Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
  • C07K5/02—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link
  • C07K5/0205—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link containing the structure -NH-(X)3-C(=0)-, e.g. statine or derivatives thereof

Definitions

• the present invention relates to a process for preparing a compound of formula I, a GH secretagogue which can be used i.a. in 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 mobilisation 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 primarily 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
• L-Dopa L-3,4-dihydroxyphenylalanine
• PACAP pituitary adenylyl cyclase activating peptide
• muscarinic receptor agonists muscarinic receptor agonists
• GHRP growth hormone releasing peptide
• WO 97/23508 discloses a method for preparing the compound of formula I. This method is very expensive and makes use of reagents which are adverse to the environment.
• the present invention relates to a method for preparing a compound of formula
• P is Troc.
• P is Boc.
• P is Fmoc.
• P is Troc.
• the agent capable of forming an amide or ester or mixed carbonic anhydride or anhydride or acidhalide is selected from benzotriazole, isobutyl chlorocarbonate, DHOBt, HOBt, HOSu, and HOAt.
• the agent capable of forming an amide or ester or mixed carbonic anhydride or anhydride or acidhalide is DHOBt.
• the de-protection is usually carried out by acidic, basic, oxidative or reductive cleavage, eg. when the protection group is Troc then reductive cleavage is carried out with Zn and acetic acid.
• P is Troc and the agent capable of forming an amide or ester or mixed carbonic anhydride or anhydride or acidhalide is DHOBt.
• the present invention relates to a compound of formula III
• P is a protecting group.
• P is Troc.
• P is Boc.
• P is Fmoc.
• P is Troc.
• the agent capable of forming an amide or ester or mixed carbonic anhydride or anhydride or acidhalide is selected from benzotriazole, isobutyl chlorocarbonate, DHOBt, HOBt, HOSu, and HOAt, preferably DHOBt.
• P is Troc and said amide or ester or mixed carbonic anhydride or anhydride or acidhalide is obtained by reacting DHOBt with the car- boxylic acid moiety in the compound of formula II.
• the present invention relates to a compound of formula V
• the compounds of the present invention may optionally be on a salt form, such as a pharmaceutically acceptable salt form e.g. the pharmaceutically acceptable acid addition salts of compounds of formula I, II, III, IV and V, which include those prepared by reacting the compound of formula I, II, III, IV and V with an inorganic or organic acid such as hydrochloric, hydrobromic, sulfuric, acetic, phosphoric, lactic, malic, maleic, mandelic phthalic, citric, glutaric, gluconic, methanesulfonic, salicylic, succinic, tartaric, toluenesulfonic, trifluoracetic, sulfamic or fumaric acid and/or water.
• a pharmaceutically acceptable salt form e.g. the pharmaceutically acceptable acid addition salts of compounds of formula I, II, III, IV and V, which include those prepared by reacting the compound of formula I, II, III, IV and V with an inorganic or organic acid such as hydrochloric, hydro
• Compound 1 is then reacted with an agent capable of forming an amide or ester or mixed carbonic anhydride or anhy- dride or acidhalide, such as benzotriazole, isobutyl chlorocarbonate, 3,4-Dihydro-3-hydroxy- 4-0x0-1 ,2,3-benzotriazine (DHOBt), N-hydroxy benzotriazol (HOBt), N-hydroxy succinimid (HOSu), and 1-hydroxy-7-azabenzotriazole (HOAt), preferably DHOBt, in a solvent selected from an organic solvent or mixture of organic solvents or a mixture of organic solvent(s) and water, such as ethylacetate, thereby producing the compound 2, which is isolated in a conventional manner, e.g.
• an agent capable of forming an amide or ester or mixed carbonic anhydride or anhy- dride or acidhalide such as benzotriazole, isobutyl chlorocarbonate, 3,4-Dihydr
• the C 1-12 -alkyl, C 1j3 -alkyl, or C M -alkyl groups specified herein are intended to include those al- kyl or alkylene groups of the designated length in either a linear or branched or cyclic configuration.
• linear alkyl are methyl, ethyl, propyl, butyl, pentyl, hexyl and heptyl.
• branched alkyl are isopropyl, sec-butyl, tert-butyl, isopentyl, isohexyl and isoheptyl.
• cyclic alkyl examples include C ⁇ -cycloalkyl such as cyclopropyl, cyclobutyl, cyclopentyl, cyclo- hexyl and cycloheptyl.
• the C 1-6 -alkoxy groups specified herein are intended to include an oxygen atom connected to a C 1-6 -alkyl as defined above. Examples are methoxy, ethoxy, isopro- pyloxy, sec-butyloxy, tert-butyloxy, cyclopropyloxy, cyclobutyloxy, etc.
• aryl is intended to include monovalent carbocyclic aromatic ring moieties, being ei- ther monocyclic, bicyclic or polycyclic, e.g. selected from the group consisting of phenyi and naphthyl, optionally substituted with one or more C 1-6 -alkyl, C 1-6 -alkoxy, halogen, amino or aryl. Such aryl groups are described in Morrison and Boyd “Organic Chemistry", 4. Ed.
• heteroaryl is intended to include monovalent heterocyclic aromatic ring moieties, being either monocyclic, bicyclic or polycyclic, e.g.
• pyridyl 1-H- tetrazol-5-yl, thiazolyl, imidazolyl, indolyl, pyrimidinyl, thiadiazolyl, pyrazolyl, oxazolyl, isoxa- zolyl, oxadiazolyl, thienyl, quinolinyl, pyrazinyl, or isothiazolyl, optionally substituted by one or more C ⁇ -alkyl, C 1-6 -alkoxy, halogen, amino or aryl.
• Such hetaryl groups are described in Morrison and Boyd "Organic Chemistry", 4. Ed.
• fused-ring aromatic system is intended to include monovalent aromatic ring moieties, being either bicyclic or polycyclic hydrocarbons, such as a system selected from the group consisting of fluorenes, e.g. flourenyl. Such fused-ring aromatic systems are described in Morrison and Boyd "Organic Chemistry", 4. Ed.
• halogen is intended to include chlorine (Cl), fluorine (F), bromine (Br) and iodine (I).
• Protecting group is intended to include any group which protect the amino group when the carboxylic group in the compound of formula II is subjected to functional derivati- zation, and which is easy to remove afterwards by cleavage.
• protecting groups are described in "Protective groups in organic chemistry", 2. Ed, Greene.T.W.; Wuts.P.G.M., John Wiley&Sons.lnc. 1991 ; and "The Peptides, Analysis, Synthesis, Biology", vol 3 "Protection of Functional Groups in Peptide synthesis", Gross.E.; Meienhofer, J.; Academic Press.
• agent capable of forming an amide or ester or mixed carbonic anhydride or anhydride or acidhalide is intended to include such agents which activates the compound by forming a functional derivative which may be used in acylation.
• agents are described in "The Peptides, Analysis, Synthesis, Biology", vol 1 "Major Methods of Bond Formation", Gross.E.; Meienhofer, J.; Academic Press, 1981.
• Suitable agents are selected from benzotri- azoie, isobutyl chlorocarbonate, DHOBt, HOBt, HOSu, and HOAt.
• de-protection is intended to include acidic, basic, oxidative or reductive cleavage as described in "The Peptides, Analysis, Synthesis, Biology", vol 1 "Major Methods of Bond Formation", Gross.E.; Meienhofer, J.; Academic Press, 1981.
• the protection group is Troc then reductive cleavage is carried out with Zn and acetic acid.
• the compounds of formula I when used for the intended purpose of releasing endogenous growth hormone may be administered in pharmaceutically acceptable acid addition salt form or, where appropriate, as a alkali metal or alkaline earth metal or lower alkylammonium salt.
• Such salt forms are believed to exhibit approximately the same order of activity as the free base forms.
• the compounds of the general formula I possess the ability to release endogenous growth hormone in vivo as mentioned in WO 97/23508.
• the compounds may therefore be used in the treatment of conditions which require increased plasma growth hormone levels such as in growth hormone deficient humans or in elderly patients or livestock.
• 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.
• Compounds of formula I are useful for stimulation of growth hormone release in the elderly, prevention of catabolic side effects of glu- cocorticoids, 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 re- nal failure or insufficiency, treatment of cardiomyopathy, treatment of wasting in connection with chronic liver disease, treatment of thrombocytopenia, treatment of growth retardation in connection with Crohn's disease, treatment of short bowel syndrome, treatment of wasting in connection with chronic obstructive pulmonary disease (COPD), treatment of complications associated with transplantation, treatment of physiological short stature including growth hor- mone deficient children and short stature associated with chronic illness, treatment of obesity and growth retardation associated with obesity, treatment of anorexia, treatment of growth retardation associated with the Prader-Willi syndrome and Turner's syndrome; increasing the growth rate of a patient having partial growth hormone insensitive syndrome, accelerating the recovery and reducing hospitalization of burn patients; treatment of intrauterine growth retarda- tion, skeletal dysplasia, hypercortisolism and Cushing's syndrome; induction of pulsatile growth hormone release; replacement of
• treatment of insulin resistance in the heart improvement of sleep quality and correction of the relative hyposomatotropism of senescence due to high increase in REM sleep and a decrease in REM latency
• treatment of hypothermia treatment of frailty associated with aging
• treatment of congestive heart failure treatment of hip fractures
• treatment of immune deficiency in individuals with a depressed T4/T8 cell ratio treatment of muscular atrophy
• treat- ment of musculoskeletal impairment in elderly enhancing the activity of protein kinase B (PKB), improvement of the overall pulmonary function, and treatment of sleep disorders.
• Treatment is also intended to include prophylactic treatment.
• the dosage will vary depending on the compound of formula I em- ployed, on the mode of administration and on the therapy desired.
• the dosage of the compounds according to this invention is suitably 0.01-500 mg/day, e.g. from about 5 to about 50 mg, such as about 10 mg per dose, when administered to patients, e.g. humans, as a drug.
• 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. Morever 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.
• dosage forms suitable for oral, nasal, pulmonal or transdermal administration comprise from about 0.0001 mg to about 100 mg, pref- erably from about 0.001 mg to about 50 mg of the compounds of formula I admixed with a pharmaceutically acceptable carrier or diluent.
• a pharmaceutical composition comprising the compound of formula I prepared by the method of the invention may be combined with one or more compounds exhibiting a differ- ent 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.
• HCI 5-Amino-5-methyl-hex-(2E)- enoic acid
• HCI salt is dissolved in 730 ml NaHC0 3 /NaOH buffer, pH 11, and 730 ml 2-PrOH, pH is approx. 9,5. Cooled to0-5°C.
• TroOSu Succinimidyl-2,2,2-trichloroethylcarbonate
• pH is maintained at 9.5-10 with 2M NaOH (approx. 75ml). Precipitation occurs. The mixture is stirred overnight at 0-5°C to complete the reaction. The reaction mixture (pH 9.1) is fil- tered. The 2-propanol (2-PrOH) content in the filtrate is distilled off under vacuum (30°C). pH is adjusted to 9.5 with 2M NaOH. 500ml methyl-tert-butylether (MTBE) are added. 3 phases occur. The two lower phases which contains the product are isolated. The organic MTBE phase is extracted with 300ml water (pH 10, adjusted with aq. NaOH).
• the aqueous phases are combined and 500ml of MTBE are added.
• the mixture is acidified to pH 2 with 4M HCI at ambient temperature. Evolution of C0 2 gas.
• the organic phase is isolated and the water phase extracted with 500ml MTBE.
• the combined organic phases are dried. Evaporation of solvent yields 131g of 5-(N-Troc)-amino-5-methyl-hex-(2E)-enoic acid (100%). It is isolated as an oil which slowly crystallises.
• a 2 L reactor is equipped with a mechanical stirrer, thermometer, heating facility and N 2 -inlet.
• NMM N-methyl morpholine
• HCI N-Methyl-N-((1 R)-1-(N-methyl-N- ((1 R)-1 -(methylcarbamoyl)-2-phenylethyl)carbamoyl)-2-(2-naphthyl)ethyl)amide
• a 1 L reactor is equipped with a mechanical stirrer, thermometer, heating/cooling facility, dropping funnel and N 2 -inlet.
• 35,3g activated Zn dust ( ⁇ 60 ⁇ m) is suspended in 162ml acetic acid.
• 5-(N-Trichloroethyloxycarbonyl)amino-5-methylhex-(2E)-enoic acid N-methyl-N-((1R)-1- (N-methyl-N-((1 R)-1-(methylcarbamoyl)-2-phenylethyl)carbamoyl)-2-(2-naphthyl)ethyl)amide dissolved in 162ml EtOAc is slowly added over 3h.
• Heat and gas is developed, especially in the beginning.
• the temperature is kept at 30-35°C for 18-24h in order to complete the reac- tion.
• the mixture is cooled to 20-25°C and 200ml EtOAc and 600ml water are added.
• H 2 is developed.
• the reaction mixture is filtered and the water phase isolated.
• the organic layer is extracted with 1x 500ml and 1x 100ml 0,01 M HCI.
• 800ml EtOAc is added to the combined water phases and the pH is adjusted to 9 with 400ml 25% NH 3 in water.
• the temperature is kept at 15-25°C.
• the organic layer is isolated and the water phase extracted with 400ml EtOAc.
• Step A At 0 °C, ethyl chloroformate (1.10 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 ,1-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 1.10 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 (1.10 g, 9.8 mmol) was added. The solution was stirred for 40 min at room temperature. A solution of 3-(tert-butoxycarbonylamino)-3-methylbutanal (1.10 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 1N hydrochloric acid (100 L). The phases were separated.
• 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 (0.120 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 L) and was extracted with tert-butyl methyl ether (2 x 100 mL).
• Step E N-Tert-butoxycarbonyl-N-methyl-D-phenylalanine (1.22 g, 4.4 mmol), 1- hydroxybenzotriazole hydrate(0.59 g, 4.4 mmol) and 1-ethyl-3-(3-dimethyl- aminopropyl)carbodiimid hydrochloride (0.88 g, 4.6 mmol) were dissolved in N,N- dimethylformamide (25 mL) and stirred for 30 min. Methylamine (0.51 g of a 40% solution in methanol, 6.6 mmol) was added and the mixture was stirred overnight. Methylene chloride (80 mL) and water (100 mL) were added and the phases were separated.
• Step F N-Methyl-N-((R)1-(methylcarbamoyl)-2-phenylethyl)carbamic acid tert-butyl ester (1.39 g, 7.23mmol) was dissolved in a mixture of trifluoroacetic acid (5 mL) and methylene chloride (10 mL) and stirred for 45 min. The volatiles were removed in vacuo and the residue was stirred with a mixture of ethyl acetate (100 mL) and water (100 mL). Sodium hydrogen carbonate (50 mL, saturated) was added and the phases were separated. The organic phase was dried (magnesium sulfate) and the solvent removed in vacuo to afford 330 mg of (R)-N- methyl-2-methylamino-3-phenylpropionamide.
• Step G (R)-Tert-butoxycarbonyl-N-methylamino-3-(2-naphthyl)propionic acid (548 mg, 1.66 mmol) was dissolved in methylene chloride (5 mL); 1-hydroxy-7-azabenzotriazole (227 mg, 1,66 mmol) was added along with N,N-dimethylformamide (2 mL). 1-Ethyl-3-(3- dimethylaminopropyl)carbodiimide hydrochloride (351 mg, 1.83 mmol) was added and the solution was stirred for 15 min.
• Step H N-Methyl-N- ⁇ (1 R)-1 -(N-methyl-N-((1 R)-1 -(methylcarbamoyl)-2-phenylethyl)carbamoyl)- 2-(2-naphthyl)ethyl ⁇ carbamic acid tert-butyl ester (600 mg, 1.19 mmol) was stirred in trifluoroacetic acid/methylene chloride (1:1, 5 mL) for 10 min and the volatiles were removed in vacuo. The residue was stripped with diethylether (2 x 5 mL) and dissolved in methanol (2 mL) and mixed with sodium hydrogen carbonate (10 mL) and ethylacetate (15 mL).
• Step I (2E)-5-(tert-Butyloxycarbonylamino)-5-methylhex-2-enoic acid (200 mg, 0.82 mmol), 1- hydroxy-7-azabenzotriazole (112 mg, 0.82 mmol) and 1-ethyl-3-(3-dimethylaminopropyl)- carbodiimide hydrochloride (173 mg, 0.90 mmol) were dissolved in a mixture of methylene chloride (10 mL) and N,N-dimethylformamide (1 mL) and strirred for 15 min.
• N-Methyl-2- methylamino-N-((1 R)-1 -(methylcarbamoyl)-2-phenylethyl)-3-(2-naphthyl)propionamide (332 mg, 0.82 mol) dissolved in methylene chloride (5 mL) and diisopropylethylamine (0.14 mL) were added and the mixture was stirred overnight under nitrogen atmosphere.
• the mixture was diluted with methylene chloride (50 mL), washed with water (50 mL), sodium hydrogen carbonate (30 mL, saturated), and sodium hydrogensulfate (30 mL, 5%). The phases were separated and the organic phase was dried with magnesium sulfate and evaporated in vacuo.
• Step J ((3E)-1 , 1 -Dimethyl-4-(methyl-((1 R)-1 -(methyl-((1 R)-1 -(methylcarbamoyl)-2- phenylethyl)carbamoyl)-2-(2-naphthyl)ethyl)carbamoyl)but-3-enyl)carbamic acid tert-butyl ester (403 mg, 0.63 mmol) was stirred in a mixture of trifluroacetic acdi (4mL) and methylene chloride (4 mL) for 10 min.

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• 1999
  • 1999-06-09 EP EP99955478A patent/EP1093463A1/en not_active Withdrawn
  • 1999-06-09 JP JP2000553462A patent/JP4574849B2/ja not_active Expired - Lifetime
  • 1999-06-09 AU AU42567/99A patent/AU4256799A/en not_active Abandoned
  • 1999-06-09 WO PCT/DK1999/000305 patent/WO1999064456A1/en active Application Filing

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