WO1992012170A1 - Inhibitions de formulation d'endotheline - Google Patents

Inhibitions de formulation d'endotheline Download PDF

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Publication number
WO1992012170A1
WO1992012170A1 PCT/EP1991/002513 EP9102513W WO9212170A1 WO 1992012170 A1 WO1992012170 A1 WO 1992012170A1 EP 9102513 W EP9102513 W EP 9102513W WO 9212170 A1 WO9212170 A1 WO 9212170A1
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WO
WIPO (PCT)
Prior art keywords
val
asn
asp
residue
gly
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/EP1991/002513
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English (en)
Inventor
Valeria Caiolfa
Moreno Zamai
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Pfizer Italia SRL
Original Assignee
Farmitalia Carlo Erba SRL
Carlo Erba SpA
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Farmitalia Carlo Erba SRL, Carlo Erba SpA filed Critical Farmitalia Carlo Erba SRL
Priority to JP92505488A priority Critical patent/JPH05506250A/ja
Publication of WO1992012170A1 publication Critical patent/WO1992012170A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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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
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P5/00—Drugs for disorders of the endocrine system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/12—Antihypertensives
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/57536—Endothelin, vasoactive intestinal contractor [VIC]
    • 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
    • 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 peptides capable of binding to human big-endothelin (bigET), to their preparation and to pharmaceutical composition containing them.
  • bigET big-endothelin
  • Endothelin is a mammalian hormone of 21 amino acids which possesses high vasoconstrictor activity.
  • the present invention relates to peptides having endothelin formation inhibitor activity, useful in the therapy of diseases which are dependent from endothelin effects.
  • the invention provides a peptide of up to 20 amino acids which is capable of binding to human big endothelin and which contains a sequence of the formula I
  • a 4 and A' 4 independently represent Val, Pro, Gly or Ala residue
  • a 5 represents Asp or Asn residue.
  • Salts of peptides according to the invention with pharmaceutically acceptable acids or bases are also within the scope of the present invention.
  • Such acid addition salts can be derived from a. variety of. inorganic and organic acids such as sulphuric, phosphoric, hydrochloric, hydrobromic, hydroiodic, nitric, sulphamic, citric, lactic, pyruvic, oxalic, maleic, succinic, tartaric, cinnamic, acetic, trifluoroacetic, benzoic, salicyclic, gluconic, ascorbic and related acids.
  • Such base addition salts can be derived from a variety of inorganic and organic bases as sodium hydroxide, potassium hydroxide, diethylamine, triethylamine and dicyclohexylamine.
  • amino acid residues are denoted by the three letter code (Eur.J.Biochem. 138. 9-37, 1984).
  • the sequence of the formula I was derived from the non-coding DNA strain of the 212 amino acid endothelin precursor and from the hydropatic map of big-endothelin according to the indeces of Kyte and Doolittle (J. Mol. Biol. 157, 105-132, 1982).
  • the peptides tipically comprise at least 5 aminoacid residues.
  • the peptides contain no more that 20 aminoacid residues, for example up to 18 aminoacid residues.
  • the peptides have the formula II: X-A 6 -A 7 -A 8 -A 9 -A 10 -A 1 -A 2 -A 3 -A 4 -A 4 -A 4 -A' 3 -A' 2 -A' 1 -A' 10 -A' 9 -A' 8
  • a 3 ,A 4 ,A 5 ,A' 4 ,A' 3 are as defined above, X represents hydrogen atom or a nitrogen protecting group,
  • a 1 and A' 1 independently represent Val, Leu, Cys or Ile residue, or a valence bond
  • a 2 and A' 2 independently represent Gly, Ala or Asp residue, or a valence bond
  • a 6 and A' 6 independently represent Val or a valence bond
  • a 7 and A' 7 independently represent Arg, Ala, Gly, Cys, Glu or
  • a 8 and A' 8 independently represent Asn, Pro, Gly, Ala residue or a valence bond
  • a 9 and A' 9 independently represent Asn, Asp, Val, Leu, Met residue or a valence bond
  • a 10 and A' 10 independently represent
  • the peptides have the formula III or IV: X-A 7 -Asn-A 9 -A 10 -A 1 -A 2 -A 3 -Val-A 5 -A ' 4 -A ' 3 -Asp-A ' 1 -A ' 1 0 -A ' 9 - ⁇ ' 8 -A ' 7 -Val-W III
  • a 1 represents Val, Leu, or Cys residue
  • A' 1 represents Val or He residue
  • a 2 represents Gly or Ala residue
  • a 3 represents Ser or Gly residue
  • A' 3 represents Asp or Asn residue.
  • A' 4 represents Pro, Gly or Ala residue
  • a 5 represents Asp or Asn residue
  • a 7 represents Arg, Ala, Gly or Cys residue
  • A' 7 represents Glu or Lys residue
  • A' 8 represents Pro, Gly or Ala residue
  • a 9 represents Asn or Asp residue
  • A' 9 represents Val, Leu or Met residue
  • a 10 represents Val, Leu or Met residue
  • A' 10 represents His or Glu residue
  • X represents hydrogen atom or a nitrogen protecting group and W represents hydroxy or amino group, or an enantiomer thereof having all the optically active amino acid residues in the D-form.
  • Preferred peptides have the sequences:
  • the synthesis of the peptides of the invention may be accomplished either by classical solution methods or by solid phase on polymeric supports.
  • the classical solution method the synthesis consists essentially in appropriate successive condensations of protected amino acids or peptides. The condensation is carried out so that the resulting peptides have the desired sequence of 5 to 20 amino acid residues.
  • the amino acids and peptides which are condensed according to methods known in themselves in polypeptide chemistry, have such of their amino and carboxy groups that are not involved in the formation of the peptide linkage blocked by a suitable protecting group.
  • the hydroxy functions of hydroxy amino acids may be protected by suitable protecting groups (throughout all the synthesis or only during a few steps) or may be kept unprotected.
  • the protecting groups are capable of being removed by acidolysis, saponification or hydrogenolysis.
  • the following protective groups may for example be employed: benzyloxycarbonyl, t-butoxycarbonyl, trityl, formyl, trifluoroacetyl, o-nitrophenylsulphenyl, 4- methoxybenzyloxycarbonyl, 9-fluorenylmethoxycarbonyl or 3,5dimethoxy- ⁇ , ⁇ '- dimethylbenzyloxycarbonyl.
  • the following protective groups may for example be employed: methyl, ethyl, t-butyl, benzyl or p-nitro-benzyl.
  • the following protecting groups may for example be used: acetyl, t-butoxycarbonyl, benzyloxycarbonyl, 2-bromobenzyloxycarbonyl, tetrahydropyranyl, t-butyl, trityl, benzyl or 2,4-dichlorobenzyl.
  • the condensation between an amino group of one molecule and a carboxyl group of another molecule to form the peptide linkage may be carried out through an activated acyl-derivative such as a mixed anhydride, an azide or an activated ester, or by direct condensation between free amino group and a free carboxyl group, in the presence of a condensing agent such as dicyclohexylcarbodiimide, alone or together with a racemization preventing agent, such as N-hydroxysuccinimide or 1-hydroxybenzotriazole.
  • an activated acyl-derivative such as a mixed anhydride, an azide or an activated ester
  • a condensing agent such as dicyclohexylcarbodiimide
  • a racemization preventing agent such as N-hydroxysuccinimide or 1-hydroxybenzotriazole.
  • Hydrazido or substituted hydrazido derivatives according to the invention are prepared by condensation of the N-protected peptide or amino acid with a suitably substituted hydrazido such as benzylcarbazate, t-butylcarbazate, adamantylcarbazate, phenylhydrazine or adamanthylhydrazine, or reacting the N-protected peptide or amino acid hydrazide with a suitable alkylating agent such as benzylchloroformate, t-butylchloroformate, di-t-butyldicarbonate or adamantylfluoroformate.
  • a suitably substituted hydrazido such as benzylcarbazate, t-butylcarbazate, adamantylcarbazate, phenylhydrazine or adamanthylhydrazine
  • the condensation may be carried out in a solvent such as dimethylformamide, pyridine, acetonitrile, tetrahydrofuran or N-methyl-2-pyrrolidone.
  • the reaction temperature may be from -30°C to room temperature.
  • the reaction time is generally from 1 to 120 hours.
  • the scheme of synthesis, the protecting groups and the condensing agents are selected so as to avoid the risk of racemization.
  • D-protecting reactions are carried out according to methods known per se in polypeptide chemistry.
  • a polymeric support is used.
  • the polymer is preferably a copolymer of styrene with from 1 to 2 percent by weight of divinyl benzene as a cross-linking agent which causes the polystyrene polymer to be completely insoluble in most organic solvents.
  • the synthesis is commenced from the C-terminal end of the peptide, by attaching the reguired amino acid to a chloromethylated resin, a hydroxymethyl resin, or a benzhydrylamine resin.
  • the amino and side chain protecting groups are those described in the classical solution synthesis.
  • an amino protected amino acid is coupled to the chloromethylated resin via caesium salt, or to hydroxymethyl or benzhydrylamine resin, with the aid of a condensing agent such as dicyclohexylcarbodiimide.
  • the amino protecting group is removed by a choice of reagents including trifluoroacetic acid or hydrogen chloride solutions in organic solvents at room temperature.
  • each protected amino acid is generally reacted in a 3-fold excess using an appropriate carboxy group activator such as dicyclohexylcarbodiimide in solution, in, for example, methylene dichloride; dimethylformamide mixtures.
  • an appropriate carboxy group activator such as dicyclohexylcarbodiimide in solution, in, for example, methylene dichloride; dimethylformamide mixtures.
  • the peptide is removed from the resin support by treatment with a reagent such as hydrogen fluoride, which not only cleaves the peptides from the resin, but also cleaves most of the remaining side-chain protecting groups.
  • a reagent such as hydrogen fluoride
  • the peptide amides can be obtained from the peptide acids by amminolysis.
  • the compounds according to the invention have an interesting pharmacological activity on inhibition of maturation process of endothelin.
  • ⁇ -chymotrypsin has been shown to cleave the human 38 residue ET precursor (bigET (1.38) in vitro, at neutral pH (E.G. McMahon Et al. BBRC 161:406-414 1989).
  • bigET is totally converted into bigET (1-31) which in turn is cleaved at the Trp(21) with formation of ET(1-21). This second fragmentation is greatly retarded by the presence of peptides of the present invention.
  • ET (1-21), bigET (1-38) and bigET (1-31) are shown in the following formula:
  • Alpha-chymotrypsin (0.014 mg/ml) was incubated with synthetic bigET (0.2 mg/ml) in glycine buffer (25 mM). The enzyme/bigET molar ratio was 1/500.
  • Table 1 Inhibition of bigET cleavage in the presence of peptides as formula I as a function of time (a). time P/S (b) P/S (b) P/S (b)
  • P/S product (ET) /substrate (bigET 1-31) values were calculated as ratio of the area of the correspondent HPLC peaks. Standard errors on P/S values were estimated less than 9%.
  • the peptides of the formula I are inhibitors of endothelin formation and can, therefore, find application in the therapy of human diseases which are caused by endothelin, either directly or in concert with other factors.
  • these peptides can be used in the management of the clinical symptoms associated with these deseases such as hypertension.
  • the compounds of the invention can be administered by the usual routes, for example, parenterally, e.g. by intravenous injection or infusion, or by intramuscular, subcutaenous, intracavity and intranasal administration.
  • the dosage depends on the age, weight and condition of the patient and on the administration route.
  • the therapeutic doses in humans will be in the range 10 ng/kg - 10 mg/kg, once to 6 times daily.
  • the invention also provides pharmaceutical compositions containing a compound of formula (I) as the active substance, in association with one or more pharmaceutically acceptable excipients.
  • compositions of the invention are usually prepared following conventional methods and are administered in a pharmaceutically suitable form.
  • solutions for intravenous injection or infusion may contain as carrier, for example, sterile water or, preferably, they may be in the form of sterile aqueous isotonic saline solutions.
  • Suspensions or solutions for intramuscular injections may contain, together with the active compound, a pharmaceutically acceptable carrier, e.g. sterile water, olive oil, ethyl oleate, glycols, e.g. propylene glycol, and, if desired, a suitable amount of lidocaine hydrochloride.
  • a pharmaceutically acceptable carrier e.g. sterile water, olive oil, ethyl oleate, glycols, e.g. propylene glycol, and, if desired, a suitable amount of lidocaine hydrochloride.
  • HPLC High performance liquid chromatography
  • a Hewlett-Packard 1084B apparatus equipped with a UV detector operating at 215 nm.
  • the peptides are separated on a 4 x 250 mm (I.D.) nucleosil 300 C 18 5 ⁇ column.
  • the following solvents are used:
  • the elution is programmed with a linear gradient from 15 to 50
  • the peptides are characterized by their retention time (RT).
  • the peptide was synthesised u ⁇ ing an adaption of the Merrifield method (Merrifield, JACS 85, 2149-2154, 1963).
  • the peptide was synthesised on a phenylacetanidomethyl resin.
  • the ⁇ -amino protecting group on each amino acid was N-tert-butyloxycarbonyl
  • the peptide was cleaved off the resin using hydrogen fluoride for 1 hour with an anisole scavenger 10%. The peptide was then ether washed, dried, dissolved in 15% acetic acid and lyophilized.

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  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Genetics & Genomics (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Molecular Biology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Veterinary Medicine (AREA)
  • Endocrinology (AREA)
  • Public Health (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • General Chemical & Material Sciences (AREA)
  • Toxicology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Heart & Thoracic Surgery (AREA)
  • Cardiology (AREA)
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  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Peptides Or Proteins (AREA)

Abstract

Peptides comprenant jusqu'à 20 acides aminés, capables de se lier à l'endothéline majeure humaine, et contenant une séquence de la formule (I): où A3 et A'3 représentent indépendamment un reste de Ser, Gly, Asp ou Asn, et A4 et A'4 représentent indépendamment un reste de Val, Pro, Gly ou Ala, et A5 représente un reste d'Asp ou Asn. On décrit également des énantiomères dans lesquels chaque acide aminé est de forme D, et des sels acceptables en pharmacologie. Lesdits peptides sont des inhibiteurs de la formation d'endothéline et trouvent pour cette raison leur application dans la thérapie de maladies humaines provoquées par l'endothéline.
PCT/EP1991/002513 1990-12-28 1991-12-27 Inhibitions de formulation d'endotheline Ceased WO1992012170A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
JP92505488A JPH05506250A (ja) 1990-12-28 1991-12-27 エンドテリン形成の阻害

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB9028140.3 1990-12-28
GB909028140A GB9028140D0 (en) 1990-12-28 1990-12-28 Peptides

Publications (1)

Publication Number Publication Date
WO1992012170A1 true WO1992012170A1 (fr) 1992-07-23

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Application Number Title Priority Date Filing Date
PCT/EP1991/002513 Ceased WO1992012170A1 (fr) 1990-12-28 1991-12-27 Inhibitions de formulation d'endotheline

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EP (1) EP0517913A1 (fr)
JP (1) JPH05506250A (fr)
GB (1) GB9028140D0 (fr)
WO (1) WO1992012170A1 (fr)

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0315118A2 (fr) * 1987-11-02 1989-05-10 Takeda Chemical Industries, Ltd. ADN codant pour endothéline et son utilisant
EP0411503A1 (fr) * 1989-07-31 1991-02-06 TECNOGEN Società Consortile per azioni Procédé pour l'identification et la synthèse des sites de liaison de protéines interagissants

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0315118A2 (fr) * 1987-11-02 1989-05-10 Takeda Chemical Industries, Ltd. ADN codant pour endothéline et son utilisant
EP0411503A1 (fr) * 1989-07-31 1991-02-06 TECNOGEN Società Consortile per azioni Procédé pour l'identification et la synthèse des sites de liaison de protéines interagissants

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS. vol. 167, no. 2, 16 March 1990, DULUTH, MINNESOTA US pages 860 - 866; R.IKEGAWA C.S.: 'EVIDENCE FOR PEPSTATIN-SENSITIVE CONVERSION OF PORCINEBIG-ET-1 TO ET-1 BY THE ENDOTHELIAL CELL EXTRACT' cited in the application *
J.MOL.BIOL. vol. 157, no. 1, 5 May 1982, LONDON pages 105 - 132; J.KYTE C.S.: 'A SIMPLE METHOD FOR DISPLAYING THE HYDROPATHIC CHARACTER OF A PROTEIN' cited in the application *
JOURNAL OF BIOLOGICAL CHEMISTRY. vol. 264, no. 19, 5 July 1989, BALTIMORE US pages 11252 - 11257; G.FASSINA C.S.: 'RECOGNITION PROPERTIES OF PEPTIDES HYDROPATHICALLY COMPLEMENTARY TO RESIDUES 356-375 OF THE C-RAF PROTEIN' THE WHOLE DOCUMENT;esp. pag. 11254, col.2,par 2 pag. 11255,col 1, par1 *

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Publication number Publication date
EP0517913A1 (fr) 1992-12-16
GB9028140D0 (en) 1991-02-13
JPH05506250A (ja) 1993-09-16

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