EP1809657A1

EP1809657A1 - Cyclic nonapeptide amides

Info

Publication number: EP1809657A1
Application number: EP05798236A
Authority: EP
Inventors: Franz Von Nussbaum; Nina Brunner; Chantal FÜRSTNER; Rainer Endermann; Jacques Ragot; Joachim Telser; Werner Schröder; Sonja Anlauf; Joachim Schuhmacher; Elke Hartmann
Original assignee: Aicuris GmbH and Co KG
Current assignee: Aicuris GmbH and Co KG
Priority date: 2004-11-05
Filing date: 2005-10-26
Publication date: 2007-07-25
Also published as: CA2602070A1; JP2008518986A; WO2006048156A1; CN101107265A; US20080070884A1; JP4843615B2; DE102004053410A1; US7718611B2

Abstract

The invention relates to cyclic nonapeptide amides, methods for the production thereof, and the use thereof for producing medicaments used for treating and/or preventing diseases, especially bacterial infectious diseases.

Description

Cyclic nonapeptyldamides

The invention relates to cyclic Nonapeptidamide and process for their preparation and their use for the preparation of medicaments for the treatment and / or prophylaxis of diseases, in particular bacterial infectious diseases.

The bacterial cell wall is synthesized by a number of enzymes (cell wall biosynthesis) and is essential for the survival or multiplication of microorganisms. The structure of this macromolecule, as well as the proteins involved in its synthesis, are highly conserved within the bacteria. Due to their essential nature and uniformity, cell wall biosynthesis is an ideal target for new antibiotics (D.W.Green, The bacterial cell wall as a source of antibacterial targets, Expert Opin. Ther. Targets, 2002, 6, 1-19).

Vancomycin and penicillins are inhibitors of bacterial cell wall biosynthesis and are successful examples of the antibiotic potency of this principle. They have been used for several decades in the clinic for the treatment of bacterial infections, especially with Gram-positive pathogens. Due to the increasing incidence of resistant germs, e.g. Methicillin-resistant staphylococci, penicillin-resistant pneumococci, and vancomycin-resistant enterococci (F. Baquero, Gram-positive resistance: challenge for the development of new antibiotics, J. 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, vancomycin for the first time. resistant staphylococci (B. Goldrick, First reported case of VRSA in the United States, Am.J. Nurs., 2002, 102, 17), these substances are increasingly losing their therapeutic efficacy.

The present invention describes a new class of cell wall biosynthesis inhibitors without cross-resistance to known antibiotic classes.

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 antibacterial activity.

The antibacterial activity of lysobactin and katanosin A is further described in O'Sullivan, J. et al., J. Antibiot. 1988, 41, 1740-1744, Bonner, DP et al, J. Antibiot. 1988, 41, 1745-1751, Shoji, J. et al, J. Antibiot. 1988, 41, 713-718 and Tymiak, AA et al, J. Org. Chem. 1989, 54, 1149-1157. The stability of an active substance is an important parameter for its suitability as a medicinal product. The stability plays u. a role in the storage and presentation of drugs. Many natural products show inadequate stability for drugs.

The antibacterial depsipeptide lysobactin hydrolyzes in aqueous neutral to basic medium (pH> 7) within days. This produces the "antibacterially inactive" "ope" lysobactin which is opened on the lactone. "Therefore, effective analogs of lysobactin with a higher ring stability are desirable.

An object of the present invention is to provide alternative compounds to lysobactin having comparable or improved antibacterial activity, better compatibility, e.g. B. lower nephrotoxicity, and improved stability in an aqueous neutral to basic environment for the treatment of bacterial diseases in humans and animals to provide.

In the context of this invention, it has surprisingly been found that lysobactinamides (cyclic nonapeptide amides) have an analogous antibacterial action as lysobactin and are stable to hydrolysis in an aqueous neutral to basic medium. Lysobactinamides are previously undescribed aza analogs of lysobactin in which the central lactone functionality is replaced by a lactam functionality.

The invention relates to compounds of the formula

in which

R is hydrogen or methyl,

R ^{7 is} a group of the formula

stands,

in which

the point of attachment to the amine means

R ^{1 is} C ₁ -C ₆ -alkyl, C ₂ -C ₆ -alkenyl, C ₃ -C ₆ -cycloalkyl or C ₆ -C ₁₀ -aryl,

wherein alkyl, alkenyl, cycloalkyl and aryl may be substituted with 0, 1, 2 or 3 substituents independently selected from the group consisting of halogen, hydroxy, amino, cyano, trimethylsilyl, Ci-C ₆ alkyl, C ₁ -C ₆ -alkoxy, benzyloxy, C ₃ -C ₆ cycloalkyl, C ₆ -C ₁₀ aryl, 5- to 7-membered heterocyclyl, 5- to 10-membered heteroaryl, Ci-C _ö alkylamino, C ₆ -C ₁₀ - arylamino, C ₁ -C ₆ - alkylcarbonylamino, C ₆ -C ₁₀ aryl-carbonyl-arnmo, Ci-Cβ-alkylcarbonyl, Ci-C ₆ - alkoxycarbonyl, C ₆ -Cio-arylcarbonyl, and Beαzyloxy-carbonylamino,

in which cycloalkyl, aryl, heterocyclyl and heteroaryl in turn may be substituted by 0, 1, 2 or 3 substituents independently aus¬ selected from the group consisting of halogen, hydroxy, amino, cyano, nitro, trifluoromethyl, C ₁ -C ₆ -AllCyI , C _r C ₆ alkoxy, phenyl and 5- to 7-membered heterocyclyl,

R ² is C _r C ₆ alkyl, C ₂ -C ₆ alkenyl, C ₃ -C ₆ -cycloalkyl or C ₆ -C represents ₀ aryl,

where alkyl, alkenyl, cycloalkyl and aryl may be substituted by 0, 1, 2 or 3 substituents independently of one another selected from the group consisting of halogen, hydroxyl, amino, cyano, trimethylsilyl, C ₁ -C ₆ -alkyl, C _r C ₆ - alkoxy, benzyloxy, C ₃ -C ₆ cycloalkyl, C ₆ -Cio-aryl, 5- to 7-membered heterocyclyl, 5- to 10-membered heteroaryl, Ci-C ₆ -alkylamino, C ₆ -C ₀ - arylamino, C _r C ₆ - alkylcarbonylamino, Cβ-Cio-arylcarbonyl-amino, C) -C ₆ alkylcarbonyl, Ci-C ₆ - Alkoxycarbonyl, Q-Cio-arylcarbonyl and benzyloxycarbonylamino,

wherein cycloalkyl, aryl, heterocyclyl and heteroaryl for their part may be substituted with 0, 1, 2 or 3 substituents independently aus¬ selected from the group consisting of halogen, hydroxy, amino, cyano, nitro, trifluoromethyl, C _r C ₆ alkyl, C _r C ₆ alkoxy, phenyl and 5- to 7-membered heterocyclyl,

R ⁴ is C, -C ₆ alkyl, C ₂ -C ₆ alkenyl, C ₃ -C ₆ -cycloalkyl or C ₆ -C ₁ 0 aryl,

where alkyl, alkenyl, cycloalkyl and aryl may be substituted by 0, 1, 2 or 3 substituents independently of one another selected from the group consisting of halogen, hydroxyl, amino, cyano, trimethylsilyl, C ₁ -C ₆ -alkyl, C ₁ -C ₆ - alkoxy,

Benzyloxy, C ₃ -C ₆ cycloalkyl, C ₆ -C ₀ aryl, 5- to 7-membered heterocyclyl, 5- to 10-membered heteroaryl, CrCe- alkylamino, C ₀ -C ₀ - arylamino, C ₁ -C ₆ - alkylcarbonylamino, C _ö -Cio-arylcarbonyl-amino, QC _ö alkylcarbonyl, C ₁ -C ₆ - alkoxycarbonyl, C ₆ -C ₀ aryl-carbonyl and benzyloxy-carbonylamino,

R ^{5 is} C 1 -C ₆ -alkyl, C ₂ -C ₆ -alkenyl, C ₃ -C ₆ -cycloalkyl or C ₆ -C ₁₀ -aryl,

where alkyl, alkenyl, cycloalkyl and aryl may be substituted by 0, 1, 2 or 3 substituents independently of one another selected from the group consisting of halogen, hydroxyl, amino, cyano, trimethylsilyl, C ₁ -C ₆ -alkyl, C ₁ -C ₆ - -alkoxy, BenΣyloxy, C ₃ -C ₆ cycloalkyl, C ₆ -C ₀ aryl, 5- to 7-membered heterocyclyl, 5- to 10-membered heteroaryl, Ci-C ₆ -alkylamino, C ₆ -C ₀ -Arylamino, C _r C ₆ -

Alkylcarbonylamino, C _δ- Cio-arylcarbonyl-amino, Cx-Qj-alkylcarbonyl, C] -C ₆ -alkoxycarbonyl, Cβ-Cio-arylcarbonyl and benzyloxy-caxbonylamino,

in which cycloalkyl, aryl, heterocyclyl and heteroaryl may in turn be substituted by 0, 1, 2 or 3 substituents independently of one another selected from the group consisting of halogen, hydroxyl, amino, cyano,

Nitro, trifluoromethyl, Ci-C ₆ alkyl, C _r C ₆ alkoxy, phenyl and 5- to 7-membered heterocyclyl, and their salts, their solvates, and the solvates of their salts.

Compounds of the invention are the compounds of the formula (I), (Ia) and (Ib) and their salts, solvates, solvates of the salts and prodrugs, the compounds of the formulas (I), (Ia) and (Ib) of the formulas mentioned below and their salts, solvates, solvates of the salts and prodrugs, and the compounds of formula (I), (Ia) and (Ib), hereinafter referred to as exemplary compounds and their salts, solvates, solvates of the salts and prodrugs, as far as they are of compounds (I), (Ia) and (Ib), compounds mentioned below are not already salts, solvates, solvates of the salts and prodrugs.

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.

If the compounds according to the invention can occur in tautomeric forms, the present invention encompasses all tautomeric forms.

Salts in the context of the present invention are physiologically acceptable salts of the compounds according to the invention. But are also included salts that are not suitable for pharmaceutical applications themselves but can be used for example for the isolation or purification of the inventive compounds or mixed salts. 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.

Physiologically acceptable salts of the compounds of the invention include acid addition salts of mineral acids, carboxylic acids and sulfonic acids, e.g. Salts of Hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, methanesulfonic acid, ethanesulfonic acid, toluenesulfonic acid, benzenesulfonic acid, naphthalenedisulfonic acid, acetic acid, trifluoroacetic acid, propionic acid, lactic acid, tartaric acid, malic acid, citric acid, fumaric acid, maleic acid and benzoic acid.

Physiologically acceptable salts of the compounds according to the invention also include salts of customary bases, such as, by way of example and by way of preference, alkali metal salts (for example sodium and potassium salts), alkaline earth salts (for example calcium and magnesium salts) and ammonium salts derived from ammonia or organic amines having from 1 to 16 carbon atoms, as exemplified and preferably, ethylamine, diethylamine, triethylamine, ethyldiisopropylamine, monoethanolamine, diethanolamine, triethanolamine, dicyclohexylamine, dimethylaminoethanol, procaine, dibenzylamine, N-methyl morpholine, arginine, lysine, ethylenediamine and N-methylpiperidine.

Solvates in the context of the invention are those forms of the compounds according to the invention which form a complex in the solid or liquid state by coordination with solvent molecules. Hydrates are a special form of solvates that coordinate with water.

Unless otherwise specified, in the context of the present invention, the substituents have the following meaning:

Alkyl per se and "Alk" and "alkyl" in alkoxy, alkylamino, alkylcarbonyl, alkoxycarbonyl and alkylcarbonylamino is a linear or branched alkyl radical having usually 1 to 6, preferably 1 to 4, particularly preferably 1 to 3 carbon atoms, by way of example and preferably methyl, ethyl, n-propyl, isopropyl, tert-butyl, 2,2-dimethylprop-1-yl, 2,2-dimethylbut-1-yl, n-pentyl and n-hexyl.

Alkoxy is, by way of example and by way of preference, methoxy, ethoxy, n-propoxy, isopropoxy, tert-butoxy, n-pentoxy and n-hexoxy.

Alkenyl represents a straight-chain or branched alkenyl radical having 2 to 6 carbon atoms. Preference is given to a straight-chain or branched alkenyl radical having 2 to 4, particularly preferably 2 to 3, carbon atoms. Examples which may be mentioned by way of example and by way of preference: vinyl, alumina, n-prop-1-en-1-yl, n-but-2-en-1-yl, 2-methylprop-1-en-1-yl and 2-methylpropyl 2-en-l-yl.

Alkylamino is an alkylamino radical having one or two (independently selected) alkyl substituents, by way of example and preferably methylamino, ethylamino, n-

Propylamino, isopropylamino, tert-butylamino, n-pentylamino, n-hexylamino, N, N-dimethylamino,

N, N-diethylamino, N-ethyl-N-methylamino, N-methyl-Nn-propylamino, N-isopropyl-N-propylamino, N-tert-butyl-N-methylamino, N-ethyl-N-II-pentylarnino and Nn-hexyl-N-methylamino. C ₁ -C ₃ -alkylamino is, for example, a monoalkylamino radical having 1 to 3 carbon atoms or a dialkylamino radical having 1 to 3 carbon atoms each

Alkyl substituent.

Arylamino is an aryl substituent bonded via an amino group, where the amino group may optionally have a further substituent, such as e.g. Aryl or alkyl, is exemplified and preferably phenylamino, Νaphthylamino, phenylmethylamino or diphenylamino.

Alkylcarbonyl is by way of example and preferably methylcarbonyl, ethylcarbonyl, n-propyl carbonyl, isopropylcarbonyl, tert-butylcarbonyl, n-pentylcarbonyl and n-hexylcarbonyl.

Alkoxycarbonyl is exemplified and preferably methoxycarboiryl, ethoxycarbonyl, n-propoxycarbonyl, isopropoxycarbonyl, terf-butoxycarbonyl, n-pentoxycarborryl and n-hexoxycarbonyl.

Arylcarbonyl is an aryl substituent bonded via a carbonyl group, by way of example and preferably phenylcarbonyl, naphthylcarbonyl and phenanthrertylcarbonyl.

Alkylcarbonylamino is by way of example and preferably methylcarbonylamino, ethylcarbonylamino, n-propylcarbonylamino, isopropylcarbonylamino, tert-butylcarbonylamino, n-pentylcarbonylamino and n-hexylcarbonylamino.

Arylcarbonylamino is by way of example and preferably phenylcarbonylamino, naphthylcarbonylamino and phenanthrenylcarbonylamino.

Cycloalkyl represents a cycloalkyl group having usually 3 to 6 Kolilenstoffatomen, by way of example and preferably cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl.

Aryl is a mono- to tricyclic aromatic, carbocyclic radical of usually 6 to 10 carbon atoms; by way of example and preferably for phenyl and naphthyl.

Heterocyclyl is a mono- or polycyclic, preferably mono- or bicyclic, heterocyclic radical having usually 5 to 7 ring atoms and up to 3, preferably up to 2 heteroatoms and / or hetero groups from the series N, O, S, SO, SO ₂ . The heterocyclyl radicals may be saturated or partially unsaturated. Preference is given to 5- to 7-membered, monocyclic saturated heterocyclyl radicals having up to two heteroatoms from the series O, N and S, such as by way of example and preferably tetrahydrofuranyl, pyrrolidinyl, pyrrolinyl, piperidinyl, tetrahydropyranyl, piperazinyl, morpholinyl and perhydroazepinyl.

Heteroaryl is an aromatic, mono- or bicyclic radical having usually 5 to 10, preferably 5 to 6 ring atoms and up to 5, preferably up to 4 heteroatoms from the series S, O and N, by way of example and preferably for thienyl, furyl , Pyrrolyl, thiazolyl, oxazolyl, imidazolyl, pyridyl, pyrimidyl, pyridazinyl, indolyl, indazolyl, benzofuranyl, benzothiophenyl, quinolinyl and isoquinolinyl.

Halogen is fluorine, chlorine, bromine and iodine, preferably fluorine and chlorine.

Description of the pictures:

Fig. 1: Single crystal X-ray structure of Example 12A, Ortep plot (50%). Fig. 2: ¹ H-NMR (500 MHz, dj-pyridine) of Example 21A

Preference is given to compounds of the formula (Ib) in which

R ^{6 is} methyl,

R ^{7 is} a group of the formula

stands,

in which

the point of attachment to the amine means

R ^{1 is} 2-methylprop-1-yl, 2,2-dimethylprop-1-yl, 2,2-dimethylbut-1-yl,

Trimethylsilylmethyl, 1-hydroxy-2-methylprop-1-yl, 1-hydroxy-2,2-dimethylprop-1-yl, 1-hydroxy-2,2-dimethyl-but-1-yl, 1-hydroxy-2-yl ethyl-2-ethylbut-1-yl, 1-hydroxy-2,2-diethylbut-1-yl, phenylmethyl, 1-hydroxy-1-phenylmethyl, 2-pyridylmethyl or 3-pyridylmethane 1 means

where 2-pyridylmethyl or 3-pyridylmethyl can be substituted by 0, 1, 2 or 3 substituents independently of one another selected from the group consisting of hydroxyl, amino, trifluoromethyl, methyl, methoxy and morpholinyl,

R ^{2 is} 2-methylprop-1-yl, 2,2-dimethylprop-1-yl, 2 ₃ 2-dimethylbut-1-yl,

Trimethylsilylmethyl, 1-hydroxy-2-methylprop-1-yl, 1-hydroxy; y-2,2-dimethylprop-1-yl, 1-hydroxy-2,2-dimethyl-but-1-yl, 1-hydroxy 2-ethyl-2-methylbut-1-yl, 1-hydroxy-2,2-diethylbut-1-yl, phenylmethyl, 1-hydroxy-1-phenylmethyl, 2-pyridylmethyl, 1 or 3 pyridine lmethy 1 means

where 2-pyridylmethyl or 3-pyridylmethyl can be substituted by 0, 1, 2 or 3 substituents independently of one another selected from the group consisting of hydroxyl, amino, trifluoromethyl, methyl, methoxy and morpholinyl, R ^{4 is} 2-methylprop-1-yl, 2,2-dimethylprop-1-yl, 2,2-dimethylbut-1-yl,

Trimethylsilylmethyl, 1-hydroxy-2-methylprop-1-yl, 1-hydroxy-2,2-dimethylpropyl

1-yl, 1-hydroxy-2,2-dimethylbut-1-yl, 1-hydroxy-2-ethyl-2-methylbut-1-yl, 1

Hydroxy-2,2-diethylbut-1-yl, phenylmethyl, 1-hydroxy-1-phenylmethyl, 2-pyridylmethyl or 3-pyridylmethyl,

where 2-pyridylniethyl or 3-pyridylmethyl can be substituted by 0, 1, 2 or 3 substituents independently of one another selected from the group consisting of hydroxyl, amino, trifluoromethyl, methyl, methoxy and morpholinyl,

R ^{5 is} 2-methylprop-1-yl, 2,2-dimethylprop-1-yl, 2,2-dimethylbut-1-yl,

Trimethylsilylmethyl, 1-hydroxy-2-methylprop-1-yl, 1-hydroxy-2,2-dimethylprop-1-yl, 1-hydroxy-2,2-dimethylbut-1-yl, 1-hydroxy-2-yl ethyl-2-methylbut-1-yl, 1-hydroxy-2,2-diethylbut-1-yl, phenylmethyl, 1-hydroxy-1-phenylmethyl, 2-pyridylmethyl or 3-pyridylmethyl,

wherein 2-pyridylmethyl or 3-pyridylmethyl may be substituted with

0, 1, 2 or 3 substituents independently selected from the group consisting of hydroxy, amino, trifluoromethyl, methyl, methoxy and morpholinyl,

and their salts, their solvates, and the solvates of their salts.

Preference is also given to compounds of the formula (Ib) in which

R ^{6 is} methyl,

R ^{7 is} a group of the formula

stands,

in which

the point of attachment to the amine means R ^{1 is} 2-methylprop-1-yl, 2,2-dimethylprop-1-yl, trimethylsilylmethyl or 3-pyridylmethyl,

where 3-pyridylmethyl may be substituted by 0, 1 or 2 substituents independently of one another selected from the group consisting of hydroxyl, amino, trifluoromethyl, methyl, methoxy and morpholinyl,

R ^{2 is} 2-methylprop-1-yl, 2,2-dimethylprop-1-yl, trimethylsilylmethyl or 3-pyridylmethyl,

R ^{4 is} 2-methylprop-1-yl, 2,2-dimethylprop-1-yl, trimethylsilylmethyl or 3-pyridylmethyl,

where 3-pyridylmethyl may be substituted by O, 1 or 2 substituents independently of one another selected from the group consisting of hydroxyl, amino, trifluoromethyl, methyl, methoxy and morpholinyl,

R ^{5 is} 2-methylprop-1-yl, 2,2-dimethylprop-1-yl, trimethylsilylmethyl or 3-pyridylmethyl,

where 3-pyridylmethyl may be substituted by O, 1 or 2 substituents independently of one another selected from the group consisting of hydroxyl, amino, trifluoromethyl, methyl, methox.y and morpholinyl,

and their salts, their solvates, and the solvates of their salts.

Preference is also given to compounds of the formula (Ib) in which

R ^{6 is} methyl,

R ^{7 is} a group of the formula

stands,

in which

* means the point of attachment to the amine,

and R ¹ and R ^{2 have} the meaning given above.

Preference is also given to compounds of the formula (Ib) in which

R ^{5 is} methyl,

R ^{7 is} a group of the formula

stands,

in which

* means the point of attachment to the amine,

and R ⁴ and R ⁵ are as defined above.

The invention also relates to compounds of the formula

in which

R ¹ is C _r C ₆ alkyl, C ₂ -C ₆ alkenyl, C ₃ -C ₆ -cycloalkyl or C ₆ -C, ₀ aryl,

wherein alkyl, alkenyl, cycloalkyl and aryl may be substituted with 0, 1, 2 or 3 substituents independently selected from the group consisting of halogen,

Hydroxy, amino, cyano, trimethylsilyl, C ₁ -C ₆ -alkyl, C ₁ -C ₆ -alkoxy, benzyloxy, C ₃ -C ₀ -

Cycloalkyl, Cβ-Cio-aryl, 5- to 7-membered heterocyclyl, 5- to 10-membered heteroaryl,

Ci-C ₆ -alkylamino, Cβ-Cio-arylamino, Ci-Cβ-alkylcarbonylamino, C ₆ -C ₁₀ amino -Arylcarbonyl-, Ci-Cβ-alkylcarbonyl, Ci-Cβ alkoxycarbonyl, Cβ-C jo-arylcarbonyl, and benzyloxy carbonylamino .

in which cycloalkyl, aryl, heterocyclyl and heteroaryl in turn may be substituted by 0, 1, 2 or 3 substituents independently of one another selected from the group consisting of halogen, hydroxyl, amixio, cyano, nitro, trifluoromethyl, C ₁ -C 6 -alkyl, C ₁ - Ce-AIkOXy, phenyl and 5- to 7-membered heterocyclyl,

R ² Ci-C ₆ alkyl, C ₂ -C ₆ alkenyl, C ₃ -C ₆ -C ₆ -cycloalkyl or represents C ₀ aryl,

where alkyl, alkenyl, cycloalkyl and aryl may be substituted by 0, 1, 2 or 3 substituents independently of one another selected from the group consisting of halogen, hydroxyl, amino, cyano, trimethylsilyl, C ₁ -C ₆ -alkyl, C ₁ -C ₆ - Alkoxy; y, benzyloxy, C ₃ -C ₆ - Cycloalkyl, C ₆ -C ₁₀ aryl, 5- to 7-membered heterocyclyl, 5- to 10-membered heteroaryl, Ci-C _ö -Alkylammo, C ₆ -Cio-Arylammo, Ci-Ce-alkylcarbonylamino, Cβ-CIO Arylcarbonylamino, C ₁ -C ₆ -alkylcarbonyl, C ₁ -C ₆ -alkoxycarbonyl, C ₆ -C 10 -arylcarbonyl and benzyloxycarbonylamino,

in which cycloalkyl, aryl, heterocyclyl and heteroaryl in turn may be substituted by 0, 1, 2 or 3 substituents independently of one another selected from the group consisting of halogen, hydroxyl, amino, cyano, nitro, trifluoromethyl, C ₁ -C ₆ -alkyl, ci C ₆ alkoxy, phenyl and 5- to 7-membered heterocyclyl,

and their salts, their solvates, and the solvates of their salts.

Preference is also given to compounds of the formula (I) in which

R ^{1 is} 2-methylprop-1-yl, 2,2-dimethylprop-1-yl, 2,2-dimethylbut-1-yl, trimethylsilylmethyl, 1-hydroxy-2-methylprop-1-yl, 1-hydroxy-2 , 2-dimethylprop-1-yl, 1-hydroxy-2,2-dimethyl-but-1-yl, 1-hydroxy-2-ethyl-2-methylbut-1-yl, 1-hydroxy-2,2-diethylbutane -l-yl, phenylmethyl, 1-hydroxy-1-phenylmethyl, 2-pyridylmethyl or 3-pyridylmethyl,

where 2-pyridylmethyl or 3-pyridylmethyl may be substituted by 0, 1, 2 or 3 substituents independently of one another selected from the group consisting of hydroxyl, amino, trifluoromethyl, methyl, methoxy and morpholinyl,

R ^{2 is} 2-methylprop-1-yl, 2,2-dimethylprop-1-yl, 2,2-dimethylbut-1-yl, trimethylsilylmethyl, 1-hydroxy-2-methylprop-1-yl, 1-hydroxy -2,2-dimethylprop-1-yl, 1-hydroxy-2,2-dimethyl-1-but-1-yl, 1-hydroxy-2-ethyl-2-methylbut-1-yl, 1-hydroxy-2 , 2-diethylbut-1-yl, phenylmethyl, 1-hydroxy-1-phenylmethyl, 2-pyridylmethyl or 3-pyridylmethyl,

and their salts, their solvates, and the solvates of their salts.

Preference is also given to compounds of the formula (I) in which

R ^{1 is} 2-methylprop-1-yl, 2,2-dimethylprop-1-yl, trimethylsilylmethyl or 3-pyridylmethyl means

where 3-pyridylmethyl can be substituted by 0, 1 or 2 substituents independently of one another selected from the group consisting of hydroxyl, amino, trifluoromethyl, methyl, methoxy and morpholinyl,

and their salts, their solvates, and the solvates of their salts.

The invention also relates to compounds of the formula

in which

R ¹ Ci-C ₆ alkyl, C ₂ -C ₆ alkenyl, C ₃ -C ₆ -cycloalkyl or C ₆ -C ₀ -aryl,

where alkyl, alkenyl, cycloalkyl and aryl may be substituted by 0, 1, 2 or 3 substituents independently of one another selected from the group consisting of halogen, hydroxyl, amino, cyano, trimethylsilyl, C _r C ₆ -alkyl, C _r C ₆ - Alkoxy, benzyloxy, C ₃ -C ₆ - Cycloalkyl, C ₆ -C ₀ aryl, 5- to 7-membered heterocyclyl, 5- to 10-membered heteroaryl, Ci-C ₆ -alkylamino, Cβ-Cio-arylamino, Ci-C _ö alkylcarbonylamino, C6-CIO Arylcarbonylamino, C 1 -C 6 -alkylcarbonyl, C 1 -C 6 -alkoxycarbonyl, C ₆ -C ₁₀ -arylcarbonyl and benzyloxycarbonylamino,

wherein cycloalkyl, aryl, heterocyclyl and heteroaryl in turn may be substituted by 0, 1, 2 or 3 substituents independently selected from the group consisting of halogen, hydroxy, amino, cyano, nitro, trifluoromethyl, Ci-Cβ-AJkyl, Ci-C _ό -alkoxy, phenyl and 5- to 7-membered heterocyclyl,

IC Ci-Ce-alkyl, C ₂ -C ₆ alkenyl, C ₃ -C ₆ -cycloalkyl or C ₆ -C represents ₀ aryl,

where alkyl, alkenyl, cycloalkyl and aryl may be substituted by 0, 1, 2 or 3 substituents independently of one another selected from the group consisting of halogen, hydroxyl, amino, cyano, trimethylsilyl, C 1 -C 6 -alkyl, C 1 -C 6 -alkoxy, benzyloxy, _C3 -C6- cycloalkyl, C _ö -Cio-aryl, 5- to 7-membered heterocyclyl, 5- to 10-membered heteroaryl, Ci-Cβ alkylamino, Coe-Cio-arylamino, Ci-Cβ-alkylcarbonylamino, C ₆ -C 10 -arylcyconebonylamino, C ₁ -C ₆ -alkylcarbonyl, C ₁ -C ₆ -alkoxycarbonyl, C ₆ -C 10 -arylcarbonyl and benzyloxycarbonylamino,

in which cycloalkyl, aryl, heterocyclyl and heteroaryl in turn may be substituted by 0, 1, 2 or 3 substituents independently of one another selected from the group consisting of halogen, hydroxyl, amino, cyano, nitro,

Trifluoromethyl, C _ό alkyl, Ci-Cβ alkoxy, phenyl and 5- to 7-glϊedriges heterocyclyl,

and their salts, their solvates, and the solvates of their salts.

The invention further provides a process for the preparation of the compounds of the formulas (Ib), wherein

[A] the compounds of the formula

in which

R ^{6 has} the meaning given above,

first with compounds of the formula

in which

R ₅ R ₅ R and R have the abovementioned meaning,

R ^{3 is} butoxy-butoxycarbonyl or benzyloxycarbonyl, and

X ^{1 is} halogen, preferably bromine, chlorine or fluorine, or hydroxy,

and subsequently reacted with an acid and / or by hydrogenolysis,

or

[B] the compounds of the formula

in which

R ^{6 has} the meaning given above,

first with compounds of the formula

in which

R, R, R and R have the abovementioned meaning,

and then in a 4-step synthesis

a) with a fluoride reagent such as tetrabutylammonium fluoride,

b) with an acid,

c) with a dehydrating reagent, gege ^~ appropriate in the presence of a base, and

d) by hydrogenolysis be implemented.

Method [A]:

If X ^{1 is} halogen, the reaction of the first stage is carried out in. Generally in inert solvents, optionally in the presence of a base, preferably in a temperature range from -30 ° C to 50 ⁰ C at atmospheric pressure.

Inert solvents are, for example, tetrahydrofuran, methylene chloride, pyridine, dioxane, dimethylacetamide, N-methylpyrrolidine or dimethylformamide, preference is given to pyridine or dimethylformamide.

Examples of bases are triethylamine, diisopropylethylamine or N-methylmorpholine, preference is given to diisopropylethylamine.

If X ^{1 is} hydroxy, the reaction of the first stage is generally carried out in inert solvents, in the presence of a dehydrating reagent, optionally in the presence of a base, preferably in a temperature range from -30 ⁰ C to 5O ⁰ C at atmospheric pressure.

Inert solvents are, for example, halogenated hydrocarbons, such as dichloromethane or trichloromethane, hydrocarbons, such as benzene, nitromethane, dioxane, dimethylformamide or acetonitrile. It is likewise possible to use mixtures of the solvents. Particularly preferred is dichloromethane or dimethylformamide.

Suitable dehydrating here, for example, carbodiimides such as N ₁ N'-diethyl-, N ₁ N ₁ dipropyl, N, N'-diisopropyl-, N, N'-dicyclohexylcarbodiimide, N- (3-di ~ methylaminoisopropyty-N'suitable ethylcarbodiimide hydrochloride (EDC), N-cyclohexylcarbodiimide-N'-propyloxymethyl-polystyrene (PS-carbodiimide) or carbonyl compounds such as carbonyldiimidazole, or 1,2-oxazolium compounds such as 2-ethyl-5-phenyl-1,2-oxazolium 3-sulphate or 2-tert-butyl-5-methylisoxazolium perchlorate, or acylamino compounds such as 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline, or propanephosphonic anhydride, or isobutylchloroformate, or bis (2-oxo -3-oxazolidinyl) -phosphorylchlorid or benzotriazolyloxy-tri (dimethylamino) - phosphonium hexafluorophosphate, or O- (benzotriazol-1 -y \) - N, N, 17 ^~ ', N-tetra methyluronium- hexafluorophosphate (HBTU), 2- (2-Oxo-1 - (2H) -pyridyl) -1,1,3,3-tetramethyluronium tetrafluoroborate (TPTU) or 0- (7-azabenzotriazol-1-yl) -N, N, N ', N 'tetramethyl-uroniumhex fluorophosphate (HATU), or 1-hydroxybenzotriazole (HOBt), or benzotriazole-l-3 / loxytris (dimethylamino) phosphonium hexafluorophosphate (BOP), or benzotriazol-1-yloxytris (pyrrolidino) phosphonium hexafluorophosphate (PyBOP) ₃ or N- Hydroxysuccinimide, or mixtures of these, with bases. Examples of bases are alkali metal carbonates, such as sodium or potassium carbonate, or hydrogencarbonates drogencarbonat, or organic bases such as trialkylamines, for example triethylamine, N ^{'methylmorpholine,} N-methylpiperidine, 4-dimethylaminopyridine or diisopropylethylamine.

Preferably, the condensation is carried out with HATU or with EDC in the presence of HOBt.

The reaction takes place with an acid in the second step of the process preferably in a temperature range from ⁰ ° C to 4O ⁰ C at atmospheric pressure.

Suitable acids in this case are hydrogen chloride in dioxane, hydrogen bromide in acetic acid or trifluoroacetic acid in methylene chloride.

The hydrogenolysis in the second stage of the process is generally carried out in a solvent in the presence of hydrogen and palladium on activated carbon, preferably in a temperature range from ⁰ ° C. to 40 ° C. under normal pressure.

Solvents are, for example, alcohols such as methanol, ethanol, n-propanol or isopropanol, in a mixture with water and acetic acid or aqueous hydrochloric acid, vorzπugt is a mixture of ethanol, water and acetic acid or a mixture of iso-propanol and aqueous hydrochloric acid.

Method [B]:

The reaction of the compounds of the formula (IV) with compounds of the formula (V) or (VII) is generally carried out in inert solvents, in the presence of a dehydrating reagent, if appropriate in the presence of a base, preferably in a temperature range from -3O ⁰ C to 50 ⁰ C at normal pressure.

Suitable dehydrating reagents here are, for example, carbodiimides, such as N, N'-diethyl, N, N-dipropyl, NN'-diisopropyl, N, N'-dicyclohexylcarbodiimide, N- (3-dimethylaminoisopropyl) -N ' ethylcarbodiimide hydrochloride (EDC), N-cyclohexylcarbodiimide-N'-propyloxymethyl-polystyrene (PS-carbodiimide) or carbonyl compounds such as carbonyldiimidazole, or 1,2-oxazolium compounds such as 2-ethyl-5-phenyl-1,2-oxazolium 3-sulphate or 2-tert-butyl-5-methylisoxazoline perchlorate, or acylamino compounds such as 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline, or propanephosphonic anhydride, or isobutylchloroformate, or bis- (2-oxo-3-oxazolidinyl) -phosphoryl chloride or benzotriazolyloxy-tri (dimethylamino) phosphonium hexafluorophosphate, or O- (benzotriazol-1-yl) -N, N, N ', N'-tetra-methyluronium hexafluorophosphate (HBTU), 2- (2-oxo-l- (2H) -pyridyl) -l, l, 3,3-tetramethyluronium tetrafluoroborate (TPTU) or ^ - (y-azaberizotriazol-1-yO-N.NN 'N'-tetramethyl-uronium hexafluorophosphate (HATU), or 1-hydroxybenzotriazole (HOBt), or benzotriazol-1-yloxytris (dimethylamino) phosphonium hexafluorophosphate (BOP), or benzotriazol-1-yloxytris (pyrrolidino) phosphonium hexafluorophosphate (PyBOP) , or N-hydroxysuccinimide, or mixtures thereof, with bases.

Bases are, for example, alkali carbonates, e.g. Sodium or potassium carbonate, or hydrogen carbonate, or organic bases such as trialkylamines e.g. Triethylamine, N-methylmorpholine, N-methylpiperidine, 4-dimethylarninopyridine or diisopropylethylamine.

Preferably, the condensation is carried out with HATU or with a mixture of EDC and HOBt.

The reaction with tetrabutyl ammonium fluoride in the first stage (a) of the further process is generally carried out in inert solvents, preferably in a temperature range from ⁰ ° C. to 40 ^° C. under normal pressure.

Inert solvents are, for example, halogenated hydrocarbons, such as dichloromethane or trichloromethane, or ethers, tetrahydrofuran or dioxane. Particularly preferred is tetrahydrofuran.

The reaction with an acid in the second stage (b) of the process is preferably carried out in a temperature range from 0 ⁰ C to 40 ⁰ C at atmospheric pressure.

The reaction in the third stage (c) of the process is analogous to the reaction of compounds of the formula (IV) with compounds of the formula (V) or (VII).

The hydrogenolysis in the fourth step (d) of the process is generally carried out in a solvent in the presence of hydrogen and palladium on activated carbon, preferably in a temperature range from ⁰ ° C. to 40 ^° C. under normal pressure.

Solvents are, for example, alcohols such as methanol, ethanol, n-propanol or isopropanol, in a mixture with water and acetic acid or aqueous hydrochloric acid, preferably a mixture of ethanol, water and acetic acid or a mixture of isopropanol and aqueous hydrochloric acid. The compound of formula (H) can be synthesized by hydrogenation, enzymatic cleavage and cyclization from lysobactin (Example IA) or katanosin A as described in the experimental section under Example 2A, 3A and 2OA to 26A. The compound of formula (FV) is an intermediate in this synthetic sequence.

The compounds of the formulas (IH), (V), (VI) and (VE) are known or can be synthesized from the corresponding starting materials by known methods or by the method described below.

The preparation of the compounds according to the invention can be illustrated by the following synthesis schemes.

Synthetic Scheme 1:

H

(1) Chymotrypsin cleavage (2) Protection group introduction

Synthesis Scheme 2:

The present invention further provides a process for the preparation of the compound of the formula 2- (trimethylsilyl) ethyl (3i?) -3-amino-N ² - [(benzyloxy) carbonyl] -L-phenylalaninate trifluoroacetate (Example 1 9A)

characterized in that in a 6-stage synthesis, the compound of the formula (rac) -3- [(t erf-butoxycarbony l) amino] -3-pheny lpropansäuremethy lester

a) in the presence of a base, preferably lithium hexamethyldisilazide, with dibenzyl azadicarboxylate,

b) with N, N, N, N, -tetramethylguanidine,

c) with hydrogen in the presence of Raney-Νickel,

d) with N-benzyloxycarbonyloxysuccinimide ester in the presence of a base, preferably N-methylmorpholine,

e) with a base, preferably lithium hydroxide, and subsequent chromatographic enantiomer separation,

and

f) with trifluoroacetic acid in dichloromethane or hydrogen chloride in dioxane

is implemented.

The detailed synthesis is in the experimental part under Example 12A to Example 19A described.

Another object of the present invention is the compound of formula

or

36A)

or

2- (Trimethylsilyl) ethyl- (35) -3-amino-N ² - [(benzyloxy) carbonyl] -D-phenylalaninate trifluoroacetate (Example 38A)

The compounds of the invention show an unpredictable, valuable spectrum of pharmacological activity. They show an antibacterial effect.

They are therefore suitable for use as medicaments for the treatment and / or prophylaxis of diseases in humans and animals.

The compounds of the invention are characterized by a lower nephrotoxicity to lysobactin.

The compounds according to the invention are distinguished by improved stability in an aqueous neutral to basic medium. This property improves the storage of the compounds according to the invention and the administration as medicaments. The described nonadepsipeptides act as inhibitors of bacterial cell wall biosynthesis.

The preparations according to the invention are particularly effective against bacteria and bacteria-like microorganisms. They are therefore particularly well suited for the prophylaxis and chemotherapy of local and systemic infections in human and veterinary medicine, which are caused by these pathogens.

In principle, the preparations according to the invention can be used against all bacteria and bacteria-like microorganisms which are in the possession of a bacterial cell wall (murein sacculus) or the associated enzyme systems, for example by the following pathogens or by mixtures of the following pathogens:

Gram-negative cocci (Neisseria gonorrhoeae) as well as Gram-negative rods such as Enterobacteriaceae, e.g. Escherichia coli, Haemophilus influenzae, Pseudomonas, Klebsiella, Citrobacter (C. freundii, C. divernis), Salmonella and Shigella; also Enterobacter (E. aerogenes, E. agglomerans), Hafnia, Serratia (S. marcescens), Providencia, Yersinia, and the genus Acinetobacter, Branhamella and Chlamydia. 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 exhibit particularly pronounced activity against gram-positive cocci, e.g. Staphylococci (S. aureus, S. epidermidis, S. haemolyticus, S. carnosus), enterococci (E. faecalis, E. faecium) and streptococci (S. agalactiae, S. pneumoniae, S. pyogenes).

The above enumeration of pathogens is merely exemplary and not to be construed as limiting. 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:

Infectious diseases in humans such as uncomplicated and complicated urinary tract infections, uncomplicated skin and surface infections, complicated skin and soft tissue infections, hospital and community-acquired pneumonia, nosocomial pneumonia, acute exacerbations and secondary bacterial infections of chronic bronchitis, acute otitis media, acute sinusitis, streptococcal Pharyngitis, bacterial meningitis, uncomplicated gonococcal and non-gonococcal urethritis / cervicitis, acute prostatitis, endocarditis, uncomplicated and complicated intra-abdominal infections, gynecological infections, pelvic inflammatory disease, bacterial vaginosis, acute and chronic osteomyelitis, acute bacterial arthritis, empiric therapy in febrile neutropenic patients, furthermore Bacteria, MRSA infections, acute infectious diarrhea, Helicobacter pylori infections, postoperative infections, odontogenic infections, ophthalmic infections, postoperative infections (including periprocal abscess, wound infections, biliary infections, mastitis and acute appendicitis), cystic fibrosis and bronchiectasis.

Apart from humans, bacterial infections can also be treated in other species. Examples include:

Pig: diarrhea, enterotoxemia, sepsis, dysentery, salmonellosis, metritis-mastitis-agalactiae syndrome, mastitis;

Ruminants (cattle, sheep, goats): diarrhea, sepsis, bronchopneumonia, salmonellosis, pasteurellosis, genital infections;

Horse: bronchopneumonia, foal disease, puerperal and postpuerperal infections, salmonellosis;

Dog and cat: bronchopneumonia, diarrhea, dermatitis, otitis, urinary tract infections, prostatitis;

Poultry (chicken, turkey, quail, pigeon, ornamental birds and others): E. co / z infections, chronic respiratory diseases, salmonellosis, pasteurellosis, psittacosis.

Likewise, bacterial diseases in the rearing and keeping of farmed and ornamental fish can be treated, wherein the antibacterial spectrum on the aforementioned pathogen addition to other pathogens such. Pasteurella, Brucella, Campylobacter, Listeria, Erysipelothris, Corynebacteria, Borellia, Treponema, Nocardia, Rikettsia, Yersinia.

Another object of the present invention is the use of the compounds of the invention for the treatment and / or prophylaxis of diseases, especially of bacterial infectious diseases.

Another object of the present invention is the use of the compounds of the invention for the treatment and / or prophylaxis of diseases, in particular the aforementioned diseases. Another object of the present invention is the use of the compounds of the invention for the manufacture of a medicament for the treatment and / or prophylaxis of diseases, in particular the aforementioned diseases.

The compounds according to the invention are preferably used for the preparation of medicaments which are suitable for the prophylaxis and / or treatment of bacterial diseases.

Another object of the present invention is a method for the treatment and / or prophylaxis of diseases, in particular the aforementioned diseases, using an antibacterially effective amount of the inventive compounds.

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.

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.

For these administration routes, the compounds according to the invention can be administered in suitable administration forms.

For the oral administration are according to the prior art functioning rapidly and / or modified compounds of the invention donating application forms containing the compounds of the invention in crystalline and / or amorphized and / or dissolved form, such as. Tablets (uncoated or coated tablets, for example with enteric or delayed-release or insoluble coatings which control the release of the compound of the invention), orally disintegrating tablets or films / wafers, films / lyophilisates, capsules (for example hard or soft gelatin capsules) in the oral cavity , Dragees, granules, pellets, powders, emulsions, suspensions, aerosols or solutions.

Parenteral administration can be done bypassing a resorption step (eg intravenously, intraarterially, intracardially, intraspinally or intralumbarly) or with involvement of resorption (eg intramuscular, subcutaneous, intracutaneous, percutaneous or intraperitoneal). For the Parenteral administration are suitable as application forms, including injection and infusion preparations in the form of solutions, suspensions, emulsions, lyophilisates or sterile powders.

For the other routes of administration are suitable, for example Inhalation medicines (including powder inhalers, nebulizers), nasal drops, solutions, sprays; lingual, sublingual or buccal tablets, films / wafers or capsules, suppositories, ear or ophthalmic preparations, vaginal capsules, aqueous suspensions (lotions, shake mixtures), lipophilic suspensions, ointments, creams, transdermal therapeutic systems (such as patches), milk, Pastes, foams, scattering powders, implants or stents.

The compounds according to the invention can be converted into the mentioned administration forms. This can be done in a conventional manner by mixing with inert, non-toxic, pharmaceutically suitable excipients. These aids include, among others. Carriers (for example microcrystalline cellulose, lactose, mannitol), solvents (eg liquid polyethylene glycols), emulsifiers and dispersing or wetting agents (for example sodium dodecyl sulfate, 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 odor remedies.

Another object of the present invention are pharmaceutical compositions containing at least one erfmdungsgemäße connection, usually together with one or more inert, non-toxic, pharmaceutically suitable excipients, and their use for the purposes mentioned above.

In general, it has been found 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.

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 manage with less than the aforementioned minimum 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. The 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. Examples

Abbreviations

Alig. General (e)

Area (peak) area calculated

BHI Brain Heart Infusion

Boc ter l -Butoxycarbonyl br. broad signal (in NMR spectra)

Example d doublet (in NMR spectra)

TLC thin layer chromatography

DCC dicyclohexylcarbodiimide

DCI direct chemical ionization (in MS)

DCM dichloromethane

DIEA N, N-diisopropylethylamine

DMAP 4-N, N-dimethylaminopyridine

DMSO dimethyl sulfoxide

DMF N, N-dimethylformamide d. Th. Of theory

EDC 1-ethyl-3- (3-dimethylaminoprop3'l) carbodiimide (also EDCI)

EDCxHCl 1 -ethyl-3 - (3-dimethylaminopropyl) carbodiimide hydrochloride

EDTA ethylenediaminetetraacetic acid (Ethylene Diamine Tetraacetic Acid)

EE ethyl acetate (ethyl acetate)

EI electron impact ionization (in MS)

ESI electrospray ionization (in MS)

Company. Company

Mp melting point found ges. saturated h hour

HATU (^ - (V-Azabenzotriazol-1-yO-A / ^' N'N'-N'-tetramethyluronium hexafluorophosphate

HOBt 1-hydroxybenzotriazole

HPLC high pressure, high performance liquid chromatography

HR High Resolution i. V. concentrated in vacuo. Concentrated

LC-MS liquid chromatography-coupled mass spectrometry

LDA lithium diisopropylamide

LLA (3-cyclohexyl) D-Leu-Leu- (3-cyclohexy I) Ala

LLF D-Leu-Leu-Phe m middle (medium) (for UV and IR spectra) m multiple «(for NMR spectra)

MALDI Matrix-Assisted Laser Desorption / Ionization

MIC Minimum inhibitory concentration min minute / minutes

MRSA Methicillin-resistant Staphylococcus aureus

MS mass spectroscopy

NCCLS National Committee for Clinical Laboratory Standards neg. Negative

NMM N-methylmorpholine

NMR Nuclear Magnetic Resonance (NMR) p.a. pro analysi

Pd palladium

Pd-C palladium on carbon pos. positive percent. Percent

PTFE polytetrafluoroethylene quant. quantitatively

RP-HPLC reverse phase HPLC

RT room temperature

Retention time (on HPLC)

S strong (in UV and IR spectra)

S singlet (in ΝMR spectra)

TBAF tetrabutylammonium fluoride

TBTU O- (benzotriazol-1-yl) -NN, N'N'-tetramethyluronium

tetrafluoroborate

TCTU 0- (1H-6-chlorobenzotriazol-1-yl) -1,3,3-tetramethyluronium

tetrafluoroborate

TFA trifluoroacetic acid

TFE 2,2,2-trifluoroethanol THF tetrahydrofuran

TMG N, N, N, N-tetramethylguanidine

TMSE 2- (trimethylsilyl) ethyl

TOF time of flight

UV ultraviolet

Visible (visible)

VRSA vancomycin-resistant Stapylococcus aureus

W weak (in UV and IR spectra) aq. aqueous (e)

Z, Cbz benzyloxycarbonyl

literature

For the nomenclature of peptides and cyclodepsipeptides, see:

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. As well as cited literature.

General methods GC-MS, LC-MS, HR-MS, HPLC and gel chromatography

Method 1 (TOF-HR-MS): TOF HR-MS-ESI + spectra are recorded using a Micromass LCT apparatus (capillary voltage: 3.2 KV, cone Spanmmg: 42 V Source Temperature: 120 ⁰ C, Desolvations Temperature: 280 ⁰ C ). For this purpose, a syringe pump (Harvard Apparatus) is used for sample introduction. The standard is leucine-enkephalin (Tyr-Gly-Gly-Phe-Leu).

Method 2 (Analytical HPLC, Synergi): Device Type HPLC: HP 1100 Series; UV DAD; S-column: Phenomenex Synergi 2μ Hydro-RP Mercury 20 mm x 4 mm; Eluent A: 1 l of water + 1 ml of 50% formic acid, eluent B: 1 l of acetonitrile; Gradient: 0.0 min 90% A -> 2.5 min 30% A -E ^ 3.0 min 5% A -> 4.5 min 5% A; Flow: 0.0 min 1 ml / min, 2.5 min / 3.0 min / 4.5 min. 2 ml / min; Oven: 5O ⁰ C; UV detection: 210 nm.

Method 3 (Analytical HPLC, Synergi): Device Type HPLC: HP 1050 Series; UV DAD; Column: Phenomenex Synergi 2μ Max-RP Mercury 20mm x 4mm; Eluent A: 1 1 water + O.05% Trifluoroacetic acid, eluent B: 1 liter acetonitrile; Gradient: 0.0 min 90% A -> 2.5 min 30% A - »3.0 min 5% A -> 4.5 min 5% A; Flow: 0.0 min 1 ml / min, 2.5 min / 3.0 min / 4.5 min. 2 ml / min; Oven: 5O ⁰ C; UV detection: 210 nm.

Method 4 (Analytical HPLC): Device Type HPLC: HP 1050 Series; UV DAD 1100 Series; Column: Kromasil C] ₈ , 60 × 2 mm, 3.-5 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, 4.5-6.5 min 90% B, 6.5-6.7 min 90- 2% B, 6.7-7.5 min 2% B; Flow: 0.75 ml / min, Oven: 3O ⁰ C, detection UV 210 nm.

Method 5 (LC-MS): Device Type MS: Micromass LCT (ESI pos./neg.); Device type HPLC: HP 1100 Series; UV DAD 1100 Series; Column SymmetryPrep ™ Ci ₈ , Waters, 50 x 2.1 mm, 3.5 μm; Eluent A: water / 0.1% formic acid, eluent B: acetonitrile / 0.1% formic acid; Gradient: 0-1 min 0% B, 1-6 min 0-90% B, 6-8 min 90-100% B, 8-10 min 100% B, 10-10.1 min 100-0% B, 10.1- 12 min 0% B, then regeneration of the chromatography column. Oven: 4O ⁰ C, flow: 0.5 ml / min (at 10.1 min in the short term to 1 ml / min), UV detection: 210 nm.

Method 6 (Preparative HPLC, Xterra, C ₁₈ , 0.1% TFA): Device: Gilson Abimed HPLC; binary pump system; Column: Xterra, Waters, 5 μm; 150 x 10 mm; Eluent A: water / 0.1% trifluoroacetic acid, eluent B: acetonitrile; Gradient: 0-7 min 5% B, 7-9 min 5-40% B, 9-11 min 40% B, 11-18 min 40-90% B, then regeneration of the chromatography column; Flow: 8 ml / min; UV detector 210 nm.

Method 7 (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 of water + 0.5 ml of 50% formic acid, eluent B: 1 l of acetonitrile + 0.5 ml of 50% formic acid; Gradient: 0.0 min 90% A -> 2.5 min 30% A -> 3.0 min 5% A -> 4.5 min 5% A; Flow: 0.0 min 1 ml / min, 2.5 min / 3.0 min / 4.5 min 2 ml / min; Oven: 5O ⁰ C; UV detection: 210 nm.

Method 8 (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 of water + 0.5 ml of 50% formic acid, eluent B: 1 l of acetonitrile + 0.5 ml of 50% formic acid; Gradient: 0.0 min 90% A -> 2.5 min 30% A ^■ »3.0 min 5% A -> 4.5 min 5% A; Flow: 0.0 min 1 ml / min, 2.5 min / 3.0 min / 4.5 min 2 ml / min; Oven: 5O ⁰ C; UV detection: 208-400 nm.

Method 9 (Amino Acid Analysis): The amino acid analyzes are carried out with an Eppendorf / Biotronik LC3000 amino acid analyzer. A slightly modified standard separation program from Eppendorf / Biotronik is used. The separation programs and the function of the analyzer are described in detail in the manual of the device (Handbook of the amino acid analyzer LC 3000, Scientific Devices GmbH Biotronik, Maintal, 1996). Method 10 (Preparative HPLC, Nucleodur, TF A): Apparatus: Gilson Abimed HPLC, binary pump system Column: Nucleodur Ci ₈ Gravity, Macherey-Nagel, 5 μm, 250 × 21 mm, eluent A: water / 0.1% trifluoroacetic acid , Eluent B: acetonitrile; gradient: 0-8 min 5% B, 8-40 min 5-60% B, 40-60 min 60% B, 60-75 min 60-100% B, 75-80 min 100% B, followed by regeneration of the chromatography column, flow: 7-15 ml / min, UV Detektox 210 nm.

Method 11 (Preparative HPLC, Nucleodur, Acetic Acid): Device: Gilson Abimed HPLC; binary pump system; Column: Nucleodur Ci ₈ Gravity, Macherey-Nagel, 5 μm; 250 x 40 mm; Eluent A: water / 0.2% acetic acid, eluent B: acetonitrile / 0.2% acetic acid; Gradient: 0-10 min 10% B, 10-24 min 10-30% B, 24-28 min 30-50% B, 28-35 min 50% B, 35-45 min 50-60% B, 45- 53 min 60-70% B, 53-60 min 60-90% B, 60-70 min 100% B, then regeneration of the chromatography column; Flow: 15-45 ml / min; UV Detettor 210 nm.

Method 12 (Preparative HPLC, Symmetry, Acetic Acid): Device: Gilson Abimed HPLC; binary pump system; Column: SymmetryPrep ™ C _{] 8} , Waters, 7 μm; 300 x 19 mm; Eluent A: water / 0.5-0.25% acetic acid, eluent B: acetonitrile; Gradient: 0-2 min 5% B, 2-60 min 5-90% B, 60-80 min 100% B, then regeneration of the chromatography column; Flow: 7 ml / min; UV detector 210 nm.

Method 13 (Preparative HPLC, Kromasil, acetic acid): Device: Gilson Abimed HPLC; binary pump system; Column: Kromasil-IOOA Ci ₈ , 5 microns; 250 x 30 mm; Eluent A: water / 0.25% acetic acid, eluent B: acetonitrile; Gradient: 0-3 min 5% B, 3-30 min 5-100% B, 30-38 min 100% B, then regeneration of the chromatography column; Flow: 25 ml / min; UV detector 210 nm.

Method 14 (gel chromatography on Sephadex LH-20): Gel chromatography is performed without pressure on Sephadex LH-20 (Pharmacia). It is fractionated after UV activity (UV detector for 254 nm, Knauer) (fraction collector ISCO Foxy 200). Column dimensions: 32 x 7 cm (1000-100 μmol scale); 30 x 4 cm (100-10 μmol scale); 25 x 2 cm (10-1 μmol scale). The eluent used is methanol or methanol / 0.2% acetic acid.

Method 15 (Chiral preparative HPLC): column (Staβht column, dimension 250x30 mm); stationary phase (chiral silica / polyamide composite KBD 5326, based on the selector poly (N-L-leucine methacryolyl dicyclopropylmethylamide); eluent ethyl acetate, isocratic; flow: 25 ml / min; temperature: 24 ⁰ C; UV detection: 225 nm Sample: Repetitive injection of 2000 μl.

Method 16 (determination of enantiomeric purity): column (steel column, dimension 250 x 4.6 mm); stationary phase (chiral silica / polyamide composite KBD 5326, based on the selector poly (N-methacryolyl-L-leucine-dicyclopropylxnethylamide); ethyl acetate eluent, isocratic; Flow: 1 ml / min; Temperature: 25 ^° C; UV detection: 220 nm; Sample: Inject 10 μl.

Method 17 (Analytical HPLC): Device Type HPLC: HP 1100 Series; UV DAD column: Zorbax Eclipse XBD-C8 (Agilent), 150 mm x 4.6 mm, 5 μm; Eluent A: 5 ml HC1O _4/1 water, eluent B: acetonitrile; Gradient: 0-1 min 10% B, 1-4 min 10-90% B, 4-5 min 90% B; Flow: 2.0 ml / min; Oven: 3O ⁰ C; UV detection: 210 and 254 nm.

Method 18 (Analytical HPLC): Column: Kromasil RP-18, 60 mm × 2 mm, 3.5 μm; Eluent A: 5 ml HCIO ₄ / l water, eluent B: acetonitrile; Gradient: 0 min 2% B, 0.5 min 2% B, 4.5 min 90% B, 9 min 90% B; Flow: 0.75 ml / min; Oven: 30 ^° C; UV detection: 210 nm.

Method 19 (Analytical HPLC): Column: Kromasil RP-18, 250 mm × 4 mm, 5 μm; Eluent A: 5 ml HCIO ₄ / l water, eluent B: acetonitrile; Gradient: 0 min 5% B, 10 min 95% B; Flow: 1 ml / min; Oven: 40 ^° C; UV detection: 210 nm.

Method 20 (Analytical HPLC): Column: Kromasil RP-18, 250 mm x 4 mm, 5 μm; Eluent A: 2 ml HClO ₄ / l of water, eluent B: acetonitrile; Isocratic: 45% B, 55% A; Flow: 1 ml / min; Oven: 40 ^° C; UV detection: 210 nm.

Method 21 (Preparative HPLC, Nucleodur, 0.05-0.1% TF A): Device: Gilson Abimed HPLC; binary pump system; Column: Nucleodur Qg Gravity, Macherey-Nagel, 5 μm, 250 × 21 mm; Eluent A: water / 0.05-0.1% TFA, eluent B: acetonitrile; Gradient: 0-8 min 5% B, 8-40 min 5-60% B, 40-60 min 60% B, 60-75 min 60-100% B, 75-80 min 100% B, then regeneration of the chromatography column ; Flow: 7-15 ml / min; UV detection: 210 nm.

Method 22 (Analytical HPLC): Device Type HPLC: HP 1050 Series; UV DAD 1100 Series; Column: Kromasil C ₁₈ , 60 mm x 2 mm, 3.5 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 mia 90-2% B, 9.2-10.0 min 2% B; Flow: 0.75 ml / min, oven: 30 ^° C., UV detection 210 nm.

Method 23 (Analytical HPLC): Agilent 1100 with DAD (G1315B), Binary Pump (G1312A), Autosampler (G1313A), Degasser (G1379A) and Column Thermostat (G1316A); Column: Synergi 4 μ. Hydro-RP 80A, 4.6 x 150 x 5 now; Eluent A: water + 0.05% 70% perchloric acid; Eluent Br acetonitrile; Gradient: 0-1 min 10% B, ramp, 4-5 min 90% B, ramp, 5.5 min 10% B; Flow z 2.00 ml / min; Oven temperature: 3 O ⁰ C.

Method 24 (Analytical HPLC): Device: Agilent 1100 with DAD (G1315B), binary pump (GI 312A), autosampler (GI 313A), solvent degasser (GI 379A) and column thermostat: (G1316A); Column: Agilent Eclipse XDB-C8 4.6 x 150 x 5 mm; Eluent A: 0.05% 70% perchloric acid in water; Eluent B: methanol; Isocratic: 0-7 min 55% B; Flow: 2.00 ml / min; Column temperature: 4O ⁰ C.

Method 25 (Analytical HPLC): Device Type HPLC: HP 1050 Series; LW BAD; Column: Zorbax 300 mSB-C18 3.5 μ, 4.6 mm x 150 mm; Eluent A: 1 l water + 0.1% trifluoroacetic acid, eluent B: 400 ml acetonitrile / 600 ml water + 0.1% trifluoroacetic acid; Gradient: 0.0 RNIN 100% A, 1.3 min 10% B, 18.0 min 80% B, 20.0 min 80% B, 21.0 min 100% B, 25.0 min 100 ^«% B, 26.0 min 0% B, 30.0 min 0% B , Flow: 1 ml / min; Oven: 40 ^° C; UV detection: 210 nm.

Method 26 (Chiral HPLC): Gilson Abimed HPLC; Column: Daicel Chiralpak AD-H 5 um; 250 mm x 20 mm; Eluent A: / so-hexane, eluent B: 0.2% acetic acid / 1% water / 2-propanol; isocratic; Flow: 15 ml / min; UV detector 212 nm.

Method 27 (determination of enantiomeric purity): Steel column: dimension 250 mm x 4.6 mm; stationary phase: Daicel Chirapak AD-H, 5 μm; Eluent: ethanol / water / acetic acid 1000: 10: 2, isocratic; Flow: 0.7 ml / min; Temperature: 45 ^° C .; UV detection: 215 nm; Sample: Inject 5 μl.

Method 28 (determination of enantiomeric purity): column (steel column, dimension 250 mm x 4.6 mm); stationary phase (Chirapak IA); Eluent iso-hexane / iso-propanol 4: 1, isocratic; Flow: 1 ml / min; Temperature: 25 ^° C; UV detection: 254 nm; Sample: Injection ^"of 10 ul.

Method 29 (LC-MS): UV detection: 210 nm. Device Type MS: Micromass ZQ; Device type HPLC: Waters Alliance 2795; Column: Phenomenex Synergi 2μ Hydro-RP Mercury 20mm x 4mm; Eluent A: 1 l of water + 0.5 ml of 50% formic acid, eluent B: 1 l of acetonitrile + 0.5 ml of 50% formic acid; Gradient: 0.0 min 90% A -> 2.5 min 30% A -> 3.0 min 5 ° / όA -> 4.5 min 5% A; Flow: 0.0 min 1 ml / min, 2.5 min / 3.0 min / 4.5 min 2 ml / min; Oven: 5O ⁰ C; UV detection: 210 nm.

Method 30 (LC-MS): Instrument: Micromass Platform LCZ with HPLC Agilent Series 1100; Column: Phenomenex Synergi 2μ Hydro-RP Mercury 20 mm x 4 mm; Eluent A .: 1 liter of water + 0.5 ml of 50% formic acid, eluent B: 1 liter of acetonitrile + 0.5 ml of 50% formic acid; Gradient: 0.0 min 90% A -> 2.5 min 30% A -> 3.0 min 5% A -> 4.5 min 5% A; Flow: 0.0 min 1 ml / min, 2.5 min / 3.0 min / 4.5 min 2 ml / min; Oven: 50 ^° C .; UV detection: 210 nm.

Method 31 (MALDI-MS): The MALDI-MS / MS examinations are performed on a 4700 proteomics analyzer (Applied Biosystems, Framingham, MA) using

TOF / TOF ion optics and 200 Hz Nd: YAG laser (355 nm). The quasi-molecules are accelerated in the ion source with 8 kV, selected with an electric deflector (MSl), and encountered in a collision cell, which is located between MSl and MS2, with argon atoms. The resulting fragment ions are post-accelerated at 15 kV and characterized by the second time-of-flight mass analyzer (MS2).

Method 32 (Chiral Preparative HPLC): Steel Column: Dimension 420 mm x 75 mm; stationary phase: silica gel phase with the sector poly (N-methacryloyl-D-leucine-dicyclopropylmethylamide); Eluent A: te / t-butyl methyl ether, eluent B: methanol; Gradient: 0-22.17 min 100% A, 22.18- 27.24 min 100% B, 27.25-40.30 min 100% A; Flow: 100 ml / min; Temperature: 24 ^° C; UV detection: 260 nm; Sample: Repetitive injection of 50000 μl.

Method 33 (Preparative HPLC, oducleodur, 0.05-0.1% TFA): Device: Gilson Abimed HPLC; binary pump system; Column: Νucleodur Qs Gravity, Macherey-Νagel, 5 μm; 250 mm x 40 mm; Eluent A: water / 0.05-0.1% trifluoroacetic acid, eluent B: acetonitrile; Gradient: 0-10 min 10% B, 10-24 min 10-30% B, 24-28 min 30-50% B, 28-35 min 50 ° / oB, 35-45 min 50- 60% B, 45 -53 min 60-70% B, 53-60 min 60-90% B, 60-70 min 10O% B, then regeneration of the chromatography column; Flow: 15-45 ml / min; UV detector ZlO nm.

Method 34 (Preparative HPLC Kromasil 0.05% TFA): Device: Gilson Abimed HPLC; UV detector 210 nm; binary pump system; Column: Kromasil C _{] 8} , 5 μm, 100 Å, 250 mm × 20 mm; Eluent A: 0.05% trifluoroacetic acid in water, eluent B: 0.05% trifluoroacetic acid in acetonitrile: flow rate: 20 ml / min; 0-3 min 10% B, ramp, 30-38 min 90% B, 38-45 min 1 0% B.

Method 35 (Preparative HPLC, oducleodur C ₁₈ Gravity, 0.05-0.1% TFA): Device: Gilson Abimed HPLC; binary pump system; Column: Νucleodur Ci ₈ Gravity, 5 μm; 250 mm x 40 mm; Eluent A: water / 0.05-0.1% trifluoroacetic acid, eluent B: acetonitrile; 0-10 min 10% B, ramp, 10.01-55 min 100% B; Flow: 10-60 ml / min; UV detector 210 nm.

Method 36 (Preparative HPLC, Kromasil, 0.05% TFA): Device: Gilson Abimed HPLC; binary pump system; Column: Kromasil-IOOA Ci ₈ , 5 microns; 250 mm x. 30 mm; Eluent A: water / 0.05% trifluoroacetic acid, eluent B: acetonitrile; 0-10 min 10% B, ramp, 10.01-55 min 100% B; Flow: 30 ml / min; UV detector 210 nm.

Method 37 (Preparative HPLC, Kromasil, 0.05% TFA): Device: Gilson Abimed HPLC; binary pump system; Column: Kromasil-100A C _{] 8} , 5 μm; 250 mm JC 21 mm; Eluent A: water / 0.05% trifluoroacetic acid, eluent B: acetonitrile; 0-10 min 10% B, ramp, 10.01-55 min 100% B; Flow: 20 ml / min; UV detector 210 nm.

Method 38 (Preparative HPLC, Kromasil, 0.05% TFA): Device: Gilson Abimed HPLC; binary pump system; Column: Kromasil-100A Ci ₈ , 5 μm; 250 mm x 21 mm; Eluent A: Water / 0.05% trifluoroacetic acid, eluent B: acetonitrile / water 50/50; 0-10 min 10% B, ramp, 10.01-55 min 100% B; Flow: 20 ml / min; UV detector 210 um.

Method 39 (Preparative HPLC, Kromasil, 0.05% TFA): Device: Gilson Abimed HPLC; binary pump system; Column: Kromasil 100A Ci ₈ , 5 μm; 250 mm x 30 mm; Eluent A: water / 0.05% trifluoroacetic acid, eluent B: acetonitrile; 0-10 min 10% B, ramp, 10.01-55 min 100% B; Flow: 10-60 ml / min; UV detector 210 nm.

Method 40 (Preparative HPLC): Device: Gilson Abimed HPLC; UV detector 210 nm; binary pump system; Column: Waters Symmetry-Prep ™ Ci ₈ , 7 μm, 300 mm x 19 mm; Eluent A: 0.05% trifluoroacetic acid in water, eluent B: 0.05% trifluoroacetic acid in acetonitrile: flow rate: 20 ml / min; 0-3 min 10% B, ramp, 30-38 min 90% B, 38-45 min 10% B.

Method 41 (Preparative HPLC, Grom-Sil, 0.1% TFA): Device: Gilson Abimed HPLC; UV detector 210 nm; binary pump system; Column: Grom-Sil SNr.4051 120 ODS-4HE, 10 μm, 250 mm x 40 mm; Eluent A: 0.1% trifluoroacetic acid in water, eluent B: 0.1% trifluoroacetic acid in acetonitrile; Flow rate: 50 ml / min; 0-3 min 10% B, 3-27 min gradient ramp, 27-35 min 95% B, 35-40 min 10% B.

Method 42 (Chiral Preparative HPLC): column (steel column, dimension 500 mm x 50 mm); stationary phase (Chirapak AD, 20 μm); Eluent A: iso-hexane, eluent B: iso-propanol; Gradient: 0- 11.74 min 15% B, 11.74-11.75 min 100% B, 15.74 min 1 00% B, 15.75 min 15% B, 21.25 min 15% B; Sample preparation: dissolve in 50 ml iso-propanol / 200 ml iso-hexane; Injection volume: 30 ml, temperature: 24 ⁰ C; UV detection: 220 nm.

Method 43 (Preparative HPLC): Device: Gilson Abimed HPLC; UV detector 254 nm; binary pump system; Column: Nucleosil RP-18, 7 μm; 250 x 5 O mm; Flow: 30 ml / min; Eluent A: water / 0.1% trifluoroacetic acid, eluent B: acetonitrile / O.1% trifluoroacetic acid; Gradient: 0-40 minutes 20-25% B, 40-60 minutes 25% B, 60-110 minutes 25-50% B, 110-120 minutes 50% B, 120-130 minutes 50-100% B, 130- 160 min 100% B, then regeneration of the chromatography column.

Method 44 (Preparative HPLC): Device: Gilson Abimed HPLC; UV detector 254 nm; binary pump system; Column: Nucleosil RP-18, 7 μm; 250 x 50 mm; Flow: 40 ml / min; Eluent A: water / 0.05% trifluoroacetic acid, eluent B: acetonitrile / 0.05% trifluoroacetic acid; Gradient: 0-105min 20-25% B, 105-111min 25% B, 111-131min 25-27 ° / l> B, 131-157min 27-35% B, 157-192min 35-40 % B, 40-45% B, 192-207 min, then regeneration of the chromatography column. General working instructions

Procedure 1 (Removal of Boc Protective Experts with TFA)

The Boc-protected compound is suspended in dichloromethane (1 / 5-1 / 10 of the Reaktions¬ solution) and then under argon inert gas atmosphere with trifluoroacetic acid in dichloro-5 methane (30%, about 1 ml / 10 mg starting material in the 100- 1 millimole scale, about 1 ml / 1 mg in 100-1 micromole scale) and stirred at room temperature until the HPLC chromatogram shows complete conversion (method 2). Then the solvent is distilled off in vacuo, the bath temperature should not exceed 3O ⁰ C. The crude product is suspended in toluene, concentrated again on a rotary evaporator and dried under high vacuum. 0 This procedure is repeated several times with toluene or with dichloromethane (two to five times).

Operating procedure 2 (removal of the N-fer ^ -butoxycarbonyl protective group, dioxane, 4% hydrogen chloride)

The N- (tert-butoxycarbonyl) -protected compound (1 mmol) is initially charged in dioxane (2-3 ml) under argon inert gas atmosphere. At RT and with vigorous stirring, 4N hydrochloric acid in 5% dioxane (30 ml) is added dropwise. The mixture is stirred until the analytical HPLC (method 2) indicates complete conversion (about 2 h). The reaction mixture is evaporated at RT in vacuo. The crude product is taken up in a little dichloromethane and freed from solvent again in vacuo. This process is repeated several times with toluene (twice) and with dichloromethane (twice). Finally, the crude product is lyophilized or directly further reacted after O high-vacuum drying.

Procedure 3 (Hydrogenolvent ester cleavage, cleavage of the Cbz protective group)

The peptidic benzyl ester or the peptidic N-Cbz-protected amine (1.2 mmol) is dissolved in methanol (60 ml) and treated under argon protective gas atmosphere with 1 Oproz. Palladium-carbon (100 mg). At RT and normal pressure, hydrogenation is carried out until analytical HPLC 5 (method 2) indicates complete conversion. The reaction mixture is filtered (eg., Via diatomaceous earth, Celite ^®), concentrated in vacuo and dried under high vacuum.

Working instruction 4 (hydrolytic ester cleavage, yserification)

The carboxylic acid ester (3 mmol) under argon protective gas atmosphere in THF / water / DMF

200/100 / 2.5 (20 ml). At 0 ^° C., under strict temperature control, powdered O lithium hydroxide (3.6 mmol, 1.2 equivalents) is added in portions to the strongly stirred solution.

If complete conversion is not observed after 2 h by analytical HPLC, the reaction is repeated Solid lithium hydroxide added (3.3 mmol, 1.1 equivalents). This process is repeated until complete conversion, whereupon the reaction mixture is adjusted to pH 3-4 at ⁰ ° C. with 0.1 N aqueous hydrochloric acid and concentrated in vacuo and then dried. The crude product can now be gel chromatographed (Method 14) and / or finely purified by preparative HPLC (Method 13).

Working instruction 5 (amide coupling)

To a solution of the amine Cy clopeptids (1.0 equivalents), the dipeptide acid (1.5 - 1.8 equivalents and NMM (1.0-2.0 equivalents) in dry DMF, HATU (1.5-1.8 equivalents) is first added at -2.0 ⁰ C under argon inert gas atmosphere. The reaction mixture is stirred (about 15 minutes) and admixed again with NMM (2.5-3.0 equivalents) The IReaktionsgemisch is slowly heated from -1O ⁰ C to RT and is stirred at this temperature until a complete conversion of the amine component The reaction mixture is admixed with solid potassium dihydrogen phosphate (10 equivalents, 500 μmol) and then concentrated by evaporation in a high vacuum and purified by chromatography.

Alternatively, the reaction after reaction with water and methane «! are added and then after purification via preparative RP-HPLC or gel chromatography, the product is obtained.

starting compounds

Example IA

Fermentation:

Culture Medium:

YM: Yeast Malt Agar: D-glucose (4 g / l), yeast extract (4 g / l), malt extract (10 g / l), 1 liter of Lewatit water. Before sterilization (at 121 ° C for 20 minutes) the pH is adjusted to 7.2.

HPM: mannitol (5.4 g / L), yeast extract (5 g / L), meat peptone (3 g / L).

Working Preservation: The lyophilized strain (ATCC 53042) is grown in 50 ml of YM medium.

Piston Fermentation: Inoculate 150 ml of YM medium or 100 ml of HPM medium in a 1 1 Erlenmeyer flask with 2 ml of the working fluid and grow on a shaker at 240 rpm for 30-48 hours at 28 ° C. 30 1 Fermentation: 300 ml of the piston fermentation (HPM medium) are used to inoculate a sterile 30 L culture medium solution (1 ml Antifoam SAG 5693/1). This culture is wm for 21 hours at 28 ⁰ C, 300 rpm and aeration with sterile air of 0.3 grown. 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 cultivation time.

Main culture (200 l): 15 x 150 ml YM medium in 1 l Erlenmeyer flask is inoculated with 2 ml of the working preserve and grown at 28 ° C for 48 hours and 240 rpm on the shaker. 2250 ml of this culture are used to inoculate a sterile 200 L culture medium 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 μm.

To control the fermentation process, samples are taken every hour (50 ml). 2 ml of this culture broth are mixed with 1 ml of methanol (0.5% trifluoroacetic acid) and filtered through a 0.45 micron filter. 30 μl of this suspension are analyzed by HPLC (Method 17 and Method 18).

After 18.5 hours, the culture broth of the main culture is separated at 17000 rpm in supernatant and sediment.

Insulation:

The supernatant (183 1) is adjusted to pH 6.5-7 with concentrated trifluoroacetic acid or sodium hydroxide solution and applied to a Lewapol column (OC 1064, 60 l content). It is then eluted with pure water, water / methanol 1: 1 and then with pure methanol (with 0.1% trifluoroacetic acid). This organic phase is concentrated in vacuo to a residual aqueous residue of 11.5 1.

The remaining aqueous phase is bound to silica gel Cis and separated (MPLC, Biotage Flash 75, 75 × 30 cm, KP-Cl 8-WP, 15-20 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 43), then by gel filtration on Sephadex LH-20 (5 × 70 cm, acetonitrile / water 1: 1, each with 0.05% trifluoroacetic acid) and another preparative HPLC ( Method 44) separately.

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 resulting lyophilizate (89.9 g) is taken up in methanol, filtered off, concentrated and separated (Method 20). Example IA is then purified by gel filtration (Sephadex LH-20, 5 x 68 cm, water / acetonitrile 9: 1 (with 0.05% trifluoroacetic acid), flow: 2.7 ml / min, fraction size 13.5 ml) to give the pure substance.

This process provides 447 mg of Example IA.

HPLC (Method 17): R ₄ = 6.19 in MS (ESIpos): m / z = 1277 (M + H) ⁺ ¹ U NMR (500.13 MHz, 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), 0.92 (d, 3H), 0.95 (d, 3H), 0.96 (i.e. , 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), 5.58 (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), 7.20 (br, 2H), 7.23 (m, 2H), 7.42 (m, 2H), 7.54 (i.e. , IH), 7.58 (d, IH), 8.32 (br., IH), 9.18 (br., IH), 9.20 (m, 2H), 9.50 (br., IH). ¹³ C NMR (125.77 MHz, J ₀ -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.

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).

Example 2A

Mixture of dihydro-lysobactin trifluoroacetate {D-Leu-Leu-Phe - [(3R) -Leu (3-OH)] - Leu-D-Arg-Ile-aThr-Gly - [(3S) -3-Asn ( 3-OHr)] - Ser-trifluoroacetate} and octahydro-lysobactin trifluoroacetate {D-leu-leu-Ala (3-cyclohexyl) - [(3J?) - Leu (3-OH)] - Leu-D-Arg- Ile-aThr-Gly - [(3, S ^r) -3-Asn (3-OH)] - Ser} trifluoroacetate

Lysobactin bistrifluoroacetate (Example IA, 500 mg, 0.33 mmol) is dissolved in isopropanol / water 2: 1 (30 ml). Under argon protective gas atmosphere, palladium on carbon (10%, 100 mg) is added. The reaction mixture is stirred (after degassing) in a pressure autoclave at 80-70 bar hydrogen pressure and RT for 48 h. Palladium on carbon (10%, 100 mg) is again added to the reaction. The reaction mixture is (after degassing) again stirred in a pressure autoclave at 80-70 bar hydrogen pressure and RT for 48 h. Now it is no longer possible to detect lysobactin by HPLC (method 2). The reaction mixture is filtered through a glass frit (pore size 4) or diatomaceous earth, concentrated in vacuo, taken up in 1% ethanol / 0.2% acetic acid, filtered through a syringe filter (Biotage, PTFE), concentrated in vacuo and dried under high vacuum. This gives 496 mg (quant.) Product (80% »Dihydrolysobactin, 20% octahydrolysobactin).

Example 3A

[(3i?) - 3-hydroxy-L-leucyl] -L-leucyl-D-arginyl-L-isoleucyl-L-allothreony-l-glycyl - [(35) -3-hydroxy-L-asparaginyl] -L serine trifluoroacetate {Edman Decomposition Trifluoroacetate}

775 mg of dihydrolysobactin and octahydrolysobactin are dissolved in 77.5 ml of methanol and then treated with 667 ml of cleavage buffer (0.1 M ammonium bicarbonate solution / 0.5 M urea pH 8). Before the enzyme addition, the solution is in a drying oven at 37 ⁰ C it warms ~. 31 mg chymotrypsin (31 ml chymotrypsin solution water / ethylene glycol 1: 1, 1 mg / ml, 1:25, 37 ° C preheated) are added and the reaction is carried out at 37 ° C. The enzyme reaction is stopped after 60 min with 30 ml of acetonitrile and about 6 ml of TFA. The pH of the solution is between 1 and 2. Until the preparative separation, the solution can be stored at -2O ⁰ C.

Preparative separation of fragments 1-3 and 4-11

Approximately 800 ml of the cleavage solution are filtered through a filter (0.2 μm) and chromatographed in two portions of approximately 400 ml each on a Source 15RPC column (40 ml) with an acetonitrile / TTA gradient. Conditions: eluent A: 0.1% TFA, eluent B: 0.1% TFA / acetoritritil; Gradient: 0% B to 45% B in 40 minutes; Flow: 2 ml / min; UV detection 210 nm; Fraction size 1.5 ml.

The fragments 4-11 (R ₄ = ca. 15 min) and 1-3 (LLF) (R ₁ = ca. 25 min) as well as 1-3 (LLA (3-cyclohexyl)) (R ₁ = ca. 30 min) are combined and lyophilized. This gives the title compound as fragment 4-11.

HPLC / UV-Vis (Method 3): R _t = 1.2 min. LC-MS (method 7): R _t = 1.0 min; MS (ESI pos.): M / z (%) = 453.5 (100) [M + 2H] ²⁺ , 906 (10) [M + H] ⁺ . MS (ES Ineg.): M / z (%) = 904 (100) [M-H] ^" LC-MS (Method 5): R ₁ = 3.6 min; MS (ESIpos.): M / z (%) = 453.5 (100) [M + 2H] ²⁺ MS (ES Ineg.): M / z (%) = 904 (100) [M - Hf. Example 4A

Methyl- (2) -2 - [(^ erf-butoxycarbonyl) amino] -4,4-dimethylpent-2-enoate

Pivalaldehyde (303.2 g, 3.41 mol, 10 equiv) and methyl {[tert-butoxycarbonyl] amhrino} (dimethoxyphosphoryl) acetate (101.5 g, 0.341 mol, 1.0 equiv.) Are dissolved in tetrahydrofuran

(800 ml) and cooled to -70 ⁰ C cooled. At -7O ⁰ C, N, N, N-tetramethylguanidine (78.7 g,

0.683 mmol, 6.95 ml, 2.0 equivalents) was slowly added dropwise and then quilted 4 h at -70 ⁰ C, followed by 4 days at RT. The reaction mixture is concentrated, then with

Extracted ethyl acetate (2 x 500 ml) against water, the combined organic phase »washed with saturated sodium chloride solution (100 ml) and dried over Νatriumsulfat. TSTach the concentration, the crude product is chromatographed (1.5 kg silica gel, eluent:

Cyclohexane / ethyl acetate 5: 1). There are obtained 67 g (76% of theory) of the title compound.

Alternatively, the crude product after the aqueous workup can be purified by crystallization from cyclohexane / ethyl acetate.

R / (silica gel, cyclohexane / ethyl acetate 4: 1) = 0.5

LC-MS (Method 7): R _t = 2.5 min;

MS (ESI pos.): M / z (%) = 158 (100) [M - Boc + H] ⁺ , 280 (5) [M + Na] ⁺ .

¹ H NMR (400 MHz, J ₆ -DMSO): δ = 1.08 (s, 9H, C (CH ₃ ) ₃ ), 1.38 (s, 9H, OC (CH ₃ ) ₃ ), 3.61 (s ₌ 3H, CO ₂ CH _3), 6.40 (s, IH, C H ^beta), 8.14 (s, IH, NH). HR-TOF-MS (Method 1): C _{] 3} H ₂₃ NO ₄ [M + H] ⁺ gef. 258.1696, calculated 258.1700. Example 5A

N-tert-ButoxycarbonyW-tert-butyl-D-alanine methylester

Methyl (2Z) -2 - [(α-butoxycarbonyl) amino] -4,4-dimethylpent-2-enoate (Example 4A, 60 g, 233.2 mmol) is dissolved in ethanol 3: 1/1000 ml (1000 ml ) solved. With a cannula is about 10 min

Passed through argon. The solution is placed in an ultrasonic bath (about 5 minutes), and it becomes (+) -

1, 2-Bis - [(2i, 5i) diethylphospholano] benzene (cyclooctadiene) rhodium (I) triflate (600 mg, 1 wt%). It is hydrogenated for 3 days at 3.5 bar hydrogen pressure and RT. The

The reaction mixture is filtered through kieselguhr and the eluate is concentrated. The crude product is chromatographed (silica gel, eluent: cyclohexane / ethyl acetate 4: 1). There are obtained 60 g (99% of theory) of the title compound.

[α] ²⁰ _Na = + 5 ° (c = 0.33 in CHCl ₃ ).

DCI-MS (NH ₃ ): m / z (%) = 221 (100), 260 (40) [M + H] ⁺ , 277 (100) [M + NH ₄ ] ⁺ . ¹ H NMR (400 MHz, CDCl _3): δ = 0.93 (s, 9H), 1:40 (m, 10H), 1.68 (dd, J = 3.7, 14.5 Hz, 1H), 3.68 (s, 3H), 4.30 ( t, J = 7.9 Hz, IH), 4.81 (d, br, J = 7.7 Hz, IH).

¹³ C NMR (126 MHz, CDCl _3): δ = 28.30 (3C), 29.52 (3C), 30.61, 46.27, 51.19, 52.17, 79.79,

155.11, 174.39.

HR-TOF-MS (method 1). C ₂₆ H _5I N ₂ O ₈ via 519.3640, obs. 519.3634 [M + H] ⁺ .

Example 6A

N-tert-butoxycarbonyl-3-tert-butyl-D-alanine

N-tert-butoxycarbonyl-3-tert-butyl-D-alanine methyl ester (Example 5A, 60 g, 231 mmol) is dissolved in tetrahydrofuran p.a. (463 ml) dissolved. At RT, a solution of lithium hydroxide monohydrate (19.4 g, 462.7 mmol) in water (463 ml) is slowly added dropwise. When the HPLC chromatogram (Method 2) shows complete conversion (about 20 h), the reaction mixture is cautiously adjusted to pH 3-4 with 1 Ν aqueous hydrochloric acid while cooling with ice. To the reaction mixture is added sodium chloride (150 g), followed by extraction with ethyl acetate (twice 500 ml). The combined organic phases are dried with saturated sodium chloride solution and then with sodium sulfate and filtered. The filtrate is concentrated on a rotary evaporator and dried under high vacuum. This gives 55.4 g (98% of theory) of the title compound.

HPLC / UV-Vis (Method 4): R, = 4.2 min.

DCI-MS (NH ₃ ): m / z (%) = 263 (100) [M + NH ₄ J ⁺ , 280 (5) [M + N ₂ H ₇ J ⁺ .

¹ H NMR (400 MHz, CDCl ₃ ): δ = 0.96 (s, 9H ₉ C (CH ₃ ) ₃ ), 1.42 (m, 10H, OC (CH ₃ ) ₃ , H ^), 1.79 ("d", J = 14.4 Hz, IH, H ^A ), 4.31 (t, J = 8.0 Hz, IH, H ^α ), 4.82 (d, J = 8.4 Hz, IH, NH).

¹³ C NMR (126 MHz, CDCl _3): δ = 28.32 (3C), 29.54 (3C), 30.74, 45.92, 51.24, 80.19, 155.41,

178.93.

HR-TOF-MS (Method 1): C ₂₄ H ₄₇ N ₂ O ₈ calc. 491.3327, et. 491.3328 [2M + HI ⁺ .

Example 7A

3-tert-Butyl 1-L-alanine methyl ester hydrochloride

HCl

For the preparation of the title compound, see SD Bull, SG Davies, AC Garner, MD O'Shea, J. Chem. Soc. Perkin Trans. I, 2001, 3281-3287.

The title compound is prepared according to protocol 2 from N-tert-butoxycarbonyl-3- / tert-butyl-L-alanine methyl ester (28.0 g, 108 mmol) and 4N hydrochloric acid in dioxane (280 ml). Yield 22 g (quantitative). The product is further reacted without purification.

¹ H NMR (400 MHz, J ₆ -DMSO): δ = 0.95 (s, br, 9H, tBu), 1.65 (dd, IH), 1.80 (dd, IH), 3.75 (s, 3H, OCH ₃ ), 3.90 (dd, IH), 8.60 (s, br, 3H).

Example 8A

N - (? ^ He butoxycarbonyl)? -3- e -f-butyl-D-alanyl-3-fer / -butyl-L-alanine-methylester

To a solution of 3-tert-butyl-L-alanine methyl ester hydrochloride (1.1 equivalents, 21 g, 107 mmol) in dichloromethane pa (1.4 l) are added successively at -2O ⁰ C HOBt (3 equivalents, 39.4 g, 292 mmol), N-methylmorpholine (3 equivalents, 32.1 mL, 291.8 mmol), N-te / t-butoxycarbonyl-3-tert-butyl-D-alanine (Example 6A, 1.0 equiv., 97.3 mmol), EDC (2 Equivalents, 37.3 g, 194.6 mmol) and again N-methylmorpholine (2 equivalents, 21.4 mL, 1 ^ 4.5 mmol). The reaction mixture warms slowly (about 12 h) to RT, with complete conversion of the amine component being monitored by HPLC. The Reaktionsgernisch is then washed with saturated aqueous sodium bicarbonate solution (300 ml), 5% aqueous citric acid (twice 500 ml), saturated aqueous sodium bicarbonate solution (500 ml) and saturated sodium chloride solution. It is dried over sodium sulfate and filtered. The mixture is evaporated to dryness in vacuo and subsequently dried in a high vacuum. This gives 36 g (96% of theory) of the title compound, which is reacted otxne further purification. DCI-MS (NH ₃ ): m / z (%) = 387 (40) [M + H] ⁺ , 404 (100) [M + NTH ₄ J ⁺ .

¹ H NMR (400 MHz, J ₆ -DMSO): δ = 0.95 (s, br, 18H, tBu), 1.35 (dd, IH), 1.45 (s, 9H, OtBu), 1.50 (dd, IH), 1.65 (dd, IH), 1.95 (dd, br, IH), 3.70 (s, 3H, OCH _3), 4.15 ( "t", br, IH), 4:55 ( "t", br, IH), 4.80 (d , IH), 6.65 (d, br, IH).

Example 9A

N- (ter ^ butoxycarbonyl) -D-leucyl-L-leucine benzyl ester

To a solution of L-leucine benzyl ester hydrochloride (1.1 equivalents, 300 mg, 1.1 mmol, see T. Wakamiya, M. Kamata, S. Kusumoto, H. Kobayashi, Y. Sai, et al., Bull be. Chem. Soc. Jpn., 1998, 71, 699-710) in dichloromethane pa (30 ml) at -10 ⁰ C successively HOBt (4 equivalents, 540 mg, 4 mmol), N-methylmorpholine (3 equivalents, 3 mmol), N-tert-butoxycarbonyl-D-leucine (1.0 equivalent, 230 mg, 1 mmol, from Bachern), EDC (2 equivalents, 380 mg, 2 mmol) and again N-methylmorpholine (2 equivalents, 2 mmol). given. The reaction mixture warms slowly (about 12 h) to RT, with complete conversion of the amine component being monitored by HPLC. The reaction mixture is concentrated in vacuo, taken up in acetonitrile and purified by preparative HPLC (Method 13). 362 mg (80% of theory) of the title compound are obtained.

HPLC / UV-Vis (Method 4): R ₁ = 5.3 min. [Ot] ²⁰ _Na = + 19 ° (c = 0.10 in methylene chloride). Example IQA

N- (ferr-butoxycarbonyl) -3-ter / -butyl-D-alanyl-3-RERT-butyl-L-alanine

N- (tert-butoxycarbonyl) -3-r-butyl-D-alanyl-3-he-? -Butyl-L-alanine methyl ester (Example 8A, 36 g, 93.1 mmol) is dissolved in tetrahydrofuran pa ( 279 ml). At about 10 ⁰ C a solution of lithium hydroxide monohydrate (7.82 g, 186.3 mmol, 2 equivalents) is added dropwise to water (187 ml) slowly. When the HPXC chromatogram (Method 2) shows complete conversion (approximately 20 h), the reaction mixture is freed from THF at 200 mbar and then extracted with methyl tert-methyl ether (200 ml). The organic phase is diluted with ethyl acetate (5O0 ml) and then treated with water * and then carefully adjusted to pH 3-4 with 1 Ν aqueous hydrochloric acid. The combined organic phases are washed with saturated aqueous common salt solution, dried over sodium sulfate, filtered, evaporated in vacuo and dried under high vacuum. This gives 97.4 g (97% of theory) of the title compound.

LC-MS (Method 7): R _t = 2.26 min; MS (ESI pos.): M / z (%) = 373 (100) [M + H] ⁺ .

HR-TOF-MS (method 1): C ₁₉ H ₃₇ N ₂ O ₅ calc. 373.2702, gef. 373.2717 [M + H] ^" * ^" . ¹ H NMR (400 MHz, J ₆ -DMSO): δ = 0.83 (s, br, 18H, tBu), 1.31 (s, 9H, OtBu>, 1.40 (d, J = 6.1 Hz, 2H ₅ ^ -CH ₂ ), 1.48 (dd, J = 14.1, 9.4 Hz, lH, yβ-CH), 1.59 (dd, J = 14.1, 2.7 Hz, lH _{5 j} S-CH ') ₅ 3.98 (m, IH, α-CH) , 4.12 (m, IH, α-CH), 6.73 (d, J = 9.1 Hz, IH, NH), 7.72 (d, J = 7.9 Hz, IH, NH), 12.42 (s, br, IH, CO ₂ H).

¹³ C NMR (125 MHz, J ₆ -DMSO): δ = 28.49 (3C), 29.66 (3C), 29.78 (3C), 30.52, 30.58, 44.63 OS-CH ₂ ), 45.24 05-CH ₂ ), 49.67 ( α-CH), 52.40 (α-CH), 78.29, 155.05, 172.97, 174.61. Example IIA

N - (ω-butoxycarbonyl) -D-leucyl-L-leucine

N- (Butoxycarbonyl) -D-leucyl-L-leucine benzyl ester (Example 9A, 308 mg, 0.69 mmol) is reacted according to procedure 3. 244.6 mg (99% of theory) of the title compound are obtained.

Example 12A

Under argon protective gas atmosphere, a 1 N solution of lithium hexamethyldisilazide (157.5 mmol, 157.5 ml, 2.2 equivalents) in THF in the reaction solvent THF (300 ml) is introduced. Ch 32 1991 [(tert-butoxycarbonyl) amino] -3-phenylpropan säuremethylester (AV Rao Rama, AK Singh, VNS Varaprasad, Tetrahedron Lett, - at -78 ⁰ C, a solution of (rac) -3.. 4393-4396) (20 g, 71.2 mmol) was slowly added dropwise. The mixture is stirred for 1 0 min at -25 ⁰ C and then cooled again to -78 ⁰ C. Dibenzylazadicarboxylate (34.2 g, 114.6 mmol, 1.6 equiv.) Is added in one portion to the reaction mixture. It is stirred for 3 h at -60 to -45 ° C. In order to stop the reaction is again cooled down to -78 ° C and (20.5 ml, 358 mmol, 5 equivalents) of acetic acid was added and then heated to RT and finally 0 ⁰ C. The reaction mixture is evaporated in vacuo and taken up in ethyl acetate (1000 ml). The suspension is washed with saturated aqueous sodium bicarbonate solution (twice), water (once), 5% aqueous citric acid (twice) and saturated aqueous sodium chloride solution (once). All aqueous phases are reextracted one at a time with ethyl acetate. All organic phases are evaporated in vacuo and taken up again in dichloromethane (2000 ml), filtered, dried over sodium sulfate, filtered again, evaporated in vacuo and dried under high vacuum. This gives 7.2 g (18% of theory) of the title compound as a solid. The filtrate of the dichloromethane phase (vide suprά) is concentrated and then recrystallized from methanol to give 13.2 g (26% of theory) of the title compound.

Alternative Synthesis Procedure: A 1 N solution of lithium hexamethyldisilazide (590.7 mmol, 590.7 ml, 2.2 equivalents) in THF in the reaction solvent THF (2500 ml) is placed under argon protective gas atmosphere Λ. At -78 ° C, a solution of (rac) -3 - [(tert-butoxycarbonyl) amino] -3-phenylpropanoic acid methyl ester (75 g, 268.5 mmol) is slowly added dropwise (internal temperature about -70 ⁰ C). The mixture is stirred for 10 min at -25 ° C and then cooled again to -70 ⁰ C. down. Dry dibenzyl azadicarboxylate (128.2 g, 429.6 mmol, 1.6 equiv.) Is added to the reaction mixture in four portions as a solid. It is stirred for 2 h at -60 to -50 ⁰ C. To stop the reaction, is cooled down again to -78 ⁰ C and treated with acetic acid (76.? Ml, 1342 mmol, 5 equivalents) was added, and then finally RT and warmed to 0 ⁰ C. The title compound precipitates out as a solid, which is dried in a vacuum drying oven (4.1 g, 2.2% of theory, of crystallizate 1). The reaction mixture is evaporated in vacuo and the residue is recrystallized from ethyl acetate to give the title compound as a solid (38.8 g, 24.3% of theory, crystallizate T). The mother liquor is diluted with ethyl acetate and then washed with water (twice) and saturated aqueous sodium chloride solution (twice). The organic phase is dried over sodium sulfate, filtered, evaporated in vacuo and dried under high vacuum. The crude product is recrystallized from ethyl acetate-diethyl ether. The title compound is obtained as a solid (50.4 g, 28% of theory, crystallization t 3). The mother liquor is evaporated and purified by flash chromatography (1.5 kg of silica gel, toluene / ethyl acetate 4: 1). This gives 4.7 g (1.7% of theory) of the title compound. Total yield 98.0 g title compound (56.2% of theory).

LC-MS (Method 5): R _t = 6.8 min;

MS (ESI pos.): M / z (%) = 578 (40) [M + H] ⁺ , 1156 (100) [2M + H] ⁺ .

MS (ES Ineg.): M / z (%) = 576 (100) [M-H] ^" .

HR-TOF-MS (Method 1): C ₃ H ₃₆ N ₃ O ₈ [M + H] ⁺ gef. 578.2490, accounted for 578.2497.

The relative stereochemistry of the title compound is confirmed by an X-ray structure (Figure L).

Example 13A

Methyl (2S *, 3i *) - N - [(benzyloxy) carbonyl] - N - [(benzyloxy) carbonylamino] -3 - [(tert-butoxycarbonyl) amino] phenylalaninate

Under argon protective gas atmosphere, a solution of methyl (2R *, 3jf? *) - N ² - [(benzyloxy) carbonyl] -N ² - [(benzyloxy) carbonylamino] -3 - [(tert-butoxycarbonyl) amino] - Phenylalaninat (Example 12A, 20.5 g, 35.5 mmol) in dry DMF pa (750 ml) at 0 ⁰ C with N, NN, N-tetramethylguanidine (50 ml, 399 mmol). The reaction mixture is allowed to thaw and stirred until the HPLC chromatogram (Method 2) indicates complete conversion (approximately 60% product) (approximately 12 h) to stop the reaction by adding acetic acid (pH 4-6). The reaction mixture is evaporated in vacuo at RT and taken up in ethyl acetate. The organic phase is washed with water (twice), 5% citric acid (twice), water (once), saturated aqueous sodium bicarbonate solution (twice), saturated aqueous sodium chloride solution (once), dried over sodium sulfate, filtered, evaporated in vacuo, under high vacuum dried. The crude product is purified by preparative HPLC (Method 11) or flash chromatography (silica gel, cyclohexane / ethyl acetate 3: 1). This gives 7.7 g (37% of theory) of the title compound and 5 g of the starting compound (25% of theory).

Alternative Synthesis Method: A solution of methyl (2i *, 3R *) - N ² - [(benzyloxy) carbonyl] -N ² - [(benzyloxy) carbonylamino] -3 - [(^ erf-butoxycarbonyl) - under argon protective gas atmosphere. amino] -phenylalaninate (Example 13A, 22.0 g, 38.1 mmol) in dry dichloromethane (540 ml) at 0 ⁰ C with N, N, N, N-tetramethylguanidine (50 ml, 399 mmol). The reaction mixture is thawed and stirred until the HPLC chromatogram indicates complete conversion (about 65% product) (about 12 h) to stop the reaction by adding acetic acid (23 ml, pH 4-6). The organic phase is washed with water (twice), saturated aqueous sodium bicarbonate solution (twice), saturated aqueous sodium chloride solution (once). The aqueous phases are reextracted with dichloromethane. The United organic phases are dried over sodium sulfate, filtered, evaporated in vacuo, dried under high vacuum. The crude product is purified by flash chromatography (6 1 silica gel, cyclohexane / ethyl acetate 3: 1). This gives 13.7 g (62% of theory, based on the recovered starting material 69%) of the title compound and 3.1 g of the starting compound (10% of theory).

HPLC / UV-Vis (method 2): R ₁ = 3.0 min. LC-MS (Method 8): R ₁ = 3.0 min;

MS (ESI pos.): Mfz (%) = 478 (100) [M -Boc + H] ⁺ , 578 (30) [M + H] ⁺ . LC-MS (Method 5): R ₁ = 7.0 min; MS (ESI pos.): Mfz (%) = 578 (40) [M + H] ⁺ , 1156 (100) [2M + H] ⁺ . MS (ES Ineg.): Mfz (%) = 576 (100) [M-H] ^" . HR-TOF-MS (Method 1): C ₃ H ₃₆ N ₃ O ₈ [M + H] ⁺ F. 578.2483, ber. 578.2497.

Example 14A

Methyl (2S *, 3R *) - 3 - [(ter ^ butoxycarbonyl) amino] phenylalaninate

Under argon protective gas atmosphere is added to a solution of methyl (25 '*, 3i? *) - N - [(benzyloxy) carbonyl] - N - [(benzyloxy) carbonylamino] -3 - [(te7t-butoxycarbonyl) amino] -phenylalaninate (Example 13A, 705 mg, 1.22 mmol) in methanol / dichloromethane 1: 1 (42 mL) of Raney-Νickel (61 mg, ca. 10 mol%). The reaction mixture is hydrogenated in a pressure autoclave at 80 bar hydrogen pressure and RT (40 h). The HPLC chromatogram shows complete conversion. The reaction mixture is filtered under argon protective gas atmosphere over a glass frit, the glass frit is washed several times with methanol / water / 0.2% acetic acid. The filtrate is evaporated in vacuo and dried under high vacuum. This gives a solid (about 3 g) which is then suspended in ethyl acetate in an ultrasonic bath. To the suspension is added a solution of EDTA (400 mg) in 7% aqueous sodium bicarbonate (400 ml). The aqueous phase is extracted with ethyl acetate (100 ml ₃ three times). The combined organic phases are then treated with saturated aqueous sodium bicarbonate. carbonate solution (once), and washed with saturated aqueous sodium chloride solution (twice). All aqueous phases are reextracted one at a time with ethyl acetate. The combined organic phases are then dried over sodium sulfate, filtered and dried under high vacuum. The product obtained is a solid (1.26 g, quant.) Which is further reacted without fine cleaning.

HPLC / UV-Vis (Method 3): R _t = 1.7 min. LC-MS (Method 5): R, = 4.1 min; MS (ESI pos.): M / z (%) = 239 (100), 295 (80) [M + H] ⁺ .

¹ H NMR (400 MHz, ^ ₆ -DMSO): δ = 1.36 (s, 9H), 3.34 (s, 3H), 3.60 (d, J = 5.3 Oz, IH), 4.89 (dd, J = 9.6, 4.9 Hz, IH), 7.20-7.34 (m, 5H), 7.41 (d, J = 9.4 Hz, IH).

¹³ C NMR (125 MHz, ^ ₆ -DMSO): δ = 28.24 (3C), 52.84, 54.91, 56.86, 79.23, 127.14 (2C), 128.44,

128.88 (2C), 137.28, 154.86, 168.16.

HR-TOF-MS (method 1): C ₁₅ H ₂₂ N ₂ O ₄ [M + H] ⁺ gef. 295.1658, ber. 295.1653.

Example 15A

Methyl (25 '*, 3R *) - N - [(benzyloxy) carbonyl] -3 - [(tert-butoxycarbonyl) amino] phenylalaninate

Under argon protective gas atmosphere is added to a solution of methyl (2S *, 3R. ^{= I =} ) -3 - [(terϊ-butoxycarbonyl) amino] phenylalaninat (Example 14A, 360 mg, 1.2 mmol) and N-Benzryloxycarbonyloxy succinimidester ( 610mg, 2.44mmol, 2equiv) in THF (25ml) at ⁰ ° C Λ ^ -methylmorpholine (260mg, 2.6mmol, 2.1equiv). The reaction mixture warms; slowly (12 h), with complete conversion observed by HPLC (Method 2). Acetic acid (0.7 ml) is then added, concentrated by evaporation in a vacuum and purified by means of preparative HPLC (Method 13). This gives 396 mg (76% of theory) of the title compound. HPLC / UV-Vis (Method 3): R ₄ = 2.7 min. LC-MS (Method 5): R _t = 6.4 min;

MS (ESI pos.): M / z (%) = 329 (100) [M -Boc + H] ⁺ , 429 (80) [IM + H] ⁺ , 858 (60) [2M + H] ⁺ . ¹ H NMR (500 MHz, DMSO): δ = 1.35 (s, 9H), 3.63 (s, 3H), 4.57 (dd, J = 9.5, 4.0 Hz, IH), 4.93 (m, 2H), 5.27 (dd, J = 10.1, 4.1 Hz, IH) ₅ 7.19-7.34 (m, 10H), 7.50 (d, J = 10.2 Hz, IH), 7.59 (d, J = 10.2 Hz, IH) ₁ ¹³ C NMR ( 126 MHz, J-DMSO): δ = 28.26 (3C), 52.33, 54.67, 59.06, 65.73, 78.73, 126.66 (2C), 127.42, 127.65 (2C), 128.00, 128.44 <2C), 128.53 (2C). 136.91, 139.26, 154.98, 156.25, 170.91. HR-TOF-MS (Method 1): C ₂₃ H ₂₈ N ₂ O ₆ Na [M + Na] ⁺ gef. 451 .1823, about 451.1845.

Example 16A

(25 *, 3i? *) - N ² - [(benzyloxy) carbonyl] -3 - [(terf-butoxycarbonyl) amino] -phenylalanine

Under argon protective gas atmosphere, a solution of methyl (2 <S '*, 3i → *) - N - [(benzyloxy) carbonyl] -3 - [(te ^ butoxycarbonyl) amino] phenylalaninate (Example 15A, 755 mg, 1.76 mmol ) in THF / water 2: 1 (30 ml). At 0 ^° C., a degassed 1% strength aqueous solution of lithium hydroxide monohydrate (86.5 mg, 3.6 mmol, 2 equivalents) is slowly added dropwise with vigorous stirring. The mixture is stirred at RT until the HPLC chromatogram (method 2) indicates complete conversion (about 1 h). It is then mixed with acetic acid (0.5 ml), the reaction mixture concentrated in vacuo and ufoerschichtet with ethyl acetate (100 ml). The aqueous phase is then acidified with 5% citric acid (pH 2-3) and then extracted with ethyl acetate (50 ml, three times). The combined organic phases are washed with saturated aqueous sodium chloride solution (20 ml, twice), dried over sodium sulfate, filtered, concentrated in vacuo and dried under high vacuum. 750 mg are obtained (quant.) Crude product of the title compound, which is finely purified by preparative HPLC (Method 13).

HPLC / UV-Vis (method 2): R, = 2.4 min.

LC-MS (Method 5): R _t = 6.1 min;

MS (ESIpos.): M / z (%) = 359 (100), 415 (60) [M + H] ⁺ , 829 (60) [2M + H] ⁺ .

MS (ESIng.): M / z (%) = 413 (100) [M-H] ^" .

¹ H NMR (500 MHz, ^ ₆ -DMSO): δ = 1.31 (s, 9H), 4.42 (dd, J = 10.0, 3.9 Hz, IH), 4.87 (s, 2H),

5.22 (dd, J = 10.0, 3.7 Hz, IH), 7.14-7.28 (m, 10H), 7.43-7.46 (m, 2H), 12.91 (s, br, IH).

¹³ C NMR (126 MHz, ^ _n -DMSO): δ = 28.33 (3C), 54.84 (C ^α), 58.94 (C ^Q), 65.54, 78.56, 126.52

(2C), 127.22, 127.53 (2C), 127.92, 128.37 (2C), 128.49 (2C), 137.03, 140.14, 155.08, 156.29,

171.60.

HR-TOF-MS (method 1): C ₂₂ H ₂₆ N ₂ O ₆ [M + H] ⁺ Found. 415.1860, ber. 415.1864.

Example 17A

(3R) -N ² - [(benzyloxy) carbonyl] -3 - [(^ erf-butoxycarbonyl) amino] -L-phenylalanine

The enantiomeric mixture of (2S *, 3i * *) - N - [(benzyloxy) carbonyl] -3-p (tert-butoxycarbonyl) -amino] phenylalanine (Example 16A, 750 mg, 1.8 mmol) is purified by preparative HPLC (Method 15 ) separated. 334 mg (98% ee, 45% of theory) of (2S, 3R) -N - [(benzyloxy) carbonyl] -3- [(tert-butoxycarbonyl) amino] phenylalanine (title compound) and 275 mg ( 98% ee, 37% of theory) (2R, 3S) - N - [(benzyloxy) carbonyl] -3 - [(tert-butoxycarbonyl) amino] phenylalanine (further enantiomer).

Enantiomer determination according to method 16. M = _Na ²⁰ + 22 ° (c = 0.50, chloroform) (title compound). M ²⁰ _Na = -20 ° (c = 0.49, chloroform) (further enantiomer).

¹ H NMR (500 MHz, J ₀ -DMSO): δ = 1.35 (s, 9H), 4.46 (dd, J = 9.6, 3.6 Hz, IH), 4.91 (s, 2H), 5.26 (dd, J = 9.9 , 3.6 Hz, IH), 7.18-7.32 (m, 10H), 7.48-7.51 (m, 2H), 12.95 (s, br, IH). ¹³ C NMR (126 MHz, δ = 28.34 (3C), 54.84, 58.96, 65.55, 78.56, 126.53 (2C), 127.23, 127.55 (2C) ₅ 127.93, 128.39 (2C), 128.51 (2C), 137.05, 140.16, 155.09, 156.30, 171.62. HR-TOF-MS (method 1): C ₂₂ H ₂₆ N ₂ O ₆ [M + H] ⁺ Found. 415.1864, ber. 415.1864.

Example 18A

2- (trimethylsilyl) ethyl (3R) -N - [(benzyloxy) carbonyl] -3 - [(^ e7t-butoxycarbonyl) amino] -L-phenylalaninate

A mixture of (3i?) - N ² - [(benzyloxy) carbonyl] -3 - [(fe / t-butoxycarbonyl) amino] -L-phenylalanine (Example 17A, 151 mg, 0.36 mmol), 2- ( Trimethylsilyl) ethanol (431 mg, 3.64 mmol, 10 equivalents) and 4 wt% molecular sieve 4 A (about 10 mg) in dry dichloromethane (2.7 ml) is stirred for 30 min at RT under argon protective gas atmosphere. Subsequently, (1 equivalent 44.5 mg, 0:36 mmol,) are (mg 150, 0.73 mmol, 2 equivalents) at -30 ⁰ C DCC and DMAP were added. The reaction mixture is thawed (about 12 h) and stirred at RT until the HPLC chromatogram indicates complete conversion (12-72 h). The reaction mixture is evaporated at RT in vacuo and purified by preparative HPLC (Method 13). 145 mg (77% of theory) of the title compound are obtained. [α] ²⁰ Na = + 7.8 ° (c = 0.45 in chloroform)

HPLC / UV-Vis (Method 3): R, = 3.3 min.

LC-MS (Method 5): R _t = 7.3 min;

MS (ESI pos.): M / z (%) = 515 (100) [M + H] ⁺ , 1030 (60) [2M + H] ⁺ .

¹ H NMR (500 MHz, J ₆ -DMSO): δ = -0.02 (s, 9H), 0.88 (t, J = 8.6 Hz, 2H), 1.31 (s, 9H), 4.01-

4.15 (m, 2H), 4.48 (dd, J = 9.5, 4.0 Hz, IH), 4.86 (d, J = 13.1 Hz, IH), 4.90 (d, J = 12.6 Hz, IH),

5.25 (dd, J = 10.0, 4.0 Hz, IH), 7.14-7.29 (m, 10H), 7.48 (d, IH, J = 10.2 Hz), 7.51 (d, IH, J =

9.5 Hz).

¹³ C NMR (126 MHz, J ₆ -DMSO): δ = -1.67 (3C), 16.78, 27.96 (3C), 54.38, 58.83, 62.95, 65.34,

78.30, 126.29 (2C), 127.06, 127.30 (2C), 127.66, 128.13 (2C), 128.20 (2C), 136.63, 139.17,

154.58, 155.95, 170.02.

HR-TOF-MS (Method 1): C ₂₇ H ₃₈ N ₂ O ₆ Si [M + H] ⁺ m.p. 515.2564, accounted for 515.2572.

Example 19A

2- (trimethylsilyl) ethyl- (3i) -3-amino-N ² - [(benzyloxy) carbonyl] -L-phenylalaninate trifluoroacetate

2- (Trimethylsilyl) ethyl- (3i?) - N - [(benzyloxy) carbonyl] -3 - [(ter? -Butoxycarbonyl) amino] -L-phenylalaninate (Example 18A, 550 mg, 1.7 mmol) is added Implemented by working procedure 1 (reaction time: 10 min). The crude product is purified by preparative HPLC (Method 21). 424 mg (75% of theory) of the title compound are obtained.

[α] ² V = -11 -2 ° (c = 0.47 in chloroform)

HPLC / UV-Vis (Method 2): R _t = 1.9 min.

LC-MS (Method 7): R _t = 2.0 min;

MS (ESI pos.): M / z (%) = 387.2 (60), 415 (100) [M + H] ⁺ .

¹ H NMR (500 MHz, J ₆ -DMSO): δ = -0.07 (s, 9H), 0.52 (rn, 2H), 3.77 (m, IH), 3.88 (m, IH), 4.50

(d, J = 9.0 Hz, IH), 4.58 ("t", J = about 8.2 Hz, IH), 5.08 (s ₌ 2H), 7.34-7.43 (m, 10H), 8.12 (d, IH,

J = 8.5 Hz, IH), 8.67 (s, br, 2H). ¹³ C NMR (126 MHz, J ₀ -DMSO): δ = -1.46 (3C), 16.51, 55.14, 57.92, 63.36, 66.28, 128.02 (2C), 128.18, 128.22 (2C), 128.56 (2C), 128.95 ( 2C), 129.54, 134.00, 136.70, 156.14, 169.06. HR-TOF-MS (method 1): C ₂₂ H ₃₀ N ₂ O ₄ Si [M + H] ⁺ Found. 415.2036, ber. 415.2048. Example 20A

N ^2; -tert-butoxycarbonyl [(3i) -3-hydroxy-L-leucyl] -L-leucyl-D-arginyl-L-isoleucyl-L-allothreonyl-glycyl - [(35) -3-hydroxy-L-asparaginyl] -L-serine trifluoFacetat

To a solution of the Edman ^{i 0} precursor (Example 3A, 690 mg, 680 .mu.mol, 1 equivalent) in water-dioxane 1: 2 (30 ml) under argon protective gas atmosphere di-tert-butyldicarboxylate (369 mg, 1.69 mmol, 2.5 Equivalents) and N-methylmorpholine (68 mg, 680 μmol, 1 equivalent). The reaction mixture is stirred until the HPLC chromatogram shows complete conversion (about 48 h). The reaction mixture is mixed with Kaliumdihydrogenphospha-t (5 equivalents), concentrated in vacuo and purified by preparative HPLC (Method 21 or Method 11 followed by re-salting the chromatography product by addition of TFA (2000 .mu.mol, as a 0.05% solution in acetonitrile-water 1 : 1)) cleaned. 531 mg (70% of theory) of the title compound are obtained.

HPLC / UV-Vis (Method 3): R ₁ = 1.7 min. LC-MS (Method 5): R, = 4.6 min;

MS (ESIpos.): M / z (%) = 453.5 (60) [M-Boc + 2H] ²⁺ , 1006 (100) [M + H] ⁺ . MS (ES Ineg.): M / z (%) = 1004 (100) [M-H]. ^" HR-TOF-MS (Method 1): C ₄₂ H ₇₇ Ni ₂ Oj ₆ [M + H] ⁺ m.p. 1005.5560, calc. 1005.5576. Example 21A

N ² - (benzyloxycarbonyl) -N ³ - {N ^2; 3-hydroxy-L-leucyl] -L-leucyl-D-arginyl-L-isoleucyl-L-allothreonyl-glycyl - [(3S) -3-hydroxy-L-asparaginyl ] -L-seryl} - (3i?) - 3-amino

L-phenylalanine (2- (trimethylsilyl) ethyl) ester trifluoroacetate

To a solution of the diamino acid (Example 19A, 1.2 equivalents, 42.5 mg, 80 .mu.mol), the octapptidic acid (Example 2OA, 1.0 equivalents, 75.0 mg, 70 .mu.mol) and N-methylmorpholine (1-0 equivalents, 70 .mu.mol ) in dry dimethylformamide (2 ml) at 0 ⁰ C untex argon inert gas atmosphere first HATU (1.5 equivalents, 38.2 mg, 100 .mu.mol) gegeten. The reaction mixture is stirred (about 15 min) and again with N-methylmorpholine (2.5 equivalents, 175 .mu.mol). The reaction mixture warmed slowly (about 12 h) to room temperature and then shows complete conversion of the amine component (HPLC control). The reaction mixture is admixed with solid potassium dihydrogen phosphate (5 equivalents, 350 μmol) and then concentrated under high vacuum and purified by chromatography (Method 21 or MC method 13 followed by re-salting of the chromatography product by addition of TFA (200 μmol, as 0.05% solution in Acetonitrile-water 1: 1)). 73.5 mg (72% of theory) of product are obtained.

HPLC / UV-Vis (method 2): R ₁ = 2.3 min. LC-MS (Method 5): R, = 5.5 min; MS (ESI pos.): M / z (%) = 637.6 (60), 1402 (100) [M + H] ⁺ . MS (ES Ineg.): M / z (%) = 1400 (100) [M-H] ^" , 1447 (20) [M-HCO ₂ H-H] ^" . Example 22A

N ² - (benzyloxycarbonyl) -N ⁵ - {N ^{2 /} -FE / '' - butoxycarbonyl [(3 /?) - 3-hydroxy-L-leucyl] -L-leucyl-D-arginyl-L-isoleucyl-L -allothreonyl-glycyl - [(3IS) -3-hydroxy-L-asparaginyl] -L-seryl} - (3JR) -3-amino-

L-phenylalanine trifluoroacetate

The nonapeptide trimethylsilyl ester (Example 21A, 70 mg, 46 μmol) and molecular sieve 4 A (about 10 mg) are initially charged in dry THF (3 ml) under argon protective gas atmosphere. At RT, a 1 N TBAF-THF solution (320 ul, 7 equivalents) is added dropwise with vigorous stirring and stirred until the HPLC chromatogram (Method 2) shows complete conversion (about 1 h). The reaction mixture is then neutralized with acetic acid (50 μl, ca. 20 equivalents). The reaction mixture is evaporated in vacuo and purified by preparative HPLC (Method 21 or Method 13 followed by re-salting the chromatography product by addition of TFA (200 .mu.mol, as a 0.05% solution in acetonitrile-water 1: 1)). 50.2 mg (77% of theory) of the title compound are obtained.

HPLC / UV-Vis (Method 2): R, = 2.1 min. LC-MS (Method 5): R _t = 5.2 min;

MS (ESI pos.): M / z (%) = 601.6 (60) [M - Boc + 2H] ²⁺ , 1302 (100) [M + H] ⁺ . MS (ES Ineg.): M / z (%) = 1300 (100) [M-H] ^" . Example 23A

N ² - (benzyloxycarbonyl) -N ⁵ - {N ^2; -tert-butoxycarbonyl [(3 /?) - 3-hydroxy-L-leucyl] -L-leucyl-D-arginyl-L-isoleucyl-L-allothreonyl-glycyl - [(3, S) -3-hydroxy-L -asparaginyl] -L-seryl} - (3i?) - 3-aniino-

L-phenylalanine (pentafluorophenyl) ester trifluoroacetate

The nonapeptide acid (Example 22A, 54.4 mg, 38 μmol) is placed under argon protective gas atmosphere in dichloromethane (1 ml). At -20 ⁰ C successively pentafluorophenol are (71 mg, 380 .mu.mol, 10 equivalents), EDC (17 mg, 192 .mu.mol, 2.3 equivalents) and DMAP (mg 1, 8 .mu.mol, 0.2 equivalents) was added. It is allowed to thaw slowly (about 3 h) and stirred at RT (about 12 h) until the HPLC chromatogram indicates complete conversion (method 2). The crude product is reacted further without purification.

LC-MS (Method 8): R, = 2.3 min;

MS (ESI pos.): M / z (%) = 685 (100) [M-Boc + 2H] ²⁺ , 1468 (10) [M + H] ⁺ .

MS (ES Ineg.): M / z (%) = 1282 (100), 1465 (40) [M-H] ^"

Example 24A

N ⁵ - (benzyloxycarbonyl) -N ³ - {[3- ^(z?) - 3-hydroxy-L-leucyl] -L-leucyl-D-arginyl-L-isoleucyl-L-allothreonyl-glycyl - [(35) 3-amino-L-phenylalanine - -3-hydroxy-L-asparaginyl] -L-seryl} - (3i?)

(Pentafluorρhιenyl) ester bishydrochloride

According to the procedure 2, the N- (tert-butoxycarbonyl) cyclopeptide (Example 23 A, 60 mg, 40 .mu.mol) is reacted under argon protective gas atmosphere. Following microphilic drying, 50 mg (96% of theory) of product are obtained, which is reacted directly without further purification.

HPLC / UV-Vis (Method 2): R _t = 1.9 min, λ _max (qualitative) = 210 nm (s), 255-270 (w). LC-MS (Method 5): R _t = 4.7 min;

MS (ESI pos.): Mk (%) = 684.3 (10O) [M + 2H] ²⁺ , 1368 (50) [M + H] ⁺ . MS (ES Ineg.): M / z (%) = 1366 (100) [M-Hf.

Example 25A

N ^2J - (benzyloxycarbonyl) - [(3i?) - 3-amino-L-phenylalanyl)] - [(ii?) - 3-hydroxy-L-leucyl] -L-leucyl-D-arginyl-L-isoleucyl- L-allothreonyl-glycyl - [(35) -3-hydroxy-L-asparaginyl] -L-serine-C ^{y ≤>} -N ⁵ -lactam-trifluoroacetate

A solution of the pentafluorophenol ester (Example 24A, 15.8 mg, 11 .mu.mol) in DMF (1 ml) is diluted with dioxane (20 ml) and then (as a suspension) under vigorous stirring under an argon inert gas atmosphere in pyrolyzed to 8O ⁰ C ( 80 ml) was added dropwise. The reaction mixture is stirred at 85 ° C. until the HPLC chromatograph (Method 2) shows complete conversion (about 1 h). It is concentrated in vacuo. The crude product is first gel chromatographed (Method 14) and then finely purified by preparative HPLC (Method 10). 4.3 mg (33% of theory) of the title compound are obtained.

Alternative Synthesis Method (Direct Cyclization of the Free Nonapeptide Amino Acid): At ⁰ ° C, a solution of the free nonapeptide (N ² - (benzyloxycarbonyl) -N ⁵ - {[(3i) -3-hydroxy-L-leucyl] -L) is added. leucyl-D-arginyl-L-isoleucyl-L-allothreonyl-glycyl - [(35) -3-hydroxy-L-asparaginyl] -L-seryl} - (3i?) - 3-amino-L-phenylalanine trifluoroacetate, 280mg, 0.22mmol) in DMF (70mL) and ΝMM (142μl, 6 equivalents) was added with HATU (245mg, 0.65mmol, 3equiv). The reaction mixture is stirred to complete conversion (1.5 h, HPLC control). The reaction mixture is then quenched with methanol (10 ml), concentrated in vacuo and purified directly by preparative HPLC (Method 35). Ϊ The product is obtained as a solid (201.5 mg, 70% of theory).

LC-MS (Method 5): R ₁ = 4.8 min;

MS (ESI pos.): M / z (%) = 592.6 (20) [M + 2H] ²⁺ , 1184 (100) [M + H] ⁺ . MS (ES Ineg.): M / z (%) = 1182 (100) [M-H]. ^" LC-MS (Method 7): R, = 1.8 min; MS (ESIpos.): M / z (%) = 592.7 (50) [M + 2H] i2 ² + ⁺ , 1184 (100) [M + H] ⁺ .

MS (ES Ineg.): M / z (%) = 536.6 (100), 1182 (50) [M-H] ^" , 1229 [M-H + HCO ₂ H] ^" .

HR-TOF-MS (method 1): C ₅₄ H ₈₃ Ni ₄ O ₁₆ [M + H] ⁺ gef. 1183.6122, b. 1183.6 106.

Example 26A

[(3i?) - 3-amino-L-phenylalanyl)] - [(3i?) - 3-hydroxy-L-leucyl] -L-leucyl-D-arginyl-L-isoleucyl-L-allothreonyl-glycyl- [ (3jS) -3-hydroxy-L-asparaginyl] -L-serine-C ^λ9 -N ^{J /} -lactam bishydrochloride

The Cbz-protected cyclopeptide (Example 25A, 4.3 mg, 3.3 μmol) is dissolved in methanol (4 ml) and treated under argon protective gas atmosphere with 10%. Palladium-carbon (100 mg) and aqueous IN hydrochloric acid (200 μl) were added. At RT and atmospheric pressure, hydrogenation is carried out (about 1 h) until analytical HPLC (method 2) indicates complete conversion. The reaction mixture is filtered (through kieselguhr, Celite ^® or a syringe filter, Fa. Biotage, PTFE), concentrated in vacuo and dried under high vacuum. 4 mg (quant.) Of the title compound are obtained.

Alternative Preparation Method (Title Compound as Trifluoroacetate) The Cbz-protected cyclopeptide (Example 25A, 330 mg, 254 μmol) is dissolved in methanol (200 ml) and treated with 10% palladium-carbon (127 mg) and hydrochloric acid (1526 μl, under argon protective gas atmosphere). It is hydrogenated for about 1 h and catalyst (100 mg) and hydrochloric acid (3 equivalents, 760 μl) are added again at RT and normal pressure is continued (about 1 h) until the analytical HPLC (method 2) the reaction mixture is filtered (on diatomaceous earth, celite), concentrated in vacuo and dried under high vacuum Crude product is purified by preparative HPLC (Method 33). 250 mg (77% of theory) of the title compound are obtained as trifluoroacetate.

HPLC / UV-Vis (Method 3): R _t = 1.4 min, ^ _x (qualitative) = 210 nm (s), 255-270 (w). LC-MS (Method 7): R _t = 1.1 min;

MS (ESI pos.): M / z (%) - 525.6 (100) [M + 2H] ²⁺ .

MS (ES Ineg.): M / z (%) = 1048 (100) [M-H] ^" , 1095 [M-H + HCO ₂ H] -

HR-TOF-MS (Method 1): C ₄₆ H ₇₇ Ni ₄ O ₁₄ [M + H] ⁺ gef. 1049.5728, accounted for 1049.5739.

Example 27A

N ^{2 y} - (tert -butoxycarbonyl) - [3-rerr-butyl-D-alanyl] - [3- / e? "F-butyl-L-alanyl] - [(3i?) - 3-amino-L - phenylalanyl)] - [(3jR) -3-hydroxy-L-leucyl] -L-leucyl-D-arginyl-L-isoleucyl-L-allothreonyl-glycyl [(3S) -3-hydroxy-L-asparaginyl] -L-serine-C ^{λ //} -N ⁵ -mactam trifluoroacetate

To a solution of the cyclopeptide (Example 26A, 1.0 equivalents, 11.0 mg, 10 μmol) of the dipeptide acid N- (fer-butoxycarbonyl) -3-tert-butyl-D-alanyl-3-tert-butyl-L-alanine (4.0 Equi¬ valente, 14.6 mg, 40 .mu.mol) and N-methylmorpholine (2.0 equivalents, 20 .mu.mol) in dry dimethylformamide (270 .mu.l) is at least HATU (4.1 equivalents, 14.6 mg, 40 .mu.mol) at ^{0 0} C under argon inert gas atmosphere ). The reaction mixture is stirred (about 15 minutes) and treated again with N-methylmorpholine (5.0 equivalents, 50 .mu.mol). The reaction mixture warmed slowly (about 12 h) to room temperature and then shows complete conversion of Amine component (HPLC control, method 3). The reaction mixture is combined with solid potassium dihydrogen phosphate (10 equivalents, 500 μmol) and then concentrated by evaporation in a high vacuum and purified by chromatography (Method 21 or Method 13 followed by re-salting the chromatography product by addition of TFA (100 μmol, as a 0.05% solution in Acetonitrile-water 1: 1)). 10.5 mg (70% of theory) of product are obtained.

HPLC / UV-Vis (Method 3): R, = 2.5 min, λ _max (qualitative) = 210 ran (s), 255-270 (w).

LC-MS (Method 5): R, = 5.5 min;

MS (ESI pos.): M / z (%) = 702.6 (30) [M + 2H] ²⁺ , 1404 (20) [M + H] ⁺ .

MS (ES Ineg.): M / z (%) = 700.7 (50) [M-2H] ^{2 "} , 1402 (10) [M-H] -, 1448 [M-H + HCO ₂ H] -.

LC-MS (Method 7): R ₄ = 2.3 min;

MS (ESI pos.): M / z (%) = 653 (100) [M - Boc + 2H] ²⁺ , 1404 (50) [M + H] ⁺ .

HR-TOF-MS (Method 1): C ₆₅ H _1I iNi ₆ O ₁₈ [M + H] ⁺ gef. 1403.8232, on. 1403.8257.

Example 28A

N ^zy - (benzyloxycarbonyl) -D-leucyl-L-leucyl - [(3i?) - 3-amino-L-phenylalanyl)] - [(3 /?) - 3-hydroxy-L-leucyl] -L-leucyl -D-arginyl-L-isoleucyl-L-allothreonyl-glycyl - [(35) -3-hydroxy-L-asparaginyl] -L-serine-C ^{/ /;} -N ^JJ- lactam trifluoroacetate

Preparation analogous to Example 27A from the compound from Example 26A (900 μg, 0.8 μmol) and N- (benzyloxycarbonyl) -D-leucyl-L-leucine (Example 3OA, 1200 μg, 3.2 μmol, 4 equivalents). The Crude product is purified by gel chromatography (method 14) to give 500 μg (41% of theory) of product as a solid after freeze-drying.

HPLC / UV-Vis (Method 3): R ₁ = 2.3 min, LC-MS (Method 5): R _t = 5.2 min; MS (ESI pos.): M / z (%) = 706.2 (100) [M + 2H] ²⁺ , 1410 (20) [M + H] ⁺ . MS (ES Ineg.): M / z (%) = 1408 (100) [M-H] ^"

Example 29A

N, benzyloxycarbonyl-D-leucyl-L-leucine methyl ester

To a solution of L-leucine methylester hydrochloride (1.1 equivalents, 753.2 mg, 4.15 mmol) in dichloromethane pa (20 ml) successively HOBt be at -10 ⁰ C (4 equivalents, 2038 mg, 8.15 mmol), N-methylmorpholine (3 equivalents, 11.3 mmol), N-benzyloxycarbonyl-D-leucine (1.0 equivalents, 1000 mg, 3.8 mmol), EDC (2 equivalents, 1441 mg, 7.5 mmol), and again added N-methylmorpholine (2 equivalents, 7.5 mmol) , The reaction mixture warms slowly (about 12 h) to RT, with complete conversion of the amine component being monitored by HPLC. The reaction mixture is concentrated, taken up in ethyl acetate (about 200 ml) and then with saturated aqueous sodium bicarbonate solution (once), 5% aqueous citric acid (twice), saturated aqueous sodium bicarbonate solution (once) and saturated sodium chloride solution washed. It is dried over sodium sulfate and filtered. The mixture is evaporated to dryness in vacuo and subsequently dried in a high vacuum. 1483 mg (93% of theory) of the title compound are obtained, which are reacted without further purification.

LC-MS (method 7): R _t = 2.59 min;

MS (ESI pos.): M / z (%) = 393 (100) [M + O] ⁺ . Example 30A

7V- <TBenzyloxycarbonyl) -D-leucyl-L-leucine

N- (benzyloxycarbonyl) -D-leucyl-L-leucine-methylester (Example 29A, 1.12 g, 2.85 mmol) at 0 ⁰ C in THF / water 2: submitted (ml 15) and then with 1 Lithϊumhydroxid monohydrate (140 mg,

5.71 mmol, 2 equivalents). When the HPLC chromatogram (Method 2) is complete

Sales shows (about 1 h), the reaction mixture is adjusted to pH 3-4 with acetic acid, cold in

Concentrated vacuum and then freed from the freeze-drying overnight of solvent. The

Crude product is purified by preparative HPLC (Method 13) to give 958 mg (88.7% of theory) of the title compound.

LC-MS (Method 7): R _t = 2.33 min;

MS (ESI pos.): M / z (%) = 379 (100) [M + H] ⁺ .

HR-TOF-MS (method 1): C ₂₀ H _3I N ₂ O ₅ [M + H] ⁺ Found. 379.2227, p. 379.2233.

Example 31A

Methyl (? 2i, 3i?) - N ² - [(ben2yloxy) carbonyl] -N ² - [(benzyloxy) carbonyl amino] -3 - [(ter ^ butoxy carbonyl) amino] -phenylalaninat

Method A (5 g scale): A 1 N solution of lithium hexamethyldisilazide (39.4 mmol, 39.4 ml, 2.2 equivalents) in THF in the reaction solvent THF (200 ml) is placed under an argon protective gas atmosphere. At -78 ⁰ C a solution of (S) -3 is - zugetropfit slowly [(tert-butoxycarbonyl) amino] -3-phenylpropansäuremethylester (5.0 g, 17.9 mmol). The mixture is stirred for 10 min at -25 ⁰ C and then cooled again to -78 ⁰ C. Dibenzylazadicarboxylate (8.54 g, 28.6 mmol, 1.6 equiv.) Is added in one portion to. Given reaction mixture. It is stirred for 3 h at -50 to -6O ⁰ C. In order to stop the reaction is again cooled to -78 ⁰ C and herunter¬ (5.1 ml, 89.5 mmol, 5 equivalents) of acetic acid was added and then heated to RT and finally 0 ⁰ C. Work-up analogously to Example 12A (Method B). This gives 4.8 g (44% of theory) of the title compound as a solid.

Method B (50 g scale): A 1 N solution of lithium hexamethyldisilazide (452.9 mmol, 452.9 ml, 2.2 equivalents) in THF in the reaction solvent THF (1800 ml) is placed under an argon protective gas atmosphere. At -7O ⁰ C a solution of (S) -3 is - (ml 600) was slowly added dropwise [(tert-butoxycarbonyl) amino] -3-phenylpropansäuremethylester (57.5 g, 205.8 mmol) in THF. The mixture is stirred for 10 min at -25 ° C and then cooled again to -7O ⁰ C down. Dibenzylazadicarboxylate (98.2 g, 329.4 mmol, 1.6 equiv.) Is added in 4 portions to the reaction mixture. It is stirred for 2 h at -50 to -60 ⁰ C gerülxrt. To stop the reaction, is cooled again to -7O ⁰ C and (61.7 ml, 1 mol, 5 equivalents) of acetic acid was added and then warmed to RT overnight. The precipitate in the reaction solution is filtered off with suction. The filter residue is dried in high vacuum at 5O ⁰ C dried (46.1 g, crude product 1). The filtered mother liquor (reaction mixture) is concentrated in vacuo (crude product 2). The two crude products 1 and 2 are recrystallized from hot ethyl acetate (2 × 500 ml) (reflux, 1 h, product fractions 1 (31.7 g) and 2 (37.4 g).) The mother liquor is again evaporated and recrystallized from tert-butyl methyl ether (370 ml). This gives product fraction 3 (14.5 g). A total of 83.6 g (70% of theory) of the title compound as a solid.

M ²⁰ Na = -13 ° (c = 0.20 in chloroform). HPLC / UV-Vis (method 2): R, = 2.88 min. LC-MS (Method 29): R, = 2.8 min;

MS (ESI pos.): M / z (%) = 478 (100) [M -Boc + H] ⁺ , 578 (70) [M + H] ⁺ , 1156 (20) [2M + H] ⁺ ; MS (ES Ineg.): M / z (%) = 576 (100) [M-H] ^" .

¹ HNMR (500 MHz, J ₆ -DMSO, 335 K, broad signals): δ = 1.35 (s, 9H), 3.15 (s, 3H), 3.30 (s, 3H), 4.90-5.15 (m, 6H), 7.20-7.40 (m, 10H), 8.30, 8.95 (3s, br, 2H).

Example 32A

Methyl (2ιS ', 3i?) - N - [(benzyloxy) carbonyl] - N - [(benzyloxy) carbonylamino] -3 - [(te ^ "? - butoxycarbonyl) -amino] -phenylalaninate

Under an argon protective gas atmosphere, a solution of methyl (2i, 3J?) - N ² - [(benzyloxy) - carbonyl] -N ² - [(benzyloxy) carbonyl amino] -3 - [(^ erf-butoxycarbonyl) amino] phen. ylalaninate (3.95 g, 6.84 mmol) in dry dichloromethane (170 ml) at ⁰ ° C with N, N, N, Λ / -Tetramethylguanidin (16.6 ml, 132 mmol). The reaction mixture is thawed and stirred until the HPLC chromatogram (Method 2) indicates complete conversion (about 60% of product) (about 12 h) and the reaction is then carried out by addition of acetic acid (10.5 ml, pH 4-6). to stop. The organic phase is washed with water (twice), saturated aqueous sodium bicarbonate solution (twice), saturated aqueous sodium chloride solution (once). The aqueous phases are with Dichloromethane reextracted. The combined organic phases are dried over sodium sulfate, filtered, evaporated in vacuo, dried under high vacuum. The crude product is purified by flash chromatography (silica gel, toluene / ethyl acetate 4: 1). This gives 2.4 g (61% of theory, based on recovered starting material 68%) of the title compound and 490 mg of the starting compound (12% of theory).

W ²⁰ _Na = -120.7 ° (c = 0.52 in chloroform). HPLC / UV-Vis (Method 2): R, = 2.99 min. LC-MS (Method 29): R _t = 2.9 min;

MS (ESI pos.): M / z (%) = 478 (100) [M -Boc + H] ⁺ , 578 (50) [M + H] ⁺ , 1156 (10) [2M + H] ⁺ ; MS (ES Ineg.): M / z (%) = 502 (100), 576 (40) [M - Hf.

HR-TOF-MS (Method 1): C ₃ H ₃₆ N ₃ O ₈ [M + H] ⁺ . 578.2505, accounted for 578.2497.

Example 33A

Methyl (2S, 3R) -3 - [(tert-butoxycarbonyl) amino] phenylalaninate

Under argon protective gas atmosphere, to a solution of methyl (2 ₎ S ', 3i?) - N - [(benzyloxy) - carbonyl] - N - [(benzyloxy) carbonylamino] -3 - [(tert-butoxycarbonyl) amino] phenylalaninate ( 600 mg, 1.04 mmol) in methanol / dichloromethane 1: 1 (12 mL) of Raney-Νickel (86 mg). The reaction mixture is hydrogenated in a pressure autoclave at 80 bar hydrogen pressure and RT (72 h). The HPLC chromatogram shows complete conversion. The reaction mixture is filtered under argon protective gas atmosphere over a (fritted with diatomaceous earth) glass frit. The glass frit is washed several times with methanol / water / 0.2% acetic acid. The filtrate is washed with water (twice) and then evaporated in vacuo. The crude product obtained rαan a solid (280 mg, 92% of theory) is reacted further without fine cleaning. For analytical purposes, a gel chromatographic fine cleaning (Method 14, methanol / O.1% TFA) can be performed.

[Cc] ²⁰ _Na = + 11 ° (c = 0.25 in chloroform) (TFA salt). HPLC / UV-Vis (method 2): R, = 1.5 min. LC-MS (Method 29): R _t = 1.2 min; MS (ESI pos.): M / z (%) = 239 (100), 295 (40) [M + H] ⁺ .

¹ H NMR (500 MHz, J ₆ -DMSO, TFA SaIz): δ = 1.36 (s, 9H), 3.50 (s, 3H), 4.30 (d, J = 7.2 Hz, IH), 5.05 ("t") , J = 8.0 Hz, IH), 7.30-7.39 (m, 5H), 7.60 (d, J = 9.7 Hz, IH).

¹³ C NMR (125 MHz, J ₀ -DMSO, TFA salt): δ = 28.25 (3C), 52.87, 54.88, 56.87, 79.29, 127.12

(2C), 128.45, 128.89 (2C), 137.18, 154.83, 168.14.

HR-TOF-MS (method 1): C ₁₅ H ₂₂ N ₂ O ₄ [M + H] ⁺ gef. 295.1663, ber. 295.1653.

Example 34A

Methyl (2 _> S ', 3i?) - N - [(benzyloxy) carbonyl] -3 - [(? - tert-butoxycarbonyl) amino] phenylalaninate

Under argon protective gas atmosphere is added to a solution of methyl (2 <S ', 3i?) - 3 - [(fer? -Bxιtoxycarbonyl) amino] phenylalaninat (250 mg, 0.62 mmol) and N-Benzyloxycarbonyloxysuccimimid- ester (180 mg, 0.71 mmol, 1.15 equivalents) in a mixture of dichloromethane (20 nxl) and water (45 ml) at 0 ⁰ C solid hydatriumhydrogencarbonat (80 mg, 0.93 mmol, 1.5 equivalents) was added. The Reaktiosgemisch slowly warms up (12 h), with complete conversion by HPLC (Method 2) is observed. The aqueous phase is reextra¬ hiert with dichloromethane and then washed with 5% aqueous citric acid, saturated aqueous Νa-trium- bicarbonate solution and saturated brine, dried over Νatriurnsulfat, filtered and concentrated in vacuo. The mixture is then purified by means of flash chromatography (silica gel, toluene / ethyl acetate 4: 1). 144 mg (47% of theory) of the title compound are obtained 51

- 81 -

[α] ²⁰ _Na = + 13.3 ° (c = 0.70 in chloroform). HPLC / UV-Vis (method 2): R, = 2.6 min. LC-MS (Method 7): R ₁ = 2.7 min;

MS (ESIpos.): M / z (%) = 329 (100) [M -Boc + H] ⁺ , 373 (80), 429 (60) [M + H] ⁺ , 858 (10) £ 2M + H ] ⁺ .

¹ HNMR (400 MHz, ^ ₆ -DMSO): δ = 1.37 (s / 9H), 3.62 (s, 3H), 4.58 (dd, J = 9.5, 4.2 Hz, 1H>, 4.94 (m, 2H), 5.27 (dd, J = 10.4, 4.0Hz, IH), 7.21-7.35 (m, 10H), 7.50 (d, J = 10.5Hz, IH), 1.59 (d, J = 9.4Hz, IH).

¹³ C NMR (126 MHz, DMSO): δ = 28.25 (3C), 52.31, 54.66, 59.03, 65.71, 78.72, 126.64- (2C), 127.40, 127.63 (2C), 127.99, 128.42 (2C), 128.51 (2C), 136.90, 139.24, 154.97, 156.23, 170. 89. HR-TOF-MS (method 1): C ₂₃ H ₂₉ N ₂ O ₆ [M + H] ⁺ Found. 429.2011, ber. 429.2021.

Example 35A

(3i?) - N ² - [(benzyloxy) carbonyl] -3 - [(ferf-butoxycarbonyl) amino] -L-phenylalanine

Under argon protective gas atmosphere, a solution of methyl (25 ^l , 3i?) N - [(benzyloxy) carbonyl] -3- [(tert-butoxycarbonyl) amino] phenylalaninate (152 mg, 0.30 mmol) in THF / water 2: 1 (22.5 ml) submitted. At ⁰ ° C., a degassed 1% strength aqueous solution of lithium hydroxide monohydrate (14.8 mg, 0.62 mmol, 2 equivalents) is slowly added dropwise with vigorous stirring. The mixture is stirred at RT until the HPLC chromatogram (method 2) indicates complete conversion (about 1 h). It is then mixed with acetic acid (0.1 ml), the reaction mixture concentrated in vacuo and the residue is freeze-dried. The crude product is finely purified by preparative HPLC (Method 13). The product obtained is 74 mg (59% of theory) of the title compound. [Ot] ²⁰ _Na = + 22 ° (c = 0.50 in chloroform) (by chiral chromatography); [α] ² ° _Na ⁼ + 21.5 ° (c = 0.51 in chloroform) (by enantioselective synthesis) LC-MS (Method 7): R _t = 2.4 min;

MS (ESIpos.): M / z (%) = 315 (90), 359 (100), 415 (80) [M + H] ⁺ , 729 (30), 829.5 (50) [2M + H] ⁺ . MS (ES Ineg.): M / z (%) = 413 (100) [M-H] ^" . Chiral HPLC / UV-Vis (Method 24): R _t = 5.4 min (narrow peak).

¹ H NMR (500 MHz, J ₀ -DMSO): δ = 1.35 (s, 9H), 4.46 (dd, J = 9.6, 3.6 Hz, IH), 4.91 (s, 2H), 5.26 (dd, J = 9.9 , 3.6 Hz, IH), 7.18-7.32 (m, 10H) ₅ 7.48-7.51 (m, 2H), 12.95 (s, br, IH). ¹³ C NMR (126 MHz, J ₆ -DMSO): δ = 28.34 (3C), 54.84, 58.96, 65.55, 78.56, 126.53 (2C), 127.23,

IO 127.55 (2C), 127.93, 128.39 (2C), 128.51 (2C), 137.05, 140.16, 155.09, 156.30, 171.62. HR-TOF-MS (method 1): C ₂₂ H ₂₆ N ₂ O ₆ [M + H] ⁺ Found. 415.1864, ber. 415.1864.

Example 36A

(3 ₁ S) -N ² - [(benzyloxy) carbonyl] -3 - [(t-t-butoxycarbonyl) amino] -D-phenylalanine

15 For illustration see example 35 A.

[Ot] ²⁰ Na = -20 ° (c = 0.49 in chloroform).

Chiral HPLC / UV-Vis (Method 24): R, = 11.4 min (broad peak). LC-MS (method 7): R ₁ = 2.5 min;

MS (ESI pos.): M / z (%) = 315 (40), 359 (40), 415 (50) [M - + H] ⁺ , 729 (20), 829.5 (10) [2M + H] ⁺ , 20 MS (ES Ineg.): M / z (%) = 413 (100) [M - Hf.

¹ H NMR (500 MHz, J ₀ -DMSO): δ = 1.35 (s, 9H), 4.45 (dd, J = 9.6, 3.2 Hz, IH), 4.91 (s, 2H), 5.26 (dd, J = 9.4 , 3.6 Hz, IH), 7.18-7.30 (m, 10H), 7.47-7.52 cm, 2H), 12.95 (s, br, IH). HR-TOF-MS (method 1): C ₂₂ H ₂₆ N ₂ O ₆ [M + H] ⁺ Found. 415.1869, ber. 415.1864.

Example 37A

2- (trimethylsilyl) ethyl (3iS) -N - [(benzyloxy) carbonyl] -3 - [(but-butoxycarbonyl) amino] -D-phenylalaninate

A mixture of (35) -N ² - [(benzyloxy) carbonyl] -3 - [(tert-butoxycarbonyl) amino] -D-phenylalanine (3.96 g, 9.55 mmol), 2- (trimethylsilyl) ethanol (11.3 g, 95.6 mmol, 10 equivalents) and molecular sieve 4 A (about 200 mg, freshly pulverized) in dry dichloromethane (77 ml) is stirred for 3 h at RT under argon protective gas atmosphere. (1 equivalent g 1.17 9:55 mmol,) are subsequently at -30 ⁰ C. DCC (3.94 g, 19.11 mmol, 2 equivalents) and DMAP added. The reaction mixture is thawed (about 12 h) and stirred at RT until the HPLC chromatogram (method 2) indicates complete conversion (about 12 h). The reaction mixture is filtered through kieselguhr and then evaporated at RT in vacuo. The crude product is pre-fractionated on a RPig cartridge (eluent water, then acetonitrile) and purified by preparative flash chromatography (1.5 kg silica gel, toluene / ethyl acetate 4: 1). 3.6 g (73% of theory) of the title compound are obtained.

[Oc] ²⁰ Na = -8.0 ° (c = 0.52 in chloroform) HPLC / UV-Vis (Method 3): R, = 3.2 min.

LC-MS (method 7): R, = 3.3 min; MS (ESI pos.): M / z (%) = 387 (100), 515 (90) [M + H] ⁺ . ¹ H NMR (500 MHz, ^ ₆ -DMSO): δ = -0.02 (s, 9H), 0.88 (t, J = 8.0 Hz, 2H), 1.31 (s, 9H), 4.02-4.15 (m, 2H) , 4.48 (dd, J = 9.6, 4.3 Hz, IH), 4.86 (d, J = 12.6 Hz, IH), 4.90 (d, J = 12.6 Hz, IH), 5.25 (dd, J = 10.0, 3.8 Hz, IH), 7.14-7.29 (m, 10H), 7.47 (d, J = 10.3 Hz, IH), 7.50 (d, J = 9.7 Hz,

IH).

HR-TOF-MS (Method 1): C ₂₇ H ₃₈ N ₂ O ₆ Si [M + H] ⁺ m.p. 515.2567, on. 515.2572.

Example 38A

2- (trimethylsilyl) ethyl- (3) S) -3-amino-N ^ - [(benzyloxy) carbonyl] -D-phenylalaninate trifluoroacetate

2- (trimethylsilyl) ethyl (3iS) -N - [(benzyloxy) carbonyl] -3 - [(tert-butoxycarbonyl) amino] -D-phenylalaninate (3600 mg, 7.0 mmol) is reacted according to procedure 1 ( Reaction time: 10 min). The crude product is purified by preparative HPLC (Method 13, Method 21). 3060 mg (83% of theory) of the title compound are obtained.

[Oc] ²⁰ Na - + 15.0 ° (c = 0.22 in chloroform) HPLC / UV-Vis (Method 3): R ₁ = 1.9 min. LC-MS (Method 8): R ₄ = 1.8 min; MS (ESI pos.): M / z (%) = 387.2 (60), 415 (100) [M + H] ⁺ . ¹ H NMR (500 MHz, J ₆ -DMSO): δ = -0.06 (s, 9H), 0.52 (m, 2H), 3.76 (rn, IH) ₃ 3.88 (m, IH), 4.49 (d, J = 9.3 Hz, IH), 4.58 ("t", J = 9.0 Hz, IH), 5.08 (s, 2H), 7.34-7.44 (m, 10H), 8.12 (d, J = 8.5 Hz, IH), 8.62 ( s, br, 2H). HR-TOF-MS (method 1): C ₂₂ H ₃₀ N ₂ O ₄ Si [M + H] ⁺ Found. 415.2042, about 415.2048.

Example 39A

(3IS) -N-Boc-y? -Phenylalanine methylester

The enantiomer mixture of (rαc) -N-Boc - / - phenylalanine methyl ester (62.8 g, 224.8 mmol) is separated into the enantiomers by preparative HPLC (Method 32). This gives 29.1 g of (3R) -N-Boc - / - phenylalanine methyl ester (95.7% ee, 46% of th., Example 40, eluting later) and 30.5 g (3S) -N-Boc- / ?. phenylalanine methyl ester (> 99% ee, 49% of theory, title compound, eluting earlier).

M ²⁰ _Na = -30 ° (c = 0.52 chloroform). HPLC / UV-Vis (Method 3): R ₄ = 2.5 min.

¹ H NMR (500 MHz, (I ₆ -DMSO): δ = 1.35 (s, 9H), 2.67 (dd, J = 15.4, 6.0 Hz, 1 H), 2.75 (dd, J = 15.7, 8.6 Hz, IH ), 3.55 (s, 3H), 4.92 (m, IH), 7.23 (m, IH), 7.29-7.33 (m, 4H), 7.48 (d, J = 8.3 Hz, IH).

¹³ C NMR (126 MHz, ^ ₆ -DMSO): δ = 28.38 (3C, (C (CH ₃ ) ₃ )), 41.19 (C 1 H ₂ ), 51 .25 (C ^α H), 51.54 (OCH ₃ ), 78.07 (C (CH ₃ ) ₃ ), 126.47 (2C), 127.17, 128.46 (2C), 142.95, 154.90, 170.86. HR-TOF-MS (Method 1): C ₁₅ H ₂₂ NO ₄ [M + H] ⁺ gef. 280.1533, about 280.1544.

Example 4OA

(3R) -N-Boc - / - phenylalanine methylester

The synthesis is carried out according to Example 39A. _η 20

[α] Na = + 30 ° (c = 0.52 in chloroform). ¹ H NMR (500 MHz, J ₀ -DMSO): δ = 1.35 (s, 9H), 2.68 (dd, J = 15.2, 6.0 Hz, IH), 2.75 (dd, J =

15.6, 8.8 Hz, IH), 3.55 (s, 3H), 4.92 (m, IH), 7.23 (m, IH), 7.30-7.33 (m, 4H), 7.48 (d, J = 8.7 Hz,

IH).

HR-TOF-MS (method 1): C ₁₅ H ₂ iNO ₄ [M + H] ⁺ gef. 280.1555, ber. 280.1544.

Example 41A

Methyl-3- (6-methylpyridin-2-yl) -Z-alaninate

A mixture of methyl N- (benzyloxycarbonyl) -3- (6-methylpyridin-2-yl) -L-alaninate (7.25 g, 22.1 mmol) [S. Jones, C.F. Palmer, J.M. Paul, P.D. Tiffin, Tetrahedron Lett. 1999, 40, 1211-1214] and 10%. Palladium on carbon (0.690 g) in ethanol (80 ml) is hydrogenated at RT and normal pressure for 1 h. The reaction mixture is passed through a syringe filter (Millipore, 0.45 μ, hydrophobic PTFE), which is washed with ethanol (twice 10 ml each time). The residue is concentrated on a rotary evaporator (drag out of solvent residues with toluene, twice) and dried under high vacuum. The product obtained is an oil (3.89 g, yield 91% of Th.).

HPLC / UV-Vis (Method 2): R ₁ = 0.3 min.

LC-MS (method 30): R, = 0.70 min, broad peak; MS (ESI pos.): M / z (%) = 195 (100) [M + H] ⁺ . ¹ H NMR (400 MHz, J _n -DMSO): δ = 1.95 (m, 2H), 2.41 (s, 3H), 2.86 (dd, J = 7.4 Hz, IH), 2.99 (dd, J = 5.9 Hz, IH), 3.59 (s, 3H), 3.73 (dd, J = 7.4, 5.7 Hz, IH), 7.02 (d, J = 7.5 Hz, IH), 7.06 (d, J = 7.1 Hz, IH), 7.56 ( t, J = 7.5 Hz, IH).

Example 42A

N- ^ he ^ butoxycarbonyl-3-to ^ -butyl-D-alanyl-3- (6-methylpyridin-2-yl) -L-alanine-methylester

To a solution of N- (tert -butoxycarbonyl) -3-ter / -butyl-D-alanine (Example 6A, 1.0 equiv., 1.6 g, 6.6 mmol) and methyl 3- (6-methylpyridin-2-yl) - L-alaninate (example 41A, 1.1 eq, 1.4 g, 7.2 mmol) in dry DMF (26 ml) are slowly added at 0 ⁰ C ΝMM (2 eq, 13.1 mmol) and finally HATU (1.3 eq, 3.2 g, 8.5 mmol) given. The reaction mixture warmed slowly to RT (about 12 h), with complete conversion being observed by HPLC (method 2). For workup, the reaction mixture in a rotary evaporator. concentrated (water bath 35 ° C) with ethyl acetate (100 ml) and then with saturated sodium bicarbonate solution (twice 20 ml), 5 percent. Citric acid (twice 20 ml), and washed with saturated aqueous sodium chloride solution. The organic phase is dried over sodium sulfate, filtered and concentrated. The product obtained is a solid (2.88 g, 99% of theory) which can be finely purified by preparative HPLC (Method 13).

HPLC / UV-Vis (Method 2): R _t = 2.1 min.

LC-MS (Method 29): R, = 1.73 min; MS CESIpos.): M / z (%) = 422 (100) [M + H] ⁺ ;

MS CESIneg.): M / z (%) = 420 (20) [M-H] ^" , 466 (100) [M-H + HCO ₂ H] ^" .

HR-TOF-MS (Method 1): C ₂ H ₃₄ N ₃ O ₅ [M + H] ⁺ calc. 408.2498, m.p. 408.2503.

¹ H NMR (400 MHz, J ₆ -DMSO): δ = 0.79 (s, 9H), 1.23-1.43 (m, HH), 2.42 (s, 3H), 2.99-3 _ 13 (m,

2H), 3.60 (s, 3H), 3.95 (dt, J = 8.9, 3.8 Hz, IH), 4.64 (m, IH), 6.83 (d, J = 8.9 Hz, IH), 7.02 (d, J = 7.8 Hz, IH), 7.07 (d, J = 7.7 Hz, IH), 7.54 (t, J = 7.7 Hz, IH), 8.18 (d, J = 7.9 Hz, IH).

Example 43 A

N-ter? -Butoxycarbonyl-3- / e / * f-butyl-Z) -alanyl-3- (6-methyl-2-pyridyl) -I-alanine

(Example 42A, 850 mg, 2.02 mmol) is reacted according to General Procedure 4. 766 mg (93% of theory) of product are obtained.

Example 44A

(^ rf-Butoxycarbonyl) - [3-tert-butyl-D-alaiiyl] - [3- (6-methylpyrid-2-yl) -L-alanyl] - [(3i?) - 3-amino-L-phenylalanyl )] - [(3R) -3-hydroxy-L-leucyl] -L-leucyl-D-arginyl-L-isoleucyl-L-allothreoyl-glycyl - [(3 ₁ S) -3-hydroxy-L- asparagmyl] -L-serine lactam bistrifluoroacetate

In accordance with protocol 5, the cyclopeptide (Example 26A_, 40.0 mg, 31.32 μmol) and the dipeptide acid (Example 43A, 22.9 mg, 56.37 μmol, 1.8 equivalents) with the aid of NMM (17.2 μl, 156.58 μmol, 5 equivalents) and HATU (21.43 mg, 56.36 μmol, 1.8 equivalents) in DMF (15 ml) within 2.5 h to the amide at -10 ⁰ C reacted. The title compound is obtained as a solid after chromatographic purification (Method 37) (29.4 mg, 55% of theory).

HPLC / UV-Vis (method 2): R ₁ = 2.25 min. LC-MS (method 7): R _t = 2.05 min;

MS (ESI pos.): M / z (%) = 1439 (5) [M + H] ⁺ , 720 (100) [M + 2H] ²⁺ ; MS (ES Ineg.): M / z (%) = 1436 (100) [M-Hf, 718 [M-2H] ² \

HR-TOF-MS (method 1): C ₆₇ H ₁₀₈ N ₁₇ O] ₈ [M + H] ⁺ calc. 1438.8053 ,, gef. 1438.8060. Example 45A

N ^{2 /} - (but-butoxycarbonyl) - [3-rer-butyl-D-alanyl] - [3- (pyrid-2-yl) -L-alanyl] - [(3i?) - 3-amino-L - phenylalanyl)] - [(3i?) - 3-hydroxy-L-leucyl] -L-leucyl-D-arginyl-L-isoleucyl-L-allothreonyl-glycyl-

[(3S) -3-hydroxy-L-asparaginyl] -L-serine-C ^{λ //} -N ^5J -lactam bistrifluoroacetate

According to protocol 5, the cyclopeptide (example 26A, 100.0 mg, 78.29 μmol) and the N- (3'-butoxycarbonyl) -4-methyl-D-leucyl-3-pyridin-2-yl-Z, -alanine ( 55.5 mg, 140.63 μmol, 1.8 equivalents) (see WO2004099239) with the aid of ΝMM (43.0 μl, 156.58 μmol, 5 equivalents) and HATU (21.43 mg, 391.46 μmol, 1.8 equivalents) in DMF (8 ml) overnight to the amide RT implemented. The title compound is obtained as a solid after chromatography (Method 35) (111.3 mg, 86% of theory).

HPLC / UV-Vis (Method 25): R _t = 19.44 min. LC-MS (method 7): R ₁ = 2.15 min;

MS (ESI pos.): M / z (%) = 1424 (25) [M + H] ⁺ , 713 (100) [M + 2H] ²⁺ ; MS (ES Ineg.): M / z (%) = 1422 (100) [M-H] ^" , 711 [M-2H] ^2" .

HR-TOF-MS (method 1). C ₆₆ H ₁₀₆ N _N O ₁₈ [M + H] ⁺ calcd 1424.7897, Found. 1424.7906. Example 46A

4-methyl-D-leucine-trifluoroacetate

As described in Procedure 1, the title compound is obtained from the Boc-protected amino acid (Example 6A, 20.0 g, 81.53 mmol) with 21.7 g (quant.) Crude yield.

MS (ESIpos): m / z (%) = 146 (100) [M + H] ⁺ .

HR-TOF-MS (Method 1): C ₇ H ₁₆ N ₂ O ₂ -C ₂ H ₃ N [M + CH ₃ CN + H] ⁺ calc. 187.1442, m.p. 187.1450. ¹ H NMR (500 MHz, ^ ₆ -DMSO): δ = 0.95 (m, 9H), 1.58 (dd, J = 14.5 Hz, 5.2 Hz, IH), 1.81 (dd, J = 14.5 Hz, 6.3 Hz, IH ), 3.79 (t, J = 5.4 Hz, IH), 8.32 (s, br., 3H). ¹³ C NMR (126 MHz, DMSO c _ö?): Δ = 29.27 (3C), 30.26, 44.30, 50.01, 172.23.

Example 47A

N - [(benzyloxy) carbonyl] -4-methyl-D-leucine

The amino acid (Example 46A, 10.0 g, 38.6 mmol) and N-benzyloxycarbonyloxysuccinimide ester (11.1 g, 44.4 mmol, 1.15 equiv) are dissolved in dichloromethane / water 4: 1 (250 mL). The reaction mixture (mmol 4.86 g, 57.9, 1.5 equivalents) at 0 ⁰ C with solid Νatriumhydrogencarbonat and (valent 1.2 g. 3.9 mmol, 0.1 Äqui¬) with solid tetra-n-butylammonium bromide was added and then stirred overnight at RT to become. The HPLC chromatogram shows complete conversion. The basic aqueous phase is washed with dichloromethane (six times). It is then adjusted to pH 2-3 with 1 N aqueous hydrochloric acid. The watery Phase is then extracted with ethyl acetate (three times). The combined organic phases are treated with conc. washed aqueous sodium chloride solution, dried over sodium sulfate, filtered and concentrated in vacuo. The resulting crude product is purified by preparative HDPLC (Method 35). This gives 6.86 g (64% of theory) of the title compound.

[α] ²⁰ _Na = + 2 ° (c = 0.17 in CHCl ₃ ).

HPLC / UV-Vis (Method 3): R, = 2.2 min.

LC-MS (Method 29): R _t = 2.0 min;

MS (ESI pos.): M / z (%) = 236 (100), 280 (50) [M + H] ⁺ ;

MS (ES Ineg): m / z (%) = 170 (100), 278 (40) [M-H] ^" . ¹ H NMR (400 MHz, ^ _6- DMSO): δ = 0.81 (d, J = 6.3 Hz, 3H), 0.86 (d, J = 6.4 Hz, 3H), 0.89 (s,

9H), 1.48 (m, 3H), 1.57 (m, 2H), 3.61 (s, 3H), 4.14 (m, IH), 4.23 (m, IH), 5.03 (s, 2H), 7.27-7.40

(m, 6H), 8.19 (d, J = 7.9 Hz, IH).

¹³ C NMR (126 MHz, ^ ₆ -DMSO): δ = 29.53 (3C), 30.50, 44.11, 51.49, 65.47, 127.75 (2C), 127.95,

128.51 (2C), 137.34, 156.03, 174.97. HR-TOF-MS (method 1): C ₁₅ H ₂₂ NO ₄ calc. 280.1544, gef. 280.1535 [M + H] ⁺ .

Example 48A

Methyl-N - [(benzyloxy) carbonyl] -4-methyl-D-leucyl-L-leucinate

Methyl L-leucinate hydrochloride (600.9 mg, 3.31 mmol, 1.1 equivalents) is initially charged under argon blanket gas atmosphere in dichloromethane (250 ml), the solution is treated with HOBt (1625.3 mg,

12.03 mmol, 4 equiv), ΝMM (992 μL, 9.02 mmol, 3 equiv), benzyloxycarbonyl protected amino acid (Example 47A, 840.0 mg, 3.0 mmol, 1 equiv), EDC (1149.7 mg,

6.01 mmol, 2 equivalents) and treated with further ΝMM (661 .mu.l, 6.02 mmol, 2 equivalents) at -10 ⁰ C and overnight is brought under stirring to complete conversion. For workup, the reaction solution is concentrated on a rotary evaporator at 30 ⁰ C bath temperature, the

Residue dissolved in ethyl acetate and saturated sodium bicarbonate solution, the Separated aqueous phase extracted with ethyl acetate, the combined organic phases washed successively with 5% citric acid solution, with saturated sodium bicarbonate solution and saturated sodium chloride solution and dried over sodium sulfate. After separation of the drying agent, the solvent is completely removed on a rotary evaporator at 3O ⁰ C bath temperature. The product is obtained after lyophilization with 1200 mg (98% of theory) yield.

HPLC (method 2): R _t = 2.65 min. LC-MS (method 7): R ₁ = 2.84 min; MS (ESI pos.): M / z (%) = 407 (100), 813 (27) [2M + H] ⁺ . ¹ H NMR (400 MHz, ^ ₆ -DMSO): δ = 0.81 (d, J = 6.3 Hz, 3H), 0.86 (d, J = 6.3 Hz, 3H), 0.89 (s, 9H), 1.42-1.55 ( m, 5H), 3.61 (s, 3H) 4.15 (m, IH), 4.23 (m, H), 5.03 (s, 2H) ₅ 7.28-7.41 (m, 6H), 8.01 (d, J = 7.9 Hz, 1H) .HR-TOF-MS (Method 1): C ₂₂ H ₃₅ N ₂ O ₅ calc. 407.2541, m.p. 407.2534 [M + H] ⁺ .

Example 49A

N - [(benzyloxy) carbonyl] -4-methyl-D-leucyl-L-leucine

The title compound is prepared from the methyl ester (Example 48A, 1.1 g, 2.71 mmol) by hydrolysis according to procedure 4. After a reaction time of 4 h, the HPLC chromatogram (Method 2) indicates almost complete conversion. For fine purification, the crude product is chromatographed on the preparative RP-HPLC (Method 36) and then 980 mg (92% of theory) of product.

HPLC (method 2): R ₁ = 2.40 min. LC-MS (method 7): R, = 2.56 min;

MS (ESI pos.): M / z (%) = 393 (100) [M + H] ⁺ , 785 (72) [2M + H] ⁺ , MS (ES Ineg): m / z (%) = 391 (57 ) [M - H] ^" , 783 (100) [2M - H] ^" . ¹ H NMR (400 MHz, J ₆ -DMSO): δ = 0.81 (d, J = 6.3 Hz, 3H), 0.86 (d, J = 6.3 Hz, 3H), 0.88 (s, 9H), 1.42-1.55 ( m, 5H), 4.11-4.23 (m, 2H), 5.03 (s, 2H), 7.27-7.39 (m, 6H), 8.01 Cd, J = 8.0 Hz, IH), 12.52 (s, br., IH) , HR-TOF-MS (method 1):. C _2I H ₃₃ N ₂ O ₅ Calculated 393.2384, obs. 393.2383 [M + H] ⁺ .

Example 5OA

N ^2; - (benzyloxycarbonyl) - [3-fer butyl-D-alanyl?] -L-leucyl - [(3R) -3-amino-L-phenylaIanyl)] - [(3i) -

3-hydroxy-L-leucyl] -L-leucyl-D-arginyl-L-isoleucyl-L-allothreonyl-glycyl - [(35) -3-hydroxy-L-asparaginyl] -L-serine-C ^{/ y /} N, ^J " ^3- lactam trifluoroacetate

In accordance with protocol 5, the cyclopeptide (Example 26A ₅ 120.0 mg, 93.95 μmol) and the dipeptide acid (Example 49A, 63.38 mg, 169.11 μmol, 1.8 equivalents) with the aid of NMM (52 μl, 469.75 μmol, 5 equivalents) and HATU (64.3 mg, 169.11 μmol, 1.8 equivalents) in DMF (15 mL) overnight to the amide at RT. The Titelverbindvmg is obtained after chromatography (Method 35) with 138 mg (96% of theory) yield.

HPLC (Method 25): R ₁ = 21.46 min. LC-MS (method 7): R 1 = 2.19 min; MS (ESIpos.): M / z (%) = 1424 (25) [M + - H] ⁺ , 712 (100) [M + 2 H] ²⁺ ,

MS (ES Ineg): m / z (%) = 1422 (22) [M-H] ^" , 710 (100) [M-2 H] ^2" .

HR-TOF-MS (Method 1): C ₆₇ Hi ₀₇ Ni ₆ Oi ₈ calc. 1423.7944, ref. 1423.7964 [M + H] ⁺ .

Example 51A

(2Z) -2 - {[(benzyloxy) carbonyl] amino} -3- (6-trifluoromethyl-pyridin-3-yl) -acrylic acid methylester

Dissolve 6-trifluoromethylpyridine-3-carbaldehyde (4.85 g, 27.70 mmol) and methyl {[benzyloxycarbonyl] -amino} (dimethoxyphosphoryl) acetate (9.17 g, 27.70 mmol, 1.0 equiv.) In THF (70 mL). 70 ⁰ C cooled. At -70 ⁰ C NN, N, N-tetramethylguanidine (6:38 g, 55.39 mmol, 6.95 mL, 2.0 equivalents) slowly zugetropfit and then 4 h at -70 ° C, then stirred for 12 h at RT. The reaction mixture is concentrated, extracted with ethyl acetate (twice 100 ml) against water. The combined organic phases are washed with saturated brine, dried over sodium sulfate and filtered. After concentration, 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 (Method 22): R _t = 4.60 min. HPLC / UV-Vis (Method 17): R _t = 4.54 min. LC-MS (Method 29): R, = 2.44 min; MS (ESI pos.): M / z (%) = 381 (100) [M + H] ⁺ ; MS (ES Ineg.): M / z (%) = 379 (100) [M-H] ^" .

¹ H NMR (400 MHz, J ₆ -DMSO): δ = 3.74 (s, 3H), 5.10 (s, 2H), 7.27 (s, IH), 7.33-7.38 (m, 5H), 7.94 (d, J = 8.5 Hz, IH), 8.27 (d, J = 8.5 Hz, IH), 8.93 (s, IH), 9.51 (s, IH). HR-TOF-MS (Method 1): C] ₈ Hi ₆ N ₂ O ₄ F ₃ [M + H] ⁺ calc. 381.1062, m.p. 381.1065. Example 52A

N - [(benzyloxy) carbonyl] -3- (6-trifluoromethyl-pyridin-3-yl) -L-alaπin-methylester

Example Compound 51A (10.15 g, 26.69 mmol) is dissolved in methanol p.a. (100 ml) dissolved. With a cannula, argon is passed through for about 5 minutes, then (-f-) -1,2-bis - [(2) S ', 5> S) diethylphospholano] benzene (cyclooctadiene) rhodium (I) triflate (289 mg, 400 μmol, 0.015 equivalents).

It is hydrogenated for 12 h at 4 bar hydrogen pressure and RT. It is then filtered through kieselguhr (methanol) and the eluate is concentrated. The crude product is chromatographed (silica gel,

Eluent: toluene: ethyl acetate 5: 1). There are obtained 9.9 g (97% of theory) of the title compound.

[Ot] ²⁰ Na = -24 ° (c = 0.093 in methanol).

HPLC / UV-Vis (Method 22): R, = 4.5 min.

HPLC / UV-Vis (method 17): R ₄ = 4.49 min.

LC-MS (Method 29): R, = 2.40 min; MS (ESI pos.): M / z (%) = 383 (100) [M + H] ⁺ ;

MS (ES Ineg.): M / z (%) = 273 (100), 381 (50) [M-H] ^" .

¹ H NMR (400 MHz, J ₁₅ -DMSO): δ = 2.99 (dd, J = 3.5 and 1 1.0 Hz ₅ IH), 3.22 (dd, J = 3.5 and

11.0 Hz, IH), 3.66 (s, 3H), 4.40 (m, IH), 4.97 (s, 2H), 7.23 (d, J = 5.5 Hz, IH), 7.29-7.33 (m, 3H),

7.83 (d, J = 6.5 Hz, IH), 7.93-7.98 (m, 2H), 8.65 (s, IH). HR-TOF-MS (method 1). C ₁₈ H ₁₈ N ₂ O ₄ F ₃ [M + H] ⁺ calcd 383.1219, obs. 383.1223. Example 53A

3 - (6-trifluoromethyl-1-pyridin-3-yl) -L-alanine methyl ester

Example Compound 52A (9.90 g, 25.89 mmol) is dissolved in methanol (100 mL). With a cannula, argon is passed for about 5 minutes, then Pd is added via C (10%, 990 mg). It is hydrogenated for 12 h at 4 bar hydrogen pressure and RT. It is then filtered through kieselguhr, concentrated and dried under high vacuum. Yield: 5.8 g (90% of theory) of the title compound.

[α] ¹⁹⁹ Na = + 3 ° (c = 0.186 in methanol).

HPLC / UV-Vis (method 22): R, = 3.34 min. HPLC / UV-Vis (Method 17): R ₁ = 3.22 min.

IR v _max (NaCl, cm ^-1 ): 3415, 1734, 1339, 1136, 1087.

LC-MS (Method 29): R ₄ = 1.74 min;

MS (ESI pos.): M / z (%) = 249 (100) [IM + H] ⁺ .

¹ H NMR (500 MHz, U ₆ -OUSO): δ = 2.85 (dd, J = 5.5 and 13.5 Hz, IH), 3.01 (dd, J = 5.5 aod 13.5 Hz, IH), 3.61 (s, 3H), 3.63-3.69 (m, IH), 7.82 (d, J = 7.5 Hz, IH), 7.93 (d, J = 7.5 Hz, IH),

8.61 (s, IH).

HR-TOF-MS (method 1): C ₁₂ Hi ₅ N ₃ O ₂ F ₃ [M + H] ⁺ calc. 290.1116, gef. 290.1122.

Example 54A

N- (tert-3-ynoxycarbonyl) -3 - (tert-butyl) -D-alanyl-3- (6-trifluoromethyl-1-pyridin-3-yl) -L-alanine methyl ester

To a solution of example compound 53A (6.34 g, 25.54 mmol) and N- (tert-butoxycarbonyl) -3-tert-butyl-D-alanine (6.27 g, 25.54 mmol, 1.0 equiv.) In dry DMF (240 mL) are added -3O ⁰ C slowly ΝMM (12.92 g, 127.72 mmol, 14.04 mL, 5 equiv) and HATU (9.71 g, 25.54 mmol, 1 equiv). The reaction mixture warmed slowly to RT (about 3 h), with complete conversion being observed by HPLC (Method 17). Potassium dihydrogen phosphate (34.76 g, 255.44 mmol, 10 equivalents) is added, and the reaction mixture is stirred for 20 min, then it is filtered, diluted with ethyl acetate ver¬ and washed with sat. 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, eluent: gradient cyclohexane: ethyl acetate 10: 1 to 2: 1) to give 9.74 g (73% of theory) of the title compound.

[Ot] ¹⁹⁹ _Na = + 7.0 ° (c = 0.044 in methanol).

HPLC / UV-Vis (Method 22): R _t = 4.89 min. HPLC / UV-Vis (method 17): R ₄ = 4.75 min.

LC-MS (Method 29): 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] ^" .

¹ H NMR (500 MHz, J ₀ -DMSO): δ = 0.74 (s, 9H), 0.97-1.O0 (m, IH), 1.20-1.25 (m, IH), 1.35 (s, 9H), 2.99 -3.05 (m, IH), 3.23-3.26 (m, IH), 3.66 (s, 3H), 3.94 (m, IH), 4.60 (m, IH), 6.82 (d, J =

8.5 Hz, IH), 7.78 (d, J = 8.0 Hz, IH) ₅ 7.94 (d, J = 8.0 Hz _* IH), 8.34 (d, J = 8.5 Hz, IH), 8.64 (s,

IH).

IR v _max (NaCl, cm ^-1 ): 2959, 1742, 1655, 1520, 1336, 1160, 1136, 1087, 1050, 1027.

HR-TOF-MS (method 1). C ₂₂ H ₃₃ N ₃ O ₅ F ₃ [M + H] ⁺ calcd 476.2372, obs. 476.2364. Example 55A

N ^{2 /} - (r? -Butoxycarbonyl) - [3-tert-butyl-D-alanyl] - [3- (6-trifluoromethylpyrid-3-yl) -L-alanyl] - [(3i?) - 3-amino -L-phenylalanyl)] - [(3i?) - 3-hydroxy-L-leucyl] -L-leucyl-D-arginyl-L-isoleucyl-L-allothreonyl-glycyl - [(35) -3-hydroxy -L-asparaginyl] -L-serine-C ^y ' ^y -N ^{5 "J-} lactam trifluoroacetate

According to procedure 5, the cyclopeptide (Example 26A, 175.0 mg, 0.14 mmol) and the dipeptide acid (Example 54A, 113.8 mg, 0:25 μmol, 1.8 equivalents) with the aid of NMM (75 μl, 0.69 mmol, 5 equivalents) and HATU (93.8 mg, 0:25 mmol, 1.8 equivalents) in DMF (15 mL) overnight to form the amide at -10 ⁰ C. The title compound is obtained after chromatography (Method 35) with 190 mg (86% of theory) of yield.

HPLC (Method 25): R _t = 21.51 min. LC-MS (method 7): R _t = 2.27 min;

MS (ESI pos.): M / z (%) = 1494 (33) [M + H] ⁺ , 747 (100) [M + 2 H] ²⁺ , MS (ES Ineg): m / z (%) = 1492 (10) [M-H] ^" , 745 (100) [M-2 H] ^{2 ~} . HR-TOF-MS (Method 1): C ₅₇ H ₁₀₅ N ₁₇ Oi ₈ F ₃ calc. 1492.7771, found 1492.7778 [M + H] ⁺ . Example 56A

Memyl- (2Z) -2 - [(tert-butoxycarbonyl) amino] -3-cyclopentyl

Methyl {[tert-butoxycarbonyl] amino} (dimethoxyphosphoryl) acetate (39.2 g, 132.00 mmol) and cyclopentanecarbaldehyde (38.9 g, 396.00 mmol, 3 equiv.) Are initially charged in THF (320 mL) and N, NNN-tetramethylguanidine (24.8 mL, 22.81 g, 198.00 mmol, 1.5 equivalents) is added at -7O ⁰ C to the solution. Thaw the cold bath, the reaction is slowly brought to RT. Within 2 days full turnover is achieved. For working up, the reaction is combined with ethyl acetate (1600 ml) and water (800 ml) and the separated organic phase is dried over sodium sulfate. The desiccant is separated by filtration and the filtrate is concentrated on a rotary evaporator. From the residue, the product is isolated by column chromatography (silica gel 60: 1 kg, eluent: cyclohexane / ethyl acetate 4: 1) with 34.8 g (98% of theory) yield.

HPLC / UV-Vis (Method 4): R, = 4.76 min. ¹ H NMR (300 MHz, CDCl ₃₎ δ = 1:28 to 1:43 (m, 2H), 1:46 (s, 9H), 1.52-1.78 (m, 4H), 1.83-1.97 (m, 2H), 2.71 (m, IH), 3.78 (s, 3H), 5.79 (s, br., IH), 6.50 (d, J = 9.9 Hz, IH).

Example 57A

Methyl-N - -3-cyclopentyl-L-alaninate (he ^ -butoxycarbonyl?)

The example compound 56A (13.0 g, 48.27 mmol) is dissolved in ethanol (220 ml), the solution is purged with argon for 10 min and placed in an ultrasonic bath for 5 min. After the addition of (+) - l, 2-bis - [(2S, 55) diethylphospholano] benzene (cyclooctadiene) rhodium (I) triflate (100.0 mg, 0.13 mmol) is rinsed again with argon for 30 min, in an ultrasonic bath for 5 min degassed and then hydrogenated for 3 days at RT at a hydrogen pressure of 3.5 bar. For workup, the solvent is completely removed on a rotary evaporator and chromatographed on silica gel 60 (300 ml, eluent: cyclohexane / ethyl acetate 2/1). The title compound is obtained with 12.9 g (94% of theory).

HPLC / UV-Vis (Method 4): R _t = 4.88 min. DCI-MS (NH ₃ ): m / z = 289 (100) [M + NH ₄ J ⁺ , 560 (15) [2M + NHJ ⁺ .

¹ H NMR (200 MHz, CDCl ₃₎ δ = 1:00 to 1:25 (m, 2H), 1:45 (s, 9H), 1.48-1.95 (m, 9H), 3.72 (s, 3H), 4.28 (m, IH) , 4.95 (d, J = 8.4 Hz, IH).

Example 58A

MethyW-cyclopentyl-L-alaninates hydrochloride

From the N-protected amino acid (Example 57A, 5.0 g, 18.43 mmol), the title compound with 3.9 g crude yield (quant.) Is obtained according to Arbeitsvor¬ 2.

LC-MS (method 30): R ₁ = 2.02 min; MS (ESI pos.): M / z (%) = 172 (100) [M + H] ⁺ . ¹ H NMR (300 MHz, d ₆ ) δ = 0.98-1.18 (m, 2H), 1.40-1.67 (m, 4H) 1.68-1.85 (m, 4H in 1.82 (d, J = 6.6 Hz, 2H)), 1.92 (m, IH), 3.75 (s, 3H) ₅ 3.90 (t, J = 8.4 Hz, IH), 8.63 (s, br., 3H). Example 59A

Methyl-N- (fer? -Butoxycarbonyl) -3-cyclopentyl-D-alaninate

From the acrylate (Example 56A, 13.0 g, 48.27 mmol) and the (+) - l, 2-bis - [(2i, 5R) diethylphospholano] benzene (cyclooctadiene) rhodium (I) triflate (10000 mg , 0.13 mmol) are prepared as described in Example 57A, 12.6 g (96% of theory) of the title compound.

MS (DCI, NH ₃ ): m / z (%) = 289 (100) [M + NH ₄ J ⁺ , 560 (15) [2M + NH _t ] ⁺ .

¹ H NMR (300 MHz, CDCl ₃₎ δ = 1:00 to 1:21 (m, 2H), 1:45 (s, 9H), 1.48-1.93 (m, 9H), 3.73 (s, 3H), 4.28 (m, IH) , 4.95 (m, IH).

Example 6OA

N- (tert-butoxycarbonyl) -3-cyclopentyl-D-alanine

The exemplary compound 59A (5.0 g, 18.43 mmol) is reacted according to protocol 4 within 2 h to the iV ^" protected amino acid to give 5.11 g (quant.) Of the title compound.

[Ot] ²⁰ Na = + 7 ° (c = 0.13 in CH ₂ Cl ₂ ). HPLC / UV-Vis (Method 2): R, = 2.33 min. LC-MS (Method 7): R _t = 2.26 min; MS (ESI pos.): M / z (%) = 158 (100) [M -C ₄ H ₈ -CO ₂ + H] ⁺ ; MS (ESIneg): m / z (%) = 256 (58) [M - H] ^{^'1} H NMR (300 MHz, C ₆ -DMSO) δ = 1:05 (m, 2H), 1:38 (s, 9H). , 1.42-1.78 (m, 8H), 1.83 (m, IH), 3.85 (m, IH), 7.04 (d, J = 8.2 Hz, IH), 12.27 (s, br., IH). Example 61A

(? -Butoxycarbonyl fer) -3-cyclopentyl-D-alanyl-3-cyclopentyl-L-alaninate Methyl-N-

The peptidic amine (Example 58A, 0.9 g, 4.27 mmol, 1.1 equivalents) is initially charged in dichloromethane (250 ml) under argon protective gas atmosphere, the solution cooled to -10 ⁰ C and then closing successively with HOBt (2.1 g, 15.54 mmol , 4 equivalents), ΝMM (1.3 ml, 1.2 g, 11.67 mmol, 3 equivalents), the peptidic acid (Example 6OA, 1.0 g, 3.89 mmol), EDC (1.5 g, 7.77 mmol, 2 equivalents) and again with ΝMM ( 0.8 ml, 0.8 g, 7.76 mmol, 2 equivalents). The reaction is slowly brought to RT in the cooling bath, completely reacted overnight. For working up the reaction, water is added, concentrated on a rotary evaporator (water bath 35 ⁰ C), treated with ethyl acetate and saturated sodium bicarbonate solution and the separated aqueous phase extracted twice with ethyl acetate, the combined organic phases successively with saturated, aqueous 5% citric acid Solution, saturated sodium bicarbonate solution and saturated sodium chloride solution. The organic phase is dried over sodium sulfate, filtered and concentrated. The crude product is 1.64 g (quant.) Of the title compound.

HPLC / UV-Vis (method 2): R. = 2.97 min.

LC-MS (method 7): R ₁ = 2.94 min;

MS (ESI pos.): M / z (%) = 411 (100) [M + H] ⁺ , 311 (57) [M -C ₄ H ₈ -CO ₂ + H] ⁺ .

Example 62A

N- (tert-butoxycarbonyl) -3-cyclopentyl-D-alanyl-3-cyclopentyl-L-alanine

The dipeptide (Example 61 A, 1.5 g, 3.65 mmol) is reacted according to Working Method 4 within 1 h to dipeptidic carboxylic acid. After the fine purification (Method 13) of the freeze-dried crude product, the title compound is isolated with 923.4 mg (64% of theory) of iso.

HPLC / UV-Vis (Method 2): R _t = 2.65 min. LC-MS (method 7): R, = 2.60 min;

MS (ESI pos.): M / z (%) = 397 (100) [M + H] ⁺ , 297 (60) [M -C ₄ H ₈ -CO ₂ + H] ⁺ , 793 (46) [2M + H] ⁺ ; MS (ES Ineg): m / z (%) = 395 (57) [M - Hf, 791 (100) [2M - Hf ¹ H NMR (4OO MHz, J ₆ -DMSO) δ = 1.05 (m, 4H) 1.38 (s, 9H), 1.43 (m, 4H), 1.55 (m, 6H), 1.61-1.86 (m, 8H), 3.96 (m, IH), 4.16 (m, IH), 6.75 (d, J = 8.5 Hz, IH), 7.96 (d, J = 7.9 Hz, IH) 12.55 (s, br, IH). HR-TOF-MS (method 1):. C _2I H ₃₈ N ₂ O ₅ Calculated 397.2702, obs. 397.2697 [M + H] ⁺ .

Example 63A

N ^2; - (^ er ^ -butoxycarbonyl) - [3-cyclopentyl-D-alanyl] - [3-cyclopentyl-L-alanyl] - [(3i?) - 3-amino-L-phenylalanyl I)] - [(3R ) -3-hydroxy-L-leucyl] -L-leucyl-L-arginyl-L-isoleucyl-L-allothreonyl-glycyne 1- [(J 1 -S -hydroxy-L-asparaginyl-L-serine-C 1-10 - N '^ - lactam trifluoroacetate

In accordance with protocol 5, the cyclopeptide (Example 26A, 30.0 mg, 23.49 μmol) and the dipeptide acid (Example 62A, 16.8 mg, 42.28 μmol, 1.8 equivalents) with the aid of NMM (13 μl, 117.43 μmol, 5 equivalents) and HATU ( 16.1 mg, 40.54 mmol, 1.8 equivalents) in DMF (2 ml) within 2.5 h to the amide at -1O ⁰ C reacted. The title compound is obtained after chromatography (Method 37) with 26.6 mg (67% of theory) yield.

HPLC (Method 2): R _t = 2.30 min. LC-MS (Method 7): R, = 2.21 min;

MS (ESI pos.): M / z (%) = 1428 (35) [M + H] ⁺ , 664 (100) [M + 2 H] ²⁺ , MS (ES Ineg): m / z (%) = 1426 (100) [M-HT ^" .

HR-TOF-MS (Method 1): C ₆₇ H _1n Ni ₆ O ₁₈ calc. 1427.8257, ref. 1427.8242 [M + H] ⁺ .

Example 64A

N - [(benzyloxy) carbonyl] -3- (tert-butyl) -D-alanyl-3- (pyridm-3-yl) -L-alanine methyl ester trifluoroacetate

3-Pyridin-3-yl-L-alanine (800 mg, 3.16 mmol) and N- (benzyloxycarbonyl) -3-ferf-butyl-D-alanine (1.47 g, 3.16 mmol, 1 equiv) are added at 0 ^° C in DMF (12 ml) dissolved. Then 4-methylmorpholine (1.74 ml, 15.8 mmol, 5 equivalents) and HATU (1.8 g, 4.74 mmol, 1.5 equivalents) are added and stirred at RT for about 3 h. It is extracted with ethyl acetate (twice) against conc. Νatriumhydrogencarbonat, the combined organic phases with 1 M citric acid and washed again with conc. Sodium bicarbonate, dried over sodium sulfate, filtered and concentrated in vacuo. After chromatographic purification (Method 39), 1.1 g (63% of theory) of the title compound are obtained.

W ¹⁹⁹ Na = + 6 ° (c = 0.13 in methanol).

HPLC (Method 17): R _t = 3.92 min.

HPLC (Method 23): R _t = 4.06 min.

LC-MS (method 7): R, = 2.03 min, MS (ESIpos.): M / z (%) = 442 (100) [M + H] ⁺ .

IR v _max (NaCl, cm ^-1 ): 3295, 2952, 2359, 1715, 1659, 1522, 1435, 1365, 1283, 1243, 1138, 1048,

1326th

HR-TOF-MS (method 1). C ₂₄ H ₃₂ N ₃ O ₅ [M + H] ⁺ calcd 442.2342, obs. 442.2342.

Example 65A

N- (Benzyloxycarbonyl) -3- (ferr-butyl) -D-alanyl-3- (pyridin-3-yl) -L-alanine

Example 64A (1.1 g, 1.98 mmol) in THF and water 3: 1 was added (48 ml) and cooled to -2O ⁰ C. Lithium hydroxide (119 mg, 2.5 mmol, 4.95 equivalents) is added and the mixture is stirred at RT for 12 h. The reaction is stopped by the addition of potassium dihydrogen phosphate (1.35 g) and concentrated in vacuo. After chromatographic purification (Method 14), 0.57 g (67% of theory) of the title compound are obtained as a solid.

M ²⁰ N _a = + 44 ° (c = 0.15 in methanol). HPLC (Method 17): R _t = 3.73 min. HPLC (Method 23): R ₁ = 3.88 min. LC-MS (Method 7): R _t = 1.72 min, MS (ESI pos.): M / z (%) = 428 (100) [M + H] ⁺ .

IR v _max (NaCl, cm ^-1 ): 3290, 2956, 1659, 1613, 1537, 1414, 1251, 1205, 1184, 1136, 1050, 1028. HR-TOF-MS (Method 1): C ₂₃ H ₂₉ N ₃ O ₅ [M + H] ⁺ calc. 428.2180, found 428.2169.

Example 66A

N ^{z /} - (benzyloxycarbonyl) - [3- (tert -butyl) -D-alanyl] - [3- (pyridin-3-yl) -L-alanyl] - [(3i?) - 3-amino-L- phenylalanyl)] - [(3i) -3-hydroxy-L-leucyl] -L-leucyl-D-arginyl-L-isoleucyl-L-allothreonyl-glycyl- [(3) S) -3-hydroxy-L- asparaginyl] -L-serine-C ^{/ ;;} -N ^3J -lactam bistrifluoroacetate

According to protocol 5, the cyclopeptide (Example 26A ₅ 20.0 mg, 1.566 mmol) and the dipeptide acid (Example 65A, 12.0 mg, 28.19 μmol, 1.8 equivalents) with the aid of NMM (9 ul, 78.3 .mu.mol, 5 equivalents) and HATU (10.7 mg, 28.19 .mu.mol, 1.8 equivalents) in DMF (6 ml) was allowed to react to form the amide at -20 ⁰ C to -10 ⁰ C within 2.5. After chromatography (eg Method 36) 16.5 mg (61% of theory) of the title compound are obtained.

HPLC (Method 25): R ₄ = 18.55 min. LC-MS (Method 29): R ₄ = 1.73 min;

MS (ESI pos.): M / z (%) = 1458 (20) [M + H] ⁺ , 730 (100) [M + 2 H] ²⁺ , MS (ES Ineg): m / z (%) = 1456 (27) [M-H] ^" , 728 (100) [M-2 H] ^2" . HR-TOF-MS (Method 1): C ₆₉ Hi ₀₄ N _n O ₁₈ calc. 1458.7740, Gef. 1458.7739 [M + - H] ⁺ .

Example 67A

Methyl 3 - (teτ-7-butyl-L) -L-alaninate trifluoroacetate

Methyl N- (tert-butoxycarbonyl) -3- (t-t-butyl) -L-alanmat (16 g, 61.70 mmol) [J. A. Bajgrowicz, et al., Tetrahedron Lett, 1984, 2759-2762] is added trifluoroacetic acid / dichloromethane 3: 1 (220 ml) and stirred for 1.5 h at RT. It is then concentrated in vacuo and the residue dried in a HV. The yield is quantitative (16.5 g).

MS (DCI ₅ NH ₃ ): m / z = 160 (100) [M + H] ⁺ .

¹ HNMR (300 MHz ₅ J ₆ -DMSO): δ = 0.92 (s, 9H), 1.59 (dd, J = 4.7, 14.5 Hz ₅ 1 Η), 1.80 (dd, J = 7.6, 14.5 Hz, IH), 3.75 (s, 3H), 3.95 (dd, J = 4.7, 7.6 Hz ₅ IH), 8.5-8.3 (br, s, 3H).

Example 68A

Methyl-N- (ter ^ butoxycarbonyl) -D-leucyl-3- (ter ^ butyl) -L-alaninate

The peptidic amine (Example 77A, 4.0 g, 14.68 mmol, 1.1 equivalents) is initially charged under argon inert gas atmosphere in dichloromethane (365 ml), the solution is washed successively with HOBt (7.2 g, 53.23 mmol, 4 equivalents), NMM (4.3 ml , 39.92 mmol, 3 equivalents), N- (tert-butoxycarbonyl) -. D-leucine (3.1 g, 13.31 mmol, 1 equivalent) [T. Kato, N. Izumiya, Bull. Chem. Soc. Jpn., 1966, 39, 2242-2249], EDC (5.1 g, 26.62 mmol, 2 equivalents) and with further NMM (3 ml, 26.61 mmol, 2 equivalents) at -10 ° C and overnight with stirring to brought complete sales. For workup, the reaction solution is concentrated on a rotary evaporator at 3O ⁰ C bath temperature and then chromatographed by preparative HPLC (eg, Method 35). This gives 5.5 g (80% of theory) of the title compound.

LC-MS (method 7): R _t = 2.72 min;

MS (ESI pos.): M / z (%) = 373 (78) [M + H] ⁺ , 273 (100) [M -C ₄ H ₈ -CO ₂ + H] ⁺ .

Example 69A

N- (fer ^ butoxycarbonyl) -D-leucyl-3- (teτt-butyl) -L-alanine

Example compound 68A (400 mg, 1.07 mmol) is reacted according to protocol 4 within 1 h to the dipeptide carboxylic acid. For work-up, the solution is combined with potassium dihydrogenphosphate (219.2 mg, 1.61 mmol, 1.5 equivalents), concentrated by evaporation in a cold rotary evaporator and finely purified by preparative HPLC (eg Method 35). The title compound is obtained as lyophilisate (282.0 mg, 65% of theory). (For analytical data see WO2004099239). Example 70A

N ^{2 '} ' - (tert-butoxycarbonyl) -D-leucyl- [3 - (tertbutyl) -L-alany 1] - [(3R) -3-amino-L-phenylalanine I)] -

[(3i?) - 3-hydroxy-L-leucyl] -L-leucyl-D-arginyl-L-isoleucyl-L-allothreonyl-glycyl - [(3. S) -3-hydroxy

L-asparaginyl] -L-serine-C ^{/ ;;} -N ^{3 5} -lactam-fluoroacetate

According to protocol 5, the cyclopeptide (Example 26A, 30.0 mg, 23.49 μmol) and the dipeptide acid (Example 69A, 15.2 mg, 42.28 μmol, 1.8 equivalents) with the aid of NMM (13 μl, 117.45 μmol, 5 equivalents) and HATU ( 16.1 mg, 42.28 μmol, 1.8 equivalents) in DMF (10 mL) overnight to the amide at RT. The title compound is obtained after chromatographing (Method 36) with 34.0 mg (96% of theory) of yield.

HPLC (Method 2): R _t = 2.25 min. LC-MS (Method 7): R _t = 2.18 min;

MS (ESI pos.): M / z (%) = 1390 (77) [M + H] ⁺ , 645 (100) [M -C ₄ H ₈ -CO ₂ + 2 H] ^{2 "} *, MS (ES Ineg) : m / z (%) = 1387 (73) [M-H] ^" , 693 (100) [M-2 H] ^2" HR-TOF-MS (Method 1): C ₆₄ H ₁₀₉ N ₁₆ O ₁₈ 1389.8101, found 1389.8101 [M -J- H] ⁺ . Example 71A and Example 72A

(2S) N- (tert-butoxycarbonyl) -3- (trimethylsilyl) -D-alanine (71A) and (2i?) - N- (tert-butoxycarbonyl) -3- (trimethylsilyl) -L-alanine (72A )

The synthesis is carried out according to M. Merget, et al., J. Organomet. Chem. 2001 628, 183-194. The enantiomers are separated by preparative HPLC on a chiral phase (method 2S). The isomers are assigned by HPLC comparison with an authentic sample of h / (tert-butoxycarbonyl) -L-3-trimethylsilylalanine (2R compound, Mercachem AMR 39.260).

Example 7 IA, N- (tert-butoxycarbonyl) -3-trimethylsilyl-D-alanine

Chiral HPLC (Method 26): R _t = 4.16 min, ee> 99%.

[α] _D ²⁰ = +1.1 (c = 0.83, methanol)

Example 72A, N- (ferf-butoxycarbonyl) -3-trimethylsilyl-L-alanine

Chiral HPLC (Method 26): R _t = 9.27 min, ee> 99%

[α] _D ²⁰ = -1.6 (c = 0.66, methanol)

Example 73A

3-trimethylsilyl-L-alanine methylester hydrochloride

The compound from Example IIA. (300 mg) is dissolved in methanol (3 ml) and cooled to 0 ^° C. Trimethylsilyl chloride (590 mg, 4.7 equivalents) is dissolved in 30 min. added dropwise and the mixture warmed to RT overnight. The volatile components are removed on a Rotavapor and then under high vacuum. The target compound (224 mg, 92% of theory) is obtained.

¹ H NMR WrDMSO, 300 MHz): δ = 0.03 (s, 9H), 0.97-1.71 (m, 2H), 3.73 (s, 3H), 3.93 (dd, J = 5.3, 10.9 Hz, IH), 8.40 ( brs, 3H).

Example 74A

N-benzyloxycarbonyl-3-trimethylsilyl-L-alanine methylester

The compound of Example 73A (2.0 g) is initially charged in THF (40 ml) under argon at 0 ° C. Triethylamine (2.3 g, 2.4 equivalents) is added and then a solution of JV-benzyloxycarbonyloxysuccinimide (2.83 g, 1.2 equivalents) in THF (20 ml) is added dropwise. The mixture is then stirred at RT overnight, concentrated by half, diluted with ethyl acetate, washed twice with water and once with saturated sodium chloride solution. The organic phase is dried over sodium sulfate and concentrated by rotary evaporation. The residue is purified by preparative HPLC (Method 41). The target compound (2.32 g, 79% of theory) is obtained.

HPLC (Method 18): R _t = 4.96 min.

MS (DCI, NH ₃ ): m / z = 327 [M-KNH ₄ J ⁺ (IOO), 310 (3) [M + H] ⁺ .

Example 75A

(2i?) - N-benzyloxycarbonyl-3-trϊmethylsilyl-L-Alaninol

To a solution of Example 74A (2.32 g, 7.5 mmol) in THF (14 mL) was added lithium chloride (0.64 g, 15 mmol) and sodium borohydride (0.57 g, 15 mmol) then ethanol (22 mL). The mixture is stirred overnight at RT. For workup, the mixture is adjusted to pH 4 under cooling with ice-water and with citric acid solution (10% in water), then diluted with 200 ml of water and extracted three times with dichloromethane. The combined organic phases are dried over magnesium sulfate, filtered and concentrated in vacuo. The residue is separated by HPLC (Method 41) and the appropriate fraction is concentrated in vacuo. The title compound is obtained as an oil (1.24 g, 59% of theory).

HPLC (Method 4): R, = 4.48 min.

MS (DCI ₅ NH ₃ ): m / z OOO), 282 (13) [M + H] ⁺ .

¹ H NMR _(CDCl3, 300 MHz): δ = 0.O3 (s, 9H), 0.58-0.84 (m, 2H), 2.13 (br.s, IH), 3:45 (br.rn, IH),

3.65 (br.m, IH), 3.83 (br.m, IH), 4.72 (br.m, IH), 5.08 (s, 2H), 7.33 (m, 5H).

Example 76A

(2i?) - N-benzyloxycarbonyl-3-trimethylsilyl-L-alaninal

A solution of pyridine-SO ₃ complex (2.04 g, 12.8 mmol) in anhydrous DMSO (12 mL) is added to a solution of Example 75A (0.36 g, 1.28 mmol) and N, N-diisopropylethylamine (1.65 g, 12.8 mmol) 7 ml of DMSO under argon at 10 ⁰ C was added. The cooling bath is removed and the mixture is left for 20 min. stirred. 400 ml of ice-water are added and the solution is extracted three times with diethyl ether. The combined organic phases are washed twice with a citric acid solution (10% in water), once with water, once with saturated bicarbonate solution and once with saturated sodium chloride solution Dried magnesium sulfate, filtered and concentrated by rotary evaporation. The title compound is obtained as an oil (0.39 g, 93% of theory) and used directly as the crude product.

MS (DCI, NH ₃ ): m / z = 297 [M + NH _t f OOO], 280 (25) [M + H] ⁺ .

¹ H NMR (CDCl ₃ , 300 MHz): δ = 0.07 (s, 9H), 0.78 (dd, J = 9.4, 1 4.0 Hz, IH), 1.12 (dd, J = 5.8, -5 14.0 Hz, IH) , 5.13 (s, 2H), 7.35 (m, 5H), 9.53 (s, IH).

Example 77A

3- (te ^ butyl) -L-alanine-methylester trifluoroacetate

The (25) -N-tert-butoxycarbonyl-3- ^ e? * T-butyl-L-alanine methyl ester (529 mg, 2.04 mmol) is stirred in 0 TFA / dichloromethane 1: 3 (3 mL) at RT, the solution is concentrated by rotary evaporation and dried under high vacuum. 540 mg (97% of theory) of product are obtained.

¹ H NMR W ₅ -DMSO, 300 MHz): δ = 0.92 (s, 9H), 1.59 (dd, J = 4.7, 14.4 Hz, IH), 1.80 (dd, J = 7.5, 14.4 Hz, IH), 3.76 (s, 3H), 3.95 (dd, J = 4.7, 7.5 Hz, IH), 8.33 (br.s, 3H).

Example 78A

5 N - [(2i?) - 2-Benzyloxycarbonylamino-3-trimethylsilyl-propyl] -3- (terf-butyl) -L-alanine methyl ester hydrotrifluoroacetate

Under argon, the compound Example 77A are (4:33 g, 15.9 mmol), Example Compound 76A (7.2 g, 20.6 mmol) and Νatriumacetat (1.3 g, 15.9 mmol) were introduced into DMEF (140 ml) at 0 ⁰ C. Natriumcyanborohydrid (1.99 g, 31.7 mmol) is added and the mixture stirred at 0 ⁰ C for 2 h. For workup, the mixture is diluted with 1 1 ethyl acetate and washed twice with a 5% solution of sodium bicarbonate. The aqueous phases are reextracted with ethyl acetate. The combined organic phases are washed with sat. Washed sodium chloride solution, dried over magnesium sulfate, filtered and concentrated. The residue is purified by preparative HPLC (Method 39). This gives 6.02 g (71% of theory) of the title compound as crystals.

HPLC (Method 4): R, = 4.8 min. ESI-MS (pos.): M / z = 423 (100) [M + HT | ⁺ . 14.7 δ = 0:03 (s, 9H), 0.76 (dd, J = 5.1, 14.7 Hz, IH), 0.94 (s, 9H), 0.95 (dd, J = 10.2, ¹ H NMR _(CDCl3, 300 MHz) Hz, IH), 1.95-1.80 (m, 2H), 2.93 (dd, J = 2.5, 12.5 Hz, IH), 3.35 (dd, J = 9.8, 11.3 Hz, IH), 3.81 (s, 3H) , 3.85 (dd, J = 4.5, 7.9 Hz, IH), 4.07-3.90 (m, IH), 5.05 (d, J = 11.5 Hz, IH), 5.16 (d, J = 11.5 Hz, IH), 5.63 ( d, J = 0.9 Hz, IH), 7.33 (s, 5H).

Example 79A

N - [(2i?) - 2-Benzyloxycarbonylamino-3-trimethylsilyl-propyl] -3- (tert-butyl) -L-alanine hydrochloricl

Example compound 78A (6.02 g, 1.2 mmol) is dissolved in methanol (225 ml) and treated at RT with a 1 Ν lithium hydroxide solution (101 ml) in water (9 equivalents). The reaction mixture is stirred at RT overnight (1 h), then acidified with 1 N hydrochloric acid and freed from methanol on a rotary evaporator. The remaining suspension is diluted with 200 ml of water and extracted three times with ethyl acetate. The combined organic phases are dried over magnesium sulfate, filtered and concentrated. The residue is dried under high vacuum. This gives 5.3 g (quantitative) of the title compound, which is reacted further without Reiniguxig.

HPLC (Method 4): R ₁ = 4.6 min.

MS (ESIpos): m / z (%) = 409 (100) [M + Hf, MS (ES Ineg): m / z (%) = 407 (10) [MH] ^" .

¹ H-NMR W ₅ -DMSO, 400 MHz): δ = 0.03 (s, 9H), 0.82 (m, 2H), 0.95 (s, 9H), 1.68 (br.d, J = 14.5, IH), 1.84 (dd, J = 9.0, 14.5, IH), 2.88 (m, IH), 3.10 (m, IH), 3.80-4.03 (m, 2H), 5.01 (d, J = 12, IH), 5.13 (d, J = 12, IH), 7.29 (d, J = 9.0, IH), 7.43-7.56 (m, 5H), 8.7-9.2 (br, 2H, NH ₂ ⁺ ).

Example 8OA

N ^2; - [(2i?) - 2- (benzyloxycarbonylamino) -3- (trimethylsilyl) -propyl] - [3- (tert -butyl) -L-alanyl] - [(3i?) - 3-amino-L-phenylalanyl) ] - [(3i?) - 3-hydroxy-L-leucyl] -L-leucyl-D-arginyl-L-isoleucyl-L-allothreonyl-glycyl - [(35) -3-hydroxy-L-asparaginyl] -L-serine-C ^{; ; o} -N ^5J -lactam bisfluoroacetate

According to protocol 5, the cyclopeptide (Example 26A, 30.0 mg, 23.49 μmol) and di & dipeptide acid (Example 69A, 18.8 mg, 42.28 μmol, 1.8 equivalents) with the aid of NMMI (13 μl, 117.45 μmol, 5 equivalents) and HATU (16.1 mg, 42.28 μmol, 1.8 equivalents) in DMF (15 ml) within 2.5 h to the amide at -20 ⁰ C to -10 ⁰ C reacted. The title compound is obtained after chromatography (Method 36) with 34.0 mg (96% of theory) yield.

HPLC (Method 25): R, = 19.59 min.

LC-MS (Method 7): R, = 1.97 min; MS (ESI pos.): M / z (%) = 720 (100) [M + 2H] ²⁺ . HR-TOF-MS (method 1): C ₆₇ Hi _I iN ₁₆ O ₁₇ calc. 1439.8077, et. 1439.8036 [M + H] ⁺ . Example 81A

(2S) -N-benzyloxycarbonyl-3-ter? -Butyl-L-alaninal

The compound is prepared analogously to the synthetic sequence of Example 75A and Example 76A from Z-ß ter /.- butyl-alanine methyl ester.

MS (DCI, NH _3): m / z = 281 [M + _NH4] ⁺ (100); 264 (20) [M + H] ⁺ .

¹ H-NMR _(CDCl3, 300 MHz): δ = 1.00 (s, 9H), 1.30 (dd, J = 8.3, 14.7 Hz, IH), 1.83 (dd, J = 3.8,

14.6 Hz, IH), 4.33 (dt, J = 3.8, 8.7 Hz, IH), 5.06 (m, IH), 5.12 (s, 2H), 7.36 (m, 5H), 9.58 (s, IH).

Example 82A

N - [(25) -2-benzyloxycarbonylamino-4,4-dimethylpentyl] -3- (tert -butyl) -L-alanine methyl ester trifluoroacetate

The compound is prepared analogously to Example 78A from Example 81 A and Example 77 A. Yield: 860 mg (36% of theory).

HPLC (Method 4): R, = 4.7 min.

MS (ESI pos): m / z = 407 (100) [M + H] ⁺ .

¹ H-NMR _(CDCl3, 300 MHz): δ = 0.91 (s, IH), 0.93 (s, 9H), 1:34 (br d, J = 15, IH), 1:53 (dd, J =

8.7, 14.7, IH), 1.68-1.95 (m, 2H), 2.89 (dd, J = 3.5, 12 Hz, IH), 3.33 (dd, J = 10.5, 12 Hz, IH), 3.84 (m, IH), 3.80 (s, 3H), 3.99 (m, IH), 5.04 (d, J = 12 Hz, IH), 5.13 (d, J = 12 Hz, IH), 5.73 (br.d , J = 8.5 Hz, IH), 7.32 (m, 5H).

Example 83A

N - [(2S) -2-Benzyloxycarbonylaπn.ino-4,4-dimethylpentyl] -3- ^ erf-butyl-L-alanine trifluoroacetate

This compound is prepared analogously to Example 79A from Example 83A (1.10 g, 2.11 mmol) and 1 Ν lithium hydroxide solution (19 ml). Yield: 895 mg (83% of theory).

HPLC (Method 4): R _t = 4.47 min.

MS (ESI pos.): M / z (%) = 393 (100) [M + H] ⁺ ; MS (ESIng.): M / z (%) = 391 (<4) [MH] ^" .

¹ H NMR WrDMSO, 300 MHz): δ = 0.89 (s, 9H), 0.93 (s, 9H), 1.30-1.45 (m, 2H), 1.64 (dd, J = 2,

14 Hz, IH), 1.81 (dd, J = 9.7, 14 Hz, IH), 2.88 (m, IH), 2.98 (m, IH), 3.85-3.99 (m, 2H), 4.99 (d,

J = 12.7 Hz, IH), 5.12 (d, J = 12.7 Hz, IH), 7.07 (d, J = 9.0 Hz, IH), 7.27-7.40 (m, 5H).

¹³ C NMR (126 MHz, ^ ₆ -DMSO): δ = 29.00 (3C), 29.38 (3C), 29.96 (2C), 42.22, 45.10, 45.64, 50.52, 56.97, 65.39, 127.56 (2C) , 127.73, 128.23 (2C), 136.83, 155.47, 171.47.

HR-TOF-MS (method 1): C ₂₂ H ₃₇ N ₂ O ₄ [M + H] ⁺ Found. 393.2753, ber. 393.2748

Example 84A

N ^{2 /} - [(26) -2- (benzyloxycarbonylamino) -3- (tert-butyl) propyl] - [3- (tert-butyl) -L-alanyl] - [(3i?) - 3-amino-L -phenylalanyl)] - [(3i) -3-hydroxy-L-leucyl] -L-leucyl-D-arginyl-L-isoleucyl-L-allothreoyl-glycyl - [(3S) -3-hydroxy-La .sparaginyl] -L-serine-C ^λyo -N ^J ' ² -lactam bistrifluoroacetate

According to protocol 5, the cyclopeptide (Example 26A, 170.0 mg, 0.13 mmol), the dipeptide acid (Example 83A, 121.4 mg, 0.24 mmol, 1.8 equivalents), NMM (73 μl, 0.67 μmol, 5 equivalents) and HATU (91.1 mg , 42.28 μmol, 1 .8 equivalents) in DMF (6 ml) within 2.5 h to amide at -2O ⁰ C to - 10 ⁰ C implemented. The title compound is chromatographed (eg, Method 35) to give 34.0 mg (96% of theory) of the title compound.

HPLC (Method 2): R _t = 2.08 min.

LC-MS (method 7): R ₁ = 1.9 min; MS (ESI pos.): M / z (%) = 712.7 (100) [M + 2H] ²⁺ , 1424 (5) [M + H] ⁺ ; MS (ESIneg.). M / z (%) - 1422.7 (100) [ML - H] ^"HR-TOF-MS (Method 1): C ₆₈ H _{1 n} Ni ₆ O ₁₇ [M + H] ⁺ Found. 1423.8318, supra 1423.8313, Example 85A

N ^{2 y} - [(2i?) - 2- (benzyloxycarbonylamino) -3 - (? R / -butyl) propyl] - [3- (ferr-butyl) -L-alanyl] - [(3 /?) -

3-amino-L-phenylalanyl)] - [(3i) -3-hydroxy-L-leucyl-L-leucyl-D-arginyl-L-isoleucyl-L-allothreonyl-glycyl - [(3S) -3 -hydroxy-L-asparaginyl] -L-serine-C ^{λ / (>} -N ³ ' ^2- lactam-bisfluoroacetate

According to protocol 5, the cyclopeptide (example 26A, 42.0 mg, 32.88 μmol), N- ((2R) -2- [[(benzyloxy) carbonyl] amino} -4,4-dimethylpentyl) -3- {tert -bxity 1) -L-alanine trifluoroacetate (29.98 mg, 59.18 μmol, 1.8 equivalents), ΝMM (18 μl, 164.40 μmol, 5 equivalents) and HATU (22.5 mg, 59.18 μmol, 1.8 equivalents) in DMF (15 ml) within 2.5 h to amide at -20 ⁰ C to -1O ⁰ C reacted. The title compound is obtained after chromatography (Method 10) (37.5 mg, 69% of theory).

The dipeptide acid, N - ((2R) -2 - {[(benzyloxy) carbonyl] amino} -4,4-dimethylpentyl) -3- (tert-butyl) -L-alanine hydrotrifluoroacetate, can be prepared analogously to the sequence Example 82A from compound 77A and N-benzyloxycarbonyl-3- (te-t-butyl) -D-alanmal and subsequent saponification (see Example 83A).

HPLC (Method 2): R _t = 2.13 min. LC-MS (method 7): R _t = 1.98 min; MS (ESIpos.): M / z (%) = 712 (100) [M + 2H] ²⁺ , MS (ES Ineg.): M / z (%) = 1422 (100) [M-H] ^" , 710 (40) [M - 2H] ^{2 ~} .

HR-TOF-MS (method 1): C ₆₈ HmNi ₆ O ₁₇ [M + H] ⁺ gef. 1423.8282, about 1423.8308.

Example 86A

Methyl-N- (fer? -Butoxycarbonyl) -3- (pyridin-3-yl) -L-alaninate EP2005 / 011451

- 121 -

(2S) -N- (ferric butoxycarbonyl) -3- (pyridin-3-yl) alanine (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. Then concentrated in vacuo, the residue is mixed with ethyl acetate and shaken out against 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. There remains an oil that crystallizes during drying in an oil pump vacuum. Yield: 23.60 g (90% of theory).

HPLC / UV-Vis (Method 17): R ₁ = 3.28 min.

LC-MS (Method 7): R, = 1.21 min, MS (ESIpos.): M / z (%) = 281 (100) [M + H] ⁺ . ¹ H NMR (400 MHz, J ₀ -DMSO) S = 1.30 (s, 9H), 2.86 (m, IH), 3.04 (m, IH), 3.63 (s, 3H), 4.22 (m, IH), 7.28 -7.39 (m, 2H), 7.69 (d, IH), 8.43 (m, 2H).

Example 87A

3 - (Pyridyne-3-yl) -L-alanine-methyl 1-ester-bis-trifluoroacetate

The compound from Example 86A (11.8 g, 42.09 mmol) is dissolved in trifluoroacetic acid in dichloromethane (160 ml, 30% solution) and stirred for 30 min. stirred at RT. Then you narrow in Vacuum. The residue is taken up in a little water and lyophilized. Then the lyophilizate is 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 (method 23): R, = 0.88 min. LC-MS (method 7): R, = 0.46 min,

MS (ESI pos.): M / z (%) = 181 (100) [M + H] ⁺ .

¹ H NMR (400 MHz, DMSO) δ = 2.79 (dd, IH), 2.92 (dd, IH), 3.60 (s, 3H), 3.63 (m, IH), 7.30

(m, IH), 7.62 (d, IH), 8.41 (m, 2H).

Example 88A

Methyl N- (tert-M3-oxycarbonyl) -3- (trimethylsilyl) -D-alanyl-3- (pyridin-3-yl) -L-alaninate

The compound from Example 87A (10.31 g, 39.4 mmol) and the compound from Example 71A (16.10 g, 39.4 mmol, 1 equivalent) are dissolved at ⁰ ° C. in DMF (186 ml). Then NMM (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 (Method 17): R, = 3.91 min. LC-MS (Method 7): R _t = 1.90 min, MS (ESI pos.): M / z (%) = 424 (100) [M + H] ⁺ .

¹ H NMR (400 MHz, efe-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), 6.6S (d, IH), 7.26 (dd, IH), 7.61 (m, IH), 8.20 (d, IH) , 8.40 (m, 2H). Example 89A

N- (fer-butoxycarbonyl) -3- (trimethylsilyl) -D-alanyl-3- (pyridin-3-yl) -L-alanine

The compound from Example 88A (7.4 g, 17.56 mmol) is taken up in THF ^" water (6: 4), cooled to ⁰ ° C., and admixed with lithium hydroxide monohydrate (1.47 g, 35.13 mmol, 2 equivalents) Stir O ⁰ C. After one hour, add another equivalent (0.74 g)

Lithium hydroxide monohydrate added and stirred for an additional hour. It is distilled

Remove most of the THF in vacuo, wash with two portions of MTBE and then adjust the aqueous

Phase by adding citric acid to pH 4. A solid precipitates. It is extracted with three portions of ethyl acetate, whereby the solid dissolves. The combined organic phases are dried over sodium sulfate, filtered and concentrated. The crude product is through

Gel chromatography (method 3, eluent: methanol). Yield: 6.67 g (93% of theory).

HPLC / UV-Vis (method 17): R ₁ = 3.73 min. LC-MS (method 7): R, = 1.68 min, MS (ESIpos.): M / z (%) = 410 (40) [M + H] ⁺ .

¹ H NMR (300 MHz, J ₀ -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) ,

Example 9OA

N- (tot-benzyloxycarbonyl) -3- (trimethylsilyl) -D-alanyl-3-pyridin-3-yl-L-alanine

The compound of Example 89A (500 mg, 1.22 mmol) is dissolved in 30% TFA in dichloromethane (20 ml) and stirred at RT for 30 min. It is then concentrated, the residue taken up in water and lyophilized. The lyophilizate (600 mg solid) is introduced (25 ml) under argon in THF, N- (benzyloxycarbonyloxy) succinimide (320 mg, 1.28 mmol, 1.15 equivalents) are then at 0 ⁰ C, and finally 4-methylmorpholine (150 uL, 1.34 mmol, 1.2 equivalents). Allow to warm to RT and stir overnight. The reaction is stopped by addition of glacial acetic acid and concentrated in vacuo. The residue is taken up in ethyl acetate and extracted twice against 0.5 M citric acid. The organic phase is dried over sodium sulfate, filtered and concentrated in vacuo. The residue is purified by chromatography (Method 34). Yield: 395 mg (0.71 mmol, 58% of theory).

HPLC (Method 17): R ₄ = 3.79 min.

LC-MS (method 7): R ₁ = 1.84 min, MS (ESIpos.): M / z C%) = 444.1 (100) [M + H] ⁺ . ¹ H NMR (400 MHz ₅ ^ ₅ -DMSO) δ = -0.07 (s, 9H), 0.60-0.75 (m, 2H), 2.97-3.03 (m, IH), 3.28 (m, IH), 3.88 (m, IH), 4.54 (m, IH), 4.98 (d, J = 12.7, IH), 4.98 (d, J = 12.7, IH), 5.07 (d, J = 12.7, IH) 7.27-7.35 (m , 6H), 7.65 (m, IH), 8.08 (d, J = 7.04, IH), 8.19 (d, J = 8.8, IH), 8.62 (s, 2H).

Example 91A

N ^{2 /} - (benzyloxycarbonyl) - [3- (trimethylsilyl) -D-alanyl] - [3- (pyrid-3-yl) -L-alanyl] - [(3R) -3-amino-L-phenylalanyl)] - [(3i?) - 3-hydroxy-L-leucyl] -L-leucyl-D-arginyl-L-isoleucyl-L-allothreonyl-glycyl [(i? -S-hydroxy-L-asparaginyl) J-L-serine -C ^ ^ -N '^ - lactaπi-bistrifluoracetat

To a solution of the cyclopeptide (Example 26A., 1.0 equivalent, 18.6 mg, 15 μmol), the diprophtic acid (Example 9OA, 1.5 equivalents, 9.7 mg, 22 μmol) and NMM (1.0 equivalent, 15 μmol) in dry DMF ( 200 .mu.l) HATU (1.6 equivalents, 8.9 mg, 23 .mu.mol) is first added at -2O ⁰ C under argon inert gas atmosphere. The reaction mixture is stirred (about 15 minutes) and NMM (2.5 equivalents, 38 μmol) is added again. The reaction mixture warmed slowly to RT (about 12 h) and then shows complete conversion of the amine component (HPLC grade).

Control, method 2). The reaction mixture is treated with solid potassium dihydrogen phosphate (10

Equivalents, 500 μmol) and then evaporated in vacuo and purified by chromatography (Method 21). 15.8 mg (63% of theory) of product are obtained.

HPLC / UV-Vis (method 2): R _t = 1.9 min, X _1113x (qualitative) = 210 nm (s), 255-270 (m).

LC-MS (Method 7): R _t = 2.0 min;

MS (ESI pos.): M / z (%) = 738 (100) [M-Boc + 2H] ²⁺ , 1475 (15) [M + H] ⁺ ;

MS (ES Ineg.): M / z (%) = 682 (100), 736 (80) [ME-2H] ^{2 "} , 1473 (60) [M-H] ^" .

Example 92A

3-? E ^ -butyl-D-alanyl-3-ter? -Butyl-L-alanine hydrochloride

Λ / (tert-butoxycarbonyl) -3-tert-butyl-D-alanyl-3-tert-butyl-L-alanine (4.5 g, 12.1 mmol, Example 1 OA) is pre-dissolved in dioxane (3 ml). At RT, a 4 N solution of hydrogen chloride in dioxane (30.2 mmol, 120 mmol, 10 equivalents) was added dropwise. The reaction mixture is stirred for 30 min, evaporated in vacuo and dried under high vacuum. The title compound is obtained as a solid (3.5 g, 99% of theory).

LC-MS (method 7): R ₄ = 1.52 min;

MS (ESI pos.): M / z (%) = 273.6 (100) [M + H] ⁺ ;

MS (ESIneg): m / z (%) = 271.5 (100) [M - H] ^{^'1} H NMR (500 MHz, J ₆ -DMSO): δ = 0.85 (s, 9H), 0.86 (s, 9H. ), 1.49 (dd, J = 14.3, 1.6 Hz, IH),

1.50 (d, J = 13.8 Hz, IH), 1.64 (dd, J = 14.3, 4.0 Hz, IH), 1.71 (dd, J = 14.3, 6.9 Hz, IH), 3.77

("T", J = 6.5 Hz, IH), 4.14 (m, IH), 8.27 (s, br, 3H), 8.94 (d, J = 8.2 Hz, IH), 12.58 (s, br, IH).

¹³ C NMR (126 MHz, ^ ₆ -DMSO): δ = 28.41 (3C), 29.50 (3C), 30.08, 30.38, 44.46, 44.80, 50.03,

50.30, 169.10, 173.98. HR-TOF-MS (method 1): C ₁₄ H ₂₉ N ₂ O ₃ calc. 273.2173, gef. 273.2167 [M + H] ⁺ .

Example 93A

7V- (Benzyloxycarbonyl) -3-tert-butyl 1-D-alanyne-1-tert-butyl-L-alanine

- 12.7 -

3-tert-Butyl-D-alanyl-3-tert-butyl-L-alanine (3.73 g, 12.1 mmol, Example 92A) is dissolved in THF (170 ml) under argon gas atmosphere. After addition of water (170 ml), benzyloxycarbonyloxysuccinimide ester (4.52 g, 18.1 mmol, 1 .5 equivalents) and N-methylmorpholine (4.28 g, 4.23 mmol, 3.5 equivalents) at ⁰ ° C., the mixture is stirred vigorously at RT until it is all Reactor is reacted (several hours, BDPLC control, method 2). The approach is done with glacial acetic acid. In the vacuum the TBDF deducted. The remaining aqueous phase is acidified with ethyl acetate with 4 Ν hydrochloric acid to pH <3 and then extracted several times with ethyl acetate. The organic phases are washed with brine, dried over sodium sulfate, filtered and concentrated. The resulting foam is stirred with acetonitrile to form a solid, which is filtered off, then washed with a little acetonitrile. This process can be repeated several times with the Fitrat - after it has been concentrated. The combined solids are dried under high vacuum to give the title compound as a solid. The remaining mother liquor is concentrated and purified by preparative HPLC (Method 10). The title compound (combined solids and purification product from HPLC separation) is obtained as a solid (3.49 g, 71% of theory).

HPLC / UV-Vis (method 2): R _t = 2.6 min.

LC-MS (Method 8): R, = 2.46 min; MS (ESI pos.): M / z (%) = 363 (60), 407 (100) [M + H] ⁺ . MS (ES Ineg): m / z (%) = 297 (100), 405.5 (40) [M-H] ^" .

¹ H NMR (500 MHz, ^ ₆ -DMSO): δ = 0.81 (s, 9H), 0.83 (s, 9H), 1.40-1.44 (m, 2H), 1.49 (dd, J = 14.3, 9.7 Hz, IH ), 1.58 (dd, J = 13.5, 1.4 Hz, IH), 4.07 (m, IH), 4.13 (m, IH), 4.94 (d, J = 12.3 Hz, IH), 4.99 (d, J = 12.5 Hz , IH), 7.25-7.32 (m, 5H), 7.92 (d, J = 8.5 Hz, IH). ¹³ C NMR (126 MHz, ^ ₆ -DMSO): δ = 29.49 (3C), 29.75 (3C), 30.41, 30.46, 44.52, 45.11, 49.55, 52.73, 65.49, 127.70 (2C), 127.91, 128.48 (2C) , 137.31, 155.59, 172.52, 174.50. HR-TOF-MS (method 1). C ₂₂ H ₃₄ N ₂ O ₅ Calculated 407.2541, obs. 407.2531 [M + H] ⁺ .

Example 94A

N ² - (Benzyloxycarbonyl- [3-fer? -Butyl-D-alanyl] -3-tert-butyl-L-alanyl) - [(3i?) - 3? -? - butylammo-L-phenylalanine] -methyl ester

To a solution of the racemic Aminosäuremethylesters (1.0 equivalents, g 4.16 10.19 mmol, Example 14) in dichloromethane pa (100 ml) at -30 ⁰ C successively HOBt (4 equivalents, 5:51 g, 40.77 mmol), N-methylmorpholine (4 Equivalents, 40.77 mmol), the N-benzyloxycarbonyl dipeptide (N- (benzyloxycarbonyl) -3-ferf-butyl-D-alanyl-3-fcrt-butyl-L-alanine, 1.1 equivalents, 4.56 g, 11.21 mmol, Example 93A) and EDC (2 equivale, 3.91 g, 20.38 mmol). The reaction mixture warmed slowly to RT (about 12 h), with complete conversion of the amine component being monitored by HPLC (method 2). The reaction mixture is quenched with potassium dihydrogen phosphate (5.0 equivalents, 6.93 g, 50.96 mmol), concentrated in vacuo, taken up in ethyl acetate (about 400 ml) and then with 5% aqueous citric acid (twice), distilled water and saturated Washed with sodium chloride solution. It is dried over sodium sulfate and filtered. The crude product is then filtered through silica gel. In vacuo, the solution is evaporated to Trockαe and then dried in a high vacuum. This gives 5.36 g (77% of theory) of the title compound as a mixture of diastereomers with relative (2.S '*, 3i * *) stereochemistry on aminophenylalanine. The diastereomers are separated from each other in a further chromatographic step (Method 42). This gives 2.87 g (41% of theory) of the title compound (> 99%, method 28). This reaction can be carried out analogously with the enantiomerically pure amino acid methyl ester. Direct diastereomerically pure product is obtained.

HPLC / UV-Vis (Method 3): R, = 3.1 min. HPLC / UV-Vis (Method 28): R, = 4.84 min. LC-MS (Method 8): R ₁ = 2.7 min;

MS (ESI pos.): M / z (%) = 583.5 (100) [M + H - CO ₂ -C ₄ Hs] ⁺ , 683.5 (60) [M + H] ⁺ ; MS (ES Ineg.): M / z (%) = 681.6 (10) [M-H] ^" , 727.6 (100) [M -H + HCO ₂ Hr. LC-MS (method 7): R ₄ = 3.1 min MS (ESI pos.): M / z (%) = 583.5 (100) [M + H - CO ₂ -C ₄ Hg] ⁺ , 683.5 (40) [M + H] ⁺ ; MS (ES Ineg.): M / z (%) = 573 (100), 681.6 (5) [M - H] ^" ; 727-6 (40) [M - H + HCO ₂ H] -

Example 95A

N ² - (benzyloxycarbonyl- [3- / erf-butyl-D-alanyl] -3- / he / butyl-C-alanyl) - [(3i?) - 3- ^ e / -f-butylamino-L- phenylalanine]

Under argon protective gas atmosphere, a solution of the tripeptide methyl ester (Example 94A, 1.0O g ₅ 1.46 mmol) in THF / water 2: 1 (300 ml) was added. At 0 ⁰ C, a degassed 5% aqueous solution of lithium hydroxide with vigorous stirring (59.6 mg, 2.5 mmol, 1.7 equivalents) was slowly added dropwise. The mixture is stirred at 0 ^° C. until the HPLC chromatogram (method 2) indicates complete conversion (about 6 h). If the reaction time is too long, there is a risk of epimerization. It is then mixed with acetic acid (0.4 ml), the reaction mixture concentrated in vacuo and overlaid with ethyl acetate (100 ml). The aqueous phase is then acidified with 5% citric acid (pH 2-3) and then extracted with ethyl acetate (50 ml, three times). The combined organic phases are washed with saturated aqueous sodium chloride solution (20 ml, twice), dried on sodium sulfate, filtered, concentrated in vacuo and dried in a high vacuum. The crude product is finely purified by preparative HPLC (Method 38). 682 mg (56% of theory) of the title compound are obtained.

HPLC / UV-Vis (Method 2): Rj = 2.9 min. HPLC / UV-Vis (method 25): R, = 24.6 min. LC-MS (method 7): R ₁ = 2.9 min;

MS (ESI pos.): M / z (%) = 569.2 (100), 669.3 (50) [M + H] ⁺ , 1337.4 (40);

MS (ES Ineg.): M / z (%) = 667.2 (100) [M - Hf, 1335.4 (80).

HR-TOF-MS (method 1): C ₃₆ H ₅₃ N ₄ O ₈ [M + H] ⁺ Found. 669.3881, ber. 669.3858.

Example 96A

N ² - (Benzyloxycarbonyl- [3-? Er? -Butyl-D-alanyl] -3-tert-butyl-L-alanyl) - [(3i?) - 3-? -? -? - butylamino-L-phenylalanine] - [2- (trimethylsilyl) ethyl)] ester

A mixture of the tripeptide acid (Example 96A, 733 mg, 1.10 mmol), 2- (trimethylsilyl) -ethanol 1.30 g, 11.0 mmol, 10 equivalents) and 4 mmol IMOLI (about 100 mg) in dry dichloromethane (6.5 ml) is stirred for 30 min at RT under argon protective gas atmosphere. On closing (1 equivalent mg 134, 1.10 mmol,) are (mg 452, 2.20 mmol, 2 equivalents) at -3O ⁰ C DCC and DMAP were added. The reaction mixture is thawed (about 12 h) and stirred at RT until the HPLC chromatogram (method 2) indicates complete conversion (about 12 h). The reaction mixture is quenched with glacial acetic acid (125 μl), then at RT Vacuum, suspended in a little dichloromethane (6.5 ml), filtered through an ec RP18-HPLC cartridge (Macherey Nagel), concentrated again and then purified by preparative HPLC (Method 35). 706 mg (84% of theory) of the title compound are obtained.

HPLC / UV-Vis (Method 2): R, = 3.4 min. LC-MS (Method 7): R ₁ = 3.4 min;

MS (ESI pos.): M / z (%) = 669.5 (80), 769.6 (100) [M + 2H] ²⁺ , 1556 (30). HR-TOF-MS (Method 1): C _4I H ₆₅ N ₄ O ₈ Si [M + H] ⁺ Found. 769.4548, ^" ber. 769.4567.

Example 97A

N ² - (benzyloxycarbonyl- [3- ert-butyl-D-alanyl?] -3- ^ erϊ-butyl-L-alanyl) - (3 _J R) -3-amino-L-phenylalanine [2- (trimethylsilyl ) ethyl)] ester trifluoroacetate

The N- (t-t-butoxycarbonyl) -protected tripeptide (720 mg, 0.94 mmol, Example 96A) is placed under an argon blanket gas atmosphere. At RT and with vigorous stirring, 4N hydrochloric acid in dioxane (40 ml) is added dropwise. The mixture is stirred until the analytical HPLC (method 2) indicates complete conversion (about 30 min). The reaction mixture is evaporated at RT in vacuo. The crude product is purified by preparative HPLC (Method 10) and lyophilized. The title compound is obtained as a lyophilisate (394 mg, 54% of theory).

HPLC / UV-Vis (method 25): R, = 25.2 min. HPLC / UV-Vis (method 2): R, = 2.4 min. LC-MS (Method 8): R ₄ = 2.3 min; MS (ESI pos.): M / z (%) = 669.6 (100) [M + H] ⁺ ; MS (ES Ineg.): M / z (%) = 703.6 (100) [M-H + HCO ₂ H]. ^" LC-MS (Method 7): R ₁ = 2.6 min;

MS (ESI pos.): M / z (%) = 669.4 (100) [M + H] ⁺ .

Example 98A

N ² - (Benzyloxycarbony l - [3-tert-butyl-1-D-alanyl] -3-tert-butyl-L-alanyfJ-N ³ - {tert-butoxycarphenyl 1- [(3i?) - 3-hydroxy] L-leucyl] -L-leucyl-D-arginyl-L-isoleucyl-L-allothreonyl-glycyl - [(35) -3-hydroxy-L-asparaginyl] -L-seryl} - (3i?) - 3-amino -L-phenylalanine [2- (trimethylsilyl) ethyl)] ester trifluoroacetate

To a solution of the Boc-octapeptide (Example 2OA, 1.0 equivalents, 114.4 mg, 0.10 mmol), the tripeptide (Example 97A, 1.0 equivalents, 80.0 mg, 0.10 mmol, Example 97A) and N-methylmorpholine (1.0 equivalent, 0:10 mmol) in dry DMF (3.0 mL) was added at -15 ⁰ C ujnter argon protective gas atmosphere initially HATU (1.5 eq, 58.3 mg, 0:15 mmol). The reaction mixture is stirred (about 15 minutes) and combined again with ΝMM (1.0 equivalents, ClO mmol). The reaction mixture is warmed to ⁰ ° C. and admixed again with HATU (2.5 equivalents, 0.25 mmol). The reaction mixture warmed slowly (about 12 h) to RT and then shows complete conversion of the amine component (HPLC control, method 3). The Reaktions¬ mixture is treated with solid potassium dihydrogen phosphate (5 equivalents, 0:50 mmol) and then evaporated under high vacuum and purified by chromatography (method 21 or method 13) followed by subsequent double decomposition of the Chromatographieproduktes & ^~ hrough additional of TFA (100 μmol, as a 0.05% solution in acetonitrile-water 1: 1). 140.5 mg (78% of theory) of product are obtained.

HPLC / UV-Vis (Method 2): R ₁ = 2.6 min. HPLC / UV-Vis (method 25): R, = 26.6 min. LC-MS (method 7): R ₁ = 2.5 min;

MS (ESI pos.): M / z (%) = 1656.8 (100) [M + H] ⁺ ;

MS (ESIng.): M / z (%) = 1655 (100) [M-H] -.

HR-TOF-MS (method 1): C ₇₈ H _13I N ₁₆ O _2I Si [M + H] ⁺ . 1655.9467, about 1655.9439.

Example 99A

^ - (benzyloxycarbonyl-tS-cetylbutyl-D-alanyl-S-ferl-butyl-L-alanyl-N ⁵ - ferric-butoxycarbonyl-

[(3i?) - 3-hydroxy-L-leucyl] -L-leucyl-D-arginyl-L-isoleucyl-L-allothreonyl-glycyl - [(3iS) -3-hydroxy-L-asparaginyl] -L-seryl } - (3J?) - 3-amino-L-phenylalanine trifluoroacetate

A solution of the TMSE ester (460 mg, 0.260 mmol, Example 98A) in dry THF (8 ml) is mixed with molecular sieve 4 A and stirred under argon protective gas atmosphere at RT (15 min). The dried solution is then added over a period of 2 h in two portions with a 1 N solution of tetrabutylammonium fluoride in THF (4.16 ml, 4.16 mmol, 16 equivalents). The mixture is stirred until complete conversion is observed (HPLC, method 2). The reaction mixture is quenched with glacial acetic acid (298 .mu.l, 5.198 mmol, 20 equivalents), concentrated in vacuo and purified directly by preparative HPLC (Method 21). After lyophilization, the product is obtained as a lyophilisate (375 mg, 86% of theory).

HPLC / UV-Vis (method 2): R ₁ = 2.4 min. LC-MS (Method 7): R _t = 2.3 min;

MS (ESI pos.): M / z (%) = 728.8 (100) [M + 2H - CO ₂ -C ₄ Hg] ²⁺ , 778.8 (30) [M + 2H] ²⁺ , 1556.9

(40) [M + H] ⁺ ;

MS (ES Ineg.): M / z (%) = 1554 (100) [M-H] ^" .

HR-TOF-MS (method 1): C ₇₃ H ₁₁₉ N ₁₆ O ₂ , [M + H] ⁺ gef. 1555.8737, about 1555.8731.

Example IQOA

N ² - (Benzyloxycarbonyl- [3-? Er? -Butyl-D-alanyl] -3- (er) -butyl-L-alanyl) -N ^J - {(3i?) - 3-hydroxy-L-leucyl- L-leucyl-D-arginyl-L-isoleucyl-L-allothreonyl-glycyl - [(iS) -3-hydroxy-L-asparaginyl] -L-seryl} - (3i?) - 3-amino-L-phenylalanyl bistrifluoroacetate

The N- (tert-butoxycarbonyl) -protected compound (100 mg, 0.06 mmol, Example 99A) is placed under an argon-protective gas atmosphere. At RT and with vigorous stirring, 4N hydrochloric acid in dioxane (15 ml) is added dropwise. The mixture is stirred until the analytical HPLC (method 2) indicates complete conversion (about 30 min). The reaction mixture is evaporated at RT in vacuo. The crude product is purified by preparative HPLC (Method 35) and lyophilized. The title compound is obtained as a lyophilisate (85.5 mg, 85% of theory).

HPLC / UV-Vis (method 2): R, = 1.9 min.

HPLC / UV-Vis (Method 25): R ₁ = 18.4 min, A ^ _13x (qualitative) = 210 nm (s), 255-270 (w).

LC-MS (Method 7): R, = 1.8 min;

MS (ESI pos.): M / z (%) = 728.7 (100) [M + 2H] ²⁺ ;

MS (ES Ineg.): M / z (%) = 1454 (100) [M-H] ^" .

HR-TOF-MS (Method 1): C ₆₈ H ₁₁₁ N] ₆ O ₁₉ [M + H] ⁺ . 1455.8192, about 1455.8206. Example IQIA

N ² - (Benzyloxycarbonyl) -N ⁵ - {[(3i) -3-hydroxy-L-leucyl] -L-leucyl-D-arginyl-L-isoleucyl-L-allothreonyl-glycyl - [(3> S) 3-hydroxy-L-asparaginyl] -L-seryl} - (3i?) - 3-ammo-L-phenylalanine trifluoroacetate

The N- (tert-butoxycarbonyl) -protected apeonapeptide (Example 22A, 1200 mg, 0.848 mmol) is initially charged in dioxane (10 ml) under an argon blanket gas atmosphere. At RT and with vigorous stirring, 4N hydrochloric acid in dioxane (230 ml) is added. The mixture is stirred until analytical HPLC (method 2) indicates complete conversion (about 1 h). The reaction mixture is concentrated on a rotary evaporator, then the residue is treated at RT with water and then evaporated in vacuo. The crude product is purified by preparative HPLC (Method 11) and lyophilized. The title compound is obtained as a colorless solid (918 mg, 95% purity, 83% of theory).

HPLC / UV-Vis (method 2): R ₁ = 1.6 min. HPLC / UV-Vis (Method 25): R, = 14.3 min. LC-MS (method 7): R ₄ = 1.4 min;

MS (ESI pos.): M / z (%) = 601.6 (100) [M + 2H] ²⁺ , 1201.7 (20) [M + H] ⁺ ; MS (ES Ineg.): M / z (%) = 1199.8 (100) [M - Hf.

Example 102A

Λ ^ ^; - (benzyloxycarbonyl) - [3- / ert-butyl-D-alanyl] - [3-ter ^ -butyl-L-alanyl] - [(3 _J R) -3-amino-L-phenyl alanyl)] - [(3i?) - 3-hydroxy-L-leucyl] -L-leucyl-D-arginyl-L-isoleucyl-L-allothreonyl-glycyl [(3S) -3-hydroxy-L-asparagine] -L-serine -C'-N'-lactam trifluoroacetate

To a solution of the cyclopeptide (Example 26A, 1.0 equivalents, 16.1 mg, 15 μmol), the diprop- tide acid N- (benzyloxycarbonyl) -3-tert-butyl-D-alanyl-3-ferf-butyl-L-alanine (9.4 mg, 23 μmol, 1.5 equivalents, Example 93A) and N-methylmorpholine (1.0 equivalent, 15 μmol) in dry DMF (250 μl) is initially HATU (1.6 equivalents, 9.4 mg, 25 μmol) at 0 ^° C. under argon protective gas atmosphere. given. The reaction mixture is stirred (about 15 min) and again with N-methylmorpholine (3.5 equivalents, 54 .mu.mol). The reaction mixture warmed slowly (about 12 h) to RT and then shows complete conversion of the amine component (HPLC control, method 3). The reaction mixture is admixed with solid potassium dihydrogen phosphate (10 equivalents, 150 μmol) and then concentrated under high vacuum and purified by chromatography (Method 10). 17.7 mg (74% of theory) of product are obtained.

Alternative preparation method: At 0 ⁰ C a solution of the free undecapeptide (N ² - (Benzyloxycarbonyl-fS-tert-butyl-D-alanylj-S-förf-butyl-L-alany ^^ - l ^^ - S-hydroxy -L-leucyl-L-leucyl-D-arginyl-L-isoleucyl-L-allothreonyl-glycyl - [(SjS) -3-hydroxy-L-asparaginyl] -L-seryl} - (3R) - 3-amino- L-phenylalanine bistrifluoroacetate, Example IO OA, 70 mg, 42 μmol) in DMF (30 mL) and ΝMM (32 μL, 6 equiv) with HATU (47 mg, 125 μM, 3 equiv.) Stirred to complete conversion (12 h, HPLC control) .Then the reaction mixture with potassium dihydrogen phosphate (IO equivalents, 56.6 mg), concentrated in vacuo and purified directly by preparative HPLC (Method 36). The product is obtained as a solid (74.4 mg, 73% of theory).

HPLC / UV-Vis (method 2): R, = 2.3 min.

HPLC / UV-Vis (Method 25): R, = 24.0 min, K _m (qualitative) = 210 nm (s), 255-270 (w). - ¹ HNMR (500 MHz, ^ -pyridine) δ 0.98-1.21 (m, 39H, 12 CH ₃ ) ,, 1.36 (m, IH), 1.52 (s, br, IH), 1.60 (d, J = 4.1 Hz , 3H, CH ₃ ), 1.89 (m, IH), 1.96-2.03 (m, 2H), 2.09-2.35 (m, 5H), 2.47 ("d", J = 15.3 Hz, IH), 3.15 (s, br, 2H) ₅ 3.87 ("d", J = 14.2 Hz, IH), 4.04 ("d", J = 9.5 Hz, IH), 4.17 (s, br, IH), 4.37 - ^. 55 (m, 4H), 4.66 (s, br, IH), 4.74 (q, J = 5.2 Hz, 1 H), 4.83 ("t", J = 9.3 Hz, IH), 4.91 (s, br, IH), 5.20 ( s, IH), 5.36-5.42 (m, 2H), 5.62 (d, J = 12.1 Hz, IH), 5.70 (d, J = 12.5 Hz, IH), 5.99-6.06 (m, 2H), 6.73 (s , br, 2H), 7.24 ("t", J = 7.6 Hz, IH), 7.28 (d, J = 6.8 Hz, IH), 7.34 ("t", J = 7.6 Hz, 2H), 7.40 (m, IH), 7.46 ("t", J = 7.2 Hz, 2H), 7.57 cm, 2H), 7.62 (s, br, IH), 7.68 (s, br, IH), 7.82 (s, br, IH), 7.97 ("d", J = 6.5 Hz, 2H), 8.19 (s, br, IH), 8.24-8.29 (m, 2H), 8.35 (s, IH), 8.52 (s, IH), 8.57 (m, IH), 8.63 (m, IH), 8.85 (s, br, IH), 9.05 (s, IH), 9.75 (s, IH), 11.30 (s, br, IH). For 8 protons no signals can be assigned. LC-MS (Method 7): R, = 2.1 min; MS (ESI pos.): M / z (%) = 719.8 (100) [M + 2H] ²⁺ , 1437.9 (30) [M + H] ⁺ ; MS (ES Ineg.): M / z (%) = 663.7 (100), 1435.9 (100) CM-HT ^" HR-TOF-MS (Method 1): C ₆₈ Hi ₀₉ Ni ₆ O ₁₈ [M + H] ⁺ gef. 1437.8135, ber. 1437.8101.

Example 103A

D-leucyl-N ^/ - {(? 3, S ', ^65', 125 ', 15s, 18i, 21S, ₂₄₎ S', 27S, 28 _J R) -6 - [(lS) -2-amino- 1-hydroxy-2-oxoethyl] -18- (3 - {[amino (imino) methyl] 1-amino} propyl] - 12- [(1S) -1-hydroxyethyl] -3- (hydroxymethyl) ) -24 - [(1R) -1-hydroxy-2-methylpropyl] -21-isobutyl-15-isopropyl 1-2,5,8,11,14,17,20,23,26-nonaoxo-28- phenyl 1-oxa-4,7,10,13,16,19,22,25-octaazacyclooctacosan-27-yl} -L-lencinamide bistrifluoroacetate

For the synthesis of catenosine A see WO 04/099239 Example 2A.

Exemplary embodiments

example 1

[3- / er ^ butyl-D-alanyl] - [3- / er / -butyl-L-alanyl] - [(3i?) - 3-ammo-L-phenylalanyl)] - [(3i) - 3-hydroxy-L-leucyl] -L-leucyl-D-arginyl-L-isoleucyl-L-allothreonyl-glycyl - [(35) -3-hydroxy-L-asparaginyl] -L-serine-C'-N ^ lactam bistrifluoroacetate

According to procedure 1, the N- (tert-butoxycarbonyl) -cyclopeptide (example 27A, 10 mg, 10 μmol) is reacted. After chromatographic purification by means of preparative HPLC (Method 10), freeze-drying gives 6.5 mg (64% of theory) of product.

In an alternative method, the title compound is prepared according to protocol 3 from the Cbz-protected cyclopeptide (Example 102A).

HPLC / UV-Vis (Method 3): R _t = 1.9 min, λ _max (qualitative) = 210 nm (s), 265 (m). LC-MS (Method 5): R ₁ = 4.51 min; MS (ESI pos.): M / z (%) = 652.8 (100) [M + 2H] ²⁺ , 1304.9 (10) [M + H] ⁺ .

MS (ESIneg.). M / z (%) = 650.7 (10) 1301.9 (10) [M - H] ^', 1347.9 [M - H + HCO ₂ H] - ¹ H NMR (500 MHz, ^ - Pyridine) δ = 0.82 (3H), 0.95 (3H), 0.99 (3H), 1.05 (9H), 1.07 (3H), 1.11 (9H), 1.12 (3H), 1.18 (3H), 1.32, 1.46, 1.61 ( 3H), 1.93, 1.97, 2.02, 2.03, 2.13 (2H), 2.19 (2H), 2.27, 2.32, 2.35, 2.35, 2.35, 3.18 (2H), 3.94, 4.02, 4.15, 4.31, 4.36, 4.37, 4.47, 4.50, 4.58, 4.59, 4.75, 4.99, 5.17, 5.33, 5.38, 6.00, 6.00, 6.40, 7.21 (2H), 7.45 (2HT), 7.64, 7.72, 7.73, 7.92, 8.02, 8.17, 8.23, 8.30, 8.50, 8.55, 8.85, 9.09, 29.11. No unique ¹ H signals are found for: tBuAla ¹ NH ₂ , tBuAla ² NH, HyLeu ⁴ NH, HyLeu ⁴ OH, Arg ⁶ N ^C H, Arg ⁶ N ^ζ H ₂ , α // oThr ⁸ OH, HyAsn ¹⁰ OH, Ser ¹¹ OH. ¹³ C NMR (126 MHz, ^ -pyridine) δ = 10.40, 16.00, 19.20, 19.20, 20_70, 21.30, 23.90, 24.70, 26.20, 26.50, 28.60, 29.40 (3C), 29.80 (3C), 30.90, 36.00, 41.10 , 41.30, 44.00, 44.20, 51.50, 52.80, 54.30, 55.50, 55.60, 56.60, 57.70, 58.40, 60.30, 60.60, 62.60, 63.00, 70.20, 71.90, 75.20, 128.20 (2C), 128.30, 129.10 (2C), 137.70, 158.23, 169.38, 171.04, 172.23, 172.43 (2C), 173.12, 174.04, 174.29, 174.66, 174.84, 175.41, 176.64. HR-TOF-MS (Method 1): C ₆₀ H ₁₆ O N _I03 _I6 [M + H] ⁺ Found. 1303.7719, about 1303.7733.

The structure is confirmed by a single-crystal X-ray structure analysis.

Example 2

D-Leucyl-L-leucyl - [(3i?) - 3-amino-L-phenylalanyl)] - [(3i?) -3-hydroxy-L-leucyl] -L-leucyl-D-arginyl-L-isoleucyl -L-allothreonyl-glycyl - [(3, S) -3-hydroxy-L-asparagmatyl] -I _^ -serine-C ^; ^{'7 /} -N ^5'5 lactam bistri- trifluoroacetate

Under an argon protective gas atmosphere, the Cbz-protected cyclopeptide (Example 28A, 800 μg,

0.5 .mu.mol) in methanol (1 ml) and then with 1 N aqueous hydrochloric acid (50 .mu.l) and 10%.

Palladium-carbon (1 mg). At RT and atmospheric pressure, hydrogenation is carried out (about 1 h) until analytical HPLC (method 2) indicates complete conversion. The reaction mixture is filtered (Syringe filter from. Biotage, PTFE), concentrated in vacuo and dried under high vacuum. The crude product was purified by preparative HPLC (Method 10). The product obtained is 600 μg (76% of theory) of a solid.

[αfV = -66.0 ° (c = 0.24 in methanol). HPLC / UV-Vis (Method 3): R, = 1.7 min, λ _max (qualitative) = 210 nm (s), 265 (m). LC-MS (Method 5): R, = 4.3 min; MS (ESI pos.): M / z (%) - 638.7 (100) [M + 2H] ²⁺ . MS (ES Ineg.): M / z (%) = 1274 (100) [M-H] ^" . ¹ H NMR (500 MHz, = 0.72 (3H), 0.77 (3H), 0.89 (3H), 0.96 (3H), 0.99 (3H), 1.02 (3H), 1.02 (3H), 1.08 (3H), 1.09 (3H), 1.22 (3H) , 1.29, 1.44, 1.58 (3H), 1.75, 1.99, 2.0O ₅ 2.09 (2H), 2.10, 2.14 (2H), 2.17, 2.21 (2H), 2.21, 2.29, 2.29, 2.39, 2.39, 3.18, 3.33, 4.00, 4.10, 4.21, 4.35, 4.35, 4.48, 4.53, 4.61, 4.71, 4.80, 5.06, 5.17, 5.35, 5.36, 6.04, 6.05, 6.63, 7.25 (2H), 7.50 (2H), 7.67, 7.70, 7.88, 8.07, 8.11, 8.24, 8.24, 8.26, 8.47, 8.54, 8.95, 9.23, 9.31, 11.26. No clear. ¹ H signals are found for: Leu ¹ NH ₂ , Leu ² NH, HyLeu ⁴ OH, Arg ⁶ N ^ς H, Arg ⁶ N ^ζ H ₂ , α // oThr ^s OH, HyAsn ¹⁰ OH, Ser ¹¹ OH- ¹³ C NMR (126 MHz, rf _r pyridine) δ = 16.27, 19.17, 19.90, 20.49, 20.87, 21.11, 21.70, 22.63, 23.40, 24.19, 24.81, 25.23, 25.23, 26.06, 26.61, 21.70, 28.97, 31.04, 36.52, 40.00 , 41.31, 41.94, 42.40, 45.43, 52.60, 53.26, 53.72, 56.09, 56.62, 57.94, 58.53, 58.75, 60.83, 60.83, 62.61, 63.47, 70.50, 72.11, 75.39, 128.09 (2C), 128.19, 128.99 (2C) , 138.52, 158.12, 169.39, 170.44, 172.25, 172.46, 173.13, 173.58, 174.16, 174.32, 174.59, 174.59, 174.69, 176.64. MALDI-MS: m / z (%) = 1275 (100) [M + H] ⁺ , 1297 (5) [M + Na] ⁺ , 1313 (5) [M + K] ⁺ . MALDI-MS / MS @ 1275: m / z (%) = 86 (100), 199 (50), 1049 (20). HR-TOF-MS (Method 1): C ₅₈ H ₉₉ N ₁₆ O ₁₆ [M + H] ⁺ gef. 1275.7424, about 1275.7420.

The structure is confirmed by a single-crystal X-ray structure analysis.

An amino acid analysis (Method 9) is carried out (Table A). Table A

dissolved in 20% methanol 1 mg / ml

LY

PR

JR_

Sum 1 _

Hy.d. No standard ßNH2Ph not stainable in the AA _

Hy.d. No standard _

Hy-Asn, Hy-Leu could not be quantified because no standard Hy-Asn is most likely included as Hy-Asp.

Example 3

[3- (trimethylsilyl) -D-alanyl] - [3- (pyrid-3-yl) -L-alanyl] - [(? 3i) - 3-amino-L-phenylalanyl)] - [(3i?) - 3-hydroxy-L-leucyl] -L-leucyl-D-arginyl-L-isoleucyl-L-allothreonyl-glycyl - [(3iS) -3-hydroxy-L-asparaginyl] -L-serine-C'-N ^ lactam tristrifluoracetat

According to protocol 3, the Cbz-protected cyclopeptide (example 91A, 19.9 mg, 12 μmol) is reacted. After chromatographic purification by preparative HPLC (method 21), freeze-drying gives 14.7 mg (99% of theory) of product.

HPLC / UV-Vis (Method 2): R ₄ = 1.4 min, λ _max (qualitative) = 210 nm (s), 265 (m). LC-MS (Method 5): R ₄ = 1.50 min;

MS (ESI pos.): M / z (%) = 671.0 (100) [M + 2H] ²⁺ , 1340.4 (10) [M + H] ⁺ ; MS (ES Ineg.): M / z (%) = 668.8 (100), 1338.4 (10) [M-H]. ^" HR-TOF-MS (Method 1): C ₆₀ H ₉₇ N ₁₇ O ₁₆ Si [M + H] ⁺ gef. 1340.7117, calc. 1340.7142.

The structure is confirmed by a single-crystal X-ray structure analysis.

Example 4

[3 - (^ r / -butyl) -D-alanyl] - [3- (pyrid-3-yl) -L-alanyl] - [(3R) -3-amino-L-phenylalanyl)] - [(^ ) -3-hydroxy-L-leucyl] -L-leucyl-D-arginyl-L-isoleucyl-L-allothreonyl-glycyl - [(35) -3-hydroxy-L-asparaginyl] -L-serine-C ^λ7; -N ^{J 5} -lactam tristrifluoroacetate

According to protocol 3, the Cbz-protected cyclopeptide (Example 66A, 15.0 mg, 8.89 μmol) is reacted. After chromatographic purification by means of preparative HPLC (Method 38), 11.7 mg (79% of theory) of product are obtained by freeze-drying.

Example 5

Analogously to the procedure 3, the Cbz-spiked cyclopeptide (Example 84A, 169.0 mg, 0.11 mmol) is reacted. The product is isolated after fine purification via preparative RP-HPLC (e.g., Method 35) with 140.0 mg (84% of theory) of yield.

HPLC / UV-Vis (Method 25): R, = 15.01 min.

LC-MS (method 7): R, = 1.54 min;

MS (ESI pos.): M / z (%) = 645 (100) [M + 2H] ²⁺ ,

MS (ES Ineg.): M / z (%) = 1288 (100) [M-H] ^" , 643.8 (16) [M -2H] ² \

HR-TOF-MS (Method 1): C ₆₀ H ₁₀₅ N ₁₆ Oi ₅ [M + H] ⁺ Found. 1289.7950, about 1289.7940.

Example 6

[3- (RERT-butyl) -D-alanyl] - [3- (6-methylpyrid-2-yl) -L-alanyl] - [(3i?) - 3-amino-L-phenylalanyl)] - [( - i)? -

3-hydroxy-L-leucyl] -L-leucyl-D-arginyl-L-isoleucyl-L-allothreonyl-glycyl - [(36) -3-hydroxy-L-asparaginyl] -L-serine-C 1-4 -N ^ ^ lactam tristrifluoracetat

Analogously to Working Procedure 1, the tert-butoxycarbonyl-protected cyclopeptide (Example 44A, 142.0 mg, 85.20 μmol) is reacted. The product is isolated after fine purification on the preparative RP-HPLC (Method 39) with 118.0 mg (82% of theory) yield.

HPLC / UV-Vis (method 25): R _t = 12.88 min. LC-MS (method 7): R _t = ^» 1.58 min;

MS (ESI pos.): M / z (%) = 1339 (5) [M + H] ⁺ , 670 (100) [M + 2H] ^{2 ~ * ~} , MS (ES Ineg.): M / z (%) = 1337 (100) [M-H] ^" , 668 (6) [M-2H] ² \ HR-TOF-MS (Method 1): C ₆₂ H 1 _O iN ₁₇ Oi ₆ [M + H] ⁺ Found 1338.7507 , about 1338.7529.

Example 7

[3 - (t-butyl) -D-alanyl] - [3- (6-trifluoromethyl-pyrid-3-yl) -L-alanyl] - [(3i?) - 3-amino-L-phenyl-alany I)] - [(3R) -3 -hy droxy-L-leucy1] -L-leucyl-D-arginy 1-L-isoleucy 1-L-allothreonyl-glycyl - [(3iS) -3-hydroxy- L-asparaginyl] -L-serine-C ^{λ /;} -N ^3J- lactam-bis-trifluoroacetate

Analogously to the procedure 1, the te? ~ / -Butoxycarbonyl-protected cyclopeptide (Example 55A, 180.0 mg, 0.11 mmol) is reacted. The product is isolated after fine purification via preparative RP-HPLC (Method 39) with 182.0 mg (97% of theory) yield.

HPLC / UV-Vis (method 2): R _t = 1.71 min. LC-MS (Method 29): R _t = 1.30 min;

MS (ESIpos.): M / z (%) = 1392 (10) [M + H] \ 697 (100) [M + 2H] ²⁺ , MS (ES Ineg.): M / z (%) = 1391 ( 20) [M-H] ^" , 695 (100) [M-2H] ² \ HR-TOF-MS (Method 1): C ₆₂ H ₉₇ N ₁₇ O ₁₆ F ₃ [M + H] ⁺ Ft 1392.7267, about 1392.7260.

Example 8

N ^z; - [(2i?) - 2-amino-3- (trimethylsilyl) -propyl] - [3- (tert-butyl) -L-alanyl] - [(3i?) - 3-amino-L-phenylalanyl )] - [(3R) -3-hydroxy-L-leucyl] -L-leucyl-D-arginyl-L-isoleucyl-L-allothreonyl-glycyl [(3S) -3-hydroxy-L-asparaginyl] -L- serine-C'-N-mactam tris-trifluoroacetate

Analogously to Working Procedure 3, the beazyloxycarbonyl-protected cyclopeptide (Example 8OA, 32 mg, 19.19 μmol) is reacted. The product is isolated after fine purification by preparative RP-HPLC (Method 39) with 21.3 mg (67% of theory) yield.

9

Analogously to the procedure 3, the benzyloxycarbonyl-protected cyclopeptide (example 7OA, 32.0 mg, 21.28 μmol) is reacted. The product is isolated after fine purification on the preparative RP-HPLC (Method 35) with 22.7 mg (70% of theory) yield.

HPLC / UV-Vis (Method 25): R, = 14.00 min. LC-MS (method 7): R _t = 1.63 min;

MS (ESI pos.): M / z (%) = 1289 (4) [M + H] ⁺ , 645 (100) [M + 2H] ²⁺ , MS (ES Ineg.): M / z (%) = 1287 (100) [M - Hf HR-TOF-MS (method 1). C ₅₉ H ₁₆ _1O iN Oi ₆ [M + H] ⁺ Found. 1289.7583, t> er. 1289.7576.

Example 10

[3-ter ^ -butyl-D-alanyl] - [3- (pyrid-2-yl) -L-alanyl] - [(3i?) - 3-amino-L-phenylalanyl)] - [(3i)? 3-hydroxy-L-leucyl] -L-leucyl-D-arginyl-L-isoleucyl-L-allothreonyl-glycyl - [(35) -3-hydroxy-L-asparaginyl] -L-serine-C ^{Λ; 7} -N ^5J- lactam tris trifluoroacetate

Analogously to working procedure 1, the terMSutoxycarbonyl-protected cyclopeptide (Example 45A, 105.0 mg, 63.53 μmol) is reacted. The product is isolated after fine purification on the preparative RP-HPLC (Method 35) with 84.4 mg (80% of theory) yield.

HPLC / UV-Vis (Method 25): R _t = 14.16 min. LC-MS (method 7): R ₄ = 1.30 min;

MS (ESI pos.): M / z (%) = 1325 (15) [M + H] ⁺ , 663 (100) [M + 2H] ²⁺ , MS (ES Ineg.): M / z (%) = 1323 (100) [M-H]. ^" HR-TOF-MS (Method 1): C" _{6 "} iH ₉₈ N ₁₇ Oi ₆ [M + H] ⁺ 1324.7351, calc. 1324.7372.

Example 11

[3- (3-Butyl-D-alanyl) -L-leucyl - [(3A) -3-amino-L-phenylalanyl)] - [(3i?) -3-hydroxy-L-leucyl] -L-leucyl -D-arginyl-L-isoleucyl-L-allothreonyl-glycyl - [(35) -3-hydroxy-L-asparaginyl] -L-serine-C ^{λ ;;} -

N ⁵ ^'5 lactam tris trifluoroacetate

Analogously to working procedure 1, the tert-butoxycarbonyl-protected cyclopeptide (example 5OA, 131.0 mg, 85.19 μmol) is reacted. The product is isolated after fine purification on the preparative RP-HPLC (Method 35) with 49.5 mg (38% of theory) yield.

HPLC / UV-Vis (Method 25): R ₁ = 14.56 min. LC-MS (Method 7): R, = 1.58 min;

MS (ESIpos.): M / z (%) = 1290 (5) [M + H] ⁺ , 645 (100) [M + 2H] ²⁺ , MS (ES Ineg.): M / z (%) = 1288 (100) [M-Hf, 643 (100) [M-2H] ^{2 "} . HR-TOF-MS (Method 1): C ₅₉ H ₁₀₁ Ni ₆ O ₁₆ [M + H] ⁺ gef. 1289.7567, calc. 1289.7576.

Example 12

[3-cyc / o-pentyl-D-alanyl] - [3-cjc / o-pentyl-L-alanyl] - [(3R) -3-amino-L-phenylalanyl)] - [(3i?) - 3 - hydroxy-L-leucyl] -L-leucyl-D-arginyl-L-isoleucyl-L-allothreonyl-glycyl - [(35) -3-hydroxy-L-asparaginyl] -J-L-serine-C 1-4 -N-mactam -bistrifluoracetat

Analogously to Working Procedure 1, the terf-butoxycarbonyl-protected cyclopeptide (Example 63A, 25.0 mg, 15 μmol) is reacted. The product is isolated after fine purification on the preparative RP-HPLC (Method 35) with 24.5 mg (quant.) Yield.

HPLC / UV-Vis (Method 25): R ₁ = 17.2 min.

LC-MS (method 7): R ₄ = 1.66 min;

MS (ESIpos.): M / z (%) = 664.8 (100) [M + 2H] ²⁺ , 1328 (10) [M + H] ⁺ ,

MS (ES Ineg.): M / z (%) = 1326 (100) [M-H] ^" .

HR-TOF-MS (method 1): C ₆₂ H ₁₀₃ N ₁₆ O ₆ [M + H] ⁺ gef. 1327.7761, about 1327.7733. B. Evaluation of physiological activity

The in vitro effect of the compounds according to the invention can be shown in the following assays:

Determination of the minimum inhibitory concentration (MIC);

The MIC is determined in the liquid dilution test according to the NCCLS guidelines. Overnight cultures of Staphylococcus aureus 133, Entercococcus faecahs 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 ml in isosensitization medium (Difco, 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 ⁰ C for 18-24 hours, S pneumoniae in Ge genwart 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 μg / ml.

Representative in vitro activity data for the compounds of the invention are given in Table B:

Table B

The suitability of the compounds according to the invention for the treatment of bacterial infections can be demonstrated in the following animal model ^* Systemic infection with Stapftylococcus aureus 133:

Cells of & aureus 133 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 infusion 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.

The properties of the compounds according to the invention with regard to kidney compatibility can be shown in the following animal model:

Mouse model for the determination of nephrotoxic effects:

Nephrotoxic side effects of the nonadepsipeptides are analyzed by histopathological investigations of the kidneys in mice after repeated administration of a specific dose. For this purpose, 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 toxicity 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 defined semiquantitatively for each animal as degrees of severity of 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. Further kidney changes such as tubular dilatation, necrosis and necrotic material accumulation are also listed. C ₁ embodiments of pharmaceutical compositions

The compounds according to the invention can be converted into pharmaceutical preparations as follows:

Tablet:

Composition:

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 Pvlagnesium stearate.

Tablet weight 212 mg. Diameter 8 mm, radius of curvature 12 mm.

production:

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.

Orally administrable suspension:

Composition:

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.

A single dose of 100 mg of the compound according to the invention corresponds to 10 ml of oral suspension.

production:

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. Intravenous solution:

Composition:

100-200 mg of the compound of Example 1, 15 g of polyethylene glycol 400 and 250 g of water for injection.

production:

The compound of Example 1 is dissolved in the Wassex together with polyethylene glycol 400 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

claims

1. Compound of the formula

in which

R is hydrogen or methyl,

R ^{7 is} a group of the formula

stands,

in which

the point of attachment to the amine means

R ^{1 is} C ₁ -C ₆ -alkyl, C ₂ -C ₆ -alkenyl, C ₃ -C ₆ -cycloalkyl or Q-Cio-aryl,

where alkyl, alkenyl, cycloalkyl and aryl may be substituted by 0, 1, 2 or 3 substituents independently selected from the group consisting of halogen, hydroxy, amino, cyano, trimethylsilyl, Ci-Cβ alkyl, C _r C ₆ -alkoxy, benzyloxy, C ₃ -C ₆ cycloalkyl, C ₆ - C ₁₀ aryl, 5- to 7-membered heterocyclyl, 5- to 10-membered heteroaryl, C] -C ₆ - alkylamino, C ₆ -Cio arylamino, Ci-C _ό alkylcarbonylamino, C ₆ -C ₀ -

Arylcarbonyl-amino, Cj-C _ö alkylcarbonyl, Ci-C ₆ alkoxycarbonyl, C ₆ -C ₀ - arylcarbonyl, and benzyloxy-carbonylamino,

in which cycloalkyl, aryl, heterocyclyl and heteroaryl may in turn be substituted by 0, 1, 2 or 3 substituents independently of one another selected from the group consisting of halides, hydroxy, amino, cyano, nitro, trifluoromethyl, C ₁ -C ₆ - Alkyl, C 1 -C ₆ -alkoxy, phenyl and 5- to 7-membered heterocyclyl,

R ² Ci-C ₆ alkyl, C ₂ -C ₆ alkenyl, C ₃ -C ₆ -cycloalkyl or C ₆ -C] ₀ aryl,

wherein alkyl, alkenyl, cycloalkyl and aryl may be substituted with 0, 1, 2 or 3 substituents independently selected from

A group consisting of halogen, hydroxy, amino, cyano, trimethylsilyl, C 1 -C ₆ -alkyl, C 1 -C ₆ -alkoxy, benzyloxy, C ₃ -C ₆ -cycloalkyl, C ₆ -C ₁₀ -aryl, 5-bis 7-membered heterocyclyl, 5- to 10-membered heteroaryl Ci-C ₆ - alkylamino, C ₆ -Cio arylamino, Ci-C ₆ alkylcarbonylamino, C ₆ -C ₀ - arylcarbonyl-amino, C] _-C6 - Alkylcarbonyl, C] -C ₆ -alkoxycarbonyl, yl, C ₆ -C] ₀ -

Arylcarbonyl and benzyloxycarbonylamino,

in which cycloalkyl, aryl, heterocyclyl and heteroaryl in turn may be substituted by 0, 1, 2 or 3 substituents independently of one another selected from the group consisting of halo, hydroxy, amino, cyano, nitro, trifluoromethyl, C ₁ -C ₆ -alkyl,

_Cj-C6 alkoxy, phenyl and 5- to 7-membered heterocyclyl,

R ⁴ Ci-C ₆ alkyl, C ₂ -C ₆ alkenyl, C ₃ -C ₆ -C ₆ -cycloalkyl or represents C ₀ aryl,

where alkyl, alkenyl, cycloalkyl and aryl may be substituted by 0, 1, 2 or 3 substituents independently of one another selected from the group consisting of halogen, hydroxyl, amino, cyano, trimethylsilyl,

C _r C ₆ alkyl, C _r C ₆ -alkoxy, benzyloxy, C ₃ -C _r cycloalkyl, C ₆ -C ₀ aryl, 5- to 7-membered heterocyclyl, 5- to 10-membered heteroaryl, C ₆ - Alkylamino, Cβ-Cio-arylamino, Ci-C _ό alkylcarbonylamino, C ₆ -C] ₀ - arylcarbonyl-amino, Cj-Ce-alkylcarbonyl, Ci-C ₆ alkoxycarbonyl, C ₆ arylcarbonyl, and -CiO- benzyloxy-carbonylamino,

in which cycloalkyl, aryl, heterocyclyl and heteroaryl may in turn be substituted by 0, 1, 2 or 3 substituents independently of one another selected from the group consisting of halides, hydroxy, amino, cyano, nitro, trifluoromethyl, C _r C ₆ - Alkyl, C ₁ -C ₆ -alkoxy, phenyl and 5- to 7-membered heterocyclyl,

R ^{5 is} C ₁ -C ₅ - alkyl, C ₂ -C ₆ alkenyl, C ₃ -C ₆ -C ₆ -cycloalkyl or represents C ₀ aryl,

wherein .Alkyl, alkenyl, cycloalkyl and aryl may be substituted with 0, 1, 2 or 3 substituents independently selected from the group consisting of halogen, hydroxy, amino, cyano, trimethylsilyl, C, -C ₆ alkyl, Ci-C ₆ -alkoxy, benzyloxy, C ₃ -C ₆ cycloalkyl, C ₆ -C ₀ aryl, 5- to 7-membered heterocyclyl, 5- to 10-membered heteroaryl, C ₆ - alkylamino, C ₆ -C ₀ arylamino, Ci-Cό alkylcarbonylamino, C ₆ -C ₀ - arylcarbonyl-amino, CpCβ-alkylcarbonyl, Ci-C ₆ alkoxycarbonyl, C ₆ arylcarbonyl, and -CiO- benzyloxy-carbonylamino,

in which cycloalkyl, aryl, heterocyclyl and heteroaryl may in turn be substituted by 0, 1, 2 or 3 substituents independently of one another selected from the group consisting of halides, hydroxy, amino, cyano, nitro, trifluoromethyl, C 1 -C 6 -alkyl -. Ci-C _ö alkoxy, phenyl and 5- to 7-membered heterocyclyl,

or one of its salts, its solvates or the solvates of its salts.

Connection according to claim 1, characterized in that

R ^{6 is} methyl,

R ^{7 is} a group of the formula

stands,

in which

* means the point of attachment to the amine,

R ¹ 2-methylprop-1-yl, 2,2-dimethylprop-1-yl, 2,2-dimethylbut-1-yl, trimethylsilylmethyl, 1-hydroxy-2-methylprop-1-yl, 1-hydroxy 2,2-dimethylprop-1-yl, 1-hydroxy-2,2-dimethyl-1-but-1-yl, 1-hydroxy-2-ethyl-1-methylbut-1-yl, 1-hydroxy-2 , 2-diethylbut-1-yl, phenylmethyl, 1-hydroxy-1-phenylmethyl, 2-pyridylmethyl or 3-pyridylmethyl,

where 2-pyridylmethyl or 3-pyridylmethyl can be substituted by 0, 1, 2 or 3 substituents selected independently of one another from the group consisting of hydroxyl, amino, trifluoromethyl, methyl, methoxy and morpholinyl,

R ⁴ 2-methylprop-1-yl, 2,2-dimethylprop-1-yl, 2,2-dimethylbut-1-yl, trimethylsilylmethyl, 1-hydroxy-2-methylprop-1-yl, 1-hydroxy 2,2-dimethylprop-1-yl, 1-hydroxy-2,2-dimethylbut-1-yl, 1-hydroxy-2-ethyl-2-methylbut-1-yl, 1-hydroxy-2,2- diethylbut-1-yl, phenylmethyl, 1-hydroxy

1-phenylmethyl, 2-pyridylmethyl or 3-pyridylmethyl,

where 2-pyridylmethyl or 3-pyridylmethyl can be substituted by 0, 1, 2 or 3 substituents independently of one another selected from the group consisting of hydroxyl, amino, trifluoromethyl, methyl, methoxy and morpholinyl, R ^{5 is} 2-methylprop-1-yl, 2,2-dimethylprop-1-yl, 2,2-dimethylbut-1-yl, trimethylsilylmethyl, 1-hydroxy-2-methylprop-1-yl, 1-hydroxy 2,2-dimethylpropyl-1-yl, 1-hydroxy-2,2-dimethylbut-1-yl, 1-hydroxy-2-ethyl-1,2-methylbut-1-yl, 1-hydroxy-2, 2-diethylbut-1-yl, phenylmethyl, 1-hydroxy-1-phenylmethyl, 2-pyridylmethyl or 3-pyridylmethyl,

where 2-pyridylmethyl or 3-pyridylmethyl can be substituted by 0, 1,

2 or 3 substituents independently aus¬ selected from the group consisting of hydroxy, amino, trifluoromethyl, methyl, methoxy and morpholinyl,

or one of its salts, its solvates or the solvates of its salts.

3. A compound according to claim 1 or 2, characterized in that

R ^{6 is} methyl,

R ^{7 is} a group of the formula

stands,

in which

* means the point of attachment to the amine,

R ^{1 is} 2-methylprop-1-yl, 2,2-dimethyl-prop-1-yl, trimethylsilylmetyl or 3-pyridylmethyl,

R ^{2 is} 2-methylprop-1-yl, 2,2-dimethylprop-1-yl, trimethylsilylmetyl or 3-pyridylmethyl, where 3-pyridylmethyl can be substituted by 0, 1 or 2 substituents independently of one another selected from the group consisting of hydroxyl, amino, trifluoromethyl, methyl, IVΪethoxy and morpholinyl,

^' R ^{4 is} 2-methylprop-1-yl, 2,2-dimethylprop-1-yl, trimethylsilylmethyl or 3-

Pyridylmethyl means

or one of its salts, its solvates or the solvates of its salts.

4. A process for the preparation of a compound of formula (Ib) according to claim 1, characterized in that

[A] a compound of the formula

in which

R ^{6 has} the meaning given in claim 1,

first with a compound of the formula

in which

R, R, R and R have the meaning given in claim 1,

R ^{3 is} tert-butoxycarbonyl or benzyloxycarbonyl, and

X ^{1 is} halogen, preferably bromine, chlorine or fluorine, or hydroxy,

and then reacted with an acid and / or by hydrogenolysis,

or

[B] a compound of the formula

in which

R ^{6 has} the meaning given in claim 1,

first with a compound of the formula

in which

R ¹ , R ² , R ⁴ and R ^{5 have} the meaning given in claim 1,

and then in a 4-step synthesis

a) with a fluoride reagent,

b) with an acid,

c) with a dehydration reagent, optionally in the presence of a base, and d) by hydrogenolysis

is implemented.

5. A compound according to any one of claims 1 to 3 for the treatment and / or prophylaxis of diseases.

6. Use of a compound according to any one of claims 1 to 3 for the manufacture of a medicament for the treatment and / or prophylaxis of diseases.

7. Use of a compound according to any one of claims 1 to 3 for the manufacture of a medicament for the treatment and / or prophylaxis of bacterial infections.

8. A pharmaceutical composition comprising a compound according to any one of claims 1 to 3 in combination with an inert, non-toxic, pharmaceutically suitable excipient.

9. Medicament according to claim 8 for the treatment and / or prophylaxis of bacterial infections.

10. 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 3, a drug according to claim 8 or a drug according to claim 6 or 7 An¬ obtained drug.

11. Process for the preparation of the compound of the formula 2- (trimethylsilyl) ethyl- (3i?) - 3-amino-N ² - [(benzyloxy) carbonyl] -L-phenylalaninate trifluoroacetate (Example 1 SA)

characterized in that in a 6-step synthesis, the compound of formula

(Rac) -3 - [(tert-butoxycarbonyl) amino] -3-phenylpropansäuremethylester

a) in the presence of a base, with dibenzyl azadicarboxylate,

b) with N, N, N, N, -tetramethylguanidine,

c) with hydrogen in the presence of Raney-Νickel,

d) with N-benzyloxycarbonyloxysuccinimide ester in the presence of a base,

e) with a base and subsequent chromatographic enantiomer separation

and

f) with trifluoroacetic acid in dichloromethane or hydrogen chloride in dioxane

is implemented.

12. Compound of the formula

(3i?) - N ² - [(benzyloxy) carbonyl] -3 - [(rf-butoxycarbonyl) amino] -L-phenylalanine (Example 17A and Example 35A)

or

2- (trimethylsilyl) ethyl (3i?) - N - [(benzyloxy) carbonyl] -3 - [(ferr-butoxycarbonyl) ammo] -L-phenylalaninate (Example 18A)

or

2- (trimethylsilyl) ethyl (3R) -3-amino-N ² - [(benzyloxy) carbonyl] -L-ph-enylalaninate trifluoroacetate (Example 19A)

or

(3 <S) -N ² - [(Benzyloxy) carbonyl] -3 - [(but-butoxycarbonyl) amino] -D-phenylalanine (Example 36A)

or

2- (trimethylsilyl) ethyl (35) -N - [(benzyloxy) carbonyl] -3 - [(tert-butoxycarbonyl) aynino] -D-phenylalaninate (Example 37A)

or 2- (trimethylsilyl) ethyl (3iS) -3-amino-N ² - [(ben2yloxy) carbonyl] -D-phenylalaninate trifluoroacetate (Example 38A)