90%B; Fluss: 0.75 mL/min; Ofen: 3O0C; UV-Detektion: 210 nm.Method 19 (HPLC): Column: Kromasil RP-18, 60 mm × 2 mm, 3.5 μm; Eluent A: 5 mL HCIO 4 / I 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: 3O 0 C; UV detection: 210 nm.
Methode 20 (HPLC): Säule: Kromasil RP-18, 250 mm x 4 mm, 5 μm; Eluent A: 5 mL HClO4/! Wasser, Eluent B: Acetonitril; Gradient: 0 min 5%B, 10 min 95%B; Fluss: 1 mL/min; Ofen: 400C; UV-Detektion: 210 nm.Method 20 (HPLC): Column: Kromasil RP-18, 250 mm x 4 mm, 5 μm; Eluent A: 5 mL HClO 4 /! Water, eluent B: acetonitrile; Gradient: 0 min 5% B, 10 min 95% B; Flow: 1 mL / min; Oven: 40 ° C; UV detection: 210 nm.
Methode 21 (HPLC): Säule: Kromasil RP-18, 250 mm x 4 mm, 5 μm; Eluent A: 2 mL HCIO4/I Wasser, Eluent B: Acetonitril; Isokratisch: 45%B, 55%A; Fluss: 1 mL/min; Ofen: 4O0C; UV- Detektion: 210 nm.Method 21 (HPLC): Column: Kromasil RP-18, 250 mm x 4 mm, 5 μm; Eluent A: 2 mL HCIO 4 / l water, eluent B: acetonitrile; Isocratic: 45% B, 55% A; Flow: 1 mL / min; Oven: 4O 0 C; UV detection: 210 nm.
Methode 22 (LC-MS): Gerätetyp MS: Micromass ZQ; Gerätetyp HPLC: HP 1100 Series; UV DAD; Säule: Grom-Sil 120 ODS-4 HE 50 x 2 mm, 3.0 μm; Eluent A: Wasser/0.025% Ameisen¬ säure/l, Eluent B: Acetonitril/0.025% Ameisensäure; Gradient: 0-2.9 min 0-70%B, 2.9-3.1 min 70- 90%B, 3.1-4.5 min 70-90%B; Ofen: 5O0C, Fluss: 0.8 mL/min, UV-Detektion: 210 nm.Method 22 (LC-MS): Device Type MS: Micromass ZQ; Device type HPLC: HP 1100 Series; UV DAD; Column: Grom-Sil 120 ODS-4 HE 50 × 2 mm, 3.0 μm; Eluent A: water / 0.025% formic acid / l, eluent B: acetonitrile / 0.025% formic acid; Gradient: 0-2.9 min 0-70% B, 2.9-3.1 min 70-90% B, 3.1-4.5 min 70-90% B; Oven: 5O 0 C, flow: 0.8 mL / min, UV detection: 210 nm.
Methode 23 (HPLC): Gerätetyp HPLC: HP 1050 Series; UV DAD 1100 Series; Säule Symmetry- Prep™Ci8, Firma Waters, 50 x 2.1 mm, 3.5 μm; Eluent A: Wasser/0.05% Trifluoressigsäure, E- luent B: Acetonitril; Gradient: 0-9 min 0-100%B, 9-11 min 100%B, 11-12 min 100-0%B, an¬ schließend Regeneration der Chromatographiesäule; Ofen: 4O0C, Fluss: 0.4 mL/min, UV- Detektion: 210 nm.Method 23 (HPLC): Device Type HPLC: HP 1050 Series; UV DAD 1100 Series; Column Symmetry-Prep ™ Ci 8 , Waters, 50 x 2.1 mm, 3.5 μm; Eluent A: water / 0.05% trifluoroacetic acid, eluent B: acetonitrile; Gradient: 0-9 min 0-100% B, 9-11 min 100% B, 11-12 min 100-0% B, subsequent regeneration of the chromatography column; Oven: 4O 0 C, flow: 0.4 mL / min, UV detection: 210 nm.
Methode 24 (quantitative 19F-NMR-Spektroskopie): Ungefähr 10 mg genau eingewogene Pro¬ bensubstanz und ungefähr 20 mg genau eingewogenes 1,4-Dibromtetrafluorbenzol werden in Pyri- din gelöst und 19F-NMR-spektroskopisch vermessen, δ - 74 (TFA) und -132.0 (1,4- Dibromtetrafluorbenzol) werden integriert und verglichen. Der TFA-Gehalt wird in Prozent TFA der Masse der Probensubstanz angegeben.Method 24 (Quantitative 19 F-NMR Spectroscopy): Approximately 10 mg of sample substance weighed in exactly and approximately 20 mg of 1,4-dibromotetrafluorobenzene weighed in exactly are dissolved in pyridine and measured by 19 F-NMR spectroscopy, δ-74 (cf. TFA) and -132.0 (1,4-dibromotetrafluorobenzene) are integrated and compared. The TFA content is expressed in percent TFA of the mass of the sample substance.
Methode 25 (Ionenchromatographie): Ionenchromatographie-System mit Suppressor-System und Leitfähigkeitsdetektor; Vorsäule: A SUPP 4/5 Guard, Trennsäule: A SUPP 5 4.0 x 250 mm; Eluent: 3.2 mM Natriumcarbonat und 2.4 mM Natriumhydrogencarbonat in Wasser; Fluss: 0.7 mL/min. Die Probe wird in Methanol (20% des finalen Probenvolumens) gelöst, im Ultraschallbad 3 min behandelt und mit Wasser aufgefüllt. Die Probe wird über ein ionenfreies Celluloseacetat- Filter (Por 0.45 μm) filtriert und injiziert. Quantifizierung gegen externe Standards (0.5 mg/L - 10 mg/L). AusgangsverbiudungenMethod 25 (Ion Chromatography): Ion chromatography system with suppressor system and conductivity detector; Guard column: A SUPP 4/5 Guard, separation column: A SUPP 5 4.0 x 250 mm; Eluent: 3.2 mM sodium carbonate and 2.4 mM sodium bicarbonate in water; Flow: 0.7 mL / min. The sample is dissolved in methanol (20% of the final sample volume), treated in an ultrasonic bath for 3 minutes and made up with water. The sample is filtered through an ion-free cellulose acetate filter (Por 0.45 μm) and injected. Quantification against external standards (0.5 mg / L - 10 mg / L). Ausgangsverbiudungen
Beispiel IAExample IA
D-Leucyl-V-ICS^ό^π^lS^lδR^liS'^^V^S^-ό-tCl^^-amino-l-hydroxy^-oxoethyll-lS- (3-{[amino(imino)methyl]amino}propyl)-12-[(liS)-l-hydroxyethyl]-3-(hydroxymethyl)-24-[(ii?)-l- hydroxy-2-methylpropyl]-21-isobutyl-15-[(l,S)-l-methylpropyl]-2,5,8,ll,14,17,20,23,26-nonaoxo- 28-phenyl- 1 -oxa-4,7, 10,13,16,19,22,25-octaazacyclooctacosan-27-yl } -L-leucinamid-bistrifluor- acetat (Lysobactin)D-Leucyl-V-ICS ^ ό ^ π ^ lS ^ lδR ^ liS '^^ V ^ S ^ -ό-tCl ^^ - amino-l-hydroxy ^ -oxoethyll-IS- (3 - {[amino (imino ) methyl] amino} propyl) -12 - [(liS) -l-hydroxyethyl] -3- (hydroxymethyl) -24 - [(ii) -l-hydroxy-2-methylpropyl] -21-isobutyl-15- [ (l, S) -l-methylpropyl] -2,5,8, ll, 14,17,20,23,26-nonaoxo-28-phenyl-1-oxa-4,7, 10,13,16,19 , 22,25-octaazacyclooctacosan-27-yl} -L-leucine amide bistrifluoroacetate (lysobactin)
Fermentation:Fermentation:
Kultur Medium:Culture Medium:
YM: Hefe-Malz Agar: D-Glucose (4 g/l), Hefe Extrakt (4 g/l), Malz Extrakt (10 g/l), 1 Liter Lewa- tit Wasser. Vor dem Sterilisieren (20 Minuten bei 1210C) wird der pH auf 7.2 eingestellt.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 (20 minutes at 121 0 C), the pH is adjusted to 7.2.
HPM: Mannit (5.4 g/l), Hefe Extrakt (5 g/l), Fleischpepton (3 g/l).HPM: mannitol (5.4 g / L), yeast extract (5 g / L), meat peptone (3 g / L).
Arbeitskonserve: Der lyophilisierte Stamm (ATCC 53042) wird in 50 mL YM Medium angezogen.Working Preservation: The lyophilized strain (ATCC 53042) is grown in 50 mL YM medium.
Kolbenfermentation: 150 mL YM Medium oder 100 mL HPM Medium in einem 1 L Erlenmeyer- kolben werden mit 2 mL der Arbeitskonserve beimpft und für 30-48 Stunden bei 280C auf einem Schüttler bei 240 Upm wachsen gelassen. 30 L Fermentation: 300 mL der Kolbenfermentation (HPM Medium) werden zum Animpfen einer sterilen 30 L Nährmedienlösung verwandt (1 mL Antifoam SAG 5693/1). Diese Kultur wird für 21 Stunden bei 280C, 300 Upm und einer Belüftung mit steriler Luft von 0.3 wm wachsen gelassen. Der pH wird mit 1 M Salzsäure auf pH = 7.2 konstant gehalten. Insgesamt werden während der Kultivierungszeit 880 mL 1 M Salzsäure zugeführt.Piston Fermentation: Inoculate 150 mL of YM medium or 100 mL of HPM medium in a 1 L Erlenmeyer flask with 2 mL of the working fluid and allow to grow for 30-48 hours at 28 0 C on a shaker at 240 rpm. 30 L 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. The pH is kept constant with 1 M hydrochloric acid to pH = 7.2. In total, 880 ml of 1 M hydrochloric acid are added during the culture time.
Hauptkultur (200 L): 15 x 150 mL YM Medium in 1 L Erlenmeyerkolben werden mit 2 mL der Arbeitskonserve beimpft und bei 280C für 48 Stunden und 240 Upm auf dem Schüttler wachsen gelassen. 2250 mL dieser Kultur werden zum Beimpfen einer sterilen 200 L Nährmedienlösung (YM) verwandt (1 mL Antifoam SAG 5693/1) und für 18.5 Stunden bei 28°C, 150 Upm und einer Belüftung mit steriler Luft von 0.3 wm wachsen gelassen.Main culture (200 L): 15 x 150 mL YM medium in 1 L Erlenmeyer flasks are inoculated with 2 mL of the working fluid and grown on the shaker at 28 0 C for 48 hours and 240 rpm. 2250 mL of this culture are used to inoculate a sterile 200 L culture media solution (YM) (1 mL Antifoam SAG 5693/1) and grown for 18.5 hours at 28 ° C, 150 rpm and a sterile air aeration of 0.3 wm.
Zur Kontrolle des Fermentationsverlaufs werden stündlich Proben (50 mL) entnommen. 2 mL dieser Kulturbrühe werden mit 1 mL Methanol (0.5% Trifluoressigsäure) versetzt und über einen 0.45 μm Filter filtriert. 30 μl dieser Suspension werden mittels HPLC analysiert (Methode 18 und Methode 19).To control the fermentation process, samples (50 mL) are taken every hour. 2 mL of this culture broth are mixed with 1 mL of methanol (0.5% trifluoroacetic acid) and filtered through a 0.45 μm filter. 30 μl of this suspension are analyzed by HPLC (Method 18 and Method 19).
Nach 18.5 Stunden wird die Kulturbrühe der Hauptkultur bei 17000 Upm in Überstand und Sedi¬ ment getrennt.After 18.5 hours, the culture broth of the main culture is separated at 17000 rpm in supernatant and sediment.
Isolierung:Insulation:
Der Überstand (183 L) wird mit konzentrierter Trifluoressigsäure bzw. Natronlauge auf pH 6.5-7 eingestellt und auf eine Lewapolsäule (OC 1064, 60 L Inhalt) aufgetragen. Anschließend wird mit reinem Wasser, Wasser/Methanol 1 :1 und anschließend mit reinem Methanol (mit 0.1% Trifluo¬ ressigsäure) eluiert. Diese organische Phase wird im Vakuum bis zu einem verbleibenden wässri- gen Rest von 11.5 L eingeengt.The supernatant (183 L) 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). The mixture 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 remaining aqueous residue of 11.5 L.
Die verbleibende wässrige Phase wird an Kieselgel Cig gebunden und aufgetrennt (MPLC, Biotage Flash 75, 75 x 30 cm, KP-C18-WP, 15-20 μm, Fluss: 30 mL; Eluent: Acetonitril/ Wasser mit 0.1% Trifluoressigsäure; Gradient: 10%, 15% and 40% Acetonitril). Die 40% Acetonitril Phase, die die Hauptmenge von Beispiel IA enthält, wird im Vakuum eingeengt und anschließend lyophilisiert (ca. 13 g). Dieses Feststoffgemisch wird in 1.2 g Portionen zunächst an einer präparativen HPLC (Methode 7), anschließend durch Gelfϊltration an Sephadex LH-20 (5 x 70 cm, Acetonitril/Wasser 1:1, jeweils mit 0.05% Trifluoressigsäure) und einer weiteren präparativen HPLC (Methode 20) getrennt.The remaining aqueous phase is bound to silica gel Cig and separated (MPLC, Biotage Flash 75, 75 × 30 cm, KP-C18-WP, 15-20 μm, 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). This solid mixture is dissolved in 1.2 g portions initially on a preparative HPLC (Method 7), then by gel filtration on Sephadex LH-20 (5 × 70 cm, acetonitrile / water 1: 1, each with 0.05% trifluoroacetic acid) and a further preparative HPLC ( Method 20) separately.
Dieser Prozess liefert 2250 mg Beispiel IA. Das Sediment wird in 4 L Aceton/Wasser 4:1 aufgenommen, mit 2 kg Celite versetzt, mit Trifluo- ressigsäure auf pH = 6 eingestellt, ausgerührt und zentrifugiert. Das Lösungsmittel wird im Vaku¬ um eingeengt und der Rückstand gefriergetrocknet. Das erhaltene Lyophilisat (89.9 g) wird in Me¬ thanol aufgenommen, abfiltriert, eingeengt und an Kieselgel (Methode 21) getrennt. Beispiel IA wird danach per Gelfϊltration (Sephadex LH-20, 5 x 68 cm, Wasser/Acetonitril 9:1 (mit 0.05% Trifluoressigsäure), Fluss: 2.7 mL/min, Fraktionsgröße 13.5 mL) zur Reinsubstanz aufgereinigt.This process provides 2250 mg of Example IA. The sediment is taken up in 4 L of acetone / water 4: 1, mixed with 2 kg of Celite, adjusted to pH = 6 with trifluoroacetic acid, stirred and centrifuged. The solvent is concentrated in vacuo and the residue is freeze-dried. The lyophilizate obtained (89.9 g) is taken up in methanol, filtered off, concentrated and separated on silica gel (Method 21). Example IA is then purified by gel filtration (Sephadex LH-20, 5 × 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.
Dieser Prozess liefert 447 mg Beispiel IA.This process provides 447 mg of Example IA.
HPLC (Methode 18): R1 = 6.19 minHPLC (Method 18): R 1 = 6.19 min
MS (ESIpos): m/z = 1277 (M+H)+ MS (ESIpos): m / z = 1277 (M + H) +
1H-NMR (500.13 MHz, 4-DMSO): δ = 0.75 (d, 3H), 0.78 (d, 6H), 0.80 (t, 3H), 0.82 (d, 3H), 0.90 (d, 3H), 0.91 (d, 3H)5 0.92 (d, 3H), 0.95 (d, 3H), 0.96 (d, 3H), 1.05 (m, IH), 1.19 (d, 3H), 1.25 (m, 2H), 1.50 (m, 4H), 1.51 (m, 2H), 1.55 (m, IH), 1.61 (m, IH), 1.65 (m, IH), 1.84 (m, IH), 1.85 (m, IH), 1.86 (m, IH), 1.89 (m, IH), 1.95 (m, IH), 2.75 (m, 2H), 3.40 (m, IH), 3.52 (m, 2H), 3.53 (dd, IH), 3.64 (m, 2H), 3.66 (m, IH), 3.68 (dd, IH), 3.73 (m, 2H), 4.00 (dd, IH), 4.02 (br., IH), 4.13 (br., IH), 4.32 (dd, IH), 4.39 (t, IH), 4.55 (m, IH), 4.75 (dd, IH), 5.19 (t, IH), 5.29 (d, IH), 5.30 (br., IH)5 5.58 (m, 2H), 6.68 (m, 3H), 6.89 (d, IH), 6.93 (m, 3H), 6.94 (br., IH), 6.98 (d, IH), 7.12 (br., IH)5 7.20 (br., 2H), 7.23 (m, 2H), 7.42 (m, 2H), 7.54 (d, IH), 7.58 (d, IH), 8.32 (br., IH), 9.18 (br., IH)3 9.20 (m, 2H), 9.50 (br., IH). 1 H-NMR (500.13 MHz, 4-DMSO): δ = 0.75 (d, 3H), 0.78 (d, 6H), 0.80 (t, 3H), 0.82 (d, 3H), 0.90 (d, 3H), 0.91 (d, 3H) 5 0.92 (d, 3H), 0.95 (d, 3H), 0.96 (d, 3H), 1.05 (m, IH), 1.19 (d, 3H), 1.25 (m, 2H), 1.50 (m, 4H), 1.51 (m, 2H), 1.55 (m, IH), 1.61 (m, IH), 1.65 (m, IH), 1.84 (m, IH), 1.85 (m, IH), 1.86 ( m, IH), 1.89 (m, IH), 1.95 (m, IH), 2.75 (m, 2H), 3.40 (m, IH), 3.52 (m, 2H), 3.53 (dd, IH), 3.64 (m , 2H), 3.66 (m, IH), 3.68 (dd, IH), 3.73 (m, 2H), 4.00 (dd, IH), 4.02 (br, IH), 4.13 (br, IH), 4.32 ( dd, IH), 4.39 (t, IH), 4.55 (m, IH), 4.75 (dd, IH), 5.19 (t, IH), 5.29 (d, IH), 5.30 (br., IH) 5 5.58 ( m, 2H), 6.68 (m, 3H), 6.89 (d, IH), 6.93 (m, 3H), 6.94 (br, IH), 6.98 (d, IH), 7.12 (br, IH) 5 7.20 (br., 2H), 7.23 (m, 2H), 7.42 (m, 2H), 7.54 (d, IH), 7.58 (d, IH), 8.32 (br., IH), 9.18 (br., IH) 3 9.20 (m, 2H), 9.50 (br., IH).
13C-NMR (125.77 MHz5 ^-DMSO): δ = 10.3, 15.3, 19.0, 19.2, 19.6, 20.0, 20.9, 22.0, 22.4, 23.0, 23.2, 24.3, 24.4, 25.0, 25.4, 26.0, 27.8, 30.9, 35.4, 39.5, 40.8, 40.9, 41.6, 44.1, 51.5, 52.7, 55.9, 56.2, 56.4, 57.9, 58.8, 60.2, 61.1, 62.6, 70.1, 71.6, 71.7, 75.5, 128.1, 128.6, 136.7, 156.8, 168.2, 170.1, 170.4, 171.2, 171.5, 171.9, 172.2, 172.4, 173.7. 13 C-NMR (125.77 MHz 5- DMSO): δ = 10.3, 15.3, 19.0, 19.2, 19.6, 20.0, 20.9, 22.0, 22.4, 23.0, 23.2, 24.3, 24.4, 25.0, 25.4, 26.0, 27.8, 30.9 , 35.4, 39.5, 40.8, 40.9, 41.6, 44.1, 51.5, 52.7, 55.9, 56.2, 56.4, 57.9, 58.8, 60.2, 61.1, 62.6, 70.1, 71.6, 71.7, 75.5, 128.1, 128.6, 136.7, 156.8, 168.2 , 170.1, 170.4, 171.2, 171.5, 171.9, 172.2, 172.4, 173.7.
Die Zuordnung der Signale erfolgte nach der in der Literatur beschriebenen Zuordnung (T. Kato, H. Hinoo, Y. Terui, J. Antibiot, 1988, 61, 719-725).The assignment of the signals was carried out according to the assignment described in the literature (T. Kato, H. Hinoo, Y. Terui, J. Antibiot, 1988, 61, 719-725).
Beispiel 2AExample 2A
Des(l-D-leucyl-2-L-leucyl)-lysobactin-bis-trifluoracetat (Edman20 -Abbauprodukt) Des (lD-leucyl-2-L-leucyl) -lysobactin bis-trifluoroacetate (Edman 20 Decomposition Product)
Lysobactin-bistrifluoracetat (60.0 g, 39.88 mmol) wird unter Argonatmosphäre in Pyridin (840 WLL) gelöst. Dann wird Phenylisothiocyanat (32.35 g, 239.28 mmol, 6 Äquivalente) zugege¬ ben und die Reaktionsmischung bei 37°C für 7 h gerührt. Anschließend wird das Lösungsmittel am Rotationsverdampfer bei 4O0C Badtemperatur abdestilliert. Der Rückstand wird mit Methyl-tert- buty lether (1400 mL) versetzt und 30 min kräftig gerührt. Dann wird über eine Glasfritte (Poren¬ weite 3, 13 cm Durchmesser) abgesaugt. Das Zwischenprodukt (EdmanOJ-Abbauprodukt) wird in einer Rohausbeute von 72 g isoliert und ohne Aufarbeitung weiter umgesetzt.Lysobactin bistrifluoroacetate (60.0 g, 39.88 mmol) is dissolved under argon atmosphere in pyridine (840 WLL). Then phenylisothiocyanate (32.35 g, 239.28 mmol, 6 equivalents) is added ben and the reaction mixture is stirred at 37 ° C for 7 h. Subsequently, the solvent is distilled off on a rotary evaporator at 4O 0 C bath temperature. The residue is treated with methyl tert-butyl ether (1400 ml) and stirred vigorously for 30 min. Then it is filtered off with suction through a glass frit (pore width 3, 13 cm diameter). The intermediate product (Edman OJ degradation product) is isolated in a crude yield of 72 g and further reacted without workup.
Dazu wird das Rohprodukt unter Argonatmosphäre in Trifluoressigsäure (1026 mL) gelöst und 30 min bei RT gerührt. Dann wird die Lösung am Rotationsverdampfer unter Vakuum bei 2O0C Badtemperatur eingeengt. Der Rückstand wird in Methyl-tert-butylether (1400 mL) aufgenommen und so lange kräftig gerührt, bis ein pulvriger amorpher Feststoff entstanden ist. Dieser wird mit Vakuum über eine Fritte (Porenweite 3, 18 cm Durchmesser) abfiltriert. Der Feststoff wird nun mit Diethy lether (1400 mL) gerührt und erneut abfiltriert. Dieselbe Prozedur wird mit 2 Portionen Dichlormethan (je 900 mL) wiederholt. Das Rohprodukt wird im Vakuum getrocknet. Man erhält 58 g rohes Des(l-D-leucyl)-lysobactin-bis-trifluoracetat (Edman; o-Abbauprodukt).For this, the crude product is dissolved under argon atmosphere in trifluoroacetic acid (1026 mL) and stirred at RT for 30 min. Then the solution is concentrated on a rotary evaporator under vacuum at 2O 0 C bath temperature. The residue is taken up in methyl tert-butyl ether (1400 ml) and stirred vigorously until a powdery amorphous solid is formed. This is filtered off with a vacuum over a frit (pore width 3, 18 cm in diameter). The solid is then stirred with diethyl ether (1400 ml) and filtered off again. The same procedure is repeated with 2 portions of dichloromethane (900 mL each). The crude product is dried in vacuo. This gives 58 g of crude des (lD-leucyl) -lysobactin-bis-trifluoroacetate (Edman , o degradation product).
Das Rohprodukt wird ohne weitere Aufreinigung unter Argonatmosphäre in Pyridin (108O mL) gelöst. Dann wird Phenylisothiocyanat (107 g, 0.80 mol, 20 Äquivalente) zugegeben und die Reak¬ tionsmischung wird bei 37 bis 4O0C für 7 h gerührt. Anschließend wird das Lösungsmittel am Ro- tationsverdampfer bei 400C Badtemperatur abdestilliert. Der Rückstand wird mit Methyl-tert- butylether (140O mL) versetzt und kräftig gerührt. Dann wird über eine Glasfritte (Porenweite 3, 13 cm Durchmesser) abgesaugt. Das Zwischenprodukt (EdmanΛ5- Abbauprodukt) wird in einer Rohausbeute von 65 g isoliert und nach dem Trocknen im Ölpumpenvakuum direkt unter Argon¬ atmosphäre in Trifluoressigsäure (1240 mL) gelöst und 30 min bei RT gerührt. Dann wird die Lö¬ sung am Rotationsverdampfer unter Vakuum bei 200C Badtemperatur eingeengt. Der Rückstand wird in Methyl-fert-butylether (1400 mL) aufgenommen und so lange kräftig gerührt, bis ein pulv¬ riger amorpher Feststoff entstanden ist. Dieser wird mit Vakuum über eine Fritte (Porenweite 3, 18 cm Durchmesser) abfiltriert. Der Feststoff wird nun zunächst mit Diethy lether (140O mL), dann mit Dichlormethan (1400 mL) gerührt und jeweils abfiltriert. Man erhält 55 g des rohen Produktes. Dieses wird durch präparative HPLC (Methode 14) gereinigt. Man erhält 28.55 g (56% d. Th.) der Titelverbindung.The crude product is dissolved without further purification under argon atmosphere in pyridine (108O mL). Then phenyl isothiocyanate is (107 g, 0.80 mol, 20 equivalents) was added and the mixture Reak¬ tion is stirred at 37 to 4O 0 C for. 7 The solvent is then distilled off tationsverdampfer at 40 0 C. bath temperature at ro. The residue is treated with methyl tert-butyl ether (140O mL) and stirred vigorously. Then a glass frit (pore size 3, 13 cm diameter) is sucked off. The intermediate product ( Edman® 5 -degradation product) is isolated in a crude yield of 65 g and, after drying in an oil pump vacuum, dissolved directly under argon atmosphere in trifluoroacetic acid (1240 mL) and stirred at RT for 30 min. Then, the solution is Lö¬ on a rotary evaporator under vacuum at 20 0 C. bath temperature concentrated. The residue is taken up in methyl tert-butyl ether (1400 ml) and stirred vigorously until a pulverulent amorphous solid is formed. This is filtered off with a vacuum over a frit (pore width 3, 18 cm in diameter). The solid is then stirred first with diethyl ether (140O mL), then with dichloromethane (1400 mL) and filtered off in each case. 55 g of the crude product are obtained. This is purified by preparative HPLC (Method 14). This gives 28.55 g (56% of theory) of the title compound.
HPLC/UV-Vis (Methode 23): R4 = 4.71 min,HPLC / UV-Vis (method 23): R 4 = 4.71 min,
X1113x (qualitativ) = 220 nm (s), 255-270 (w).X 1113x (qualitative) = 220 nm (s), 255-270 (w).
LC-MS (Methode 22): R4 = 1.65 min;LC-MS (Method 22): R 4 = 1.65 min;
MS (ESIpos.): m/z (%) = 526 (100) [M + 2H]2+, 1051 (15) [M + H]+.MS (ESI pos.): M / z (%) = 526 (100) [M + 2H] 2+ , 1051 (15) [M + H] + .
Beispiel 3AExample 3A
(22)-2- { [(Benzyloxy)carbony 1] amino} -3 -(6-trifluormethyl-pyridin-3 -yl)-acry lsäuremethy lester(22) -2- {[(Benzyloxy) carbonyl] 1] amino} -3- (6-trifluoromethyl-pyridin-3-yl) -acryic acid methyl ester
6-Trifluormethyl-pyridin-3-carbaldehyd (4.85 g, 27.70 mmol) und Methyl- {[(benzyloxy)carbony I]- amino}(dimethoxyphosphoryl)acetat (9.17 g, 27.70 mmol, 1.0 Äquivalente) werden in THF (7O mL) gelöst und auf -700C gekühlt. Bei -700C wird N,N,N,N-Tetramethylguanidin (6.38 g, 55.39 mmol, 6.95 mL, 2.0 Äquivalente) langsam zugetropft und dann 4 h bei -700C, anschließend 12 h bei RT gerührt. Das Reaktionsgemisch wird eingeengt, dann wird mit Ethylacetat (2 x 100 mL) gegen Wasser ausgeschüttelt, die vereinigten organischen Phasen werden mit gesättigter Kochsalzlösung gewaschen und über Νatriumsulfat getrocknet. Nach dem Einengen im Vakuum wird das Rohprodukt chromatographiert (Kieselgel, Eluent: Toluol, dann Toluol/Ethylacetat 10:1). Es werden 6.93 g (66% d. Th.) der Titelverbindung erhalten.6-Trifluoromethylpyridine-3-carbaldehyde (4.85 g, 27.70 mmol) and methyl {[(benzyloxy) carbonyl] amino] (dimethoxyphosphoryl) acetate (9.17 g, 27.70 mmol, 1.0 equiv.) Are dissolved in THF (70 mL ) and cooled to -70 0 C. At -70 0 C N, N, N, N-tetramethylguanidine (6:38 g, 55.39 mmol, 6.95 mL, 2.0 equivalents) was added dropwise slowly and then stirred for 4 h then at -70 0 C for 12 h at RT. The reaction mixture is concentrated, then shaken out with ethyl acetate (2 × 100 ml) against water, the combined organic phases are washed with saturated common salt solution and dried over sodium sulfate. After concentration in vacuo the crude product is chromatographed (silica gel, eluent: toluene, then toluene / ethyl acetate 10: 1). There are obtained 6.93 g (66% of theory) of the title compound.
HPLC/UV-Vis (Methode 8): R4 = 4.60 min.HPLC / UV-Vis (method 8): R 4 = 4.60 min.
HPLC/UV-Vis (Methode 9): R1 = 4.54 min.HPLC / UV-Vis (Method 9): R 1 = 4.54 min.
1H-NMR (400 MHz, cfc-DMSO): δ = 3.74 (s, 3H, OMe), 5.10 (s, 2H5 CH2), 7.27 (s, IH, PyrH), 7.33-7.38 (m, 5H, ArH), 7.94 (d, J= 8.5 Hz, IH, PyrH), 8.27 (d, J= 8.5 Hz, IH, PyrH), 8.93 (s, IH, ß-CH), 9.51 (S5 IH5 NH). 1 H-NMR (400 MHz, cfc-DMSO): δ = 3.74 (s, 3H, OMe), 5.10 (s, 2H 5 CH 2 ), 7.27 (s, IH, PyrH), 7.33-7.38 (m, 5H , ArH), 7.94 (d, J = 8.5 Hz, IH, PyrH), 8.27 (d, J = 8.5 Hz, IH, PyrH), 8.93 (s, IH, β-CH), 9.51 (S 5 IH 5 NH ).
LC-MS (Methode 7): Rt = 2.44 min; MS (ESIpos.): m/z (%) = 381 (100) [M + H]+; MS (ESIneg.): m/z (%) = 379 (100) [M - H]".LC-MS (Method 7): R t = 2.44 min; MS (ESI pos.): M / z (%) = 381 (100) [M + H] + ; MS (ES Ineg.): M / z (%) = 379 (100) [M-H] " .
HR-TOF-MS (Methode 1): C18H16N2O4F3 [M + H]+ ber. 381.1062, gef. 381.1065.HR-TOF-MS (method 1). C 18 H 16 N 2 O 4 F 3 [M + H] + calcd 381.1062, obs. 381.1065.
Beispiel 4AExample 4A
N-[(Benzyloxy)carbonyl]-3-(6-trifluormethyl-pyridin-3-yl)-L-alanin-methylesterN - [(benzyloxy) carbonyl] -3- (6-trifluoromethyl-pyridin-3-yl) -L-alanine-methylester
Die Verbindung aus Beispiel 3A (10.15 g, 26.69 mmol) wird in Methanol p.a. (10O mL) gelöst. Mit einer Kanüle wird etwa 5 min Argon hindurchgeleitet, dann wird (+)-l,2-Bis-[(2>Sr,5)S)diethyl- phospholano]benzen(cyclooctadien)rhodium(I)-Triflat (289 mg, 400 μmoL 0.015 Äquivalente) zugegeben. Es wird über 12 h bei 4 bar Wasserstoffdruck und RT hydriert. Dann wird es über Kie¬ selgur filtriert (Methanol) und das Eluat eingeengt. Das Rohprodukt wird chromatographiert (Kie¬ selgel, Eluent: Toluol/Ethylacetat 5:1). Es werden 9.9 g (97% d. Th.) der Titelverbindung erhalten.The compound of Example 3A (10.15 g, 26.69 mmol) is dissolved in methanol (10 mL). With a cannula, argon is passed through for about 5 minutes, then (+) - l, 2-bis - [(2> S r , 5 ) S) diethylphospholano] benzene (cyclooctadiene) rhodium (I) triflate (289 mg , 400 μmol 0.015 equivalent). It is hydrogenated for 12 h at 4 bar hydrogen pressure and RT. It is then filtered through Kie selgur (methanol) and the eluate is concentrated. The crude product is chromatographed (Kie selgel, eluent: toluene / ethyl acetate 5: 1). There are obtained 9.9 g (97% of theory) of the title compound.
[α]20 Na = -24° (c = 0.093 in Methanol).[α] 20 Na = -24 ° (c = 0.093 in methanol).
HPLC/UV-Vis (Methode 8): R, = 4.50 min.HPLC / UV-Vis (method 8): R, = 4.50 min.
HPLC/UV-Vis (Methode 9): R, = 4.49 min. 1H-NMR (400 MHz, J6-DMSO): δ = 2.99 (dd, J= 3.5, 11.0 Hz, IH, ß-CH), 3.22 (dd, J= 3.5, 11.0 Hz, IH, ß-CH), 3.66 (s, 3H, OMe), 4.40 (m, IH, α-CH), 4.97 (s, 2H, CH2), 7.23 (m, 2H), 7.29-7.33 (m, 3H), 7.83 (d, J= 6.5 Hz, IH), 7.93-7.98 (m, 2H), 8.65 (s, IH, NH).HPLC / UV-Vis (Method 9): R, = 4.49 min. 1 H-NMR (400 MHz, J 6 -DMSO): δ = 2.99 (dd, J = 3.5, 11.0 Hz, IH, β-CH), 3.22 (dd, J = 3.5, 11.0 Hz, IH, β-CH ), 3.66 (s, 3H, OMe), 4.40 (m, IH, α-CH), 4.97 (s, 2H, CH 2 ), 7.23 (m, 2H), 7.29-7.33 (m, 3H), 7.83 ( d, J = 6.5 Hz, IH), 7.93-7.98 (m, 2H), 8.65 (s, IH, NH).
LC-MS (Methode 7): R1 = 2.40 min; MS (ESIpos.): m/z (%) = 383 (100) [M + H]+; MS (ESIneg.): m/z (%) = 273 (100), 381 (50) [M - H]".LC-MS (method 7): R 1 = 2.40 min; MS (ESI pos.): M / z (%) = 383 (100) [M + H] + ; MS (ES Ineg.): M / z (%) = 273 (100), 381 (50) [M-H] " .
HR-TOF-MS (Methode 1): C18Hi8N2O4F3 [M + H]+ ber. 383.1219, gef. 383.1223.HR-TOF-MS (Method 1): C 18 Hi 8 N 2 O 4 F 3 [M + H] + calc. 383.1219, eq. 383.1223.
Beispiel 5AExample 5A
3-(6-Trifluormethyl-pyridin-3-yl)-L-alanin-methylester3- (6-trifluoromethyl-pyridin-3-yl) -L-alanine-methylester
Die Verbindung aus Beispiel 4A (9.90 g, 25.89 mmol) wird in Methanol (100 mL) gelöst. Mit einer Kanüle wird etwa 5 min Argon hindurchgeleitet, dann wird Pd auf Kohle (10%, 990 mg) zugegeben. Es wird über 12 h bei 4 bar Wasserstoffdruck und RT hydriert. Dann wird über Kiesel¬ gur filtriert, eingeengt und im Ölpumpenvakuum getrocknet. Ausbeute: 5.8 g (90% d. Th.) der Titelverbindung.The compound from Example 4A (9.90 g, 25.89 mmol) is dissolved in methanol (100 mL). With a cannula, argon is passed for about 5 minutes, then Pd on charcoal (10%, 990 mg) is added. It is hydrogenated for 12 h at 4 bar hydrogen pressure and RT. It is then filtered through kieselguhr, concentrated and dried in an oil pump vacuum. Yield: 5.8 g (90% of theory) of the title compound.
[α]199 Na = +3° (c = 0.186 in Methanol).[α] 199 Na = + 3 ° (c = 0.186 in methanol).
HPLC/UV-Vis (Methode 8): R4 = 3.34 min.HPLC / UV-Vis (Method 8): R 4 = 3.34 min.
HPLC/UV-Vis (Methode 9): Rt = 3.22 min.HPLC / UV-Vis (Method 9): R t = 3.22 min.
IR vmax (NaCl, cm"1): 3415, 1734, 1339, 1136, 1087.IR v max (NaCl, cm -1 ): 3415, 1734, 1339, 1136, 1087.
1H-NMR (500 MHz, J6-DMSO): δ = 2.85 (dd, J= 5.5, 13.5 Hz, IH, ß-CH), 3.01 (dd, J= 5.5, 13.5 Hz, IH, ß-CH), 3.61 (s, 3H, OMe), 3.63-3.69 (m, IH, α-CH), 7.82 (d, J= 7.5 Hz, IH), 7.93 (d, J= 7.5 Hz, IH), 8.61 (s, IH). 1 H NMR (500 MHz, J 6 -DMSO): δ = 2.85 (dd, J = 5.5, 13.5 Hz, IH, β-CH), 3.01 (dd, J = 5.5, 13.5 Hz, IH, β-CH ), 3.61 (s, 3H, OMe), 3.63-3.69 (m, IH, α-CH), 7.82 (d, J = 7.5 Hz, IH), 7.93 (d, J = 7.5 Hz, IH), 8.61 ( s, IH).
LC-MS (Methode 7): Rt = 1.74 min; MS (ESIpos.): m/z (%) = 249 (100) [M + H]+.LC-MS (Method 7): R t = 1.74 min; MS (ESI pos.): M / z (%) = 249 (100) [M + H] + .
HR-TOF-MS (Methode 1): Ci2Hi5N3O2F3 [M + CH3CN + H]+ ber. 290.1116, gef. 290.1122. Beispiel 6AHR-TOF-MS (Method 1): Ci 2 Hi 5 N 3 O 2 F 3 [M + CH 3 CN + H] + calc. 290.1116, m.p. 290.1122. Example 6A
N-(/ert-Butoxycarbonyl)-3-(^er^-butyl)-D-alanyl-3-(6-trifluormethyl-pyridin-3-yl)-L-alanin- methylesterN - (tert-butoxycarbonyl) -3- (tert-butyl) -D-alanyl-3- (6-trifluoromethylpyridin-3-yl) -L-alanine methyl ester
Zu einer Lösung der Verbindung aus Beispiel 5A (6.34 g, 25.54 mmol) und N-(tert- Butoxycarbonyl)-3-fer?-butyl-D-alanin (6.27 g, 25.54 mmol, 1.0 Äquivalente) in trockenem DMF (24O mL) werden bei -3O0C langsam N-Methylmorpholin (12.92 g, 127.72 mmol, 14.04 mL, 5 Äquivalente) und HATU (9.71 g, 25.54 mmol, 1 Äquivalente) gegeben. Das Reaktionsgemisch erwärmt sich langsam (ca. 3 h) auf RT, wobei vollständiger Umsatz mittels HPLC (Methode 9) be- obachtet wird. Kaliumdihydrogenphosphat (34.76 g, 255.44 mmol, 10 Äquivalente) wird zugege¬ ben, und das Reaktionsgemisch wird 20 min gerührt, anschließend wird es filtriert, mit Ethylacetat verdünnt und mit gesättigter wässriger Νatriumhydrogencarbonatlösung (10 mL) gewaschen. Die organische Phase wird mittels Νatriumsulfat getrocknet, filtriert und eingeengt. Das Rohprodukt wird mittels Flashchromatographie (Kieselgel, Gradient Cyclohexan/Ethylacetat 10:1 bis 2:1) auf- gereinigt, wobei man 9.74 g (73% d. Th.) der Titelverbindung erhält.To a solution of the compound of Example 5A (6.34 g, 25.54 mmol) and N- (tert-butoxycarbonyl) -3-fer? -Butyl-D-alanine (6.27 g, 25.54 mmol, 1.0 equiv.) In dry DMF (24 mL ) are slowly added at -3O 0 C N-methylmorpholine (12.92 g, 127.72 mmol, 14.04 mL, 5 equivalents) and HATU (9.71 g, 25.54 mmol, 1 eq). The reaction mixture warmed slowly to RT (about 3 h), with complete conversion being observed by means of HPLC (Method 9). Potassium dihydrogen phosphate (34.76 g, 255.44 mmol, 10 equiv.) Is added, and the reaction mixture is stirred for 20 min, then it is filtered, diluted with ethyl acetate and washed with saturated aqueous sodium bicarbonate solution (10 mL). The organic phase is dried by means of sodium sulfate, filtered and concentrated. The crude product is purified by flash chromatography (silica gel, gradient cyclohexane / ethyl acetate 10: 1 to 2: 1) to give 9.74 g (73% of theory) of the title compound.
[α]199Νa = +7.0° (c = 0.044 in Methanol).[α] 199 Νa = + 7.0 ° (c = 0.044 in methanol).
HPLC/UV-Vis (Methode 8): Rt = 4.89 min.HPLC / UV-Vis (Method 8): R t = 4.89 min.
HPLC/UV-Vis (Methode 9): R, = 4.75 min.HPLC / UV-Vis (Method 9): R, = 4.75 min.
IR vmax (NaCl, cm"1): 2959, 1742, 1655, 1520, 1336, 1160, 1136, 1087, 1050, 1027.IR v max (NaCl, cm -1 ): 2959, 1742, 1655, 1520, 1336, 1160, 1136, 1087, 1050, 1027.
1H-NMR (500 MHz, ^6-DMSO): δ = 0.74 (s, 9H, tBu), 0.97-1.00 (m, IH, ß-CB2), 1.20-1.25 (m, lH, ;β-CH2), 1-35 (s, 9H, OtBu), 2.99-3.05 (m, lH, ß-CH2), 3.23-3.26 (m, lH, yß-CH2), 3.66 (s, 3H, OMe), 3.94 (m, IH, α-CH), 4.60 (m, IH, α-CH), 6.82 (d, J= 8.5 Hz, IH, NH), 7.78 (d, J= 8.0 Hz, IH3 PyrH), 7.94 (d, J= 8.0 Hz, IH, PyrH), 8.34 (d, J= 8.5 Hz, IH, NH), 8.64 (s, IH5 PyrH). 1 H NMR (500 MHz, ^ 6 -DMSO): δ = 0.74 (s, 9H, tBu), 0.97-1.00 (m, IH, β-CB2), 1.20-1.25 (m, 1H, β-CH2 ), 1-35 (s, 9H, OtBu), 2.99-3.05 (m, 1H, β-CH2), 3.23-3.26 (m, 1H, yβ-CH2), 3.66 (s, 3H, OMe), 3.94 (m, IH, α-CH), 4.60 (m, IH, α-CH), 6.82 (d, J = 8.5 Hz, IH, NH), 7.78 (d, J = 8.0 Hz, IH 3 PyrH), 7.94 (d, J = 8.0 Hz, IH, PyrH), 8.34 (d, J = 8.5 Hz, IH, NH), 8.64 (s, IH 5 PyrH).
LC-MS (Methode 7): R4 = 2.67 min; MS (ESIpos.): m/z (%) = 476 (100), [M + H]+; MS (ESIneg.): m/z (%) = 400 (80), 474 (40) [M - H]" LC-MS (method 7): R 4 = 2.67 min; MS (ESI pos.): M / z (%) = 476 (100), [M + H] + ; MS (ES Ineg.): M / z (%) = 400 (80), 474 (40) [M-H] "
HR-TOF-MS (Methode 1): C22H33N3O5F3 [M + H]+ ber. 476.2372, gef. 476.2364.HR-TOF-MS (method 1). C 22 H 33 N 3 O 5 F 3 [M + H] + calcd 476.2372, obs. 476.2364.
Beispiel 7AExample 7A
N-ter?-Butoxycarbonyl-3-fer^-butyl-D-alanyl-3-(6-trifluormethyl-pyridin-3-yl)-L-alaninN-ter? -Butoxycarbonyl-3-fer ^ -butyl-D-alanyl-3- (6-trifluoromethyl-pyridin-3-yl) -L-alanine
Zu einer Lösung der Verbindung aus Beispiel 6A (9.2 g, 1.0 Äquivalente, 19.35 mmol) in THF (360 mL) und Wasser (100 mL) wird bei -2O0C eine Lösung von Lithiumhydroxid-hydrat (1.16 g, 2.5 Äquivalente, 48.37 mmol) in Wasser (20 mL) gegeben. Das Reaktionsgemisch erwärmt sich (ca. 1.5 h) auf +15°C, wobei vollständiger Umsatz mittels HPLC (Methode 9) beobachtet wird. Zur Aufarbeitung wird Kaliumdihydrogenphosphat (26.33 g, 10 Äquivalente, 193.5 mmol) zugegeben (ca. pH 7). Das Reaktionsgemisch wird filtriert und im Vakuum aufkonzentriert. Das Rohprodukt wird mittels Gelchromatographie (Methode 10, Laufmittel Methanol/Aceton 4:1) aufgereinigt, wo¬ bei man 4.72 g (53% d. Th.) Produkt erhält.To a solution of the compound from Example 6A (9.2 g, 1.0 equivalents, 19.35 mmol) in THF (360 mL) and water (100 mL) at -2O 0 C is a solution of lithium hydroxide hydrate (1.16 g, 2.5 equivalents, 48.37 mmol) in water (20 mL). The reaction mixture is warmed (about 1.5 h) to + 15 ° C, with complete conversion by HPLC (Method 9) is observed. For workup, potassium dihydrogen phosphate (26.33 g, 10 equivalents, 193.5 mmol) is added (about pH 7). The reaction mixture is filtered and concentrated in vacuo. The crude product is purified by gel chromatography (Method 10, eluent methanol / acetone 4: 1), where 4.72 g (53% of theory) product.
[Ot]20 Na = +51.3° (c = 0.402 in Methanol).[Ot] 20 Na = + 51.3 ° (c = 0.402 in methanol).
HPLC/UV-Vis (Methode 8): R4 = 4.63 min.HPLC / UV-Vis (method 8): R 4 = 4.63 min.
HPLC/UV-Vis (Methode 9): Rt = 4.55 min.HPLC / UV-Vis (Method 9): R t = 4.55 min.
IR vmax (NaCl, cm"1): 3305, 2959, 1663, 1519, 1336, 1173, 1134, 1086. 1H-NMR (500 MHz, J15-DMSO): δ - 0.77 (s, 9H, tBu), 1.06-1.13 (m, IH, ß-CH2), 1.23-1.26 (m, IH, ß-CH2), 1.34 (s, 9H, OtBu), 3.01 (tapp, J= 11.0 Hz, IH, ß-CB2), 3.23 (br d, J = 11.0 Hz, IH, ß-CW), 3.94 (t, J= 8.0 Hz, IH, α-CH), 4.42 (br s, IH, α-CH), 6.90 (d, J= 8.5 Hz, IH, NH), 7.74 (d, J= 7.5 Hz, IH, PyrH), 7.86 (d, J= 7.5 Hz, IH, PyrH), 8.00 (br s, IH, NH), 8.56 (s, IH, PyrH).IR v max (NaCl, cm -1 ): 3305, 2959, 1663, 1519, 1336, 1173, 1134, 1086. 1 H-NMR (500 MHz, J 15 -DMSO): δ-0.77 (s, 9H, tBu), 1.06-1.13 (m, IH, β-CH 2), 1.23-1.26 (m, IH, β-CH 2) , 1.34 (s, 9H, OtBu), 3.01 (t app , J = 11.0 Hz, IH, ß-CB2), 3.23 (br d, J = 11.0 Hz, IH, ß-CW), 3.94 (t, J = 8.0 Hz, IH, α-CH), 4.42 (brs, IH, α-CH), 6.90 (d, J = 8.5 Hz, IH, NH), 7.74 (d, J = 7.5 Hz, IH, PyrH), 7.86 (d, J = 7.5Hz, IH, PyrH), 8.00 (brs, IH, NH), 8.56 (s, IH, PyrH).
LC-MS (Methode 7): Rt = 2.42 min; MS (ESIpos.): m/z (%) = 406 (100), 462 (85) [M + H]+; MS (ESIneg.): m/z (%) = 460 (100) [M - H]".LC-MS (Method 7): R t = 2.42 min; MS (ESI pos.): M / z (%) = 406 (100), 462 (85) [M + H] + ; MS (ES Ineg.): M / z (%) = 460 (100) [M-H] " .
HR-TOF-MS (Methode 1): C21H3IN3O5F3 [M + H]+ ber. 462.2216, gef. 462.2203.HR-TOF-MS (method 1). C 21 H 3I N 3 O 5 F 3 [M + H] + calcd 462.2216, obs. 462.2203.
Beispiel 8AExample 8A
N-ter^-Butoxycarbonyl-3-fer^-butyl-D-alanyl-3-(6-trifluormethyl-pyridin-3-yl)-L-alanyl- des( 1 -D-leucyl-2-L-leucy l)lysobactin-trifluoracetatN-tert -butoxycarbonyl-3-fer-butyl-D-alanyl-3- (6-trifluoromethyl-pyridin-3-yl) -L-alanyl-des (1-D-leucyl-2-L-leucyl ) lysobactin trifluoroacetate
Zu einer Lösung der Verbindung aus Beispiel 2A (7.00 g, 1.0 Äquivalente, 5.48 mmol) und Bei¬ spiel 7A (3.03 g, 1.2 Äquivalente, 6.57 mmol) in trockenem DMF (119 mL) werden bei -3O0C langsam N-Methylmorpholin (2.77 g, 3.01 mL, 5 Äquivalente, 27.38 mmol) und HATU (4.37 g, 2.1 Äquivalente, 11.50 mmol) gegeben. Das Reaktionsgemisch erwärmt sich langsam (ca. 1 h) auf RT, wobei vollständiger Umsatz mittels HPLC/UV-Vis (Methode 9) beobachtet wird. Die Reakti¬ on wird mit Kaliumdihydrogenphosphat (7.45 g, 10.0 Äquivalente, 54.76 mmol) gequencht. Das Reaktionsgemisch wird mittels Gelchromatographie (Methode 10, Laufmittel Methanol/Aceton 4:1) aufgereinigt, wobei man 12.63 g (quant.) Produkt erhält. HPLC/UV-Vis (Methode 8): R4 = 4.78 min.To a solution of the compound from Example 2A (7.00 g, 1.0 equivalents, 5.48 mmol) and Example 7A (3.03 g, 1.2 equivalents, 6.57 mmol) in dry DMF (119 mL) at -3O 0 C slowly N-methylmorpholine (2.77 g, 3.01 mL, 5 eq, 27.38 mmol) and HATU (4.37 g, 2.1 eq, 11.50 mmol). The reaction mixture warmed slowly to RT (about 1 h), with complete conversion being observed by means of HPLC / UV-Vis (Method 9). The reaction is quenched with potassium dihydrogen phosphate (7.45 g, 10.0 equivalents, 54.76 mmol). The reaction mixture is purified by gel chromatography (method 10, eluent methanol / acetone 4: 1) to give 12.63 g (quant.) Product. HPLC / UV-Vis (Method 8): R 4 = 4.78 min.
HPLC/UV-Vis (Methode 9): R1 = 4.35 min.HPLC / UV-Vis (Method 9): R 1 = 4.35 min.
LC-MS (Methode 7): Rt = 2.28 min; MS (ESIpos.): m/z (%) = 697 (100) [M + 2H]2+, 1493 (15) [M + H]+; MS (ESIneg.): m/z (%) = 745 (100) [M - 2H]2", 1491 (5) [M - H]".LC-MS (method 7): R t = 2.28 min; MS (ESI pos.): M / z (%) = 697 (100) [M + 2H] 2+ , 1493 (15) [M + H] + ; MS (ES Ineg.): M / z (%) = 745 (100) [M-2H] 2 " , 1491 (5) [M-H] " .
HR-TOF-MS (Methode 1): C67H]04N16O19F3 [M + H]+ ber. 1493.7616, gef. 1493.7594.HR-TOF-MS (Method 1): C 67 H] 04 N 16 O 19 F 3 [M + H] + calc. 1493.7616, m.p. 1493.7594.
Beispiel 9A und Beispiel IQAExample 9A and Example IQA
(2,S)-N-(rerr-Butoxycarbonyl)-3-(trimethylsilyl)alanin und (2i?)-N-(fert-Butoxycarbonyl)-3- (trimethylsilyl)alanin(2, S) -N- (rerr-butoxycarbonyl) -3- (trimethylsilyl) alanine and (2i?) - N- (tert-butoxycarbonyl) -3- (trimethylsilyl) alanine
Die Synthese erfolgt nach M. Merget, K. Günther, M. Bernd, E. Günther, R. Tacke, J. Organomet. Chem. 2001 628, 183-194. Die Trennung der Enantiomere erfolgt durch präparative HPLC an chiraler Phase: Gilson Abimed HPLC; Säule: Daicel Chiralpak AD-H 5 μm; 250 x 20 mm; E- luent A: ώo-Hexan, Eluent B: 0.2% Essigsäure/1% Wasser/2-Propanol; isokratisch; Fluss: 15 mL/min; UV-Detektor 212 nm. Die Zuordnung der Isomere erfolgt durch HPLC-Vergleich mit einer authentischen Probe von N-(ter^Butoxycarbonyl)-L-3-trimethylsilylalanin (2R Verbindung, Mercachem AMR 39.260).The synthesis is carried out according to M. Merget, K. Günther, M. Bernd, E. Günther, R. Tacke, J. Organomet. Chem. 2001 628, 183-194. The enantiomers are separated by preparative HPLC on a chiral phase: Gilson Abimed HPLC; Column: Daicel Chiralpak AD-H 5 μm; 250 x 20 mm; Eluent A: hexane, eluent B: 0.2% acetic acid / 1% water / 2-propanol; isocratic; Flow: 15 mL / min; UV detector 212 nm. The assignment of the isomers by HPLC comparison with an authentic sample of N- (ter ^ butoxycarbonyl) -L-3-trimethylsilylalanine (2R compound, Mercachem AMR 39.260).
Beispiel 9AExample 9A
N-(ter£-Butoxycarbony l)-D-3 -trimethylsilylalanin (2S Verbindung)N- (tert -butoxycarbonyl) -D-3-trimethylsilylalanine (2S compound)
Chirale HPLC (Methode 15): R4 = 4.16 min, e.e. > 99%.Chiral HPLC (Method 15): R 4 = 4.16 min, ee> 99%.
[α]D 20 = +1.1 (c = 0.83 in Methanol) Beispiel IQA[α] D 20 = +1.1 (c = 0.83 in methanol) Example IQA
N-(ter^Butoxycarbonyl)-L-3-trimethylsilylalanin (2R Verbindung)N- (tert-butoxycarbonyl) -L-3-trimethylsilylalanine (2R compound)
Chirale HPLC (Methode 15): R1 = 9.27 min, e.e. > 99%.Chiral HPLC (Method 15): R 1 = 9.27 min, ee> 99%.
[α]D 20 = -1.6 (c = 0.66 in Methanol)[α] D 20 = -1.6 (c = 0.66 in methanol)
Beispiel IIAExample IIA
Methyl-N-(fert-butoxycarbonyl)-3-(pyridin-3-yl)-L-alaninatMethyl-N- (tert-butoxycarbonyl) -3- (pyridin-3-yl) -L-alaninate
Die Herstellung erfolgt analog B. Neises, W. Steglich, Org. Synth. 1985, 63, 183-187.The preparation is analogous to B. Neises, W. Steglich, Org. Synth. 1985, 63, 183-187.
(2.S)-N-(tert-Butoxycarbonyl)-3-(pyridin-3-yl)alanm (25.00 g, 93.88 mmol) wird unter Argon in 300 mL Dichlormethan gelöst. Methanol (11.4 mL, 9.02 g, 281 mmol, 3 Äquivalente) und ein Körnchen DMAP werden hinzugefügt. Dann wird die Mischung auf O0C gekühlt. EDC (19.80 g, 103 mmol, 1.1 Äquivalente) wird zugesetzt. Nach 5 min. entfernt man das Eisbad und lässt über 1 h bei RT rühren. Dann engt man im Vakuum ein, versetzt den Rückstand mit Ethylacetat und schüttelt gegen gesättigte Natriurnhydrogencarbonatlösung aus. Die wässrige Phase wird einmal mit Ethylacetat nachextrahiert, dann werden die vereinigten organischen Phasen mit 0.5 M Citro- nensäure und anschließend nochmals mit gesättigter Natriurnhydrogencarbonatlösung gewaschen. Die organische Phase wird über Natriumsulfat getrocknet, filtriert und im Vakuum eingeengt. Es verbleibt ein klares Öl, das beim Trocknen im Ölpumpenvakuum kristallisiert. Ausbeute: 23.60 g (90% d. Th.).(2.S) -N- (tert-Butoxycarbonyl) -3- (pyridin-3-yl) alanm (25.00 g, 93.88 mmol) is dissolved under argon in 300 mL dichloromethane. Methanol (11.4 mL, 9.02 g, 281 mmol, 3 equiv) and a granule of DMAP are added. Then the mixture is cooled to 0 ° C. EDC (19.80 g, 103 mmol, 1.1 equiv.) Is added. After 5 min. remove the ice bath and stir for 1 h at RT. It is then concentrated in vacuo, the residue is combined with ethyl acetate and extracted by shaking with saturated sodium bicarbonate solution. The aqueous phase is back-extracted once with ethyl acetate, then the combined organic phases are washed with 0.5 M citric acid and then again with saturated sodium bicarbonate solution. The organic phase is dried over sodium sulfate, filtered and concentrated in vacuo. It remains a clear oil that crystallizes during drying in an oil pump vacuum. Yield: 23.60 g (90% of theory).
HPLC/UV-Vis (Methode 9): Rt = 3.28 min.HPLC / UV-Vis (Method 9): R t = 3.28 min.
LC-MS (Methode 7): R4= 1.21 min, MS (ESIpos.): m/z (%) = 281 (100) [M + H]+.LC-MS (Method 7): R 4 = 1.21 min, MS (ESIpos.): M / z (%) = 281 (100) [M + H] + .
1H-NMR (400 MHz, J6-DMSO): δ = 1.30 (s, 9H), 2.86 (m, IH), 3.04 (m, IH)3 3.63 (s, 3H), 4.22 (m, IH), 7.28 - 7.39 (m, 2H), 7.69 (d, IH), 8.43 (m, 2H). 1 H NMR (400 MHz, J 6 -DMSO): δ = 1.30 (s, 9H), 2.86 (m, IH), 3.04 (m, IH) 3 3.63 (s, 3H), 4.22 (m, IH) , 7.28 - 7.39 (m, 2H), 7.69 (d, IH), 8.43 (m, 2H).
Beispiel 12AExample 12A
3 -(Pyridin-3 -y l)-L-alanin-methylester-bistrifluoracetat3 - (pyridin-3-yl) -L-alanine methyl ester bistrifluoroacetate
Die Verbindung aus Beispiel I IA (11.8 g, 42.09 mmol) wird in Trifluoressigsäure in Dichlor- methan (160 mL; 30%ige Lösung) gelöst und 30 min. bei RT gerührt. Dann engt man im Vakuum ein. Der Rückstand wird in etwas Wasser aufgenommen und lyophilisiert. Dann wird das Lyophili¬ sat mit Toluol versetzt und im Vakuum eingeengt. Schließlich wird bis zur Gewichtskonstanz im Ölpumpenvakuum getrocknet. Ausbeute: 17.15 g (quant).The compound from Example I IA (11.8 g, 42.09 mmol) is dissolved in trifluoroacetic acid in dichloromethane (160 mL, 30% solution) and 30 min. stirred at RT. Then you narrow in a vacuum. The residue is taken up in a little water and lyophilized. The lyophilizate is then treated with toluene and concentrated in vacuo. Finally, it is dried to constant weight in an oil pump vacuum. Yield: 17.15 g (quant).
HPLC/UV-Vis (Methode 9): R1 = 0.88 min.HPLC / UV-Vis (Method 9): R 1 = 0.88 min.
LC-MS (Methode 7): R1= 0.46 min, MS (ESIpos.): m/z (%) = 181 (100) [M + H]+.LC-MS (Method 7): R 1 = 0.46 min, MS (ESIpos.): M / z (%) = 181 (100) [M + H] + .
1H-NMR (400 MHz, J0-DMSO): δ = 2.79 (dd, IH), 2.92 (dd, IH)5 3.60 (s, 3H), 3.63 (m, IH), 7.30 (m, IH), 7.62 (d, IH), 8.41 (m, 2H). Beispiel 13A 1 H-NMR (400 MHz, J 0 -DMSO): δ = 2.79 (dd, IH), 2.92 (dd, IH) 5 3.60 (s, 3H), 3.63 (m, IH), 7.30 (m, IH) , 7.62 (d, IH), 8.41 (m, 2H). Example 13A
Methyl-N-(fert-butoxycarbonyl)-3-(trimethylsilyl)-D-alanyl-3-(pyridin-3-yl)-L-alaninatMethyl-N- (tert-butoxycarbonyl) -3- (trimethylsilyl) -D-alanyl-3- (pyridin-3-yl) -L-alaninate
Die Verbindung aus Beispiel 9A (10.31 g, 39.4 mmol) und die Verbindung aus Beispiel 12A (16.10 g, 39.4 mmol, 1 Äquivalent) werden bei O0C in DMF (186 mL) gelöst. Dann werden N-Methylmorpholin (17.34 mL, 16.00 g, 4 Äquivalente) und HATU (22.49 g, 59.16 mmol, 1.5 Äquivalente) zugesetzt. Der Ansatz wird zwei Stunden bei RT gerührt. Man versetzt mit tert- Butylmethylether und wäscht mit gesättigter Νatriumcarbonatlösung. Die wässrige Phase wird einmal mit tert-Burylmethylether nachextrahiert, dann werden die vereinigten organischen Phasen mit 1 M wässriger Citronensäure sowie abermals mit gesättigter Νatriumcarbonatlösung gewa¬ schen, über Νatriumsulfat getrocknet, filtriert und im Vakuum eingeengt. Es wird über Kieselgel filtriert (Cyclohexan/Ethylacetat 2:1). Ausbeute: 14.1 g (84% d. Th.).The compound from Example 9A (10.31 g, 39.4 mmol) and the compound from Example 12A (16.10 g, 39.4 mmol, 1 equivalent) are dissolved at 0 ° C. in DMF (186 ml). Then N-methylmorpholine (17.34 mL, 16.00 g, 4 equiv) and HATU (22.49 g, 59.16 mmol, 1.5 equiv) are added. The batch is stirred for two hours at RT. It is mixed with tert-butyl methyl ether and washed with saturated sodium carbonate solution. The aqueous phase is back-extracted once with tert-butyl methyl ether, then the combined organic phases are washed with 1 M aqueous citric acid and again with saturated sodium carbonate solution, dried over sodium sulfate, filtered and concentrated in vacuo. It is filtered through silica gel (cyclohexane / ethyl acetate 2: 1). Yield: 14.1 g (84% of theory).
HPLC/UV-Vis (Methode 9): Rt = 3.91 min.HPLC / UV-Vis (Method 9): R t = 3.91 min.
LC-MS (Methode 7): Rt = 1.90 min, MS (ESIpos.): m/z (%) = 424 (100) [M + Hf.LC-MS (Method 7): R t = 1.90 min, MS (ESI pos.): M / z (%) = 424 (100) [M + Hf.
1H-NMR (400 MHz, ^-DMSO): δ = -0.09 (s, 9H), 0.56 - 0.75 (m, 2H), 1.47 (s, 9H), 2.90 (dd, IH), 3.09 (dd, IH), 3.62 (s, 3H), 3.98 (m, IH), 4.49 (m, IH), 6.68 (d, IH), 7.26 (dd, IH), 7.61 (m, IH), 8.20 (d, IH), 8.40 (m, 2H). 1 H-NMR (400 MHz, ^ -DMSO): δ = -0.09 (s, 9H), 0.56-0.75 (m, 2H), 1.47 (s, 9H), 2.90 (dd, IH), 3.09 (dd, IH), 3.62 (s, 3H), 3.98 (m, IH), 4.49 (m, IH), 6.68 (d, IH), 7.26 (dd, IH), 7.61 (m, IH), 8.20 (d, IH ), 8.40 (m, 2H).
Beisniel 14AExample 14A
N-(tert-Butoxy carbonyl)-3 -(trimethylsilyl)-D-alany 1-3 -(pyridin-3 -yl)-L-alaninN- (tert-butoxycarbonyl) -3- (trimethylsilyl) -D-alany 1-3 - (pyridin-3-yl) -L-alanine
Die Verbindung aus Beispiel 13A (7.4 g, 17.56 mmol) wird in THF/Wasser (6:4) aufgenommen, auf 00C gekühlt und mit Lithiumhydroxid-Monohydrat (1.47 g, 35.13 mmol, 2 Äquivalente) ver¬ setzt. Man lässt bei 00C rühren. Nach einer Stunde wird ein weiteres Äquivalent (0.74 g) Lithium¬ hydroxid-Monohydrat zugesetzt und eine weitere Stunde gerührt. Man destilliert den Großteil des THF im Vakuum ab, wäscht mit zwei Portionen Methyl-ter?-butylether und stellt dann die wässri- ge Phase durch Zusatz von Citronensäure auf pH 4. Ein Feststoff fällt aus. Es wird mit drei Portio- nen Ethylacetat extrahiert, wobei sich der Feststoff löst. Die vereinigten organischen Phasen wer¬ den über Natriumsulfat getrocknet, filtriert und eingeengt. Das Rohprodukt wird durch Gelchroma¬ tographie (Methode 3, Fließmittel: Methanol) gereinigt. Ausbeute: 6.67 g (93% d. Th.).The compound from Example 13A (7.4 g, 17:56 mmol) in THF / water (6: 4) was added, cooled to 0 0 C, and (g 1:47, 35.13 mmol, 2 equivalents) of lithium hydroxide monohydrate is ver¬. The mixture is stirred at 0 ° C. After one hour, another equivalent (0.74 g) of lithium hydroxide monohydrate is added and the mixture is stirred for a further hour. The majority of the THF is distilled off in vacuo, washed with two portions of methyl tert-butyl ether and then the aqueous phase is adjusted to pH 4 by addition of citric acid. A solid precipitates out. It is extracted with three portions of ethyl acetate, whereby the solid dissolves. The combined organic phases wer¬ dried over sodium sulfate, filtered and concentrated. The crude product is purified by gel chromatography (Method 3, mobile phase: methanol). Yield: 6.67 g (93% of theory).
HPLC/UV-Vis (Methode 9): Rt = 3.73 min.HPLC / UV-Vis (Method 9): R t = 3.73 min.
LC-MS (Methode 7): Rt = 1.68 min, MS (ESIpos.): m/z (%) = 410 (40) [M + H]+.LC-MS (Method 7): R t = 1.68 min, MS (ESI pos.): M / z (%) = 410 (40) [M + H] + .
1H-NMR (300 MHz, ^6-DMSO): δ = -0.090 (s, 9H), 0.56 - 0.75 (m, 2H), 1.35 (s, 9H), 2.90 (dd, IH), 3.09 (dd, IH), 3.98 (m, IH), 4.41 (m, IH), 6.70 (d, IH), 7.26 (dd, IH), 7.60 (m, IH), 8.00 (d, IH), 8.37 (m, 2H). 1 H-NMR (300 MHz, ^ 6 -DMSO): δ = -0.090 (s, 9H), 0.56-0.75 (m, 2H), 1.35 (s, 9H), 2.90 (dd, IH), 3.09 (dd , IH), 3.98 (m, IH), 4.41 (m, IH), 6.70 (d, IH), 7.26 (dd, IH), 7.60 (m, IH), 8.00 (d, IH), 8.37 (m, 2H).
Beispiel 15AExample 15A
N-(ter/-Butoxycarbonyl)-3-(trimethylsilyl)-D-alanyl-3-(pyridin-3-yl)-L-alanyl-des(l-D-leucyl-2-L- leucy^-lysobactin-trifluoracetatN- (tert-butoxycarbonyl) -3- (trimethylsilyl) -D-alanyl-3- (pyridin-3-yl) -L-alanyl-des (1-leucyl-2-L-leucyl-lysobactin trifluoroacetate
Die Verbindung aus Beispiel 2A (3.00 g, 2.35 mmol) und die Verbindung aus Beispiel 14A (1.44 g, 3.52 mmol, 1.5 Äquivalente) werden in DMF (50 mL) gelöst und auf O0C gekühlt. Dann werden zunächst 4.7 mL (4.7 mmol, 2 Äquivalente) einer 1 M Lösung von 4-Methylmorpholin in DMF zugegeben. Anschließend wird sofort HATU (1.52 g, 3.99 mmol, 1.7 Äquivalente) zugegeben und 15 min bei O0C gerührt. Dann werden weitere 4.7 mL (4.7 mmol, 2 Äquivalente) der 1 M Lösung von 4-Methylmorpholin in DMF zugetropft. Der Ansatz wird dann 2 h bei RT gerührt. Das Roh¬ produkt wird gelchromatographiert (Methode 3). Das Produkt wird ohne Feinreinigung weiter um¬ gesetzt. Ausbeute: 3.6 g (82% d. Th.).The compound from Example 2A (3.00 g, 2.35 mmol) and the compound from Example 14A (1.44 g, 3.52 mmol, 1.5 equivalents) are dissolved in DMF (50 mL) and cooled to 0 ° C. Then, firstly 4.7 mL (4.7 mmol, 2 equivalents) of a 1 M solution of 4-methylmorpholine in DMF are added. Subsequently, HATU (1.52 g, 3.99 mmol, 1.7 equivalents) is added immediately and stirred at 0 ° C. for 15 min. Then another 4.7 mL (4.7 mmol, 2 equivalents) of the 1 M solution of 4-methylmorpholine in DMF are added dropwise. The batch is then stirred for 2 h at RT. The crude product is gel-chromatographed (Method 3). The product is further reacted without fine cleaning. Yield: 3.6 g (82% of theory).
HPLC (Methode 9): Rt = 3.90 min.HPLC (Method 9): R t = 3.90 min.
LC-MS (Methode 7): R1 = 2.00 min, MS (ESIpos.): m/z (%) = 721.8 (100) [M + 2 H]2+; 1442.1 (5) [M + H]+.LC-MS (Method 7): R 1 = 2.00 min, MS (ESIpos.): M / z (%) = 721.8 (100) [M + 2 H] 2+ ; 1442.1 (5) [M + H] + .
Alternatives Verfahren: Die Verbindung aus Beispiel 2A (14.00 g, 10.95 mmol) und die Verbin¬ dung aus Beispiel 14A (5.38 g, 13.14 mmol, 1.2 Äquivalente) werden in DMF (280 mL) gelöst und auf - 200C gekühlt. Dann wird N-Methylmorpholin (5.54 g, 6.02 mL, 5 Äquivalente) zügegeben und anschließend HATU (6.66 g, 17.52 mmol, 1.6 Äquivalente). Der Ansatz wird langsam auf RT erwäπnt und über Nacht (ca. 16 h) gerührt. Dann wird Kaliumdihydrogenphosphat (14.91 g, 10 Äquivalente) unter Rühren zugesetzt und 30 Minuten weitergerührt Das Rohprodukt wird gelch- romatographiert (Methode 3, Fließmittel: Methanol). Das Produkt wird ohne Feinreinigung weiter umgesetzt. Ausbeute: 14.35 g (61% d. Th.).Alternative method: The compound of Example 2A (14.00 g, 10.95 mmol) and the Verbin¬ dung from Example 14A (5:38 g, 13:14 mmol, 1.2 equivalents) are dissolved in DMF (280 mL) and - cooled to -20 0 C. Then N-methylmorpholine (5.54 g, 6.02 mL, 5 equiv.) Is added followed by HATU (6.66 g, 17.52 mmol, 1.6 equiv.). The approach is slow to RT erwäπnt and stirred overnight (about 16 h). Then potassium dihydrogen phosphate (14.91 g, 10 equivalents) is added with stirring and stirring is continued for 30 minutes. The crude product is subjected to gel chromatography (Method 3, mobile phase: methanol). The product is further reacted without fine cleaning. Yield: 14.35 g (61% of theory).
Beispiel 16AExample 16A
2,2-Dimethyl-l-butanal2,2-dimethyl-l-butanal
2,2-Dimethyl-l-butanol (4.0 g, 39 mmol) wird in Dichlormethan (136 mL) gelöst und mit Alumi¬ niumoxid (7.98 g, 78 mmol, 2 Äquivalente) und mit Pyridiniumchlorochromat (16.88 g, 78 mmol, 2 Äquivalente) versetzt. Der Ansatz wird bei RT 1 h gerührt und dann über eine Schicht Kieselgel filtriert. Das Filtrat wird vorsichtig eingeengt, und der Rückstand wird bei Normaldruck destilliert (Siedepunkt: 1020C (990 mbar)). Ausbeute: 2.97 g (75% d. Th.).2,2-Dimethyl-1-butanol (4.0 g, 39 mmol) is dissolved in dichloromethane (136 mL) and washed with alumina (7.98 g, 78 mmol, 2 equivalents) and with pyridinium chlorochromate (16.88 g, 78 mmol, 2 Equivalents). The batch is stirred at RT for 1 h and then filtered through a layer of silica gel. The filtrate is carefully concentrated and the residue is distilled at normal pressure (boiling point: 102 0 C (990 mbar)). Yield: 2.97 g (75% of theory).
GC-MS (Methode 17): R, = 2.21 min, MS (ESIpos.): m/z (%) = 99.9 (5) [M]+;GC-MS (Method 17): R, = 2.21 min, MS (ESIpos.): M / z (%) = 99.9 (5) [M] + ;
1H NMR (400 MHz, CDCl3) δ 0.83 (t, 3H), 1.03 (s, 6H), 1.51 (q, 2H), 9.42 (s, IH). 1 H NMR (400 MHz, CDCl3) δ 0.83 (t, 3H), 1:03 (s, 6H), 1:51 (q, 2H), 9:42 (s, IH).
Beispiel 17AExample 17A
Methyl-(2Z)-2-{[(benzyloxy)carbonyl]amino}-4,4-dimethylhex-2-enoatMethyl (2Z) -2 - {[(benzyloxy) carbonyl] amino} -4,4-dimethylhex-2-enoate
Die Verbindung aus Beispiel 16A (2.55 g, 25.46 mmol) und Methyl- {[(benzyloxy)carbony I]- amino}(dimethoxyphosphoryl)-acetat (8.43 g, 25.46 mmol) werden in 50 mL THF gelöst und auf 00C gekühlt. N,N,N\N'-Tetramethylguanidin wird zugetropft, dann wird erst 15 min. bei 00C und anschließend 5 Tage bei RT gerührt. Der Ansatz wird mit etwa 20 g Kieselgel versetzt, eingeengt und chromatographiert (Kieselgel Biotage 4OM, ZIF-SIM, Cyclohexan /Ethylacetat 87:13). Ausbeute: 1.20 g (13% d. Th.).The compound from Example 16A (2.55 g, 25.46 mmol) and methyl {[(benzyloxy) carbonyl I] - amino} (dimethoxyphosphoryl) acetate (8:43 g, 25.46 mmol) are dissolved in 50 mL THF and cooled to 0 0 C , N, N, N'-tetramethylguanidine is added dropwise, then only 15 min. at 0 0 C and then stirred for 5 days at RT. The batch is mixed with about 20 g of silica gel, concentrated and chromatographed (silica gel Biotage 4OM, ZIF-SIM, cyclohexane / ethyl acetate 87:13). Yield: 1.20 g (13% of theory).
HPLC (Methode 9): R1 = 3.71 min.HPLC (Method 9): R 1 = 3.71 min.
MS (DCI): m/z (%) = 323.3 (100) [M + NH4].MS (DCI): m / z (%) = 323.3 (100) [M + NH 4 ].
1H-NMR (400 MHz, CDCl3): δ = 0.83 (m, 3H), 1.13 (s, 6H), 1.49 (q, 2H), 3.75 (br s, 3H), 5.72 (br s, IH), 6.58 (br s, IH), 5.12 (s, 2H), 7.36 (m, 5H). 1 H-NMR (400 MHz, CDCl 3 ): δ = 0.83 (m, 3H), 1.13 (s, 6H), 1.49 (q, 2H), 3.75 (br s, 3H), 5.72 (br s, IH) , 6.58 (br s, IH), 5.12 (s, 2H), 7.36 (m, 5H).
Beispiel 18AExample 18A
Methyl-N-[(benzyloxy)carbonyl]-4,4-dimethyl-D-norleucinatMethyl-N - [(benzyloxy) carbonyl] -4,4-dimethyl-D-norleucinat
Die Verbindung aus Beispiel 17A (1.2 g, Rohprodukt, 3.26 mmol) wird in Ethanol p.a. (60 mL) gelöst. Mit einer Kanüle wird etwa 5 min Argon hindurchgeleitet, dann wird (+)-l,2-Bis-[(2i?,5i?)- diethylρhospholano]benzen(cyclooctadien)rhodium(I)-Triflat (28 mg, 0.04 mmol, 0.012 Äquiva¬ lente) zugegeben und im Ultraschallbad gelöst. Es wird über 24 h bei 3 bar Wasserstoff-druck und RT hydriert. Der Ansatz wird eingeengt und chromatographiert (Kieselgel Biotage 25M, Cyclohe- xan/Ethylacetat 9:1). Ausbeute: 920 mg (92% d. Th.).The compound from example 17A (1.2 g, crude product, 3.26 mmol) is dissolved in ethanol p.a. (60 mL) dissolved. With a cannula, argon is passed through for about 5 minutes, then (+) - l, 2-bis - [(2i, 5i) -diethylphospholano] benzene (cyclooctadiene) rhodium (I) triflate (28 mg, 0.04 mmol, 0.012 equivalents) was added and dissolved in an ultrasonic bath. It is hydrogenated for 24 h at 3 bar hydrogen pressure and RT. The batch is concentrated and chromatographed (Kieselgel Biotage 25M, cyclohexane / ethyl acetate 9: 1). Yield: 920 mg (92% of theory).
HPLC (Methode 9): R1 = 4.96 min.HPLC (Method 9): R 1 = 4.96 min.
LC-MS (Methode 7): R1 = 2.76 min; MS (ESIpos.): m/z (%) = 308 (25) [M + H]+.LC-MS (method 7): R 1 = 2.76 min; MS (ESI pos.): M / z (%) = 308 (25) [M + H] + .
1H-NMR (400 MHz, CDCl3): δ = 0.80 (t, 3H), 0.86 (s, 6H), 1.29 (q, 2H), 1.41 (dd, IH)5 1.73 (dd, IH), 3.72 (s, 3H), 4.40 (m, IH), 5.02 (d, IH), 5.11 (m, 2H), 7.35 (m, 5H). Beispiel 19A 1 H-NMR (400 MHz, CDCl 3 ): δ = 0.80 (t, 3H), 0.86 (s, 6H), 1.29 (q, 2H), 1.41 (dd, IH) 5 1.73 (dd, IH), 3.72 (s, 3H), 4.40 (m, IH), 5.02 (d, IH), 5.11 (m, 2H), 7.35 (m, 5H). Example 19A
N-[(Ben2yloxy)carbonyl]-4,4-dimethyl-D-norleucinN - [(Ben2yloxy) carbonyl] -4,4-dimethyl-D-norleucine
In THF (12 mL) wird die Verbindung aus Beispiel 18A (915 mg, 2.98 mmol) gelöst. Die Lösung wird auf O0C gekühlt, dann werden 3.7 mL (7.4 mmol, 2.5 Äquivalente) einer 2 M Lösung von Lithiumhydroxid-Monohydrat in Wasser zugegeben und 1 h kräftig gerührt. Anschließend wird Citronensäure (1 M) bis zur sauren Reaktion zugetropft und das Gemisch mit Ethylacetat extra¬ hiert. Der organische Extrakt wird über Νatriumsulfat getrocknet, eingeengt und chromatographiert (Methode 16). Ausbeute: 434 mg (50% d. Th.).In THF (12 mL), the compound from Example 18A (915 mg, 2.98 mmol) is dissolved. The solution is cooled to 0 ° C., then 3.7 mL (7.4 mmol, 2.5 equivalents) of a 2 M solution of lithium hydroxide monohydrate in water are added and stirred vigorously for 1 h. Then citric acid (1 M) is added dropwise until the acid reaction and the mixture is extracted with ethyl acetate. The organic extract is dried over sodium sulfate, concentrated and chromatographed (Method 16). Yield: 434 mg (50% of theory).
HPLC (Methode 9): R, = 4.54 min.HPLC (Method 9): R, = 4.54 min.
LC-MS (Methode 7): Rt = 2.44 min, MS (ESIpos.): m/z (%) = 294 (20) [M + H]+.LC-MS (Method 7): R t = 2.44 min, MS (ESI pos.): M / z (%) = 294 (20) [M + H] + .
1H-NMR (400 MHz, J6-DMSO): δ = 0.80 (t, 3H), 0.83 (s, 6H), 1.21 (q, 2H), 1.53 (dd, IH), 1.60 (dd, IH), 3.99 (m, IH), 5.02 (s, 2H), 7.35 (m, 5H), 7.58 (d, 2H), 12.52 (br s, IH). 1 H NMR (400 MHz, J 6 -DMSO): δ = 0.80 (t, 3H), 0.83 (s, 6H), 1.21 (q, 2H), 1.53 (dd, IH), 1.60 (dd, IH) , 3.99 (m, IH), 5.02 (s, 2H), 7.35 (m, 5H), 7.58 (d, 2H), 12.52 (br s, IH).
Beispiel 2OAExample 2OA
Methyl-N-[(benzyloxy)carbonyl]-4,4-dimethyl-D-norleucyl-3-(pyridin-3-yl)-L-alaninatMethyl-N - [(benzyloxy) carbonyl] -4,4-dimethyl-D-norleucyl-3- (pyridin-3-yl) -L-alaninate
Die Verbindung aus Beispiel 19A (430 mg, 1.47 mmol) und die Verbindung aus Beispiel 12A (809 mg, 1.47 mmol, 1 Äquivalent) werden bei O0C in DMF (5 mL) gelöst, dann werden 4- Methylmorpholin (644 μl, 5.86 mmol, 4 Äquivalente) und HATU (836 mg, 2.20 mmol, 1.5 Äqui¬ valente) zugesetzt. Der Ansatz wird drei Stunden bei RT gerührt. Man versetzt mit Ethylacetat und wäscht mit gesättigter Natriumhydrogencarbonatlösung. Die wässrige Phase wird einmal mit E- thylacetat extrahiert, dann werden die vereinigten organischen Phasen mit 1 M wässriger Citronen- säure sowie abermals mit gesättigter Natriumhydrogencarbonatlösung gewaschen, über Natrium¬ sulfat getrocknet, filtriert und im Vakuum eingeengt. Der Rückstand wird chromatographiert (Me¬ thode 16). Ausbeute: 496 mg (74% d. Th.).The compound from Example 19A (430 mg, 1.47 mmol) and the compound from Example 12A (809 mg, 1.47 mmol, 1 equivalent) are dissolved at 0 ° C. in DMF (5 mL), then 4- Methylmorpholine (644 .mu.l, 5.86 mmol, 4 equivalents) and HATU (836 mg, 2.20 mmol, 1.5 equivalents) was added. The batch is stirred for three hours at RT. It is mixed with ethyl acetate and washed with saturated sodium bicarbonate solution. The aqueous phase is extracted once with ethyl acetate, then the combined organic phases are washed with 1 M aqueous citric acid and again with saturated sodium bicarbonate solution, dried over sodium sulfate, filtered and concentrated in vacuo. The residue is chromatographed (method 16). Yield: 496 mg (74% of theory).
HPLC (Methode 9): R1 = 3.94 min.HPLC (Method 9): R 1 = 3.94 min.
LC-MS (Methode 7): R4 = 1.85 min; MS (ESIpos.): m/z (%) = 456 (100) [M + H]+.LC-MS (method 7): R 4 = 1.85 min; MS (ESI pos.): M / z (%) = 456 (100) [M + H] + .
1H-NMR (300 MHz, ^0-DMSO): δ = 0.80 (m, 9H), 1.10 (m, 3H), 1.30 (dd, IH), 2.91 (dd, IH), 3.12 (dd, IH), 3.30 (s, 3H), 4.02 (m, IH), 4.51 (m, IH), 5.01 (d, IH), 5.06 (d, IH), 7.22 (dd, IH), 7.30 (m, 5H), 7.63 (m, IH), 8.40 (m, 2H). 1 H-NMR (300 MHz, ^ 0 -DMSO): δ = 0.80 (m, 9H), 1.10 (m, 3H), 1.30 (dd, IH), 2.91 (dd, IH), 3.12 (dd, IH) , 3.30 (s, 3H), 4.02 (m, IH), 4.51 (m, IH), 5.01 (d, IH), 5.06 (d, IH), 7.22 (dd, IH), 7.30 (m, 5H), 7.63 (m, IH), 8.40 (m, 2H).
Beispiel 21 AExample 21A
N-[(Benzyloxy)carbonyl]-4,4-dimethyl-D-norleucyl-3-(pyridin-3-yl)-L-alaninN - [(benzyloxy) carbonyl] -4,4-dimethyl-D-norleucyl-3- (pyridin-3-yl) -L-alanine
In THF (5 mL) wird die Verbindung aus Beispiel 2OA (490 mg, 1.08 mmol) gelöst. Die Lösung wird auf 00C gekühlt, dann werden 1.35 mL (2.7 mmol, 2.5 Äquivalente) einer 2 M Lösung von Lithiumhydroxid-Monohydrat in Wasser zugegeben und 1 h kräftig gerührt. Dann wird Citronen- säure (1 M in Wasser) bis zur sauren Reaktion zugetropft und das Gemisch mit Ethylacetat extra¬ hiert. Der organische Extrakt wird über Νatriumsulfat getrocknet und eingeengt. Ausbeute: 484 mg (quant).In THF (5 mL), the compound from Example 2OA (490 mg, 1.08 mmol) is dissolved. The solution is cooled to 0 ° C., then 1.35 ml (2.7 mmol, 2.5 equivalents) of a 2 M solution of lithium hydroxide monohydrate in water are added and the mixture is stirred vigorously for 1 h. Citric acid (1 M in water) is then added dropwise until the reaction is acid, and the mixture is extracted with ethyl acetate. The organic extract is dried over sodium sulfate and concentrated. Yield: 484 mg (quant).
HPLC (Methode 9): Rt = 3.76 min.HPLC (Method 9): R t = 3.76 min.
LC-MS (Methode 7): R1 = 1.88 min; MS (ESIpos.): m/z (%) = 442 (100) [M + H]+. 1H-NMR (400 MHz, Cf6-DMSO): δ = 0.70 (m, 9H)5 1.14 (m, 3H), 1.30 (dd, IH), 2.64 (d, IH), 2.75 (d, IH), 2.89 (dd, IH), 3.11 (dd, IH), 4.03 (m, IH), 4.45 (m, IH), 4.98 (d, IH), 5.05 (d, IH), 7.22 (dd, IH), 7.30 (m, 5H), 7.61 (m, IH), 8.40 (m, 2H).LC-MS (method 7): R 1 = 1.88 min; MS (ESI pos.): M / z (%) = 442 (100) [M + H] + . 1 H NMR (400 MHz, Cf 6 -DMSO): δ = 0.70 (m, 9H) 5 1.14 (m, 3H), 1.30 (dd, IH), 2.64 (d, IH), 2.75 (d, IH) , 2.89 (dd, IH), 3.11 (dd, IH), 4.03 (m, IH), 4.45 (m, IH), 4.98 (d, IH), 5.05 (d, IH), 7.22 (dd, IH), 7.30 (m, 5H), 7.61 (m, IH), 8.40 (m, 2H).
Beispiel 22AExample 22A
N-[(Benzyloxy)carbonyl]-4,4-dimethyl-D-norleucyl-3-(pyridin-3-yl)-L-alanyl-des(l-D-leucyl-2-L- leucyO-lysobactin-trifluoracetatN - [(benzyloxy) carbonyl] -4,4-dimethyl-D-norleucyl-3- (pyridin-3-yl) -L-alanyl-des (1-leucyl-2-L-leucyl-lysobactin trifluoroacetate
Die Verbindung aus Beispiel 2A (0.28 g, 0.22 mmol) und die Verbindung aus Beispiel 21A (148 mg, 0.33 mmol, 1.5 Äquivalente) werden in DMF (4 mL) gelöst und auf O0C gekühlt. Dann werden zunächst 0.47 mL (0.44 mmol, 2 Äquivalente) einer 1 M Lösung von N-Methylmorpholin in DMF zugegeben. Anschließend wird sofort HATU (141 mg, 0.37 mmol, 1.7 Äquivalente) zugegeben und 15 min bei 00C gerührt. Dann werden weitere 0.44 mL (0.47 mmol, 2 Äquivalente) der 1 M Lösung von 4-Methylmorpholin in DMF zugetropft. Der Ansatz wird dann über Nacht bei RT gerührt. Der Ansatz wird an Sephadex LH 20 gereinigt (Methode 3, Fließmittel: Methanol). Das Rohprodukt wird mittels präparativer HPLC (Methode 13) aufgereinigt. Ausbeute: 149 mg (43% d. Th.). HPLC (Methode 9): Rt = 3.93 min.The compound from Example 2A (0.28 g, 0.22 mmol) and the compound from Example 21A (148 mg, 0.33 mmol, 1.5 equivalents) are dissolved in DMF (4 mL) and cooled to 0 ° C. Then first 0.47 mL (0.44 mmol, 2 equivalents) of a 1 M solution of N-methylmorpholine in DMF is added. Then immediately HATU (141 mg, 0.37 mmol, 1.7 equivalents) was added and stirred at 0 0 C for 15 min. Then another 0.44 mL (0.47 mmol, 2 equivalents) of the 1 M solution of 4-methylmorpholine in DMF are added dropwise. The mixture is then stirred overnight at RT. The batch is purified on Sephadex LH 20 (method 3, flow agent: methanol). The crude product is purified by preparative HPLC (Method 13). Yield: 149 mg (43% of theory). HPLC (Method 9): R t = 3.93 min.
LC-MS (Methode 7): R4 = 2.13 min, MS (ESIpos.): m/z (%) = 737.4 (100) [M + 2H]2+, 1473 (2) [M + H]+. LC-MS (method 7): R 4 = 2.13 min, MS (ESIpos.): M / z (%) = 737.4 (100) [M + 2H] 2+ , 1473 (2) [M + H] + .
AusführangsbeispieleAusführangsbeispiele
Beispiel 1example 1
3-ter/-Butyl-D-alanyl-3-(6-trifluormethyl-pyridin-3-yl)-L-alanyl-des(l-D-leucyl-2-L-leucyl)- lysobactin-bistrifluoracetat3-tert -Butyl-D-alanyl-3- (6-trifluoromethyl-pyridin-3-yl) -L-alanyl-des (1- (D-leucyl-2-L-leucyl) -lysobactin-bistrifluoroacetate
Zu einer Lösung der Verbindung aus Beispiel 8A (10.3 g, 1.0 Äquivalente, 4.48 mmol, Rohpro¬ dukt) in Dichlormethan (150 mL) wird bei RT langsam Trifluoressigsäure (150 mL). zugetropft. Das Reaktionsgemisch wird bei RT gerührt (10 min), wobei vollständiger Umsatz mittels HPLC (Methode 9) beobachtet wird. Das Reaktionsgemisch wird am Rotationsverdampfer eingeengt und mittels präparativer HPLC (Methode 11) aufgereinigt, wobei man 3.48 g (48% d. Th.) Produkt erhält.To a solution of the compound from Example 8A (10.3 g, 1.0 equivalents, 4.48 mmol, crude product) in dichloromethane (150 ml), trifluoroacetic acid (150 ml) is slowly added at RT. dropwise. The reaction mixture is stirred at RT (10 min), with complete conversion being monitored by HPLC (Method 9). The reaction mixture is concentrated on a rotary evaporator and purified by preparative HPLC (Method 11) to give 3.48 g (48% of theory) of product.
HPLC/UV-Vis (Methode 8): Rt = 4.03 min.HPLC / UV-Vis (Method 8): R t = 4.03 min.
HPLC/UV-Vis (Methode 9): R1 = 3.64 min.HPLC / UV-Vis (Method 9): R 1 = 3.64 min.
LC-MS (Methode 7): R, = 1.69 min; MS (ESIpos.): m/z (%) = 697 (100) [M + 2H]2+, 1393 (5) [M + H]+; MS (ESIneg.): m/z (%) = 695 (100) [M - 2H]2~ 1391 (20) [M - H]".LC-MS (method 7): R, = 1.69 min; MS (ESI pos.): M / z (%) = 697 (100) [M + 2H] 2+ , 1393 (5) [M + H] + ; MS (ES Ineg.): M / z (%) = 695 (100) [M-2H] 2 ~ 1391 (20) [M-H] " .
19F-NMR (400 MHz, ^-Pyridin): δ = -67 (Ar-CF3), -74 (CF3COOH), -132 (1,4- Dibromtetrafluorbenzol als Referenz). Gehalt TFA: 14.3 Gew.-%. HR-MS (Methode 1): C62H96Ni6O17F3 [M + H]+ ber. 1393.7091, gef. 1393.7119. 19 F-NMR (400 MHz, ^ -pyridine): δ = -67 (Ar-CF 3 ), -74 (CF 3 COOH), -132 (1,4-dibromotetrafluorobenzene as reference). TFA content: 14.3% by weight. HR-MS (Method 1): C 62 H 96 Ni 6 O 17 F 3 [M + H] + calc. 1393.7091, m.p. 1393.7119.
Beispiel 2Example 2
3-(Trimethylsilyl)-D-alanyl-3-(pyridin-3-yl)-L-alanyl-des(l-D-leucyl-2-L-leucyl)-lysobactin-tris-tri- fluoracetat3- (trimethylsilyl) -D-alanyl-3- (pyridin-3-yl) -L-alanyl-des (1- (D-leucyl-2-L-leucyl) -lysobactin tris-trifluoroacetate
Die Verbindung aus Beispiel 15A (9.12 g, 4.93 mmol, Rohprodukt) wird in Trifluoressigsäure in Dichlormethan (65 mL; 30%ige Lösung) aufgenommen. Der Ansatz wird bei RT für 20 min ge¬ rührt. Das Lösungsmittel wird abdestilliert. Der Rückstand wird im Ölpumpenvakuum getrocknet und dann chromatographisch gereinigt (Methode 14). Ausbeute: 5.54 g (67% d. Th.).The compound from Example 15A (9.12 g, 4.93 mmol, crude product) is taken up in trifluoroacetic acid in dichloromethane (65 mL, 30% solution). The batch is stirred at RT for 20 min. The solvent is distilled off. The residue is dried in an oil pump vacuum and then purified by chromatography (Method 14). Yield: 5.54 g (67% of theory).
HPLC (Methode 9): Rt = 3.32 min.HPLC (Method 9): R t = 3.32 min.
LC-MS (Methode 7): R» = 1.41 min, MS (ESIpos.): m/z (%) = 671.7 (100) [M + 2 H] 2+LC-MS (Method 7): R »= 1.41 min, MS (ESIpos.): M / z (%) = 671.7 (100) [M + 2 H] 2+
HR-TOF-MS (Methode 1): C60H97N16O17Si [M + H]+ ber. 1341.6987, gef. 1341.7019.HR-TOF-MS (method 1): C 60 H 97 N 16 O 17 Si [M + H] + calc. 1341.6987, rec. 1341.7019.
1H-NMR (500 MHz, d5-?yήάm): δ = -0.172 (s, 9H), 0.611 (d, J = 6.9 Hz, 3H), 0.881 (d, J = 7.9 Hz, 3H), 0.948 (d, J= 6.3 Hz, 3H), 0.954 - 0.997 (m, 6H), 1.135 (d, J= 6.0 Hz, 3H), 1.208 (m, 2H), 1.361 (d, J= 5.4 Hz, 3H), 1.439 (m, IH), 1.497 (m, IH), 1.953 (m, 2H), 2.04 (m, IH), 2.154 (m, 3H), 2.372 (m, 3H), 3.111 (m, IH), 3.266 (m, IH), 3.563 (d, J= 14.95 Hz, IH), 3.726 (dd, J = 12.2, 14.95 Hz, IH), 3.840 (d, J= 9.6 Hz, IH), 3.960 (m, IH), 4.152 (m, IH), 4.198 (m, IH), 4.278 (m, IH), 4.382 (m, IH), 4.488 (m, IH), 4.565 (dd, J= 9.5, 9.6 Hz, IH), 4.628 (m, IH), 4.630 (m, IH), 4.779 (d, J= 12.2 Hz, IH), 5.069 (m, IH), 5.159 (dd, J= 9.3 Hz, IH), 5.264 (m, IH)5 5.362 (s, IH), 5.98 (d, J = 9.9 Hz, IH), 6.351 (dd, J= 8.5 Hz, J= 8.7 Hz, IH), 7.169 (m, IH), 7.246 (m, IH), 7.382 (d, J= 9.9 Hz, IH), 7.512 (m, 2H), 7.583 - 7.614 (m, 2H), 7.728 (m, 2H), 7.90 (d, J = 8.7 Hz, IH), 8.126 (m, 3H), 8.341 (m, IH), 8.576 (d, J = 3.6 Hz, IH), 8.695 (m, 2H), 8.793 (m, IH), 9.139 (br s, IH), 9.715 (m, IH), 10.957 (br s, IH), 11.268 (br s, IH). 1 H-NMR (500 MHz, d 5 -? Ym): δ = -0.172 (s, 9H), 0.611 (d, J = 6.9 Hz, 3H), 0.881 (d, J = 7.9 Hz, 3H), 0.948 (d, J = 6.3Hz, 3H), 0.954-0.997 (m, 6H), 1.135 (d, J = 6.0Hz, 3H), 1.208 (m, 2H), 1.361 (d, J = 5.4Hz, 3H) , 1.439 (m, IH), 1.497 (m, IH), 1.953 (m, 2H), 2.04 (m, IH), 2.154 (m, 3H), 2.372 (m, 3H), 3.111 (m, IH), 3,266 (m, IH), 3,563 (d, J = 14.95 Hz, IH), 3,726 (dd, J = 12.2, 14.95 Hz, IH), 3,840 (d, J = 9.6 Hz, IH), 3,960 (m, IH ), 4,152 (m, IH), 4,198 (m, IH), 4,278 (m, IH), 4,382 (m, IH), 4,488 (m, IH), 4,565 (dd, J = 9.5, 9.6 Hz, IH) , 4,628 (m, IH), 4,630 (m, IH), 4.779 (d, J = 12.2 Hz, IH), 5.069 (m, IH), 5.159 (dd, J = 9.3 Hz, IH), 5.264 (m, IH) 5 5.362 (s, IH), 5.98 ( d, J = 9.9 Hz, IH), 6.351 (dd, J = 8.5 Hz, J = 8.7 Hz, IH), 7.169 (m, IH), 7.246 (m, IH), 7.382 (d, J = 9.9 Hz, IH), 7.512 (m, 2H), 7.583-7.614 (m, 2H), 7.728 (m, 2H), 7.90 (d, J = 8.7Hz, IH), 8.126 (m, 3H), 8.341 (m, IH ), 8,576 (d, J = 3.6 Hz, IH), 8,695 (m, 2H), 8,793 (m, IH), 9,139 (brs, IH), 9,715 (m, IH), 10,957 (brs, IH) , 11.268 (brs, IH).
13C-NMR (126 MHz, Jj-Pyridin): δ = -1.8, 11.08, 15.84, 18.8, 18.8, 19.79, 20.89, 20.93, 21.65, 23.38, 24.78, 26.57, 26.88, 28.80, 31.04, 34.06, 36.84, 40.95, 41.25, 44.38, 50.94, 52.82, 55.99, 56.10, 56.74, 58.40, 59.10, 60.34, 60.72, 62.33, 62.55, 70.72, 72.06, 75.58, 75.65, 123.66, 128.25, 128.95, 129.80, 132.39, 136.77, 137.18, 149.17, 158.11, 162.15, 162.41, 162.70, 162.96, 169.01, 169.69, 170.21, 172.57, 173.27, 173.44, 173.66, 174.07, 174.36, 175.34, 175.56. 13 C-NMR (126 MHz, Jj-pyridine): δ = -1.8, 11.08, 15.84, 18.8, 18.8, 19.79, 20.89, 20.93, 21.65, 23.38, 24.78, 26.57, 26.88, 28.80, 31.04, 34.06, 36.84, 40.95, 41.25, 44.38, 50.94, 52.82, 55.99, 56.10, 56.74, 58.40, 59.10, 60.34, 60.72, 62.33, 62.55, 70.72, 72.06, 75.58, 75.65, 123.66, 128.25, 128.95, 129.80, 132.39, 136.77, 137.18, 149.17, 158.11, 162.15, 162.41, 162.70, 162.96, 169.01, 169.69, 170.21, 172.57, 173.27, 173.44, 173.66, 174.07, 174.36, 175.34, 175.56.
19F-NMR (400 MHz, cf5-Pyridin): δ = -74 (CF3COOH), -132 (1,4-Dibromtetrafluorbenzol als Referenz). Gehalt TFA: 19.3 Gew.-%. 19 F-NMR (400 MHz, cf 5 -pyridine): δ = -74 (CF 3 COOH), -132 (1,4-dibromotetrafluorobenzene as reference). Content TFA: 19.3% by weight.
Die Struktur ist durch eine Einkristall-Röntgenstrukturanalyse bestätigt.The structure is confirmed by a single-crystal X-ray structure analysis.
Beispiel 3Example 3
3-(Trimethylsilyl)-D-alanyl-3-(pyridin-3-yl)-L-alanyl-des(l-D-leucyl-2-L-leucyl)-lysobactin-tris- methansulfonat3- (trimethylsilyl) -D-alanyl-3- (pyridin-3-yl) -L-alanyl-des (1- (D-leucyl-2-L-leucyl) -lysobactinetris-methanesulfonate
3-(Trimethylsilyl)-D-alanyl-3-(pyridin-3-yl)-L-alanyl-des(l-D-leucyl-2-L-leucyl)-lysobactin-tris-tri- fluoracetat (447 mg, 0.27 mmol) wird in 22 mL Wasser gelöst. Methansulfonsäure (70%ig) wird zugegeben. Der Ansatz wird kräftig gerührt und dann lyophilisiert. Das Lyophilisat wird mit Was¬ ser (3.4 mL) aufgenommen und 70%ige Natriummethansulfonatlösung (0.9 mL) wird zugesetzt. Es wird 10 min bei RT gerührt. Dann wird das Produkt abzentrifugiert. Man erhält 472 mg Rohpro¬ dukt. 3- (Trimethylsilyl) -D-alanyl-3- (pyridin-3-yl) -L-alanyl-des (ID-leucyl-2-L-leucyl) -lysobactin tris-trifluoroacetate (447 mg, 0.27 mmol ) is dissolved in 22 mL of water. Methanesulfonic acid (70%) is added. The batch is stirred vigorously and then lyophilized. The lyophilizate is taken up with water (3.4 mL) and 70% sodium methanesulfonate solution (0.9 mL) is added. It is stirred for 10 min at RT. Then the product is centrifuged off. This gives 472 mg Rohpro¬ product.
Aus mehreren Ansätzen vereinigte Rohprodukte (insgesamt 1053 mg, 0.63 mmol) werden in Was¬ ser (5 mL) suspendiert und bei RT für 4 Tage gerührt. Es wird erneut abzentrifugiert und der erhal¬ tene Feststoff im Vakuum getrocknet. Man erhält 575 mg (55% d. Th.) des Produktes.Crude products composed of several batches (total 1053 mg, 0.63 mmol) are suspended in water (5 mL) and stirred at RT for 4 days. It is again centrifuged off and the solid obtained is dried in vacuo. 575 mg (55% of theory) of the product are obtained.
HPLC (Methode 9): Rt = 3.30 min.HPLC (Method 9): R t = 3.30 min.
LC-MS (Methode 7): R1 = 1.49 min, MS (ESIpos.): m/z (%) = 671.8 (100) [M + 2 H]2+, 1342.1 (5) [M + H]+.LC-MS (method 7): R 1 = 1.49 min, MS (ESIpos.): M / z (%) = 671.8 (100) [M + 2 H] 2+ , 1342.1 (5) [M + H] + ,
1H-NMR (500 MHz, di-Pyridin): δ = -0.170 (s, 9H), 0.702 (d, J = 6.3 Hz, 3H), 0.867 (d, J = 6.1 Hz, 3H), 0.972 (d, J= 6.2 Hz, 3H), 0.966 - 1.069 (m, 9H), 1.186 (d, J= 6.5 Hz, 3H), 1.315 (m, 2H), 1.448 - 1.501 (m, 6H), 2.010 (m, IH) , 2.084 (m, 3H), 2.180 - 2.365 (m, 4H), 2.540 (m, IH), 3.082 (s, 9H), 3.166 (m, IH), 3.325 (m, IH), 3.623 (d, J= 14.05 Hz, IH), 3.865 (dd, J= 13.7, 14.05 Hz, IH), 3.967 - 3.986 (m, 2H), 4.257 (m, IH), 4.339 (m, 2H), 4.410 (m, IH), 4.566 (m, IH), 4.591 - 4.686 (m, 2H), 4.75 (d, 1 H, J = 12.1 Hz, IH), 4.859 (m, IH), 5.086 (m, IH)5 5.229 (m, IH)5 5.348 (m, IH), 5.375 (s, IH) 6.02 (d, J = 10.0 Hz, IH), 6.403 (m, IH)5 7.068 (d, J = 9.8 Hz, IH), 7.222 (m, 3H)5 7.544 (m, 3H)5 7.625 (m, IH)5 7.681 (m, IH)5 7.830 (d5 J = 7.6 Hz, IH), 7.921 (d, J = 9.35 Hz, IH), 8.071 (m, 2H)5 8.188 (br S5 IH), 8.285 (br s, IH), 8.420 (d, J = 8.4 Hz5 IH), 8.614 (d5 J= 4.0 Hz, IH)5 8.701 (m, IH)5 8.874 (br s, IH), 10.175 (m, IH)5 10.279 (m, IH)5 10.941 (br s, IH). 1 H-NMR (500 MHz, di-pyridine): δ = -0.170 (s, 9H), 0.702 (d, J = 6.3 Hz, 3H), 0.867 (d, J = 6.1 Hz, 3H), 0.972 (i.e. , J = 6.2 Hz, 3H), 0.966 - 1.069 (m, 9H), 1.186 (d, J = 6.5 Hz, 3H), 1.315 (m, 2H), 1.448 - 1.501 (m, 6H), 2.010 (m, IH), 2,084 (m, 3H), 2,180 - 2,365 (m, 4H), 2,540 (m, IH), 3,082 (s, 9H), 3,166 (m, IH), 3,325 (m, IH), 3,623 (i.e. , J = 14.05 Hz, IH), 3.865 (dd, J = 13.7, 14.05 Hz, IH), 3,967 - 3,986 (m, 2H), 4,257 (m, IH), 4,339 (m, 2H), 4,410 (m, IH), 4,566 (m, IH), 4,591 - 4,686 (m, 2H), 4.75 (d, 1H, J = 12.1 Hz, IH), 4.859 (m, IH), 5.086 (m, IH) 5 5.229 ( m, IH) 5 5.348 (m, IH), 5.375 (s, IH) 6:02 (d, J = 10.0 Hz, IH), 6.403 (m, IH) 5 7.068 (d, J = 9.8 Hz, IH), 7.222 (m, 3H) 5 7.544 (m, 3H) 5 7.625 (m, IH) 5 7.681 (m, IH) 5 7.830 (d 5 J = 7.6 Hz, IH), 7.921 (d, J = 9.35 Hz, IH) , 8.071 (m, 2H) 5 8.188 (br S 5 IH), 8.285 (br s, IH), 8.420 (d, J = 8.4 Hz 5 IH), 8.614 (d 5 J = 4.0 Hz, IH) 5 8.701 ( m, IH) 5 8,874 (brs, IH), 10,175 (m, IH) 5 10,279 (m, IH) 5 10,941 (brs, IH).
19F-NMR (Pyridin, 400 MHz, Methode 24): δ -74.0 (s, TFA5 0.20), -132.0 (s, 1,4-Dibromtetra- fluorbenzol, 1000.O)5 TFA = 0.02 Gew.%. 19 F NMR (pyridine, 400 MHz, method 24): δ -74.0 (s, TFA 5 0.20), -132.0 (s, 1,4-dibromotetrafluorobenzene, 1000.O) 5 TFA = 0.02 wt%.
Alternatives Verfahren: 32 g Dowex 1X8-400 (HCl-Form) werden in eine Säule gepackt (35 mm Durchmesser). Ca. 60 mL 1 M Natriumhydroxidlösung werden über die Säule gegeben, gefolgt von 60 mL HPLC-Wasser. Dann wird mit 60 mL 1 M Methansulfonsäure konditioniert und anschlie¬ ßend mit ungefähr 100 mL Wasser bis zur neutralen Reaktion des Eluats gewaschen. (Macherey & Nagel Tritest). Dann werden 2.00 g (1.19 mmol) 3-(Trimethylsüyl)-D-alanyl-3-(pyridin-3-yl)-L- alanyl-des(l-D-leucyl-2-L-leucyl)-lysobactin-tris-trifluoracetat (Beispiel 2) in 90 mL Wasser gelöst und die Lösung aufgegeben und langsam eluiert. Die Säule wird mit ca. 20 mL Wasser nachge- spült. Produkthaltige Eluate werden vereinigt, feinfiltriert (Porengröße 0.20 μm) und lyophilisiert. Die Säule wird erneut konditioniert und wiederverwendet. Man erhält 1.79 g (1.10 mmol, 92% d. Th.) Beispiel 3 aus 2000 mg (1.19 mmol) Beispiel 2.Alternative method: 32 g of Dowex 1X8-400 (HCl form) are packed in a column (35 mm diameter). Approximately 60 mL 1 M sodium hydroxide solution is passed through the column, followed by 60 mL HPLC water. It is then conditioned with 60 ml of 1 M methanesulfonic acid and subsequently washed with approximately 100 ml of water until neutral reaction of the eluate. (Macherey & Nagel Tritest). Then 2.00 g (1.19 mmol) of 3- (trimethylsilyl) -D-alanyl-3- (pyridin-3-yl) -L-alanyl-des (ID-leucyl-2-L-leucyl) -lysobactin tris-trifluoroacetate (Example 2) dissolved in 90 mL of water and the solution was abandoned and slowly eluted. The column is refilled with approx. 20 mL of water. flushes. Product-containing eluates are combined, finely filtered (pore size 0.20 μm) and lyophilized. The column is conditioned again and reused. This gives 1.79 g (1.10 mmol, 92% of theory) of Example 3 from 2000 mg (1.19 mmol) of Example 2.
Beispiel 4Example 4
4,4-Dimethyl-D-norleucyl-3-(pyridin-3-yl)-L-alanyl-des(l-D-leucyl-2-L-leucyl)-lysobactin-tris-tri- fluoracetat4,4-Dimethyl-D-norleucyl-3- (pyridin-3-yl) -L-alanyl-des (1- (D-leucyl-2-L-leucyl) -lysobactin tris-trifluoroacetate
Die Verbindung aus Beispiel 22A (149 mg, 0.09 mmol) wird in Methanol/enthaltend 0.05% Trifluoressigsäure (10 mL) gelöst. Palladium auf Aktivkohle (10%ig; 20 mg) wird zugegeben, dann wird insgesamt 2.5 h bei RT und Normaldruck mit Wasserstoff hydriert. Das Rohprodukt wird vom Katalysator abfiltriert und das Filtrat eingeengt. Der Rückstand wird chromatographisch gereinigt (Methode 13). Ausbeute: 68 mg (46% d. Th.).The compound from Example 22A (149 mg, 0.09 mmol) is dissolved in methanol / containing 0.05% trifluoroacetic acid (10 mL). Palladium on activated carbon (10%, 20 mg) is added, then a total of 2.5 h is hydrogenated at RT and atmospheric pressure with hydrogen. The crude product is filtered off from the catalyst and the filtrate is concentrated. The residue is purified by chromatography (Method 13). Yield: 68 mg (46% of theory).
HPLC (Methode 9): R1 = 3.29 min.HPLC (Method 9): R 1 = 3.29 min.
LC-MS (Methode 7): R4 = 1.49 min, MS (ESIpos.): m/z (%) = 671.0 (100) [M +2H]2+, 1340 (5) [M + H]+.LC-MS (method 7): R 4 = 1.49 min, MS (ESIpos.): M / z (%) = 671.0 (100) [M + 2H] 2+ , 1340 (5) [M + H] + .
HR-TOF-MS (Methode 1): C62H98N16O17 [M + H]+ ber. 1339.7369, gef. 1339.7368. Beispiel 5 (Vergleichsbeispiel)HR-TOF-MS (Method 1): C 62 H 98 N 16 O 17 [M + H] + calc. 1339.7369, m.p. 1339.7368. Example 5 (Comparative Example)
3-tert-Butyl-D-alanyl-3-(pyridin-3-yl)-L-alanyl-des(l-D-leucyl-2-L-leucyl)lysobactin-tristrifluor- acetat3-tert-butyl-D-alanyl-3- (pyridin-3-yl) -L-alanyl-des (1-leucyl-2-L-leucyl) lysobactin tristrifluoroacetate
Beispiel 6Example 6
(Trimethylsilyl)-D-alanyl-3-(pyridin-3-yl)-L-alanyl-des(l-D-leucyl-2-L-leucyl)-lysobactin-tris- hydrochlorid (Trimethylsilyl) -D-alanyl-3- (pyridin-3-yl) -L-alanyl-des (ID-leucyl-2-L-leucyl) -lysobactinetris hydrochloride
32 g Dowex 1X8-400 (HCl-Form) werden in eine Säule gepackt (35 mm Durchmesser). Ca 60 mL 1 M Natriumhydroxidlösung werden über die Säule gegeben, gefolgt von 60 mL HPLC- Wasser. Dann wird mit 60 mL 1 M Salzsäure konditioniert und anschließend mit ungefähr 100 mL Wasser bis zur neutralen Reaktion des Eluats gewaschen. (Macherey & Nagel Tritest). Dann werden 2.00 g (1.19 mmol) 3-(Trimethylsilyl)-D-alanyl-3-(pyridin-3-yl)-L-alanyl-des(l-D-Ieucyl-2-L-leucyl)- lysobactin-tris-trifluoracetat (Beispiel 2) in 90 mL Wasser gelöst und die Lösung aufgegeben und langsam eluiert. Die Säule wird mit ca. 20 mL Wasser nachgespült. Produkthaltige Eluate werden vereinigt, feinfiltriert (Porengröße 0.20 μm) und lyophilisiert. Die Säule wird erneut konditioniert und wiederverwendet. Man erhält 1.54 g (1.06 mmol, 89% d. Th.) Beispiel 6 aus 2000 mg (1.19 mmol) Beispiel 2.32 g of Dowex 1X8-400 (HCl form) are packed in a column (35 mm diameter). Approximately 60 mL 1 M sodium hydroxide solution is passed through the column, followed by 60 mL HPLC water. It is then conditioned with 60 mL of 1 M hydrochloric acid and then washed with approximately 100 mL of water until neutral reaction of the eluate. (Macherey & Nagel Tritest). Then, 2.00 g (1.19 mmol) of 3- (trimethylsilyl) -D-alanyl-3- (pyridin-3-yl) -L-alanyl-des (ID-leucyl-2-L-leucyl) -lysobactin tris-trifluoroacetate (Example 2) dissolved in 90 mL of water and the solution was abandoned and slowly eluted. The column is rinsed with approx. 20 mL of water. Product-containing eluates are combined, finely filtered (pore size 0.20 μm) and lyophilized. The column is conditioned again and reused. This gives 1.54 g (1.06 mmol, 89% of theory) of Example 6 from 2000 mg (1.19 mmol) of Example 2.
HPLC (Methode 9): Rt = 3.30 min.HPLC (Method 9): R t = 3.30 min.
LC-MS (Methode 7): R, = 1.47 min, MS (ESIpos.): m/z (%) = 671.5 (100) [M+2 H]2+, 1341.4 (20), [M+Hf.LC-MS (Method 7): R, = 1.47 min, MS (ESIpos.): M / z (%) = 671.5 (100) [M + 2 H] 2+ , 1341.4 (20), [M + Hf.
Ionenchromatographie (Methode 25): CY (ber.) - 7.43 %, Cl" gef. = 7.1 %; TFA (gef.) < 0.1 %.Ion Chromatography (Method 25): CY (calc.) - 7.43%, Cl " = 7.1%; TFA (%) <0.1%.
1H-NMR (500 MHz, d5-?yήdiή) δ = -0.170 (s, 9H), 0.580 (d, J= 6.3 Hz, 3H), 0.772 (d, J= 5.7 Hz, 3H), 0.920 (d, J= 5.8 Hz, 3H), 0.974 - 0.985 (m, 6H), 1.147 (d, J= 6.4 Hz, 3H), 1.250 - 1.358 (m, 2H), 1.411 (d, J = 5.7 Hz, 3H), 1.468 (m, IH)3 1.604 (m, IH), 1.959 - 2.049 (m, 3H), 2.154 - 2.228 (m, 3H), 2.380 (m, 3H), 2.935 (m, IH), 3.120 (ms IH), 3.300 (m, IH), 3.607 (d, J= 14.9 Hz, IH), 3.762 (dd, J= 14.7 Hz, IH), 3.882 (d, J = 9.1 Hz, IH), 3.938 (m, IH), 4.204 (m, IH), 4.253 (m, IH), 4.389 (m, IH)3 4.465 (m, IH), 4.589 - 4.661 (m, 4H), 4.806 (d, J= 12.1 Hz, IH), 5.027 (m, IH), 5.202 (dd, J= 9.4 Hz, IH), 5.289 (m, IH), 5.390 (s, IH), 6.000 (d, J= 9.5 Hz, IH)3 6.394 (dd, J= 9.0 Hz, IH), 7.173 (m, 2H), 7.428 (m, IH), 7.54 (d, J = 7.0 Hz, IH), 7.664 - 7.973 (m, 3H), 7.767 (d, J = 8.6, IH), 7.918 - 7.973 (m, 3H), 8.038 (br s, 2H), 8.098 (br s, IH), 8.185 (br s, IH), 8.253 (d, J = 8.6 Hz, IH), 8.582 (m, IH), 8.684 (d, J = 9.8 Hz, IH), 8.741 (br s, IH), 8.782 (m, IH), 9.928 (br s, IH), 10.272 (d, J= 8.3 Hz, IH), 10.886 (br s, IH), 11.135 (br s, IH). 1 H-NMR (500 MHz, d 5 -? Yήdiή) δ = -0.170 (s, 9H), 0.580 (d, J = 6.3 Hz, 3H), 0.772 (d, J = 5.7 Hz, 3H), 0.920 ( d, J = 5.8Hz, 3H), 0.974-0.985 (m, 6H), 1.147 (d, J = 6.4Hz, 3H), 1250-1358 (m, 2H), 1.411 (d, J = 5.7Hz, 3H ), 1,468 (m, IH) 3 1,604 (m, IH), 1,959 - 2,049 (m, 3H), 2,154 - 2,228 (m, 3H), 2,380 (m, 3H), 2,935 (m, IH), 3,120 ( m s IH), 3,300 (m, IH), 3,607 (d, J = 14.9 Hz, IH), 3.762 (dd, J = 14.7 Hz, IH), 3.882 (d, J = 9.1 Hz, IH), 3.938 (m, IH), 4.204 (m, IH), 4.253 (m, IH), 4.389 ( m, IH) 3 4.465 (m, IH), 4.589 - 4.661 (m, 4H), 4.806 (d, J = 12.1 Hz, IH), 5.027 (m, IH), 5.202 (dd, J = 9.4 Hz, IH ), 5,289 (m, IH), 5,390 (s, IH), 6,000 (d, J = 9.5 Hz, IH) 3 6,394 (dd, J = 9.0 Hz, IH), 7,173 (m, 2H), 7,428 (m , IH), 7.54 (d, J = 7.0 Hz, IH), 7,664 - 7,973 (m, 3H), 7,767 (d, J = 8.6, IH), 7,918 - 7,973 (m, 3H), 8,038 (br s, 2H), 8,098 (brs, IH), 8,185 (brs, IH), 8,253 (d, J = 8.6 Hz, IH), 8,582 (m, IH), 8,684 (d, J = 9.8 Hz, IH), 8,741 (brs, IH), 8,782 (m, IH), 9,928 (brs, IH), 10,272 (d, J = 8.3 Hz, IH), 10,886 (brs, IH), 11,135 (brs, IH) ,
19F-NMR (Pyridin, 400 MHz, Methode 24): TFA < Nachweisgrenze. 19 F NMR (pyridine, 400 MHz, Method 24): TFA <detection limit.
Beispiel 7Example 7
(Trimethylsilyl)-D-alanyl-3 -(pyridin-3 -yl)-L-alanyl-des( 1 -D-leucyl^-L-leucyty-lysobactin-tris-L- lactat(Trimethylsilyl) -D-alanyl-3 - (pyridin-3-yl) -L-alanyl-des (1-D-leucyl) -L-leucocyte lysobactin tris-L-lactate
32 g Dowex 1X8-400 (HCl-Form) werden in eine Säule gepackt (35 mm Durchmesser). Ca. 60 mL32 g of Dowex 1X8-400 (HCl form) are packed in a column (35 mm diameter). Approximately 60 mL
1 M Natriumhydroxidlösung werden über die Säule gegeben, gefolgt von 60 mL HPLC- Wasser. Dann wird mit 60 mL 1 M Milchsäure konditioniert und anschließend mit ca. 100 mL Wasser bis zur neutralen Reaktion des Eluats gewaschen. (Macherey & Nagel Tritest). Dann werden 2.00 g (1.19 mmol) S-CTrimethylsilyO-D-alanyl-S-Cpyridin-S-y^-L-alanyl-desCl -D-leucyl-2-L-leucyl)- lysobactin-tris-trifluoracetat (Beispiel 2) in 90 mL Wasser gelöst und die Lösung aufgegeben und langsam eluiβrt. Die Säule wird mit ca. 20 mL Wasser nachgespült. Produkthaltige Eluate werden vereinigt, feinfiltriert (Porengröße 0.20 μm) und lyophilisiert. Die Säule wird erneut konditioniert und wiederverwendet. Man erhält 1903 mg (1.18 mmol, 99% d. Th.) Beispiel 7 aus 2000 mg (1.19 mmol) Beispiel 2.1 M sodium hydroxide solution is passed through the column, followed by 60 mL of HPLC water. It is then conditioned with 60 mL 1 M lactic acid and then washed with approx. 100 mL water until the reaction of the eluate is neutral. (Macherey & Nagel Tritest). Then 2.00 g (1.19 mmol) S-CTrimethylsilyO-D-alanyl-S-cpyridine-Sy ^ -L-alanyl-desCl-D-leucyl-2-L-leucyl) -lysobactin tris-trifluoroacetate (Example 2) dissolved in 90 mL water and the solution is given up and slowly eluted. The column is rinsed with approx. 20 mL of water. Product-containing eluates are combined, finely filtered (pore size 0.20 μm) and lyophilized. The column is conditioned again and reused. 1903 mg (1.18 mmol, 99% of theory) of Example 7 are obtained from 2000 mg (1.19 mmol) of Example 2.
HPLC (Methode 9): Rt = 3.29 min.HPLC (Method 9): R t = 3.29 min.
LC-MS (Methode 7): R, = 1.38 min, MS (ESIpos.): m/z (%) = 671.7 (100) [M+2 H]2+, 1341.8 (20), [M+H]+.LC-MS (Method 7): R, = 1.38 min, MS (ESIpos.): M / z (%) = 671.7 (100) [M + 2 H] 2+ , 1341.8 (20), [M + H] + .
1H-NMR (500 MHz, ^-Pyridin): δ = -0.170 (s, 9H), 0.754 (d, J = 6.3 Hz, 3H), 0.836 (d, J = 6.1 Hz, 3H), 0.922 (d, J= 6.2 Hz, 3H), 0.922 - 0.957 (m, 6H), 1.054 (d, J = 6.4 Hz, 3H), 1.190 (m, 2H), 1.416 (d, J = 5.8 Hz, 3H), 1.439 - 1.662 (m, 4H), 1.529 (d, J = 7.0 Hz, 3H), 1.598 (d, J = 6.5 Hz, 3H), 1.669 (d, J= 6.9 Hz, 3H), 1.923 -2.280 (m, 9H), 3.057 (m, IH), 3.266 (m, IH), 3.564 (d, J= 14. 5 Hz, IH), 3.761 (dd, J= 13.7 Hz, IH), 3.840 (m, IH), 4.049 (m, IH), 4.109 (m, IH), 4.164 (m, IH), 4.202 (m, IH), 4.410 (m, IH), 4.484 (m, IH), 4.616 (dd, J = 6.7, 13.6 Hz, IH), 4.656 (m, IH), 4.669 (dd, J= 7.0, 13.9 Hz, IH), 4.780 (d, J= 13.0 Hz, IH), 4.921 (m, IH), 5.144 (dd, J= 9.6 Hz, IH), 5.281 (m, IH), 5.363 - 5.426 (m, 3H) 5.362 (s, IH), 5.989 (d, J = 9.8 Hz, im, 6.323 (m, IH), 7.122 (m, IH), 7.180 (m, IH), 7.381 (m, IH), 7.596 (m, IH), 7.671 - 7.925 (m, 6H), 8.036 (br s, IH), 8.208 (m, 3H), 8.412 (m, IH), 8.551 (d, J= 3.6 Hz, IH), 8.714 (m, 2H), 8.794 (m, IH), 10.298 (br s, IH), 10.938 (br s, IH). 1 H-NMR (500 MHz, ^ -pyridine): δ = -0.170 (s, 9H), 0.754 (d, J = 6.3 Hz, 3H), 0.836 (d, J = 6.1 Hz, 3H), 0.922 (i.e. , J = 6.2 Hz, 3H), 0.922-0.957 (m, 6H), 1054 (d, J = 6.4 Hz, 3H), 1.190 (m, 2H), 1.416 (d, J = 5.8 Hz, 3H), 1.439 - 1.662 (m, 4H), 1.529 (d, J = 7.0 Hz, 3H), 1.598 (d, J = 6.5 Hz, 3H), 1.669 (d, J = 6.9 Hz, 3H), 1.923-2.280 (m, 9H), 3,057 (m, IH), 3,266 (m, IH), 3,564 (d, J = 14.5 Hz, IH), 3,761 (dd, J = 13.7 Hz, IH), 3,840 (m, IH), 4.049 (m, IH), 4.109 (m, IH), 4.164 (m, IH), 4.202 (m, IH), 4.410 (m, IH), 4.484 (m, IH), 4.616 (dd, J = 6.7, 13.6 Hz, IH), 4.656 (m, IH), 4.669 (dd, J = 7.0, 13.9 Hz, IH), 4,780 (d, J = 13.0 Hz, IH), 4.921 (m, IH), 5.144 (dd, J = 9.6 Hz, IH), 5.281 (m, IH), 5.363 - 5.426 (m, 3H) 5.362 (s, IH), 5.989 (d, J = 9.8 Hz, im, 6.323 (m, IH), 7.122 ( m, IH), 7,180 (m, IH), 7,381 (m, IH), 7,596 (m, IH), 7,671 - 7,925 (m, 6H), 8,036 (brs, IH), 8,208 (m, 3H), 8,412 (m, IH), 8,551 (d, J = 3.6Hz, IH), 8,714 (m, 2H), 8,794 (m, IH), 10,298 (brs, IH), 10,938 (br s, IH).
19F-NMR (Pyridin, 400 MHz, Methode 24): TFA < Nachweisgrenze. 19 F NMR (pyridine, 400 MHz, Method 24): TFA <detection limit.
Beispiel 8Example 8
(Trimethy lsilyl)-D-alany 1-3 -(pyridin-3 -y l)-L-alany l-des( 1 -D-leucy 1-2-L-leucy l)-lysobactin-tris-D- tartrat (Trimethylsilyl) -D-alany 1-3- (pyridin-3-yl) -L-alanyl-des (1-D-leucate 1-2-L-leucyl) -lysobactin-tris-D-tartrate
Konditionieren des Ionentauschers: 5 g DOWEX 1X8 400 (HCl-SaIz) wird mit 1 M Natronlauge gewaschen, bis das Eluat alkalisch reagiert. Dann wäscht man mit 2 Säulenvolumen Wasser und anschließend mit 1 M D- Weinsäure, bis das Eluat sauer reagiert. Dann wäscht man mit Wasser, bis das Eluat neutral ist (Macherey & Nagel Tritest).Conditioning of the ion exchanger: 5 g of DOWEX 1X8 400 (HCl salt) is washed with 1 M sodium hydroxide solution until the eluate is alkaline. Then wash with 2 column volumes of water and then with 1 M D-tartaric acid until the eluate is acidic. Then wash with water until the eluate is neutral (Macherey & Nagel Tritest).
100 mg (59 μmol) Beispiel 2 werden dann in 10 mL Wasser gelöst und auf die Säule aufgetragen. Anschließend wäscht man in mehreren Portionen von ca. 10 mL Wasser nach. Das Edukt löst sich nur mäßig und verbleibt zum Teil auf der Säule. Produkthaltige Eluatfraktionen werden vereinigt und lyophilisiert. Man erhält 57 mg (31 μmol, 52% d. Th.) des Produktes. Die Integration des 1H- NMR Spektrums deutet auf eine Stöchiometrie 1 :3 für (Trimethylsilyl)-D-alanyl-3-(pyridin-3-yl)-L- alanyl-des(l-D-leucyl-2-L-leucyl)-lysobactin : D- Weinsäure hin.100 mg (59 μmol) of Example 2 are then dissolved in 10 mL of water and applied to the column. Then it is washed in several portions of about 10 mL of water. The educt dissolves only moderately and remains partly on the column. Product-containing eluate fractions are combined and lyophilized. 57 mg (31 μmol, 52% of theory) of the product are obtained. The integration of the 1 H-NMR spectrum indicates a stoichiometry 1: 3 for (trimethylsilyl) -D-alanyl-3- (pyridin-3-yl) -L-alanyl-des (ID-leucyl-2-L-leucyl) -lysobactin: D-tartaric acid.
HPLC (Methode 9): R1 = 3.29 min.HPLC (Method 9): R 1 = 3.29 min.
LC-MS (Methode 7): R1 = 1.37 min, MS (ESIpos.): m/z (%) = 671.7 (100) [M+2 H]2+, 1341.9 (20), [M+H]+.LC-MS (method 7): R 1 = 1.37 min, MS (ESIpos.): M / z (%) = 671.7 (100) [M + 2 H] 2+ , 1341.9 (20), [M + H] + .
1H-NMR (500 MHz, rfj-Pyridin): δ = -0.172 (s, 9H), 0.597 (d, J = 6.3 Hz, 3H), 0.776 (d, J = 5.7 Hz, 3H), 0.924 (d, J= 5.8 Hz, 3H), 0.954 - 0.997 (m, 6H), 1.173 (d, J= 5.7 Hz, 3H), 1.303 (m, 2H), 1.434 (d, J= 5.7 Hz, 3H), 1.468 (m, IH), 1.632 (m, IH), 1.972 (m, 2H), 2.051 (IH, m), 2.174 (m, 3H)5 2.402 (m, 3H), 3.129 (m, IH), 3.305 (m, IH), 3.602 (d, J= 15.2 Hz, IH), 3.799 (dd, J= 14.6 Hz, IH), 3.893 (d, J= 12.3 Hz, IH), 3.967 (m, IH), 4.228 (m, IH), 4.276 (m, IH), 4.409 (m, IH), 4.468 (m, IH), 4.578 - 4.669 (m, 4H), 4.791 (d, J^ = 11.5 Hz, IH), 5.044 (m, IH), 5.226 (dd, J= 9.4 Hz, IH), 5.190 (m, IH), 5.362 (s, IH), 5.417 (s, 6H), 5.997 (d, J= 9.7 Hz, IH), 6.403 (dd, J= 8.9 Hz, IH), 7.182 (m, IH), 7.551 - 8.210 (m, 13H), 8.589 (d, J= 3.6 Hz, IH), 8.711 (d, J = 10.0 Hz, IH), 8.782 (m, 2H), 9.902 (br s, IH), 10.358 (m, IH), 10.882 (br s, IH), 11.093 (br s, IH). 1 H-NMR (500 MHz, rfj-pyridine): δ = -0.172 (s, 9H), 0.597 (d, J = 6.3 Hz, 3H), 0.776 (d, J = 5.7 Hz, 3H), 0.924 (i.e. , J = 5.8 Hz, 3H), 0.954 - 0.997 (m, 6H), 1.173 (d, J = 5.7 Hz, 3H), 1.303 (m, 2H), 1,434 (d, J = 5.7Hz, 3H), 1,468 (m, IH), 1,632 (m, IH), 1,972 (m, 2H), 2,051 (IH, m), 2,174 (m, 3H) 5 2.402 (m, 3H), 3.129 (m, IH), 3.305 (m, IH), 3.602 (d, J = 15.2 Hz, IH), 3.799 (dd, J = 14.6 Hz, IH), 3.893 (d, J = 12.3 Hz, IH), 3.967 (m, IH), 4.228 (m, IH), 4.276 (m, IH), 4.409 (m, IH), 4.468 (m, IH), 4.578 - 4.669 (m, 4H) , 4.791 (d, J ^ = 11.5 Hz, IH), 5.044 (m, IH), 5.226 (dd, J = 9.4 Hz, IH), 5.190 (m, IH), 5.362 (s, IH), 5.417 (s , 6H), 5.997 (d, J = 9.7 Hz, IH), 6.403 (dd, J = 8.9 Hz, IH), 7.182 (m, IH), 7.551 - 8.210 (m, 13H), 8.589 (d, J = 3.6 Hz, IH), 8,711 (d, J = 10.0 Hz, IH), 8,782 (m, 2H), 9,902 (brs, IH), 10,358 (m, IH), 10,882 (brs, IH), 11,093 ( br s, IH).
Beispiel 9Example 9
(Trimethylsilyl)-D-alanyl-3-(pyridin-3-yl)-L-alanyl-des( 1 -D-leucyl-2-L-leucyl)-lysobactin(Trimethylsilyl) -D-alanyl-3- (pyridin-3-yl) -L-alanyl-des (1-D-leucyl-2-L-leucyl) -lysobactin
Polymergebundenes Hydrogencarbonat PL-HCO3 (Polymer Labs, Kapazität 1.8 mmol/g) wird in eine Säule gepackt. Zur Umsetzung von 100 mg (59 μmol) der Verbindung Beispiel 2 werden 396 mg (entspricht 12 Äquivalenten Hydrogencarbonat) des Harzes verwendet. Das Harz wird mit Pyridin gespült. Dann wird eine Lösung von Beispiel 2 (100 mg, 59 μmol) in Pyridin (0.75 mL) auf die Säule gegeben und langsam eluiert. Die Säule wird zunächst mit weiterem Pyridin (2 mL) und dann mit Wasser (ca. 2 mL) gespült. Alle Eluate werden vereinigt und ohne Wärmezufuhr im Ölpumpenvakuum eingeengt. Der glasartige Rückstand wird in einigen Tropfen Wasser gelöst und unverzüglich gefriergetrocknet. Man erhält die Titelverbindung als farbloses amorphes Pulver. HPLC (Methode 9): R4 = 3.30 min.Polymer-bound bicarbonate PL-HCO 3 (Polymer Labs, capacity 1.8 mmol / g) is packed in a column. For the reaction of 100 mg (59 μmol) of compound Example 2, 396 mg (corresponding to 12 equivalents of bicarbonate) of the resin are used. The resin is rinsed with pyridine. Then, a solution of Example 2 (100 mg, 59 μmol) in pyridine (0.75 mL) is added to the column and slowly eluted. The column is first flushed with more pyridine (2 mL) and then with water (about 2 mL). All eluates are combined and concentrated without heat in an oil pump vacuum. The glassy residue is dissolved in a few drops of water and immediately freeze-dried. The title compound is obtained as a colorless amorphous powder. HPLC (Method 9): R 4 = 3.30 min.
LC-MS (Methode 7): R1 = 1.49 min, MS (ESIpos.): m/z (%) = 671.8 (100) [M+2 H]2+, 1341.9 (10), [M+H]+, MS (ESIneg): m/z (%) = 669.9 (100) [M-2 H]2\ 1340.0 (100) M-H]-.LC-MS (method 7): R 1 = 1.49 min, MS (ESIpos.): M / z (%) = 671.8 (100) [M + 2 H] 2+ , 1341.9 (10), [M + H] + , MS (ES Ineg): m / z (%) = 669.9 (100) [M-2 H] 2 \ 1340.0 (100) MH] -.
19F-NMR (Pyridin, 400 MHz, Methode 24): TFA 2.9%. 19 F NMR (pyridine, 400 MHz, Method 24): TFA 2.9%.
Beispiel 10Example 10
(Trimethylsilyl)-D-alanyl-3-(pyridin-3-yl)-L-alanyl-des(l-D-leucyl-2-L-leucyl)-lysobactin-mesylat- trifluoracetat(Trimethylsilyl) -D-alanyl-3- (pyridin-3-yl) -L-alanyl-des (1-leucyl-2-L-leucyl) -lysobactin-mesylate trifluoroacetate
Durch Umkristallisation von Beispiel 3 aus mit TFA dotiertem Wasser bei RT kann die Titelver- bindung als kristalline Substanz erhalten werden (langsames Eindampfen der Lösung).By recrystallization of Example 3 from TFA-doped water at RT, the title compound can be obtained as a crystalline substance (slow evaporation of the solution).
LC-MS (Methode 6): Rt = 1.41 min, MS (ESIpos.): m/z (%) = 671.9 (100) [M+2 H i]2+ , 1342.1 (10), [M+H]+, MS (ESIneg): m/z (%) = 669.9 (100) [M-2 Hf, 1340.1 (80) M-H]".LC-MS (Method 6): R t = 1.41 min, MS (ESI pos.): M / z (%) = 671.9 (100) [M + 2 H i] 2+, 1342.1 (10), [M + H ] + , MS (ES Ineg): m / z (%) = 669.9 (100) [M-2 Hf, 1340.1 (80) MH] " .
HR-TOF-MS (Methode 1): C60H97N16O17Si [M + H]+ ber. 1341.6982, gef. 1341.7006.HR-TOF-MS (method 1): C 60 H 97 N 16 O 17 Si [M + H] + calc. 1341.6982, et. 1341.7006.
Die Struktur und die Salzform ist durch eine Einkristall-Röntgenstrukturanalyse bestätigt. B. Bewertung der physiologischen WirksamkeitThe structure and the salt form are confirmed by a single-crystal X-ray structure analysis. B. Evaluation of physiological activity
Die in vitro- Wirkung der erfindungsgemäßen Verbindungen kann in folgenden Assays gezeigt werden:The in vitro activity of the compounds according to the invention can be shown in the following assays:
Bestimmung der minimalen Hemmkonzentration (MHK):Determination of Minimum Inhibitory Concentration (MIC):
Die MHK wird im Flüssigdilutionstest gemäß der NCCLS-Richtlinien bestimmt. Übernachtkultu¬ ren von Staphylococcus aureus 133, Entercococcus faecalis 27159, E. faecium 4147 und Strepto¬ coccus pneumoniae G9a werden mit den beschriebenen Testsubstanzen in einer 1 :2 Verdünnungs¬ reihe inkubiert. Die MHK-Bestimmung wird mit einer Zellzahl von 105 Keimen pro 1 mL in Iso- sensitest-Medium (Fa. Difco, Irvine/USA) durchgeführt, mit Ausnahme von S. pneumoniae, der in BHI-Bouillon (Fa. Difco, Irvine/USA) mit 10% Rinderserum bei einer Zellzahl von 106 Keimen pro mL getestet wird. Die Kulturen werden bei 37°C für 18-24 Stunden inkubiert, S. pneumoniae in Gegenwart von 10% CO2.The MIC is determined in the liquid dilution test according to the NCCLS guidelines. Overnight cultures of Staphylococcus aureus 133, Entercococcus faecalis 27159, E. faecium 4147 and Streptococcus pneumoniae G9a are incubated with the described test substances in a 1: 2 dilution series. The MIC determination is carried out with a cell count of 10 5 germs per 1 ml in Iso-sensitest medium (Difco Co., Irvine / USA), with the exception of S. pneumoniae, which is in BHI broth (Difco, Irvine / USA) is tested with 10% bovine serum at a cell count of 10 6 germs per mL. The cultures are incubated at 37 ° C for 18-24 hours, S. pneumoniae in the presence of 10% CO 2 .
Die jeweils niedrigste Substanzkonzentration, bei der kein sichtbares Bakterienwachstum mehr auftritt, wird als MHK definiert. Die MHK- Werte werden in μg/mL angegeben.The lowest substance concentration at which no visible bacterial growth occurs is defined as MIC. The MIC values are given in μg / mL.
Repräsentative in-vitro- Wirkdaten und fu-Daten für die erfindungsgemäßen Verbindungen sind in Tabelle A wiedergegeben:Representative in vitro activity data and f u data for the compounds according to the invention are reproduced in Table A:
Tabelle ATable A
Die Eignung der erfindungsgemäßen Verbindungen zur Behandlung von bakteriellen Infektionen kann im folgenden Tiermodell gezeigt werden: The suitability of the compounds according to the invention for the treatment of bacterial infections can be demonstrated in the following animal model:
Svstemische Infektion mit Staphylococcus aureus 133:Svstemic infection with Staphylococcus aureus 133:
Zellen von S. aureus 133 werden über Nacht in BHI-Bouillon (Fa. Oxoid, New York/ USA) ange- züchtet. Die Übernachtkultur wird 1:100 in frische BHI-Bouillon verdünnt und für 3 Stunden in¬ kubiert. Die dann in der logarithmischen Wachstumsphase befindlichen Zellen werden abzentrifu- giert und zweimal mit gepufferter, physiologischer Kochsalzlösung gewaschen. Danach wird pho¬ tometrisch eine Zellsuspension in Kochsalzlösung mit einer Extinktion von 50 Einheiten einge¬ stellt. Nach einem Verdünnungsschritt (1 :15) wird diese Suspension 1 : 1 mit einer 10%igen Mucin- lösung gemischt. Von dieser Infektionslösung werden 0.25 mL/20 g Maus intraperitoneal (entspre¬ chend 1x106 Keimen/Maus) appliziert. Die Therapie erfolgt intraperitoneal oder intravenös 30 Minuten nach der Infektion. Für den Infektionsversuch werden weibliche CFWl -Mäuse verwen¬ det. Das Überleben der Tiere wird über 6 Tage protokolliert.S. aureus 133 cells are grown overnight in BHI broth (Oxoid, New York / USA). The overnight culture is diluted 1: 100 in fresh BHI broth and incubated for 3 hours. The cells, which are then in the logarithmic growth phase, are centrifuged off and washed twice with buffered physiological saline. Thereafter, a cell suspension in saline solution with an extinction of 50 units is adjusted by photometry. After a dilution step (1:15), this suspension is mixed 1: 1 with a 10% mucin solution. 0.25 ml / 20 g of mouse are administered intraperitoneally from this infectious solution (corresponding to 1 × 10 6 germs / mouse). Therapy is intraperitoneal or intravenous 30 minutes after infection. For the infection experiment, female CFW1 mice are used. The survival of the animals is recorded over 6 days.
Die Eigenschaften der erfindungsgemäßen Verbindungen im Hinblick auf die Nierenverträglich- keit können im folgenden Tiermodell gezeigt werden:The properties of the compounds according to the invention with regard to kidney compatibility can be shown in the following animal model:
Maus-Modell zur Bestimmung nephrotoxischer Effekte;Mouse model for the determination of nephrotoxic effects;
Nephrotoxische Nebenwirkungen der Nonadepsipeptide werden durch histopathologische Unter¬ suchungen der Nieren in Mäusen nach mehrfacher Gabe einer bestimmten Dosierung analysiert. Dafür werden 5-6 Tiere täglich entweder intravenös (i.v.) oder intraperitoneal (i.p.) mit Substanzen behandelt, die in wässriger Lösung oder unter Zusatz von Solutol gelöst werden. Nierentoxische Effekte werden durch lichtmikroskopische Auswertung Hämatoxilin und Eosin (H&E) gefärbter Paraffinschnitte der Nieren bestimmt. Eine 'Periodic-Acid Schiff (PAS)-Reaktion wird wahlweise zur besseren Darstellung von Glykoproteinen durchgeführt. Nephrotoxische Effekte werden semi¬ quantitativ für jedes Tier als Schweregrade der auftretenden tubulären Basophilie und Degenerati- on/Regeneration festgelegt (Schweregrade: 0 = kein Effekt; 1 = minimaler Effekt; 2 = leichter Effekt; 3 = moderater Effekt; 4 = schwere Läsionen). Der durchschnittliche Schweregrad der tubu¬ lären Degeneration/Regeneration sowie die Inzidenz (Anzahl der betroffenen Tiere) wird pro Tier¬ gruppe oder Derivat berechnet. Darüber hinausgehende Nierenveränderungen wie tubuläre Dilata¬ tion sowie Nekrosen und Ansammlung nekrotischen Materials werden ebenfalls aufgeführt. Ratten-Modell zur Bestimmung nephrotoxischer Effekte:Nephrotoxic side effects of nonadepsipeptides are analyzed by histopathological investigations of the kidneys in mice after repeated administration of a specific dose. 5-6 animals are treated daily either intravenously (iv) or intraperitoneally (ip) with substances which are dissolved in aqueous solution or with the addition of solutol. Kidney toxic effects are determined by light microscopic analysis of hematoxylin and eosin (H & E) stained paraffin sections of the kidneys. A Periodic Acid Schiff (PAS) reaction is optionally carried out to better display glycoproteins. Nephrotoxic effects are determined semi¬ quantitatively for each animal as degrees of severity of the occurring tubular basophilia and degeneration / regeneration (severity levels: 0 = no effect, 1 = minimal effect, 2 = slight effect, 3 = moderate effect, 4 = severe lesions) , The average severity of tubular degeneration / regeneration and the incidence (number of affected animals) are calculated per animal group or derivative. Further kidney changes such as tubular dilatation, necrosis and accumulation of necrotic material are also listed. Rat model for the determination of nephrotoxic effects:
Nephrotoxische Nebenwirkungen der Nonadepsipeptide werden durch histopathologische Unter¬ suchungen der Nieren in Ratten nach mehrfacher Gabe einer bestimmten Dosierung analysiert. Dafür werden 5 Tiere täglich intravenös (i.v.) mit Substanzen behandelt, die in Saline oder Ringer- Lactat-Lösung gelöst werden. Nierentoxische Effekte werden durch lichtmikroskopische Auswer¬ tung Hämatoxilin und Eosin (H&E) gefärbter Paraffinschnitte der Nieren bestimmt. Eine 'Periodic- Acid Schiff (PAS)-Reaktion wird wahlweise zur besseren Darstellung von Glykoproteinen durch¬ geführt. Nephrotoxische Effekte werden semiquantitativ für jedes Tier als Schweregrade der auf¬ tretenden tubulären Basophilie und Degeneration/Regeneration festgelegt (Schweregrade: 0 = kein Effekt; 1 = minimaler Effekt; 2 = leichter Effekt; 3 = moderater Effekt; 4 = schwere Läsionen). Der durchschnittliche Schweregrad der tubulären Degeneration/Regeneration sowie die Inzidenz (Anzahl der betroffenen Tiere) wird pro Tiergruppe oder Derivat berechnet. Darüber hinausgehen¬ de Nierenveränderungen wie tubuläre Dilatation sowie Nekrosen und Ansammlung nekrotischen Materials werden ebenfalls aufgeführt.Nephrotoxic side effects of the nonadepsipeptides are analyzed by histopathological investigations of the kidneys in rats after repeated administration of a specific dose. For this purpose, 5 animals are treated intravenously (i.v.) daily with substances which are dissolved in saline or Ringer's lactate solution. Kidney toxic effects are determined by light microscopic evaluation of hematoxylin and eosin (H & E) stained paraffin sections of the kidneys. A periodic acid Schiff (PAS) reaction is optionally carried out for the better presentation of glycoproteins. Nephrotoxic effects are defined semiquantitatively for each animal as severity of onset tubular basophilia and degeneration / regeneration (severity levels: 0 = no effect, 1 = minimal effect, 2 = mild effect, 3 = moderate effect, 4 = severe lesions). The average severity of tubular degeneration / regeneration and the incidence (number of affected animals) are calculated per animal group or derivative. Beyond kidney changes such as tubular dilatation and necrosis and accumulation of necrotic material are also listed.
Prinzip der Bestimmung der freien Fraktion via Transil:Principle of determining the free fraction via Transil:
Die hier beschriebene Methode zur Bestimmung der freien Fraktion (fu) einer Prüfsubstanz glie¬ dert sich in 2 Teile:The method described here for determining the free fraction (f u ) of a test substance is divided into 2 parts:
a) Bestimmung des Transil®/Puffer- Verteilungsverhältnisses (MAbUffer) durch Inkubation der Prüf¬ substanz in einer Transil®-Puffer (pH 7.4)-Dispersion und anschließende Bestimmung der Kon- zentration in der Dispersion und im Puffer-Überstand.a) determination of the Transil ® / buffer distribution ratio (MA buffer) by incubating the Prüf¬ substance in a Transil ® buffer (pH 7.4) dispersion and subsequent determination of the concentra- tion of the dispersion and in the buffer supernatant.
b) Bestimmung des Transil®/Plasma-Verteilungsverhältnisses (MApiasma) durch Inkubation der Prüfsubstanz in einer Transil®-Plasma-Dispersion und anschließende Bestimmung der Konzentra¬ tion in der Dispersion und im Plasma.b) determination of the Transil ® / plasma distribution ratio (MA p asma i) by incubating the test substance in a Transil ® -plasma dispersion and subsequent determination of Konzentra¬ tion in the dispersion and in the plasma.
Der Quotient der beiden Verteilungsverhältnisse ergibt fu.The quotient of the two distribution ratios gives f u .
Bei hochproteingebundenen Substanzen wird das Plasma in der Regel mit isotonischem Phosphat¬ puffer (pH 7.4) verdünnt und anschließend mit Transil® suspendiert. Die Bestimmung von fu' (freie Fraktion in verdünntem Plasma) in dieser verdünnten Proteinlösung erfolgt analog zur Bestim¬ mung von fu. Die freie Fraktion in unverdünntem Plasma wird aus fu' und dem Verdünnungsfaktor berechnet. Vergleich zu dieser Methode auch: Schuhmacher, Joachim; Kohlsdorfer, Christian; Buehner, Klaus; Brandenburger, Tim; Kruk, Renate, "High-throughput determination of the free fraction of drugs strongly bound to plasma proteins." Journal ofPharmaceutical Sciences 2004, 93, 816-830.In highly protein-bound substances, the plasma is usually diluted with isotonic phosphate-buffer (pH 7.4) and then suspended with Transil ®. The determination of f u '(free fraction in diluted plasma) in this diluted protein solution is carried out analogously to the determination of f u . The free fraction in undiluted plasma is calculated from f u 'and the dilution factor. Comparison to this method also: Schuhmacher, Joachim; Kohlsdorfer, Christian; Buehner, Klaus; Brandenburger, Tim; Kruk, Renate, "High-throughput determination of the free fraction of drugs bound to plasma proteins." Journal of Pharmaceutical Sciences 2004, 93, 816-830.
Bestimmung der Membranaffinität einer Prüfsubstanz nach Verteilung zwischen Transil® αnd Puffer (MAh,,»,,):Determining the membrane affinity of a test substance on the distribution between Transil ® αnd buffer (MAh ,, ",,):
Alle Inkubationen werden in geeigneten Glasgefäßen, z. B. Glasvials, Schliffreagenzgläsern, durchgeführt. In der Regel beträgt das Gesamtvolumen 0.5-5 mL, das Transil® Volumen 10-100 μl. Im Falle von hohen erwarteten Membranaffinitäten kann die Transil® Dispersion mit Phosphatpuf¬ fer pH 7.4 z.B. Dulbeccos PBS bis zu 20fach verdünnt werden. Phosphatpuffer pH 7.4 wird in die Inkubationsgefaße vorgelegt und das Transil® nach gründlichem Mischen zupipettiert. Die Prüf¬ substanz wird in einer Konzentration von z.B. 200 ng/mL, n = 6, zupipettiert. Der Anteil an orga¬ nischem Lösungsmittel sollte <2% sein. Die Ansätze werden 30 min bei Raumtemperatur z.B. auf einem Mini-Shaker im Winkel von ca. 45° bei ca. 400 UPM inkubiert. Zur Bestimmung des 100% Wertes wird mindestens ein Aliquot von z.B. 100 μl entnommen, der restliche Ansatz wird ca. 10 min bei ca. 1800 g zentrifugiert. Von jeder Probe werden mindestens 2 Aliquote (z.B. 100 μl) des Überstandes für die Konzentrationsbestimmung entnommen.All incubations are carried out in suitable glass jars, e.g. B. glass vials, grinding test tubes performed. In general, the total volume of 0.5-5 mL is that Transil ® volume 10-100 ul. In the case of high expected membrane affinities of the Transil ® dispersion with Phosphatpuf¬ fer pH 7.4, for example Dulbecco's PBS can be diluted up to 20 times. Phosphate buffer pH 7.4 is added to the incubation vessels and the Transil ® is pipetted in after thorough mixing. The test substance is pipetted in a concentration of, for example, 200 ng / ml, n = 6. The proportion of organic solvent should be <2%. The mixtures are incubated for 30 min at room temperature, for example on a mini shaker at an angle of about 45 ° at about 400 rpm. To determine the 100% value, at least one aliquot of, for example, 100 .mu.l is taken, the remaining batch is centrifuged for about 10 min at about 1800 g. From each sample, at least 2 aliquots (eg, 100 μl) of the supernatant are taken for concentration determination.
Das Gesamtinkubationsvolumen und das zugesetzte Transil® Volumen hängen von der erwarteten freien Fraktion ab. In der Regel beträgt das Gesamtvolumen 0.5 —1 mL, das Transil® Volumen 10- 100 μl. Im Falle von sehr niedrigen freien Fraktionen wird das Plasma der zu untersuchenden Spe¬ zies mit isotonischer Pufferlösung, pH 7.4 z.B. 10 -400fach verdünnt und anschließend mit Tran¬ sil® versetzt. Das weitere Vorgehen erfolgt wie oben für die Bestimmung der MAbUffer Werte be¬ schrieben.The total incubation and the added Transil ® volume will depend on the expected free fraction. In general, the total volume of 0.5 mL -1 is that Transil ® volume 10- 100 ul. In the case of very low free fractions of the plasma to be tested is Spe¬ zies with isotonic buffer solution, pH 7.4, for example, 10 -400fach diluted and then added with Tran¬ sil ®. The further procedure is as described above for the determination of the MAb Uffer values.
Prinzip der Bestimmung der freien Fraktion via Ultrafiltration:Principle of determination of the free fraction via ultrafiltration:
Das Plasma der zu untersuchenden Spezies wird über eine semipermeable Membran filtriert. Die Substanzkonzentration im Filtrat wird gemessen und daraus die freie Fraktion fu berechnet. Das Centrifree micropartition System von Millipore/Amicon wird verwendet. Die Ultrafiltrations¬ membranen haben eine Ausschlussgröße von 30 000 Da. Die Substanz wird zu 1 mL Plasma in einer Konzentration von ca. 1 μg/mL zudotiert. Der Lösungsmittelanteil sollte < 2 % sein. Nach 30 minütiger Inkubation bei Raumtemperatur wird das Plasma in das Ultrafiltrationssystem pipettiert und 10 Minuten bei 1800 g zentrifugiert. Die Substanzkonzentration im Ultrafϊltrat (Cu; ungebun- dene Substanzkonzentration) und im Plasma vor der Zentrifugation (C; Gesamtsubstanzkonzentra¬ tion) wird gemessen. Die freie Fraktion errechnet sich nach der Formel: fu (%) = CxJC *100. The plasma of the species to be examined is filtered through a semipermeable membrane. The concentration of the substance in the filtrate is measured and from this the free fraction fu is calculated. The Centrifree micropartition system from Millipore / Amicon is used. The ultrafiltration membranes have an exclusion size of 30,000 Da. The substance is added to 1 mL plasma at a concentration of approx. 1 μg / mL. The solvent content should be <2%. After 30 minutes incubation at room temperature, the plasma is pipetted into the ultrafiltration system and centrifuged for 10 minutes at 1800 g. The substance concentration in the ultrafiltrate (C u, unbound) dene substance concentration) and in the plasma before centrifugation (C; Gesamttsubstanzkonzentra¬ tion) is measured. The free fraction is calculated according to the formula: f u (%) = C x JC * 100.
C. Ausführungsbeispiele für pharmazeutische ZusammensetzungenC. Embodiments of Pharmaceutical Compositions
Die erfindungsgemäßen Verbindungen können folgendermaßen in pharmazeutische Zubereitungen überfuhrt werden:The compounds according to the invention can be converted into pharmaceutical preparations as follows:
Tablette:Tablet:
Zusammensetzung:Composition:
100 mg der Verbindung von Beispiel 1, 50 mg Lactose (Monohydrat), 50 mg Maisstärke (nativ), 10 mg Polyvinylpyrolidon (PVP 25) (Fa. BASF, Ludwigshafen, Deutschland) und 2 mg Magnesi- umstearat.100 mg of the compound of Example 1, 50 mg of lactose (monohydrate), 50 mg of corn starch (native), 10 mg of polyvinylpyrrolidone (PVP 25) (BASF, Ludwigshafen, Germany) and 2 mg of magnesium stearate.
Tablettengewicht 212 mg. Durchmesser 8 mm, Wölbungsradius 12 mm.Tablet weight 212 mg. Diameter 8 mm, radius of curvature 12 mm.
Herstellung:production:
Die Mischung aus Wirkstoff, Lactose und Stärke wird mit einer 5%-igen Lösung (m/m) des PVPs in Wasser granuliert. Das Granulat wird nach dem Trocknen mit dem Magnesiumstearat für 5 min gemischt. Diese Mischung wird mit einer üblichen Tablettenpresse verpresst (Format der Tablette siehe oben). Als Richtwert für die Verpressung wird eine Presskraft von 15 kN verwendet.The mixture of active ingredient, 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 min. This mixture is compressed with a conventional tablet press (for the tablet format see above). As a guideline for the compression, a pressing force of 15 kN is used.
Oral applizierbare Suspension;Orally administrable suspension;
Zusammensetzung:Composition:
1000 mg der Verbindung von Beispiel 1, 1000 mg Ethanol (96%), 400 mg Rhodigel (Xanthan gum der Fa. FMC, Pennsylvania, USA) und 99 g Wasser.1000 mg of the compound of Example 1, 1000 mg of ethanol (96%), 400 mg of Rhodigel (xanthan gum from FMC, Pennsylvania, USA) and 99 g of water.
Einer Einzeldosis von 100 mg der erfindungsgemäßen Verbindung entsprechen 10 mL orale Sus- pension.A single dose of 100 mg of the compound according to the invention corresponds to 10 ml of oral suspension.
Herstellung:production:
Das Rhodigel wird in Ethanol suspendiert, der Wirkstoff wird der Suspension zugefügt. Unter Rühren erfolgt die Zugabe des Wassers. Bis zum Abschluss der Quellung des Rhodigels wird ca. 6h gerührt. Intravenös applizierbare Lösung;The rhodigel is suspended in ethanol, the active ingredient is added to the suspension. While stirring, the addition of water. Until the swelling of the Rhodigels is complete, it is stirred for about 6 hours. Intravenously administrable solution;
Zusammensetzung:Composition:
100-200 mg der Verbindung von Beispiel 1, 15 g Polyethylenglykol 400 und 250 g Wasser für Injektionszwecke.100-200 mg of the compound of Example 1, 15 g of polyethylene glycol 400 and 250 g of water for injection.
Herstellung:production:
Die Verbindung von Beispiel 1 wird zusammen mit Polyethylenglykol 400 in dem Wasser unter Rühren gelöst. Die Lösung wird sterilfiltriert (Porendurchmesser 0.22 μm) und unter aseptischen Bedingungen in hitzesterilisierte Infusionsflaschen abgefüllt. Diese werden mit Infusionsstopfen und Bördelkappen verschlossen. The compound of Example 1 is dissolved together with polyethylene glycol 400 in the water with stirring. The solution is sterile-filtered (pore diameter 0.22 μm) and filled under aseptic conditions into heat-sterilized infusion bottles. These are closed with infusion stoppers and crimp caps.

Claims

Patentansprüche claims
1. Verbindung der Formel1. Compound of the formula
in welcherin which
R1 Wasserstoff bedeutet undR 1 is hydrogen and
R2 2,2-Dimethylbut-l-yl, 2-Ethyl-2-methylbut-l-yl, 2,2-Diethyl-but-l-yl, 2,2- Dimethylpent-1-yl oder Trimethylsilylmethyl bedeutet,R 2 is 2,2-dimethylbut-1-yl, 2-ethyl-2-methylbut-1-yl, 2,2-diethyl-but-1-yl, 2,2-dimethylpent-1-yl or trimethylsilylmethyl,
oderor
R1 Trifluormethyl bedeutet undR 1 is trifluoromethyl and
R2 2,2-Dimethylprop-l-yl, 2,2-Dimethylbut-l-yl, 2-Ethyl-2-methylbut-l-yl, 2,2- Diethyl-but-l-yl, 2,2-Dimethylpent-l-yl oder Trimethylsilylmethyl bedeutet,R 2 2,2-dimethylprop-1-yl, 2,2-dimethylbut-1-yl, 2-ethyl-2-methylbut-1-yl, 2,2-diethyl-but-1-yl, 2,2- Dimethylpent-1-yl or trimethylsilylmethyl,
oder eines ihrer Salze, ihrer Solvate oder der Solvate ihrer Salze.or one of its salts, its solvates or the solvates of its salts.
2. Verbindung nach Anspruch 1, dadurch gekennzeichnet, dass2. A compound according to claim 1, characterized in that
R1 Wasserstoff bedeutet undR 1 is hydrogen and
R2 2,2-Dimethylbut-l-yl oder Trimethylsilylmethyl bedeutet, oderR 2 is 2,2-dimethylbut-1-yl or trimethylsilylmethyl, or
R1 Trifluormethyl bedeutet undR 1 is trifluoromethyl and
R2 2,2-Dimethylprop-l-yl, 2,2-Dimethylbut-l-yl oder Trimethylsilylmethyl bedeutet.R 2 is 2,2-dimethylprop-1-yl, 2,2-dimethylbut-1-yl or trimethylsilylmethyl.
3. Verbindung nach Anspruch 1, dadurch gekennzeichnet, dass3. A compound according to claim 1, characterized in that
R1 Wasserstoff bedeutet undR 1 is hydrogen and
R2 2,2-Dimethylbut-l-yl, 2-Ethyl-2-methylbut-l-yl, 2,2-Diethyl-but-l-yl oder Tri¬ methylsilylmethyl bedeutet.R 2 is 2,2-dimethylbut-1-yl, 2-ethyl-2-methylbut-1-yl, 2,2-diethyl-but-1-yl or trimethylsilylmethyl.
4. Verbindung nach Anspruch 1 mit folgender Struktur 3-(Trimethylsilyl)-D-alanyl-3- (pyridin-3-yl)-L-alanyl-des( 1 -D-leucyl-2-L-leucyl)-lysobactin4. A compound according to claim 1 having the following structure: 3- (trimethylsilyl) -D-alanyl-3- (pyridin-3-yl) -L-alanyl-des (1-D-leucyl-2-L-leucyl) -lysobactin
oder eines ihrer Salze, ihrer Solvate oder der Solvate ihrer Salze.or one of its salts, its solvates or the solvates of its salts.
5. Verfahren zur Herstellung einer Verbindung der Formel (I) nach Anspruch 1, dadurch gekennzeichnet, dass die Verbindung der Formel 5. A process for preparing a compound of formula (I) according to claim 1, characterized in that the compound of formula
mit einer Verbindung der Formelwith a compound of the formula
in welcherin which
R1 und R2 die in Anspruch 1 angegebene Bedeutung haben, undR 1 and R 2 have the meaning given in claim 1, and
X1 Halogen, bevorzugt Brom, Chlor oder Fluor, oder Hydroxy bedeutet,X 1 is halogen, preferably bromine, chlorine or fluorine, or hydroxy,
umgesetzt wird.is implemented.
6. Verbindung nach einem der Ansprüche 1 bis 4 zur Behandlung und/oder Prophylaxe von Krankheiten.6. A compound according to any one of claims 1 to 4 for the treatment and / or prophylaxis of diseases.
7. Verwendung einer Verbindung nach einem der Ansprüche 1 bis 4 zur Herstellung eines Arzneimittels zur Behandlung und/oder Prophylaxe von Krankheiten. 7. Use of a compound according to any one of claims 1 to 4 for the manufacture of a medicament for the treatment and / or prophylaxis of diseases.
8. Verwendung einer Verbindung nach einem der Ansprüche 1 bis 4 zur Herstellung eines Arzneimittels zur Behandlung und/oder Prophylaxe von bakteriellen Infektionen.8. Use of a compound according to any one of claims 1 to 4 for the manufacture of a medicament for the treatment and / or prophylaxis of bacterial infections.
9. Arzneimittel enthaltend eine Verbindung nach einem der Ansprüche 1 bis 4 in Kombinati¬ on mit einem inerten, nichttoxischen, pharmazeutisch geeigneten Hilfsstoff.9. Medicament comprising a compound according to any one of claims 1 to 4 in combination with an inert, non-toxic, pharmaceutically suitable excipient.
10. Arzneimittel nach Anspruch 9 zur Behandlung und/oder Prophylaxe von bakteriellen In¬ fektionen.10. Medicament according to claim 9 for the treatment and / or prophylaxis of bacterial infections.
11. Verfahren zur Bekämpfung von bakteriellen Infektionen in Menschen und Tieren durch Verabreichung einer antibakteriell wirksamen Menge mindestens einer Verbindung nach einem der Ansprüche 1 bis 4, eines Arzneimittels nach Anspruch 9 oder eines nach An- Spruch 7 oder 8 erhaltenen Arzneimittels. 11. A method for combating bacterial infections in humans and animals by administering an antibacterially effective amount of at least one compound according to any one of claims 1 to 4, a drug according to claim 9 or a obtained according to claim 7 or 8 drug.
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