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WO1993000359A1 - Peptides modifies pouvant etre transportes vers le systeme nerveux central - Google Patents

Peptides modifies pouvant etre transportes vers le systeme nerveux central Download PDF

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Publication number
WO1993000359A1
WO1993000359A1 PCT/US1992/004968 US9204968W WO9300359A1 WO 1993000359 A1 WO1993000359 A1 WO 1993000359A1 US 9204968 W US9204968 W US 9204968W WO 9300359 A1 WO9300359 A1 WO 9300359A1
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Prior art keywords
group
leu
carbon atoms
pro
tyr
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PCT/US1992/004968
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English (en)
Inventor
Argyrios Arvantis
Gary Avonn Cain
Thomas Eugene Christos
Pasquale Nicholas Confalone
Richard Scott Pottorf
William Koch Schmidt
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The Du Pont Merck Pharmaceutical Company
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Publication of WO1993000359A1 publication Critical patent/WO1993000359A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/02—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link
    • C07K5/0207—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link containing the structure -NH-(X)4-C(=0), e.g. 'isosters', replacing two amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • C07K7/083—Neurotensin

Definitions

  • This invention relates to modified peptides and their pharmaceutically acceptable salts which can effectively penetrate the blood-brain barrier and, in particular, to peptides of no more than six amino acid residues modified by attachment of a lipophilic group to a suitable functional group on the peptide. Also of concern are pharmaceutical compositions containing these peptides and methods of treatment using such compositions.
  • Peptides which are capable of imitating or blocking the effects of a specific biologically active peptide are, in principle, potential therapeutic agents.
  • Neurotensin is an endogenous neurotridecapeptide which is unstable ia vivo. It exhibits analgesic, antipsychotic, cognition activating, and other effects on the central nervous system (CNS) when injected into the brain or surrounding cerebrospinal fluid. In contrast when neurotensin is administered intravenously, intramuscularly, subcutaneously, or orally it does not exhibit any effects on the CNS because it is metabolically unstable and is unable to cross the blood- brain barrier.
  • CNS central nervous system
  • MEDI 15 discloses H-D-Lys 8 -Arg 9 -Pro 10 - Trp 11 -Tle 1 2-Leu* 1 -3-0H, a neurotensin (8-13) hexapeptide analog as a subcutaneously active psychotropic substance.
  • the blood-brain barrier In addition to metabolic instability, penetration of the blood-brain barrier is another hurdle which researchers face in making neurologically active peptides.
  • the blood-brain barrier severely limits the bioavailability of such peptides to the nervous system.
  • the nature of the blood-brain barrier and problems associated with transport of peptides and proteins therethrough is set forth in Pardridge, Endocrine Reviews, 7(3): 314-330 (March 1986).
  • the blood-brain barrier serves as a highly important protective device for the extremely sensitive neural tissue. This barrier acts as a system-wide cellular membrane which separates the brain interstitial space from the blood.
  • the unique morphologic characteristics of the brain capillaries which constitute this barrier are the following: (a) epithelial-like high resistance tight junctions which literally cement all endothelia of brain capillaries together, and (b) scanty pinocytosis or transendothelial channels, which are abundant in endothelia of peripheral organs.
  • peptide latentiation or conversion of water-soluble functional groups on the peptide to lipid-soluble derivatives One form of latentiation of dipeptides is formation of the cyclized derivative, or diketopiperazine.
  • the coupling of the terminal carboxy and amino groups to form the diketopiperazine results in log order increases in the lipid solubility of the compound owing to the loss of several hydrogen bond-forming functional groups on the parent molecule.
  • U.S. Patent No. 4,933,324 issued to Shashoua on June 12, 1990, describes the formation of a prodrug from a fatty acid carrier and a neuroactive drug.
  • the prodrug is described as being preferably inactive until such time as it is hydrolyzed. Once in the central nervous system, the prodrug is hydrolyzed into the fatty acid carrier and the drug.
  • Y is a lipophilic moiety having the structure L-C(O)-, or R-(CH 2 )p-C(0)-(CH 2 ) r -, provided that when Y is L-C(O)- then L is selected from the group consisting of (i) at least one alkyl group having 1-16 carbon atoms, said alkyl group can be branched or unbranched, unsubstituted or substituted with at least one cyclic moiety selected from the group consisting of a cycloalkyl group having 3-8 carbon atoms, a heterocyclic group having 5-7 atoms in which the heteroatom is N, 0, or S, or an aryl group having 6-15 carbon atoms wherein said aryl group can be unsubstituted or substituted with at least one alkyl group having 1-4 carbon atoms, (ii) perfluoroalkyl having 1-10 carbon atoms which can be unsubstituted or substituted with at least one cyclic group selected from the
  • W is an amino acid residue selected from the group consisting of arginine, lysine, ornithine, homoarginine, 2,4-diaminobutyric acid, 2, 3-diaminopropionic acid, norleucine, N-methylnorleucine, D-arginine, D-lysine, proline, and 4-aminocyclohexylalanine;
  • X is an amino acid residue selected from the group consisting of arginine, lysine, ornithine, homoarginine, 2,4-diaminobutyric acid, 2, 3-diaminopropionic acid, norleucine, N-methylnorleucine, D-arginine, D-lysine, proline, 4-aminocyclohexylalanine, alanine, or an alpha- amino acid residue substituted at the alpha carbon with at least one alkyl group having 1-6 carbon atoms, or said alpha-carbon atom is part of a cyclic moiety selected from the group consisting of cycloalkyl having 3-8 carbon atoms or heterocyclic having 3-8 atoms in which the heteroatom is N, 0, or S; m and n are independently 0 or 1, provided that m and n are not both 0 unless L is R 1 -NH-R 2 ;
  • A', A, C, and E are independently selected from the group consisting of -C0NH-, -C0N(CH3)-, -N(CH3)C0-, -NHCR'R"-, -CR , R n NH-, -S0 2 NR'R"-, -NR'R"S ⁇ 2-, "CH 2 NH-, -CH 2 0-, -CH 2 S-, -NHCH 2 -, -0CH 2 -, -CSNH-, -NHC0NH-, -S(0)CH 2 -, -S(0) 2 CH 2 -, -NHSC-, -CH 2 S(0)-, -CH 2 S(0) 2 -r
  • H is an amino acid residue selected from the group consisting of proline or N-methylaminobutyric acid
  • B is an amino acid residue selected from the group consisting of tyrosine, phenylalanine, tryptophan, naphthylalanine, phenylglycine, and beta-phenylproline;
  • D is an amino acid residue selected from the group consisting of isoleucine, leucine, tert-leucine, and phenylglycine;
  • F is an amino acid residue selected from the group consisting of leucine, valine, and methionine; and Z is OH or OR 3 wherein R 3 is an alkyl group having 1-6 carbon atoms .
  • a preferred embodiment of the invention is one wherein Y is a lipophilic moiety having the structure L-C(O)- or R-(CH2)p-C(0)-(CH 2 ) r -r provided that when Y is L-C(O)- then L is selected from the group consisting of (i) alkyl, branched or unbranched, having 1-16 carbon atoms, (ii) perfluoroalkyl having 1-10 carbon atoms, (iii) cycloalkyl having 3-8 carbon atoms, (iv) bicycloalkyl having 6-18 carbon atoms, (v) tricycloalkyl having 6-18 carbon atoms, (vi) R 1 -NH-R 2 - wherein R 1 is H or alkyl having 1-4 carbon atoms, R 2 is selected from the group consisting of alkanediyl, branched or unbranched having 1-16 carbon atoms, alkylaryl substituted with at least one moiety selected from the group consisting of al
  • W is an amino acid residue selected from the group consisting of arginine, lysine, ornithine, 2,4- diaminobutyric acid, norleucine, N-methylnorleucine, D-arginine, 4-aminocyclohexylalanine, or proline;
  • X is an amino acid residue selected from the group consisting of arginine, lysine, ornithine, 2,4- diaminobutyric acid, norleucine, N-methylnorleucine, D-arginine, proline, 4-aminocyclohexylalanine, alanine, or an alpha-amino acid residue in which the alpha carbon is part of cyclic moiety selected from the group consisting of cycloalkyl having 3-8 carbon atoms or heterocyclic having 3-8 atoms in which the hetero atom is N, 0, or S; m and n are independently 0 or 1, provided that m and n are not both 0 unless L is R 1 -NH-R 2 -;
  • H is an amino acid residue selected from the group consisting of proline or N-methylaminobutyric acid
  • B is an amino acid residue selected from the group consisting of tyrosine, phenylalanine, tryptophan, naphthylalanine, phenylglycine, and beta-phenylproline
  • D is an amino acid residue selected from the group consisting of isoleucine, leucine, tert-leucine, and phenylglycine;
  • F is an amino acid residue selected from the group consisting of leucine, valine, and methionine; and Z is OH or OR 3 wherein R 3 is alkyl having 1-6 carbon atoms.
  • R 3 is alkyl having 1-6 carbon atoms.
  • Y is selected from the group consisting of acetyl, pivaloyl, neopentylcarbonyl, n-perfluoroctanoyl, 1-bicyclo[3.3.0]octanecarbonyl, 2-bicyclo[2.2.1] eptane- acetyl, 1-adamantanecarbonyl, 2-pyrrolidinecarbonyl (prolyl) , 2-(5-pyrrolid-5-one)-carbonyl[pyroglutamyl] , benzoyl, 4-tert-butylbenzoyl, 4-phenylbenzoyl, nicotinoyl, 2-benzyl-5-aminopentanoyl, trans-4-
  • X is an amino acid residue selected from the group consisting of arginine, lysine, ornithine, 4- aminocyclohexylalanine, 4-aminopiperidine-4-carboxylic acid, 1-aminocyclopentanecarboxylic acid, 1- aminocyclobutanecarboxylic acid, or 1-amino- cyclopropanecarboxylic acid;
  • m and n are independently 0 or 1, provided that m and n are not both zero, except when Y is 2-benzyl-5- aminopentanoyl then m and n can be zero, and further provided that when Y is acetyl then m and n are 1;
  • A', C, and E are -C0NH-;
  • A is -CONH- or -CH 2 NH-;
  • H is a proline residue
  • B is an amino acid residue selected from the group consisting of tyrosine and tryptophan;
  • D is an amino acid residue selected from the group consisting of isoleucine, tert-leucine, and phenylglycine;
  • F is a leucine residue
  • Z is OH or 0CH 3 .
  • Y is selected from the group consisting of 1- adamantanecarbonyl, 2-benzyl-5-aminopentanoyl, benzoyl, nicotinoyl, and acetyl;
  • W is an arginine residue
  • X is an amino acid residue selected from the group consisting of arginine, lysine, and ornithine
  • m and n are independently 0 or 1, provided that m and n are not both zero, except when Y is l-benzyl-5- aminopentanoyl then m and n can be zero, and further provided that when Y is acetyl, both and n are 1;
  • A*, A, C, and E are -CONH-;
  • H is a proline residue
  • B is an amino acid residue selected from the group consisting of tyrosine and tryptophan
  • D is an amino acid residue selected from the group consisting of isoleucine, tert-leucine, and phenylglycine
  • F is a leucine residue
  • Z is OH or 0CH 3 .
  • N°*-alkyl amino acids are represented by the following abbreviations :
  • Lys lysine
  • Trp tryptophan
  • amino acid as used herein means an organic compound containing both a basic amino group and an acidic carboxyl group. Included within this term are modified and unusual amino acids as well as amino acids which are known to occur biologically in free or combined form but usually do not occur in proteins .
  • amino acid residue as used herein means that portion of an amino acid (as defined herein) that is present in a peptide/pseudopeptide.
  • peptide as used herein means a linear compound that consists of two or more ⁇ -amino acids (as defined herein) that are linked by means of peptide or pseudopeptide bonds.
  • peptide refers to both peptides and pseudopeptides.
  • a pseudopeptide is a compound which mimics the appearance of a peptide either by using linking groups other than amide linkages between the residual units and/or by using unnatural amino acids as described above and/or an amino acid residue modified in such a way as to render it stable to enzymatic hydrolysis and make it bioavailable.
  • peptide bond means a covalent link formed by splitting out a molecule of water between the carboxyl group of one amino acid and the amino group of a second amino acid.
  • This term includes "pseudopeptide bonds” or peptide bond isosteres which are substitutes for the normal amide linkage. These substitute linkages are formed from combinations of atoms not normally found in peptides or proteins which mimic the spatial requirements of the amide bond and which should stabilize the molecule to enzymatic degradation.
  • peptides consisting of up to six amino acid residues can be effectively transported into the central nervous system from the circulatory system by attaching a lipophilic group to a suitable position of the peptide, e.g., to a functional group such as amino, carboxylic acid, or hydroxyl or directly to an appropriate carbon atom.
  • a lipophilic group may protect susceptible peptide bonds from enzymatic cleavage by virtue of their size.
  • compounds of the invention can be stabilized against enzyme degradation by incorporating: (1) non- hydrolyzable equivalents of the amide bond present as the linking unit between amino acid residues, and/or (2) by using modified or unusual amino acids which are not susceptible to enzymatic degradation.
  • non-hydrolyzable equivalents of the amide bond include, but are not limited to, the following: -CON(CH 3 )-, -N(CH 3 )C0-, -NHCR'R"-, -CR'CR"NH-, -S ⁇ 2 NR'R"-, -NR'R"S0 2 -, -CH2NH-, -CH2O-, -CH2S-, -NHCH2 ⁇ r -OCH2-, -CSNH-, -NHCONH-, -S(0)CH 2 ⁇ ,
  • Non-hydrolyzable equivalents such as monofluorovinyl linkers can be incorporated into peptides using a process such as that described by Allmendinger et al. in Tetrahedron Letters, 31: 7297- 7300 (1990) .
  • Modified or unusual amino acids which can be used to practice the invention include, but are not limited to, hydroxylysine, 4-hydroxyproline, ornithine, 2,4- diaminobutyric acid, homoarginine, norleucine, N- methylammobutyric acid, naphthylalanine, phenylglycine.
  • the compounds of the invention are made as described below.
  • Biological evaluations involve a combination of in vitro and in vivo assays as described below.
  • One embodiment of this invention concerns compounds which are analogs of neurotensin, in particular, analogs of the C-terminal fragment of neurotensin, for which the amino acid sequences (positions 8-13) and (positions 9- 13) are known to possess substantially all the biological activity of the intact natural tridecapeptide.
  • Introduction of these two peptides into the brain by intracerebroventricular administration produces profound analgesia of limited duration in laboratory animals such as mice and rats.
  • These peptides are substantially devoid of this activity when administered by oral, subcutaneous, or intravenous routes. This inactivity indicates lack of bioavailability except by direct application to the site of action.
  • NT (8-13) (arginine) and NT (9-13) (arginine) or analogs having other N-terminal amino acids such as lysine, ornithine, etc. are protected by attach:.: g a lipophilic group, such as 1- ad; * mantanecarbonyl, these peptides become more lipophilic and, then, exhibit analgesic effects both by intracerebroventricular and intravenous routes of administration.
  • NT (9-13) is inactive when administered intravenously.
  • a 1- adamantanecarbonyl derivative is active and has an ED50 value of 2.2 mg/kg.
  • the protected ⁇ -amino group of the N-terminal acid can be replaced entirely by a lipophilic moiety, such as a benzyl group, to produce a derivative having substantially the same intravenous analgesic activity.
  • the duration of the analgesic effects is increased using the neurotensin analog. Indeed, the duration exceeds 160 minutes when administered intravenously or intracerebroventricularly whereas the natural peptide exhibits such effects for only a few minutes when administered by the intracerebro- ventricular route.
  • Neurotensin analogs of the invention can also be used as antipsychotic agents to treat diseases such as schizophrenia. Because neurotensin modulates dopaminergic neurons without blockade of the receptor system, especially in the nigrostriatal region, neurotensin has antipsychotic properties and is devoid of the adverse motor effects known as extrapyramidal symptoms which are characteristic of typical neuroleptics such as chlorpromazine and haloperidol.
  • compositions of the compounds of the invention can be prepared by reacting the free acid or base forms of these peptides with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two; generally, nonaqueous media like ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred. Lists of suitable salts are found in A. R. Gennaro, ed.. Remington's Pharmaceutical Sciences. 17th ed., Mack Publishing Company, Easton, PA, 1985, p. 1418, the disclosure of which is hereby incorporated by reference.
  • peptides are elongated by deprotecting the ⁇ -amine of the C-terminal residue and coupling the next suitably protected amino acid through a peptide linkage using the methods described. This deprotection and coupling procedure is repeated until the desired sequence is obtained.
  • This coupling can be performed with the constituent amino acids in a stepwise fashion, or by condensation of fragments (two to several amino acids), or combination of both processes, or by solid phase peptide synthesis according to the method originally described by Merrifield, J. Am. Chem. Soc.. 1963, 85, 2149-2154, the disclosure of which is hereby incorporated by reference.
  • compounds of the invention can be synthesized using automated peptide synthesizing equipment.
  • peptide syntheses are described in Stewart and Young, "Solid Phase Peptide Synthesis", 2nd ed., Pierce Chemical Co., Rockford, IL (1984) ; Gross, Meienhofer, Udenfriend, Eds., "The Peptides: Analysis, Synthesis, Biology", Vol 1, 2, 3, 5, and 9, Academic Press, New York, 1980-1987; Bodanszky, “Peptide Chemistry: A Practical Textbook", Springer-Verlag, New York (1988); and Bodanszky, et al. "The Practice of Peptide Synthesis” Springer-Verlag, New York (1984), the disclosures of which are hereby incorporated by reference.
  • Coupling between two amino acids, an amino acid and a peptide, or two peptide fragments can be carried out using standard coupling procedures such as the azide method, mixed carbonic acid anhydride (isobutyl chloroformate) method, carbodiimide (dicyclohexylcarbo ⁇ diimide, diisopropylcarbodiimide, or water-soluble carbodiimide) method, active ester (p-nitrophenyl ester, N-hydroxysuccinic imido ester) method, Woodward reagent K method, carbonyldiimidazole method, phosphorus reagents such as BOP-C1, or oxidation-reduction method. Some of these methods (especially the carbodiimide method) can be enhanced by adding 1-hydroxybenzo- triazole. These coupling reactions can be performed in either solution (liquid phase) or solid phase.
  • the functional groups of the constituent amino acids must be protected during the coupling reactions to avoid formation of undesired bonds.
  • the protecting groups that can be used are listed in Greene, "Protective Groups in Organic Synthesis", John Wiley & Sons, New York (1981) and "The Peptides: Analysis, Synthesis, Biology", Vol. 3, Academic Press, New York (1981) , the disclosure of which is hereby incorporated by reference.
  • the ⁇ -carboxyl group of the C-terminal residue is usually protected by an ester that can be cleaved to give the carboxylic acid.
  • Protecting groups which can be used include: 1) alkyl esters such as methyl and t- butyl, 2) aryl esters such as benzyl and substituted benzyl, or 3) esters which can be cleaved by mild base treatment or mild reductive means such as trichloroethyl and phenacyl esters.
  • an insoluble carrier usually polystyrene
  • These insoluble carriers contain a group which will react with the carboxyl group to form a bond which is stable to the elongation conditions but readily cleaved later. Examples of which are: chloro- or bromomethyl resin, hydroxymethyl resin, and aminomethyl resin. Many of these resins are commercially available with the desired C-terminal amino acid already incorporated.
  • ⁇ -amino group of each amino acid must be protected. Any protecting group known in the art can be used. Examples of which include: 1) acyl types such as formyl, trifluoroacetyl, phthalyl, and p-toluene- sulfonyl; 2) aromatic carbamate types such as benzyloxycarbonyl (Cbz or Z) and substituted benzyloxycarbonyls, 1-(p-biphenyl)-1-methylethoxy- carbonyl, and 9-fluorenylmethyloxycarbonyl (Fmoc) ; 3) aliphatic carbamate types such as tert- butyloxycarbonyl (Boc) , ethoxycarbonyl, diisopropyl- methoxycarbonyl, and allyloxycarbonyl; 4) cyclic alkyl carbamate types such as cyclopentyloxycarbonyl and adamantyloxycarbonyl; 5) alkyl
  • the ⁇ -amino protecting group is cleaved prior to the coupling of the next amino acid.
  • the methods of choice are trifluoroacetic acid, neat or in dichloromethane, or HCl in dioxane.
  • the resulting ammonium salt is then neutralized either prior to the coupling or in situ with basic solutions such as aqueous buffers, or tertiary amines in dichloromethane or dimethylformamide.
  • the reagents of choice are piperidine or substituted piperidines in dimethylformamide, but any secondary amine or aqueous basic solutions can be used.
  • the deprotection is carried out at a temperature between 0°C and room temperature.
  • any of the amino acids bearing side chain functionalities must be protected during the preparation of the peptide using any of the above-described groups.
  • Those skilled in the art will appreciate that the selection and use of appropriate protecting groups for these side chain functionalities depends upon the amino acid and presence of other protecting groups in the peptide. The selection of such a protecting group is important in that it must not be removed during the deprotection and coupling of the ⁇ -amino group.
  • Boc when Boc is chosen for the ⁇ -amine protection the following protecting groups are acceptable: p-toluenesulfonyl (tosyl) moieties and nitro for arginine; benzyloxycarbonyl, substituted benzyloxycarbonyls, or tosyl for lysine; benzyl or alkyl esters such as cyclohexyl for glutamic and aspartic acids; benzyl ethers for serine and threonine; benzyl ethers, substituted benzyl ethers or 2- bromobenzyloxycarbonyl for tyrosine; p-methylbenzyl, p- methoxybenzyl, acetamidomethyl, benzyl, or t- butylsulfonyl for cysteine; and the indole of tryptophan can either be left unprotected or protected with a formyl group.
  • tert-butyl based protecting groups are acceptable.
  • Boc can be used for lysine, tert-butyl ether for serine, threonine and tyrosine, and tert-butyl ester for glutamic and aspartic acids.
  • the cleavage of the peptide can also be accomplished by other acid reagents such as trifluoromethanesulfonic acid/trifluoroacetic acid mixtures. If the Fmoc protection scheme is used the N- terminal Fmoc group is cleaved with reagents described earlier. The other protecting groups and the peptide are cleaved from the resin using solutions of trifluoroacetic acid and various additives such as anisole, etc.
  • Compounds of general formula (I) can be prepared by standard solution phase peptide synthesis.
  • Scheme I outlines the coupling process wherein Compound (IV) , an unprotected amine peptide residue is allowed to react with the mixed anhydride form, Compound (V) , of the N- protected amino acid residue.
  • Compound (V) is prepared by treatment of the acid (VI) with isobutyl chloroformate at a low temperature, such as -15°C, in the presence of a base.
  • the resultant dipeptide of formula (VII) is deprotected at the N-terminus, as described in Scheme II, by treatment with 4 M HCl in dioxane to afford the hydrochloride salt of formula (VIII) .
  • Peptide residue W is a differentially protected diamino residue such as, but not limited to, lysine, protected by a t-butyloxycarbonyl group at the ⁇ -amine and a benzyloxycarbonyl (Z) group at the ⁇ —amine.
  • the ⁇ - amine is then deprotected as described in Scheme II to provide the hydrochloride salt of the partially protected pentapeptide of formula (X) .
  • Terminal is an ⁇ -Substituted ⁇ -Amino Acid
  • MeOBzl - 4-Methoxybenzyi Tyr(Bzl) Tyrosine-O-benzyl ether
  • Leu-OBzl Leucine benzyl ester
  • R ls R 2 alkyl of 1- 6 carbons
  • P NH, O, S
  • the starting amino acids used below were purchased from one of four commercially available sources: Sigma (St. Louis, MO), Bachem Bioscience, Inc. (Philadelphia, PA), Advanced Chemtech (Louisville, KY) ; and/or Peninsula Laboratories, Inc. (Belmont, CA) .
  • amino acids were coupled in turn as their N-Boc derivatives using dicyclohexylcarbo ⁇ diimide/1-hydroxybenzotriazole in dimethylformamide in a 2.5 fold excess.
  • side chain functionalities of Tyr and Arg were protected by 2-bromobenzyloxycarbonyl and tosyl respectively.
  • the 1-adamantanecarbonyl group was coupled to the resin bound peptide by treatment with a five fold excess of 1-adamantanecarbonyl chloride and one equivalent of diisopropylethylamine in dimethylformamide. This treatment was repeated until all peptide amine groups were blocked with the adamantanecarbonyl groups.
  • the peptide was cleaved from the resin and the protecting groups were removed by treating the peptide resin with a solution of anisole in anhydrous HF (1:10) . Purification of the deprotected peptide was accomplished by reverse phase high performance liquid chromatography.
  • This peptide was prepared according to the procedure described above in Example 1.
  • N-Boc-isoleucine N-Boc-Ile-0.5 H2O
  • 5.0 g (20.8 mmol) was dissolved in 110 ml of dry THF and chilled to -15°C in a dry ice acetone bath.
  • a separate flask was charged with 8.19 g (20.8 mmol) of leucine benzyl ester tosylate (Tos"Leu(OBzl) ) , 40 ml DMF then chilled to -15°C.
  • Step B Isoleucylleucine, Benzyl Ester, Hydrochloride (Ile-Le (OBzl) « HC1)
  • Step C N-Boc-Tyr(OBzl) Ile-Leu(OBzl)
  • N-Boc-Tyr(OBzl) was coupled to the compound synthesized in Step B above using the procedure described in Step A above to yield 89.8% of N-Boc- Tyr(OBzl)-Ile-Le (OBzl) .
  • Step D Tyr(OBzl)-Ile-Leu (OBzl) -HCl 9.5 g (13.8 mmol) of the compound synthesized in Step C above was deprotected according to the procedure described in Step B above to yield 8.5 g of Tyr(OBzl)- Ile-Leu (OBzl) «HC1.
  • Step E N-Boc-Pro-Tyr(OBzl)-Ile- Leu (OBzl) (SEQ ID NO: 9)
  • N-Boc-Pro-Tyr(OBzl)-Ile-Le (OBzl) SEQ ID NO: 9 .
  • Step F Pro-Tyr(OBzl) -Ile-Leu(OBzl) (SEQ ID NO:10)
  • Step G N°*-Boc,N ⁇ (Z)-Lys-Pro-Tyr(OBzl)- Ile-Leu(OBzl) (SEQ ID NO:11)
  • Step H N ⁇ (Z)-Lys-Pro-Tyr (OBzl)-Ile- Leu(OBzl) »HC1 (SEQ ID NO:12) 3.1 g (2.96 mmol) of the product synthesized as described in Part G above was deprotected according to the procedure described in Step B above to yield 2.9 g (99.6% yield) of SEQ ID NO:12.
  • Step I N 0 *—(1-Adamantanecarbonyl) ,N ⁇ (Z)-Lys-Pro- Tyr(OBzl)-Ile-Leu(OBzl) (SEQ ID NO:13)
  • Step J N ⁇ - (1-Adamantanecarbonyl)-Lys- Pro-Tyr-Ile-Leu (SEQ ID NO:3) 1.05 g (0.95 mmol) of the product synthesized in Step I above was mixed with 50 ml ethanol, 10 ml of cyclohexene, 0.054 ml (0.95 mmol) of glacial acetic acid and 100 mg 20% palladium hydroxide on carbon. Reaction was refluxed for 24 hours. After which the reaction mixture was cooled and catalyst removed by filtration through a pad of Celite®.
  • Step A N ⁇ - (2-Norbornaneacetyl) ,N ⁇ (Z)-Lys-Pro- Tyr(OBzl)-Ile-Leu(OBzl) (SEQ ID NO:14) 0.15 g (0.15 mmol) of the product from Example 3, Step H above was coupled to 2-norbornaneacetyl chloride according to the procedure de ⁇ —ibed above for the preparation of the compound of ..xample 3, Step I. 0.15 g (92% yield) of N ⁇ - (2-norbornaneacetyl)-N ⁇ - (Z) -
  • Step B N 0 *—(2-Norbornaneacetyl)-Lys-Pro-Tyr-Ile-Leu, Acetic Acid Salt (SEQ ID NO:15) 0.14 g (0.13 mmol) of the compound synthesized above in Example 4, Step B was deprotected according to the procedure described above in Example 3, Step J to give 0.11 g (100% yield) of N ⁇ (2-norbornaneacetyl)-Lys- Pro-Tyr-Ile-Leu (SEQ ID NO:15) acetic acid salt.
  • Step A N ⁇ -(CF 3 (CF 2 )6CO) ,N ⁇ (Z)-Lys-Pro-Tyr(OBzl)-Ile- Leu(OBzl) (SEQ ID NO:16) 0.1 g (0.1 mmol) of the product synthesized above in Example 3, Step H was coupled to perfluorooctanoyl chloride according to the procedure as described above for the preparation of the compound synthesized in Example 4, Step A above.
  • N ⁇ - (perfluorooctanoyl) -N ⁇ (Z)-Lys-Pro-Tyr(OBzl)-Ile- Leu(OBzl) (SEQ ID NO:16) was isolated.
  • N ⁇ (CF 3 (CF 2 ) 6CO) N ⁇ ( Z ) -Lys-Pro-Tyr (Bzl) -Ile- Leu (OBzl) (SEQ ID NO : 16 )
  • Step B N ⁇ (CF3(CF2) 6CO) -Lys-Pro-Tyr-Ile-Leu, Acetic Acid Salt (SEQ ID NO:17) 0.13 g of the product synthesized in Example 5,
  • Step A above was deprotected according to the procedure described above Example 3, Step J to produce 90 mg (85.4% yield) of N ⁇ - (perfluorooctanoyl)-Lys-Pro-Tyr-Ile- Leu (SEQ ID NO: 17), acetic acid salt.
  • Example 6 N ⁇ - (cis-Bicyclo (3.3.0)Octane-2-Carbonyl)-Lys-Pro-Tyr- Ile-Leu, Acetic Acid Salt (SEQ ID NO:5) Step A: N ⁇ (cis-Bicyclo (3.3.0)Octane-2-carbonyl) , N ⁇ (Z)-Lys-Pro-Tyr (OBzl)-Ile-
  • Step B N 0 *- (cis-Bicyclo(3.3.0)-Octane-2-Carbonyl)- Lys-Pro-Tyr-Ile-Leu, Acetic Acid Salt (SEQ ID NO:5) 0.15 g (0.14 mmol) of the product synthesized in Example 6, Step A was deprotected according to the procedure described above in Example 3, Step J to yield 0.12 g of N°-—(cis-bicyclo(3.3.0)-octane-2-carbonyl)-Lys- Pro-Tyr-Ile-Leu (SEQ ID NO:5), acetic acid salt.
  • Step B N-Boc-Pro- ⁇ [CH2NH]Tyr(OBzl)-
  • Ile-Le (OBzl) (SEQ ID NO:19) 0.7 g (3.5 mmol) of N-Boc-prolinal, 2.0 g (3.2 mmol) of the product of Example 3, Step D, and 0.287 g (3.5 mmol) of sodium acetate were added to 50 ml of a 2% acetic acid/DMF solution. 2.0 g (32 mmol) of NaBH 3 CN was then added slowly and reaction allowed to stir at room temperature for 24 hours. Reaction mixture poured into aqueous K2C0 3 (pH 10) and extracted 3 x 100 ml with ethyl acetate.
  • Step C Pro- ⁇ [CH2NH]Tyr (OBzl)-Ile-Leu(OBzl) , Hydrochloride Salt (SEQ ID NO:20)
  • Step C Pro- ⁇ [CH 2 NH]Tyr(OBzl)-Ile- Leu(OBzl) -HCl (SEQ ID NO:20)
  • Step D N ⁇ -Boc,N ⁇ (Z)-Lys-Pro- ⁇ [CH2NH]Tyr(OBzl)-
  • Step E N ⁇ (Z) -Lys-Pro- ⁇ [CH2NH] Tyr (OBzl) -Ile- Leu(OBzl) » HC1 (SEQ ID NO:22)
  • Step F N ⁇ (1-Adamantanecarbonyl) ,N ⁇ (Z)-Lys- Pro- ⁇ [CH 2 NH] yr(OBzl)-Ile-Leu (OBzl) (SEQ ID NO:23)
  • Step G N ⁇ (1-Adamantanecarbonyl)-Lys-Pro- ⁇ [CH 2 NH]Tyr- Ile-Leu-CH 3 C ⁇ 2H (SEQ ID NO:4)
  • Example 8 N ⁇ _(i-Adamantanecarbonyl)-Orn-Pro-Tyr-He- Leu, Acetic Acid Salt (SEQ ID NO:6)
  • Step A The partially protected tetrapeptide Pro- Tyr(Bzl)-Ile-Leu(OBzl) --HCl made in Example 3, Step F (SEQ ID NO:10) was coupled to N ⁇ - (Z) ,N ⁇ -Boc-Ornithine using standard isobutyl chloroformate/N-methyl morpholine chemistry to yield N ⁇ -(Z) ,N ⁇ -Boc-Orn-Pro- Tyr(OBzl)-Ile-Leu(OBzl) (SEQ ID NO:24) (62%) as a colorless solid after silica gel chromatography (EtOAc/hexanes) .
  • Step B The fully protected pentapeptide synthesized in Step A above was treated with 4 M HCl in dioxane under standard conditions, then precipitated by dilution with diethyl ether to yield N°- (Z)-Orn-Pro-Tyr(OBzl)-
  • Step C The amine salt synthesized above in Step B (0.30 g, 0.31 mmol), triethylamine (95 ⁇ L, 0.68 mmol), 1-adamantanecarbonyl chloride (65 mg, 0.31 mmol), and dichloromethane were stirred at ambient temperature for 1.75 hour. The mixture was diluted with ethyl acetate (30 mL) , and extracted with 1 M HCl (2 mL) , water (3 mL), sat. aq. sodium carbonate (3 mL) , and brine (3 mL) , then dried (MgSO-j), filtered, and concentrated in vacuo.
  • N°- (z) ,N ⁇ - (1- adamantanecarbonyl)-Orn-Pro-Tyr(OBzl)-Ile-Leu (OBzl) (SEQ ID NO:26), 0.34 g (quantitative) was obtained as a colorless solid.
  • Step D The protected N ⁇ -l-adamantanecarbonyl pentapeptide synthesized above in Step B (0.33 g, 0.30 mmol) was deprotected using the standard catalytic transfer hydrogenation conditions (20% palladium hydroxide on carbon, cyclohexene, acetic acid, ethanol at reflux) to provide N ⁇ - (1-adamantanecarbonyl)-Orn-Pro- Tyr-Ile-Leu*HOAc (SEQ ID NO: 6) (0.24 g, 95%) as a colorless solid.
  • the standard catalytic transfer hydrogenation conditions (20% palladium hydroxide on carbon, cyclohexene, acetic acid, ethanol at reflux
  • Step A Ile-Leu(OBzl) » HC1 synthesized above in Example 3, Step B was coupled to N-Boc-Trp under standard isobutyl chloroformate/N-methyl morpholine conditions to yield N-Boc-Trp-Ile-Leu(OBzl) (97%) as a colorless solid after silica gel chromatography (ethyl acetate/hexanes) .
  • Step B The N-Boc group of the compound described above in Step A (2.8 g, 4.5 mmol) was cleaved by stirring with 4 M HCl in dioxane/anisole/1,2- ethanedithiol (98:1:1, 10 mL) at ambient temperature under a Drierite tube for 1 hour. The product was precipitated by dilution with diethyl ether, collected by filtration, and dried at 56°C under high vacuum for 2 hours to yield Trp-Ile-Leu(OBzl) -HCl (2.24 g, 89%) as a white solid.
  • Step C The partially protected tripeptide synthesized above in Step B was coupled to N-Boc-Pro under standard conditions to yield N-Boc-Pro-Trp-Ile-Leu(OBzl) (SEQ ID NO:27) (96%) as a colorless solid after silica gel chromatography (ethyl acetate) .
  • Step D The N-Boc group of tetrapeptide prepared in Step C above was cleaved as in the previous example to yield Pro-Trp-Ile-Leu (OBzl) -HCl (SEQ ID N0:28) (88%) as a solid which contained -10% of an unknown impurity.
  • MS-DCI (NH3) : 721 (M+H, est . 10%, unknown by-product) , 617 (M+H, base, est. 90%, desired product) .
  • Step E The crude tetrapeptide amine salt prepared above in Step D was coupled to N ⁇ -Cbz,N ⁇ -Boc-Lys under standard conditions to yield N ⁇ - (Z) ,N ⁇ -Boc-Lys-Pro-Trp- Ile-Leu(OBzl) (SEQ ID NO:29) (66%) as a colorless solid after silica gel chromatography (ethyl acetate/hexanes) .
  • MS-DCI (NH3) : 997 (M+NH4, 66%), 980 (M+H, base), 880
  • Step F The N-Boc group of pentapeptide prepared above in Step E was cleaved as described above to yield N ⁇ - Cbz-Lys-Pro-Trp-Ile-Leu(OBzl) 'HCl (SEQ ID NO:30) (87%) as a light brown solid.
  • Step G The pentapeptide amine salt prepared above in
  • Step F was acylated by 1-adamantanecarbonyl chloride as described above to yield N ⁇ -Cbz,N ⁇ - (1-adamantane ⁇ carbonyl)-Lys-Pro-Trp-Ile-Leu(OBzl) (SEQ ID NO:31) (quant.) as a tan solid.
  • MS-DCI (NH3) : 1059 (M+NH4, 15%), 1042 (M+H, base)
  • Rf 5% methanol/chloroform
  • Step H The compound prepared in Step G above was deprotected using standard catalytic transfer hydrogenation conditions to yield N ⁇ -(1- adamantanecarbonyl)-Lys-Pro-Trp-Ile-Leu"HOAc (SEQ ID NO:
  • Step B The N-Boc group of the compound prepared above in Step A was cleaved under standard 4 M HCl in dioxane conditions to yield (S)-2-phenylglycyl-Leu(OBzl) •HCl ' (quant.) . m.p. 200-201°C
  • Step C Dipeptide salt of the compound prepared above in Step B was coupled to N-Boc-Tyrosine Benzyl Ether (N- Boc-Tyr(OBzl) ) under standard conditions to yield N-Boc- Tyr(OBzl)- (S)-2-phenylglycyl-Leu(OBzl) (89%) as a glassy solid.
  • Step D The N-Boc group of tripeptide prepared above in Step C was cleaved under standard conditions to yield Tyr(OBzl)- (S)-2-phenylglycyl-Leu (OBzl) »HC1 (93%) as a colorless solid.
  • MS-DCI (NH3) : 608 (M+H, base) [ ⁇ ]D 25 +34.8° (c - 0.630, methanol) Anal. Calc'd. for C37H41N3O5.HCI:
  • Step E Tripeptide amine salt prepared above in Step D was coupled to N-Boc-Pro under standard conditions to yield N-Boc-Pro-Tyr (OBzl)- (S)-2-phenylglycyl-Leu (OBzl) (SEQ ID NO:32) (99%) as a colorless solid after silica gel chromatography (ethyl acetate/hexanes) .
  • Step F The N-Boc group of tetrapeptide prepared above in Step E was cleaved under standard conditions to yield Pro-Tyr(OBzl)-(S)-2-phenylglycyl-Leu(OBzl) -HCl (SEQ ID NO:33) (95%) as a colorless solid.
  • Anal. Calc'd. for C 2H48N ⁇ 6 ⁇ HCl » l 2 H2O:
  • Step G The tetrapeptide amine salt prepared above in Step F was coupled with N ⁇ -Cbz-N ⁇ -Boc-Lys under standard conditions to yield N ⁇ -(Z) ,N ⁇ -Boc-Lys-Pro-Tyr(OBzl)- (S)- 2-phenylglycyl-Leu(OBzl) (SEQ ID NO:34) (76%) as a colorless solid after silica gel chromatography (ethyl acetate/hexanes) .
  • MS-DCI (NH3) : 1084 (M+NH4, 52%), 1067 (M+H, 35%), 967
  • Step H The N° ⁇ Boc group of pentapeptide prepared above in Step G was cleaved under standard conditions to yield N ⁇ -Cbz-Lys-Pro-Tyr(OBzl)- (S)-2-phenylglycyl- Leu(OBzl) «HC1 (SEQ ID NO:35) (91%) as a colorless solid.
  • Step I The pentapeptide amine salt prepared above in Step H was acylated with 1-adamantanecarbonyl chloride under usual conditions to yield N ⁇ -(Z) ,N ⁇ - (1- adamantanecarbonyl)-Lys-Pro-Tyr(OBzl)- (S)-2- phenylglycyl-Leu (OBzl) (SEQ ID NO:36) (quant.) as a colorless solid.
  • Step J Deprotection of the product prepared above in Step I under standard catalytic transfer hydrogenation conditions provided N 0 *—(1-adamantanecarbonyl)-Lys-Pro- Tyr- (S)-2-phenylglycyl-Leu-HOAc (SEQ ID NO:8) (99%) as a colorless solid.
  • Example 11 4-(1'-Adamantanecarbamido)-4-Piperidinecarbonyl- Pro-Tyr-Ile-Leu, Acetic Acid Salt (SEQ ID NO:40)
  • Step A l-Benzyl-4-cyano-4-aminopiperidine l-Benzyl-4-piperidone, 10.5 g (56.7 mmoles), was treated with 8 ml NH4OH, 3.34 g (62.36 mmoles), 3.74 g (58 mmoles) KCN in 10 ml water at 60°C for 16 hours and then at 80°C for 4.5 hours.
  • Step B l-Benzyl-4-(1'-Adamantane)Carbamidopiperidine- 4-Carboxylic Acid 0.5 g (2.32 mmoles) of the product of Step A was treated with 0.475 g (2.32 mmoles) 1-adamantanecarbonyl chloride in the presence of 2.32 (mmoles) N- methyImorpholine in CH 2 CI 2 at 25°C for 16 hours and then at reflux for 2 hours. Then the reaction mixture was poured into a separatory funnel containing 200 ml EtOAc and 100 ml 5% NaHC0 3 .
  • Step C l-Benzyl-4-(1'-Adamantane)Carbamidopiperidine- 4-Carboxy-Pro-Tyr(OBzl)-Ile-Leu(OBzl) (SEQ ID NO:39) Five hundred milligrams (0.69 mmoles) HCl»Pro-
  • Step D 4- (1 '-Adamantane)Carbamidopiperidine-4- Carbonyl-Pro-Tyr-Ile-Leu-»AcOH (SEQ ID NO:40)
  • Example 13 N ⁇ (nicotinoyl)Lys-Pro-Tyr-Ile-Le -HOAc (SEQ ID NO: 3) Step A: N ⁇ (nicotinoyl)Lys-Pro-Tyr(OBzl) Ile-Leu(OBzl) (SEQ ID NO:42) 2.0 g (2.04 mmol) of the compound of Example 3, Step H was coupled with 0.36 g (2.04 mmol) of nicotinoyl chloride hydrochloride in the same manner as described above in Example 4, Step H. 1.39 (65% yield) of a colorless solid was isolated.
  • Step B N ⁇ (nicotinoyl)Lys-Pro-Tyr-Ile-Leu ⁇ OAc (SEC ID NO:43) 200 mg (0.19 mmol) of the product from Step A described above was deprotected according to the same procedure as that described above in Example 3, Step J to give 150 mg (99% yield) of a colorless solid.
  • Step B N ⁇ (Boc)Orn-Pro- ⁇ [CH2NH]Tyr-Ile- Leu-HOAc (SEQ ID NO:4 ' 5) 0.14 g (0.14 mmol) of product obtained above in Example 14, Step A was deprotected using the same procedure as described above in Example 3, Step J to give 90 mg (84% yield) of product.
  • Step B Tyr- (OBzl)- ⁇ [CH2NH]-Ile-Leu(OBzl) « HC1 0.5 g (0.7 mmol) of the compound made in Step A above was deprotected in the same manner as that described above in Example 3, Step B to give 0.45 g (quantitative) of a crystalline solid.
  • Step C (Boc)Pro-Tyr (OBzl) - ⁇ [CH2NH]- Ile-Leu (OBzl) (SEQ ID NO:46)
  • Step B 0.45 g (0.7 mmol) of the compound made in Example 15, Step B was coupled to Boc-L-proline (177 mg, 0.82 mmol) in the same manner as that described above in Example 3, Step B to give 0.47 g (87% yield) of a colorless solid.
  • Step D Pro-Tyr(OBzl)- ⁇ [CH 2 NH]-Ile- Leu(OBzl) -HCl (SEQ ID NO:47) 0.47 g of the compound made in Example 15, Step C was deprotected in the same manner as that described for Example 3, Step B to give 0.4 g of a crystalline solid.
  • Step E N ⁇ (Boc) ,N ⁇ (Z) Orn-Pro-Tyr(OBzl)- ⁇ [CH2NH]- Ile-Leu(OBzl) (SEQ ID NO:48) 0.2 g (0.28 mmol) of the compound made in Example 15, Step D was coupled to 0.103 g (0.28 mmol) of N ⁇ (Boc) N (Z)Orn in the same manner as that described above in Example 3, Step G to give 0.1 g (35% yield) of a colorless solid.
  • Step F N ⁇ (Boc)-Orn-Pro-Tyr ⁇ [CH2NH]-Ile- Leu-HOAc (SEQ ID NO: 49)
  • Step E 0.1 g of the compound made in Example 15, Step E was deprotected in the same manner as that described in Example 3, Step J to give 0.08 g of a white crystalline solid.
  • Example 22 N ⁇ [CH 3 (CH ) i ⁇ CO]Lys-Pro-Tyr-Ile-Leu, Acetic Acid Salt (SEQ ID NO:57)
  • Example 22 was prepared according to the synthetic procedure described above in Example 3 by using the appropriate acid chloride.
  • R f (chloroform/methanol 20:1) 0.40.
  • Step B N ⁇ BocN ⁇ (Z)-Lys ⁇ [CH 2 N]Pro-Tyr(OBzl)Ile- Leu(OBzl) (SEQ ID NO:59) .
  • the aldehyde from Step A was coupled to the product from Example 3, Step F using the synthetic procedure described in Example 7, Step B.
  • Step D N ⁇ (l-Adamantanecarbonyl)N ⁇ (Z)-Lys ⁇ [CH 2 N]Pro- Tyr(OBzl) Ile-Leu (OBzl) (SEQ ID NO: 61)
  • Step E N ⁇ (1-Adamantanecarbonyl)Lys ⁇ [CH2N]Pro-Tyr-
  • Example 24 N ⁇ (1-Adamantanecarbonyl) Lys-Pro-Tyr ⁇ [CH2NH] Ile- Leu, Acetic Acid Salt (SEQ ID NO: 62) Step A: N ⁇ (Boc)N ⁇ (Z)-Lys-Pro-Ty (OBzl)-Ile- Leu (OBzl) (SEQ ID NO: 63)
  • Step D Example 15, Step D above in the same manner described above for Example 3, Step G.
  • MS - DCI (NH 3 ) 1033 (M+H) R f (chloroform/methanol 20:1) 0.82.
  • Step B N ⁇ (Z)Lys-Pro-Tyr(OBzl) ⁇ [CH 2 NH]Ile-Leu(OBzl) Hydrochloride Salt (SEQ ID NO: 64)
  • MS - DCI (NH 3 ) 933 (M+H, Base) R f (chloroform/methanol 20:1) 0.30.
  • Step C N ⁇ (1-Adamantanecarbonyl)N ⁇ (Z)Lys-Pro- Tyr(OBzl) ⁇ [CH NH] Ile-Le (OBzl)
  • Step D N ⁇ (1-Adamantanecarbonyl)Lys-Pro- Tyr ⁇ [CH 2 NH] Ile-Leu, Acetic Acid Salt (SEQ ID NO:
  • N-Boc-Ala (0.95 g) was coupled using the procedure described above for Example 3, Step A to Pro-Tyr(OBzl)- Ile-Leu(OBzl) -HCl (3.6 g) [SEQ ID NO:10] to yield 1.9 g (44% yield) of N-Boc-Ala-Pro-Tyr-(OBzl)-Ile-Leu(OBzl) (SEQ ID NO:79)-l/2 H 2 0 as a colorless solid.
  • Step B N-t-Boc-Ala-Pro-Tyr-Ile-Leu (SEQ ID NO:78) N-Boc-Ala-Pro-Tyr(OBzl)-Ile-Leu(OBzl) (SEQ ID NO:79) (1.2 g) was deprotected using the method of Example 3, Step J (except no acetic acid was added) to yield 1.1 g of N-t-Boc-Ala-Pro-Tyr-Ile-Leu «l/2 H 2 0 (SEQ ID NO:78) as a white solid. MS (Calc ' d) MS ( found)
  • Example 31 N-[2-(aminomethyl)benzoyl]-Pro-Tyr-Ile- Leu-HCl (SEQ ID NO:84) This compound was prepared according to the procedure described above in Example 3.
  • Step A l-tert-Butoxycarbonyl-2-(S)- propenonepyrrolidine
  • 6 g (27.88 mmoles) Boc-Proline was dissolved in 400 ml dry THF and cooled to -78°C.
  • 17 ml of 1.6 M solution nBuLi in hexanes (27.2 mmoles) was added to the mixture followed by 60 ml (60 mmoles) vinylmagnesium bromide, 1 M solution in THF (60 mmoles) . Then it was allowed to warm to 25°C, and stirred for 3 hours .
  • Step B l-tert-Butoxycarbonyl-2-(S)-(1'-(S)- hydroxyprop-2-ene)pyrrolidine
  • a 200 ml flask 4.55 g (20.2 mmoles) of 1-tert- butoxycarbonyl-2-(S)-propenonepyrrolidine was dissolved in 100 ml MeOH, 6.6 g (18 mmoles) CeCl 3 »7H 2 ⁇ was added, and cooled to -78°C.
  • 680 mg (17.8 mmoles) NaBH 4 was added, stirred at -78°C for 4 hours, allowed to warm to -10°C and quenched with 50 ml 10% HCl.
  • Step C l-tert-Butoxycarbonyl-2-(S)-(1'-(S)-0(-2"- hexahydropyrane)prop-2-ene)pyrrolidine
  • a 300 ml flask 3.8 g 16.72 (mmoles) 1-tert- butoxycarbonyl-2-(S)-(!'-(S)-hydroxyprop-2- ene)pyrrolidine was dissolved in 90 ml dry CH 2 CI 2 containing 2.7 ml (29.7 mmoles) tetrahydropyrane and 50 mg (2 mmoles) pyridinium paratoluenesulfonate.
  • Step D l-tert-Butoxycarbonyl-2-(S)-(l'-(S)-0(-2' '- hexahydropyrane) 2-carboxaldehyde)pyrrolidine
  • 1-tert- butoxycarbonyl-2-(S)- (1'-(S)-O(-2 ' '-hexahydro ⁇ pyrane)prop-2-ene)pyrrolidine was dissolved in 300 ml CH 2 CI 2 containing 2.88 ml pyridine and cooled to -78°C. To that 0 3 was passed through until it turned pale blue. The excess 0 3 was removed with oxygen, 4 ml methyl sulfide was added, and the mixture was allowed to warm to 25°C and stirred for 3 hours. The solvent was stripped in vacuo and the resulting oil was used directly for the next reaction.
  • Step E l-tert-Butoxycarbonyl-2- (S)-(1'-(S)-O(-2* '- hexahydropyrane) 4 '-carbomethoxybut-2 '- ,ene)pyrrolidine
  • a 300 ml flask 834 mg (19.2 mmoles) of 60% NaH in oil was washed with 60 ml hexanes, suspended in 75 ml THF, and cooled to 0°C. In this 3.4 ml (21 mmoles), (CH3O) 2 -? (0)CH 2 C0 2 CH 3 was added and the mixture stirred for 45 minutes.
  • Step F l-tert-Butoxycarbonyl-2- (S)- (1 '- (S) -hydroxy-4 '- carbomethoxybut-2 '-ene)pyrrolidine
  • a 100 flask 2.86 g (7.72 mmoles) of l-tert- butoxycarbonyl-2- (S)- (1'- (S)-0(-2"-hexahydropyrane) 4 *- carbomethoxybut-2 '-ene)pyrrolidine was dissolved in 50 ml MeOH containing 150 mg (0.66 mmoles) camphorsulfonic acid.
  • the reaction was stirred at 25°C for 3 hours, quenched with 5 ml 5% NaHC0 3 , 200 mg NaHC0 3 and stripped in vacuo. The remaining was dissolved in 200 ml EtOAc and washed with 30 ml water, and brine, dried and the solvent was stripped in vacuo.
  • the product was purified by silica gel chromatography to give 1.2 g of product, a 54% yield.
  • Step G l-tert-Butoxycarbonyl-2 (S) (1'- (S) -O-methane- sulfonyl-4 '-carbomethoxybut-2 '-ene)pyrrolidine
  • l-tert-butoxycarbonyl-2 (S)- (1 '- (S)-hydroxy-4 '-carbomethoxybut- 2 '-ene)pyrrolidine dissolved in 10 ml CH2CI2 was cooled to 0°C, was treated with 0.57 ml (3.27 mmoles)diiso- propylethyl amine followed by 0.25 ml (3.27 mmoles) methanesulfonyl chloride.
  • Step H Methyl trans-2-(S)-Benzyloxybenzyl-4-(1'-tert- butoxycarbonyl-2' (S)-pyrrolidine)buten-3-ate
  • benzyloxybenzyl chloride was treated with 400 mg in 10 ml THF at 0°C for 3 hours. Then it was transferred via cannula into a flask containing 486 mg (6.5 mmoles)CuCN suspended in 10 ml THF and cooled to -40°C in a CH 3 CN/dry ice bath.
  • Step I trans-2- (S)-Benzyloxybenzyl-4- (1 '-tert- butoxycarbonyl-2 ' (S)-pyrrolidine)buten-3-oic acid
  • Step J trans-2- (S)-Benzyloxybenzyl-4-(1'-tert- butoxycarbonyl-2 '- (S)-pyrrolidine)buten- 3-ate-Ile-Leu(OBzl) (SEQ ID NO:102)
  • a 35 ml flask 250 mg (0.56 mmoles) of trans-2- (S)-benzyloxybenzyl-4- (1 '-tert-butoxycarbonyl-2 ' (S)- pyrrolidine)but-3-enoic acid, in 6 ml THF was cooled to -10°C and 0.078 ml (0.69 mmoles) N-methyImorpholine, followed by 0.09 ml (0.69 mmoles) isobutyl chloroformate was added.
  • Step K trans-2- (S)-Benzyloxybenzyl-4- (2*- (S)- pyrrolidine)buten-3-ate-Ile-Le (OBzl) hydrochloride (SEQ ID NO:103)
  • trans-2-(S)- benzyloxybenzyl-4- (1'-tert-butoxycarbonyl-2' (S)- pyrrolidine)buten-3-ate-Ile-Leu(OBzl) (SEQ ID NO:102) was dissolved in 2 ml CH 2 CI2 and 0.5 ml 4.5 M HCl solution in dioxane was added. After stirring at 25°C for 3 hours the solvent was stripped in vacuo and the hydrochloride was precipitated from ether, filtered and dried to give 120 mg of product which was used for the next reaction without purification.
  • Step L (N ⁇ Boc) (N ⁇ CBZ)Lys-trans-2-(S)-4'-
  • Step B Ethyl 5-di(p-methoxybenzyl) amino valerate
  • a 500 ml flask 19 g crude 5-di (p- methoxybenzyl)aminovaleric acid was dissolved in 300 ml ethanol and 3 ml cone. H2SO 4 was added. The mixture was heated to reflux for 5 hours, and then stirred at 25°C for 16 hours. The acid was neutralized with solid
  • Step C Ethyl 2-benzyl-5-di(p-methoxy- benzyl)amino valerate
  • Step D 2-Benzyl-5-di (p-methoxybenzyl)amino valeric acid
  • Step E 2-Benzyl-4-di(p-methoxybenzyl) aminopentane- carbonyl-Pro-Tyr(OBzl)-Ile-Leu(OBzl) (SEQ ID NO:89)
  • HCl-Pro-Tyr- (OBzl)-Ile-Leu(OBzl) (SEQ ID NO:10) and 510 mg (1.14 mmoles) 2-benzyl-5-di(p-methoxybenzyl)-amino valeric acid were coupled with 236 mg (1.14 mmoles) DCC, 177 mg (1.14 mmoles) 1-hydroxybenzotriazole, and 0.148 ml (1.14 mmoles*) N-methyImorpholine as in Example 21.
  • the product was purified by chromatography on silica gel using 0.1% NH 4 OH/l% CH 3 OH/CH2Cl2 to give 800
  • Step F 2-Benzyl-5-aminopentanecarbonyl-Pro-Tyr-Ile- Leu (SEQ ID NO:88)
  • Example 36 4-t-Butoxycarbamidopiperidine-4-carbonyl)- Pro-Tyr-Ile-Leu (SEQ ID NO: 90)
  • Step A l-Benzyl-4-aminopiperidine-4-carboxylic acid
  • Benzyl-4-cyano-4-aminopiperidine 3.0 g (13.95 mmoles)
  • HCl HCl
  • 20 ml water was added and the pH was adjusted at 5-6 by addition of solid NaHC0 3 .
  • Step B l-Benzyl-4-t-butoxycarbamidopiperidine-4- carboxylic acid l-Benzyl-4-aminopiperidine-4-carboxylic acid, 2.0 g (7.4 mmoles), was dissolved in a mixture of 30 ml 0.5 M LiOH and 30 ml dioxane and was treated with 1.8 (10 mmoles) of (Boc)2 ⁇ at 25°C for 16 hours and then with 3.6 g (20 mmoles) at 55°C for 11 hours. Then it was filtered at 25°C, neutralized with 10% KHS0 4 , and the solvent was stripped off. The resulting solid was extracted with 100 ml hot methanol and filtered at 25°C.
  • Step C (l-Benzyl-4-t-butoxycarbamidopiperidine-4- carbonyl)-Pro-Tyr(OBzl)-Ile-Leu(OBzl) (SEQ ID NO:37) Boc-Pro-Tyr(OBzl)-Ile-Leu(OBzl) «HC1 (SEQ ID NO:9) 322 mg (0.5 mmoles), 167.5 mg (0.5 mmoles) l-benzyl-4-t- butoxycarbamidopiperidine-4-carboxylic acid, 103 mg (0.5 mmoles) DCC and 76.5 mg (0.5 mmoles) 1-hydroxybenzo- triazole hydrate were dissolved in 4 ml DMF at -10°C.
  • Step D (4-t-Butoxycarbamidopiperidine-4-carbonyl)- Pro-Tyr-Ile-Leu (SEQ ID NO: 90)
  • 270 mg (0.27 mmoles) (l-benzyl-4- t-butoxycarbamidopiperidine-4-carbonyl)-Pro- Tyr(OBzl) Ile-Leu (OBzl) (SEQ ID NO:37) was dissolved in 10 ml ethanol, 5 ml cyclohexene and 15 ⁇ l AcOH.
  • 27 mg 20% Pd(OH)2/C was added and the mixture was heated to reflux for 4 hours under vigorous stirring.
  • Step A 1-t-Butoxycarbamido-l-cyclopentanecarboxylic acid
  • 1-amino-l-cyclopentanecarboxylic acid, 2 g (15.49 mmoles) was dissolved in a mixture of 32 ml 0.5 M NaOH and 32 ml water and 3.75 g (17.04 mmoles) (Boc)2 ⁇ was added.
  • Step B (1-t-Butoxycarbamido-l-cyclopentanecarbonyl)- Pro-Tyr(OBzl)-Ile-Leu(OBzl) (SEQ ID NO:97) Pro-Tyr(OBzl)-Ile-Leu(OBzl) -HCl (SEQ ID NO:10) 910 mg (1.26 mmoles), 289 mg (1.26 mmoles), 1-t-butoxy- carbamido-1-cyclopentanecarboxylic acid 260 mg (1.26 mmoles), 1,3 dicyclohexylcarbodiimide and 193 mg (1.26 mmoles) 1-hydroxybenzotriazole hydrate were dissolved in 4 ml DMF at -10°C. To that 0.06 ml N-methyImorpholine was added
  • Step C (1-amino-l-cyclopentanecarbonyl)-Pro- Tyr(OBzl)Ile-Leu(OBzl) «HC1 (SEQ ID NO:93) (1-t-Butoxycarbamido-l-cyclopentanecarbonyl)-Pro- Tyr(OBzl)-Ile-Leu(OBzl) (SEQ ID NO:97) 450 mg (0.5 mmoles), was dissolved in 3 ml 4.5 M HCl in dioxane and stirred at 25°C for 3 hours. Then ether was added, and the precipitating salt was filtered under vacuum, to give 260 mg of product, a 62% yield. The product was used without further purification.
  • Step D ⁇ -Boc- ⁇ -CBZ-Lys- (1-amino-l-cyclopentane carbonyl) -Pro-Tyr(OBzl)-Ile-Leu (OBzl) (SEQ ID NO: 92) 1,3 Dicyclohexylcarbodiimide 64 mg (0.31 mmoles), ⁇ -Boc- ⁇ -CBZ-Lys 118 mg (0.31 mmoles), (1-amino-l- cyclopentanecarbonyl)-Pro-Tyr(OBzl)-Ile-Leu (OBzl) •HCl (SEQ ID NO:93) 260 mg (0.31 mmoles) and 1-hydroxybenzo ⁇ triazole hydrate 48 mg (0.33 mmoles) were dissolved in 2.5 ml DMF at -10°C.
  • N-methylmorpholine was added and the reaction was continued at 25°C for 48 hours.
  • the reaction was quenched with 50 ml 5% NaHC0 3 and extracted with 75 ml ethyl acetate.
  • the EtOAc extracts were washed with 10% HCl, 5% NaHC0 3 and brine, dried, and evaporated to dryness in vacuo leaving a white solid. This was chromatographed on silica gel using 1% CH 3 OH/CH2Cl 2 , to give 200 mg, a 55% yield of product.
  • Step E ⁇ -CBZ-Lys- (1-amino-l-cyclopentanecarbonyl)-
  • Pro-Tyr (OBzl)-Ile-Leu(OBzl) (SEQ ID NO: 92) 420 mg (0.36 mmoles) was dissolved in 1 ml 4.5 M HCl in dioxane and 2 ml CH 2 CI 2 and stirred at 25°C for 1 hour. Then ether was added and the product was precipitated as a white solid 350 mg, an 89% yield. This was used for the next reaction without further purification.
  • Step F ⁇ -(1-Adamantanecarbonyl)- ⁇ -CBZ-Lys-(1-amino- l-cyclopentanecarbonyl)-Pro-Tyr(OBzl)-Ile- Leu(OBzl) (SEQ ID NO:91) ⁇ -CBZ-Lys-(1-amino-l-cyclopentanecarbonyl)-Pro- Tyr(OBzl)-Ile-Leu(OBzl) •HCl (SEQ ID NO:95) 350 mg (0.32 mmoles) which was dissolved in 5 ml CH 2 CI2 and cooled to 0°C.
  • Step G ⁇ - (1-Adamantanecarbonyl)-Lys- (1-amino- l-cyclopentanecarbonyl)-Pro-Tyr-Ile-Leu (SEQ ID NO:96) ⁇ -(1-Adamantanecarbonyl)- ⁇ -CBZ-Lys- (1-amino-l- cyclopentanecarbonyl)-Pro-Tyr(OBzl)-Ile-Leu(OBzl) (SEQ ID NO:91) 210 mg (0.17 mmoles) was dissolved in a mixture of 8 ml EtOH and 4 ml cyclohexene containing 10% w/w of 20% Pd(0H)2/C on carbon and 0.015 ml acetic acid.
  • This compound can be prepared according to the procedure described above in Example 3 .
  • Pro-Tyr (OBzl)-Ile-Leu(OBzl) (SEQ ID NO: 92) 190 mg (0.164 mmoles) was dissolved in a mixture of 6 ml EtOH and 3 ml cyclohexene containing 20 mg of 20% Pd(OH)2/C on carbon and 0.018 ml acetic acid. The mixture was heated to reflux for 4 hours, then filtered through Celite® and stripped in vacuo. The product was crystallized from methanol, mp 166-167°C.
  • Example 42
  • Step A (l-Benzyl-4- (1 '-adamantaneoxy) carbamido- piperidine-4-carboxylic acid l-Benzyl-4-amino-4-piperidine-carboxylic acid hydrochloride hydrate 0.5 g (1.68 mmoles) was dissolved in a mixture of 3.5 ml 1M LiOH and 3.5 ml water. In this solution 7 ml dioxane and 333 mg (1.68 mmoles) 1- adamantyl fluoroformate was added and the resulting mixture was stirred at 25°C for 16 hours. The precipitated white solid was filtered off and dried, and used for the next reaction without purification.
  • Step B (l-Benzyl-4-(1'-adamantaneoxy)carbamido- piperidine-4-carbonyl-Pro-Tyr(OBzl)-Ile- Leu(OBzl) (SEQ ID NO:101) (l-Benzyl-4- (1'-adamantaneoxy)carbamidopiperidine- 4-carboxylic acid crude 300 mg (0.71 mmoles), HCl*Pro- Tyr(OBzl)-Ile-Leu(OBzl) (SEQ ID NO:10) 500 mg (0.69 mmoles), DCC 142.5 mg (0.69 mmoles), and 1-hydroxy ⁇ benzotriazole 108 mg (0.69 mmoles) were dissolved in 6 ml DMF and cooled to -10°C.
  • N-methyImorpholine was added and the reaction was stirred at 25°C for 72 hours . Then it was quenched with NaHC0 3 (30 ml) , and extracted with EtOAc (2x100 ml) . The EtOAc was dried and stripped in vacuo. The resulting solid was chromatographed on silica gel using 0.25% NH OH/2.5% MeOH/CH 2 Cl2 as eluent to give 540 mg of product, a 72% yield, mp 79.5-81.5°C.
  • Step C 4-(1'-adamantaneoxy)carbamidopiperidine-4- carbonyl-Pro-Tyr-Ile-Leu*AcOH (SEQ ID NO:98)
  • Brain membrane preparations were prepared according to the method described in Tarn (Proc. Natl. Acad. Sci.
  • the homogenate was centrifuged at 920 x g for 10 minutes.
  • the supernatant was centrifuged at 47,000 x g for 20 minutes.
  • the resulting membrane pellet was resuspended in 10 volumes (original wt/vol) of 50 m Tris-HCl (pH 7.4) and incubated at 37°C for 45 minutes to degrade and dissociate bound endogenous ligands.
  • the membranes were then centrifuged at 47,000 x g for 20 minutes and resuspended at a concentration of 2 brains per 90 ml of buffer, containing 50 mM Tris-HCl, pH 7.4, 1 m EDTA, 0.1% bovine serum albumin, and 50 mg/ml bacitracin.
  • Neurotensin receptor binding was performed according to the method of Goedert et al. (Brain Research. 1984, 304: 71-81). One ml of the brain membrane suspension containing 3 n [ ⁇ H]neurotensin with or without test compounds was incubated at 25°C for 20 minutes. Nonspecific binding was defined by binding in the presence of 1 mM neurotensin. At the end of the incubation, the tubes were incubated at 4°C for 10 minutes and filtered rapidly under reduced pressure through Whatman GF/B glass fiber filters which have been pretreated for 3 hours with 0.2% polyethyleneimine in water. Each sample was washed 3 times with 5 ml ice cold incubation buffer. Radioactivity was determined by liquid scintillation spectrometry.
  • PQW phenylquinone writhing test
  • Intracerebroventricular (i.e.v.) injections were made according to the method of T. J. Haley and W. G. McCormick, Br. J. Pharmacol. r 1957, 12, 12-15.
  • Test compounds for i.v. administration were suspended in an aqueous vehicle containing 2% by volume of Tween® 80, a pharmacological dispersant manufactured by Fisher-Scientific Company and containing 100% polysorbate 80 and 0.25% by weight of Methocel® A15 powder, a suspending agent manufactured by Dow Chemical Company and containing 100% methylcellulose.
  • I.V. doses were administered in a volume of 10 ml/kg body weight and are expressed as mg/kg doses.
  • compounds were dissolved in 100% dimethylsulfoxide (DMSO).
  • DMSO dimethylsulfoxide
  • Unilateral i.e.v. doses were injected in a volume of 5 microliters per mouse and are expressed as microgram/mouse doses.
  • Test compounds were administered i.e.v. or i.v. to fasted (17-21 hours) male white mice (CF1) , 5-15 animals per graded dose. After 5-25 minutes, aqueous 0.01% phenyl-p.-benzoquinone, 0.125 mg/kg, was injected intraperitoneally. After an additional 5 minutes, mice were observed 10 minutes for the characteristic, stretching or writhing syndrome which is indicative of pain produced by phenylquinone. The effective analgesic dose in 50% of the mice (ED 50 ) was calculated by the moving average method of W. R. Thompson, Bac. Rev.. 1947, 11, 115-145. The mouse analgesic data are summarized in Table 1.
  • the compounds of this invention may be administered ⁇ by any means that produces contact of the active agent with the agent's site of action in the body of a mammal. They can be administered by any conventional means available for use in conjunction with pharmaceuticals, either as individual therapeutic agents or in a combination of therapeutic agents . They can be administered alone, but are generally administered with a pharmaceutical carrier selected on the basis of the chosen route of administration and standard pharmaceutical practice.
  • the dosage administered will, of course, vary depending upon known factors such as the pharmacodynamic characteristics of the particular agent, and its mode and route of administration; age, health, and weight of the recipient; nature and extent of symptoms, kind of concurrent treatment, frequency of treatment, and the effect desired.
  • a daily dosage of active ingredient can be about 0.1 to 100 milligrams per kilogram of body weight.
  • 0.5 to 50, and preferably 1 to 10 milligrams per kilogram per day given in divided doses 1 to 6 times a day or in sustained release form is effective to obtain desired results.
  • Dosage forms (compositions) suitable for internal administration contain from about 1 milligram to about 500 milligrams of active ingredient per unit.
  • the active ingredient will ordinarily be present in an amount of about 0.5-95% by weight based on the total weight of the composition.
  • the active ingredient can be administered orally in solid dosage forms, such as capsules, tablets, and powders, or in liquid dosage forms, such as elixirs, syrups, and suspensions. It can also be administered parenterally, in sterile liquid dosage forms, by inhalation in the form of a nasal spray or lung inhaler, or topically as an ointment, cream or lotion.
  • Gelatin capsules contain the active ingredient and powdered carriers, such as lactose, sucrose, mannitol, starch, cellulose derivatives, magnesium stearate, stearic acid, and the like. Similar diluents can be used to make compressed tablets. Both tablets and capsules can be manufactured as sustained release products to provide for continuous release of medication over a period of hours . Compressed tablets can be sugar coated or film coated to mask any unpleasant taste and protect the tablet from the atmosphere, or enteric coated for selective disintegration in the gastrointestinal tract.
  • powdered carriers such as lactose, sucrose, mannitol, starch, cellulose derivatives, magnesium stearate, stearic acid, and the like. Similar diluents can be used to make compressed tablets. Both tablets and capsules can be manufactured as sustained release products to provide for continuous release of medication over a period of hours . Compressed tablets can be sugar coated or film coated to mask any unpleasant taste and protect the tablet from the atmosphere, or enteric coated for selective disintegration
  • Liquid dosage forms for oral administration can contain coloring and flavoring to increase patient acceptance.
  • water a suitable oil, saline, aqueous dextrose (glucose) , and related sugar solutions and glycols such as propylene glycol or polyethylene glycols are suitable carriers for parenteral solutions.
  • saline aqueous dextrose (glucose)
  • glycols such as propylene glycol or polyethylene glycols
  • Solutions for parenteral administration contain the active ingredient, suitable stabilizing agents, and if necessary, buffer substances .
  • Antioxidizing agents such as sodium bisulfite, sodium sulfite, or ascorbic acid either alone or combined are suitable stabilizing agents.
  • citric acid and its salt and sodium EDTA are suitable stabilizing agents.
  • parenteral solutions can contain preservatives, such as benzalkonium chloride, methyl- or propy1-paraben, and chlorobutanol.
  • Suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences. A. Osol, a standard reference text in this field.
  • Useful pharmaceutical dosage forms for administration of the compounds of this invention can be illustrated as follows:
  • Capsules A large number of unit capsules are prepared by filling standard two-piece hand gelatin capsules each with 50 milligrams of powdered active ingredient, 175 milligrams of lactose, 24 milligrams of talc, and 6 milligrams of magnesium stearate.
  • Soft Gelatin Capsules A mixture of active ingredient in soybean oil is prepared and injected by means of a positive displacement pump into gelatin to form soft gelatin capsules containing 50 milligrams of the active ingredient . The capsules are washed in petroleum ether and dried.
  • Tablets A large number of tablets are prepared by conventional procedures so that the dosage unit is 50 milligrams of active ingredient, 6 milligrams of magnesium stearate, 70 milligrams of microcrystalline cellulose, 11 milligrams of cornstarch and 225 milligrams of lactose. Appropriate coatings may be applied to increase palatability or delay absorption.
  • a parenteral composition suitable for administration by injection is prepared by stirring 1.5% by weight of active ingredient in 10% by volume propylene glycol and water. The solution is sterilized by commonly used techniques.
  • aqueous suspension is prepared for oral administration so that each 5 milliliters contain 25 milligrams of finely divided active ingredient, 200 milligrams of sodium carboxymethyl cellulose, 5 milligrams of sodium benzoate, 1.0 grams of sorbitol solution, U.S.P., and 0.025 milliliters of vanillin.
  • Nasal Spray aqueous suspension is prepared for oral administration so that each 5 milliliters contain 25 milligrams of finely divided active ingredient, 200 milligrams of sodium carboxymethyl cellulose, 5 milligrams of sodium benzoate, 1.0 grams of sorbitol solution, U.S.P., and 0.025 milliliters of vanillin.
  • An aqueous solution is prepared such that each 1 milliliter contains 10 milligrams of active ingredient, 1.8 milligrams methylparaben, 0.2 milligrams propylparaben and 10 milligrams methylcellulose.
  • the solution is dispensed into 1 milliliter vials.
  • a homogeneous mixture of the active ingredient in polysorbate 80 is prepared such that the final concentration of the active ingredient will be 10 milligrams per container and the final concentration of polysorbate 80 in the container will be 1% by weight.
  • the mixture is dispensed into each can, the valves are crimped onto the can and the required amount of dichlorotetrafluoroethane is added under pressure.
  • An ointment for topical administration may be prepared by adding the active ingredient to a mixture of 48% by weight white petrolatum, 10% liquid petrolatum, 8% glycerol monostearate, 3% isopropyl myristate and 20% lanolin at 70°C. After thorough mixing, a warm solution of methyl and propyl parabens in water containing sodium acetone bisulfite is added such that the final concentrations of each paraben is 0.15%, of water is 8% and of sodium acetone bisulfite is 0.5%. The mixture is stirred until it has reached room temperature.

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Abstract

Cette invention se rapporte à des peptides modifiés ainsi qu'à des sels pharmaceutiquement acceptables de ces peptides, lesquels sont susceptibles de traverser la barrière hémato-encéphalique. On décrit aussi des compositions pharmaceutiques contenant ces peptides et des procédés de traitement consistant à les utiliser.
PCT/US1992/004968 1991-06-27 1992-06-18 Peptides modifies pouvant etre transportes vers le systeme nerveux central WO1993000359A1 (fr)

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WO1994022906A1 (fr) * 1993-03-26 1994-10-13 Warner-Lambert Company Inhibiteurs de l'enzyme convertissant l'endotheline
US5962664A (en) * 1993-05-13 1999-10-05 Friedhoff; Arnold J. Psychosis protecting nucleic acid, peptides, compositions and method of use
US6939989B2 (en) 2001-03-01 2005-09-06 Bayer Aktiengesellschaft Side-chain halogenated amino dicarboxylic acid derivatives as medicaments for treating cardiovascular diseases
US7067694B2 (en) 2001-03-01 2006-06-27 Bayer Aktiengesellschaft Halogen-substituted amino dicarboxylic acid derivatives as medicaments for treating cardiovascular diseases
US7087644B1 (en) 1999-09-13 2006-08-08 Bayer Aktiengesellschaft Derivatives of dicarboxylic acid having pharmaceutical properties
WO2006103057A1 (fr) * 2005-03-31 2006-10-05 Ucb Pharma, S.A. Compounds comprising an oxazoline or thiazoline moiety, processes for making them, and their uses
US8362068B2 (en) 2009-12-18 2013-01-29 Idenix Pharmaceuticals, Inc. 5,5-fused arylene or heteroarylene hepatitis C virus inhibitors
US20160333070A1 (en) * 2013-12-20 2016-11-17 Yissum Research Development Company Of The Hebrew University Of Jerusalem Ltd. Pro-angiogenic peptides and peptide conjugates

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CA2137087A1 (fr) * 1992-06-01 1993-12-09 U. Prasad Kari Peptides a action biologique substitues en position n-terminale

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EP0333071A2 (fr) * 1988-03-11 1989-09-20 Eisai Co., Ltd. Polypeptides, méthodes pour leur préparation, les compositions pharmaceutiques les contenant et leur emploi

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203RD ACS NAT. MEETING, S. FRANCISCO, CALIFORNIA, APRIL 5-10,1992, ABSTRACT PAPERS: ABSTRACT NO. 84 vol. 203, no. 1-3, G A CAIN ET AL. 'neurotensin based analgesic identification of minimally active fragment : enhancement of potency, duration of action, and transport properties' *
BIOCHEMICAL PHARMACOLOGY vol. 36, no. 6, 1987, GB pages 869 - 874 K S KANBA ET AL. 'comparison of the stimulation of inositol phospholipid hydrolysis and of cGMP formation by neurotensin, some of its analogs and neuromedin N in neuroblastoma clone NIE-115' *

Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994022906A1 (fr) * 1993-03-26 1994-10-13 Warner-Lambert Company Inhibiteurs de l'enzyme convertissant l'endotheline
US5962664A (en) * 1993-05-13 1999-10-05 Friedhoff; Arnold J. Psychosis protecting nucleic acid, peptides, compositions and method of use
US7517896B2 (en) 1999-09-13 2009-04-14 Bayer Aktiengesellschaft Aminodicarboxylic acid derivatives having pharmaceutical properties
US7781470B2 (en) 1999-09-13 2010-08-24 Bayer Schering Pharma Aktiengesellschaft Aminodicarboxylic acid derivatives having pharmaceutical properties
US7087644B1 (en) 1999-09-13 2006-08-08 Bayer Aktiengesellschaft Derivatives of dicarboxylic acid having pharmaceutical properties
US7067694B2 (en) 2001-03-01 2006-06-27 Bayer Aktiengesellschaft Halogen-substituted amino dicarboxylic acid derivatives as medicaments for treating cardiovascular diseases
US7700653B2 (en) 2001-03-01 2010-04-20 Bayer Schering Pharma Aktiengesellschaft Halogen-substituted aminodicarboxylic acid derivatives
US6939989B2 (en) 2001-03-01 2005-09-06 Bayer Aktiengesellschaft Side-chain halogenated amino dicarboxylic acid derivatives as medicaments for treating cardiovascular diseases
WO2006103057A1 (fr) * 2005-03-31 2006-10-05 Ucb Pharma, S.A. Compounds comprising an oxazoline or thiazoline moiety, processes for making them, and their uses
US7863450B2 (en) 2005-03-31 2011-01-04 Ucb Pharma, S.A. Compounds comprising an oxazoline or thiazoline moiety, processes for making them, and their uses
US8362068B2 (en) 2009-12-18 2013-01-29 Idenix Pharmaceuticals, Inc. 5,5-fused arylene or heteroarylene hepatitis C virus inhibitors
US9187496B2 (en) 2009-12-18 2015-11-17 Idenix Pharmaceuticals Llc 5,5-fused arylene or heteroarylene hepatitis C virus inhibitors
US20160333070A1 (en) * 2013-12-20 2016-11-17 Yissum Research Development Company Of The Hebrew University Of Jerusalem Ltd. Pro-angiogenic peptides and peptide conjugates
US9873724B2 (en) * 2013-12-20 2018-01-23 Yissum Research Development Company Of The Hebrew University Of Jerusalem Ltd. Pro-angiogenic peptides and peptide conjugates

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