AU711104B2 - Compounds with growth hormone releasing properties - Google Patents

Compounds with growth hormone releasing properties Download PDF

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Publication number
AU711104B2
AU711104B2 AU61882/96A AU6188296A AU711104B2 AU 711104 B2 AU711104 B2 AU 711104B2 AU 61882/96 A AU61882/96 A AU 61882/96A AU 6188296 A AU6188296 A AU 6188296A AU 711104 B2 AU711104 B2 AU 711104B2
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phe
2nal
acid
aib
compound according
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Birgit Sehested Hansen
Thomas Kruse Hansen
Nils Langeland Johansen
Jesper Lau
Behrend Friedrich Lundt
Kjeld Madsen
Bernd Peschke
Henning Thoegersen
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Novo Nordisk AS
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/60—Growth hormone-releasing factor [GH-RF], i.e. somatoliberin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • A61P13/02—Drugs for disorders of the urinary system of urine or of the urinary tract, e.g. urine acidifiers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61P5/00—Drugs for disorders of the endocrine system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P5/00—Drugs for disorders of the endocrine system
    • A61P5/06—Drugs for disorders of the endocrine system of the anterior pituitary hormones, e.g. TSH, ACTH, FSH, LH, PRL, GH
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C237/00—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups
    • C07C237/02—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton
    • C07C237/22—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton having nitrogen atoms of amino groups bound to the carbon skeleton of the acid part, further acylated
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • C07K5/06008—Dipeptides with the first amino acid being neutral
    • C07K5/06078—Dipeptides with the first amino acid being neutral and aromatic or cycloaliphatic
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/08—Tripeptides
    • C07K5/0802—Tripeptides with the first amino acid being neutral
    • C07K5/0812—Tripeptides with the first amino acid being neutral and aromatic or cycloaliphatic
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • C07K5/1002—Tetrapeptides with the first amino acid being neutral
    • C07K5/1005—Tetrapeptides with the first amino acid being neutral and aliphatic
    • C07K5/101—Tetrapeptides with the first amino acid being neutral and aliphatic the side chain containing 2 to 4 carbon atoms, e.g. Val, Ile, Leu
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/02—Linear peptides containing at least one abnormal peptide link
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

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Description

WO 97/00894 PCT/DK96/00266 1 COMPOUNDS WITH GROWTH HORMONE RELEASING PROPERTIES FIELD OF INVENTION The present invention relates to novel peptide derivatives, 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, 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, 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 necessitates parenteral administration.
Furthermore, other directly acting natural secretagogues, GHRH and PACAP, are polypeptides of high molecular weight for which reason parenteral administration is preferred.
WO 97/00894 PCT/DK96/00266 2 The use of shorter peptides for increasing the levels of growth hormone in mammals has previously been proposed, e.g. in EP 18 072, EP 83 864, WO 89/07110, WO 89/01711, WO 89/10933, WO 88/9780, WO 83/02272, WO 91/18016, WO 92/01711 and WO 93/04081.
The structure of growth hormone releasing peptides or peptide derivatives is important for their growth hormone releasing potency as well as their bioavailability. It is therefore the object of the present invention to provide novel peptides with growth hormone releasing properties which have improved properties relative to known peptides of this type.
SUMMARY OF THE INVENTION A compound of general formula I A-B-C-D(-E)p 1 wherein p is 0 or 1; A is hydrogen or R 1 -(CH2)q-(X) r
(CH
2 wherein q is 0 or an integer selected from the group: 1, 2, 3, 4, ris 0 or1; s is 0 or an integer selected from the group: 1, 2, 3, 4,
R
1 is hydrogen, imidazolyl, guanidino, piperazino, morpholino, piperidino or N(R2)-
R
3 wherein each of R 2 and R 3 is independently hydrogen or lower alkyl optionally substituted by one or more hydroxyl, pyridinyl or furanyl groups; and X, when r is 1, is -CH 2 -CH=CH-, -C(R 16
)(R
17 'Aa. or wherein each of R 16 and R 17 is independently hydrogen or lower alkyl; B is wherein each of t and u independently is 0 or 1; WO 97/00894 PCT/DK96/00266 3 G and H are amino acid residues selected from the group consisting of natural Lamino acids or their corresponding D-isomers, or non-natural amino acids such as 1,4-diaminobutyric acid, amino-isobutyric acid, 1,3-diaminopropionic acid, 4aminophenylalanine, 3-pyridylalanine, 1 ,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, 1 ,2,3,4-tetrahydronorharman-3-carboxylic acid, N-methylanthranilic acid, anthranilic acid, N-benzylglycine, 3-aminomethylbenzoic acid, 3-amino-3-methyl butanoic acid, sarcosine, nipecotic acid or iso-nipecotic acid; and wherein, when both t and u are 1, the amide bond between G and H is optionally replaced by Y-NR 1 wherein Y is -CO- or -CH 2 and R 18 is hydrogen, lower alkyl or lower aralkyl; C is a D-amino acid of formula -NH-CH((CH 2 )w-R 4 wherein w is 0, 1 or 2; and
R
4 is selected from the group consisting of ,x or each of which is optionally substituted with halogen, lower alkylamino, amino or hydroxy; lower alkyl, lower alkyloxy, D, when p is 1, is a D-amino acid of formula
-NR
20
-CH((CH
2 )k-R 5
)-CO-
or, when p is 0, D is -NR 20
-CH((CH
2 )l-R 5
)-CH
2
-R
6 or -NR 20
-CH((CH
2 )m-R 5
)-CO-R
6 wherein k is 0, 1 or 2; I is 0, 1 or2; m is 0, 1 or 2; R20 is selected from the group consisting of lower alkyl or lower aralkyl; is selected from the group consisting of WO 97/00894 PCT/DK96/00266 4 A ,or each of which is optionally substituted with halogen, lower alkyl, lower alkyloxy amino or hydroxy; and
R
6 is piperazino, morpholino, piperidino, -OH or -N(R 7
)-R
8 wherein each of R 7 and R 8 is independently hydrogen or lower alkyl; E, when p is 1, is -NH-CH(R 1
)-(CH
2 )v-R 9 wherein v is 0 or an integer selected from the group: 1, 2, 3, 4, 5, 6, 7, 8;
R
9 is hydrogen, imidazolyl, guanidino, piperazino, morpholino, piperidino, -N(R 11
R
12
R
1 9 wherein n is 0, 1 or 2, and R 19 is hydrogen or lower alkyl,
R
11 R1 (CH2)-N (CH2)-N R 12 or 2 NR 12 wherein o is an integer selected from the group: 1, 2, 3, each of R 11 and R 12 is independently hydrogen or lower alkyl, or *o each of which is optionally substituted with halogen, lower alkyl, lower alkyloxy, amino, alkylamino, hydroxy, or the Amadori rearrangement product from an amino group and a hexapyranose or a hexapyranosyl-hexapyranose and WO 97/00894 WO 9700894PCT/DK96/00266
R
1 0, when p is 1, is selected from the group consisting of -COOH, -CH 2
-R
1 3,
-CO-R
1 3 or -CH 2 -OH, wherein R13is piperazino, morpholino, piperidino, -OH or -N(R 1 4
)-R
1 5 wherein each of
R
1 4 and R 1 5 is independently hydrogen or lower alkyl; all amide bonds within formula I with the exception of the bond between C and D may independently be replaced by -Y-NR 1 wherein Y is -CO- or -CH 2 and R 18 is hydrogen, lower alkyl or lower aralkyl; or a pharmaceutically acceptable salt thereof;, and with the exception of the compounds (3-Arninomethylbenzoyl)-D-2Nal-N-Me-D-Phe-Lys-N
H
2 H-Aib-His-D-2Nal-N-Me-D-Phe-Lys-NH 2 H-Aib-His-N-Me-D-2Nal-N-Me-D-Phe-Lys-NH 2 3-(H-Aib-His-D-2Nal-N-Me-D-Phe-NH)-1 -morpholinopropane 2-(H-Aib-His-D-2Nal-N-Me-D-Phe-NH)-2-(1 -methyl-2-pyrrolidinyl)ethane ((3R)-Piperidinecarbonyl)-N-Me-D-2Nal-N-Me-D-Phe-Lys-NH 2 3-((3-Aminomethylbenzoyl )-D-2Nal-N-Me-D-Phe-N H)-1 -morpholinopropane 2-(H-Aib-His-N-Me-D-2Nal-N-Me-D-Phe-N H)-1 -methyl-2-pyrrol idinyl)ethane 2-(((3R)-Piperidinecarbonyl)-N-Me-D-2Nal-N-Me-D-Phe-N H)-1 -methyl-2pyrrolidinyl)ethane 2-((3-Aminomethylbenzoyl)-N-Me-D-2Nal-N-Me-D-Phe-NH)-1 -methyl-2pyrrolidinyl)ethane 3-(H-Aib-His-N-Me-D-2Nal-N-Me-D-Phe-NH)-1 -morpholinopropane 3-(((3R)-Piperidinecarbonyl )-N-Me-D-2Nal-N-Me-D-Phe-NH)-1 -morpholinopropane 3-((3-Arninomethylbenzoyl )-N-Me-D-2Nal-N-Me-D-Phe-N H)-1 -morpholinopropane 2-((3-Arninomethylbenzoyl)-D-2Nal-N-Me-D-Phe-NH)-1 -methyl-2pyrrolidinyl)ethane 2-(((3R)Piperidinecarbonyl)-D-2Nal-N-Me-D-Phe-N H)-1 -methyl-2- WO 97/00894 PCT/DK96/00266 6 pyrrolidinyl)ethane.
The peptide derivatives of formula I exhibit an improved resistance to proteolytic degradation by enzymes due to the presence of adjacent D-amino acids in the peptide sequence, optionally combined with the substitution of an amide bond CO-NH-) by -Y-NR 18 as indicated above, e.g. aminomethylene (-CH 2
-NH-)
and/or modification at the N- or C-terminal end of the peptide. The increased bioavailability of the peptide derivatives of the invention compared to that of the peptides suggested in the prior art literature is, i. thought to be caused by their resistance to proteolytic degradation combined with small size.
In the above structural formulas and throughout the present specification, the following terms have the indicated meanings: The lower alkyl moities specified above are intended to include those alkyl moities, preferably with 1-6 carbon atoms, 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, tertbutyl, isopentyl, and isohexyl. Examples of cyclic alkyl are cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl.
The lower alkoxy moities specified above are intended to include those alkoxy moities preferably with 1-6 carbon atoms, 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.
The lower alkylamino moities specified above are intended to include those alkylamino moities preferably with 1-6 carbon atoms, 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.
WO 97/00894 PCT/DK96/00266 7 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 C-6-alkyl, C6-alkoxy, halogen, amino or aryl. Aryl is preferably phenyl, thienyl, imidazolyl, pyridyl, indolyl, quinoline or naphthyl optionally substituted with halogen, amino, hydroxy, C1-alkyl or CI-s-alkoxy.
The lower aralkyl moities specified above are composed of a lower alkyl moity and a aryl moiety, wherein the lower alkyl moiety and aryl moiety are as defined above.
The term "halogen" is intended to include CI, F, Br and I.
The common three-letter code is used for natural amino acids, e.g. Ala for alanine.
WO 97/00894 WO 9700894PCT/DK96/00266 8 DETAILED DESCRIPTION OF THE INVENTION In a preferred embodiment of the compound of formula I, A is hydrogen, 3-N-Me- AMB, -3-AMB or Aib. When t is 1, G in the compund of formula I is preferably Ala, Gly, sarcosine, 3-aminomethylbenzoyl, R-nipecotinyl, nipecotic acid or isonipecotic acid, more preferably 3-aminomethylbenzoyl, R-nipecotinyl, nipecotic acid or isonipecotic acid. When u is 1, H is preferably His, Phe, Tic, Phe(4-NH 2 3-Pya, Gly, Ala, Sar, Pro, Tyr, Arg, Omn, 3-aminomethylbenzoic acid or D-Phe, more preferably H is His, Phe or Ala, most preferably H is His or Ala. C in the compound of formula I is preferably D-2-naphthylalanine (D-2NaI), D-1-naphthylalanine (D- 1 Nal), D-Phe or D-Trp, more preferably D-2NaI or D-Phe and most preferably N- Me-D-2Nal, D-2Nal, D-Phe, or N-Me-D-Phe. D in the compound of formula I is preferably -NR 20
_CH((CH
2 )k-R 5 wherein k is preferably 1 and R 20 is lower alkyl, more preferably D is D-Phe or D-2Nal. Most preferably D is N-Me-D-Phe-ol, N-Me-D-Phe, N-Me-D-2Nal-ol, N-Me-D-Phe-NH 2 N-Me-D-Phe-NH-Me, or N-Me-D-.
(4-l)Phe-NH-Me.
When p is 1 in the compound of formula 1, E is preferably Lys-N H 2 Ser-N H 2
NH-
(2-(lI-piperazino)ethyl), 1-morphol ino)propyl), NH-(2-aminoethyl), N H-(4aminomethylbenzyl), NH-(benzyl), Lys-OH, N H-(l1-hydroxy-6-amino-2S-hexyl),
NH-
(2-(lI-methyl-2-pyrrolidinyl)ethyl), or 3-N, N-dimethyl-aminopropyl, most preferably E is NH-(2-(1 -methyl-2-pyrrolidinyl)ethyl), 3-N, N-dimethyl-aminopropyl, Lys-NH 2 or Ser-NH- 2 or R 4 in the compound of formula I is preferably 2-naphthyl. R 5 is preferably phenyl. v is preferably 2-6, and R 9 is NH- 2 ,2-morpholinoethyl 3-morpholinopropyl or (1-methylpyrrolidinyl)ethyl. Rio is preferably -COOH, -CH 2 -OH, -CONH 2 or
-CON(CH
3 2 Examples of specific compounds of the present invention are (2)2(3Aioehlezy)--eD2a--e--2npty~rpnl WO 97/00894 PTD9/06 PCT/DK96/00266 HN kL 0 (3-Aminomethylbenzoyl)-N-Me-D-2Na-N-Me-D-Phe-NH 2 CH 0 H 2 N, I 3
NH-
0 CH 1 3-((3-Aminomethylbenzoyl)-N-Me-D-2NaI-NMe-D-Phe-NH)-N,
N-
dimethylaminopropane: CH 0
H
2 NN. N 0 CH 3 0 H-Aib-His-N-Me-D-2Nal-N-Me-D-Phe-NH 2 H3- NH0!
H
3
C
(3-Aminomethylbenzoyl)-N-Me-D-2Nal-N-Me-D-Phe-Lys-N
H
2 WO 97/00894 WO 9700894PCT/DK96/00266 CH3 I I~
H
2 N, Nj H-Aib-Ala-D-2NaI-N-Me-D-Phe-Lys-NH 2 H-i-i--N lNM h -H 2-((3-Aminomethylbenzoyl)-N-Me-D-2NaI-N-Me-D-Phe-N H)-1 -morpholinoethane: HNCH 3 0 e' HN N ryfNH.-NT 0 CH 3 O 0 00 (3-Arninomethylbenzoyl)-N-Me-D-2NaI-N-Me-D-Phe-NH-Me:
NH
CH
3 6H 3 0 3-((3-Methylaminomethylbenzoyl)-N-Me-D-2NaI-N-Me-D-Phe-N
N-
dimethylaminopropane: WO 97/00894 WO 9700894PCT/DK96/00266
HNCH
H 3 c 0 CHO (3-Aminomethylbenzoyl)-N-Me-D-2Nal-N-Me-D-Phe-N-Me 2 CH 3 0 H-Aib-His-N-Me-D-2Nal-N-Me-D-Phe-NHMe:
H
2
N
0 WO 97/00894 12 3 -methylaminomethyl-Nme-D-2Na-Nm-DPhe-NHCH 3 PCT/DK96/00266 Piperidine-4-carboxylic acid-N-(( I R)-1 R)-2-(4-iodophenyl)1 (methylcarbamoyl )ethyl)-N-methylcarbamoyl)-2-(2-naphthy )ethyl methylamide .NH-CH3 Structures of non-natural amino acid residues: 0 2NaI 3Pyal Alb
H
0 WO 97/00894 PCT/DK96/00266 13 Abbreviation used for peptide bond substitution: -N-Me- 0
CH,
Compounds of formula I may be prepared by conventional methods of solution or solid phase peptide synthesis. For instance, solid phase synthesis may be carried out substantially as described by Stewart and Young, Solid Phase Peptide Synthesis, 2nd. Ed., Rockford, Illinois, USA, 1976. Solution peptide synthesis may for instance be carried out substantially as described by Bodansky et al., Peptide Synthesis, 2nd. Ed., New York, New York, USA, 1976.
Aminomethylene as a substitution of an amide bond may be introduced according to the method described by Y. Sasaki and D.H. Coy, Peptides 1987, pp. 119- 121. Peptide derivatives containing a mono- or di-hexapyranose derivatised amino group may be prepared by an Amadori rearrangement substantially by the method described by R. Albert et al., Life Sciences 53, 1993, pp. 517-525. Examples of suitable mono- or di-hexapyranoses are glucose, galactose, maltose, lactose or cellobiose. Derivatives used as starting materials in the synthesis may either be obtained commercially and, when required, provided with suitable protecting groups, or starting materials used to prepare the moiety in general formula I may be prepared by well-known methods and optionally protected in a manner known per se.
WO 97/00894 PCT/DK96/00266 14 Abbreviations used for protecting groups: Trt- Dad- Bom Fmoc
HC-
0
/A
SHc- 0 a -z- Boc- Cc Pharmaceutically acceptable acid addition salts of compounds of formula I include those prepared by reacting the peptide with an inorganic or organic acid such as hydrochloric, hydrobromic, sulfuric, acetic, phosphoric, lactic, maleic, phthalic, citric, glutaric, gluconic, methanesulfonic, salicylic, succinic, tartaric, oxalic, toluenesulfonic, trifluoracetic, sulfamic and 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, patches 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 and water.
WO 97/00894 PCT/DK96/00266 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.
A typical tablet which may be prepared by conventional tabletting techniques may contain: Core: Active compound (as free compound or salt thereof) 100 mg Colloidal silicon dioxide (Aerosil) 1.5 mg Cellulose, microcryst. (Avicel) 70 mg Modified cellulose gum (Ac-Di-Sol) 7.5 mg Magnesium stearate Coating: HPMC approx. 9 mg *Mywacett 9-40 T approx. 0.9 mg *Acylated monoglyceride used as plasticizer for film coating.
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.
For nasal or pulmonary 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 such as bile acid salts polyethylene glycols, polypropylene glycols or polyoxyethylene higher alcohol ethers, absorption enhancers such as lecithin (phosphatidylcholine) or cyclodextrin, or preservatives such as parabenes.
WO 97/00894 PCT/DK96/00266 16 For transdermal administration the preparation may be in a form suitable for patches or iontophoresis.
Generally, the compounds of the present invention are dispensed in unit dosage form comprising 0.0001-100 mg of active ingredient together with a pharmaceutically acceptable carrier per unit dosage.
The dosage of the compounds according to this invention is suitably 1-500 mg/day, e.g. about 100 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.
The compounds of formula I have interesting pharmacological properties.
Examples of such properties are the stimulation of release of growth hormone from the pituitary which has similar effects or uses as growth hormone itself. The uses of WO 97/00894 PCT/DK96/00266 17 growth hormone may be summarized as follows: stimulation of growth hormone release in the elderly; prevention of catabolic side effects of glucocorticoids, 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 bum 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 may 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 per day may be administered to patients and animals to obtain effective release of endogenous growth hormone. Usually, dosage forms suitable for oral or nasal 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.
WO 97/00894 PCT/DK96/00266 18 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, an antibiotic or other pharmacologically active material. This might be another secretagogue, such as GHRP (1 or 6) or GHRH or an analogue thereof, growth hormone or an analogue thereof or a somatomedin such as IGF-1 or IGF-2.
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.
The 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.
Pharmacological Methods Compounds of formula I may be evaluated in vitro for their efficacy and potency to release growth hormone in primary rat somatotrophs.
Rat primary somatotrophs may be prepared essentially as described previously (Chen et al., Endocrinology 1991, 129, 3337-3342 and Chen et al., Endocrinology WO 97/00894 PCT/DK96/00266 19 1989, 124, 2791-2798). Briefly, rats are killed by decapitation. The pituitary is quickly removed. The pituitaries are digested with 0.2 collagenase n 0.2 hyalurinidase in Hanks balanced salt solution. The cells are resuspended in Dulbecco's Modified Eagle's medium containing 0.37 NaHCO3, 10 horse serum, 2.5 fetal calf serum, 1 nonessential amino acids, 1 glutamine and 1 penicillin/streptomycin and adjusted to 1.5 x 105 cells/ml. One ml of this suspension is placed in each well of 24-well trays and left for 2-3 days before release experiments are performed.
On day one of the experiments, cells are washed twice with the above medium containing 25 mM HEPES, pH 7.4. Growth hormone release initiated by addition of medium containing 25 mM HEPES and test compound. Incubation is carried out for minutes at 370C. After incubation growth hormone released to the medium is measured by a standard RIA.
Compounds of formula I may be evaluated for their in vivo effects on growth hormone release in pentobarbital anaesthetized female rats as described previously (Bercu et al. Endocrinology 1991, 129, 2592-2598). Briefly, adult male Sprague-Dawley rats are anesthetized with pentobarbital 50 mg/kg ip. After the rats had been fully anaesthesized the rats are implanted with a tracheal cannula and catheters in the carotid artery and the jugular vein. After a 15 minute recovery, a blood sample is taken at time 0. The pituitary secretagogues are administered iv and artery blood samples are put on ice for 15 minutes and then centrifuged for 2 minutes at 12,000 xg. The serum is decanted and amount of growth hormone determined using a standard RIA.
The invention is further illustrated in the following example which is not in any way intended to limit the scope of the invention as claimed.
The compound prepared in the following example was isolated as the trifluoroacetic acid (TFA) salt.
WO 97/00894 PCT/DK96/00266 EXAMPLE 1 Preparation of 2(R)-2-((3-Aminomethylbenzoyl)-N-Me-D-2Nal-N-Me)- 3-phenylpropanol.
P0 0 CH 165.7 mg of Boc-N-Me-D-2Nal-OH and 165.2 mg of (R)-methylamino-3-phenylpropan-1-ol (prepared from H-N-Me-D-Phe-OH according to McKennon, M. J.; Meyers, A. I. J. Org. Chem. 1993, 58, 3568-71) and 68.1 mg of HOAt was dissolved in a mixture of 2 ml of DMF and 4 ml of DCM at 0 115 mg EDAC was added and the mixture was stirred 1h at 0 OC and then 18h at r.t.
The DCM was then removed from the mixture by a stream of nitrogen before 50 ml of EtOAc was added and the resulting mixture was extracted sequentially with 100 ml of 5% aqueous NaHCO 3 100 ml H 2 0, 100 ml 5% aqueous KHSO 4 and 100 ml
H
2 0. The resulting organic phase was dried with Na 2
SO
4 and concentrated in vacuum on a rotary evaporator to an oil.
502.6 mg 3-Boc-aminomethylbenzoic acid was dissolved in 10 ml DCM by addition of 2 drops of DMF and then converted to the symmetrical anhydride by stirring with 191.6 mg EDAC for 10 min.
A solution of the above lyophilized 2(R)-(H-N-Me-D2Nal-N-Me)-3-phenylpropanol and 342 Il DIEA in 5 ml DCM was added to this mixture and then reacted for 20 h at r.t. The reaction mixture was then concentrated to an oil and redissolved in 50 ml EtOAc. This solution was extracted sequentially with 100 ml 5% aqueous NaHCO 3 100 ml H 2 0, 100 ml 5% aqueous KHSO 4 and 100 ml H 2 0. The resulting organic phase was dried with Na 2
SO
4 and concentrated in vacuum on a rotary evaporator to an oil. The oil was then dissolved in 4 ml DCM TFA 1:1 and stirred. After min the mixture was concentrated by a stream of nitrogen and the resulting oil was redissolved in 20 ml 70% CH 3 CN 0.03 M HCI and 480mi H 2 0 was added.
The crude product was then purified by semipreparative HPLC in seven runs on a mm x 250 mm column packed with 7p C-18 silica which was preequilibrated -with 28% CH 3 CN in 0.05M (NH4)2SO 4 which was adjusted to pH 2.5 with 4M
H
2 S0 4 WO 97/00894 PCT/DK96/00266 21 The column was eluted with a gradient of 28% 38% CH 3 CN in 0.05M (NH 4 2
SO
4 pH 2.5 at 10 ml/min during 47 min at 40 °C and the peptide containing fractions were collected, diluted with 3 volumes of H 2 0 and applied to a Sep-Pak® C18 cartridge (Waters part. #:51910 which was equilibrated with 0.1% TFA The peptide was eluted from the Sep-Pak® cartridge with 70% CH 3 CN 0.1% TFA and isolated from the eluate by lyophilisation after dilution with water.
The final product obtained was characterised by analytical RP-HPLC (retention time) and by Plasma desorption mass spectrometry (molecular mass). Mass spectrometry agreed with the expected structure within the experimental error of the method (mass spectrometry 0.9 amu).
The RP-HPLC analysis was performed using UV detection at 214 nm and a Vydac 218TP54 4.6mm x 250mm 5p C-18 silica column (The Separations Group, Hesperia) which was eluted at 1 ml/min at 42 OC. Two different elution conditions were used: Al: The column was equilibrated with 5% CH 3 CN in a buffer consisting of 0.1M (NH4) 2 S0 4 which was adjusted to pH 2.5 with 4M H 2 S0 4 and eluted by a gradient of 5% to 60% CH 3 CN in the same buffer during 50 min.
B1: The column was equilibrated with 5% CH 3 CN 0.1% TFA/ H 2 0 and eluted by a gradient of 5% CH 3 CN 0.1% TFA H 2 0 to 60% CH 3 CN 0.1% TFA H 2 0 during 50 min.
The retention time using elution conditions Al and B1 was found to be 29.90 min and 31.52 min, respectively.
Synthesis of 3-Boc-aminomethylbenzoic acid g 3-cyanobenzoic acid was dissolved in 70 ml 25% NH 3
/H
2 0 and 200 ml H 2 0 and 5g 10% Pd/C was added under nitrogen The mixture was hydrogenated at atmospheric pressure at r.t. while pH was continously adjusted to 10.5 by addition of 12% NHa/H 2 0. After absorption of approximately 41 H 2 during 18 h, the reaction was stopped and the catalyst removed by filtration. The filtrate was concentrated in vac. to 20 ml and unreacted starting material was removed by extraction with ethyl acetate after acidification with 200 ml 1.5 M hydrochloric acid. The aqueous phase was concentrated to dryness and redissolved in 400 ml THF and 343 ml 1 M NaOH. A solution of 30g Boc-anhydride in 100 ml THF was added and the mixture was stirred overnight. Then the reaction mixture was acidified to pH 3 with 1 N HCI WO 97/00894 WO 9700894PCT/DK96/00266 22 and extracted with 3 x 300 ml of EtOAc. The organic phase was evaporated to a foam. The yield was 22 g.
Abbreviations: r.t. room temperature
EDAC:
EtOAc: Boc: N-Me-D-2NaI:
DCM:
DIEA:
DMF:
HOAt: .N-Me-D-Phe-ol:
TFA:
THE:
N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride ethyl acetate t-butyloxycarbonyl N-methyl-D-2-naphtylalanine dichloromethane diisopropylethyl amine N, N-dimethylformamide 1 -hydroxy-7-azabenzotriaztole N-methyl-D-phenylalaninol trifluoroacetic acid tetrahydrofuran EXAMPLE 2 3-((3-Arninomethylbenzoyl))-N-Me-D-2Nal-N-Me-D-Phe-N H) 7 N, N-dimethylaminopr opane H N cHo CH 3 N N NH 0 CH 3 O 0 l Boc-N-Me-D-Phe-OH (279 mg) was dissolved in DMF (4 ml) and stirred 10 min with HOBt (168 mg) and EDAC (230 mg 3-Dimethylamino-1 -propylamine (188 ill) was added and the mixture was stirred 18h at r.t. Then 5% aqueous sodium hydrogen carbonate (50 ml) was added and the resulting mixture was extracted WO 97/00894 PCT/DK96/00266 23 with EtOAc (50 ml) and the organic phase was dried over Na 2
SO
4 and concentrated in vacuum to an oil.
This oil was stirred 10 min at r.t. with TFA/DCM 1:1 (6 ml). After this the TFA DCM was evaporated by a stream of nitrogen and the resulting oil was dissolved in a mixture of 70% CH 3 CN (10 ml), 1 N HCI (3 ml) and water (37 ml) and the resulting mixture was immediately frozen and lyophilized.
The product from the lyophilisation was dissolved in DMF (6 mi) and DCM (12 ml).
To this mixture was added during stirring Boc-N-Me-D-2Nal-OH (494 mg), HOAt (204 mg), DIEA (171 and after cooling to 0 OC EDAC (288 mg). After stirring for 18h at r.t. the DCM was evaporated by a stream of nitrogen and EtOAc (100 ml) was added. This mixture was extracted two times with 5% aqueous sodium hydrogen carbonate (100 ml) and with water (100 ml) and dried over Na 2
SO
4 and concentrated in vacuum to an oil (480 mg).
This oil was stirred 10 min at r.t. with TFA DCM 1:1 (6 ml). After this the TFA DCM was evaporated by a stream of nitrogen and the resulting oil was dissolved in
CH
3 CN (10 ml). 1 N HCI (1 ml) and water (47 ml) was added and the resulting mixture was immediately frozen and lyophilized to an oil (2 HCI, H-N-Me-D-2Nal-N- Me-D-Phe-NH-(CH 2 )3-N(CH 3 2 Half of the above oil (2 HCI, H-N-Me-D-2Nal-N-Me-D-Phe-NH-(CH 2 3
-N(CH
3 2 was dissolved in DCM (9 ml) and 2 drops of DMF and DIEA (342 gl) were added.
This solution was added to a solution of Boc-3AMB-OH (503 mg) and EDAC (192 mg) in DCM (5 ml) which had been stirred for 15 min at r.t.
After stirring for 20 h the reaction mixture was concentrated to an oil with a stream of nitrogen and stirred for 15 min with 5% aqueous sodium hydrogen carbonate (100 ml).
Then EtOAc (50 ml) was added, the organic phase separated and extracted with aqueous sodium hydrogen carbonate (100 ml) and with water (100 ml) and then dried over Na 2
SO
4 and concentrated in vacuum to an oil (340 mg).
This oil was stirred 10 min at r.t. with TFA DCM 1:1 (6 ml). After this the TFA DCM was evaporated by a stream of nitrogen and the resulting oil was dissolved in
CH
3 CN (10 ml) and diluted with water to a final volume of 50 ml.
This crude product was then purified by semipreparative HPLC in eight runs and lyophilized using similar procedures as described in example 1.
The final product obtained was characterised by analytical RP-HPLC (retention time) and by Plasma desorption mass spectrometry (molecular mass). The molecular mass found (MH*:608.2 amu) agreed with the expected structure (teor.
WO 97/00894 PCT/DK96/00266 24 MH 608.8 amu) within the experimental error of the method.
The RP-HPLC retention time using elution conditions Al and B1 as defined in example 1. was found to be 25.23 min and 26.58 min, respectively.
EXAMPLE 3 3-(((3R)-3-Piperidinecarbonyl)-N-Me-D-2Nal-N-Me-D-Phe-NH)-1- N,N-dimethylaminopropane rt O 0. CH 3 N H N N N
H
CH
O CH O0 Half of the 2 HCI, H-N-Me-D-2Nal-N-Me-D-Phe-NH-(CH 2 3
-N(CH
3 2 which was obtained as an oil in example 2, was dissolved in DCM (9 ml) and 2 drops of DMF and DIEA (342 pl) were added.
This solution was added to a solution of Boc-(R)-nipecotic acid (459 mg) and EDAC (192 mg) in DCM (5 ml) which had been stirred for 15 min at r.t.
After stirring for 20 h the reaction mixture was concentrated to an oil with a stream of nitrogen and stirred for 15 min with 5% aqueous sodium hydrogen carbonate (100 ml) Then EtOAc (50 ml) was added, the organic phase separated and extracted with aqueous sodium hydrogen carbonate (100 ml) and with water (100 ml) and then dried over Na 2
SO
4 and concentrated in vacuum to an oil.
This oil was stirred 10 min at r.t. with TFA DCM 1:1 (6 ml). After this the TFA DCM was evaporated by a stream of nitrogen and the resulting oil was dissolved in
CH
3 CN (10 ml) and diluted with water to a final volume of 50 ml.
This crude product was then purified by semipreparative HPLC in five runs and yophilized using similar procedures as described in example 1.
The final product obtained was characterised by analytical RP-HPLC (retention WO 97/00894 PCT/DK96/00266 time) and by Plasma desorption mass spectrometry (molecular mass). The molecular mass found 586.3 amu) agreed with the expected structure (teor.
MH+ 585.8 amu) within the experimental error of the method.
The RP-HPLC retention time using elution conditions Al and B1 as defined in example 1 was found to be 25.33 min and 26.35 min, respectively.
EXAMPLE 4 2-(((3R)-3-Piperidinecarbonyl)-N-Me-D-2Nal-N-Me-D-Phe-NH)-1-(1-methyl-2pyrrolidinyl)ethane 1 CH,O 0 CH, NH> N H NH N SCH, 0 Boc-N-Me-D-Phe-OH (279 mg) was dissolved in DMF (10 ml) and stirred 10 min with HOBt (168 mg) and EDAC (384 mg 2-(Aminoethyl)-l-methyl-pyrrolidine (290 and DIEA (171 pl) were added and the mixture was stirred for 20 h at r.t.
Then the mixture was concentrated to an oil which was dissolved in 50 ml water and lyophilized. The product was redissolved in 25 ml water and then applied to a Sep-Pak® C18 cartridge (Waters part. #:43345 which was equilibrated with 0.03 N hydrochloric acid The product was eluted from the Sep-Pak® cartridge with
CH
3 CN in 0.03 N hydrochloric acid and isolated from the eluate by lyophilisation after dilution with water. The resulting material is stirred 10 min at r.t.
with TFA DCM 1:1 (6 ml). After this the TFA DCM was evaporated by a stream of nitrogen and the resulting oil was dissolved in 70% CH 3 CN (10 ml) and 1 N hydrochloric acid (2 ml) was added. The product was isolated by lyophilisation after dilution with water (50 ml).
The resulting material was dissolved in DMF (3 ml) and stirred 18h at r.t. after addition of Boc-N-Me-D-2Nal-OH (329 mg), HOAt (136 mg), EDAC (230 mg) and DIEA (1714p). Then EtOAc 50 ml) was added and this mixture was extracted with WO 97/00894 PCT/DK96/00266 26 aqueous sodium hydrogen carbonate (50 ml), with 5% aqueous potassium hydrogen sulphate (50 ml) and with water (50 ml). The organic phase was dried with sodium sulphate and concentrated in vac. to an oil.
This oil was stirred 10 min at r.t. with TFA DCM 1:1 (6 ml). After this the TFA DCM was evaporated by a stream of nitrogen and the resulting oil was dissolved in
CH
3 CN (10 ml) and 1 N hydrochloric acid (3 ml) was added. The product was isolated by lyophilisation after dilution with water (50 ml).
286 mg of this lyophilized product was dissolved in DCM (15 ml) and DIEA (171 pl).
This solution was added to a solution of Boc-(R)-nipecotic acid (459 mg) and EDAC (192 mg) in DCM (10 ml) which had been stirred for 25 min at r.t.
After stirring for 20 h the reaction mixture was concentrated to an oil by a stream of nitrogen and then redissolved in EtOAc (100 ml) and extracted with 5% aqueous sodium hydrogen carbonate (50 ml), with 5% aqueous potassium hydrogen sulphate (50 ml) and with water (50 ml). The organic phase was dried with sodium sulphate and concentrated in vac. to an oil.
This oil was stirred 10 min at r.t. with TFA DCM 1:1 (6 ml). After this the TFA DCM was evaporated by a stream of nitrogen and the resulting oil was dissolved in
CH
3 CN (10 ml) and diluted with water to a final volume of 50 ml.
This crude product was then purified by semipreparative HPLC in three runs and lyophilized using similar procedures as described in example 1.
The final product obtained was characterised by analytical RP-HPLC (retention time) and by Plasma desorption mass spectrometry (molecular mass). The molecular mass found 612.2 amu) agreed with the expected structure (teor.
MH': 612.39 amu) within the experimental error of the method.
The RP-HPLC retention time using elution condition Al as defined in example 1.
was found to be 25.80 min.
WO 97/00894 WO 9700894PCT/DK96/00266 27 EXAMPLE (2R)-2-((3-Arninomethylbenzoyl)-N-Me-D-2Nal-N-Me)-3-(2-naphthyl)propano (R)-2-(N-tert-Butoxycarbonyl-N-methylamino)-3-(2-naphthyl) propionic acid methyl ester.
Boc-N~> CH3 CH 3 (R)-2-tert-Butoxycarbonylamino-3-(2-naphthyl) propionic acid (5,0 g 16,4 mmol) was dissolved in dry DMF (50 ml). lodomethane (6,2 ml; 98,4 mmol) and silver(l)oxide (13,3 g ;57,4 mmol) were added and the mixture was stirred overnight. The reaction mixture was filtered and the filtrate was extracted with methylene chloride (200 ml). The organic phase was washed with potassium cyanide (2 x 50 ml; and water (3 x 75 ml). The organic phase was dried (MgSO 4 and the solvent removed in vacuo. The residue was chromatographed (Silica, 5 x 40cm) using ethyl acetate and heptane as eluent to afford 4,98 g of N-tert-butoxycarbonyl-N-methylamino)-3-(2-naphthyl) propionic acid methyl ester.
1 H-NMR (COCl 3 1.30, 1.35 (two s, 9H); 2.71, 2.75 (two s, 3H); 3.19, 3.47 (two m, 2H); 3.74, 3.77 (two s, 3H); 4.65, 5.05 (two dd, 1IH); 7.29-7.82 (in, 7H) (mixture of rotamers) WO 97/00894 PCT/DK96/00266 28 (R)-2-(N-tert-Butoxycarbonyl-N-methylamino)-3-(2-naphthyl) propionic acid.
Boc-N Yi 0
OH
CH,
(R)-2-(N-tert-Butoxycarbonyl-N-methylamino)-3-(2-naphthyl) propionic acid methyl ester (21,73 g ;65,57 mmol) was dissolved in 1,4-dioxan (200 ml) and water (20 ml) was added. The reaction mixture was cooled on an icebath and lithium hydroxide (1,73 g; 72,13 mmol) was added. After 15 min water (140 ml) was added and the reaction mixture was then stirred for additional 3 hours at room temperature. Ethyl acetate (400 ml) and water (300 ml) were added and pH was adjusted to 2,5 with sodium hydrogen sulfate 1 M (110 ml). The phases was separated and the aqueous phase was extracted with ethyl acetate (200 ml). The combined organic phases were washed with water (300 ml), dried (MgSO 4 and the solvent was removed in vacuo to afford 20,1 g af 2-(N-tert-butoxycarbonyl-N-methylamino)- 3-(2-naphthyl) propionic acid.
'H-NMR (DMSO) 1.18, 1.21 (two s, 9H); 2.62, 2.66 (two s, 3H); 3.11-3.58 2H); 4.75, 4.90 (two dd, 1H); 7.48-7.88 7H); 1.85 (s 1H)(mixture of rotamers) 2-Formylamino-3-(2-naphthyl) propionic acid.
(R)-2-amino-3-(2-naphthyl) propionic acid (18,11 g; 84,14 mmol) was dissolved in formic acid (204 ml) and acetic acid anhydride (70 ml) was added dropwise. The reaction mixture was heated to 550C and stirred 3 1 hours at room temperature.
Icecold water (70 ml) was added dropwise and stirred at 0°C for 20 min. The reaction mixture was filtrated and washed with icecold water (20 ml) to afford 20,26 g af 2-formylamino-3-(2-naphthyl) propionic acid.
WO 97/00894 WO 9700894PCT/DK96/00266 29 'H-NMR (DMS0): 3.05 (dd, 1H); 3.27 (dd, 1H); 4.64 (in, 1H); 7.48-7.87 (in, 7H); 7.95 1IH); 8.45 1 12.9 (s 1IH).
(R)-Methylamino-3-(2-naphthyl) propan-1 -oi.
0 (R)-2-Formylamino-3-(2-naphthyl)propionic acid (4,37 g; 18 mmoi) was dissolved in dry tetrahydrofuran (100 ml), and sodium borohydride (1,6 g; 43,2 mmcl) was added. Jodine (4,57 g; 18 mmol) was dissolved in dry tetrahydrofuran (40m1) and added dropwise to the reaction mixture at below 40 0 C. After addition the reaction mixture was heated to reflux for 12 hours. Potassium hydroxide (50 ml; 20%) was added. The aqueous phase was extracted with methyl tertbutyl ether (4 x 50 ml).
The combined organic layers were washed with saturated sodium chloride (150 ml), dried (MgSO 4 and the solvent was removed in vacuo. The residue was chromatographed (Silica; 5 x 40cm) using 0CM! methanol/ammonia (100: 10: 1) to afford 1,81 g of methylamino-3-(2-naphthyl)propan1-cio.
1 H-NMR (CDC 3 2.43 3H); 2.88-3.05 (in, 3H); 3.10 (s 2H); 3.42 (dd, 1H); 3.69 (dd, 1 7.30-7.82 (in, 7H).
R)-1 1 R)-2-Hydroxy-1 2 -naphthyl)methyl)ethyl)-N-methylcarbamoyl]-2 (2-naphthyl)ethyl}-N-methylcarbamic acid tert-butyl ester.
02< c--
CH
2
CN
(R)-(N-tert-Butoxycarbonyl-Nmethylamino)3(2-naphthyl) propionic acid (0,55 g; 1,67 mmcl) and methylamino-3-(2-naphthy) propan-1 -ol (0,38 g; 2,00 mmol) WO 97/00894 PCT/DK96/00266 were dissolved in methylene chloride (15 mi) and dimethylformamide (7.5 ml). The reaction mixture was cooled on an icebath. 1-Hydroxy-7-azabenzotriazole (0,24 g; 2,09 mmol) and N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (0,38 g; 2,0 mmol) were added. The reaction mixture was stirred 12 hours at room temperature. The reaction mixture was concentrated in vacuo. Ethyl acetate (200 mi) was added and the organic solution was washed with water (100 mi), sodium hydrogen carbonatelsodium carbonate (pH 9) (75 mi), sodium hydrogen sulfate ml; 10%) water (100 ml) and dried (MgSO4). The solvent was removed in vacuo and the residue was chromatographed (Silica, 2 x 45cm) using ethyl acetate to afford 0,25g of R)-2-hydroxy-1 -((2-naphthyl)methyl)ethyl)-Nmethylcarbamoyl]-2-(2-naphthyl)ethyl}-N-methylcarbamic acid tert-butyl ester.
'H-NMR (DMSO): 0.80-1.99 (several s, 9H); 2.454.20 12 4.70-5.12 2H) (selected peaks, mixture of rotamers) R)-2-hydroxy-1 -((2-naphthyl)methyl)ethyl)-N-methyl-2-methylamino-3-(2naphthyl) propionamide.
r"~X~c
I
2CH C H,
O
R)-1 R)-2-hydroxy-1 -((2-naphthyl)methyl)ethyl)-N-methylcarbamoyl]-2- (2-naphthyl)ethyl}-N-methylcarbamic acid tert-butyl ester (0,25 g; 0,475 mmol) was dissolved in DCM (3 mi). Triflouroacetic acid (1 mi) was added and the reaction mixture was stirred for 20 min. The solvent was removed in vacuo. DCM (5 ml) was added and removed in vacuo and repeated. The residue was dissolved in methanol (5 mi). Sodium hydrogen carbonate sodium carbonate (5 ml; pH 9) was added and the solution was extracted with ethyl acetate (2 x 10mi). The organic phase was dried (MgSO 4 and the solvent was removed to afford 0,22 g of (2R)-N- R)-2-hydroxy-1 2 -naphthyl)methyl)ethyl)-N-methyl-2-methylamino-3-(2 naphthyl)propionamide.
WO 97/00894 PCT/DK96/00266 31 'H-NMR (CDCI1): 1.70, 2.37, 2.45, 2.93 (four s, 6H); 2.56-3.05 2H), 3.52-3.85 7H); 4.25, 4.97 (two m, 1H); 6.86-7.78 14 H) (selected peaks, mixture of rotamers) (2R)-2-((3-Aminomethylbenzoyl)-N-Me-D-2Nal-N-Me)-3-(2-naphtyl)propanol HN H, N q H 0 o 6.
3-Boc-aminomethylbenzoic acid (502.6 mg) was dissolved in DCM (6 ml) and then converted to the symmetrical anhydride by stirring with EDAC (191.6 mg) for min.
A solution of (2R)-N-((1R)-2-hydroxy-1-((2-naphthyl)methyl)ethyl)-N-methyl-2methylamino-3-(2-naphthyl)propionamide (200 mg) in DCM (5 ml) was added to this mixture and then reacted for 20 h at r.t.
The reaction mixture was then concentrated to an oil and redissolved in EtOAc (100 ml). This solution was extracted sequentially with 5% aqueous NaHCO 3 (2 x ml), 5% aqueous KHSO 4 (2 x 50 ml) and H 2 0 (2 x 50 ml). The resulting organic phase was dried with Na 2
SO
4 and concentrated in vacuum on a rotary evaporator to an oil. The oil was then dissolved in DCM TFA 1:1 (6 ml) and stirred. After min the mixture was concentrated by a stream of nitrogen and the resulting oil was redissolved in 70% CH 3 CN 0.1% TFA (5 ml) and diluted with water to a volume of 100 ml.
This crude product was then purified by semipreparative HPLC in two runs and lyophilized using similar procedures as described in example 1.
The final product obtained was characterised by analytical RP-HPLC (retention time) and by Plasma desorption mass spectrometry (molecular mass). The molecular mass found 559.5 amu) agreed with the expected structure (teor.
MH+* 560.72 amu) within the experimental error of the method.
The RP-HPLC retention time using elution conditions Al and B1 as defined in example 1 was found to be 33.07 min and 34.63 min, respectively.
WO 97/00894 PCT/DK96/00266 32 EXAMPLE 6 H-Aib-His-D-2Nal-N-Me-D-Phe-NH 2
N
NH
c 0 The peptide was synthesized according to the Fmoc strategy on an Applied Biosystems 431A peptide synthesizer in 0.22 mmol scale using the manufacturer supplied FastMoc UV protocols which employ HBTU mediated couplings in NMP and UV monitoring of the deprotection of the Fmoc protection group. The starting resin used for the synthesis was cat. D-1675 from Bachem Feinchemikalien AG, Bubendorf, Switzerland (427) mg which is a Fmoc-2,4-dimethoxy-4'- (carboxymethyloxy)-benzhydryl-amine linked to amino methyl polystyrene resin through an amide bond. The substitution capacity was 0.55 mmol g The protected amino acid derivatives used were Fmoc-N-Me-D-Phe-OH, Fmoc-D-2Nal- OH, Fmoc-His(Trt) and Fmoc-Aib-OH. The coupling of Fmoc-N-Me-D-Phe-OH was carried out as a double coupling. After the synthesis the peptide was cleaved from 750 mg of the peptide resin by stirring for 180 min at room temperature with a mixture of 8 mi TFA, 600 mg phenol, 200 ip. ethanedithiol, 400 p. thioanisole and 400 l H 2 0. The cleavage mixture was filtered and the filtrate was concentrated to approximately 2 ml by a stream of nitrogen. The crude peptide was precipitated from this oil with 50 ml diethyl ether and washed 2 times with 50 ml diethyl ether.
The crude peptide was dried and purified by semipreparative HPLC in one run and lyophilized using similar procedures as described in example 1.
The final product obtained was characterised by analytical RP-HPLC (retention time) and by Plasma desorption mass spectrometry (molecular mass). The molecular mass found (MH+:598.5 amu) agreed with the expected structure (teor.
MH+: 598.73 amu) within the experimental error of the method.
The RP-HPLC retention time using elution conditions Al and B1 as defined in example 1 was found to be 24.68 min and 25.58 min, respectively.
WO 97/00894 PCT/DK96/00266 33 EXAMPLE 7 H-Aib-His-D-2Nal-N-Me-D-Phe-Ser-NH 2 This compound was synthesized using similar procedures as described in example 6. The final product obtained was characterised by analytical RP-HPLC (retention time) and by Plasma desorption mass spectrometry (molecular mass). The molecular mass found (MH':685.6 amu) agreed with the expected structure (teor.
MH': 685.81 amu) within the experimental error of the method.
The RP-HPLC retention time using elution conditions Al and B1 as defined in example 1. was found to be 24.42 min and 25.92 min, respectively.
EXAMPLE 8 (3-Aminomethylbenzoyl)-D-2Nal-N-Me-D-Phe-NH 2 iII This compound was synthesized using similar procedures as described in example 6. The only exception was that the coupling of Fmoc-D-2Nal-OH was performed using HATU as the activating reagent. H-N-Me-D-Phe-resin (0.23 mmol) was coupled for 150 min with 1 mmol Fmoc-D-2Nal-OH using 1 mmol HATU in the presence of DIEA (2 mmol).
The final product obtained was characterised by analytical RP-HPLC (retention time) and by Plasma desorption mass spectrometry (molecular mass). The molecular mass found (MH+:511.2 amu) agreed with the expected structure (teor.
WO 97/00894 PCT/DK96/00266 34 MH"+ 509.6 amu) within the experimental error of the method.
The RP-HPLC retention time using elution conditions Al and B1 as defined in example 1. was found to be 30.73 min and 32.47 min, respectively.
EXAMPLE 9 (4-Piperidinecarbonyl)-D-2Nal-N-Me-D-Phe-NH 2 This compound was synthesized using similar procedures as described in example 8. The final product obtained was characterised by analytical RP-HPLC (retention time) and by Plasma desorption mass spectrometry (molecular mass). The molecular mass found (MH':486.8 amu) agreed with the expected structure (teor.
MH: 487.6 amu) within the experimental error of the method.
The RP-HPLC retention time using elution conditions Al and B1 as defined in example 1 was found to be 27.03 min and 28.48 min, respectively.
EXAMPLE ((3R)-3-Piperidinecarbonyl)-D-2Nal-N-Me-D-Phe-NH 2
C
WO 97/00894 PCT/DK96/00266 This compound was synthesized using similar procedures as described in example 8. The final product obtained was characterised by analytical RP-HPLC (retention time) and by Plasma desorption mass spectrometry (molecular mass). The molecular mass found (MH':486.9 amu) agreed with the expected structure (teor.
MH+: 487.6 amu) within the experimental error of the method.
The RP-HPLC retention time using elution conditions Al and B1 as defined in example 1 was found to be 28.03 min and 29.50 min, respectively.
EXAMPLE 11 (3-Aminomethylbenzoyl)-N-Me-D-2Nal-N-Me-D-Phe-NH 2 YH ?o 0 ICH, 0 This compound was synthesized using similar procedures as described in example 8. with the exceptions that the last residue is introduced by using a symmetrical anhydride coupling. Boc-3-Aminomethylbenzoic acid (251 mg) is stirred for 15 min with EDAC (96 mg) in DCM. Then the resin (429 mg) is added and stirring is continued for 18 h. The other exception is that the time used to cleave the peptide from the resin was reduced to 60 min. The final product obtained was characterised by analytical RP-HPLC (retention time) and by Plasma desorption mass spectrometry (molecular mass). The molecular mass found (MH :522.9 amu) agreed with the expected structure (teor. MH+: 523.6 amu) within the experimental error of the method.
The RP-HPLC retention time using elution conditions Al and B1 as defined in example 1 was found to be 28.83 min and 30.13 min, respectively.
WO 97/00894 PCT/DK96/00266 36 EXAMPLE 12 H-Aib-His-N-Me-D-2Nal-N-Me-D-Phe-NH 2 °1 HC 0 This compound was synthesized using similar procedures as described in example 8; where both Fmoc-N-Me-D-2Nal-OH and Fmoc-His(Trt) were coupled using HATU and the time used to cleave the peptide from the resin was reduced to min. The final product obtained was characterised by analytical RP-HPLC (retention time) and by Plasma desorption mass spectrometry (molecular mass).
The molecular mass found (MH+:612.3 amu) agreed with the expected structure (teor. MH': 612.8 amu) within the experimental error of the method.
The RP-HPLC retention time using elution conditions Al and B1 as defined in example 1 was found to be 24.33 min and 26.20 min, respectively.
EXAMPLE 13 (3-Aminomethylbenzoyl)-D-Phe-N-Me-D-Phe-Lys-NH 2 This compound was synthesized using similar procedures as described in example 8. The final product obtained was characterised by analytical RP-HPLC (retention time) and by Plasma desorption mass spectrometry (molecular mass). The molecular mass found (MH':587.2 amu) agreed with the expected structure (teor.
WO 97/00894 PCT/DK96/00266 37 MH+: 586.74 amu) within the experimental error of the method.
The RP-HPLC retention time using elution conditions Al and B1 as defined in example 1. was found to be 21.13 min and 22.60 min, respectively.
EXAMPLE 14 (3-Aminomethylbenzoyl)-N-Me-D-Phe-N-Me-D-Phe-Lys-NH 2 HN N NH
NH
00 0 CH6 0 1 This compound was synthesized using similar procedures as described in example 11. The final product obtained was characterised by analytical RP-HPLC (retention time) and by Plasma desorption mass spectrometry (molecular mass). The molecular mass found (MH':601.6 amu) agreed with the expected structure (teor.
MH'+ 601.77 amu) within the experimental error of the method.
The RP-HPLC retention time using elution conditions Al and B1 as defined in example 1. was found to be 20.40 min and 21.70 min, respectively.
EXAMPLE ((3R)-3-Piperidinecarbonyl)-N-Me-D-Phe-N-Me-D-Phe-Lys-NH 2 SCH 0 0 NH
NH,
0 CH, 0
NH,
WO 97/00894 PCT/DK96/00266 38 This compound was synthesized using similar procedures as described in example 11. The final product obtained was characterised by analytical RP-HPLC (retention time) and by Plasma desorption mass spectrometry (molecular mass). The molecular mass found (MH':579.4 amu) agreed with the expected structure (teor.
MH 579.8 amu) within the experimental error of the method.
The RP-HPLC retention time using elution conditions Al and B1 as defined in example 1. was found to be 19.88 min and 21.20 min, respectively.
EXAMPLE 16 H-Aib-His-N-Me-D-Phe-N-Me-D-Phe-Lys-NH 2
N
H"
H C CH, 0
NH,
This compound was synthesized using similar procedures as described in example 12. The final product obtained was characterised by analytical RP-HPLC (retention time) and by Plasma desorption mass spectrometry (molecular mass). The molecular mass found (MH :690.6 amu) agreed with the expected structure (teor.
MH'+ 690.9 amu) within the experimental error of the method.
The RP-HPLC retention time using elution conditions Al and B1 as defined in example 1. was found to be 15.71 min and 17.82 min, respectively.
WO 97/00894 PCT/DK96/00266 39 EXAMPLE 17 ((3R)-3-Piperidinecarbonyl)-N-Me-D-2Nal-N-Me-D-Phe-NH 2 0 CH, 0 This compound was synthesized using similar procedures as described in example 11. using Fmoc-N-Me-D-Phe-OH, Fmoc-N-Me-D-2Nal-OH and Boc-(R)-Nipecotic acid, where both Fmoc-N-Me-D-2Nal-OH and Boc-(R)-Nipecotic acid were coupled using HATU. The final product obtained was characterised by analytical RP-HPLC (retention time) and by Plasma desorption mass spectrometry (molecular mass).
The molecular mass found (MH+:500.7 amu) agreed with the expected structure (teor. MH*: 501.7 amu) within the experimental error of the method.
The RP-HPLC retention time using elution conditions Al and B1 as defined in example 1. was found to be 28.18 min and 29.55 min, respectively.
EXAMPLE 18 H-Aib-Ala-D-2Nal-N-Me-D-Phe-Lys-NH 2 O HC 0 0 HX\C N H N H
NH,
HC 0 CH, 0 I N
H
This compound was synthesized using similar procedures as described in example 6. The final product obtained was characterised by analytical RP-HPLC (retention WO 97/00894 PCT/DK96/00266 time) and by Plasma desorption mass spectrometry (molecular mass). The molecular mass found (MH':660.7 amu) agreed with the expected structure (teor.
MH+: 660.8 amu) within the experimental error of the method.
The RP-HPLC retention time using elution conditions Al and B1 as defined in example 1. was found to be 25.63 min and 26.75 min, respectively.
EXAMPLE 19 H-3-Aminomethylbenzoyl-N-Me-D-2Nal-N-Me-D-Phe-N
H-CH
3 0 CH 3 0
H
2 N N N CNH 3 SCH, II
CH
3 0 Boc-3AMB-OH (115 mg, 0.458 mmol), 1-hydroxy-7-azabenzotriazole (62 mg, 0.458 mmol) and 1-ethyl-3(3-dimethylaminopropyl)carbodiimide hydrochloride (97 mg, 0.504 mmol) were dissolved in DCM (8 ml) and DMF (1 ml) and stirred for 15 min.
N-methyl-2-methylamino-N-((1R)-1 -(methylcarbamoyl)-2-phenylethyl)-3-(2naphthyl)propionamide (185 mg, 0.458 mmol) dissolved in DCM (5 ml) was added followed by addition of diisopropylethylamine (80 ml, 0.458 mmol) and the mixture was stirred for 20 hours.
The organic phase was washed with sodium hydrogen carbonate (50 ml, H 2 0 ml) and saturated NaCI/H 2 0 (50 ml) and dried with sodium sulfate and evaporated in vacuo. The residue was dissolved in DCM (2 ml) and treated with TFA (2 ml) for 10 min. With a stream of N 2 the volatiles was removed. The residue was dissolved in 50 ml of 20% MeCN and diluted to 500 ml with H 2 0.
WO 97/00894 PCT/DK96/00266 41 Semipreparative HPLC milmin., 5 runs, 30-40% MeCN/0,1 M (NH 4 2 S0 4 pH Detection 276 nm, Sep-Pals, 70% MeCN/IO,1 TFA, Lyophilization PD-MS Theory 536,7 Found 535,7±t 1 HPLC Al rt 31,20 min B1 rt 36,35 min EXAMPLE H-Aib-His-N-Me-D-2Nal-N-Me-D-Phe-NHMe
N
O H 3 C O 0 N NH
H
2 N NH N 'CH3 2 NH I I HC O0 CH, O Fmoc-L-His(Trityl)-OH (1,54 g, 2.48 mmol) (BACHEM B-1570) and 1-hydroxyazabenzotriazol (338 mg, 2.48 mmol) were dissolved in 9 ml of DMF, cooled to 0-4 0 C and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (475 mg, 2.48 mmol) was added. The reaction mixture was stirred for 15 min. at 04 C.
N-Methyl-2-methylamino-N-((1 R)-1 -(Methylcarbamoyl)-2-phenylethyl)-3-(2naphthyl)propionamid (500 mg, 1.24 mmol) dissolved in methylen chloride (18 mi) was cooled to 0-40 C and added and stirred for 1 hour at 0-40 C followed by addition of diisopropylethylamine (0.425 ml, 2.48 mmol). The temperature of the mixture was slowly raised to room temperature and the mixture was stirred for 72 hours. DCM was evaporated in a stream of N 2 and to the mixture was added 100 ml ethyl acetate and washed with sodium hydrogen carbonate (2 x 100 ml, and potassium hydrogen WO 97/00894 PCT/DK96/00266 42 sulfate (100 ml, The phases were separated and the organic phase was dried with sodium sulfate and evaporated in vacuo. The residue was dissolved in DMF (8 ml) and treated with piperidine for 15 min., diluted with H 2 0 (100 ml) and quenched with acetic acid (1,5 ml). Acetonitrile was added and the mixture was diluted with H 2 0 to 250 ml. The clear solution was applied to a 10 g "Seppaks" #Water, washed with H 2 0/0,03 m HCI and eluted with 50 ml 35% MeCN/0,03 M HCI. Deluted with H 2 0 to 200 ml and lyophilized.
Boca aminoisobutyric acid (756 mg, 3.72 mmol), 1-hydroxyazabenzotriazole hydrate (506 mg, 3.72 mmol) and 1-ethyl-3(3-dimethylaminopropyl)carbodiimide hydrochloride (713 mg, 3.72 mmol) were dissolved in DMF (6 ml) and after 15 min. was added H-L- His(trityl)-NMeD2Nal-NMeDPhe-NHCH 3 2 HCL dissolved in DCM (12 ml) followed by addition of diisopropylethylamine (0.637 ml) and stirred for 72 hours. DCM was evaporated in a stream of N 2 and the mixture was added 100 ml ethyl acetate and washed with sodium hydrogen carbonate (2 x 50 ml, and potassium hydrogen sulfate (50 ml, The phase were separated and the organic phase was dried with sodium sulfate and evaporated in vacuo. The residue was dissolved in DCM (6 ml), cooled to 0-40 C and treated with TFA (6 ml) for 10 min. at 0-40 C. With a stream of N 2 the volatiles was removed. The oily residue was dissolved in 35 ml of 70% acetonitrile diluted with H 2 0 to 50 ml and added 10 ml of concentrated hydrochloric acid (12 molar) and stirred for 72 hours. The mixture was diluted to 200 ml with H 2 0 and neutralized with solid sodium carbonate, finally diluted to 400 ml with H 2 0.
Semipreparative HPLC PD-MS, theory: 550,7, found 550,1 HPLC Al rt. 31,75 min.
B1 rt. 36,15 min.
WO 97/00894 PCT/DK96/00266 43 EXAMPLE 21 3-methylaminomethylbenzoyl-N-Me-D-2Nal-N-Me-D-Phe-NH-CH 3 0 H 3 C 0 HIC C IH
H
3 C'NH J N NHCH CH, O Boc-NMe3AMB-OH (658 mg, 2.48 mmol), 1-hydroxyazabenzotriazole hydrate (338 mg, 2.48 mmol) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (475 mg, 2.48 mmol) were dissolved in 6 ml of DMF and stirred for 15 min.
N-Methyl-2-methylamino-N-((1 R)-1-(methylcarbamoyl)-2-phenylethyl)-3-(2naphthyl)propionamid (500 mg, 1.24 mmol) dissolved in methylene chloride (12 ml) was added, followed by addition of diisopropylethylamine (0,425 ml, 2.48 mmol).
The mixture was stirred for 20 hours.
DCM was evaporated in a stream of N 2 and to the mixture was added 75 ml of ethyl acetate and washed with sodium hydrogencarbonate (2 x 50 ml, and potassium hydrogen sulfate (50 ml, The phases were separated and the organic phase was dried with sodium sulfate and evaporated in vacuo. The residue was dissolved in 10 ml methylene chloride, cooled to 0-40 C and treated with TFA (10 ml) for 10 min. at 0-40 C. With a stream of N 2 the volatiles were removed. The oily residue was dissolved in 25 ml of 70% MeCN/0,1 TFA and diluted to 600 ml with H 2 0.
WO 97/00894 WO 9700894PCT/DK96/00266 44 Semipreparative HPLC Large column, 40 mI/min., 8 runs 28-40& P 11 (NH4) 2 S0 4 276 nM.
Seppak, Lyophilization PD-MS Theory: 550,7, found 550,1 HPLC Al rt 31,75 min 13I rt 36,15 min EXAMPLE 22 Piperidine-4-carboxylic acid R)-1 R)-2-(4-iodophenyl)-l (methylcarbamoyl)ethyl)-N-methylcarbamoyl)-2-(2-naphthy)ethy)N-.
methylamide 0 CH 3 0
NHH
HNO H 3 C 0 2
R)-
2 -(N-tert-butoxycarbony-Nmethylamino)-3(4-iodophenyl)propionic acid Prepared according to Can J. Chem. (1977), 55, 906.
'H-NMR: (CDCI 3 d 1.34 1.38 4.5H), 2.70 2.75 2.85-3.10 (in, 1 3.2-3.4 (in, 1IH); 4.4-4.6 (in, 6.9-7.0 (in, 2H), 7.62 (d, J =10 Hz, 2 Hz), 9.5-10 (bs, 1lH) WO 97/00894 PCTIDK96/00266 I R)-2-(4-Iodophenyl)-1 -(methylcarbamoyl)ethyl)-N-methylcarbamic acid tert-butylester
CH
3 0 K- N
N~ICH
CH
3 0 2
R)-
2 -(N-tert-butoxycarbonyl-Nmethylamino)3(4-iodophenyl)propionic acid (2.00 g, 4.9 mmol) was dissolved in methylene chloride (20 ml).
Hydroxybenzotriazole hydrate (0.67 g, 4.9 mmol) and 1 -ethyl-3-(3dimethylaminopropyl)-carbodiimid hydrochloride (0.99 g, 4.9 mmol) were added and the mixture was stirred for 15 minutes. Methylamine (0.38 g of a solution in methanol, 4.9 mmol) was added and the mixture was stirred overnight. Methylene chloride (40 ml) was added and the mixture was washed with a saturated aqueous solution of sodium hydrogen carbonate (50 ml) and a solution of sodium hydrogen sulphate 50 ml). The organic phase was dried (MgSO 4 and the solvent was removed in vacuo. The residue was chromatographed on silica (2.5 x 20 cm) using ethyl acetate/heptane as eluent to afford 1.77 g of I R)-2-(4-iodophenyl)-1 ethyl carba moyl) ethy )-N-methylcarba m ic acid tert-butyl ester.
'H-NMR: (CDCI 3 (selected peaks for major rotamer) d 1.39 9H); 2.75 (s, 3H); 2.80 3H); 3.29 (dd, 1IH); 4.88 1 H).
2
R)-
3 4 -lodophenyl)-N-methyl2(methylamino)propionamide
H
3 C-K NH NHf '6H 3 0 WO 97/00894 PCT/DK96/00266 46 R)-2-(4-lodophenyl)-1 -(methylcarbamoyl)ethyl)-N-methylcarbamic acid tert-butylester (1.7 g; 4.0 mmol) was dissolved in methylene chloride (10 ml) and trifluoroacetic acid (5 ml) was added. The mixture was stirred for 1 h.
Methylene chloride (30 ml) and water (30 mi) was added. Solid sodium hydrogen carbonate was added to pH 8. The organic phase was separated, dried (MgSO 4 and evaporated in vacuo to afford 1.22 g of 2
R)-
3 4 -iodophenyl)-N-methyl-2-(methylamino)propionamide H-NMR: (CDCI 3 d 2.28 3H); 2.68 (dd, 1H); 2.81 3H); 3.08-3.19 2H); 6.95 2H); 7.63 2H) N-Methyl-N-((l R)-1 -(N-methyl-N-((l R)-1 -(methylcarbamoyl)-2-(4iodophenyl)ethyl)carbamoyl)-2-(2-naphthyl)ethyl)carbamic acid tert-butylester
CH
3 0 H 3 C O O N N NHCH CH3 O L30 (2R)-2-(N-tert-butoxycarbonyl -N-methylamino)-3-(2-naphthyl)propionicacid (1.10 g; 3.30 mmol) was dissolved in methylene chloride (10 mi) and HOAt (0.45 g, 3.1 mmol) and EDAC (0.66 g, 3.5 mmol) were added. After stirring for min, 2
R)-
3 -(4-iodophenyl)-N-methyl-2-(methylamino)propionamide (1.0 g, 3.1 mmol) and diisopropylethylamine (0.45 g, 3.4 mmole) were added and the mixture was stirred overnight. Methylene chloride (30 mi) was added and the mixture was washed with a saturated aqueous solution of sodium hydrogen carbonate (30 mi) and a solution of sodium hydrogen sulphate 30 ml).
The organic phase was dried (MgSO 4 and the solvent was removed in vacuo.
The residue was chromatographed on silica (2.5 x 20 cm) using ethyl acetate/heptane as eluent to afford 1.74 g of N-methyl-N-((1R)-1-(Nmethyl-N-((1 R)-1 -(methylcarbamoyl)-2-(4- WO 97/00894 WO 9700894PCT/DK96/00266 47 iodophenyl)ethyl)carbamoyl)-2-(2-naphthyl)ethyl)carbamic acid tert-butylester.
'H-NMR: (CDCI 3 (selected peaks for major rotamer) d 1.38 9H); 2.18 (d, 3H); 2.45 3H); 2.75 3H)5.05 (in, 1 5.42 (in, 1 H).
R)-2-(4-lodophenyl)-1 -(methylcarbamoyl)ethyl)-N-methyl-2methylamino-3-(2-naphthyl)propionamide H3C 0
H
3 C. %KF. NH H 0 N-Methyl-N-((1 R)-1 -(N-methyl-N-((1 R)-1 -(methylcarbamoyl)-2-(4iodophenyl)ethyl)carbamoyl)-2-(2-naphthyl)ethyl)carbamic acid tert-butylester was dissolved in a mixture of methylene chloride and trifluoroacetic acid and stirred for 15 min.
Methylene chloride (20 ml) and water (30 ml) were added. Solid sodium hydrogen carbonate was added to pH 8. The organic phase was separated, dried (MgSO 4 and evaporated in vacuo to afford 1.40 g of R)-2-(4-iodophenyl)-1 -(methylcarbamoyl)ethyl)-N-methyl-2methylamino-3-(2-naphthyl )propionamide.
'H-NMR: (CDCI 3 (selected peaks for major rotamer) d 1.79 3H); 2.02 (d, 3H); 2.55 3H); 3.78 (dd, 1 5.44 (dd, 1IH).
I R)-1 1 R)-2-(4-lodophenyl)-1 -(methylcarbomoyl)ethyl)-Nmethylcarbamoyl)-2-(2-naphthyl)ethyl )-N-methylcarbamoyl)piperidine-1 -carbox: ylic acid tert-butylester WO 97/00894 PCT/DK96/00266 48 O CH 3 0 N N NHCH 3 O N
CH
3
O
HC
CH
3 1-tert-butoxycarbonylpiperidine-4-carboxylic acid (143 mg, 0.66 mmol) was dissolved in methylene chloride (10 ml) and HOAt (90 mg, 0.66 mmol) and EDAC (140 mg, 0.73 mmol) were added. After 15 min of stirring R)-2-(4-iodophenyl)-1 -(methylcarbamoyl)ethyl)-N-methyl-2methylamino-3-(2-naphthyl)propionamide (350 mg, 0.66 mmol) and diisopropylethylamine (85 mg, 0.66 mmol) were added and the mixture was stirred overnight. Methylene chloride (20 mi) was added and the mixture was washed with a saturated aqueous solution of sodium hydrogen carbonate mi) and a solution of sodium hydrogen sulphate 20 ml). The organic phase was dried (MgSO 4 and the solvent was removed in vacuo. The residue was chromatographed on silica (2.5 x 20 cm) using ethyl acetate as eluent to afford 412 mg of R)-1 R)-2-(4-iodophenyl)-1 -(methylcarbomoyl)ethyl)-Nmethylcarbamoyl)-2-(2-naphthyl)ethy)-N-methylcarbamoyl)piperidine-1 carboxylic acid tert-butylester.
R)-1 R)-2-(4-lodophenyl)-1 -(methylcarbomoyl )ethyl)-Nmethylcarbamoyl)-2-(2-naphthyl)ethyl)-N-methylcarbamoyl)piperidine-1 carboxylic acid tert-butyl ester (412 mg 0.56 mmol) was dissolved in a mixture of methylene chloride (5 ml) and trifluoroacetic acid (5 mi) and stirred for 5 min.
Methylene chloride (20 ml) and a saturated aqueous solution of sodium hydrogencarbonate (20 mi) was added. Solid sodium hydrogen carbonate was added to pH 8. The phases were separated and the organic phase was dried WO 97/00894 PTD9/06 PCT/DK96/00266 49 (MgSO 4 and evaporated to afford 255 mg of the title compound.
'H-NMR: (CDC 13) (selected peaks for major rotamer) d 2.32 3H); 2.58 (s, 3H); 2.68 5.33 (in, 1IH); 5.84 1IH) HPLC: rt 33.35 min (Al) PDMS: mlz 640.8 Abbreviations
HBTU
NMP
HATU
Trt-
HOBT
3-AmB N-Me-3-AMB O-(Benzotriazol-1 -yl)-l 3-tetramethyluronium hexafluorophosphate N-methyl pyrrolidone O-(7-Azabenzotriazol--l 1,3, 3-tetramethyluronium hexafluorophosphate Trityl 7-hydroxybenzotriozole hydrate 3-Aminomethylbenzoy 3-methylaminomethylbenzoyl

Claims (13)

1. A compound of general formula I A-B-C-D(-E)p wherein p is 0 or 1; A is hydrogen or R 1 -(CH 2 r (CH 2 wherein q is 0 or an integer selected from the group: 1, 2, 3, 4, r is 0 or 1; s is 0 or an integer selected from the group: 1, 2, 3, 4, R 1 is hydrogen, imidazolyl, guanidino, piperazino, morpholino, piperidino or N(R2)- R 3 wherein each of R 2 and R 3 is independently hydrogen or lower alkyl optionally substituted by one or more hydroxyl, pyridinyl or furanyl groups; and X, when r is 1, is -CH 2 -CH=CH-, -C(R 16 )(R 17 or .wherein each of R 16 and R 17 is independently hydrogen or lower alkyl; B is wherein each of t and u independently is 0 or 1; G and H are amino acid residues selected from the group consisting of natural L- amino acids or their corresponding D-isomers, or non-natural amino acids such as 1,4-diaminobutyric acid, amino-isobutyric acid, 1,3-diaminopropionic acid, 4- aminophenylalanine,
3-pyridylalanine, 1,2, 3 ,4-tetrahydroisoquinoline-3-carboxylic acid, 1, 2 3 4 -tetrahydronorharman-3-carboxylic acid, N-methylanthranilic acid, anthranilic acid, N-benzylglycine, 3-aminomethylbenzoic acid, 3-amino-3-methyl butanoic acid, sarcosine, nipecotic acid or iso-nipecotic acid; and wherein, when both t and u are 1, the amide bond between G and H being optionally replaced by Y-NR 18 wherein Y is -CO- or -CH 2 and R 18 is hydrogen, lower alkyl or lower aralkyl; 51 C is a D-amino acid of formula -NH-CH((CH 2 )w-R 4 )-CO-wherein W is 0, 1 or 2; and R 4 is selected from the group consisting of N H X8- or I each of which is optionally substituted with halogen, lower alkyl, lower alkyloxy, lower alkylamino, amino or hydroxy; when p is 1, is a D-amino acid of formula -NR 20 -CH((CH 2 )k-R)-CO- or, when p is 0, D is-NR 20 -CH((CH 2 1 -R 5 )-CH 2 -R 6 or -NR-CH((CH 2 )m-R)-COR 6 wherein 10 kis 0, 1 or 2; 1 lisO, 1 or2; mis0, 1 or2; R 20 is selected from the group consisting of lower alkyl or lower aralkyl; i' R 5 is selected from the group consisting of S S each of which is optionally substituted with halogen, lower alkyl, lower alkyloxy amino or hydroxy; and R 6 is piperazino, morpholino, piperidino, -OH or -N(R 7 )-R 8 wherein each of R 7 and R is S> independently hydrogen or lower alkyl; 2 E, when when p is 1, is -NH-CH(R)-(CH2)v-R 9 wherein [R \LIBAA]06320.doc:mcc 51a V is 0 or an integer selected from the group: 1, 2, 3, 4, 5, 6, 7, 8; R 9 is hydrogen, imidazolyl, guanidino, piperazino, morpholino, piperidino, -N(R 1 12 a. a. a a a. a a a a a a.. -v [RALIBAA]06320.doc:mcc WO 97/00894 PCT/DK96/00266 52 (CH )n or n R19 wherein n is 0, 1 or 2, and R 19 is hydrogen or lower alkyl, R11 R 1 1 CH2)-N/ (H2)-N R 12 or R12 wherein o is an integer selected from the group: 1, 2, 3, each of R 1 1 and R 12 is independently hydrogen or lower alkyl, or 0 C/ k or each of which is optionally substituted with halogen, lower alkyl, lower alkyloxy, amino, alkylamino, hydroxy, or the Amadori rearrangement product from an amino group and a hexapyranose or a hexapyranosyl-hexapyranose and R 1 0 when p is 1, is selected from the group consisting of H, -COOH, -CH 2 -R 13 -CO-R 13 or -CH 2 -OH, wherein R 13 is piperazino, morpholino, piperidino, -OH or -N(R 1 4 )-R 1 5 wherein each of R 14 and R 15 is independently hydrogen or lower alkyl; all amide bonds within formula I with the exception of the bond between C and D may independently be replaced by -Y-NR 18 wherein Y is -CO- or -CH 2 and R 1 8 is hydrogen, lower alkyl or lower aralkyl; or a pharmaceutically acceptable salt thereof; and with the exception of the compounds 3 -Aminomethylbenzoyl)-D-2Nal-N-Me-D-Phe-Lys-NH 2 H-Aib-His-D-2Nal-N-Me-D-Phe-Lys-NH 2 H-Aib-His-N-Me-D-2Nal-N-Me-D-Phe-Lys-NH 2 53 3 -(H-Aib-His-D-2Nal-N-Me-D-Phe-MI). 1-morpholinopropane, 2-(H-Aib-His-D-2Nal- N-Me-D-Phe-NI{)-( 1 -methyl-2-pyrrolidinyl)ethane, ((3R)-Piperidinecarbonyl)-N-Me-D- 2Nal-N-Me-D-Phe-Lys-NH 2 3 -Aminomethyib enzoyl)-D-2Nal-N-Me-D-Phe-N-H) 1- morpholinopropane, 2-(H-Aib-His-N-Me-D-2Na1-N-Me-D-Phe-NH)-(l1-methyl-2-pyrrolidinyl)ethane, 2-(((3R)-Piperidinecarbony)-N-MeD2Na-NMeDPhe-NH>( 1 -methyl-2- pyrrolidinyl)ethane, 2-((3-Aminomethybenzoy)-NMeD2Na-NMeDPheII>( 1 -methyl-2- pyrrolidinyl)ethane, 3 -(H-Aib-His-N-Me-D-2Nal-N-Me-D-Phe-NH)-l1-morpholinopropane, 3-(((3R)-Piperidinecarbony1)-N-Me-D-2Na-NMeDPhe-Nfjj 1-morpholinopropane, 3-((3-Aminomethylbenzoyl)-N-Me-D-2Nal-N-Me-D-Phe-NHy 1-morpholinopropane, -Aminomethylbenzoyl)-D-2Nal-N-MeDPhe-NH)- 1 -methyl-2-pyrrolidinyl)ethane, 2-(((3R)Piperidinecarbony1)-D-2Na1-N-Me-D-Phe-NH-( 1 -methyl-2-pyrrolidinyl)ethane, 15 and with the exceptions of the compounds 2 H* *N N.9H NN [R:\LIBAAj06320.doc:mcc -NH 2 p. p p p. p p. p p p p p. p. p p p p. *ppp.. p p p p p *Ppp p p p.. p p pp pp.. p p. p p p pp pp.. \LIBAA]063 20.do: mcc 33uwzou-oovygff\:d] HH Pu e ec.. a a a a a a a a a a a a a a a. a a. a a a a a. a a. a a p a a a. a a. a a a 53c 2. A compound according to claim 1, wherein A is hydrogen, 3-AMB, N-Me-3-AMB or Aib. 3. A compound according to claim 1, wherein t is 1 and u is 0 and G is selected from the group consisting of 3-aminomethylbenzoyl, nipecotic acid and isonipecotic acid.
4. A compound according to claim 1, wherein t is 1, u is 1, G is Aib, and H is selected from the group consisting of His, Phe and Ala. A compound according to claim 1, wherein C is selected from the grop consisting of D-2-Nal and D-Phe.
6. A compound according to claim 1, wherein, when p is 1, D is D-Phe or D-2Nal.
7. A compound according to claim 1, wherein, when p is 0, D is D-Phe- NH 2 or D-2Nal-NH2. *@9 C° [R:\LIBAA]06320.doc:mcc WO 97/00894 WO 9700894PCT/DK96/00266 54
8. A compound according to claim 1, wherein E, when p is 1, is -NH-CH(R 1 0 (CH 2 9 wherein v is 0 or an integer selected from the group: 1,2,3,4; R 9 is hydrogen, morpholino, piperidino, N(R 11 1 2 or (CH )n wherein n is 0, 1 or 2, and R' 9 is hydrogen or lower alkyl, each of R 1 1 and R 12 is independently hydrogen or lower alkyl, and R' 0 when p is 1, is selected from the group consisting of -COOH, -CH 2 -R 13 -CO-R 1 3 or -CH 2 -OH, wherein R 13is piperazino, morpholino, piperidino, -OH or -N(R 14 1 5 wherein each of R 1 4 and R 1 5 is independently hydrogen or lower alkyl.
9. A compound according to claim 1, wherein at least one of the amide bonds between A and B, between B and C, between D and (E)p and between G and H is replaced by -CO-N(CH 3 A compound selected from the group consisting of (R--(-mnmtybnol--eD2a--e--hnlrpnl or the TFA salt thereof; 3-((3-Aminomethylbenzoyl))NMeD2NalNMeDPhe-NH)-1 -1 N, N- dimethylaminopropane, or the TFA salt thereof;, 3-(((3R)-3-Piperidinecarbonyl)-N-Me-D-2NalNMeDPhe-NH)-I N, N-dimethylaminopropane, or the TFA salt thereof; 2 -(((3R)-3-Piperidinecarbonyl)-N-Me-D-2NalNMeDPhe-NH)-(1 -methyl-2- pyrrolidinyl)ethane, or the TFA salt thereof;, H-Aib-His-D-2Nal-N-Me-D-Phe-Ser-NH 2 or the TFA salt thereof;, (3-Aminomethylbenzoyl)-D-2Nal-N-MeD-Phe-Lys.N H 2 or the TEA salt thereof; 4 -Piperidinecabonyl)o2NalNMeD-Phe-N H 2 or the TFA salt thereof;, 3 R)-3-Piperidinecarbonyl)-D-2Nal-N-Me-D-Phe-NH 2 or the TFA salt thereof; 3 -Aminomethylbenzoyl)D-Phe-NMeD-Phe-NH 2 or the TFA salt thereof;, (3Aioehlezy)NMeDPeNM--h-y-H 2 or the TFA salt thereof, WO 97/00894 PCT/DK96/00266 3 R)- 3 -Piperidinecarbonyl)NMeDPheNMeDPhe-Lys-NH2 or the TEA salt thereof;, H-Aib-His-N-MeDPheNMe-D-Phe-Lys-NH 2 or the TFA salt thereof; 3 R)- 3 -Piperidinecabonyl)NMeD2NaINMeD-Phe-NH2 or the TFA salt thereof;, 2 R)- 2 3 Aminomethylbenzoyl).NMe.D2NalNMe)3(2naphthyl)propal, or the TFA salt thereof;, 3 -Aminomethylbenzoyl)NMeD2NalNMeD-Phe-NH2 or the TFA salt thereof;, 3 3 -Aminomethylbenzoyl)NMeD-Phe-NH)1 -N,N-dimethylaminopropane, H-Aib-His-N-Me-D-2Nal-NMe.D-Phe.NH 2 or the TFA salt thereof; 3 -Aminomethylbenzoyl)NMe-D2NaIN-MeD-Phe-Lys-NH2 H-Aib-Ala-D-2Nal-N-MeD-Phe.Lys.NH 2 or the TEA salt thereof; H-Aib-His-D-2Nal-N-Me.D..Phe.NH 2 or the TEA salt thereof; 2 3 -AminomethylbenzoyNMeD2NalNMeDPhe-NH)-1 -morpholinoethane, (3-AminomethylbenzoylNMeD2Naj-N-Me-DPhe-NH-Me, 3 3 -Methylaminomethylbenzoyl)NMeD2NaNMeDPhe-NH)l N- dimethylaminopropane, 3 -Aminomethylbenzoyl)NMeD2NalIN-Me-D-Phe-N-Me2 H-Aib-His-N-Me-D-2Nal.N-.Me..D-.Phe-NH2 3-mnmtybnolNM--NlNM--h-HC3 or the TEA salt thereof;, 3 -methylaminomethylbenzoyl.N-MeD2NaNMeDPhe-NHCH3 or the TEA salt thereof; H-Aib-His-N-Me-D-2Nal..N-Me.D..Phe-NHMe, or the HCI salt thereof; and Piperidine-4-carboxylic acid-N-((1 R)-1 R)-2-(4-iodophenyl)-1 (methylcarbamoyl)ethyl)Nmethylcarbamoyl)2(2naphthyl)ethyl)-N methylamide, or a pharmaceutically acceptable salt thereof.
11. A pharmaceutical composition comprising, as an active ingredient, a compound according to any one of claims 1 to 10 or a pharmaceutically acceptable salt thereof together with a pharmaceutically acceptable carrier or diluent.
12. A composition according to claim 11 in unit dosage form, comprising from about 10 to about 200 mg of the compound according to any one of claims 1 to or a pharmaceutically acceptable salt thereof.
13. A pharmaceutical composition for stimulating the release of growth hormone from the pituitary, the composition comprising, as an active ingredient, a compound according to any one or claims 1 to 10 or a pharmaceutically acceptable salt thereof together with a pharmaceutically acceptable carrier or diluent.
14. A pharmaceutical composition according to any one of claims 11, 12 or 13 for oral, transdermal, nasal, pulmonary, or parenteral administration. A method of stimulating the release of growth hormone from the pituitary, the method comprising administering to a subject an effective amount of a compound according to any one of claims 1 to 10 or a pharmaceutically acceptable salt thereof, or of a composition according to any one of the claims 11, 12 or 14.
16. A method according to claim 15, wherein the effective amount of the compound according to any one of claims 1 to 10 or pharmaceutically acceptable salt or ester thereof is in the range of from 0.0001 to about 100 mg/kg body weight per day. 15 17. A method according to claim 16, wherein the amount of the compound :i or the salt or ester thereof is in the range of from about 0.001 to about 50mg/kg body weight per day. *99999
918. A method to increase the rate and extent of growth of animals, to increase the milk or wool production of animals, or for the treatment of ailments, the 9: 20 method comprising administering to a subject an effective amount of a compound ~according to any one of claims 1 to 10 or a pharmaceutically acceptable salt thereof, or of a composition according to any one of the claims 11, 12 or 14. *.9 19. A compound according to any one of claims 1 to 10 or a pharmaceutically acceptable salt thereof for use as a medicament. 20. Use of a compound according to any one of claims 1 to 10 or a pharmaceutically acceptable salt thereof for the preparation of a medicament for stimulating the release of growth hormone from the pituitary. 21. Use of a compound according to any one of claims 1 to 10 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 or wool production, or for the treatment of ailments. 22. A compound with growth hormone releasing properties, substantially as hereinbefore described with reference to any one of the Examples. [R:ALIBAA]06320.doc:mcc 57 23. A compound according to any one of claims 1 to 10 or a pharmaceutically acceptable salt thereof, or a composition according to any one of claims 11, 12 or 14 when used to stimulate the release of growth hormone from the pituitary. 24. A compound according to any one of claims 1 to 10 or a pharmaceutically acceptable salt thereof, or a composition according to any one of claims 11, 12 or 14 when used to increase the rate and extent of growth of animals, to increase the milk or wool production of animals or for the treatment of ailments. A medicament for stimulating the release of growth hormone from the pituitary, resulting from the use of claim 26. A medicament for administration to animals to increase their rate and extent of growth, to increase their milk or wool production, or for the treatment of ailments, resulting from the use of claim 21. Dated 3 August, 1999 Novo Nordisk A/S s15 Patent Attorneys for the Applicant/Nominated Person *SPRUSON FERGUSON .'.SPRUSON FERGUSON [R:\LIBAA]06320.doc:mcc
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Families Citing this family (26)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU3938797A (en) * 1996-08-29 1998-03-19 Novo Nordisk A/S Transdermal delivery of peptides
US6127341A (en) * 1997-06-20 2000-10-03 Novo Nordisk A/S Compounds with growth hormone releasing properties
AU7906998A (en) 1997-06-20 1999-01-04 Novo Nordisk A/S Compounds with growth hormone releasing properties
CN1213036C (en) 1997-08-22 2005-08-03 科研制药株式会社 Novel amide derivatives
ATE281467T1 (en) 1998-01-16 2004-11-15 Novo Nordisk As COMPOUNDS WITH GROWTH HORMONE RELEASING PROPERTIES
US6528529B1 (en) 1998-03-31 2003-03-04 Acadia Pharmaceuticals Inc. Compounds with activity on muscarinic receptors
EE200000660A (en) * 1998-05-12 2002-04-15 Warner-Lambert Company Combinations of protein farnesyl transferase and HMG CoA reductase inhibitors and their use in the treatment of cancer
RU2298547C2 (en) * 1998-06-30 2007-05-10 Ново Нордиск А/С Compounds and pharmaceutical composition possessing property for growth hormone releasing, method for stimulation of secretion of growth hormone from mammal hypophysis
US6919315B1 (en) 1998-06-30 2005-07-19 Novo Nordisk A/S Compounds with growth hormone releasing properties
AU5671099A (en) 1998-08-14 2000-03-06 The Administrators Of The Tulane Eductional Fund Compounds having growth hormone releasing activity
KR100699404B1 (en) 1999-02-18 2007-03-23 가켄 세야쿠 가부시키가이샤 Novel Amide Derivatives as Growth Hormone Secretagogues
EP1159964B1 (en) 2000-05-31 2009-10-28 Pfizer Products Inc. Use of growth hormone secretagogues for stimulating gastrointestinal motility
EP1343751A2 (en) 2000-12-20 2003-09-17 Bristol-Myers Squibb Company Cyclic derivatives as modulators of chemokine receptor activity
CA2432908A1 (en) 2000-12-20 2002-06-27 Bristol-Myers Squibb Pharma Company Diamines as modulators of chemokine receptor activity
TWI331922B (en) 2002-08-09 2010-10-21 Ipsen Pharma Sas Growth hormone releasing peptides
US7476653B2 (en) 2003-06-18 2009-01-13 Tranzyme Pharma, Inc. Macrocyclic modulators of the ghrelin receptor
EP2258358A3 (en) 2005-08-26 2011-09-07 Braincells, Inc. Neurogenesis with acetylcholinesterase inhibitor
CA2620333A1 (en) 2005-08-26 2007-03-01 Braincells, Inc. Neurogenesis by muscarinic receptor modulation
CU23558A1 (en) 2006-02-28 2010-07-20 Ct Ingenieria Genetica Biotech COMPOUNDS ANALOG TO THE PEPTIDIC SECRETAGOGS OF THE GROWTH HORMONE
AU2008241532A1 (en) 2007-02-09 2008-10-30 Tranzyme Pharma, Inc. Macrocyclic ghrelin receptor modulators and methods of using the same
CA2710039C (en) 2007-12-26 2018-07-03 Critical Outcome Technologies, Inc. Semicarbazones, thiosemicarbazones and related compounds and methods for treatment of cancer
EP3023426A1 (en) 2008-07-17 2016-05-25 Critical Outcome Technologies, Inc. Thiosemicarbazone inhibitor compounds and cancer treatment methods
EP2552915B1 (en) 2010-04-01 2017-07-19 Critical Outcome Technologies Inc. Compounds for the treatment of hiv
WO2013190520A2 (en) 2012-06-22 2013-12-27 The General Hospital Corporation Gh-releasing agents in the treatment of vascular stenosis and associated conditions
US9119832B2 (en) 2014-02-05 2015-09-01 The Regents Of The University Of California Methods of treating mild brain injury
US20170121385A1 (en) 2015-10-28 2017-05-04 Oxeia Biopharmaceuticals, Inc. Methods of treating neurodegenerative conditions

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU1272495A (en) * 1993-12-23 1995-07-10 Novo Nordisk A/S Compounds with growth hormone releasing properties
AU4164496A (en) * 1994-11-16 1996-06-06 Genentech Inc. Low molecular weight peptidomimetic growth hormone secretagogues

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5663146A (en) * 1991-08-22 1997-09-02 Administrators Of The Tulane Educational Fund Polypeptide analogues having growth hormone releasing activity

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU1272495A (en) * 1993-12-23 1995-07-10 Novo Nordisk A/S Compounds with growth hormone releasing properties
AU4164496A (en) * 1994-11-16 1996-06-06 Genentech Inc. Low molecular weight peptidomimetic growth hormone secretagogues

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