Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
  • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
  • C07K5/10—Tetrapeptides
  • C07K5/1002—Tetrapeptides with the first amino acid being neutral
  • C07K5/1005—Tetrapeptides with the first amino acid being neutral and aliphatic
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
  • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
  • C07K5/10—Tetrapeptides
  • C07K5/1002—Tetrapeptides with the first amino acid being neutral
  • C07K5/1005—Tetrapeptides with the first amino acid being neutral and aliphatic
  • C07K5/1013—Tetrapeptides with the first amino acid being neutral and aliphatic the side chain containing O or S as heteroatoms, e.g. Cys, Ser
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
  • A61K38/07—Tetrapeptides
• • 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 new farnesyl transferase inhibitors of general formula:
• Inhibition of famesyl transferase and, therefore, famesylation of the ras protein blocks the ability of the mutated ras protein to transform normal cells into cancer cells.
• the C-terminal sequence of the ras gene contains the motif "CAAX” or "Cys-Aaa] -Aaa2-Xaa” in which Aaa represents an aliphatic amino acid and Xaa represents any amino acid.
• tetrapeptides with a CAAX sequence can inhibit famesylation of the ras protein.
• peptides inhibitors of the famesyl transferase Cys-Aaa ⁇ -Aaa2-Xaa which are more particularly represented by the peptides Cys-Val-Leu- Ser, Cys-Val-Ile-Met and Cys-Val-Val-Met which manifest their inhibitory activity at concentrations close to 10 " ⁇ M or 10 -7 M.
• Rj represents a radical of general formula YSA ⁇ - in which Y represents a hydrogen atom, or an amino acid residue or a fatty acid residue or an alkyl or alkoxycarbonyl radical, or a radical R4-S- in which R4 represents a radical alkyl containing 1 to 6 carbon atoms optionally substituted by a phenyl radica, or radical of general formula
• R2 represents a straight or branched alkyl radical containing 1 to 4 carbon atoms optionally substituted by a cyclohexyl radical
• R'2 represents a hydrogen atom or a straight or branched alkyl radical containing 1 to 6 carbon atoms
• R3 represents an alkyl radical containing straight or branched containing 1 to 4 carbon atoms optionally substituted by a hydroxy radical, alkoxy containing 1 to carbon atoms, mercapto, alkylthio containing 1 to 4 carbon atoms, alkylsul finyl containing 1 to 4 atoms carbon or alkylsulfonyl containing 1 to 4 carbon atoms, it being understood that, when R3 represents an alkyl radical substituted by a hydroxy radical, R3 can form with the carboxy radical in this a lactone, R'3 represents a hydrogen atom or a straight or branched alkyl radical containing 1 to 6 carbon atoms,
• X represents an oxygen or sulfur atom
• R represents a hydrogen atom or an alkyl radical optionally substituted by an alkoxy radical containing 1 to 4 carbon atoms, alkylthio containing 1 to carbon atoms, alkylsulfinyl containing 1 to 4 carbon atoms, alkylsulfonyl containing 1 to 4 carbon atoms, phenyl, phenoxy, phenylthio, phenylsulfinyl, phenylsulfonyl, alkylamino containing 1 to 4 carbon atoms or dialkylamino each alkyl part of which contains 1 to 4 carbon atoms, or a phenyl radical optionally substituted by one or more identical or different atoms or radicals chosen from halogen atoms and alkyl, alkoxy, alkylthio or alkanoyl radicals containing 1 to 4 carbon atoms. More specifically,
• R] represents a radical of formula YS-Aj- in which Y represents a hydrogen atom or a lysine residue or a fatty acid residue containing up to 20 carbon atoms and A] represents an optionally substituted ethylene or propylene radical by an amino radical,
• R'2 represents a hydrogen atom or a methyl radical
• R'3 represents a hydrogen atom or a methyl radical
• X represents an oxygen atom
• R represents a hydrogen atom or an alkyl radical containing 1 to 4 carbon atoms optionally substituted by an alkoxy radical, or a phenyl radical. More particularly still,
• Rj represents a radical of formula YS-Aj- in which Y represents a hydrogen atom and Aj represents an ethylene or propylene radical optionally substituted by an amino radical, Xj and Y] each represent a hydrogen atom or form together with l carbon atom to which they are linked a group> CO,
• R2 represents an isopropyl, 1-methylpropyl, tert-butyl or cyclohexylmethyl radical
• R'2 represents a hydrogen atom
• R3 represents a methyl or ethyl radical substituted by a hydrox methoxy, mercapto or methylthio radical
• R'3 represents a hydrogen atom
• R represents a hydrogen atom or an alkyl radical containing 1 to 4 carbon atoms.
• R ⁇ represents a 2-mercapto-ethyl or amino-1-mercapto-ethyl radical
• X and Y each represent a hydrogen atom or together form the carbon atom to which they are linked a group> CO
• R2 represents an isopropyl radical
• R ' represents an atom of hydrogen
• R3 represents a 2-methylthio-ethyl or 2-methylsulfinyl-ethyl radical
• R'3 represents a hydrogen atom
• R represents a hydrogen atom.
• the present invention also relates to the stereoisomeric forms of products of general formula (I).
• the amino acid residues represented by R 1 C (X 1 ) (Y ⁇ ), R2CH (NR ' 2 ) [C (X 2 ) (Y2)] and R 3 CH (NR' 3 ) CO-OH preferably have the configuration natural amino acids.
• the present invention also relates to the mineral or organic salts as well as the esters of the products of general formula (I).
• the new products of general formula (I) can be obtained by solid phase synthesis using a synthesis strategy "9-fluorenylmethoxycarbonyl (FMOC)".
• FMOC 9-fluorenylmethoxycarbonyl
• the thiol groups are protected by trityl or acetamidomethyl groups
• the amino functions by Boc groups (t.butoxycarbonyl) and the acid functions in the form of t.butyl ester
• the synthesis can be carried out on a resin confined in 3 cm3 solid phase extraction syringes made of high density polyethylene provided with teflon filters.
• the syringes are mounted on a two-way teflon valve and closed with a disposable wing plug of high density polyethylene.
• the syringes are shaken on a rotary device for hemolysis tubes.
• the washing and filtration operations are carried out on a solid phase extraction workstation.
• the syntheses are then carried out on 50 ⁇ moles of resin.
• Amino acid couplings are carried out by treating the resin for 1 hour with 250 ⁇ mol of the amino acid suitably protected in the presence of 250 ⁇ mol of 2- (1H-benzotriazole-1-yl) -l, l, 3.3- tetramethyluronium, hexafluorophosphate (HBTU), 250 ⁇ moles of N-hydoxybenzotriazole and 750 ⁇ moles of diisopropylethylamine in 1.2 cm3 of an N-methylpyrrolidone-2 (NMP) / dimethylformamide mixture (1/1 by volume).
• NMP N-methylpyrrolidone-2
• the products are separated by treating the resin with 10 cm 3 of a trifluoroacetic acid-phenol-ethanedithiol-thioanisole-water mixture (40-3-1-2-2 by volume) for 1 hour 30 minutes.
• the resin is then removed by filtration.
• the filtrate is concentrated under reduced pressure by means of a centrifugal evaporator (RC10-10 Jouan) equipped with a vane pump and a trap at -90 ° C for 1.5 hours, the temperature of the evaporation being maintained at 50 ° C.
• the final volume of the concentrate is approximately 1 cm3.
• the product is then precipitated by adding 15 cm 3 of a mixture of methyl tert-butyl ether and ether of petroleum (2-1 by volume) then it is collected by centrifugation.
• the pellet is then dissolved in 1 cm3 of trifluoroacetic acid, precipitated by addition of 15 cm3 of methyl-tert-butyl ether and then washed with 15 cm3 of methyl-tert-butyl ether.
• the product is then dried under reduced pressure (3.5 kPa).
• the product is finally purified by high performance liquid chromatography (HPLC) on a column C] 8 100 (250 x 10 mm, BioRad) eluted with an acetonitrile gradient containing 0.07% trifluoroacetic acid (by volume) in water containing 0.07% trifluoroacetic acid (by volume) at a flow rate of 6 cm3 / min and then lyophilized.
• HPLC high performance liquid chromatography
• the products obtained are characterized by their mass spectra (electrospray).
• the introduction of the protective group FMOC on an amino acid e carried out by the action of the amino acid on the chloroformate of 9-fluorenylmethy (FMOC-chloride) in the presence of a base.
• FMOC-Met-chlorotrityl resin can be obtained by reacting 250 ⁇ mol of chlorotritylchloride resin (Novabiochem®) with 1 mmol of FMOC-Methionine in 2 cm3 of dichloromethane and 0.5 cm3 of diisopropylethylamine for 30 minutes. After adding 2 cm 3 of methanol, the reaction is continued for a further 30 minutes. The resin is then washed with 4 cm3 of dichloromethane at times and then dried.
• the famesyltransferase activity is determined by the amount of ( 3 H) famésy transferred from H) famesylpyrophosphate [( ⁇ H) FPP) to the p2 H-ras protein.
• the standard reaction mixture is composed, for a final volume of 60 ⁇ l of 50 mM Tris-HCl, 5 mM MgCl2, 5 mM dithiotreitol, 0.2% octyl- ⁇ -D-glucopyranosid, p21 H-ras 200 picomoles, ( 3 H) FPP (at 61,000 dpm / picomole) 4.5 picomoles.
• the reaction is initiated by the addition of approximately 5 ng of human famesyltransferas purified from cultures of THP1 cells. After incubation for 20 minutes at 37 ° C. in a microtitration plate containing 96 holes of 1 cm 3 per plate (Titer Plate®, Beckman), the reaction is stopped by adding 0.4 cm 3 of 0.1% SDS in methanol to 0 ° C. 0.4 cm 3 of trichloroacetic acid (TCA) 30% in methanol is then added to the mixture. The plates are left hanging for 1 hour in the ice. The precipitated content is then retained on a fiberglass membrane.
• TCA trichloroacetic acid
• the activity unit is defined by 1 picomole of ( 3 H) FPP transferred to p21 H-ras in 20 minutes.
• the inhibition percentages are obtained by comparison of the tests with and without inhibitor after deduction of the blanks, the IC50 being measured from the inhibitions obtained with 9 different concentrations using the Enzfitter® or Graf it® software.
• the new peptides of general formula (I) can be in the form of non-toxic and pharmaceutically acceptable salts.
• These non-toxic salts include the salts with mineral acids (hydrochloric, sulfuric, hydrobromic, phosphoric, nitric) or with organic acids (acetic, propionic, succinic, maleic, hydroxymaleic, benzoic, fumaric, methanesulfonic, trifluoroacetic or oxalic acids) or with mineral bases (soda, potash, lithine, lime) or organic bases (tertiary amines such as triethylamine, piperidine, benzylamine) depending on the nature of the amino acids which constitute the peptide of general formula (I).
• the new peptides according to the invention which inhibit famesyltransferase and famesylation of the Ras protein, are remarkable anticancer agents which act both in solid and liquid tumors.
• the present invention also relates to pharmaceutical compositions which contain at least one peptide of general formula (I) in combination with a or more pharmaceutically acceptable diluents or adjuvants whether inert or physiologically active.
• compositions can be administered orally, parenterally or rectally.
• the compositions for oral administration include tablets, pills, powders or granules.
• the active product according to the invention is mixed with one or more inert diluents such as sucrose, lactose or starch.
• these compositions can include substances other than diluents, for example a lubricant such as magnesium stearate.
• As liquid compositions for oral administration can be used pharmaceutically acceptable emulsions, solutions, suspensions, syrups, elixirs containing inert diluents such as water or paraffin oil.
• These compositions can also include substances other than diluents, for example wetting, sweetening or flavoring products.
• compositions according to the invention for parenteral administration can be sterile aqueous or non-aqueous solutions, suspensions or emulsions.
• solvent or vehicle propylene glycol, a polyethylene glycol, vegetable oils, in particular olive oil or injectable organic esters, for example ethyl oleate.
• These compositions can also contain adjuvants, in particular wetting agents, emulsifiers and dispersants. Sterilization can be done in several ways, for example using a bacteriological filter, incorporating sterilizing agents into the composition or by heating. They can also be prepared in the form of sterile solid compositions which can be dissolved at the time of use in sterile water or any other sterile injectable medium.
• compositions for rectal administration are suppositories which may contain, in addition to the active product, excipients such as cocoa butter.
• compositions according to the invention are particularly useful in human therapy in the treatment of cancers of various origins.
• the doses depend on the desired effect, on the duration of the treatment and on the factors specific to the subject to be treated.
• the doses are, in humans, between 0.1 and 20 mg / kg per day intraperitoneally.

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• Chemical & Material Sciences (AREA)
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• Proteomics, Peptides & Aminoacids (AREA)
• Genetics & Genomics (AREA)
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• Molecular Biology (AREA)
• Pharmacology & Pharmacy (AREA)
• Animal Behavior & Ethology (AREA)
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• Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
• Engineering & Computer Science (AREA)
• General Chemical & Material Sciences (AREA)
• Chemical Kinetics & Catalysis (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Gastroenterology & Hepatology (AREA)
• Immunology (AREA)
• Epidemiology (AREA)
• Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
• Peptides Or Proteins (AREA)
• Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
• Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)
• Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)

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• 1995
  • 1995-02-09 FR FR9501489A patent/FR2730491B1/fr not_active Expired - Fee Related
• 1996
  • 1996-02-07 EP EP96903061A patent/EP0808328A1/de not_active Ceased
  • 1996-02-07 CA CA002210955A patent/CA2210955A1/fr not_active Abandoned
  • 1996-02-07 US US08/875,752 patent/US5856439A/en not_active Expired - Fee Related
  • 1996-02-07 SK SK1087-97A patent/SK108797A3/sk unknown
  • 1996-02-07 BR BR9607317A patent/BR9607317A/pt not_active Application Discontinuation
  • 1996-02-07 KR KR1019970705443A patent/KR19980702048A/ko not_active Withdrawn
  • 1996-02-07 JP JP8524037A patent/JPH10513467A/ja active Pending
  • 1996-02-07 WO PCT/FR1996/000198 patent/WO1996024611A1/fr not_active Ceased
  • 1996-02-07 CZ CZ972498A patent/CZ249897A3/cs unknown
  • 1996-02-07 AU AU47227/96A patent/AU4722796A/en not_active Abandoned
  • 1996-02-07 TR TR97/00727T patent/TR199700727T1/xx unknown
  • 1996-02-07 CN CN96191867A patent/CN1173874A/zh active Pending
  • 1996-02-07 PL PL96321675A patent/PL321675A1/xx unknown
  • 1996-02-09 ZA ZA961072A patent/ZA961072B/xx unknown
• 1997
  • 1997-08-05 NO NO973608A patent/NO973608L/no unknown
  • 1997-08-08 FI FI973278A patent/FI973278A0/fi not_active Application Discontinuation

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