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WO1992011285A1 - Nouveaux peptides constituant des facteurs de necrose tumorale - Google Patents

Nouveaux peptides constituant des facteurs de necrose tumorale Download PDF

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Publication number
WO1992011285A1
WO1992011285A1 PCT/EP1991/002405 EP9102405W WO9211285A1 WO 1992011285 A1 WO1992011285 A1 WO 1992011285A1 EP 9102405 W EP9102405 W EP 9102405W WO 9211285 A1 WO9211285 A1 WO 9211285A1
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WO
WIPO (PCT)
Prior art keywords
fmoc
protecting group
group
amino
chq
Prior art date
Application number
PCT/EP1991/002405
Other languages
German (de)
English (en)
Inventor
Hans-Joachim Boehm
Lothar Daum
Andreas Haupt
Dagmar Pettig
Nigel Walker
Daniela Maennel
Reiner Frank
Rainer Stiemer
Original Assignee
Basf Aktiengesellschaft
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 Basf Aktiengesellschaft filed Critical Basf Aktiengesellschaft
Publication of WO1992011285A1 publication Critical patent/WO1992011285A1/fr

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Classifications

    • 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
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/525—Tumour necrosis factor [TNF]
    • 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 invention relates to new peptides derived from tumor necrosis factor (TNF), their production and their use as medicaments.
  • TNF tumor necrosis factor
  • TNF is one of the main participants in inflammatory reactions (Pharmac. Res. 5, 129, 1988).
  • the invention relates to compounds of the formula I.
  • A, B, D, E and 3 hydrogen atoms or one of the groups
  • F means Asp, Asn or His
  • T 1 -T 6 are hydrogen atoms or methyl groups
  • Z 1 -Z 6 each represent an oxygen atom or two hydrogen atoms
  • GR-NH-CHM-CO- or GR-NH-CHM-CO-W- means and Y -P, -NH-CHQ-CO-P, -V-NH-CHQ-CO-P,
  • Carboxyl protecting group stands or
  • G and P together also mean a covalent bond or the group -CO- (CH 2 ) a -NH-, where a is a number from 1 to 12, K, R, U, V and W peptide chains from 1-5 naturally occurring represent ⁇ -amino acids,
  • L, M and Q are hydrogen atoms or one of the groups -CH 3 , -CH 2 -CH (CH 3 ) 2 , -CH 2 -CO-NH 2 ,
  • A, B, D, M, L and Q in pairs one or two
  • the peptides of formula I are made up of L-amino acids, but they can contain 1 to 2 D-amino acids or N-methyl-amino acids.
  • the side chains of the trifunctional amino acids can carry protective groups or be unprotected.
  • physiologically compatible acids hydrochloric acid, citric acid, tartaric acid, lactic acid,
  • the new compounds can be prepared by methods known in peptide chemistry.
  • the peptide chain is gradually extended by one amino acid each, starting at the C terminus.
  • Fragment coupling can link fragments of different lengths to one another, the fragments in turn by sequential construction from amino acids or in turn by
  • Fragment coupling can be obtained.
  • the cyclic peptides are obtained after synthesis of the open-chain peptides by a cyclization reaction carried out in high dilution. Both in the sequential structure and in the fragment coupling, the building blocks must be linked by forming an amide bond. Enzymatic and chemical methods are suitable for this.
  • activators in particular dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC), 1-ethoxycarbonyl-2-ethoxy-1, are particularly preferred.
  • EEDQ 2-dihydroquinoline
  • EDCI 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride
  • PPA n-propanephosphonic anhydride
  • BOP-C1 N, N-bis (2-oxo-3-oxazolidinyl) amidophosphoric acid chloride
  • DPPA Diphenylphosphoryl azide
  • BOP O-benzotriazolyl-N, N, N ', N'-tetramethyluronium salts
  • TBTU 2,5-diphenyl-2,3-dihydro-3-oxo 4-hydroxythiophene dioxide
  • Bromo-tris-pyrrolidino-phosphonium hexafluorophosphate (PyBroP) and 1,1'-carbonyl-diimidazole (CDI).
  • the coupling reagents can be used alone or in combination with additives such as N, N'-dimethyl-4-aminopyridine (DMAP), N-hydroxybenzotriazole (HOBt), N-hydroxybenzotriazine (HOOBt), N-hydroxysuccinimide (HOSu) or 2-hydroxypyridine .
  • While protective groups can normally be dispensed with in enzymatic peptide synthesis, reversible protection of the reactive functional groups of the two reactants which are not involved in the formation of the amide bond is required for chemical synthesis.
  • three protecting group techniques known from the literature are preferred: the benzyloxycarbonyl (Z), the t-butyloxycarbonyl (Boc) and the 9-fluorenylmethyloxycarbonyl (Fmoc) protective group technique.
  • the protective group of the ⁇ -amino function of the chain-extending building block is designated in each case.
  • the side chain protecting groups of the trifunctional amino acids are chosen so that they are not necessarily split off together with the ⁇ -amino protecting group. Müller, Methods of Organic Chemistry Vol XV / 1, pp 20 - 906, Thieme Verlag, Stuttgart, 1974 provides a detailed overview of amino acid protecting groups.
  • the building blocks which serve to build up the peptide chain can be in solution, in suspension or by a similar method as described by Merrifield in 3 Amer. Chem. Soc. 85, 2149, 1963. Particular preference is given to processes in which peptides are built up sequentially or by fragment coupling using the Z, Boc or Fmoc protective group technique, the reactants being reacted in solution, and processes in which, similar to the Merrifield technique mentioned, a reaction partner is bound to an insoluble polymeric carrier (hereinafter also called resin).
  • the peptide is typically built up sequentially on the polymeric support using the Boc or Fmoc protective group technique, the growing peptide chain at the C-terminus being covalently linked to the insoluble resin particles (see Figs. 1 and 2). This procedure allows reagents and by-products to be removed by filtration, making recrystallization of intermediate products unnecessary.
  • the protected amino acids can be attached to any suitable
  • Polymers are bound, which are only insoluble in the solvents used and must have a stable physical form that enables easy filtration.
  • the polymer must contain a functional group to which the first protected amino acid can be bound by a covalent bond.
  • Various polymers are suitable for this purpose, e.g.
  • cellulose polyvinyl alcohol, polymethacrylate, sulfonated polystyrene, chloromethylated copolymer of styrene and divinylbenzene
  • Merrifield resin 4-methylbenzhydrylamine resin (MBHA resin), phenylacetamidomethyl resin (Pam resin), p -Benzyloxybenzyl alcohol resin, benzhydrylamine resin (BHA resin), 4- (hydroxymethyl) benzoyloxymethyl resin, resin according to Breipohl et al. (Tetrahedron Lett. 28, 565, 1987; Fa.
  • BACHEM BACHEM
  • HYCRAM resin ORPEGEN
  • SASRIN resin BACHEM
  • RINK resin RINK resin
  • BlOHELLAS o-chloro-trityl resin
  • All solvents which prove inert under the reaction conditions are suitable for peptide synthesis in solution, in particular water, N, N'-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), acetonitrile, dichloromethane (DCM), 1,4-dioxane, Tetrahydrofuran (THF), N-methyl-2-pyrrolidone (NMP) and mixtures of the solvents mentioned.
  • the peptide synthesis on the polymeric support can be carried out in all inert organic solvents in which the amino acid derivatives used are soluble; however, preference is given to solvents which additionally have resin-swelling properties, such as DMF, DCM, NMP,
  • the peptide is split off from the polymeric carrier.
  • the conditions under which the various types of resin can be split off are known from the literature. Acid and palladium-catalyzed cleavage reactions are most common
  • TNF activity Some of the new peptides show TNF activity. These are TNF agonists. Another part of the peptides has a high affinity for the cellular TNF receptor, but without having an activity comparable to TNF. So you pose
  • TNF antagonists In competition with natural TNF, they bind to the cellular TNF receptor and thus suppress the
  • the new peptides prove to be valuable Medicines used to treat neoplastic and autoimmune diseases, as well as to fight and prevent infections, inflammation and rejection
  • Transplants can be used.
  • TNF-sensitive indicator cells compete.
  • the agonistic evaluation of the new peptides is based on their TNFmRNA inducing effect in human monocytes.
  • the test J. Immunol. 144, 1144 (1990) was carried out as follows:
  • the antagonistic evaluation of the new peptides is based on their ability to inhibit rHuTNF-induced induction of TNFmRNA in this test system.
  • the agonistic effect of the new peptides is based on their induction of radical oxygen release (oxygen burst) in human granulocytes.
  • the test (Methods in Enzymology 133, 449 (1986) was carried out as follows:
  • the antagonistic evaluation of the new peptides is based on their ability to inhibit the rHuTNF-induced radical oxygen release in this test system. ad IV: cytotoxicity test
  • the agonistic evaluation of the new peptides is based on their cytotoxic effects on TNF-sensitive tumor cells. The test was carried out as follows:
  • the peptide concentration was determined in a proliferation test. After 24 hours of incubation, the incorporation of 3 H- ⁇ dR into the DNA of the MCF-7 cells was determined 50% inhibits.
  • the antagonistic evaluation of the new peptides is based on their ability to inhibit the cytotoxic effects of rHuTNF in this test system.
  • proteogenic amino acids are abbreviated in the examples with the well-known three-letter code.
  • proteogenic amino acids are abbreviated in the examples with the well-known three-letter code.
  • the peptide resin obtained according to Ia was dried in vacuo and in a reaction vessel of a Teflon HF apparatus
  • Hydrogen fluoride condensed (10 ml / g resin). The mixture was allowed to warm to 0 ° C and stirred at this temperature for 45 min. The hydrogen fluoride was then stripped off in vacuo and the residue was washed with ethyl acetate in order to remove remaining scavengers. The peptide was extracted with 30% acetic acid, filtered and the filtrate lyophilized.
  • the peptide resin (Pam or Merrifield resin) was suspended in DMF (15 ml / g resin) and, after addition with hydrazine hydrate (20 equivalents), stirred for 2 days at room temperature. For working up, the resin was filtered off and the filtrate was evaporated to dryness. The residue was crystallized from DMF / Et 2 O or MeOH / Et 2 O. III. Processing of the peptide resins obtained according to Ib
  • the peptide resin obtained according to Ib was dried in vacuo and then, depending on the amino acid composition, subjected to one of the following cleavage procedures (Wade, Tregear, Howard Florey Fmoc-Workshop Manual, Melbourne 1985).
  • the purity of the end products obtained was determined using
  • Boc-Asn-OH Boc-Cys (pMB) -OH
  • Boc-Asp (OCHx) -OH implemented.
  • the peptide resin obtained was dried in vacuo; the yield was 1.79 g.
  • the crude peptide obtained after TFA cleavage according to AIII was dissolved in 500 ml of degassed DMF. After adding 210 mg of NaHCO 3 and 440 mg of BOP, the mixture was stirred at room temperature for three days. The mixture was then evaporated to dryness and the crude peptide was purified by gel chromatography (SEPHADEX ® LH 20). The isolated monomer (150 mg) was subjected to an HF cleavage according to All and the deprotected peptide was purified by medium pressure chromatography (cf. AIV; 55 to 75% A; 0.25% min -1 ). 37 mg of pure product were obtained.
  • the cyclized product (420 mg) was subjected to HF cleavage according to All.
  • the lyophilized crude product was cyclized as described above and by gel chromatography (SEPHADEX ® G-25) and medium pressure chromatography (cf. AIV;. 55 to 75% A, 0.25% min -1) purified. 38 mg of pure product were obtained.
  • the crude peptide (853 mg) obtained after the TFA cleavage according to AIII was dissolved in 500 ml degassed DMF. After adding 240 mg of NaHCO3 and 440 mg of BOP, the mixture was stirred at room temperature for 3 days.
  • Fmoc-Asn (Trt) -OH implemented.
  • the resin was suspended in DMF containing 1% acetic acid, 3 equivalents of Fmoc-leucinal (produced from Fmoc-leucine O, N-dimethylhydroxamate according to Castro, B ., Fehrentz, J.-A. (1983) Synthesis, 676-678) and 3 equivalents of NaBH 3 CN.
  • 3 h negative ninhydrin test
  • washing was carried out intensively and according to Alb with 1 mmol each

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  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Zoology (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Toxicology (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

L'invention concerne des peptides répondant à la formule (I), dans laquelle A, B, D, E, F, J, T, X, Y, Z ont la signification donnée dans la description, ainsi que leur production. Ces nouveaux peptides sont utiles pour traiter des maladies.
PCT/EP1991/002405 1990-12-21 1991-12-13 Nouveaux peptides constituant des facteurs de necrose tumorale WO1992011285A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE19904041187 DE4041187A1 (de) 1990-12-21 1990-12-21 Neue tnf-peptide
DEP4041187.7 1990-12-21

Publications (1)

Publication Number Publication Date
WO1992011285A1 true WO1992011285A1 (fr) 1992-07-09

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WO (1) WO1992011285A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015044900A1 (fr) * 2013-09-27 2015-04-02 Aurigene Discovery Technologies Limited Composés immunomodulateurs thérapeutiques

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0347728A2 (fr) * 1988-06-20 1989-12-27 Denki Kagaku Kogyo Kabushiki Kaisha Anticorps monoclonal contre la lymphotoxine humaine et son utilisation
WO1990006942A1 (fr) * 1988-12-12 1990-06-28 Basf Aktiengesellschaft Nouveaux peptides derives du facteur de necrose de tumeurs (tnf)

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0347728A2 (fr) * 1988-06-20 1989-12-27 Denki Kagaku Kogyo Kabushiki Kaisha Anticorps monoclonal contre la lymphotoxine humaine et son utilisation
WO1990006942A1 (fr) * 1988-12-12 1990-06-28 Basf Aktiengesellschaft Nouveaux peptides derives du facteur de necrose de tumeurs (tnf)

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
Chemical Abstracts, Band 45, Nr. 17, 28. Oktober 1991, (Columbus, Ohio, US), A.E. POSTLETHWAITE et al.: "Stimulation of fibroblast chemotaxis by human recombinant tumor necrosis factor alpha (TNF-alpha) and a synthetic TNF-alpha 31-68 peptide", siehe Seite 716, Zusammenfassung Nr. 181051d, & J. EXP. MED. 1990, 172(6), 1749-56, siehe die Zusammenfassung *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015044900A1 (fr) * 2013-09-27 2015-04-02 Aurigene Discovery Technologies Limited Composés immunomodulateurs thérapeutiques

Also Published As

Publication number Publication date
DE4041187A1 (de) 1992-06-25

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