EP0527181A1 - Composes d'ethenediyle heterocyclique qui inhibent la kinase de tyrosine de recepteur egf - Google Patents

Composes d'ethenediyle heterocyclique qui inhibent la kinase de tyrosine de recepteur egf

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Publication number
EP0527181A1
EP0527181A1 EP91908779A EP91908779A EP0527181A1 EP 0527181 A1 EP0527181 A1 EP 0527181A1 EP 91908779 A EP91908779 A EP 91908779A EP 91908779 A EP91908779 A EP 91908779A EP 0527181 A1 EP0527181 A1 EP 0527181A1
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EP
European Patent Office
Prior art keywords
disorder
patient
cell proliferation
patient suffering
pharmaceutically acceptable
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.)
Withdrawn
Application number
EP91908779A
Other languages
German (de)
English (en)
Other versions
EP0527181A4 (en
Inventor
Alfred P. Spada
Paul E. Persons
Alexander Levitzki
Chaim Gilon
Aviv Gazit
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.)
Yissum Research Development Co of Hebrew University of Jerusalem
Original Assignee
Yissum Research Development Co of Hebrew University of Jerusalem
Rorer International Holdings Inc
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Filing date
Publication date
Application filed by Yissum Research Development Co of Hebrew University of Jerusalem, Rorer International Holdings Inc filed Critical Yissum Research Development Co of Hebrew University of Jerusalem
Publication of EP0527181A1 publication Critical patent/EP0527181A1/fr
Publication of EP0527181A4 publication Critical patent/EP0527181A4/en
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D233/00—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings
    • C07D233/54—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings having two double bonds between ring members or between ring members and non-ring members
    • C07D233/64—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings having two double bonds between ring members or between ring members and non-ring members with substituted hydrocarbon radicals attached to ring carbon atoms, e.g. histidine
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D209/00—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom
    • C07D209/02—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom condensed with one carbocyclic ring
    • C07D209/04—Indoles; Hydrogenated indoles
    • C07D209/08—Indoles; Hydrogenated indoles with only hydrogen atoms or radicals containing only hydrogen and carbon atoms, directly attached to carbon atoms of the hetero ring
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D209/00—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom
    • C07D209/02—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom condensed with one carbocyclic ring
    • C07D209/04—Indoles; Hydrogenated indoles
    • C07D209/10—Indoles; Hydrogenated indoles with substituted hydrocarbon radicals attached to carbon atoms of the hetero ring
    • C07D209/18—Radicals substituted by carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
    • C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
    • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D213/24—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with substituted hydrocarbon radicals attached to ring carbon atoms
    • C07D213/54—Radicals substituted by carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals
    • C07D213/57—Nitriles
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D231/00—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings
    • C07D231/02—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings not condensed with other rings
    • C07D231/10—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members
    • C07D231/12—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached to ring carbon atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D317/00—Heterocyclic compounds containing five-membered rings having two oxygen atoms as the only ring hetero atoms
    • C07D317/08—Heterocyclic compounds containing five-membered rings having two oxygen atoms as the only ring hetero atoms having the hetero atoms in positions 1 and 3
    • C07D317/44—Heterocyclic compounds containing five-membered rings having two oxygen atoms as the only ring hetero atoms having the hetero atoms in positions 1 and 3 ortho- or peri-condensed with carbocyclic rings or ring systems
    • C07D317/46—Heterocyclic compounds containing five-membered rings having two oxygen atoms as the only ring hetero atoms having the hetero atoms in positions 1 and 3 ortho- or peri-condensed with carbocyclic rings or ring systems condensed with one six-membered ring
    • C07D317/48—Methylenedioxybenzenes or hydrogenated methylenedioxybenzenes, unsubstituted on the hetero ring
    • C07D317/62—Methylenedioxybenzenes or hydrogenated methylenedioxybenzenes, unsubstituted on the hetero ring with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to atoms of the carbocyclic ring

Definitions

  • This invention relates to the inhibition of cell proliferation. More specifically, this invention relates to the use of heterocyclicethenediyl compounds in inhibiting cell proliferation, including compounds which are useful protein tyrosine kinase (PTK) inhibitors.
  • PTK protein tyrosine kinase
  • Normal cellular reproduction is believed to be triggered by the exposure of the cellular substrate to one or more growth factors, examples of which are insulin, epidermal growth factor (EGF) and platelet-derived growth factor (PDGF) .
  • growth factors are typically specific for corresponding growth factor receptors which are imbedded in and which penetrate through the cellular membrane.
  • EGF epidermal growth factor
  • PDGF platelet-derived growth factor
  • Such growth factors are typically specific for corresponding growth factor receptors which are imbedded in and which penetrate through the cellular membrane.
  • This growth factor-receptor binding alters the chemical characteristics of that portion of the receptor which exists within the cell and which functions as an enzyme to catalyze phosphorylation of either an intracellular substrate or the receptor itself, the latter being referred to as autophosphorylation.
  • phosphorylation enzymes include tyrosine kinases, which catalyze phosphorylation of tyrosine amino acid residues of substrate proteins.
  • diseased states are characterized by the uncontrolled reproduction of cells. These diseased states involve a variety of cell types and include disorders such as leukemia, cancer, psoriasis, atherosclerosis and restenosis injuries.
  • the inhibition of tyrosine kinase is believed to have utility in the control of uncontrolled cellular reproduction, i.e., cellular proliferative disorders.
  • Initiation of autophosphorylation i.e., phosphorylation of the growth factor receptor itself, and of the phosphorylation of a host of intracellular substrates are some of the biochemical events which are involved in mitogenesis and cell proliferation.
  • Autophosphorylation of the insulin receptor and phosphorylation of substrate proteins by other receptors are the earliest identifiable biochemical hormonal responses.
  • PTK protein tyrosine kinase
  • EGF epidermal growth factor
  • EGF receptors inhibit EGF-induced proliferation of A431/clone 15 cells with little or no effect on the proliferation of such cells when induced by other growth factors. It has been reported also that erbstatin inhibits the autophosphorylation of the EGF receptor in membranes of A431 cells. Low concentrations of erbstatin are reguired to inhibit EGF receptor autophosphorylation, whereas much higher concentra ⁇ tions of erbstatin are required to inhibit cyclic adenosine 3' ,5'-monophosphate (cAMP)-dependent protein kinase.
  • cAMP cyclic adenosine 3' ,5'-monophosphate
  • compositions comprising, in admixture with a pharmaceutically acceptable carrier, a pharmaceutically- effective amount of the aforementioned type of compound.
  • Still another aspect of the present invention relates to novel compounds of the aforementioned type.
  • each R ⁇ - is independently hydrogen, alkyl, hydroxy, alkoxy or halo
  • each R 7 is independently alkyl, hydroxy, alkoxy, halo, nitro, carboxy or carbalkoxy;
  • n 1 to about 7;
  • Alkyl means a saturated aliphatic hydrocarbon which may be either straight- or branch-chained containing from about 1 to about 6 carbon atoms.
  • “Lower alkyl” means an alkyl group as above, having 1 to about 4 carbon atoms which may be straight- or branch-chained such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl or tert-butyl.
  • Alkoxy means an alkyl-oxy group in which "alkyl” is as previously described. Lower alkoxy groups are preferred. Exemplary groups include methoxy, ethoxy, n-propoxy, i-propoxy and n-butoxy.
  • Aryl means an unsaturated or partially unsaturated ring system. Preferred aryl groups are pyridyl and indolyl.
  • acyl means an organic radical derived from an organic acid, a carboxylic acid, by the removal of its acid hydroxyl group.
  • Preferred acyl groups are lower alkyl carboxylic acid groups such as acetyl and propionyl. Benzoyl is also preferred.
  • Halo means a halogen.
  • Preferred halogens include chloride, bromide and fluoride.
  • Preferred aralkyl groups are benzyl and phenethyl.
  • PTK inhibiting compounds should be competitive with the substrate of EGF receptor tyrosine kinase (EGFRK) and not with adenosine triphosphate (ATP) .
  • EGFRK EGF receptor tyrosine kinase
  • ATP adenosine triphosphate
  • the PTK inhibitors quercetin and genistein, which compete with ATP, inhibit other protein kinases and as a result are highly cytotoxic.
  • compounds which inhibit EGFRK better than they inhibit insulin receptor kinase (IRK) and/or PDGF receptor kinase are of considerable value.
  • solubility of the compounds of the present invention both in water and in mildly hydrophobic solvents will enhance the probability that they traverse the cell membrane.
  • Various insoluble compounds have exhibited significant EGFRK inhibition in jLn vitro testing.
  • a preferred class of compounds useful in the practice of the present invention include those described by Formula I where:
  • W is a 5- or 6-membered monocyclic aryl ring including 1 or 2 N, 0 or S atoms, or a 9- or 10-membered bicyclic aryl ring including 1 to 4 N, 0 or S atoms, said ring optionally substituted with one to about three R 4 groups;
  • R- is -CN, -CONRR, -CSNRR or -COOR;
  • R is hydrogen, alkyl, or aralkyl
  • R 2 is -W, -CN, -CONHR-, -CONRR, -COOR, -CSNRR or
  • R 3 is hydrogen; ⁇ each R, is independently alkyl, hydroxy, alkoxy or halo;
  • R 5 is amino, -CONH 2 ,
  • each Rg is independently hydrogen or alkyl
  • each R 7 is independently alkyl, hydroxy, alkoxy, halo, nitro, carboxy or carbalkoxy;
  • n 0-4;
  • n 2-6;
  • W is furyl, pyrrolyl, thienyl, thiazolyl, pyridyl, imidazolyl, isoimidazolyl, pyridazinyl, pyrimdinyl, benzofuran l, indolyl, indolinyl, indolinonyl, benzothienyl, benzothiazolyl, quinolinyl, isoquinolinyl, chromenyl, 1,3-benzodioxolyl or 2,3-dihydro-l,4-benzodioxinyl.
  • W is pyridyl, indolyl, imidazolyl, benzothiazolyl, 1,3- benzodioxolyl or 2,3-dihydro-l,4-benzodioxinyl;
  • R- is -CN, -CONRR, -CSNRR or -COOR;
  • R is hydrogen, alkyl, or aralkyl
  • R 2 is -W, -CN, -CONHR-, -CONRR, -COOR, -CSNRR or
  • each R 4 is independently lower alkyl, hydroxy, lower alkoxy or halo
  • each R 7 is independently lower alkyl, hydroxy, alkoxy, halo, nitro, carboxy or carbalkoxy;
  • n 0-4;
  • n 2-6.
  • Acid addition salts may be formed and are simply a more convenient form for use; in practice, use of the salt form inherently amounts to use of the base form.
  • the acids which can be used to prepare the acid addition salts include preferably those which produce, when combined with the free base, pharmaceutically acceptable salts, that is, salts whose anions are non-toxic to . the animal organism in pharmaceutical doses of the salts, so that the beneficial properties inherent in the free base are not vitiated by side effects ascribable to the anions.
  • Pharmaceutically acceptable salts within the scope of the invention include those derived from the following acids: mineral acids such as hydrochloric acid, sulfuric acid, phosphoric acid and sulfamic acid; and organic acids such as acetic acid, citric acid, lactic acid, tartaric acid, malonic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, cyclohexylsulfamic acid, quinic acid, and the like.
  • mineral acids such as hydrochloric acid, sulfuric acid, phosphoric acid and sulfamic acid
  • organic acids such as acetic acid, citric acid, lactic acid, tartaric acid, malonic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, cyclohexylsulfamic acid, quinic
  • the corresponding acid addition salts comprise the following: hydrochloride, sulfate, phosphate, sulfamate, acetate, citrate, lactate, tartarate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, cyclohexylsulfamate and quinate, respectively.
  • the acid addition salts of the compounds of this invention are prepared either by dissolving the free base in aqueous or aqueous-alcohol solution or other suitable solvents containing the appropriate acid and isolating the salt by evaporating the solution, or by reacting the free base and acid in an organic solvent, in which case the salt separates directly or can be obtained by concentration of the solution.
  • Knoevenagel condensation of a heterocyclic aldehyde of formula W in a polar media with an active methylene compound of the formula R,CH 2 R 2 in the presence of ammonia or amines such as piperidine and raised heat results in the products of this invention.
  • the corresponding ketone starting material is used. Reaction temperatures in the range of 25°C to reflux and reaction times vary depending on the materials being used in the condensation.
  • Primary alcohols can be oxidized by oxidizing agents known in the art to form carboxylic acids or aldehydes, and secondary alcohols can be oxidized to form ketones.
  • substitution or alteration reactions can be employed to provide a variety of substituents throughout the molecule of the starting material, intermediates, or the final product.
  • Compounds within the scope of this invention exhibit significant activity as protein tyrosine kinase inhibitors and possess therapeutic value as cellular antiproliferative agents for the treatment of certain conditions including, for example, psoriasis and restenosis injuries. It is expected that the invention will be particularly applicable to the treatment of atherosclerosis. With regard to the treatment of some conditions, for example, atherosclerosis, certain people may be identified as being at high risk, for example, due to genetic, environmental or historical factors. Compounds within the scope of the present invention can be used in preventing or delaying the occurrence or reoccurrence of such conditions or otherwise treating the condition.
  • the tablets, troches, pills, capsules and the like may also contain the following: A binder such as gum tragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, lactose or saccharin may be added or a flavoring agent such as peppermint, oil of wintergreen, or cherry flavoring.
  • a binder such as gum tragacanth, acacia, corn starch or gelatin
  • excipients such as dicalcium phosphate
  • a disintegrating agent such as corn starch, potato starch, alginic acid and the like
  • a lubricant such as magnesium stearate
  • a sweetening agent such as sucrose, lactose or saccharin may be added or a flavoring agent such as peppermint,
  • tablets, pills, or capsules may be coated with shellac, sugar or both.
  • a syrup or elixir may contain the active compound, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavoring such as cherry or orange flavor.
  • any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed.
  • the active compound may be incorporated into sustained-release preparations and formulations.
  • the pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
  • the form must be sterile and must be fluid to the extent that easy syringability exists. It may be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like) , suitable mixtures thereof, and vegetable oils.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • the prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by use of agents delaying absorption, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions are prepared by incorporating the active compound in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the various sterilized active ingredient into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum drying and the freeze drying technique which yield a powder of the active ingredient plus any additional desired ingredient from previously sterile-filtered solution thereof.
  • the therapeutic compounds of this invention may be administered to a mammal alone or in combination with pharmaceutically acceptable carriers, as noted above, the proportion of which is determined by the solubility and chemical nature of the compound, chosen route of administration and standard pharmaceutical practice.
  • the dosage of the present therapeutic agents which will be most suitable for prophylaxis or treatment will vary with the form of administration, the particular compound chosen and the physiological characteristics of the particular patient under treatment. Generally, small dosages will be used initially and if necessary, will be increased by small increments until the optimum effect under the circumstances is reached.
  • the therapeutic human dosage based on physiological studies using rats, will generally be from about 0.01 mg to about 100 mg/kg of body weight per day or from about 0.4 mg to about 10 g or and higher although it may be administered in several different dosage units from once to several times a day. Oral administration requires higher dosages.
  • Examples l- 11 below are illustrative of compounds within the scope of the present invention.
  • Examples 1-5 illustrate various unsubstituted heteroaryl malononitriles, i.e., R, and R 2 are each -CN and the other R substituents are all hydrogen.
  • Example 7 illustrates a nitro-substituted heteroaryl malononitrile, i.e., R t and R 2 are each -CN, R 3 is hydrogen and R 4 is nitro.
  • Example 8 illustrates a diheteroarylethenediyl compound, i.e., R t is -CN, R 2 is pyridyl and the other R substituents are all hydrogen.
  • Example 9 illustrates a heteroarylethenediyl carbocyclic compound, i.e., R- is hydrogen, R 2 is phenanthryl, R 3 is -CN and there are no !_, substituents.
  • Examples 10 and 11 illustrate heteroarylethenediyl aryl compounds, i.e., R- is -CN,. R 2 is 3,4-dihydroxybenzoyl and R 3 is -H.
  • the supernatant is loaded onto a WGA-agarose column (100 ⁇ l of packed resin per 2 x 10 7 cells) and shaken for 2 hours at 4°C.
  • the unabsorbed material is removed and the resin washed twice with HTN buffer (50 mmol Hepes, pH 7.6, 0.1% Triton X-100, 150 mmol NaCl) , twice with HTN buffer containing 1 M NaCl, and twice with HTNG buffer (50 mmol Hepes, pH 7.6, 0.1% Triton X- 100, 150 mmol NaCl, and 10% glycerol).
  • the EGF receptor is eluted batchwise with HTNG buffer containing 0.5 M N-acetyl-D- glucosamine (200 ⁇ l per 2 x 10 7 cells) .
  • the eluted material is stored in aliquots at -70°C and diluted before use with TMTNG buffer (50 mmol Tris-Mes buffer, pH 7.6, 0.1% Triton X-100, 150 mmol NaCl, 10% glycerol) .
  • WGA-purified EGFR (0.25 ⁇ g/assay) is preactivated with
  • results of this assay indicate that the compounds of the present invention are effective in inhibiting substrate phosphorylation catalyzed by EGFRK.
  • WGA-purified EGF receptor from A431 cells (0.5 ⁇ g/assay) is activated with EGF (800 nM) for 20 minutes at 4°C.
  • the reaction is initiated by the addition of Mg(Ac) 2 (60 mmol) , Tris-Mes buffer, pH 7.6 (50 mmol), and [ 32 P]ATP (20 ⁇ M, 5 ⁇ Ci/assay) .
  • the reaction is conducted at either 4 or 15°C and terminated by addition of sodium dodecyl sulfate (SDS) sample buffer (10% glycerol, 50 mmol Tris, pH 6.8, 5% ⁇ -mercapto- ethanol, and 3% (SDS) .
  • SDS sodium dodecyl sulfate
  • WGA-purified EGF receptor from A431 cells 0.5 ⁇ g/assay is activated with EGF (0.85 ⁇ M) for 20 minutes at 4°C.
  • the assay is performed at 15°C and initiated by addition of Mg(Ac) 2 (60 mmol), Tris-Mes buffer, pH 7.6 (50 mmol), [ 3 P]ATP (carrier free, 5 ⁇ Ci/assay) , and increasing concentrations of nonradioactive ATP.
  • the assay is terminated after 10-s by addition of SDS sample buffer.
  • the samples are run on a 6% SDS polyacrylamide gel. The gel is dried and autoradiographed as described above.
  • the relevant radioactive bands are cut and counted in the Cerenkov mode, the K m for ATP determined in this fashion is found to be 7.2 ⁇ M. With use of the 10-s assay protocol, the EGF concentration dependence of EGFRK autophosphorylation is determined.
  • Rat liver membranes are prepared from the livers of 6- week-old rats as described by Cuatrecasas.
  • WGA-purified insulin receptor is prepared according to Zick et al.
  • WGA- purified rat liver InsRK (1.25 ⁇ g) is preincubated with or without 330 nM insulin in 50 mmol Tris-Mes buffer, pH 7.6, for 30 minutes at 22°C.
  • the assay is performed at 22°C and initiated by addition of a mixture which contains Mg(Ac) 2 (60 mmol), NaV0 3 (40 ⁇ M) , [7- 3 P]ATP (125 ⁇ M, 3-5 ⁇ Ci/assay) , and poly(GT) [poly(Glu 4 Tyr) ] at three concentrations: whenever an inhibitor is tested, it is added at the proper concentration.
  • the final concentration of insulin in the assay is 125 nM.
  • the total volume of the assay is 40 ⁇ l. After 20 minutes, aliquots of 30 ⁇ l are applied on Whatman 3-mm paper and soaked in cold 10% TCA, containing 0.01 M sodium pyrophosphate. After being washed overnight, the papers are dried and counted, measuring Cerenkov radiation.
  • the InsRk-catalyzed phosphorylation of poly(GT) obeys Michaelis-Menten kinetics.
  • Example EGF PDGF 8 1 20 The results of this assay show that the compounds of the present invention inhibit EGF receptor kinase better than they inhibit PDGF receptor kinase.
  • Costar dishes pre-coated with human fibronectin by incubating for 30 minutes at room temperature with 10 ⁇ g/0.5 ml/well) .
  • the cells were grown to confluence for 2 days.
  • the medium was changed to DMEM containing 0.5 calf serum for 36-48 hours and the cells were then incubated with EGF (Toyobo, New York, NY) (20 ng/ l) or serum (10% calf serum) and different concentrations of the inhibitory compounds.
  • EGF Toyobo, New York, NY
  • serum 10% calf serum
  • HER 14 and K721A were prepared by transfecting N1H3T3 cells (clone 2.2) (From C. Fryling, NCI, NIH) , which lack endogenous EGF-receptors, with cDNA constructs of wild-type EGF-receptor or mutant EGF-receptor lacking tyrosine kinase activity (in which Lys 721 at the ATP- binding site was replaced by an Ala residue, respectively) . All cells were grown in DMEM with 10% calf serum (Hyclone, Logan, Utah) .

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Abstract

Des composés hétéroaryléthènediyle ou hétéroaryléthènediyle aryle dans lesquels le groupe hétéroaryle peut être un hétéroaryle monocyclique ou bicyclique et le groupe aryle peut être hétéroaryle monocyclique ou bicyclique ou carbocyclique bicyclique ou tricyclique, ledit composé étant éventuellement substitué ou polysubstitué, à condition que le groupe hétéroaryle ne soit pas furyle ou thiényle lorsque le groupe éthènediyle possède des substituants cyano géminés, et des compositions pharmaceutiques comprenant ces composés, et leur utilisation pour inhiber la prolifération de cellules chez un patient souffrant de cette maladie.
EP19910908779 1990-04-16 1991-04-16 Heterocyclicethenediyl compounds which inhibit egf receptor tyrosine kinase Withdrawn EP0527181A4 (en)

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US50998190A 1990-04-16 1990-04-16
US509981 1990-04-16

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EP0527181A1 true EP0527181A1 (fr) 1993-02-17
EP0527181A4 EP0527181A4 (en) 1993-04-07

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ES2108120T3 (es) * 1991-05-10 1997-12-16 Rhone Poulenc Rorer Int Compuestos bis arilicos y heteroarilicos mono- y biciclicos que inhiben tirosina quinasa receptora de egf y/o pdgf.
ATE201398T1 (de) * 1993-11-17 2001-06-15 Kyowa Hakko Kogyo Kk Propenon-derivate
GB9406137D0 (en) * 1994-03-28 1994-05-18 Erba Carlo Spa N-substituted beta-aryl- and betaheteroaryl-alpha-cyanoacrylamide derivatives and process for their preparation
CA2193666A1 (fr) * 1995-05-10 1996-11-14 Shun-Ichi Ikeda Derives de propenone
US5710164A (en) * 1995-06-06 1998-01-20 American Home Products Corporation Diheterocyclic styryl nitriles
US6444694B1 (en) * 1995-06-06 2002-09-03 Wyeth Styryl benzimidazole derivatives
EP0833629A4 (fr) * 1995-06-19 1998-09-16 Ontogen Corp Derives d'acide aryl-acrylique convenant comme inhibiteurs de proteine-tyrosine-phosphatase
EP0812825B1 (fr) * 1995-12-01 2004-02-11 Kyowa Hakko Kogyo Co., Ltd. Derives du propenone
EP1701941B1 (fr) 2003-12-11 2012-05-30 Board of Regents, The University of Texas System Composes pour traiter des maladies associees a une proliferation cellulaire
RU2456265C2 (ru) 2006-03-31 2012-07-20 Дзе Борд Оф Риджентс Оф Дзе Юниверсити Оф Техас Систем Биологически доступная для перорального применения кофейная кислота, относящаяся к противоопухолевым лекарственным средствам
PL2307367T3 (pl) 2008-07-08 2015-03-31 Univ Texas Nowe inhibitory proliferacji i aktywacji białek przekazujących sygnał i aktywujących transkrypcję (STAT)
JP2019510088A (ja) * 2016-03-17 2019-04-11 ザ ジョンズ ホプキンス ユニバーシティーＴｈｅ Ｊｏｈｎｓ Ｈｏｐｋｉｎｓ Ｕｎｉｖｅｒｓｉｔｙ Ｐａｒｉｓのファルネシル化によってパーキンソン病を予防または治療する方法

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EP0322738A2 (fr) * 1987-12-24 1989-07-05 Yissum Research Development Company Of The Hebrew University Of Jerusalem Dérivés de la benzylidène pour l'inhibition des processus de prolifération dans les cellules des mammifères
WO1991013055A2 (fr) * 1990-02-28 1991-09-05 Farmitalia Carlo Erba S.R.L. Nouveaux derives d'aryl- et heteroarylethenylene, et leur procede de preparation

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See also references of WO9116305A1 *

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JPH05507072A (ja) 1993-10-14
WO1991016305A1 (fr) 1991-10-31
EP0527181A4 (en) 1993-04-07
AU7756891A (en) 1991-11-11
AU662480B2 (en) 1995-09-07
IL97873A0 (en) 1992-06-21

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