WO1996024580A1 - Compounds with growth hormone releasing properties - Google Patents

Compounds with growth hormone releasing properties Download PDF

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Publication number
WO1996024580A1
WO1996024580A1 PCT/DK1996/000058 DK9600058W WO9624580A1 WO 1996024580 A1 WO1996024580 A1 WO 1996024580A1 DK 9600058 W DK9600058 W DK 9600058W WO 9624580 A1 WO9624580 A1 WO 9624580A1
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Prior art keywords
compound
propyl
morpholin
benzyl
general formula
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PCT/DK1996/000058
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French (fr)
Inventor
Michael Ankersen
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Novo Nordisk A/S
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Priority to AU45345/96A priority Critical patent/AU4534596A/en
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
    • C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
    • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D213/60—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D213/72—Nitrogen atoms
    • C07D213/75—Amino or imino radicals, acylated by carboxylic or carbonic acids, or by sulfur or nitrogen analogues thereof, e.g. carbamates
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C335/00—Thioureas, i.e. compounds containing any of the groups, the nitrogen atoms not being part of nitro or nitroso groups
    • C07C335/04—Derivatives of thiourea
    • C07C335/16—Derivatives of thiourea having nitrogen atoms of thiourea groups bound to carbon atoms of six-membered aromatic rings of a carbon skeleton
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D215/00—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems
    • C07D215/02—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen atoms or carbon atoms directly attached to the ring nitrogen atom
    • C07D215/16—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen atoms or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D215/38—Nitrogen atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D257/00—Heterocyclic compounds containing rings having four nitrogen atoms as the only ring hetero atoms
    • C07D257/02—Heterocyclic compounds containing rings having four nitrogen atoms as the only ring hetero atoms not condensed with other rings
    • C07D257/04—Five-membered rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D285/00—Heterocyclic compounds containing rings having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by groups C07D275/00 - C07D283/00
    • C07D285/01—Five-membered rings
    • C07D285/02—Thiadiazoles; Hydrogenated thiadiazoles
    • C07D285/04—Thiadiazoles; Hydrogenated thiadiazoles not condensed with other rings
    • C07D285/06—1,2,3-Thiadiazoles; Hydrogenated 1,2,3-thiadiazoles

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 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
  • arginine, L-3,4-dihydroxyphenylalanine (L-Dopa), glucagon, vasopressin, PACAP (pituitary adenylyl cyclase activating peptide), muscarinic receptor agonists and a synthethic hexapeptide, GHRP (growth hormone releasing peptide) release endogenous growth hormone either by a direct effect on the pituitary or by affecting the release of GHRH and/or somatostatin from the hypothalamus.
  • the protein nature of growth hormone makes anything but parenteral administration non-viable.
  • other directly acting natural secretagogues e.g., GHRH and PACAP, are longer polypeptides for which reason oral administration of them is not viable.
  • the present invention relates to a compound of general formula I
  • n 0, 1 or 2
  • A, R 1 or R 2 is an aryl or branched or linear C 1-6 -alkyl substituted with aryl;
  • R 3 , R 4 , R 5 , and R 6 are independently hydrogen, aryl or C 1-6 -alkyl optionally substituted with halogen, amino, hydroxy or aryl;
  • U is -O-, -S- or a valence bond
  • o and r are independently 0, 1, 2, 3 or 4,
  • s is 0 or 1
  • r + s is 1, 2, 3 or 4; or a pharmaceutically acceptable salt thereof, and the compounds of formula I comprise any optical isomers thereof, in the form of separated, pure or patially purified optical isomers or racemic mixtures thereof.
  • C 1-6 -alkyl groups are the C 1-3 -alkyl groups.
  • Preferred C 1-3 -alkyl groups are methyl, ethyl, isopropyl, and cyclopropyl.
  • the C 1-6 -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 alkyloxy 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.
  • C 1-6 -alkoxy groups are the C 1-3 -alkoxy groups.
  • Preferred C 1-3 -alkoxy groups are methoxy, ethoxy, isopropoxy, and cyclopropyloxy.
  • aryl is intended to include aromatic rings, such as carbocyclic and heterocyclic aromatic rings selected from the group consisting of phenyl, naphthyl, pyridyl, 1-H-tetrazol-5-yl, thiazolyl, imidazolyl, indolyl, pyrimidinyl, thiadiazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiopheneyl, quinolinyl, pyrazinyl, or isothiazolyl, optionally substituted by one or more C 1-6 -alkyl, C 1-6 -alkoxy, halogen, amino or aryl.
  • aromatic rings such as carbocyclic and heterocyclic aromatic rings selected from the group consisting of phenyl, naphthyl, pyridyl, 1-H-tetrazol-5-yl, thiazolyl, imidazolyl, indolyl, pyrimidinyl,
  • Compounds of formula I may be prepared from natural or non-natural amino acid residues as shown in one of the following reaction schemes.
  • the non-natural amino acid residues may be prepared according to methods known to those skilled in the art.
  • Compounds of formula I may be prepared by the method shown in reaction scheme I starting with a primary amine 1, which may be either commercially available or prepared by methods known to those skilled in the art, e.g. peptide coupling methodologies described in the art (e.g. DCC coupling in DMF), and an aldehyde under reductive conditions e.g. with sodium cyanoborohydride in methanol/acetic acid to give the compound 2.
  • the compound 4 may be prepared from 2 and e.g. an isothiocyanate 3 in an appropriate solvent such as tetrahydrofuran to give the compound 4.
  • the compound 4 may be deprotonated by a base such as sodium hydride in an appropriate solvent such as tetrahydrofuran and alkylated with an appropriate alkylhalogen such as benzylbromide to give the compound 5 which is a compound of the formula I.
  • a base such as sodium hydride
  • an appropriate solvent such as tetrahydrofuran
  • an appropriate alkylhalogen such as benzylbromide
  • Functional groups in intermediates in reaction scheme I may be protected and deprotected using a strategy known in the art and described by e.g. T.W. Greene (Protective Groups in Organic Synthesis, 2nd Ed., John Wiley and Sons 1991).
  • Compounds of formula I may be prepared by the method shown in reaction scheme II starting with a primary amine 6 which under basic conditions such as lithium diisopropylamide in an appropriate solvent such as tetrahydrofuran may be alkylated with an alkyl halogenide 7 such as 2-bromo-3-phenyl-propanoic acid methyl ester to give the secondary amine 8.
  • the compound 10 may be prepared from 8 and e.g. and isothiocyanate 9 in an appropriate solvent such as tetrahydrofuran to give the compound 10.
  • the compound 10 may be deprotonated by a base such as sodium hydride in an appropriate solvent such as tetrahydrofuran and alkylated with an appropriate alkylhalogenide such as benzylbromide to give the compound 5 which is a compound of the formula I.
  • a base such as sodium hydride
  • an appropriate solvent such as tetrahydrofuran
  • an appropriate alkylhalogenide such as benzylbromide
  • Pharmaceutically acceptable acid addition salts of compounds of formula I include those prepared by reacting the compound with an inorganic or organic acid such as hydrochloric, hydrobromic, sulfuric, acetic, phosphoric, lactic, maleic, phthalic, citric, glutaric, gluconic, methanesulfonic, salicylic, succinic, tartaric, toluenesulfonic, trifluoracetic, sulfamic or fumaric acid.
  • an inorganic or organic acid such as hydrochloric, hydrobromic, sulfuric, acetic, phosphoric, lactic, maleic, phthalic, citric, glutaric, gluconic, methanesulfonic, salicylic, succinic, tartaric, toluenesulfonic, trifluoracetic, sulfamic or fumaric acid.
  • an inorganic or organic acid such as hydrochloric, hydrobromic, sulfuric, acetic, phosphoric, lactic
  • compositions containing a compound of the present invention may be prepared by conventional techniques, e.g. as described in Remington's Pharmaceutical Sciences, 1985.
  • 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 comprising 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 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, stimulation of the immune system, acceleration of wound healing, accelerating bone fracture repair, treatment of growth retardation, treating renal failure or insufficiency resulting from growth retardation, 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, 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 patients, treatment of osteochondrodysplasias, Noonan's syndrome, schizophrenia, depressions, Alzheimer's disease, delayed wound healing and psychosocial deprivation, treatment of pulmonary dysfunction and ventilator dependency, attenuation of
  • 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.
  • 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 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 alkylammonium salt. Such salt forms are believed to exhibit approximately the same order of activity as the free base forms.
  • 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.
  • rat pituitary cells may be evaluated in vitro for their efficacy and potency to release growth hormone in rat pituitary primary cultures.
  • 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 animals/cage) and placed in rooms with 12 hour light cycle. The room temperature varied from 19-24oC 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 medium (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 37oC 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 pipet, 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 043-0503OH), 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 ⁇ 10 5 cells/ml.
  • the cells were seeded into microtiter plates (Nunc, Denmark), 200 ml/well, and cultured for 3 days at 37oC 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 37oC.
  • 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 37oC and 5% CO 2 .
  • the medium was decanted 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 instrument 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%.
  • TLC thin layer chromatography and THF is tetrahydrofuran, CDCl 3 is deuterio chloroform, DMSO-d 6 is hexadeuterio dimethylsulfoxide and CD 3 OD is tetradeuterio methanol.
  • the structure of the compounds are confirmed by either elemental analysis or NMR, where peaks assigned to characteristic protons in the title compounds are presented where appropriate. 1 H NMR shift (d H ) are given in parts per million (ppm). M.p. is melting point and is given in oC and is not corrected. Column chromatography was carried out usin the technique described by W.C. Still et al., J. Org. Chem.
  • hydrochloride The hydrochloride was dissolved in 600 ml of water and methanol (1:1) and 200 ml of saturated sodium bicarbonate was added. The solution was concentrated in vacuo to a minimum and extracted 4 times with 200 ml ethylacetate, dried over magnesium sulphate and concentrated in vacuo to give 9.9 g (86%) of N-benzyl-N-((naphth-2-yl)methyl)amine.
  • N-Benzyl-N-((naphth-2-yl)methyl)amine (1.0 g, 4.0 mmol) in 20 ml of THF was added to a solution of 3-(morpholin-4-yl)propyl isothiocyanate (750 mg, 4.0 mmol) in 30 ml of THF at -78oC over a period of 10 minutes.
  • the mixture was stirred at room temperature overnight and the solvent was removed in vacuo and the obtained oil was chromatographed on 500 ml of silica gel in 10% methanol/methylene chloride to give an oil which was dissolved in 20 ml ethyl acetate to which was added 10 ml of 3 M C1 in ethyl acetate.
  • isothiocyanate (0.9 g, 4.7 mmol) in 20 ml of THF was added over a period of 10 minutes, and the mixture was stirred at room temperature overnight.
  • the solution was concentrated in vacuo and the residue was chromatographed on 500 ml of silic gel with 10 % methanol/methylene chloride to an oil, which was dissolved in 15 ml of ethyl acetate to which was added 15 ml of 3M HCl in ethyl acetate.
  • n 0, 1 or 2
  • A, R 1 or R 2 is an aryl or branched or linear C 1-6 -alkyl substituted with aryl;
  • A is hydrogen, -CONR 3 R 4 , -CONR 3 CHR 4 CONR 5 R 6 , -COOR 3 , -CH 2 NR 3 R 4 or -CH 2 OR 3 ,
  • R 3 , R 4 , R 5 , and R 6 are independently hydrogen, aryl or C 1-6 -alkyl optionally substituted with halogen, amino, hydrox or aryl;
  • R 7 is hydrogen or C 1-6 -alkyl
  • R 8 , R 9 , R 10 and R 11 are independently hydrogen or C 1-6 -alkyl optionally substituted with halogen, amino, hydroxy or aryl, R 8 and R 9 , R 10 and R 11 , R 8 and R 10 or R 9 and R 11 optionally forming -(CH 2 ) i -U-(CH 2 ) j -, wherein i and j are independently 1 or 2,
  • U is -O-, -S- or a valence bond
  • o and r are independently 0, 1, 2, 3 or 4 ,
  • s is 0 or 1
  • r + s is 1, .2, 3 or 4; which compounds of formula I promote the release of growth hormone in humans and animals. This property can be utilized to promote the growth of food animals to render the
  • compositions containing such compounds of formula I as the active ingredient thereof, methods of stimulating the release of growth hormone as well as use of such compounds of formula I are also disclosed.

Abstract

There are disclosed novel compounds of general formula (I) which compounds of formula (I) promote the release of growth hormone in humans and animals. This property can be utilized to promote the growth of food animals to render the production of edible meat products more efficient, and in humans, to increase the status of those afflicted with a lack of a normal secretion of natural gowth hormone. Growth promoting compositions containing such compounds of formula (I) as the active ingredient thereof, methods of stimulating the release of growth hormone as well as use of such compounds of formula (I) are also disclosed.

Description

COMPOUNDS WITH GROWTH HORMONE RELEASING PROPERTIES
FIELD OF INVENTION
The present invention relates to novel compounds, compositions containing them, and their use for treating medical disorders resulting from a deficiency in growth hormone. BACKGROUND OF THE INVENTION
Growth hormone is a hormone which stimulates growth of all tissues capable of growing. In addition, 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 primarily via specific receptors located in the pituitary. Other compounds which stimulate the release of growth hormone from the pituitary have also been described. For example arginine, L-3,4-dihydroxyphenylalanine (L-Dopa), glucagon, vasopressin, PACAP (pituitary adenylyl cyclase activating peptide), muscarinic receptor agonists and a synthethic hexapeptide, GHRP (growth hormone releasing peptide) release endogenous growth hormone either by a direct effect on the pituitary or by affecting the release of GHRH and/or somatostatin from the hypothalamus. In disorders or conditions where increased levels of growth hormone is desired, the protein nature of growth hormone makes anything but parenteral administration non-viable. Furthermore, other directly acting natural secretagogues, e.g., GHRH and PACAP, are longer polypeptides for which reason oral administration of them is not viable.
The use of certain compounds for increasing the levels of growth hormone in mammals has previously been proposed, e.g. in
EP 18072, EP 83 864, WO 89/07110, WO 89/01711, WO 89/10933, WO 88/9780, WO 83/02272, WO 91/18016, WO 92/01711, WO 93/04081 WO 95/17422, WO 95/17423 and WO 95/14666.
The composition of growth hormone releasing compounds is important for their growth hormone releasing potency as well as their bioavailability. It is therefore the object of the present invention to provide compounds with growth hormone releasing properties which have improved properties relative to known compounds of this type.
SUMMARY OF THE INVENTION
Accordingly, the present invention relates to a compound of general formula I
Figure imgf000004_0001
wherein
m is 0, 1 or 2,
R1 and R2 are independently hydrogen, aryl or C1-6-alkyl optionally substituted with halogen, amino, hydroxy or aryl, W is =S, =O, =NH or =N(CN),
with the proviso that at least one of A, R1 or R2 is an aryl or branched or linear C1-6-alkyl substituted with aryl;
A is hydrogen, -CONR3R4, -CONR3CHR4CONR5R6, -COOR3, -CH2NR3R4 or -CH2OR3,
wherein R3, R4, R5, and R6 are independently hydrogen, aryl or C1-6-alkyl optionally substituted with halogen, amino, hydroxy or aryl;
B is
Figure imgf000005_0005
.
Figure imgf000005_0001
Figure imgf000005_0002
or
Figure imgf000005_0004
Figure imgf000005_0003
Figure imgf000005_0006
optionally substituted with halogen, carboxamido, tetrazolyl, oxadiazolyl, thiadiazolyl, amino, hydroxy, C1-6-alkyl or C1-6-alkoxy, wherein R7 is hydrogen or C1-6-alkyl; D is
Figure imgf000006_0001
wherein R8, R9, R10 and R11 are independently hydrogen or C1-6-alkyl optionally substituted with halogen, amino, hydroxy or aryl, Rβ and R9, R10 and R11, R8 and R10 or R9 and R11 optionally forming -(CH2)i-U-(CH2)j-, wherein i and j are independently 1 or 2,
U is -O-, -S- or a valence bond,
o and r are independently 0, 1, 2, 3 or 4,
s is 0 or 1, and
r + s is 1, 2, 3 or 4; or a pharmaceutically acceptable salt thereof, and the compounds of formula I comprise any optical isomers thereof, in the form of separated, pure or patially purified optical isomers or racemic mixtures thereof.
It is believed that compounds of formula I exhibit an improved bioavailability because they contain no amide bonds susceptible to cleavage by proteolytic enzymes. The increased resistance to proteolytic degradation combined with the reduced size of the compounds of the invention in comparison with known growth hormone releasing compounds is expected to improve their bioavailability compared to that of the compounds suggested in the prior literature.
In the above structural formulas and throughout the present specification, the following terms have the indicated meanings: The C1-6-alkyl groups specified above are intended to include those alkyl groups of the designated length in either a linear or branched, or cyclic configuration. Examples of linear alkyl are methyl, ethyl, propyl, butyl, pentyl, and hexyl. Examples of branched alkyl are isopropyl, sec-butyl, tert-butyl, isopentyl, and isohexyl. Examples of cyclic alkyl are cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl.
Especially preferred C1-6-alkyl groups are the C1-3-alkyl groups. Preferred C1-3-alkyl groups are methyl, ethyl, isopropyl, and cyclopropyl.
The C1-6-alkoxy groups specified above are intended to include those alkoxy groups of the designated length in either a linear or branched or cyclic configuration. Examples of linear alkyloxy are methoxy, ethoxy, propoxy, butoxy, pentoxy, and hexoxy. Examples of branched alkoxy are isopropoxy, sec-butoxy, tert-butoxy, isopentoxy, and isohexoxy. Examples of cyclic alkoxy are cyclopropyloxy, cyclobutyloxy, cyclopentyloxy and cyclohexyloxy.
Especially preferred C1-6-alkoxy groups are the C1-3-alkoxy groups. Preferred C1-3-alkoxy groups are methoxy, ethoxy, isopropoxy, and cyclopropyloxy.
The C1-6-alkylamino groups specified above are intended to include those alkylamino groups of the designated length in either a linear or branched or cyclic configuration. Examples of linear alkylamino are methylamino, ethylamino, propylamino, butylamino, pentylamino, and hexylamino. Examples of branched alkylamino are isopropylamino, sec-butylamino, tert-butylamino, isopentylamino, and isohexylamino. Examples of cyclic alkylamino are cyclopropylamino, cyclobutylamino, cyclopentylamino and cyclohexylamino. Especially preferred C1-6-alkylamino groups are the C1-3-alkylamino groups. Preferred C1-3-alkylamino groups are methylamino, ethylamino, isopropylamino, and cyclopropylamino.
In the present context, the term "aryl" is intended to include aromatic rings, such as carbocyclic and heterocyclic aromatic rings selected from the group consisting of phenyl, naphthyl, pyridyl, 1-H-tetrazol-5-yl, thiazolyl, imidazolyl, indolyl, pyrimidinyl, thiadiazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiopheneyl, quinolinyl, pyrazinyl, or isothiazolyl, optionally substituted by one or more C1-6-alkyl, C1-6-alkoxy, halogen, amino or aryl. Aryl is preferably phenyl, thienyl, imidazolyl, pyridyl, indolyl, guinoline or naphthyl optionally substituted with halogen, amino, hydroxy, C1-6-alkyl or C1-6-alkoxy. The term "halogen" is intended to include Cl, F, Br and I. The compounds of the present invention may have one or more asymmetric centres and stereoisomers in the form of separated, pure or partially purified stereoisomers or racemic mixtures thereof are intended to be included in the scope of the invention. DETAILED DESCRIPTION OF THE INVENTION
Examples of specific compounds of the present invention are
1-Benzyl-3-(3-(morpholin-4-yl)propyl)-1-(naphth-2-yl)methylthiourea
Figure imgf000008_0001
1-Benzyl-3-(3-dimethylaminopropyl)-1-phenylthiourea
Figure imgf000009_0001
2-[3-(3-(Morpholin-4-yl)propyl)-1-(naphth-2-yl)methyl- thioureido]-3-phenylpropionamide
Figure imgf000009_0002
N-(4-Aminobutyl)-2-[3-((3-amino-3-methyl)butyl)-1-(naphth-2-yl)methylthioureido]-3-phenylpropionamide
Figure imgf000009_0003
N-(4-Aminobutyl)-2-(N-(naphth-2-yl)methyl-N'-(piperidin-3-yl)methyl-guanidino)-3-phenyl-propionamide
Figure imgf000010_0002
N-(4-Aminobutyl)-2-[1-methyl-3-(naphth-2-yl)methyl-3-(2-(piperidin-2-yl)ethyl)-thioureido]-3-(naphth-2-yl)propionamid
Figure imgf000010_0001
3-(3-(Morpholin-4-yl)propyl)-1-(naphth-2-yl)methyl-1-[2'-(1H-tetrazol-5-yl)-biphenyl-4-ylmethyl]thiourea
Figure imgf000011_0001
N-((1-Carbamoyl-2-phenyl)ethyl-N-methyl-2-[3-((3-morpholin-4-yl)propyl)-thioureido]-3-(naphth-2-yl)propionamide
Figure imgf000011_0002
3-(3-(Dimethylamino)propyl)-1-(naphth-1-yl)methyl-1-phenylthiourea
Figure imgf000012_0003
1-Benzyl-3-(3-(morpholin-4-yl)propyl)-1-phenylthiourea
Figure imgf000012_0002
1,1-Dibenzyl-3-(3-(morpholin-4-yl)propyl)thiourea
Figure imgf000012_0001
1-Benzyl-3-(3-(dimethylamino)propyl)-1-((naphth-2-yl)methyl)thiourea
Figure imgf000013_0001
1-Benzyl-3-(3-(morpholin-4-yl)propyl)-1-(pheneth-2-yl)thiourea
Figure imgf000013_0002
1-Benzyl-3-(3-(morpholin-4-yl)propyl)-1-(guinolin-3-yl)thiourea
Figure imgf000014_0001
1-Benzyl-3-(3-(morpholin-4-yl)propyl)-1-(pyridin-2-yl)thiourea
Figure imgf000014_0002
1-Benzyl-3-(3-(morpholin-4-yl)propyl)-1-(4-methoxyphenyl)thiourea
Figure imgf000015_0002
1-Benzyl-3-(3-(morpholin-4-yl)propyl)-1-(4-([1,2,3]thiadiazol-4-yl)benzyl)thiourea
Figure imgf000015_0001
3-(3-Dimethylaminopropyl)-1-((naphth-2-yl)methyl)-1-phenylthiourea
Figure imgf000016_0001
Compounds of formula I may be prepared from natural or non-natural amino acid residues as shown in one of the following reaction schemes. The non-natural amino acid residues may be prepared according to methods known to those skilled in the art.
General Method A
Figure imgf000017_0001
Compounds of formula I may be prepared by the method shown in reaction scheme I starting with a primary amine 1, which may be either commercially available or prepared by methods known to those skilled in the art, e.g. peptide coupling methodologies described in the art (e.g. DCC coupling in DMF), and an aldehyde under reductive conditions e.g. with sodium cyanoborohydride in methanol/acetic acid to give the compound 2. The compound 4 may be prepared from 2 and e.g. an isothiocyanate 3 in an appropriate solvent such as tetrahydrofuran to give the compound 4. The compound 4 may be deprotonated by a base such as sodium hydride in an appropriate solvent such as tetrahydrofuran and alkylated with an appropriate alkylhalogen such as benzylbromide to give the compound 5 which is a compound of the formula I. Functional groups in intermediates in reaction scheme I may be protected and deprotected using a strategy known in the art and described by e.g. T.W. Greene (Protective Groups in Organic Synthesis, 2nd Ed., John Wiley and Sons 1991).
General Method B
Figure imgf000018_0001
Compounds of formula I may be prepared by the method shown in reaction scheme II starting with a primary amine 6 which under basic conditions such as lithium diisopropylamide in an appropriate solvent such as tetrahydrofuran may be alkylated with an alkyl halogenide 7 such as 2-bromo-3-phenyl-propanoic acid methyl ester to give the secondary amine 8. The compound 10 may be prepared from 8 and e.g. and isothiocyanate 9 in an appropriate solvent such as tetrahydrofuran to give the compound 10. The compound 10 may be deprotonated by a base such as sodium hydride in an appropriate solvent such as tetrahydrofuran and alkylated with an appropriate alkylhalogenide such as benzylbromide to give the compound 5 which is a compound of the formula I. In cases where the compounds 6,7,8,9 or/and 10 contains any primary or secondary amino functionalities an appropriate protection and deprotection strategy known in the art and described by e.g. T.W. Greene (Protective Groups in Organic Synthesis, 2nd Ed., John Wiley and Sons 1991) is used.
Pharmaceutically acceptable acid addition salts of compounds of formula I include those prepared by reacting the compound with an inorganic or organic acid such as hydrochloric, hydrobromic, sulfuric, acetic, phosphoric, lactic, maleic, phthalic, citric, glutaric, gluconic, methanesulfonic, salicylic, succinic, tartaric, toluenesulfonic, trifluoracetic, sulfamic or fumaric acid. In another aspect, 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. Pharmaceutical compositions containing a compound of the present invention may be prepared by conventional techniques, e.g. as described in Remington's Pharmaceutical Sciences, 1985. 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. Examples of solid carriers are lactose, terra alba, sucrose, cyclodextrin, talc, gelatin, agar, pectin, acacia, magnesium stearate, stearic acid or lower alkyl ethers of cellulose. Examples of liquid carriers are syrup, peanut oil, olive oil, phospholipids, fatty acids, fatty acid amines, polyoxyethylene or water.
Similarly, 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.
If a solid carrier is used for oral administration, 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. If a liquid carrier is used, 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:
Core:
Figure imgf000020_0001
*Acylated monoglyceride used as plasticizer for film coating.
For nasal administration, the preparation may contain a compound of formula I dissolved or suspended in a liquid carrier, in particular an aqueous carrier, for aerosol application. 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.
Generally, the compounds of the present invention are dispensed in unit dosage form comprising 50-200 mg of active ingredient together with a pharmaceutically acceptable carrier per unit dosage.
The dosage of the compounds according to this invention is suitably 0.1-500 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.
It has been demonstrated that compounds of the general formula I possess the ability to release endogenous growth hormone in vivo. 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.
Thus, in a particular aspect, 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. In a further aspect, 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.
In a still further aspect, 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.
To those skilled in the art, it is well known that the current and potential uses of growth hormone in humans are varied and multitudinous. Thus, 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, stimulation of the immune system, acceleration of wound healing, accelerating bone fracture repair, treatment of growth retardation, treating renal failure or insufficiency resulting from growth retardation, 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, 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 patients, treatment of osteochondrodysplasias, Noonan's syndrome, schizophrenia, depressions, Alzheimer's disease, delayed wound healing and psychosocial deprivation, treatment of pulmonary dysfunction and ventilator dependency, attenuation of protein catabolic responses after major surgery, reducing cachexia and protein loss due to chronic illness such as cancer or AIDS; treatment of hyperinsulinemia including nesidioblastosis, adjuvant treatment for ovulation induction; to stimulate thymic development and prevent the age-related decline of thymic function, treatment of immunosuppressed patients, improvement in muscle strength, mobility, maintenance of skin thickness, metabolic homeostasis, renal homeostasis in the frail elderly, stimulation of osteoblasts, bone remodelling and cartilage growth, stimulation of the immune system in companion animals and treatment of disorder of aging in companion animals, growth promoter in livestock and stimulation of wool growth in sheep.
For the above indications the dosage will vary depending on the compound of formula I employed, on the mode of administration and on the therapy desired. However, generally 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. Usually, 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 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 alkylammonium salt. Such salt forms are believed to exhibit approximately the same order of activity as the free base forms.
Optionally, 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.
Apart from the pharmaceutical use of the compounds of formula I, they may be useful in vitro tools for investigating the regulation of growth hormone release.
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.
A further use of 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. Pharmacological Methods
Compounds of formula I may be evaluated in vitro for their efficacy and potency to release growth hormone in rat pituitary primary cultures. 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 Møllegaard, Lille Skensved, Denmark. The rats were housed in group cages (four animals/cage) and placed in rooms with 12 hour light cycle. The room temperature varied from 19-24ºC 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 medium (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 37ºC in a 95/5% atmosphere of O2/CO2. The tissue was then washed three times in the above buffer. Using a standard pasteur pipet, 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 043-0503OH), 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 T3 (Sigma T-2752) and 40 mg/L dexamethasone (Sigma D-4902) pH 7.3 , to a density of 2 × 105 cells/ml. The cells were seeded into microtiter plates (Nunc, Denmark), 200 ml/well, and cultured for 3 days at 37ºC and 8% CO2. Compound testing
After culturing, 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 37ºC. 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 37ºC and 5% CO2. The medium was decanted and analyzed for GH content in an rGH SPA test system.
All compounds were tested in doses ranging from 10 pM to 100 mM. A dose-response relation was constructed using the Hill equation (Fig P, Biosoft). The efficacy (maximal GH released, Emax) was expressed in % of the Emax of GHRP-6. The potency (EC50) was determined as the concentration inducing half maximal stimulation of the GH release.
Compounds of formula I may be evaluated for their metabolic stability.
Compounds were dissolved at a concentration of 1 mg/ml in water. 25 ml of this solution is added to 175 ml of the respective enzyme-solution (resulting in an enzyme:substrate ratio (w/w) of approximately 1:5). The solution is left at 37°C overnight. 10 ml of the various degradation solutions is analyzed against a corresponding zero-sample using flow injection electrospray mass spectrometry (ESMS) with selected ion monitoring of the molecular ion. If the signal has decreased more than 20% compared to the zero-sample, the remainder of the solution is analyzed by HPLC and mass spectrometry in order to identify the extent and site(s) of degradation precisely. Several standard peptides (ACTH 4-10, Angiotensin 1-14 and Glucagon) have been included in the stability tests in order to verify the. ability of the various solutions to degrade peptides.
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 (before and after degradation) was done on the API III instrument 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. 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) .
Figure imgf000029_0001
Any novel feature or combination of features described herein is considered essential to this invention.
The invention is further illustrated in the following
examples which are not in any way intended to limit the scope of the invention as claimed.
The present invention is further illustrated in the following examples which are not in any way intended to limit the scope of the invention as claimed. EXAMPLES
Herinafter, TLC is thin layer chromatography and THF is tetrahydrofuran, CDCl3 is deuterio chloroform, DMSO-d6 is hexadeuterio dimethylsulfoxide and CD3OD is tetradeuterio methanol. The structure of the compounds are confirmed by either elemental analysis or NMR, where peaks assigned to characteristic protons in the title compounds are presented where appropriate. 1H NMR shift (dH) are given in parts per million (ppm). M.p. is melting point and is given in ºC and is not corrected. Column chromatography was carried out usin the technique described by W.C. Still et al., J. Org. Chem. (1978), 43, 2923-2925 on Merck silica gel 60 (Art. 9385). HPLC analysis was performed using a 5mm C18 4x250 mm column, eluting with 20-80 % gradient of 0.1 % trifluoroacetic acid/acetonitrile and 0.1 % trifluoroacetic acid/water over 30 minutes at 35 ºC. All reactions were carried out under an atmosphere of nitrogen. THF was distilled over sodium and benzophenone before use. Compounds used as starting material are either known compounds or compounds which can readily be prepared by methods known per se.
Example 1 1-Benzyl-3- ( 3- (morpholi n-4-yl) propyl ) -1- ( (naphth-2-yl)methyl) thiourea-hydrochl oride
Figure imgf000030_0001
A solution of benzylamine (5.0 g, 47 mmol) and 2-naphthaldehyde (7.3 g, 47 mmol) in 200 ml of 99.9 % ethanol was refluxed overnight and cooled to room temperature. Sodium borohydride (1.8 g, 47 mmol) was added in small portion over a period of 15 minutes. After 30 minutes 400 ml of water was added and the solution was concentrated in vacuo to a minimum and extracted 4 times with 200 ml ethyl acetate and THF
(1:1). The combined organic layer was dried over magnesium sulfate, filtered and concentrated in vacuo to give an oil which was dissolved in 100 ml of ethyl acetate and 50 ml of 3 M C1 in ethyl acetate was added and a white solid
precipitated. The precipitate was washed four times with 50 ml of ethyl acetate and dried overnight in vacuo to give 11.3 g (86 %) of N-benzyl-N-((naphth-2-yl)methyl)amine
hydrochloride. The hydrochloride was dissolved in 600 ml of water and methanol (1:1) and 200 ml of saturated sodium bicarbonate was added. The solution was concentrated in vacuo to a minimum and extracted 4 times with 200 ml ethylacetate, dried over magnesium sulphate and concentrated in vacuo to give 9.9 g (86%) of N-benzyl-N-((naphth-2-yl)methyl)amine.
N-Benzyl-N-((naphth-2-yl)methyl)amine (1.0 g, 4.0 mmol) in 20 ml of THF was added to a solution of 3-(morpholin-4-yl)propyl isothiocyanate (750 mg, 4.0 mmol) in 30 ml of THF at -78ºC over a period of 10 minutes. The mixture was stirred at room temperature overnight and the solvent was removed in vacuo and the obtained oil was chromatographed on 500 ml of silica gel in 10% methanol/methylene chloride to give an oil which was dissolved in 20 ml ethyl acetate to which was added 10 ml of 3 M C1 in ethyl acetate. The solvent was removed in vacuo to give 1.4 g (73%) of 1-benzyl-3-(3-(morpholin-4-yl)propyl)- 1-((naphth-2-yl)methyl)thiourea-hydrochloride as a white solid. M.p. 57ºC (decomp.). 1H NMR (400 MHZ, CDCl3) : d 1.70 (t, 2H), 2.15 (d, 4H), 2.25 (t, 2H), 3.25 (d, 2H), 3.80 (q, 2H) , 4.90 (s, 2H) , 5.10 (s, 2H), 7.10-7.90 (m, 12H).
Calculated for C26H31N3OS , HCl , H2O:C, 63.9 %; H, 6.4 %; N, 8.6 % Found: C, 63.4 %; H, 7.2 %; N, 8.5 %
Reverse Phase HPLC: 28 min.
Example 2
3-(3-(Dimethylamino)propyl)-1-(naphth-1-yl)methyl-1-phenylthiourea-hydrochloride
Figure imgf000032_0001
A solution of aniline (7.0 g, 75 mmol) and 1-naphthaldehyde (11.7 g, 75 mmol) in 250 ml ethanol was stirred overnight at room temperature. Then small portions of sodium borohydride (2.8 g, 75 mmol) were added over a period of 20 minutes and the mixture was left to stir for 2 h. Then 250 ml of water was added and the mixture was concentrated in vacuo to a minimum and extracted 4 times with 200 ml of ethyl acetate, dried over magnesium sulfate and concentrated in vacuo to 15.9 (89%) of N-((naphth-1-yl)methyl) aniline as an oil. N-((naphth-1-yl))methylaniline (1.0 g, 4.3 mmol) was
dissolved in 20 ml of THF at -78ºC and lithium
diisopropylamide (2.4 ml of a 2.0 M solution in THF) was slowly added and a solution of (3-(dimethylamino)propyl)isothiocyanate in 15 ml of THF was added. The mixture was stirred overnight and concentrated in vacuo and the resulting oil was chromatographed on 250 ml of silica gel with 20% methanol/methylene chloride to give an oil which was
dissolved in 20 ml ethyl acetate to which was added 10 ml of 3 M HCl in ethyl acetate. The solution was concentrated in vacuo to give 670 mg (38 %) of 3-(3-(dimethylamino)propyl)-1-(naphth-1-yl)methyl-1-phenyl-thiourea-hydrochloride as a hygroscopic foam which rapidly turned oily.
1H NMR (400 MHz, CDCl3, free base): d 2.00 (t, 2H), 2.90 (s, 6H), 3.12 (t, 2H), 3.72 (t, 2H), 5.90 (s, 2H), 6.8-8.3 (m, 12H).
Calculated for C23H27N3S,HCl,3/2 H2O; 62.6 %; H, 7.0 %; N, 9.5 % Found: C, 62.6 %; H, 7.3 %; N, 9.2 %
Reverse Phase HPLC: 23 min. Example 3
1-Benzyl-3- ( 3- (morpholin-4-yl ) propyl ) -1-phenylthiourea
Figure imgf000033_0001
A solution of N,N-benzylphenylamine (0.5 g, 2.7 mmol) and 3-(morpholin-4-yl)propyl isothiocyanate ( 0.5 g, 2.7 mmol) in THF was heated at reflux for 20 minutes and cooled to room temperature. The solvent was removed in vacuo and the
remaining oil was chromatographed on 300 ml of silicagel wit 20 % methanol/methylene chloride to give 170 mg (17%) of 1-benzyl-3-(3-(morpholin-4-yl)propyl)-1-phenylthiourea as an oil.
1H NMR (400 MHz, CDCl3, free base): d 2.00 (t, 2H), 3.07 (t, 2H), 3.25 (m, 4H), 3.70 (t, 2H), 3.85 (m, 4H), 5.45 (s, 2H), 7.00-7.5 (m, 10H).
Reverse Phase HPLC: 19 min.
Example 4
1-Benzyl-3-(3-(dimethylamino)prooyl)-1-phenylthioureahydrochloride
Figure imgf000034_0001
To a solution of 3- (dimethylamino) propyl isothiocyanate (1.5 g, 17.8 mmol) in 25 ml of THF at -78ºC was added N-benzylaniline (3.3 g, 17.8 mmol) in 20 ml of THF over a period of 15 minutes. The mixture was allowed to warm to room temperature and stirred overnight. The solvent was removed in vacuo and the resulting oil was chromatographed on 300 ml of silica gel with 20 % methanol/methylene chloride to give an oil which was dissolved in 30 ml of ethyl acetate to which was added 30 ml of 3M HCl in ethyl acetate. The solution was concentrated in vacuo to give 2.5 g (38 %) of 1-benzyl-3-(3-(dimethylamino)propyl)-1-phenylthiourea-hydrochloride as a white solid. M.p. 180-181 ºC.
1H NMR (200 MHz, CDCl3, free base): d 2.10 (t, 2H), 2.55 (s, 6H), 2.85 (t, 2H), 3.75 (t, 2H), 5.49 (s, 2H), 6.3 (b, 1H), 6.9-7.5 (m, 10H) .
Calculated for C19H25N3S, HCl:
C, 62.6 %; H, 7.2 %; N, 11.5 %; Cl, 9.7 %
Found: C, 62.1 %; H, 7.3 %; N, 11.3 %; Cl, 9.3 %
Reverse Phase HPLC: 19 min.
Example 5
1,1-Dibenzyl-3-(3-(morpholin-4-yl)propyl)thiourea¬hydrochloride
Figure imgf000036_0001
To a solution of 3-((morpholin-4-yl)propyl) isothiocyanate (1.0 g, 5.4 mmol) in 20 ml of THF at -78 ºC was added N,N-dibenzylamine (1.1 g, 5.4 mmol) in 15 ml of THF over a period of 15 min. The mixture was allowed to warm to room
temperature and stirred overnight. The solvent was removed in vacuo and the residue was chromatographed on 300 ml of silicagel with 20 % methanol/methylene chloride to give an oil, which was dissolved in 30 ml of ethyl acetate to which was added 30 ml of 3M HCl in ethyl acetate. The solution was concentrated in vacuo to give 2.1 g (88 %) of 1,1-dibenzyl-3-(3-(morpholin-4-yl)propyl)thiourea-hydrochloride as an amorphous powder.
1H NMR (200 MHz, CD3OD) : d 2.00 (t, 2H), 3.00 (t, 4H), 3.45 (t, 2H), 3.7 (t, 4H), 4.05 (t, 2H), 5.00 (s, 4H), 7.2-7.4 (m, 10H) . Calculated for C22H30N3OS, HCl :
C, 62.7 %; H, 7.4 %; N, 9.9 %; Cl, 8.4 % Found : C , 62 . 6 % ; H , 7 . 4 % ; N , 9 . 9 % ; Cl , 8 . 4 %
Reverse Phase HPLC: 20 min.
Example 6
1-Benzyl-3-(3-(dimethylamino)propyl)-1-((naohth-2-yl)methyl)thiourea-hydrochloride
Figure imgf000037_0001
To a solution of 3-(dimethylamino)propyl isothiocyanate (0.34 g, 4.0 mmol) in 20 ml of THF at -78 ºC was added N-benzyl-N-((naphth-2-yl)methyl)amine (1.0 g, 4.0 mmol, prepared as in example 1) in 10 ml of THF over a period of 10 min. The mixture was allowed to warm to room temperature and stirred overnight. The solvent was removed in vacuo and the residue was chromatographed on 300 ml of silica gel with 10 %
methanol/methylene chloride to give an oil which was
dissolved in 30 ml of ethyl acetate to which was added 30 ml of 3M HCl in ethyl acetate. The solution was concentrated in vacuo to give 0.56 g (33 %) of 1-benzyl-3-(3-(morpholin-4-yl)propyl)-1-((naphth-2-yl)methyl)thiourea-hydrochloride as an amorphous powder. 1H NMR (200 MHz, CDCl3, free amine): d 1.65 (t, 2H), 1.70 (s, 6H), 2.30 (t, 2H), 3.70 (t, 2H), 4.90 (s, 2H), 5.05 (s, 2H), 7.2-7.9 (m, 12H).
Calculated for C22H29N3S,HCl, 1/3 H2O:
C, 66.3 %; H, 7.1 %; N, 9.7 %
Found: C, 66.1 %; H, 7.7 %; N, 9.5 %
Reverse Phase HPLC: 23 min.
Example 7
1-Benzyl-3-(3-(morpholin-4-yl)propyl)-1-(pheneth-2-yl)thiourea-hydrochloride
Figure imgf000038_0001
To a solution of N-benzyl-N-(pheneth-2-yl) amine (3.0 g, 9.7 mmol) in 10 ml of THF at 0ºC was slowly added 3-(morpholin-4-yl)propyl isothiocyanate in 10 ml of THF. After 15 minutes the solvent was removed and the brownish oil was
chromatographed on 450 g of silica gel with 10 %
methanol/methylene chloride to give a clear oil which was dissolved in 10 ml of ethyl acetate to which was added 10 ml of 3 M HCl in ethyl acetate to give 1.8 g (40 %) of 1-benzyl-3-(3-(morpholin-4-yl)propyl)-1-(pheneth-2-yl)thioureahydrochloride as a white solid. M.p. 159-160 ºC.
1H NMR (400 MHz, CDCl3, free amine): d 1.70 (t, 2H), 2.30 (t, 4H), 2.35 (t, 2H), 3.00 (t, 2H), 3.40 (t, 4H), 3.70 (t, 2H), 3.95 (t, 2H), 4.70 (s, 2H), 6.80 (b, 1H), 7.1-7.4 (m, 10H).
Reverse Phase HPLC: 22 min.
Example 8
1-Benzyl-3- (3- (morpholin-4-yl )propyl ) -1- (quinolin-3-yl) thiourea-dihydrochloride
Figure imgf000039_0001
To a solution of 3-aminoquinoline (3.0 g, 21 mmol) in 100 ml of ethanol, which had been adjusted to pH=5 with HCl in ethanol, was added benzaldehyde (2.2 g, 21 mmol) in 50 ml of ethanol and stirred overnight. Then sodium borohydride (0.8 g, 21 mmol) was added in small portions at reflux and stirred for 1 hour. The mixture was cooled to room temperature, 300 ml of water was added and the solution was concentrated to a minimum in vacuo. The remaining aqueous layer was extracted 4 times with ethyl acetate and the combined extracts were drie over magnesium sulphate and concentrated in vacuo to an oil. This oil was chromatographed on 500 ml of silicagel with 10 ethyl acetate/heptane to give 3.73 g (78 %) of N-benzyl-N-(quinoline-3-yl)amine.
To a solution of N-benzyl-N-(quinoline-3-yl)amine (1.0 g, 4.3 mmol) in 30 ml of THF at -78 ºC was added lithium
diisopropylamide (2.4 ml of a 2.0 M solution in THF) and the mixture was allowed to stir for 10 minutes. Then 3-(morpholin-4-yl)propyl isothiocyanate (0.8 g, 4.3 mmol) in 10 ml of THF was added over a period of 10 minutes and stirred at room temperature overnight. The solution was concentrated in vacuo and the residue was chromatographed on 500 ml of silicagel with 5 % methanol/methylene chloride to an oil, which was dissolved in 15 ml of ethyl acetate to which was added 15 ml of 3M HCl in ethyl acetate. The solution was concentrated in vacuo to give 1.2 g (62 %) of 1-benzyl-3-(3-(morpholin-4-yl)propyl)-1-(quinolin-3-yl)thiourea-dihydrochloride as an amorphous powder. 1H NMR (400 MHz , CDCl3, free amine): d 1.70 (t, 2H), 2.15 (t, 4H), 2.45 (t, 2H), 3.15 (t, 4H), 3.70 (m, 4H), 5.60 (s, 2H), 6.45 (s , 1H), 7.2-7.8 (m, 9H), 8.05 (d, 1H), 8.60 (s, 1H).
Calculated for C24H28N4OS,2 HCl, H2O:
C, 56.5 %; H, 6.3 %; N, 11.0 % Found: C, 56.4 %; H, 7.1 %; N, 11.1 %
Reverse Phase HPLC: 15 min.
Example 9
1-Benzyl-3-(3 (morpholin-4-yl)propyl)-1-(pyridin-2- yl)thiourea-hydrochloride
Figure imgf000041_0001
To a solution of 2-(benzylamino)pyridine (1.0 g, 5.4 mmol) in 30 ml of THF at -78 ºC was added lithium diisopropylamide (3.0 ml of a 2.0 M solution in THF) and the mixture was left to stir for 10 minutes. Then 3- (morpholin-4-yl)propyl
isothiocyanate (1.0 g, 5.4 mmol) in 10 ml of THF was added over a period of 10 minutes and stirred at room temperature overnight. The solution was concentrated in vacuo, and the residue was chromatographed on 500 ml of silica gel with 10 % methanol/methylene chloride to give an oil which was
dissolved in 15 ml of ethyl acetate to which was added 15 ml of 3M HCl in ethyl acetate. The solution was concentrated in vacuo to give 1.7 g (79 %) of 1-benzyl-3-(3-(morpholin-4-yl)propyl)-1-(pyridin-2-yl)thiourea-hydrochloride as an amorphous powder.
1H NMR (400 MHz, CDCl3, free amine): d 1.95 (t, 2H), 2.43 (t,
4H), 2.46 (t, 2H), 3.65 (t, 4H), 3.85 (t, 2H), 5.85 (s, 2H),
6.95-7.10 (m, 2H), 7.20-7.35 (m, 5H), 7.55 (t, 1H) , 8.25 (d, 1H).
Calculated for C20H26N4OS,HCl,2½H2O:
C, 53.2 %; H, 7.1 %; N, 12.4 %; Found: C, 53.5 %; H, 7.3 %; N, 12.3 %;
Reverse Phase HPLC: 15 min.
Example 10
1-Benzyl-3-(3-(morpholin-4-yl)propyl)-1-(4-methoxγphenyl)thiourea-hydrochloride
Figure imgf000042_0001
To a solution of N-benzyl-4-methoxyaniline (1.0 g, 4.7 mmol) in 30 ml of THF at -78 ºC was added lithium diisopropylamide (2.6 ml of a 2.0 M solution in THF) and the mixture was left to stir for 10 minutes. Then 3-(morpholin-4-yl)propyl
isothiocyanate (0.9 g, 4.7 mmol) in 20 ml of THF was added over a period of 10 minutes, and the mixture was stirred at room temperature overnight. The solution was concentrated in vacuo and the residue was chromatographed on 500 ml of silic gel with 10 % methanol/methylene chloride to an oil, which was dissolved in 15 ml of ethyl acetate to which was added 15 ml of 3M HCl in ethyl acetate. The solution was concentrated in vacuo to give 172 mg (8 %) of 1-benzyl-1-(4-methoxyphenyl)-3-(3-(morpholin-4-yl)propyl)thiourea-hydrochloride as an amorphous powder.
1H NMR (400 MHz, CDCl,, free amine): d 1.70 (t, 2H), 2.25 (m, 6H), 2.45 (t, 2H), 3.50 (t, 2H), 3.70 (t, 2H), 3.80 (s, 3H), 5.50 (s, 2H), 5.80 (b, 1H), 6.80-6.95 (m, 4H), 7.20-7.35 (m, 5H).
Calculated for C22H29N3O2S, HCl , 2H2O; 55.8 %; H, 7.2 %; N, 8.9 %; Found: C, 55.8 %; H, 7.2 %; N, 9.5 %;
Reverse Phase HPLC: 20 min.
Example 11
1-Benzyl-3-(3-(morpholin-4-yl)propyl)-1-(4- ([1.2.31thiadiazol-4-yl)benzyl)thiourea-hydrochloride
Figure imgf000043_0001
To a solution of benzylamine (1.7, 16 mmol) in 60 ml of THF was added 4-(4-bromomethylphenyl)-1,2,3-thiadiazole (1.0 g, 39 mmol) in 40 ml of THF over a period of 15 minutes. The mixture was stirred at room temperature overnight, 50 ml of saturated sodium hydrogencarbonate and 50 ml of water were added and the mixture was extracted 4 times with 50 ml ethyl acetate. The combined extracts were dried over magnesium sulphate and concentrated in vacuo to an oil. The crude product was chromatographed on 600 ml of silicagel with 35 % ethyl acetate/heptane to give 1.05 g (95 %) of N-benzyl-N-(4-([1,2,3]thiadiazol-4-yl)benzyl)amine.
To a solution of N-benzyl-N-(4-([1,2,3]thiadiazol-4-yl)benzyl)amine (0.95 g, 34 mmol) in 30 ml of THF at -78 ºC was added 3-(morpholin-4-yl)propyl isothiocyanate in 30 ml of THF over a period of 10 minutes. The mixture was stirred at room temperature for 2 hours and concentrated in vacuo to an oil. The crude mixture was chromatographed on 400 ml of silicagel with 10 % methanol/methylene chloride to give an oil which was dissolved in 20 ml of ethyl acetate to which was added 20 ml of 3 M of HCl in ethyl acetate and
concentrated in vacuo to give 1.37 g (81 %) of 1-benzyl-3-(3-(morpholin-4-yl)propyl)-1-(4-([1,2,3]thiadiazol-4-yl)benzyl)thiourea-hydrochloride as an amorphous powder.
1H NMR (400 MHz, CDCl3, free amine): d 2.00 (t, 2H), 3.00 (t,
4H), 3.45 (d, 2H), 3.75 (t, 4H), 4.00 (d, 2H), 5.05 (s, 2H), 5.10 (s, 2H), 7.20-7.45 (m, 7H), 8.05 (d, 2H), 9.20 (s, 1H).
Calculated for C^H^OSj,HCl,H2C; 55.2 %; H, 6.1 %; N, 13.4 % Found: C, 55.3 %; H, 6.4 %; N, 13.3 %
Reverse Phase HPLC: 23 min.
Example 12 3-(3-(Morpholin-4-yl)propyl)-1-((naphth-2-yl)methyl)-1-[2'-(1H-tetrazol-5-yl)-biphenyl-4-ylmethyl]thiourea-hydrochlorid
Figure imgf000045_0001
To a solution of 5-(4'-bromomethyl-biphenyl-2-yl)-1-trityl-1H-tetrazole (0.6 g, 1.1 mmol) prepared according to W.R. Schoen et al. (J. Med. Chem. (1994), 37, 897-906) in 40 ml of THF was added benzylamine (0.45 g, 4.3 mmol) in 30 ml of THF, and the mixture was stirred overnight. The solvent was removed in vacuo, and the remaining oil was chromatographed on 500 ml of silica gel with 70 % ethyl acetate/heptane to give 460 mg (73 %) of N-benzyl-N-[2'-(1-trityl-1H-tetrazol-5-yl)-(biphenyl-4-yl)methyl]amine.
To a solution of N-benzyl-N-[2'-(1-trityl-1H-tetrazol-5-yl)-(biphenyl-4-yl)methyl]amine (0.46 g, 0.79 mmol) in 20 ml of THF at -78 ºC was added 3-(morpholin-4-yl)propyl
isothiocyanate over a period of 10 minutes and the mixture was stirred for 2 hours at room temperature. The mixture was concentrated in vacuo to an oil which was dissolved in 20 ml of ethyl acetate, added 20 ml of 3M HCl in ethyl acetate and stirred for 90 minutes. The mixture was concentrated in vacuo and chromatographed on 300 ml of silica gel with 20 %
methanol/79.5 % methylene chloride/0.5 % ammonia to give an oil which was dissolved in 20 ml of ethyl acetate to which was added 20 ml of 3 M HCl in ethyl acetate. This solution was concentrated in vacuo to give 240 mg (54 %) of 3-(3-(morpholin-4-yl)propyl)-1-(naphth-2-yl)methyl-1-[2'-(1H-tetrazol-5-yl)-biphenyl-4-ylmethyl]thiourea- hydrochloride as an amorphous powder. 1H NMR (400 MHz, CDCl3, free amine): d 1.75 (t, 2H), 2.65 (t, 2H), 3.10 (t, 4H), 3.60 (t, 4H), 3.75 (t, 2H), 4.65 (s, 2H), 5.35 (s, 2H), 7.10-7.60 (m, 13H) .
Reverse Phase HPLC: 21 min.
Example 13 3-(3-Dimethylaminopropyl)-1-((naphth-2-yl)methyl)-1-phenylthiourea-hydrochloride
Figure imgf000046_0001
A solution of 2-naphthaldehyde (5.0 g, 32 mmol) and aniline (3.0 g, 32 mmol) in 200 ml of ethanol was refluxed for 6 hours. Then small portions of sodium borohydride (1.2 g, 32 mmol) was added, and the mixture was stirred for 1 hour at room temperature. Then 200 ml of water was added and the solution was concentrated to a minimum and was extracted 4 times with 200 ml of ethyl acetate. The combined extracts were dried over magnesium sulphate and concentrated in vacuo. The obtained oil was chromatographed on 700 ml of silicagel with 5 % ethyl acetate/heptane to give 5.2 g (70 %) of N-(naphth-2-yl)methyl-N-phenylamine. To a solution of N-(naphth-2-yl)methyl-N-phenylamine (0.70 g, 3.0 mmol) in 30 ml of THF at -78 ºC was added lithium diisopropylamine (1.7 ml of a 2 M solution in THF) and the mixture was stirred for 15 min. Then 3-(dimethylamino)propyl isothiocyanate (0.44 g, 3.0 mmol) in 20 ml of THF was added over a period of 10 minutes, and the mixture was left to stir overnight. The mixture was concentrated in vacuo and the residue was chromatographed on 500 ml of silicagel with 10 % methanol/methylene chloride to give 250 mg (20 %) of 3-(3-dimethylaminopropyl)-1-(ηaphth-2-yl)methyl-1-phenyl-thiourea as an oil which was dissolved in 20 ml of ethyl acetate to which was added 20 ml of 3M HCl in ethyl acetate to give 3-(3-dimethylaminopropyl)-1-(naph-2-yl)methyl-1-phenylthiourea-hydrochloride as an amorphous powder.
1H NMR (400 MHz, CDCl3, free amine): d 1.60 (t, 2H), 1.70 (s, 6H), 2.20 (t, 2H), 3.75 (t, 2H), 5.65 (s, 2H), 6.9-7.9 (m, 12H).
Calculated for C23H29N3S,HCl : C, 66.7 %; H, 6.8 %; N, 10.1 %; Found: C, 62.6 %; H, 6.9 %; N, 9.5 %;
Reverse Phase HPLC: 25 min.
ABSTRACT
There are disclosed novel compounds of the general formula I
Figure imgf000058_0001
I
wherein
m is 0, 1 or 2,
R1 and R2 are independently hydrogen, aryl or C1-6-alkyl optionally substituted with halogen, amino, hydroxy or aryl, W is =S, =O, =NH or =N(CN),
with the proviso that at least one of A, R1 or R2 is an aryl or branched or linear C1-6-alkyl substituted with aryl;
A is hydrogen, -CONR3R4, -CONR3CHR4CONR5R6, -COOR3, -CH2NR3R4 or -CH2OR3,
wherein R3, R4, R5, and R6 are independently hydrogen, aryl or C1-6-alkyl optionally substituted with halogen, amino, hydrox or aryl;
B is
Figure imgf000059_0001
Figure imgf000059_0002
Figure imgf000059_0003
Figure imgf000059_0005
or
Figure imgf000059_0004
Figure imgf000059_0006
optionally substituted with halogen, carboxamido, tetrazolyl, oxadiazolyl, thiadiazolyl, amino, hydroxy, C1-6-alkyl or C1-6-alkoxy, and R7 is hydrogen or C1-6-alkyl;
D is
Figure imgf000059_0007
wherein R8, R9, R10 and R11 are independently hydrogen or C1-6-alkyl optionally substituted with halogen, amino, hydroxy or aryl, R8 and R9, R10 and R11, R8 and R10 or R9 and R11 optionally forming -(CH2)i-U-(CH2)j-, wherein i and j are independently 1 or 2,
U is -O-, -S- or a valence bond, o and r are independently 0, 1, 2, 3 or 4 ,
s is 0 or 1, and
r + s is 1, .2, 3 or 4; which compounds of formula I promote the release of growth hormone in humans and animals. This property can be utilized to promote the growth of food animals to render the
production of edible meat products more efficient, and in humans, to increase the status of those afflicted with a lack of a normal secretion of natural growth hormone. Growth promoting compositions containing such compounds of formula I as the active ingredient thereof, methods of stimulating the release of growth hormone as well as use of such compounds of formula I are also disclosed.
4397.204-WO, LBKj/LSDu - 06.02.96

Claims

1. A compound of general formula I
Figure imgf000048_0001
I
wherein
m is 0, 1 or 2,
R1 and R2 are independently hydrogen, aryl or C1-6-alkyl optionally substituted with halogen, amino, hydroxy or aryl, W is =S, =O, =NH or =N(CN),
with the proviso that at least one of A, R1 or R2 is an aryl or branched or linear C1-6-alkyl substituted with aryl;
A is hydrogen, -CONR3R4, -CONR3CHR4CONR5R6, -COOR3, -CH2NR3R4 or -CH2OR3,
wherein R3, R4, R5, and R6 are independently hydrogen, aryl o C1-6-alkyl optionally substituted with halogen, amino, hydrox or aryl;
B is
Figure imgf000049_0001
Figure imgf000049_0002
.
Figure imgf000049_0003
Figure imgf000049_0005
Figure imgf000049_0004
Figure imgf000049_0006
optionally substituted with halogen, carboxamido, tetrazolyl, oxadiazolyl, thiadiazolyl, amino, hydroxy, C1-6-alkyl or C1-6-alkoxy, and R7 is hydrogen or C1-6-alkyl;
D is
Figure imgf000049_0007
wherein R8, R9, R10 and R11 are independently hydrogen or C1-6-alkyl optionally substituted with halogen, amino, hydroxy or aryl, R8 and R9, R10 and R11, R8 and R10 or R9 and R11 optionally forming -(CH2)i-U-(CH2) j-, wherein i and j are independently 1 or 2,
U is -O-, -S- or a valence bond,
o and r are independently 0, 1, 2, 3 or 4, s is 0 or 1 , and
r + s is 1 , 2 , 3 or 4 ; or a pharmaceutically acceptable salt thereof, and the compounds of formula I comprise any optical isomers thereof, in the form of separated, pure or patially purified optical isomers or racemic mixtures thereof.
2. A compound of the general formula I according to claim 1 wherein W is =S or =O;
and A, B, R1, R2, D and m are defined as in the preceding claim 1.
3. A compound of the general formula I according to any one of the preceding claims, wherein A is hydrogen or CH2OR3; and B, R1, R2, R3, D, W and m are defined as in the preceding claims. 4. A compound of the general formula I according to any one of the preceding claims, wherein B is
or
Figure imgf000050_0004
Figure imgf000050_0003
Figure imgf000050_0002
Figure imgf000050_0001
optionally substituted with halogen, carboxamido, tetrazolyl oxadiazolyl, thiadiazolyl, amino, hydroxy, C1-6-alkyl or C1-6- alkoxy;
and A, R1, R2, D, W and m are defined as in the preceding claims.
5. A compound of the general formula I according to any one of the preceding claims, wherein D is
Figure imgf000051_0001
wherein R10 and R11 are hydrogen;
R8 and R9 are independently hydrogen or C1-6-alkyl optionally substituted with halogen, amino, hydroxy or aryl;
R8 and R9, optionally forming -(CH2)i-U-(CH2)j-, wherein i and j are independently 1 or 2,
U is -O-, -S- or a valence bond,
o and r are independently 0, 1, 2, 3 or 4,
s is 0 or 1, and
r + s is 1, 2, 3 or 4;
and A, B, R1, R2, W and m are defined as in the preceding claims. 6. A compound according to claim 1, 2, 3, 4, or 5 selected from the group consisting of
1-Benzyl-3-(3-(morpholin-4-yl)propyl)-1-(naphth-2-yl)methylthiourea, or the hydrchloride salt thereof;
1-Benzyl-3-(3-dimethylaminopropyl)-1-phenyl-thiourea, or the hydrochloride salt thereof;
2-[3-(3-(Morpholin-4-yl)propyl)-1-(naphth-2-yl)methylthioureido]-3-phenyl-propionamide;
N-(4-Aminobutyl)-2-[3-((3-amino-3-methyl)butyl)-1-(naphth-2-yl)methyl-thioureido]-3-phenyl-propionamide;
N-(4-Aminobutyl)-2-(N-(naphth-2-yl)methyl-N'-(piperidin-3-yl)methyl-guanidino)-3-phenyl-propionamide;
N-(4-Aminobutyl)-2-[1-methyl-3-(naphth-2-yl)methyl-3-(2- (piperidin-2-yl) ethyl) -thioureido]-3- (naphth-2-yl)propionamide;
3-(3-(Morpholin-4-yl)propyl)-1-(naphth-2-yl)methyl-1-[2'-(1H-tetrazol-5-yl)-biphenyl-4-ylmethyl]-thiourea, or the
hydrochloride
salt thereof;
N-((1-Carbamoyl-2-phenyl)ethyl-N-methyl-2-[3-((3-morpholin-4-yl)propyl)- thioureido]-3-(naphth-2-yl)propionamide;
3-(3-(Dimethylamino)propyl)-1-(naphth-1-yl)methyl-1-phenylthiourea, or the hydrochloride salt thereof;
1-Benzyl-3-(3-(morpholin-4-yl)propyl)-1-phenylthiourea;
1,1-Dibenzyl-3-(3-(morpholin-4-yl)propyl)thiourea, or the hydrochloride salt thereof;
1-Benzyl-3-(3-(dimethylamino)propyl)-1-((naphth-2-yl)methyl)thiourea, or the hydrochloride salt thereof;
1-Benzyl-3-(3-(morpholin-4-yl)propyl)-1-(pheneth-2-yl)thiourea, or
the hydrochloride salt thereof;
1-Benzyl-3-(3-(morpholin-4-yl)propyl)-1-(quinolin-3-yl)thiourea, or
the dihydrochloride salt thereof;
1-Benzyl-3-(3-(morpholin-4-yl)propyl)-1-(pyridin-2-yl)thiourea, or
the hydrochloride salt thereof;
1-Benzyl-3-(3-(morpholin-4-yl)propyl)-1-(4-methoxyphenyl)thiourea, or the hydrochloride salt thereof;
1-Benzyl-3-(3-(morpholin-4-yl)propyl)-1-(4- ([1,2,3]thiadiazol-4-yl)benzyl)thiourea, or the hydrochlorid salt thereof; or
3-(3-Dimethylaminopropyl)-1-((naphth-2-yl)methyl)-1-phenylthiourea, or the hydrochloride salt thereof.
7. A compound of the general formula 5
wherein A, B, R1, R2, D, and m are as defined in claim 1;
or a pharmaceutically acceptable salt thereof, and the compounds of formula I comprise any optical isomers thereof, in the form of separated, pure or patially purified optical isomers or racemic mixtures thereof.
8. The compound according to claim 7, wherein W is =O or =S.
59. The compound according to any one of the claims 7 or 8, wherein A is hydrogen or CH2OR3;
and B, R1, R2, D, W and m are defined as in the preceding claims.
10. The compound according to any one of the claims 7, 8 or 109, wherein B is
Figure imgf000053_0001
or
Figure imgf000053_0002
Figure imgf000053_0003
Figure imgf000053_0004
optionally substituted with halogen, carboxamido, tetrazolyl, oxadiazolyl, thiadiazolyl, amino, hydroxy, C1-6-alkyl or C1-6- alkoxy;
5 and A, R1, R2, D, W and m are defined as in the preceding claims.
11. The compound according to any one of the claims 7, 8, 9, or 10, wherein D is
Figure imgf000054_0001
wherein R10 and R11 are hydrogen;
R8 and R9 are independently hydrogen or C1-6-alkyl optionally 5 substituted with halogen, amino, hydroxy or aryl;
R8 and R9, optionally forming -(CH2)j-U-(CH2)j-, wherein i and j are independently 1 or 2,
U is -O-, -S- or a valence bond,
o and r are independently 0, 1, 2, 3 or 4,
10 s is 0 or 1, and
r + s is 1, 2 , 3 or 4 ;
and A, B, R1, R2, W and m are defined as in the preceding claims.
12. A compound of the general formula 11
15 wherein A, B, R1, R2, D, and m are as defined in claim 1;
or a pharmaceutically acceptable salt thereof, and the compounds of formula I comprise any optical isomers thereof, in the form of separated, pure or patially purified optical isomers or racemic mixtures thereof.
2013. The compound according to claim 12, wherein W is =O or =S.
14. The compound according to any one of the claims 12 or 13, wherein A is hydrogen or CH2OR3;
and B, R1, R2, D, W and m are defined as in the preceding 25 claims.
15. The compound according to any one of the claims 12, 13 or 14, wherein B is
or
Figure imgf000055_0001
Figure imgf000055_0002
Figure imgf000055_0003
Figure imgf000055_0004
optionally substituted with halogen, carboxamido, tetrazolyl, 5 oxadiazolyl, thiadiazolyl, amino, hydroxy, C1-6-alkyl or C1-6- alkoxy;
and A, R1, R2, D, W and m are defined as in the preceding claims.
16. The compound according to any one of the claims 12, 13, 1014, or 15, wherein D is
Figure imgf000055_0005
wherein R10 and R11 are hydrogen;
R8 and R9 are independently hydrogen or C1-6-alkyl
optionally substituted with halogen, amino, hydroxy
15 or aryl;
R8 and R9, optionally forming -(CR2)i-U-(CH2)j-, wherein i and j are independently 1 or 2,
U is -O-, -S- or a valence bond,
o and r are independently 0, 1, 2, 3 or 4, s is 0 or 1 , and
r + s is 1 , 2 , 3 or 4 ;
and A, B, R1, R2, W and m are defined as in the preceding claims.
517. A pharmaceutical composition comprising, as an active ingredient, a compound of the general formula I according to any one of the claims 1-16 or a pharmaceutically acceptable salt thereof together with a pharmaceutically acceptable carrier or diluent.
1018. A composition according to claim 17 in unit dosage form, comprising from about 10 to about 200 mg of the compound of the general formula I or a pharmaceutically acceptable salt thereof.
19. A pharmaceutical composition for stimulating the release 15 of growth hormone from the pituitary, the composition
comprising, as an active ingredient, a compound of the general formula I according to any one of the claims 1-16 or a pharmaceutically acceptable salt thereof together with a pharmaceutically acceptable carrier or diluent.
2020. 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 according to any one of the claims 1-1 or a pharma-ceutically acceptable salt thereof.
2521. A method according to claim 20, wherein the effective amount of the compound of the general formula I or
pharmaceutically acceptable salt or ester thereof is in the range of from about 0.0001 to about 100 mg/kg body weight pe day, preferably from about 0.001 to about 50 mg/kg body
30 weight per day.
22. Use of a compound of the general formula I according to any one of the claims 1-16 or a pharmaceutically acceptable salt thereof for the preparation of a medicament.
23. Use of a compound of the general formula I according to any one of the claims 1-16 or a pharmaceutically acceptable salt thereof for the preparation of a medicament for
stimulating the release of growth hormone from the pituitary.
24. Use of a compound of the general formula I according to any one of the claims 1-16 or a pharmaceutically acceptable salt thereof for the preparation of a medicament for
administration to animals to increase their rate and extent of growth, to increase their milk and wool production, or for the treatment of ailments.
PCT/DK1996/000058 1995-02-09 1996-02-06 Compounds with growth hormone releasing properties WO1996024580A1 (en)

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AU45345/96A AU4534596A (en) 1995-02-09 1996-02-06 Compounds with growth hormone releasing properties

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ZA (1) ZA961007B (en)

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US6107306A (en) * 1995-12-28 2000-08-22 Pfizer Inc. Heterocyclic compounds
EP1159964A2 (en) 2000-05-31 2001-12-05 Pfizer Products Inc. Compositions and methods for stimulating gastrointestinal motility
US6638927B2 (en) 2000-08-31 2003-10-28 Chiron Corporation Guanidinobenzamides
US6716840B2 (en) 2001-04-09 2004-04-06 Chiron Corporation Guanidino compounds
US7034033B2 (en) 2002-05-23 2006-04-25 Chiron Corporation Substituted quinazolinone compounds
WO2007098716A1 (en) 2006-02-28 2007-09-07 Centro De Ingeniería Genética Y Biotecnología Compounds analogous to growth hormone peptide secretagogues and preparations containing them
US7368453B2 (en) 2003-11-19 2008-05-06 Chiron Corporation Quinazolinone compounds with reduced bioaccumulation
EP1930021A2 (en) 1999-02-18 2008-06-11 Kaken Pharmaceutical Co., Ltd. Novel amide derivatives as growth hormone secretagogues
US7625909B2 (en) 2003-05-23 2009-12-01 Novartis Vaccines And Diagnostics, Inc. Substituted quinazolinone compounds
EP2457893A1 (en) 2004-06-18 2012-05-30 Tranzyme Pharma, Inc. Intermediates for macrocyclic modulators of the ghrelin receptor
EP2644618A1 (en) 2007-02-09 2013-10-02 Tranzyme Pharma, Inc. tether intermediates for the synthesis of macrocyclic ghrelin receptor modulators
WO2013190520A2 (en) 2012-06-22 2013-12-27 The General Hospital Corporation Gh-releasing agents in the treatment of vascular stenosis and associated conditions
WO2017075535A1 (en) 2015-10-28 2017-05-04 Oxeia Biopharmaceuticals, Inc. Methods of treating neurodegenerative conditions
US10105416B2 (en) 2014-02-05 2018-10-23 The Regents Of The University Of California Methods of treating mild brain injury

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
No relevant documents have been disclosed. *

Cited By (29)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6110932A (en) * 1995-12-28 2000-08-29 Pfizer Inc. Growth hormone secretagogues
US6124264A (en) * 1995-12-28 2000-09-26 Pfizer Inc. Heterocyclic compounds
US6278000B1 (en) 1995-12-28 2001-08-21 Pfizer Inc. Growth-hormone secretagogues
US6306875B1 (en) 1995-12-28 2001-10-23 Pfizer Inc. Growth-hormone secretagogues
US6313140B1 (en) 1995-12-28 2001-11-06 Pfizer Inc. Method of treatment using certain growth-hormone secret agogues
US6482825B2 (en) 1995-12-28 2002-11-19 Pfizer Inc. Growth-hormone secretagogues
US6107306A (en) * 1995-12-28 2000-08-22 Pfizer Inc. Heterocyclic compounds
EP1930021A2 (en) 1999-02-18 2008-06-11 Kaken Pharmaceutical Co., Ltd. Novel amide derivatives as growth hormone secretagogues
EP1159964A2 (en) 2000-05-31 2001-12-05 Pfizer Products Inc. Compositions and methods for stimulating gastrointestinal motility
US6995269B2 (en) 2000-08-31 2006-02-07 Chiron Corporation Guanidinobenzamides
US6638927B2 (en) 2000-08-31 2003-10-28 Chiron Corporation Guanidinobenzamides
US6960582B2 (en) 2001-04-09 2005-11-01 Chiron Corporation Guanidino compounds
US7189727B2 (en) 2001-04-09 2007-03-13 Chiron Corporation Guanidino compounds
US6716840B2 (en) 2001-04-09 2004-04-06 Chiron Corporation Guanidino compounds
US7456183B2 (en) 2001-04-09 2008-11-25 Novartis Vaccines And Diagnostics, Inc. Guanidino compounds
US7034033B2 (en) 2002-05-23 2006-04-25 Chiron Corporation Substituted quinazolinone compounds
US7858641B2 (en) 2002-05-23 2010-12-28 Novartis Vaccines And Diagnostics, Inc. Substituted dihydroisoquinolinone compounds
US7858631B2 (en) 2002-05-23 2010-12-28 Novartis Vaccines And Diagnostics, Inc. Substituted pyrido [2,3-d] pyrimidinone compounds
US7625909B2 (en) 2003-05-23 2009-12-01 Novartis Vaccines And Diagnostics, Inc. Substituted quinazolinone compounds
US7368453B2 (en) 2003-11-19 2008-05-06 Chiron Corporation Quinazolinone compounds with reduced bioaccumulation
EP2457893A1 (en) 2004-06-18 2012-05-30 Tranzyme Pharma, Inc. Intermediates for macrocyclic modulators of the ghrelin receptor
EP2457925A1 (en) 2004-06-18 2012-05-30 Tranzyme Pharma, Inc. Process for preparing a macrocyclic modulator of the ghrelin receptor and intermediates
WO2007098716A1 (en) 2006-02-28 2007-09-07 Centro De Ingeniería Genética Y Biotecnología Compounds analogous to growth hormone peptide secretagogues and preparations containing them
EP2644618A1 (en) 2007-02-09 2013-10-02 Tranzyme Pharma, Inc. tether intermediates for the synthesis of macrocyclic ghrelin receptor modulators
WO2013190520A2 (en) 2012-06-22 2013-12-27 The General Hospital Corporation Gh-releasing agents in the treatment of vascular stenosis and associated conditions
US10105416B2 (en) 2014-02-05 2018-10-23 The Regents Of The University Of California Methods of treating mild brain injury
US10617740B2 (en) 2014-02-05 2020-04-14 The Regents Of The University Of California Methods of treating mild brain injury
US11241483B2 (en) 2014-02-05 2022-02-08 The Regents Of The University Of California Methods of treating mild brain injury
WO2017075535A1 (en) 2015-10-28 2017-05-04 Oxeia Biopharmaceuticals, Inc. Methods of treating neurodegenerative conditions

Also Published As

Publication number Publication date
IL117075A0 (en) 1996-06-18
ZA961007B (en) 1996-08-29
AU4534596A (en) 1996-08-27

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