Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
  • C07K7/04—Linear peptides containing only normal peptide links
  • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P5/00—Drugs for disorders of the endocrine system
  • A61P5/10—Drugs for disorders of the endocrine system of the posterior pituitary hormones, e.g. oxytocin, ADH
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
  • C07K5/02—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link
  • C07K5/0207—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link containing the structure -NH-(X)4-C(=0), e.g. 'isosters', replacing two amino acids
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
  • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
  • C07K5/08—Tripeptides
  • C07K5/0827—Tripeptides containing heteroatoms different from O, S, or N
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• the present invention relates to novel compounds, compositions containing them, and their use for treating medical disorders resulting from a deficiency in growth hormone.
• Growth hormone is a hormone which stimulates growth of all tissues capable of growing.
• growth hormone is known to have a number of effects on metabolic processes, e.g., stimulation of protein synthesis and free fatty acid mobilization and to cause a switch in energy metabolism from carbohydrate to fatty acid metabolism.
• Deficiency in growth hormone can result in a number of severe medical disorders, e.g., dwarfism.
• Growth hormone is released from the pituitary. The release is under tight control of a number of hormones and neurotransmitters either directly or indirectly. Growth hormone release can be stimulated by growth hormone releasing hormone (GHRH) and inhibited by somatostatin. In both cases the hormones are released from the hypothalamus but their action is mediated pri- marily via specific receptors located in the pituitary. Other compounds which stimulate the release of growth hormone from the pituitary have also been described.
• GHRH growth hormone releasing hormone
• somatostatin somatostatin
• PACAP pituitary adenylyl cy- clase activating peptide
• muscarinic receptor agonists and a synthethic hexapeptide
• GHRP growth hormone releasing peptide
• the composition of growth hormone releasing compounds is important for their growth hormone releasing potency as well as their bioavailability. It is therefore an object of the present invention to provide novel compounds with growth hormone releasing properties. Moreover it is an object to provide growth hormone releasing compounds with no or substantially no side- effects. It is also an object to provide compounds which have good oral bioavailability.
• growth hormone releasing compounds can be utilised in vitro as unique research tools for understanding, inter alia, how growth hormone secretion is regulated at the pituitary level.
• the growth hormone releasing compounds of the present invention can also be administered in vivo to increase growth hormone release.
• the present invention relates to a compound of general formula
• R 3 , R 4 , R 5 , X, Y, M, T and Q are as defined below, or a pharmaceutically acceptable salt thereof, having growth hormone releasing properties.
• the present invention relates to a compound of general formula
• R 3 , R 4 and R 5 independently of each other are hydrogen, or C ⁇ alkyl optionally substitued with d alkyl,
• X is aryl optionally substituted with halogen, C 6 alkyl or phenyl,
• Y is aryl or hetaryl optionally substituted with halogen, ⁇ alkyl, C.,- 6 alkoxy or phenyl,
• R 1 and R 12 independently of each other are hydrogen, C ⁇ alkyl optionally substituted with halogen, amino, hydroxyl, C ⁇ alkyl, C ⁇ alkoxy or phenyl; or benzyl, optionally substituted with halogen, amino, hydroxyl, C ⁇ alkyl, C ⁇ alkoxy or phenyl,
• Q is hydrogen, Q 1 , Q 2 or Q 3 ,
• R 5 cannot be hydrogen
• M cannot be M 1 , or
• R 3 , R 4 and R 5 independently of each other are hydrogen, or C ⁇ alkyl optionally substitued with
• X is aryl optionally substituted with halogen, C ⁇ alkyl or phenyl
• Y is aryl or hetaryl optionally substituted with halogen, C 1-6 alkyl, C ⁇ alkoxy or phenyl
• 6 alkyl d. 6 alkyl optionally substituted with halogen, amino, hydroxyl, C ⁇ alkyl, C ⁇ alkoxy or phenyl; or benzyl, optionally substituted with halogen, amino, hydroxyl, C,. 6 alkyl, C ⁇ alkoxy or phenyl,
• T is hydrogen, T 1 , T 2 or T 3 , wherein T 1 is -(CH 2 ) n -NH 2 , wherein n is 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10,
• R 1 and R 12 independently of each other are hydrogen, C
• Q is hydrogen, Q 1 , Q 2 or Q 3 ,
• R 5 cannot be hydrogen
• the compounds of formula I comprise any optical isomers thereof, in the form of separated, pure or partially purified optical isomers or racemic mixtures thereof. For instance, whenever one or more chiral carbonatoms are present such chiral center(s) may be in the R- or S- configuration, or a mixture of R and S.
• hetaryl is connected through a meth- ylen group to a chiral carbonatom, the hetaryl-CH 2 -moiety being in either R- or S- configuration, preferably S-configuration.
• R 16 and R 17 are independently of each other C ⁇ alkyl, preferably methyl, and in a particular embodiment R 16 and R 17 are both methyl.
• D 1 is preferably phenylene, which may be attached to the carbonyl group through any of its five ring positions, the 3- position being preferred.
• M ⁇ R 6 and R 7 are independently of each other hydrogen or C ⁇ alkyl, preferably hydrogen or methyl.
• M 4 is M 3 .
• R 6 and R 7 are both hydrogen or one is hydrogen and the other is d- ⁇ alkyl, preferably methyl.
• R 6 and R 7 are independently of each other hydrogen or C ⁇ alkyl, preferably hydrogen or ethyl.
• A is a saturated heterocyclic ring containing 5 ring members, one ring member being a nitrogen atom (N), and most preferred A is pyrrolidi- nyl, such as pyrrolidin-2-yl in the S-configuration.
• R 3 , R 4 and R 5 independently of each other are hydrogen or C ⁇ . 6 alkyl, preferably hydrogen or methyl.
• R 3 is methyl, R 4 is methyl and R 5 is methyl, or R 3 is methyl, R 4 is methyl and R 5 is hydrogen, or R 3 is hydrogen, R 4 is methyl and R 5 is hydrogen, or R 3 is hydrogen, R 4 is hydrogen and R 5 is hydrogen.
• X is aryl optionally substituted with d. 6 alkyl or phenyl.
• X is naphthyl, such as 1-naphthyl or 2-naphthyl, or phenyl substituted with C ⁇ alkyl or phenyl, preferably naphthyl or phenyl substituted with phenyl, which C ⁇ alkyl or phenyl may be attached to phenyl through any one of the five ring positions, the 4-position being preferred.
• Especially preferred X is 2-naphthyl or biphenyl-4-yl.
• aryl such as phenyl or naphthyl
• aryl-CH 2 -moiety being in either R- or S-configuration, preferably R- configuration.
• Y is hetaryl or phenyl optionally substituted with halogen, such as chloro or fluoro.
• Y is phenyl optionally substituted with halogen, such as fluoro; or thienyl.
• Y is thienyl it may be attached to the methylene group through any one of its four ring positions, the 2-position being preferred.
• Especially preferred Y is phenyl, 2-thienyl or 4-flouro-phenyl.
• hetaryl or phenyl is connected through a methylene group to a chiral carbonatom, the (hetaryl or phenyl)-CH 2 -moiety being in either R- or S-configuration, preferably R-configuration.
• R 13 is preferably C 1 . 4 alkyl, such as methyl.
• n is preferably 1 , 2, 3, 4 or 5.
• R 8 and R 9 are independently of each other C M alkyl, preferably methyl, and n is 1 , 2, 3, 4, 5 or 6. In particular when T is T 2 , n is 1 , 2 or 3.
• n is preferably 1 , 2, 3, 4 or 5, most preferred 3 or 4.
• (2E)-5-Amino-5-methylhex-2-enoic acid N-((1 R)-1 - ⁇ N-[(1 R)-1 -(((1 S)-5-amino-1 - carbamoylpentyl)carbamoyl)-2-(2-thienyl)ethyl]-N-methylcarbamoyl ⁇ -2-(2- naphthyl)ethyl ⁇ amide
• (2E)-5-Amino-5-methylhex-2-enoic acid N-((1 R)-1 - ⁇ N-[(1 R)-1 -(((1 S)-5-acetylamino-1 - carbamoylpentyl)carbamoyl)-2-phenylethyl]-N-methylcarbamoyl ⁇ -2-(biphenyl-4- yl)ethyl)amide
• (2E)-5-Amino-5-methylhex-2-enoic acid N-((1 R)-1- ⁇ N-[(1 R)-1-((1S)-5-acetylamino-1- carbamoylpentylcarbamoyl)-2-(2-thienyl)ethyl]-N-methylcarbamoyl ⁇ -2-(2- naphthyl)ethyl ⁇ amide
• (2E)-5-Amino-5-methylhex-2-enoic acid N-((1 R)-1 - ⁇ N-[(1 R)-1 -(3-aminopropylcarbamoyl)-2- phenylethyl]-N-methylcarbamoyl ⁇ -2-(biphenyl-4-yl)ethyl)-N-methylamide
• (2E)-5-Amino-5-methylhex-2-enoic acid ((1 R)-1- ⁇ N-[(1 R)-1-(5-aminopentylcarbamoyl)-2- phenylethyl]-N-methylcarbamoyl ⁇ -2-(2-naphthyl)ethyl)amide,
• (2E)-5-Amino-5-methylhex-2-enoic acid N-((1 R)-1 - ⁇ N-[(1 R)-1 -(5-aminopentylcarbamoyl)-2- phenylethyl]-N-methylcarbamoyl ⁇ -2-(2-naphthyl)ethyl)-N-methylamide, N-((1 R)-1- ⁇ N-[(1 R)-1-(4-Aminobutylcarbamoyl)-2-phenylethyl]-N-methylcarbamoyl ⁇ -2- (biphenyl-4-yl)ethyl)-3-aminomethyl-N-methylbenzamide,
• the compounds of formula I exhibit an improved resistance to proteolytic degradation by enzymes compared to that of the peptides suggested in the prior literature, due to the lack of natural peptide bonds.
• the increased resistance to proteolytic degradation of the compounds of the invention in comparison with known growth hormone releasing peptides is expected to improve their bioavailability compared to that of the peptides suggested in the prior literature.
• the d-e-alkyl, C ⁇ s-alkylene, C ⁇ -alkyl or C ⁇ -alkylene groups specified above are intended to include those alkyl or alkylene groups of the designated length in either a linear or branched or cyclic configuration.
• linear alkyl are methyl, ethyl, propyl, butyl, pentyl, and hexyl and their corresponding divalent moieties, such as ethylene.
• Examples of branched alkyl are isopropyl, sec-butyl, tert-butyl, isopentyl, and isohexyl and their corresponding divalent moieties, such as isopropylene.
• cyclic alkyl examples include Cs ⁇ -cycloalkyl such as cyclopropyl, cy- clobutyl, cyclopentyl and cyclohexyl and their corresponding divalent moieties, such as cyclo- propylene.
• the d.g-alkoxy groups specified above are intended to include those alkoxy groups of the designated length in either a linear or branched or cyclic configuration.
• linear alkoxy are methoxy, ethoxy, propoxy, butoxy, pentoxy, and hexoxy.
• branched alkoxy are isopropoxy, sec-butoxy, tert-butoxy, isopentoxy, and isohexoxy.
• cyclic alkoxy are cyclopropyloxy, cyclobutyloxy, cyclopentyloxy and cyclohexyloxy.
• aryl is intended to include monovalent carbocyclic aromatic ring moieties, being either monocyclic, bicyclic or polycyclic, e.g. selected from the group con- sisting of phenyl and naphthyl, optionally substituted with one or more C ⁇ -alkyl, C ⁇ -alkoxy, halogen, amino or aryl.
• arylene is intended to include divalent carbocyclic aromatic ring moieties, being either monocyclic, bicyclic or polycyclic, e.g. selected from the group con- sisting of phenylene and naphthylene, optionally substituted with one or more C ⁇ -alkyl, C ⁇ . 6 - alkoxy, halogen, amino or aryl.
• heterocyclic aro- matic ring moieties being either monocyclic, bicyclic or polycyclic, e.g. selected from the group consisting of pyridyl, 1-H-tetrazol-5-yl, thiazolyl, imidazolyl, indolyl, pyrimidinyl, thiadiazolyl, pyrazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thienyl, quinolinyl, pyrazinyl, or isothiazolyl, optionally substituted by one or more C ⁇ . 6 -alkyl, C ⁇ -alkoxy, halogen, amino or aryl.
• heterocyclic aromatic ring moieties being either monocyclic, bicyclic or polycyclic, e.g. selected from the group consisting of pyridinediyl, 1-H-tetrazolediyl, thiazoldiyl, imidazolediyl, indolediyl, pyrimidinediyl, thiadiazolediyl, pyrazolediyl, oxazolediyl, isoxazolediyl, oxadiazolediyl, thiophenediyl, quinolin- ediyl, pyrazinediyl, or isothiazolediyl, optionally substituted by one or more C ⁇ -alkyl, C-,. 6 - alkoxy, halogen, amino or aryl.
• halogen is intended to include chlorine (Cl), fluorine (F), bromine (Br) and iodine (I).
• the compounds of the present invention may have one or more asymmetric centres and it is intended that stereoisomers, as separated, pure or partially purified stereoisomers or racemic mixtures thereof are included in the scope of the invention.
• the compounds of the present invention may optionally be on a pharmaceutically acceptable salt form such as the pharmaceutically acceptable acid addition salts of compounds of formula I which include those prepared by reacting the compound of formula I with an inorganic or organic acid such as hydrochloric, hydrobromic, sulfuric, acetic, phosphoric, lactic, maleic, man- delic phthalic, citric, glutaric, gluconic, methanesulfonic, salicylic, succinic, tartaric, toluenesul- fonic, trifluoracetic, sulfamic or fumaric acid.
• an inorganic or organic acid such as hydrochloric, hydrobromic, sulfuric, acetic, phosphoric, lactic, maleic, man- delic phthalic, citric, glutaric, gluconic, methanesulfonic, salicylic, succinic, tartaric, toluenesul- fonic, trifluoracetic, sulfamic or fum
• the compounds of formula I may be administered in pharmaceutically acceptable acid addition salt form or, where appropriate, as a alkali metal or alkaline earth metal or lower alkylammo- nium salt. Such salt forms are believed to exhibit approximately the same order of activity as the free base forms.
• the present invention relates to a pharmaceutical composition comprising, as an active ingredient, a compound of the general formula I or a pharmaceutically acceptable salt thereof together with a pharmaceutically acceptable carrier or diluent.
• compositions containing a compound of the present invention may be prepared by conventional techniques, e.g. as described in Remington's Pharmaceutical Sciences. 1985 or in Remington: The Science and Practice of Pharmacy, 19th Edition (1995).
• the compositions may appear in conventional forms, for example capsules, tablets, aerosols, solutions, suspensions or topical applications.
• the pharmaceutical carrier or diluent employed may be a conventional solid or liquid carrier.
• solid carriers are lactose, terra alba, sucrose, cyclodextrin, talc, gelatin, agar, pectin, acacia, magnesium stearate, stearic acid or lower alkyl ethers of cellulose.
• liquid carriers are syrup, peanut oil, olive oil, phospholipids, fatty acids, fatty acid amines, polyoxyethylene or water.
• the carrier or diluent may include any sustained release material known in the art, such as glyceryl monostearate or glyceryl distearate, alone or mixed with a wax.
• the preparation may be tabletted, placed in a hard gelatin capsule in powder or pellet form or it can be in the form of a troche or lozenge.
• the amount of solid carrier will vary widely but will usually be from about 25 mg to about 1 g.
• the preparation may be in the form of a syrup, emulsion, soft gelatin capsule or sterile injectable liquid such as an aqueous or non-aqueous liquid suspension or solution.
• a typical tablet which may be prepared by conventional tabletting techniques may contain:
• the preparation may contain a compound of formula I dissolved or suspended in a liquid carrier, in particular an aqueous carrier, for aerosol application.
• a liquid carrier in particular an aqueous carrier
• the carrier may contain additives such as solubilizing agents, e.g. propylene glycol, surfactants, absorption enhancers such as lecithin (phosphatidylcholine) or cyclodextrin, or preservatives such as parabenes.
• the compounds of the present invention are dispensed in unit dosage form com- prising 50-200 mg of active ingredient together with a pharmaceutically acceptable carrier per unit dosage.
• the present invention relates to a pharmaceutical composition for stimulating the release of growth hormone from the pituitary, the composition comprising, as an active ingredient, a compound of the general formula I or a pharmaceutically acceptable salt thereof together with a pharmaceutically acceptable carrier or diluent.
• the present invention relates to a method of stimulating the release of growth hormone from the pituitary, the method comprising administering to a subject in need thereof an effective amount of a compound of the general formula I or a pharmaceutically acceptable salt thereof.
• the present invention relates to the use of a compound of the general formula I or a pharmaceutically acceptable salt thereof for the preparation of a medicament for stimulating the release of growth hormone from the pituitary.
• growth hormone in humans are varied and multitudinous.
• compounds of formula I can be administered for purposes stimulating release of growth hormone from the pituitary and would then have similar effects or uses as growth hormone itself.
• the uses of growth hormone may be summarized as follows: stimulation of growth hormone release in the elderly, prevention of catabolic side effects of glucocorticoids, prevention and treatment of osteoporosis, treatment of chronic fatigue syndrom (CFS), treatment of acute fatigue syndrom and muscle loss following election surgery, stimulation of the immune system, acceleration of wound healing, accelerating bone fracture repair, accelerating complicated fractures, e.g.
• disctraction osteogenesis treatment of wasting secondary to fractures, treatment of growth retardation, treating growth retardation resulting from renal failure or insufficiency, treatment of cardiomyopathy, treatment of chronic liver disease, treatment of thrombocytopenia, treatment of Crohn's disease, treatment of short bowel syndrome, treatment of chronic obstructive pulmonary disease (COPD), treatment of complications associated with transplantation, treatment of physiological short stature including growth hormone deficient children and short stature associated with chronic illness, treatment of obesity and growth retardation associated with obesity, treatment of anorexia, treating growth retardation associated with the Prader-Willi syndrome and Turner's syndrome; accelerating the recovery and reducing hospitalization of burn patients; treatment of intrauterine growth retardation, skeletal dysplasia, hypercortisolism and Cushing's syndrome; induction of pulsatile growth hormone release; replacement of growth hormone in stressed pa- tients, treatment of osteochondrodysplasias, Noonan's syndrome, schizophrenia, depressions, Alzheimer's disease, delayed wound healing and psychosocial
• the compounds of formula I may be used in the treatment of insulin resistance, including NIDDM, in mammals, e.g. humans. It is furthermore believed that the present compounds of formula I may improve sleep quality and correct the relative hy- posomatotropism of senescence due to high increase in REM sleep and a decrease in REM latency.
• the dosage will vary depending on the compound of formula I employed, on the mode of administration and on the therapy desired.
• An effective amount of the compounds according to this invention will depend, in part, on the particular condition to be treated, age, weight, and general health of the patient, and other factors evident to those skilled in the art.
• dosage levels between 0.0001 and 100 mg/kg body weight daily are administered to patients and animals to obtain effective release of endogenous growth hormone.
• the compounds of formula I have no or substantially no side-effects, when administered in the above dosage levels, such side-effects being e.g. release of cortisol, LH and/or prolactin.
• dosage forms suitable for oral, nasal, pulmonal or transdermal administration comprise from about 0.0001 mg to about 100 mg, preferably from about 0.001 mg to about 50 mg of the compounds of formula I admixed with a pharmaceutically acceptable carrier or diluent.
• the pharmaceutical composition of the invention may comprise a compound of formula I combined with one or more compounds exhibiting a different activity, e.g., an antibiotic or other pharmacologically active material.
• the route of administration may be any route which effectively transports the active compound to the appropriate or desired site of action, such as oral, nasal, pulmonary, transdermal or parenteral, the oral route being preferred.
• Compounds of formula I may also be useful in vivo tools for evaluating the growth hormone releasing capability of the pituitary. For example, serum samples taken before and after administration of these compounds to humans can be assayed for growth hormone. Comparison of the growth hormone in each serum sample would directly determine the ability of the patients pituitary to release growth hormone.
• Compounds of formula I may be administered to commercially important animals to increase their rate and extent of growth, and to increase milk production.
• growth hormone secretagogue compounds of formula I is in combination with other secretagogues such as GHRP (2 or 6), GHRH and its analogues, growth hormone and its analogues or somatomedins including IGF-1 and IGF-2.
• Compounds of formula I may be evaluated in vitro for their efficacy and potency to release growth hormone in rat pituitary primary cultures, and such evaluation may be performed as de- scribed below.
• rat pituitary cells The isolation of rat pituitary cells is a modification of O. Sartor et al., Endocrinology 116. 1985, pp. 952-957.
• Male albino Sprague-Dawley rats 250 +/- 25 grams were purchased from M ⁇ llegaard, Lille Skensved, Denmark. The rats were housed in group cages (four ani- mals/cage) and placed in rooms with 12 hour light cycle. The room temperature varied from 19-24°C and the humidity from 30 - 60%.
• the rats were decapitated and the pituitaries dissected.
• the neurointermediate lobes were removed and the remaining tissue was immediately placed in icecold isolation buffer (Gey's me- dium (Gibco 041-04030) supplemented with 0.25% D-glucose, 2% non-essential amino acids (Gibco 043-01140) and 1% bovine serum albumine (BSA) (Sigma A-4503)).
• the tissue was cut into small pieces and transferred to isolation buffer supplemented with 3.8 mg/ml of trypsin (Worthington #3707 TRL-3) and 330 mg/ml of DNase (Sigma D-4527).
• This mixture was incubated at 70 rotations/min for 35 min at 37°C in a 95/5% atmosphere of O 2 /CO 2 .
• the tissue was then washed three times in the above buffer. Using a standard pasteur pipette, the tissue was then aspirated into single cells. After dispersion, cells were filtered through a nylon filter (160 mm) to remove undigested tissue.
• the cell suspension was washed 3 times with isolation buffer supplemented with trypsin inhibitor (0.75 mg/ml, Worthington #2829) and finally resuspended in culture medium; DMEM (Gibco 041-01965) supplemented with 25 mM HEPES (Sigma H-3375), 4 mM glutamine (Gibco 043-05030H), 0.075% sodium bicarbonate (Sigma S- 8875), 0.1% non-essential amino acid, 2.5% fetal calf serum (FCS, Gibco 011-06290), 3% horse serum (Gibco 034-06050), 10% fresh rat serum, 1 nM T 3 (Sigma T-2752) and 40 mg/l dexamethasone (Sigma D-4902) pH 7.3, to a density of 2 x 10 5 cells/ml.
• the cells were seeded into microtiter plates (Nunc, Denmark), 200 ml/well, and cultured for 3 days at 37°C and 8% CO 2 .
• the cells were washed twice with stimulation buffer (Hanks Balanced Salt Solution (Gibco 041-04020) supplemented with 1% BSA (Sigma A-4503), 0.25% D-glucose (Sigma G-5250) and 25 mM HEPES (Sigma H-3375) pH 7.3) and preincubated for 1 hour at 37°C.
• the buffer was exchanged with 90 ml stimulation buffer (37°C).
• Ten ml test compound solution was added and the plates were incubated for 15 min at 37°C and 5% CO 2 .
• the medium was de- canted and analyzed for GH content in an rGH SPA test system.
• Standard peptides (angiotensin 1-14, ACTH 4-10 and glucagon) were purchased from Sigma, MO, USA)
• Enzymes (trypsin, chymotrypsin, elastase aminopeptidase M and carboxypeptidase Y and B) were all purchased from Boehringer Mannheim GmbH (Mannheim, Germany)
• Pancreatic enzyme mix trypsin, chymotrypsin and elastase in 100 mM ammoniumbicarbonate pH 8.0 (all concentrations 0.025 mg/ml).
• Carboxypeptidase mix carboxypeptidase Y and B in 50 mM ammoniumacetate pH 4.5 (all concentrations 0.025 mg/ml).
• Aminopeptidase M solution aminopeptidase M (0.025 mg/ml) in 100 mM ammoniumbicarbonate pH 8.0
• Mass spectrometric analysis was performed using two different mass spectrometers.
• a Sciex API III triple quadrupole LC-MS instrument (Sciex instruments, Thornhill, Ontario) equipped with an electrospray ion-source and a Bio-Ion 20 time-of-flight Plasma Desorption instrument (Bio-Ion Nordic AB, Uppsala, Sweden).
• Quantification of the compounds was done on the API III in- strument using single ion monitoring of the molecular ion in question with flow injection of the analyte.
• the liquid flow (MeOH-water 1 :1) of 100 ml/min was controlled by an ABI 140B HPLC unit (Perkin-Elmer Applied Biosystems Divisions, Foster City, CA).
• the instrument parameters were set to standard operation conditions, and SIM monitoring was performed using the most intense molecular ion (in most cases this corresponded to the doubly charged molecular ion).
• Identification of degradation products furthermore involved the use of plasma desorption mass spectrometry (PDMS) with sample application on nitrocellulose coated targets and standard instrumental settings.
• PDMS plasma desorption mass spectrometry
• the accuracy of the hereby determined masses is generally better than 0.1%.
• Separation and isolation of degradation products was done using a HY-TACH C-18 reverse phase 4.6x105 mm HPLC column (Hewlett-Packard Company, Palo Alto, CA) with a standard acetonitril: TFA separation gradient.
• the HPLC system used was HP1090M (Hewlett-Packard Company, Palo Alto, CA).
• the column was equilibrated with a mixture composed of 20% of solvent system I and 95% of solvent system II. After injection of the sample a gradient of 20% to 80% of solvent system I in solvent system II was run over 30 minutes. The gradient was then extended to 100% of solvent system I over 5 minutes followed by isocratic elution with 100% of this system for 5 minutes.
• the RP-analysis was performed using UV detections at 214, 254, 276, and 301 nm on a 218TP544.6 mm x 250 mm 5m C-18 silica column (The Seperations Group, Hesperia), which was eluted at 1 mL/min at 42°C.
• the column was equilibrated with 5% acetonitrile in a buffer consisting of 0.1 M ammonium sulfate, which was adjusted to pH 2.5 with 4M sulfuric acid, after injection the sample was eluted by a gradient of 5% to 60% acetonitrile in the same buffer during 50 min.
• the RP-analysis was performed using UV detections at 214, 254, 276, and 301 nm on a 218TP54 4.6 mm x 250 mm 5m C-18 silica column (The Seperations Group, Hesperia), which was eluted at 1 mL/min at 42°C.
• the column was equilibrated with 5% (acetonitrile + 0J % TFA) in an aqueous solution of TFA in water (0.1%). After injection the sample was eluted by a gradient of 5% to 60% (acetonitrile + 0J % TFA) in the same aqueous buffer during 50 min.
• Boc tert butyloxycarbonyl
• Z benzyloxycarbonyl
• THF tetrahydrofuran
• EDAC N-ethyl-N'-dimethylaminopropylcarbodiimide hydrochloride
• Step A At 0 °C, ethyl chloroformate (1 JO mL, 11.5 mmol) was given dropwise to a solution of 3-tert-butoxycarbonylamino-3-methylbutanoic acid (2.50 g, 11.5 mmol) and triethylamine (1.92 mL, 13.8 mmol) in tetrahydrofuran (10 mL). The solution was stirred for 40 min at 0 °C. The formed precipitate was filtered off and washed with tetrahydrofuran (20 mL). The liquid was immediately cooled to 0 °C.
• Step B DMSO (1.22 mL, 17.2 mmol) was added to a solution of oxalyl chloride (1.1 mL, 12.9 mmol) at -78 °C in dichloromethane (15 mL). The mixture was stirred for 15 min at -78 °C. A solution of 3-hydroxy-1 J-dimethylpropylcarbamic acid tert-butyl ester (1.75 g, 8.6 mmol) in dichloromethane (10 mL) was added dropwise over a period of 15 min. The solution was stirred at -78 °C for another 15 min. Triethylamine (6.0 mL, 43 mmol) was added.
• the solution was stirred at -78 °C for 5 min and then warmed to room temperature.
• the solution was diluted with dichloromethane (100 mL) and extracted with 1N hydrochloric acid (100 mL).
• the aqueous phase was extracted with dichloromethane (50 mL).
• the combined organic layers were washed with saturated sodium hydrogen carbonate solution (100 mL) and dried over magnesium sulfate.
• the solvent was removed in vacuo.
• the crude product was purified by column chromatography on silica (140 g) with ethyl acetate/heptane (1 :3) to give 1 J0 g of 3- (tert-butoxycarbonylamino)-3-methylbutanal.
• Step C Triethylphoshonoacetate (1.96 mL, 9.8 mmol) was dissolved in tetrahydrofuran (30 mL). Potassium tert-butoxide (1 J0 g, 9.8 mmol) was added. The solution was stirred for 40 min at room temperature. A solution of 3-(tert-butoxycarbonyiamino)-3-methylbutanal (1 J0 g, 5.5 mmol) in tetrahydrofuran (6 mL) was added. The solution was stirred at room temperature for 75 min. It was diluted with ethyl acetate (100 mL) and 1 N hydrochloric acid (100 mL). The phases were separated.
• the aqueous phase was extracted with ethyl acetate (2 x 50 mL).
• the combined organic phases were washed with saturated sodium hydrogen carbonate solution (60 mL) and dried over magnesium sulfate.
• the solvent was removed in vacuo.
• the crude product was purified by column chromatography on silica (90 g) with ethyl acetate/hepatane (1 :4) to give 1.27 g of ethyl (2E)-5-(tert-butoxycarbonylamino)-5-methylhex-2-enoate.
• Step D Ethyl (2E)-5-(tert-butoxycarbonylamino)-5-methylhex-2-enoate (1.233 g, 4.54 mmol) was dissolved in dioxane (20 mL). Lithium hydroxide (0J20 g, 5.00 mmol) was added as a solid. Water (10 mL) was added, until a clear solution was reached. The solution was stirred 16 h at room temperature. The solution was diluted with water (70 mL) and was extracted with tert-butyl methyl ether (2 x 100 mL).
• the aqueous phase was acidified to pH 4 with 1 M aqueous hydrogen chloride, extracted with ethyl acetate (2 x 100 ml) and the combined organic layers were dried (magnesium sulfate) and concentrated in vacuo to give 3.6 g of (2S)-2- (((carboxy)methoxy)methyl)-pyrrolidin-1 -carboxylic acid tert-butyl ester.
• the crude product was chromatographed on silica (300 g) with pentane/ethyl acetate 7:3 as eluent to give 4.3 g of (2-(tert-butoxycarbonylamino)butoxy)acetic acid ethylester.
• the product was dissolved in of 1 M lithium hydroxide in water/methanol 1 :3 (40 ml) and stirred at room temperature for 4 hours. The mixture was concentrated in vacuo and water (100 mL) was added and the solution was washed with ether (20 mL).
• the aqueous phase was acidified to pH 4 with 1 M aqueous hydrogen chloride and extracted with ethyl acetate (200 ml), dried over magnesium sulfate and concentrated in vacuo to give 2.46 g of (2- (tert butoxycarbonylamino)butoxy)acetic acid.
• the aqueous phase was adjusted to pH 11 with solid potassium hydroxide, and extracted with dichloromethane (2 x 100 mL). The combined organic phases were dried (magnesium sulfate) and concentrated in vacuo. A concentrated solution of hydrogen chloride in ethyl acetate added (100 mL) was, and the solution was concentrated in vacuo. The residue was dissolved in ethanol (25 mL) and sulphuric acid (9N, 25 mL) was added. After 16 hours at room temperature and 2 hours at reflux temperature the ethanol was removed by evaporation in vacuo and the residual aqueous mixture was adjusted to pH > 8 using solid potassium hydroxide.
• the solvent was removed in vacuo and the residue was dissolved in water (100 ml) and washed once with diethyl ether (50 ml) and acidified to pH 3 with a solution of aqueous hydrogen sulphate.
• the aqueous phase was extracted with methylene chloride (3 x 100 ml) and the combined organic phases were dried (magnesium sulphate) and the solvent was removed in vacuo to afford the Troc protected buildingblock.
• Boc-D-4-F-Phe-OH Boc-N- e-D-4-F-Phe-OH
• BocLys(Z)OSu 50 g; 0J0 mol
• ammonium hydrogencarbonate 25 g; 0.32 mole
• DMF 300 ml
• Water 1000 ml was added and the precipitate was isolated by filtration and washed with water (3 x 200 ml).
• the precipitate was dissolved in methanol (500 ml) and palladium on carbon (15 g; wet, 10%) was added.
• the mixture was hydrogenated for 4 h at ambient pressure, filtered and the solvent was removed in vacuo.
• aqueous phase was extracted with methylene chloride (4 X 700 ml) and the phases were sepa- rated.
• the organic phase was dried (magnesium sulphate) and the solvent was removed in vacuo to afford 11.0 g of [(5S)-5-carbamoyl-5-((2R)-2-methylamino-3-(2-thienyl)- propionylamino)-pentyl]-carbamic acid 2,2,2-trichloroethyl ester.
• the peptide containing fractions were collected, diluted with 3 volumes of water and applied to a Sep-Pak s C18 cartridge (Waters part. #: 51910) which was equilibrated with 0.1% trifluoroacetic acid.
• the peptide was eluted from the Sep-Pak ® cartridge with 70% acetonitrile in a 0.1% trifluoroacetic acid solution in water.
• the product was lyophilized to afford 61 mg of the title compound as a trifluoroacetate salt
• the title compound was prepared as in example 1 , except that the Z-protecting group was not transferred into the Troc-protecting group in the first step.
• the title compound was prepared as in example 1 , omitting acidic washes and using N- methyl-N'-dimethyldiaminopropane, buildingblock 7, Boc-N-Me.D-Phe-OH and Boc-N-Me-D- Bip-OH as the starting materials.
• the title compound was prepared as in example 1 , omitting acidic washes and using N- methyl-N'-dimethyldiaminopropane, Boc-N-Me-D-Phe-OH and Boc-N-Me-D-Bip-OH and Boc-AEH-OH as the starting materials
• (2E)-5-Amino-5-methylhex-2-enoic acid ((1 R)-1 - ⁇ N-[(1 R)-1 -(((1 S)-5-amino-1 - (dimethylcarbamoyl)pentyl)carbamoyl)-2-phenylethyl]-N-methylcarbamoyl ⁇ -2-(2- naphthyl)ethyl)amide
• the title compound was prepared as in example 1. Dimethylamine was used instead of am- monium hydrogencarbonate. Boc-N-Me-D-Phe-OH, Boc-N-Me-D-2-Nal-OH and Boc-AEH- OH were used as starting materials.
• the title compound was prepared as in example 1. Dimethylamine was used instead of ammonium hydrogencarbonate. Boc-N-Me-D-Phe-OH, Boc-N-Me-D-Bip-OH and Boc-AMB-OH were used as starting materials.
• Fmoc deprotection procedure The resin is swelled and shaken in 20% piperidine in DMF ( 5 ml) for 3 min. The resin is drained and the deprotection process is repeated, with 15 min reaction time.
• the drained resin is washed using the following standard washing procedure: The resin is repeatedly swelled, shaken and drained three times with 5mL of DMF, methylene chloride, DMF, 2-propanol, methanol and finally with diethyl ether.
• Rink amide resin (Novabiochem 01-64-0013, 1g , 0,46 mmol) is deprotected using the method described above followed by the standard washing procedure.
• the resin is drained and the washed using the standard procedure described above.
• the resin is deprotected using the method described above followed by the standard wash- ing procedure.
• the resin is drained and the washed using the standard procedure described above.
• the resin is deprotected using the method described above followed by the standard washing procedure.
• the resin is drained and the washed using the standard procedure described above.
• the resin is deprotected using the method described above followed by the standard wash- ing procedure.
• the resin which has been treated as described above is swelled and shaken in a mixture of 5% water in trifluoro acetic acid (10mL) for 1h.
• the resin is then drained and repeatedly washed with triflouro acetic acid (2x5mL) and DCM (4x5mL). All drained solutions are combined and concentrated under nitrogen to a 4mL volume.
• Et 2 O (50mL) is the heterogeneous mixture is centrifuged. The centrifuge pellet is washed with Et 2 O (50mL) and centrifuged. This treatment is repeated, and the final compound is dried in exicator for 18h.
• (2E)-5-Amino-5-methylhex-2-enoic acid N-((1 R)-1 - ⁇ N-[(1 R)-1 -(((1 S)-5-amino-1 - carbamoylpentyl)carbamoyl)-2-phenylethyl]-N-methylcarbamoyl ⁇ -2-(biphenyl-4-yl)ethyl ⁇ amide:
• Buildingblock 2 Fmoc-NMe-D-Phe-OH Buildingblock 3: Fmoc-D-(4-biphenyi)-Ala-OH Buildingblock 4: Boc-AEH-OH
• Buildingblock 1 Fmoc-Lys-OH
• Buildingblock 2 Fmoc-NMe-D-Phe-OH
• Buildingblock 3 Fmoc-D-(4-biphenyl)-Ala-OH
• Buildingblock 4 Boc-3-AMH-OH
• (2E)-5-Amino-5-methylhex-2-enoic acid N-((1 R)-1- ⁇ N-[(1 R)-1-(((1S)-5-amino-1- carbamoylpentyl)carbamoyl)-2-(2-thienyl)ethyl]-N-methylcarbamoyl ⁇ -2-(biphenyl-4- yl)ethyl ⁇ amide:
• Buildingblock 1 Fmoc-Lys-OH
• Buildingblock 2 Fmoc-NMe-D-(2-thienyl)-Ala-OH
• Buildingblock 3 Fmoc-D-(4-biphenyl)-Ala-OH
• Buildingblock 4 Boc-AEH-OH
• Buildingblock 1 Fmoc-Lys-OH
• Buildingblock 2 Fmoc-NMe-D-(2-thienyl)-Ala-OH
• Buildingblock 3 Fmoc-D-(4-biphenyl)-Ala-OH
• Buildingblock 4 Boc-3-AMH-OH
• Acetylation of the Lysine epsilon amino group of example 26-29 was performed using the methodology described below.
• the compound to be acetylated at the Lysine epsilon amino group (0J6mmol) is disolved or suspended in 2% Na 2 CO 3 aqueous solution (40mL) and acetic acid anhydride (O. ⁇ mmoi, 75mL) is added. This mixture is heavily stirred at 20°C for 18h. Additional acetic acid anhydride (O. ⁇ mmol, 75mL) and THF (2.5mL) are added. After 2h the reaction mixture is concentrated to 30mL, and extracted with DCM (2x20mL). The combined organic phases are concentrated in vacuo, and the crude product is purified on silica (35g) using 10% ammonia in ethanol and DCM (7:3) as eluent to give the monoactylated compound.
• This compound was prepared by acetylation of the epsilon amino group in the lysine fragment using the general method described above and using the compound prepared in example 19 as starting material.
• (2E)-5-Amino-5-methylhex-2-enoic acid N-((1 R)-1- ⁇ N-[(1 R)-1-(((1S)-5-acetylamino-1- carbamoylpentyl)carbamoyl)-2-(2-thienyl)ethyl]-N-methylcarbamoyl ⁇ -2-(biphenyl-4- yl)ethyl)amide:
• This compound was prepared by acetylation of the epsilon amino group in the lysine fragment using the general method described above and using the compound prepared in example 24 as starting material.
• This compound was prepared by acetylation of the epsilon amino group in the lysine fragment using the general method described above and using the compound prepared in example 20 as starting material.
• This compound was prepared by acetylation of the epsilon amino group in the lysine fragment using the general method described above and using the compound prepared in example 22 as starting material.
• Fmoc deprotection procedure The resin is swelled and shaken in 20% piperidine in DMF ( 5 ml) for 3 min. The resin is drained and the deprotection process is repeated, with 15 m reaction time.
• the drained resin is washed using the following standard washing procedure: The resin is repeatedly swelled, shaken and drained three times with 5mL of DMF, methylene chloride, DMF, 2-propanol, methanol and finally with diethyl ether Standard protocol for solid phase synthesis of example 30-33.
• the resin is drained and the washed using the standard procedure described above.
• the resin is deprotected using the method described above followed by the standard washing procedure.
• the resin is drained and the washed using the standard procedure described above.
• the resin is deprotected using the method described above followed by the standard washing procedure.
• the resin is deprotected using the method described above followed by the standard washing procedure.
• Diaminohexyl trityl resin was employed.
• Diaminobutyl trityl resin was employed.
• Diaminopropyl trityl resin was employed.
• (2E)-5-Amino-5-methylhex-2-enoic acid N-((1 R)-1- ⁇ N-[(1 R)-1-(2-aminoethylcarbamoyl)-2- phenylethyl]-N-methylcarbamoyl ⁇ -2-(biphenyl-4-yl)ethyl)-N-methylamide
• Diaminoethyl trityl resin was employed.
• the title compound was prepared as in example 1 , omitting acidic washes and using N- methyl-N'-dimethyldiaminopropane as the starting material. Buildingblock 7 and Boc-N-Me- D-2-Nal-OH was employed. The two stereoisomers could not be separated by HPLC. However buildingblock 7 was readily separated into two enantiomers by chiral HPLC and by repeating the procedure above the two stereoisomers were obtained.
• Example 35 3-(1-Aminoethyl)-NJ(1 R)-1-(N-((1 R)-1-(N-((dimethylcarbamoyl)methyl)-N- methylcar- bamoyl)-2-phenylethyl)-N-methylcarbamoyl)-2-(2-naphthyl )ethyl)-N-methylbenzamide.
• the mixture was stirred for 20 h and was diluted with TFA in water (0.2 %, 290 ml) and purified by preparative HPLC in three consecutive runs on a 25 X 250 mm 10u RP-18 column at 40°C with a gradient of 20-40% 0.1% TFA/acetonitrile in 0.1 % aqueous TFA.
• the peptide containing fractions were pooled and lyophilised to afford 114 mg of the title compound.
• (2S)-6-Amino-2-((2R)-2- ⁇ (2R)-2-[(2S)-2-(2-amino-2-methylpropionylamino)-3-(3H-imidazol-4- yl)propionylamino]-3-(2-naphthyl)propionylamino ⁇ -3-phenylpropionylamino)hexanoic acid amide (712 mg, 1.0 mmol) (prepared as in WO9517423) was suspended in water (10 ml) and di-tert-butyldicarbonate (240 mg, 1.0 mmol) was added.
• the mixture was stirred for 20 h and was diluted with TFA in water (0.2 %, 185 ml) and purified on a Sep-Pac (Waters #36925) RP-18 column washed with 28 % acetonitrile in 0.1% aqueous TFA and eluted with 35% acetonitrile in 0.1% aqueous TFA.
• the peptide containing fractions were pooled, diluted and lyophilised to afford 408 mg of the title compound.
• (2E)-5-Amino-5-methylhex-2-enoic acid ((1 R)-1 - ⁇ N-[(1 R)-1 -(5-aminopentylcarbamoyl)-2- phenylethyl]-N-methylcarbamoyl ⁇ -2-(2-naphthyl)ethyl)amide
• (2E)-5-Amino-5-methylhex-2-enoic acid N-[(1 R)-1-(N- ⁇ (1 R)-1-[N-(2-dimethylaminoethyl)-N- methylcarbamoyl]-2-phenylethyl ⁇ -N-methylcarbamoyl)-2-(2-naphthyl)ethyl]-N-methylamide
• (2E)-5-Amino-5-methylhex-2-enoic acid N-((1 R)-1 - ⁇ N-[(1 R)-1 -((1 S)-5-(acetylamino)-1 ⁇ (dimethylcarbamoyl)pentylcarbamoyl)-2-phenylethyl]-N-methylcarbamoyl ⁇ -2-(2- naphthyl)ethyl)-N-methylamide
• N-(3-Dimethylaminopropyl)-N ' -ethylcarbodiimide hydrochloride (824 mg, 4.30 mmol) was added at 0 °C to a solution of (2R)-2-(N-(tert-butoxycarbonyl)-N-methylamino)-3- phenylpropionic acid (1.20 g, 4.30 mmol) and 1-hydroxybenzotriazole hydrate (659 mg, 4.30 mmol) in dichloromethane (15 ml) and N,N-dimethylformamide (15 ml). The reaction mixture was stirred for 20 min at 0 °C.
• N-(3-Dimethylaminopropyl)-N ' -ethylcarbodiimide hydrochloride (594 mg, 3J mmol) was added at 0 °C to a solution of (2R)-2-(N-(tert-butoxycarbonyl)-N-methylamino)-3-(2- naphthyl)propionic acid (1.02 g, 3J mmol) and 1-hydroxy-7-azabenzotriazole (422 mg, 3J mmol) in dichloromethane (10 ml) and N,N-dimethylformamide (5 ml). The reaction mixture was stirred for 20 min at 0 °C.
• N-(3-Dimethylaminopropyl)-N ' -ethylcarbodiimide hydrochloride (279 mg, 1.46 mmol) was added at 0 °C to a solution of (2E)-5-(tert-butyoxycarbonylamino)-5-methylhex-2-enoic acid (355 mg, 1.46 mmol) and 1-hydroxy-7-azabenzotriazole (199 mg, 1.46 mmol). The reaction mixture was stirred for 20 min at 0 °C.
• Trifluoroacetic acid (4 ml) was added. The reaction mixture was stirred for 35 min at 0 °C. A saturated aqueous solution of sodium hydrogen carbonate (10 ml) was added. Solid sodium hydrogen carbonate was added until pH 7. Water (50 ml) and dichloromethane (30 ml) were added. The phases were separated. The aqueous phase was extracted with dichloromethane (5 x 40 ml). The combined organic layers were dried over magnesium sulfate. The solvent was removed in vacuo.
• the crude product was purified by flash chromatography on silica (120 g), using dichloro- methane/methanol/25% aqueous ammonia (first: 100:10:1 , then 50:10:1), as eluent to give 408 mg of the title compound.
• Example 57 For biological testing, the title compound was transferred into its acetate salt by lyophilization with 0.5 M acetic acid (40 ml).
• Example 57 For biological testing, the title compound was transferred into its acetate salt by lyophilization with 0.5 M acetic acid (40 ml).
• N-(3-Dimethylaminopropyl)-N ' -ethylcarbodiimide hydrochloride (411 mg, 2.30 mmol) was added at 0 °C to a solution of(2R)-2-(N-(tert-butoxycarbonyl)-N-methylamino)-3- phenylpropionic acid (643 mg, 2.30 mmol) and 1 -hydroxybenzotriazole hydrate (311 mg, 2.30 mmol) in dichloromethane (10 ml) and N,N-dimethylformamide (5 ml). The reaction mixture was stirred for 20 min at 0°C.
• N-(3-Dimethylaminopropyl)-N ' -ethylcarbodiimide hydrochloride (313 mg, 1.63 mmol) was added at 0 °C to a solution of (2R)-2-(N-(tert-butoxycarbonyl)-N-methylamino)-3-(2- naphthyl)propionic acid (537 mg, 1.63 mmol) and 1-hydroxy-7-azabenzotriazole (222 mg, 1.63 mmol) in dichloromethane (20 ml) and N,N-dimethylformamide (10 ml). The reaction mixture was stirred for 20 min at 0 °C.
• N-(3-Dimethylaminopropyl)-N ' -ethylcarbodiimide hydrochloride (142 mg, 0.74 mmol) was at 0 °C added to a solution of (2E)-5-(tert-butoxycarbonylamino)-5-methylhex-2-enoic acid (180 mg, 0.74 mmol) and 1-hydroxy-7-azabenzotriazole (100 mg, 0.74 mmol) in dichloromethane (10 ml) and N,N-dimethylformamide (5 ml). The reaction mixture was stirred at 0 °C for 20 min.
• Trifluoroacetic acid (8 ml) was added. The reaction mixture was stirred for 60 min at 0 °C. It was diluted with dichloromethane (30 ml). A saturated aqueous solution of sodium hydrogen carbonate (30 ml) was added carefully. Solid sodium hydrogen carbonate was added until pH 7. The phases were separated. The aqueous phase was extracted with dichloromethane (3 x25 ml). The combined organic layers were dried over magnesium sulfate. The solvent was removed in vacuo.
• the crude product was purified by flash chromatography on silica (40 g), using dichloromethane/methanol/25% aqueous ammonia (first: 100:10:1 , then 50:10:1) as eluent to give 124 mg of the title compound.
• the title compound was transferred into its acetate salt, by lyophiliza- tion with 0.5 M acetic acid (40 ml).

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