WO1992002545A1 - Bombesin antagonists - Google Patents
Bombesin antagonists Download PDFInfo
- Publication number
- WO1992002545A1 WO1992002545A1 PCT/GB1991/001289 GB9101289W WO9202545A1 WO 1992002545 A1 WO1992002545 A1 WO 1992002545A1 GB 9101289 W GB9101289 W GB 9101289W WO 9202545 A1 WO9202545 A1 WO 9202545A1
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- Prior art keywords
- ala
- phe
- pro
- phenyl
- alahisd
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- 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
- C07K7/086—Bombesin; Related peptides
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Definitions
- the present invention relates to polypeptide compounds which possess antagonist properties against bombesin, or bombesin-like peptides, and are useful in the treatment of disease, particularly human small-cell lung cancer, Zollinger-Ellison syndrome or pancreatic cancer.
- the invention thus provides the polypeptides, processes - for preparing them, pharmaceutical compositions containing them, and their use in medicine.
- Bombesin is a tetradecapeptide originally isolated from the skin of a frog. It has the formula
- Gastrin releasing peptide is a 27 amino acid peptide isolated from the porcine gut.
- the last ten amino acids at the C-terminus of gastrin releasing peptide correspond with one amino acid alteration (3) to the last ten amino acids of bombesin, viz:
- the compounds of the present invention inhibit the production of gastrin releasing peptide in mammalian cells and are therefore useful in controlling the clinical symptoms of diseases which cause the secretion of hypersecretion of gastrin releasing peptide (e.g., SCLC).
- SCLC gastrin releasing peptide
- the present invention provides a polypeptide of the formula (I) :
- X is a group X Arg(or £-Arg)X X a and X is des NH remindPro,TyrPro,des NH-TyrPro, Ada, Pro, D-Pro or is deleted;
- X is Asn, Phe, D-Phe, or Phe or D-Phe substituted by one or more halo atoms; or X is a group A-(CH distract) -CO- in which A is a group containing 1 to 3 rings of which at least one ring is aromatic, each ring system being Optionally substituted; and the alkylene group is optionally substituted by one to four groups selected from amino, hydroxy C. , alkoxy and C.
- alkyl optionally substituted by halo and n is 0 to 4, or X is a group A-(CHdonating) -CO- in which A is an optionally substituted aromatic residue containing 1 to 3 rings and the alkylene group is optionally substituted by one to four groups selected from amino, C. , alkoxy and C. , alkyl optionally substituted by halo and n is 1 to , o or X is cyclopentylcarbonyl substituted by a group X Arg (or D-Arg)
- X is His, ThiAla or is deleted
- X is Ala, D-Ala, CPenc, D-tBuGly or Pro;
- X is Val or Val substituted by one or more halo atoms
- X is Gly, Ala, D-Ala, Sarcosine, Pro, D-Pro or D-Phe;
- X 5 is His or ThiAla
- X is D-Pro ⁇ , Pro ⁇ , 2-pyrrolidinyl-3-hydroxypropionyl or D-Pro;
- X is Nle,Leu,Phe,Val,Mox, D-Phe or Phe, or D-Phe substituted by one or more halo atoms or naphthylAla or naphthyl D-Ala or a hydrophobic, substituted aromatic amino acid or aralkylamine or is deleted.
- A is phenyl, naphthyl, phenothiazinyl or indolyl.
- A is phenyl or naphthyl.
- Suitable substituents for the aromatic ring Ar include hydroxy, phenyl, halo, C. , alkyl or C. , alkoxy optionally substituted by halo.
- n is 2.
- X terminates in a des NH memo moiety.
- X is des NH ? Tyr or des NH.Pro.
- X is Gly or D-Ala.
- X is D-Phe.
- X is des ttt Phe, des NH remindTyr, des
- X is His or ThiAla
- X is Ala, Pro
- X is Val or hexa luorovaline
- X is D-Ala, D-Phe;
- X 5 is His, ThiAla
- X is n-Pro ⁇ , Pro ⁇ , Pro, D-Pro;
- X is Nle or Phe, Leu, Methoxinine, 2-Naphthyl-2-
- Preferred polypeptides of the present invention include:
- compositions of the formula (I) are- also included within the scope of the present invention.
- Suitable pharmaceutically acceptable salts are acid addition salts when the polypeptide is sufficiently basic e.g., contains one or more basic residues such as histidine.
- a suitable pharmaceutically-acceptable acid-addition salt of the invention may be formed with an inorganic acid, for example hydrochloric acid, hydrobromic acid, sulphuric acid or phosphoric acid, or with an organic acid, for example acetic acid, citric acid, maleic acid, fumaric acid, succinic acid, tartaric acid or trifluoroacetic acid.
- an inorganic acid for example hydrochloric acid, hydrobromic acid, sulphuric acid or phosphoric acid
- organic acid for example acetic acid, citric acid, maleic acid, fumaric acid, succinic acid, tartaric acid or trifluoroacetic acid.
- the invention provides, as a further feature, any one or more of the preferred compounds together with their pharmaceutically acceptable acid-addition salts.
- polypeptides of the invention may be prepared by any process well known in the art of peptide chemistry to be applicable to the synthesis of analogous compounds.
- a polypeptide of the invention may be obtained by procedures analogous to those disclosed in "Solid Phase Peptide Synthesis” by Stewart and Young (published by the Pierce Chemical Company, Illinois, 1984), “Principles of Peptide Synthesis” (published by Springer-Verlag, Berlin, 1984) , “Practice of Peptide Synthesis” (published by Springer-Verlag, Berlin, 1984), and "The Synthesis of a Tetrapeptide (J.Am.Chem.Soc, 83 2149(1963)).
- Preferred processes for the manufacture of a polypeptide of the invention include, for example: -
- protecting groups in the starting material there may be as many protecting groups in the starting material as there are radicals which may require protection, for example some or all of those groups which exist in the product as free hydroxy groups or basic amino groups (whether primary or secondary amino groups) .
- the protecting group or groups may be chosen from those described in the standard text books on peptide chemistry stated above. Various methods for the removal of the protecting group or groups are also described in those books.
- a suitable protecting group for a basic amino group is, for example, an arylmethoxycarbonyl group, which may be removed by hydrogenation over a catalyst, for example palladium-on-charcoal or it may be removed by treatment with an inorganic acid, for example anhydrous hydrogen fluoride or hydrogen bromide.
- a particularly suitable protecting group for a basic amino group is, for example, an alkoxycarbonyl group, for example a Boc-group, which may be removed by treatment with an organic acid, for example trifluoroacetic acid, or it may be removed by treatment with an inorganic acid, for example anhydrous hydrogen chloride or hydrogen bromide; or for example a 9-fluorenylmethoxycarbonyl group, which may be removed by treatment with an organic base, for example piperidine.
- an alkoxycarbonyl group for example a Boc-group
- an organic acid for example trifluoroacetic acid
- an inorganic acid for example anhydrous hydrogen chloride or hydrogen bromide
- 9-fluorenylmethoxycarbonyl group which may be removed by treatment with an organic base, for example piperidine.
- a particularly suitable protecting group for the basic amino group in the side chain of Histidine is, for example, an arylsulphonyl group, for example a tosyl group, which may be removed by treatment with hydroxylamine, for example an N-hydroxytriazole, particularly 1-hydroxybenzotriazole, benzyloxymethyl or t-butyloxy- methyl.
- a suitable protecting group for a hydroxy group is, for example, an arylmethyl group, for example a benzyl group, which may be removed by treatment with an inorganic acid, for example anhydrous hydrogen fluoride, or it may be removed by hydrogenation over a catalyst, for example palladium-on-charcoal; or it may be for example, an esterifying group, for example an acetyl or benzoyl group, which may be removed by hydrolysis with a base, for example sodium hydroxide.
- an inorganic acid for example anhydrous hydrogen fluoride
- a catalyst for example palladium-on-charcoal
- an esterifying group for example an acetyl or benzoyl group, which may be removed by hydrolysis with a base, for example sodium hydroxide.
- a suitable protecting group for a carboxy group is, for example, an esterifying group, for example an arylmethyl group, for example a benzyl group, which may be removed by treatment with an inorganic acid, for example anhydrous hydrogen fluoride, or it may be removed by hydrogenation over a catalyst, for example palladium-on- charcoal; or, an alkyl group, C. . alkyl, for example a tert-butyl group which may be removed by treatment with an organic acid, for example trifluoroacetic acid.
- an esterifying group for example an arylmethyl group, for example a benzyl group
- an inorganic acid for example anhydrous hydrogen fluoride
- a catalyst for example palladium-on- charcoal
- an alkyl group, C. . alkyl for example a tert-butyl group which may be removed by treatment with an organic acid, for example trifluoroacetic acid.
- any one- of the standard peptide coupling reactions may be used, for example those described in the standard text books on peptide chemistry stated above.
- process (b) it is to be understood that a peptide unit may contain- just one protected or unprotected amino acid.
- a suitable coupling reaction is, for example, a solu ⁇ tion-phase coupling reaction, for example an active ester coupling, an azide coupling or a coupling involving N,N' -dicyclohexylcarbodiimide, 1-hydroxybenzotriazole and BOP(Benzotriazole-l-yl-oxy-tris-(dimethyl- amino)-phosphonium hexafluorophosphate.
- a suitable reactive derivative of the peptide unit containing a carboxylic acid group is, for example, an acyl halide, for example an acyl chloride formed by the reaction of the acid and an inorganic acid chloride, for example thionyl chloride; a mixed anhydride, for example an anhydride formed by the reaction of the acid and a haloformate, for example isobutyl chloroformate; or an acyl azide, for example an azide formed by the reaction of the acid and am azide such as diphenylphosphoryl azide.
- an acyl halide for example an acyl chloride formed by the reaction of the acid and an inorganic acid chloride, for example thionyl chloride
- a mixed anhydride for example an anhydride formed by the reaction of the acid and a haloformate, for example isobutyl chloroformate
- an acyl azide for example an azide formed by the reaction of the acid and am azide such as diphen
- a particularly suitable reactive derivative of the peptide unit containing a carboxylic acid group is, for example, the product of the reaction of the acid and a carbodiimide, for example Hifi'dicyclohexylcarbodiimide or J ⁇ .N' -diisopropylcarbodii ide, or it is the product of the reaction of the acid, an N-hydroxytriazole, for example 1-hydroxybenzotriazole, and a carbodiimide, for example N,£J' -dicyclohexylcarbodiimide or _ ⁇ ,__[' -diisopropylcarbodiimide.
- a preferred strategy is, for example, to use a solid-phase synthesis wherein the amino acid which is to become the C-terminus amino acid of a polypeptide of the invention is protected at the alpha amino group and, if necessary, in the side-chain and coupled to a solid support, for example a resin, for example a hydroxymethylated or a methylbenzhdrylamine styrene-divinylbenzine crosslinked resin via an ester or amide linkage respectively, whereafter the protecting group on the alpha-amino group is removed.
- a resin for example a hydroxymethylated or a methylbenzhdrylamine styrene-divinylbenzine crosslinked resin via an ester or amide linkage respectively, whereafter the protecting group on the alpha-amino group is removed.
- the amino acid which is to be attached to the C-terminus amino acid is protected at the alpha-amino group and, if necessary, in the side-chain and coupled to the C-terminus amino acid which remains attached to the solid support.
- the step-wise process of deprotection of the alpha-amino group and coupling to the next amino acid is repeated to give a protected or unprotected polypeptide attached to the solid support.
- the protected or unprotected polypeptide may be released from the hydroxymethylated resin solid support, for example, hydrolysis, for example acid hydrolysis with, for example, an organic acid, for example trifluoroacetic acid or with, for example, an inorganic acid, for example anhydrous hydrogen fluoride or hydrogen bromide; or the polypeptide is released by, for example, alcoholysis, for example methanolysis, in the presence of a base, for example an organic base, for example diisopropylethylamine whereafter, if necessary, the protecting groups are removed using process (a) above.
- hydrolysis for example acid hydrolysis with, for example, an organic acid, for example trifluoroacetic acid or with, for example, an inorganic acid, for example anhydrous hydrogen fluoride or hydrogen bromide
- the polypeptide is released by, for example, alcoholysis, for example methanolysis, in the presence of a base, for example an organic base, for example diisopropylethylamine whereafter
- the protected or unprotected polypeptide may be released from the solid support, for example by treatment with an inorganic acid, for example hydrogen fluoride, whereafter, if necessary the protecting groups are removed using process (a) above.
- an inorganic acid for example hydrogen fluoride
- a further preferred strategy is, for example, to use a solid-phase synthesis wherein an amino acid which is to become a link within the chain of amino acids forming a polypeptide of the invention is protected at the alpha-amino group and, if necessary, in the side-chain and coupled to a solid support, for example a resin as described above, whereafter the protecting group on the alpha-amino group is removed.
- the amino acid which is to be attached to the amino acid which has been coupled to the solid support is protected at the alpha-amino group and, if necessary, in the side-chain and coupled to the amino acid which remains coupled to the solid support.
- the step-wise process of deprotection of the alpha-amino group and coupling to the next amino acid is repeated to give a protected or unprotected polypeptide attached to the solid support.
- the protected or unprotected polypeptide may be released from the solid support, for example, using one of the methods described above whereafter a further peptide unit can be coupled using a solution- phase coupling reaction as described for process (b) above, and whereafter, if necessary, the protecting groups are removed using process (a) above.
- polypeptides of the present invention have a bombesin antagonist effect, which may be demonstrated by their ability to inhibit Bombesin
- composition which comprises a polypeptide of formula I, or a pharmaceutically acceptable salt thereof, in association with a pharmaceutically acceptable diluent or carrier.
- the composition may be in a form suitable for oral use, for example a tablet, capsule, aqueous or oily solution, suspension or emulsion; for nasal use, for example, a snuff, nasal spray or nasal drops; for vaginal or rectal use, for example a suppository; for administration by inhalation, for example as a finely divided powder or a liquid aerosol; for sub-lingual or buccal use, for example a tablet or capsule; or for parenteral use (including intravenous, subcutaneous, intramuscular, intravascular or infusion), for example a sterile aqueous or oily solution or suspension.
- parenteral use including intravenous, subcutaneous, intramuscular, intravascular or infusion
- compositions may be prepared in a conventional manner using conventional excipients.
- a composition for oral administration it may be convenient for the composition to include a coating to protect the polypeptide active ingredient from the actions of enzymes in the stomach.
- a composition of the invention may also contain, in addition to the polypeptide of the invention, one or more known antitumour substances selected from, for example, mitotic inhibitors, for example vinblastine; alkylating agents, for example cis-platin, carboplatin and cyclophosphamide; antimetabolites, for example, 5-fluorouracil, cytosine arabinoside and hydroxyuea; intercalating antibiotics, for example adriamycin and bleomycin; enzymes, for example asparaginase, topoisomerase inhibitors, for example etoposide and biological response modifiers, for example interferon.
- mitotic inhibitors for example vinblastine
- alkylating agents for example cis-platin, carboplatin and cyclophosphamide
- antimetabolites for example, 5-fluorouracil, cytosine arabinoside and hydroxyuea
- intercalating antibiotics for example adriamycin and bleomycin
- a preferred composition of the invention is, for example, one suitable for oral administration in unit dosage form, form example a tablet or capsule which contains from 2.5 to 500 mg, and preferably 10 to 100 mg, of polypeptide in each unit dose, or one suitable for parenteral administration which contains from 0.5 to 100 mg of polypeptide per ml, and preferably 1 to 10 mg of polypeptide per ml of solution.
- a parenteral composition is preferably a solution in isotonic saline or isotonic dextrose buffered if necessary to a pH of 5 to 9.
- the parenteral composition may be one designed for slow release in which case the amount of polypeptide per unit dose is in general greater than that required when a conventional injectable formulation is used.
- a preferred slow release formulation is, for example, a continuous release formulation, for example a formulation of the type described in US4767628 and US5004602.
- a preferred slow release parenteral formulation contains from 10 to 100 mg of polypeptide per unit dose.
- Another preferred slow release formulation is a micro-encapsulated polypeptide using a biodegradable biocompitible copolymer.
- preparations are preferably administered intravenously, although administration may also be effected by means of subcutaneous, intramuscular, or intradermal injection.
- Polypeptides are therapeutic agents requiring specialized pharmaceutical formulations for effective, safe and convenient use by patients. Since even small oligopeptides like thyrotrophin releasing hormone (TRH) have very low oral activity, and larger molecules are inactivated by endopeptidases in the gastrointestinal tract, long-term treatment requires daily injection or administration from small portable infusion pumps.
- TRH thyrotrophin releasing hormone
- TRH thyrotrophin releasing hormone
- long-term treatment requires daily injection or administration from small portable infusion pumps.
- self-administered methods e.g. nasal application, suppositories
- controlled-release formulations such as injectable microcapsule suspensions and implants.
- Medicaments suitable for transdermal administration may take the form of an optionally buffered aqueous solution of a compound of formula (I) and may be delivered by passive diffusion or by electrically-assisted transport, for example, iontophoresis (see, for example, Pharmaceutical Research 1(6) , 318 (1986)).
- composition of the invention will normally be administered such that a daily oral dose will be from 0.1 mg/kg, to 50 mg/kg, and most preferably from 0.1 mg kg to 25 mg/kg and a daily parenteral dose, will be from 20 micrograms/kg to 10 mg/kg more preferably from 100 micrograms to 10 mg/kg.
- a method for producing a bombesin antagonist effect in a warm-blooded animal, such as man, in need of such treatment which comprises administering to said animal an effective amount of a polypeptide of formula I or a pharmaceutically-acceptable salt thereof.
- the invention also provides the use of such a polypeptide of formula I or a pharmaceutically-acceptable salt thereof in the production of a new medicament for use in the treatment of a disease or medical condition mediated by bombesin or a bombesin-like peptide.
- the compounds of the present invention are useful for inhibiting the binding of gastrin releasing peptide in cells.
- the compounds of the present invention also inhibit the growth of cancer cells.
- a polypeptide of the invention is useful in the treatment of malignant disease, for example malignant disease in the lung, such as human small cell lung cancer, for example, malignant disease in the pituitary gland, adrenal gland, pancreas or within the skin.
- a polypeptide of this invention is useful for the symptomatic relief and/or treatment of exocrine pancreatic adenocarcinoma.
- a polypeptide of the invention is useful in the treatment of conditions associated with the over-production of bombesin or bombesin-like peptides (such as gastrin releasing factor) , for example the over-production of gastrin in the gut.
- the production of gastrin in animals has been linked to the suppression of the release of growth hormone and prolactin.
- polypeptides of the invention may therefore be used to promote the availability of growth hormone in man or animals in need of such treatment.
- the polypeptides of the invention may also be used in the treatment of conditions associated with the failure of normal physiological control of the regulation of gastric acid secretion.
- compositions containing polypeptides of the invention at least one pharmaceutical carrier or excipient and, optionally, one or more other therapeutic ingredients;
- a method for inhibiting the growth of cells that are sensitive to the growth promoting activity of gastrin releasing peptide in a mammal (such as a human) in need of such treatment which comprises the administration to said mammal of a growth inhibiting amount of a polypeptide of the invention to said mammal.
- N-Boc-L-im-CBZ-L-Histidine, N-Boc- ⁇ -Thienyl-L-Alanine, and 3-(2-Naph- thyl)-D-Alanine were acquired from Bachem, Inc., 3132 Kashiwa Street, Torrance, CA 90505, U.S.A.
- L-Methoxinine (O-Methyl-L-Homoserine) was obtained from Biohellas S.A., lO.Parnithos Street, 154 52 P. Phsychiko, Athens-Greece.
- Leucine methyl ester hydrochloride, (+)-6-Methoxy- ⁇ -methyl-2-Naphthal- eneacetic acid and (S)-6-Methoxy- ⁇ -methyl-2-Naphthaleneacetic acid were supplied from the Sigma Chemical Company, P.O. Box 14508, St. Louis, MO 63178, U.S.A.
- Di-tert-butyl Dicarbonate, 4-Fluorophenylalanine, 3-(4-Hydroxyphenyl)- propionic acid N-hydroxysuccinimide ester, and both (R) - and (S)-3-Phenylbutyric acid were obtained from Fluka Chemical Corp. , 980 South Street, Ronkonkoma, NY 11779, U.S.A.
- Boc-fi-prolinol (10 g, 50 mmoles, Advanced Chemtech) was dissolved in 60 ml dry CH-C1- and chilled to -60°C in a 500 mL round-bottomed flask, under N law.
- the N caution line was set up in series to accommodate both flasks.
- the oxalyl chloride/DMSO mixture was gradually added to the alcohol solution by NRON-pressurized transfer through a double-tipped needle. By manually controlling the réelle flow rate, the contents were transferred from one flask to the other, very slowly, but not dropwise. Upon complete addition, this mixture was stirred for 20 minutes.
- 20 ml of triethylamine (140 mmoles, Kodak) was added slowly, while at -60 C. The mixture was then allowed to warm to room temperature.
- the BOP coupling reagent Benzotriazol-1-yl-oxy-tris-(dimethylamino)- phosphonium hexafluorophosphate, was obtained from Richelieu Biotechnologies, Inc., 5726 Whyr Blvd., St-Hyacinthe, QC. , J2S 3V8 Canad .
- Hydrofluoric acid was acquired from Matheson Gas Products, P.O. Box 85, 932 Paterson Plank Road, East Rutherford, NJ 07073, U.S.A.
- the aqueous mixture was acidified with 1 N HCl until pH - 2, whereupon crystals formed, in most cases, at 60% yield.
- the solid was filtered and characterized by TLC, NMR, and MS as generally pure enough to continue without further purification.
- the product in which the product that dropped out of the acidic, aqueous solution was a liquid, the product was removed from the aqueous solution with ethyl acetate.
- the organics were then dried with anhydrous sodium sulphate and the ethyl acetate was evaporated.
- the liquid residue was dissolved in a small portion of ethyl acetate. anhydrous sodium sulfate, the solvent was removed by rotary evaporation. The residue was dried on a vacuum pump for 3 hours.
- the resin was next treated with two equivalents each of Boc-nor- leucine (Bachem, Torrance, CA) , benzotriazol-l-yloxy-tris(di- methylamino) phosphoniu hexafluorophosphate (BOP, Richelieu Biotechnologies of Canada) , and 4-methylmorpholine (Aldrich) ; all dissolved in 50 ml of N,N-dimethylformamide (DMF) . This mixture was shaken for 1 hour and then rinsed twice with CHreciC1 ownership. Coupling was verified by a Kaiser test (colourless) , compared to the previous Kaiser result (blue) .
- the resin was next shaken for 2 hours with 2 equivalents of Boc-D-prolinal in 50 ml of 2% glacial acetic acid in DMF. Over the entire 2 hours, three equivalents of sodium cyanoborohydride (Aldrich) were added slowly and gradually. After rinsing twice with CH pupilC1-, coupling was checked with ninhydrin (colourless). The resin was now ready for standard solid phase peptide synthesis.
- Boc-D-Pro ⁇ (CH worthNH)X-OH may be synthesized by the procedures of Martinez, et al. , in J. Med. Chem. , 28, 1874 (1985), or D. Tourwe, et al. , in Peptides 1988: Proceedings of the 20th European Peptide Symposium. , Ed. Jung, Bayer; Walter de Gruyter, p.562-4.
- This resin may be prepared by coupling Boc-D-Pro ⁇ (CH-NH)X-0H to MBHA resin by shaking with BOP and 4-Methylmorpholine (or 1-Methylimidazole) in N-Methyl-pyrrolidone for 2 hours.
- Freshly activated zinc powder (0.79g, 12mmol, Aldrich) and benzene (50mL) were placed into a 250-mL two neck round bottom flask under N conscious. This flask was attached to a Dean-Stark apparatus and 25mL of benzene was distilled into the trap. Under reflux, a solution of Boc-D-Proli- nal (1.90g, 9.5mmol) and ethylbromoacetate (2.0g, 12mmol, Aldrich) was added dropwise. A crystal of iodine was added to initiate the reaction after half of the dropping solution was added. After complete addition, the mixture was refluxed for 3 hours, cooled, and carefully washed with 0.5n HCl.
- the peptides were synthesised using an improved version of the solid phase method described by R.B. Merrifield, "Solid Phase Peptide Synthesis I. The Synthesis of a Tetrapeptide” , J. Am. Chem. Soc, 83, 2149(1963), using an Applied Biosystems Model 430A peptide synthesizer.
- Boc-protected amino acids were coupled to the resin using a modified program to suit the BOP coupling procedure, as described by Dung Le-Nguyen, Annie Heik, and Bertrand Castro, J. Chem. Soc, Perkins Trans. 1, 1915(1987).
- the coupling protocol involved dissolving ImMole of Boc-protected amino acid, lmMole BOP, and 1ml of 1M 1-Methylimidazole in 7 ml of DMF. The mixture was added to 0.5 mMoles of resin, mixed for 1 hour, and filtered. Afterwards, a series of DMF and CH-Cl- washes are performed.
- the peptide was assembled on the resin, it was deblocked and cleaved from the resin with liquid HF containing 10% anisole, in a variation of the method described by S. Sakakibara, et al., in Bull. Chem Soc. Jap., 40, 2164(1967).
- the peptide and resin were next washed with ethyl acetate and then the peptide was extracted from the resin with an aqueous 1% acetic acid solution. The peptide solution was then lyophilized to obtain the dry, solid peptide.
- the peptides were then purified by reverse-phase liquid chromatography using a Vydac 218TP1022 column on a Waters Delta Prep 3000 system equipped with a Gilson Model 116 ultraviolet detector. Purification was achieved by equilibrating the column with 0.1% TFA in water and developing with a linear gradient of acetonitrile from 1-40% in 20 minutes at a flow rate of 20 mL/min. Samples were collected manually and checked for purity on a Spectra-Physics analytical HPLC system (including SP8700, SP8440, SP8780, and SP4200) utilizing a Vydac 218TP54 column. A flow rate of 1.5 mL/min. was employed using a 0.1% TFA/acetonitrile gradient from 10-60% ACN in 10 minutes.
- Methylbenzhydrylamine (MBHA) resin (5.0g., 4.7mmoles, Advanced Chemtech) was washed twice (3 & 5 min.) with 10% diisopropylethylamine (DIEA, Aldrich) in dichloromethane (DCM) on a Milligen Peptide Shaker. The resin was then washed with DCM and N,N-dimethylformamide (DMF). A solution containing 2.5g of Boc-Phe (9.4mmol., Chemtech), 4.3g of BOP Reagent (9.4mmol., Richilieu Biotechnologies), and 0.95mL of 4-Methylmorpholine (8.6mmol., Aldrich) were shaken on the resin for lh. The resin was then washed successively with methanol and DCM and coupling was verified via a Kaiser Ninhydrin qualitative test.
- DIEA diisopropylethylamine
- DCM dichloromethane
- DMF N,N-d
- Boc-Phe-MBHA (3.06g., 2.8mmol) was deprotected by treating with 50% trifluoro-acetic acid (TFA, Chemtech) in DCM for 5 and 20 min. periods. The resin was washed twice with DCM. The acid was then neutralized by shaking for 3 and 5 min. with 10% DIEA solution and washed with DCM and DMF. The H ⁇ N-Phe-MBHA was shaken in a 1% acetic acid/N-methylpyrrolidone solution containing 1.2g of Boc- ⁇ Prolinal ( ⁇ .Ommol., synthesis described previously) over 2h.
- TFA trifluoro-acetic acid
- the peptide (approximately l.Og.) was cleaved from the MBHA resin by treating the peptide-resin with hydrogen fluoride (approximately lOmL) at 0 C for lh.
- the peptide was precipitated and filtered with the MBHA resin by ethyl acetate.
- the peptide was then extracted from the resin using 1% aqueous acetic acid and isolated upon freeze-drying this extract.
- a portion of the peptide (lOOmg) was purified on a Vydac C-18 preparative column (Chemtech) utilizing a 0.1% TFA/H_0:0.1% TFA/acetonitrile gradient. The collected fractions were verified by an analytical Vydao C-18 column (Chemtech) , and those samples reflecting pure peptide were combined and freeze-dried. Approximately 15mg of peptide was isolated. After characterization by FAB-Mass Spectrum (MH + - 1081.7) and Amino Acid Analysis [Ala(2.19), His(1.68), Val(1.12)], 12.2mg remained (12% yield based on HPLC) .
- Purity was monitored by analytical HPLC using a Spectra-Physics analytical HPLC system, including SP8700, SP8440, SP8780, and SP4200.
- a Vydac 218TP54 column was utilized with a flow rate of 1.5 mL/min. of 1 0.1% TFA/acetonitrile gradient.
- FAB Fast atom bombardment
- the peptides were evaluated in their activity to inhibit GRP binding to Swiss 3T3 cells. Antagonistic activity was measured by inhibition of the mitogenic stimulation of quiescent 3T3 ROZ cells. 3T3 ROZ cells were obtained from Enrique Rozengurt, Imperial Cancer Research
- N-((3-Phenyl)Propionyl)-HisTrpAla 1.76x10 1139.6 N/A ValD-AlaHis(3-(2-Pyrrolidiny1- 3Hydroxy)Propionyl)-Phe-NH 2 ((Isoquinolyly-Carbonyl) -HisTrp 3.44x10-9 1104.5 Ala(1.82), AlaValD-AlaHisD-Pro ⁇ Phe-NH remind His(1.32), Val(l.OO)
Abstract
Description
Claims
Priority Applications (6)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CS9380A CZ8093A3 (en) | 1990-07-31 | 1991-07-30 | Bombesin antagonists |
PL91297652A PL167322B1 (en) | 1990-07-31 | 1991-07-30 | Method of obtaining polypeptidic compounds |
AU83111/91A AU653544B2 (en) | 1990-07-31 | 1991-07-30 | Bombesin antagonists |
SK3893A SK3893A3 (en) | 1990-07-31 | 1991-09-30 | Bombesine antagonists |
NO930262A NO930262D0 (en) | 1990-07-31 | 1993-01-26 | Bombesin ANTAGONISTS |
FI930411A FI930411A0 (en) | 1990-07-31 | 1993-01-29 | ANTAGONISTER OF BOMBES |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
GB9016810.5 | 1990-07-31 | ||
GB909016810A GB9016810D0 (en) | 1990-07-31 | 1990-07-31 | Peptides |
Publications (1)
Publication Number | Publication Date |
---|---|
WO1992002545A1 true WO1992002545A1 (en) | 1992-02-20 |
Family
ID=10679953
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/GB1991/001289 WO1992002545A1 (en) | 1990-07-31 | 1991-07-30 | Bombesin antagonists |
Country Status (20)
Country | Link |
---|---|
EP (1) | EP0541654A1 (en) |
JP (1) | JPH05509100A (en) |
AU (1) | AU653544B2 (en) |
CA (1) | CA2088166A1 (en) |
CZ (1) | CZ8093A3 (en) |
FI (1) | FI930411A0 (en) |
GB (1) | GB9016810D0 (en) |
HU (1) | HUT63178A (en) |
IE (1) | IE912671A1 (en) |
IL (1) | IL99009A0 (en) |
MC (1) | MC2312A1 (en) |
MY (1) | MY107031A (en) |
NO (1) | NO930262D0 (en) |
NZ (1) | NZ239183A (en) |
PL (1) | PL167322B1 (en) |
PT (1) | PT98498A (en) |
SK (1) | SK3893A3 (en) |
TW (1) | TW234130B (en) |
WO (1) | WO1992002545A1 (en) |
ZA (1) | ZA915978B (en) |
Cited By (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0606464A1 (en) * | 1992-07-27 | 1994-07-20 | Medical Research Foundation Of Oregon | Peptides capable of controlling acrosome reaction |
US5620955A (en) * | 1993-06-18 | 1997-04-15 | Peptide Technologies Corporation | Bombesin receptor antagonists and uses thereof |
US5723578A (en) * | 1987-09-24 | 1998-03-03 | The Administrators Of Tulane Educational Fund | Peptide analogs of bombesin |
US6307017B1 (en) | 1987-09-24 | 2001-10-23 | Biomeasure, Incorporated | Octapeptide bombesin analogs |
EP1883627A1 (en) * | 2005-05-18 | 2008-02-06 | Aegera Therapeutics Inc. | Bir domain binding compounds |
US8063095B2 (en) | 2005-10-25 | 2011-11-22 | Pharmascience Inc. | IAP BIR domain binding compounds |
US9284350B2 (en) | 2010-02-12 | 2016-03-15 | Pharmascience Inc. | IAP BIR domain binding compounds |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
AU618029B2 (en) * | 1987-11-02 | 1991-12-12 | Imperial Chemical Industries Plc | Polypeptide compounds |
-
1990
- 1990-07-31 GB GB909016810A patent/GB9016810D0/en active Pending
-
1991
- 1991-07-30 WO PCT/GB1991/001289 patent/WO1992002545A1/en not_active Application Discontinuation
- 1991-07-30 IL IL99009A patent/IL99009A0/en unknown
- 1991-07-30 CA CA002088166A patent/CA2088166A1/en not_active Abandoned
- 1991-07-30 PT PT98498A patent/PT98498A/en not_active Application Discontinuation
- 1991-07-30 PL PL91297652A patent/PL167322B1/en unknown
- 1991-07-30 IE IE267191A patent/IE912671A1/en unknown
- 1991-07-30 NZ NZ239183A patent/NZ239183A/en unknown
- 1991-07-30 EP EP91914148A patent/EP0541654A1/en not_active Withdrawn
- 1991-07-30 MC MC912312D patent/MC2312A1/en unknown
- 1991-07-30 TW TW080105951A patent/TW234130B/zh active
- 1991-07-30 MY MYPI91001370A patent/MY107031A/en unknown
- 1991-07-30 AU AU83111/91A patent/AU653544B2/en not_active Ceased
- 1991-07-30 ZA ZA915978A patent/ZA915978B/en unknown
- 1991-07-30 HU HU93239A patent/HUT63178A/en unknown
- 1991-07-30 CZ CS9380A patent/CZ8093A3/en unknown
- 1991-07-30 JP JP3513218A patent/JPH05509100A/en active Pending
- 1991-09-30 SK SK3893A patent/SK3893A3/en unknown
-
1993
- 1993-01-26 NO NO930262A patent/NO930262D0/en unknown
- 1993-01-29 FI FI930411A patent/FI930411A0/en not_active Application Discontinuation
Non-Patent Citations (2)
Title |
---|
Comprehensive Medicinal Chemistry, vol. 3, Membranes & Receptors, Edited by John C. Emmett, Pergamon Press, 1990, see page 929 * |
Life Sciences, vol. 44, 1989, Pergamon Press plc, New York, US; S. Mahmoud et al.: "Small cell lung cancer bombesin receptors are antagonized by reduced peptide bond analogues", pages 367-373, see pages 371-372, discussion * |
Cited By (12)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5723578A (en) * | 1987-09-24 | 1998-03-03 | The Administrators Of Tulane Educational Fund | Peptide analogs of bombesin |
US6307017B1 (en) | 1987-09-24 | 2001-10-23 | Biomeasure, Incorporated | Octapeptide bombesin analogs |
EP0606464A1 (en) * | 1992-07-27 | 1994-07-20 | Medical Research Foundation Of Oregon | Peptides capable of controlling acrosome reaction |
EP0606464A4 (en) * | 1992-07-27 | 1995-10-25 | Oregon Medical Res Found | Peptides capable of controlling acrosome reaction. |
US5620955A (en) * | 1993-06-18 | 1997-04-15 | Peptide Technologies Corporation | Bombesin receptor antagonists and uses thereof |
EP1883627A1 (en) * | 2005-05-18 | 2008-02-06 | Aegera Therapeutics Inc. | Bir domain binding compounds |
JP2008545629A (en) * | 2005-05-18 | 2008-12-18 | アエゲラ セラピューティクス インコーポレイテッド | BIR domain binding compound |
EP1883627A4 (en) * | 2005-05-18 | 2010-02-03 | Aegera Therapeutics Inc | Bir domain binding compounds |
US7772177B2 (en) | 2005-05-18 | 2010-08-10 | Aegera Therapeutics, Inc. | BIR domain binding compounds |
US8575113B2 (en) | 2005-05-18 | 2013-11-05 | Pharmascience Inc. | BIR domain binding compounds |
US8063095B2 (en) | 2005-10-25 | 2011-11-22 | Pharmascience Inc. | IAP BIR domain binding compounds |
US9284350B2 (en) | 2010-02-12 | 2016-03-15 | Pharmascience Inc. | IAP BIR domain binding compounds |
Also Published As
Publication number | Publication date |
---|---|
EP0541654A1 (en) | 1993-05-19 |
FI930411A (en) | 1993-01-29 |
CA2088166A1 (en) | 1992-02-01 |
ZA915978B (en) | 1993-04-28 |
FI930411A0 (en) | 1993-01-29 |
NO930262L (en) | 1993-01-26 |
AU653544B2 (en) | 1994-10-06 |
MY107031A (en) | 1995-08-30 |
PL297652A1 (en) | 1992-07-13 |
TW234130B (en) | 1994-11-11 |
MC2312A1 (en) | 1993-09-27 |
IL99009A0 (en) | 1992-07-15 |
IE912671A1 (en) | 1992-02-12 |
HUT63178A (en) | 1993-07-28 |
PT98498A (en) | 1992-05-29 |
SK3893A3 (en) | 1993-07-07 |
AU8311191A (en) | 1992-03-02 |
NO930262D0 (en) | 1993-01-26 |
PL167322B1 (en) | 1995-08-31 |
NZ239183A (en) | 1993-07-27 |
GB9016810D0 (en) | 1990-09-12 |
HU9300239D0 (en) | 1993-04-28 |
CZ8093A3 (en) | 1994-01-19 |
JPH05509100A (en) | 1993-12-16 |
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