EP0871436A1 - UTILISATION DE COMPOSES SPECIFIQUES DU DOMAINE Stat 6 SH2 POUR LE TRAITEMENT DES REACTIONS ALLERGIQUES - Google Patents

UTILISATION DE COMPOSES SPECIFIQUES DU DOMAINE Stat 6 SH2 POUR LE TRAITEMENT DES REACTIONS ALLERGIQUES

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Publication number
EP0871436A1
EP0871436A1 EP96924322A EP96924322A EP0871436A1 EP 0871436 A1 EP0871436 A1 EP 0871436A1 EP 96924322 A EP96924322 A EP 96924322A EP 96924322 A EP96924322 A EP 96924322A EP 0871436 A1 EP0871436 A1 EP 0871436A1
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European Patent Office
Prior art keywords
domain
binding affinity
binds
stat
human
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EP96924322A
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German (de)
English (en)
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EP0871436A4 (fr
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Damien John Dunnington
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SmithKline Beecham Corp
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SmithKline Beecham Corp
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/54—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one sulfur as the ring hetero atoms, e.g. sulthiame
    • A61K31/542—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one sulfur as the ring hetero atoms, e.g. sulthiame ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/545—Compounds containing 5-thia-1-azabicyclo [4.2.0] octane ring systems, i.e. compounds containing a ring system of the formula:, e.g. cephalosporins, cefaclor, or cephalexine
    • A61K31/546—Compounds containing 5-thia-1-azabicyclo [4.2.0] octane ring systems, i.e. compounds containing a ring system of the formula:, e.g. cephalosporins, cefaclor, or cephalexine containing further heterocyclic rings, e.g. cephalothin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/12—Ketones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/16—Amides, e.g. hydroxamic acids
    • A61K31/165—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/16—Amides, e.g. hydroxamic acids
    • A61K31/165—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide
    • A61K31/166—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide having the carbon of a carboxamide group directly attached to the aromatic ring, e.g. procainamide, procarbazine, metoclopramide, labetalol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/4353—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/437—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a five-membered ring having nitrogen as a ring hetero atom, e.g. indolizine, beta-carboline
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/50—Pyridazines; Hydrogenated pyridazines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/06—Antiasthmatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/08—Antiallergic agents

Definitions

  • PTK regulatory protein tyrosine kinases
  • tyrosine kinases Aberrant protein tyrosine kinase activity has been implicated or is suspected in a number of pathologies such as diabetes, atherosclerosis, psoriases, septic shock, bone loss, anemia, many cancers and other proliferative diseases. Accordingly, tyrosine kinases and the signal transduction pathways which they are part of are potential targets for drug design. For a review, see Levitzki et al. in Science 267. 1782-1788 (1995).
  • SH2 domains which are conserved non- catalytic sequences of approximately 100 amino acids found in a variety of signalling molecules such as non-receptor PTKs and kinase target effector molecules and in oncogenic proteins, play a critical role.
  • the SH2 domains are highly specific for short phosphotyrosine-containing peptide sequences found in autophosphorylated PTK receptors or intracellular tyrosine kinases. Approximately 60 proteins having distinct catalytic or other functional domains yet sharing conserved SH2 domains, conserved sequences of approximately 100 amino acids, have been identified.
  • SH2 domain-ligand/compound interactions are highly specific such that minor modifications in the structure of the ligand/compound will significantly alter the selectivity with which the ligand/compound binds to the various SH2 domains.
  • the Stat (signal transduction and activation of transcription) proteins are SH2 containing intracellular proteins that transmit a signal from cytokine receptors to the nucleus and activate transcription of specific target genes (Darnell J. et al., Science 264. 1415-1421 (1994)). These proteins are recruited to phosphorylation sites on the receptor via their SH2 domains and are themselves phosphorylated on tyrosine residues by receptor-associated Janus (JAK) tyrosine kinases. Phosphorylation of Stat tyrosine residues provides additional binding ligands for the Stat SH2 domains which are displaced from the receptor and bind to the Stat phosphotyrosine motifs forming dimers.
  • the dimeric Stats translocate to the nucleus where they bind to DNA and to other accessory proteins and activate transcription of target genes.
  • Individual Stats or Stat heterodimers transduce signals from different cytokine receptors, for example, Stat 6 mediates interleukin 4 signalling while Stat 5 transduces signals from the erythropoietin (EPO) receptor (Hou J. et al.. Science 265. 1701-1706 (1994); Penta K. et al.. J. Biol. Chem. 270. 31282-31287 (1995)).
  • EPO erythropoietin
  • Stat proteins In disease states involving dysregulation of cytokine activity, compounds that activate or inhibit Stat proteins would be useful therapeutic agents. For example, inhibition of Stat 6 would block IL-4 and IL-13-mediated up-regulation of the IgE receptor (Izuhara K.et al., J. Biol. Chem. 271. 619-22 (1996)) and be useful in the treatment of allergic reactions. Conversely, activation of Stat 5 would mimic the effects of EPO and alleviate anemia.
  • the SH2 domain of Stat proteins provides a means to activate or inhibit their function. Activation can be achieved by inducing dimer formation by means of a dyad-symmetric ligand for the relevant SH2 domain, while a monomeric ligand would inhibit Stat function. Discovery of such ligands requires targeting of the SH2 domains of specific Stat proteins to induce or inhibit the desired homo- or heterodimer formation.
  • the Stat 6 SH2 domain and the Stat 5 SH2 domain are structurally similar, possessing a high degree of conservation between the domains.
  • Antagonism of the Stat 6 SH2 domain (discussed herein) is indicated as treating allergic reactions while activation of the Stat 5 SH2 domain is indicated as increasing red blood cell production. Therefore, an activator of Stat 5 with Stat 6 cross-reactivity would exacerbate allergic reactions, whereas a Stat 6 inhibitor cross-reacting with Stat 5 would inhibit erythrocyte production.
  • selective Stat 6 SH2 domain antagonists can be identified by binding assays against the subset of SH2 domains consisting of; the src SH2 domain, the Stat 6 SH2 domain, the lck SH2 domain, the Stat 5 SH2 domain, the fyn SH2 domain, the SHPTP2 SH2 domain, the p85 domain, the Grb2 SH2 domain and the hep SH2 domain.
  • the present invention provides a method of treating allergic reactions in a subject which comprises administering to the subject a therapeutically effective amount of a compound which (a) binds to a human Sat 6 SH2 domain with a binding affinity greater than fifty-fold higher than the binding affinity with which the compound binds to a human Stat 5 SH2 domain, (b) binds to a human hep SH2 domain, a human Grb2 SH2 domain, a human SH-PTP2 SH2 domain and a human p85 SH2 domain with a binding affinity which is greater than fifty-fold lower than the binding affinity with which the compound binds to such Stat 6 SH2 domain, and (c) binds to a human src SH2 domain, a human lck SH2 domain and a human fyn SH2 domain with a binding affinity which is greater than fifty-fold lower than the binding affinity with which the compound binds to such Stat 6 SH2 domain.
  • the term "allergic reaction” means any adverse physical reaction which is exacerbated or caused by IL-4, IL-l 3 or both IL-4 and IL-l 3 mediated up-regulation of the IgE receptor.
  • Preferred allergic reactions which are treated according to the present invention are asthma, allergic asthma, allergic rhinitis and atopic dermatitis.
  • Particularly preferred allergic reactions which are treated according to the present invention are allergic asthma and allergic rhinitis.
  • the term "treating" and derivatives thereof means prophylactic or therapeutic therapy.
  • compound means a nonpeptide chemical compound.
  • Stat 6 SH2 domain antagonists means a compound which (a) binds to a human Stat 6 SH2 domain with a binding affinity greater than fifty-fold higher, preferably greater than one hundred- fold higher, than the binding affinity with which the compound binds to a human Stat 5 SH2 domain, (b) binds to a human hep SH2 domain, a human Grb2 SH2 domain, a human SH-PTP2 SH2 domain and a human p85 SH2 domain with a binding affinity which is greater than fifty-fold lower, preferably greater than one hundred-fold lower, than the binding affinity with which the compound binds to such Stat 6 SH2 domain, and (c) binds to a human src SH2 domain, a human lck SH2 domain and a human fyn SH2 domain with a binding affinity which is greater than fifty-fold lower, preferably greater than one hundred-fold lower, than the binding affinity with which the compound binds to such Stat 6 SH2 domain, and (c) binds to a
  • the present invention provides a method of treating allergic reactions in a subject which comprises administering to the subject a therapeutically effective amount of a compound which (a) binds to a human Stat 6 SH2 domain with a binding affinity greater than fifty-fold higher than the binding affinity with which the compound binds to a human Stat 5 SH2 domain, (b) binds to a human hep SH2 domain, a human Grb2 SH2 domain, a human SH-PTP2 SH2 domain and a human p85 SH2 domain with a binding affinity which is greater than fifty-fold lower than the binding affinity with which the compound binds to such Stat 6 SH2 domain, and (c) binds to a human src SH2 domain, a human lck SH2 domain and a human fyn SH2 domain with a binding affinity which is greater than fifty-fold lower than the binding affinity with which the compound binds to such Stat 6 SH2 domain.
  • a preferred aspect of the invention provides a method of treating allergic reactions in a subject which comprises administering to the subject a therapeutically effective amount of a compound which (a) binds to a human Stat 6 SH2 domain with a binding affinity greater than fifty-fold higher than the binding affinity with which the compound binds to a human Stat 5 SH2 domain.
  • a preferred aspect of the invention provides a method of treating allergic reactions in a subject which comprises administering to the subject a therapeutically effective amount of a compound which (a) binds to a human Stat 6 SH2 domain with a binding affinity greater than one hundred-fold higher than the binding affinity with which the compound binds to a human Stat 5 SH2 domain, (b) binds to a human hep SH2 domain, a human Grb2 SH2 domain, a human SH-PTP2 SH2 domain and a human p85 SH2 domain with a binding affinity which is greater than one hundred ⁇ fold lower than the binding affinity with which the compound binds to such Stat 6 SH2 domain, and (c) binds to a human src SH2 domain, a human lck SH2 domain and a human fyn SH2 domain with a binding affinity which is greater than one hundred-fold lower than the binding affinity with which the compound binds to such Stat 6 SH2 domain.
  • a preferred aspect of the invention provides a method of treating allergic reactions in a subject which comprises administering to the subject a therapeutically effective amount of a compound which (a) binds to a human Stat 6 SH2 domain with a binding affinity greater than one hundred-fold higher than the binding affinity with which the compound binds to a human Stat 5 SH2 domain.
  • the inhibitory activity of compounds at the different human SH2 domains is determined in vitro using SH2 domains expressed as fusion proteins either in I coli or in baculovirus as further described in detail in Example 11 below.
  • the data shown in the accompanying Tables 1 and 2 indicate the ability of the stated compounds to antagonize various SH2 domains.
  • Compounds indicated as selective Stat 6 SH2 domain antagonist from assays as demonstrated in Example 11 are tested in known assays for there ability to treat allergic reactions.
  • Preferred assays include: 1 ) Carballido et al. , Int. Arch. Allergy Immunol. 107: ( 1995) 304-307,
  • Activity in these assays is recognized in the art as correlating with efficacy in treating allergic reactions in vivo. Activity in these assays is also recognized in the art as correlating with efficacy in treating asthma in vivo. Activity in these assays is also recognized in the art as correlating with efficacy in treating allergic asthma in vivo. Activity in these assays is also recognized in the art as correlating with efficacy in treating allergic rhinitis in vivo. Activity in these assays is also recognized in the art as correlating with efficacy in treating atopic dermatitis in vivo.
  • the present invention therefore provides a method of treating allergic reactions, which comprises administering a quantity of a Stat 6 SH2 domain antagonists defined as herein in a quantity effective to treat allergic reactions.
  • the drug may be administered to a patient in need of treatment for allergies by any conventional route of administration, including, but not limited to, intravenous, intramuscular, oral, subcutaneous, intradermal, and parenteral.
  • the quantity effective to induce immunosuppression is from about 0.001 mg per kg to about 10.0 mg per kg of subject body weight.
  • the selected dose will be an efficacious, nontoxic quantity selected from about 0.001 mg per kg to about 10.0 mg per kg of subject body weight.
  • the selected dose will be administered from about 1-6 times daily.
  • the method of treating allergic reactions disclosed in the present invention may also be carried out using a pharmaceutical composition comprising a Stat 6 SH2 domain antagonists defined herein and a pharmaceutically acceptable carrier.
  • compositions suitable for oral administration include solid forms, such as pills, capsules, granules, tablets, and powders, and liquid forms, such as solutions, syrups, elixers, and suspensions.
  • forms useful for parenteral administration include sterile solutions, emulsions, and suspensions.
  • the drug may otherwise be prepared as a sterile solid composition which may be dissolved or suspended at the time of administration using sterile water, saline, or other appropriate sterile injectable medium.
  • Carriers are intended to include necessary and inert binders, suspending agents, lubricants, flavorants, sweeteners, preservatives, dyes and coatings.
  • Optimal dosages to be administered may be readily determined by those skilled in the art, and will vary with the particular Stat 6 SH2 domain antagonist in use, the strength of the preparation, the mode of administration, and the advancement of the disease condition. Additional factors depending on the particular patient being treated will result in a need to adjust dosages, including patient age, weight, diet, and time of administration.
  • the invention also provides for the use of a Stat 6 SH2 domain antagonists in the manufacture of a medicament for use in treating allergic reactions.
  • the invention also provides for a pharmaceutical composition for use in the treatment of allergic reactions which comprises a Stat 6 SH2 domain antagonists.
  • ° means °C.
  • L-3,5-Dibromotyrosine can be prepared by methods known in the art, for example as described in "Thyoid Hormones and Analogues. I. Synthesis, Physical Properties and Theoretical Calculations" E. C. Jorgensen, Hormonal Proteins and Peptides, Vol. VI, 1978, Academic Press, N.Y. and references cited therein.
  • L-3,5-dibromo-N-trifluoroacetyl-tyrosine methyl ester (for use in Example 2 (e) and in Example 2B (b)) can be prepared according to the following procedure.
  • L-3,5-Dibromotyrosine 500 g was suspended in methanol (5 liters) and dry hydrogen chloride passed through the stirred suspension for 5 hours. The reaction mixture was evaporated to dryness, the residue suspended in water (4 liters), and the pH adjusted to 6 with 40% sodium hydroxide. The precipitate was collected and washed with water to give L-3,5-dibromotyrosine methyl ester (467 g, 90%), m.p. 20P-203 0 . The ester (768 g) was suspended in chloroform (2.7 liters) and ethyl acetate (2.7 liters), then trifluoroacetic anhydride (565 g) was added over 0.5 hour, keeping the temperature below 35°.
  • the amino group of 4-trans-aminomethyl-cyclohexyl-carboxylic acid 1 is protected with a standard protective group such as with a Boc group (Boc anhydride, NaOH, H 2 O, dioxane) to form 2, then is coupled to Kaiser oxime resin (Kaiser, E.T.; et alJAm Chem Soc 1985, 107, 7087-7092) using a coupling reagent such as DCC to form 3.
  • the amine is then deprotected under standard conditions (25% TFA, methylene chloride) to form 4, then is acylated with standard conditions (such as with HBTU, NMM in DMF or DCC or DIC in DMF or NMP) to form 5.
  • the compound is then cleaved from the resin with various amines to form the final desired product 6.
  • the title compound was prepared by reaction of neat N,N'- diisopropylcarbodiimide (1.0 equiv) with 2-methyl-2-propanol (1.15 equiv) in the presence of CuCl (0.01 equiv) for 1 day at room temperature, according to the procedure of Santini et al. ( J. Org. Chem. 1994, 59, 2261).
  • Example l(i) (351 mg, 1 mmol), tert-butyl 7-amino-3-[2-(5-methyl- 1,3,4- thiadiazolyl)thiomethyl] ⁇ 3 -cephem-4-carboxylate of Example l(j) (368 mg, 1 mmol) and DCC (212 mg, 1 mmol) in dry dichloromethane was stirred at room temperature for 3 hours. The dicyclohexylurea was filtered off and the filtrate was evaporated to dryness.
  • Example 2(c)(2.35 g) was dissolved in dry dichloromethane (20 ml) and cooled with stirring to -50°. Boron tribromide (3 ml) was then added dropwise, and the solution was allowed to warm to room temperature. After 0.5 hours the orange reaction mixture was poured into ice/water (200 ml) and acetone added to dissolve the precipitated solid. The mixture was extracted with dichloromethane, the organic extracts were separated, washed with water, dried, and evaporated. The residue was recrystallised from ethyl acetate and petroleum spirit to give 2-(6-chloro-3- pyridazinylmethyl)-phenol (1.75 g, 80%), m.p. 132°- 132.5°. Anal. Found: C, 59.61; H, 4.13; N, 12.47; Cl, 16.09; C. ⁇ CIN.O Requires: C, 59.87; H, 4.11; N, 12.70; Cl, 16.07%.
  • Aqueous sodium hydroxide (IN, 100 ml, 100 mmol) was added to a solution of 4-trans-aminomethyl-cyclohexyl-carboxylic acid (9.0g, 60 mmol), in dioxane (100 ml), water (100 ml) at 0 degrees C. Boc anhydride (15.9 g, 66 mmol) was added and the reaction was warmed to rt and stirred overnight. The solution was concentrated to 50 ml, then was diluted with EtOAc (100 ml) and acidified to pH 2 with adqueous KHSO 4 (IN). The organic layer was then extracted with water (100 ml) two times, and the organics were concentrated in vacuo. The solid was recrystallized from EtOAc/ hexanes to yield 9.2g + 3.4g (second crop) of a white solid. (80% yield). MS (ES) m/e 242 [M+H] ⁇
  • Kaiser oxime resin (20 g, 0.7 mmol/g loading, Advanced Chem Tech) was added to a solution of N-t-butyloxy carbonyl-trans-4-aminomethyl cyclohexyl carboxylic acid (5.0 g, 20 mmol) and DCC (4.4 g, 20 mmol) in methylene chloride (200 ml) and was gentle mixed at it overnight. The solid was filtered and collected, then washed with methylene chloride (5 x 100 ml).
  • Trans-4-aminomethyl cyclohexyl (Kaiser oxime resin) carboxylate 200 mg was suspended in DMF (3.0 ml) and N-methyl morpholine (0.2 ml) and 4, 4'- benzophenone dicarboxylic acid (190 mg, 0.7 mmol) and HBTU (265 mg, 0.7 mmol) was added and the reaction was gently mixed for 3 h.
  • the solid was filtered and collected, then was washed with with DMF (3 x 20 ml), then water (3 x 20 ml), then was resuspended in DMF (3.0 ml) and N-methyl morpholine (0.1 ml) and 4,4'- benzophenone dicarboxylic acid (0.35 mmol) and HBTU (0.35 mmol) was added and the reaction was gently mixed for 3 h.
  • the solid was filtered and collected, then was washed with with DMF (3 x 20 ml), then water (3 x 20 ml), then methylene chloride (5 x 20 ml), then was dried under vacuum.
  • SH2 Domains The activity of compounds at the different human SH2 domains is determined in vitro using SH2 domains expressed as fusion proteins either in E. coli or baculovirus.
  • the SH2 domains used herein were the human forms of the Stat 6 SH2 domain, src SH2 domain, Grb2 SH2 domain, lck SH2 domain, Stat 5 SH2 domain, fyn SH2 domain, SH-PTP2 SH2 domain, p85 SH2 domain and hep SH2 domain.
  • DET1 defined epitope tag 1
  • HV-l Human Immunodeficiency Virus Type 1 envelope protein gpl 20 (or gpl 60).
  • Monoclonal antibodies to various epitopes of HIV-l gpl 20 (or gpl 60) are known in the art, see, for example U.S. Patent 5,166,050.
  • monoclonal antibody 178.1 (see, e.g., Thiriart et al., J. Immunol.. 143:1832-1836 (1989)), which was prepared by immunization of mice with a yeast-expressed HIV-l gpl60 molecule from strain BH10 (Ratner et al., Nature. 313:277-284 (1985)). This tag was used for detection of expression (by Western blot), for purification of the protein (by affinity chromatography), and for configuring assays in which the fusion protein is captured or immobilized using the 178.1 antibody.
  • DET2 is a hexa- histidine sequence tag (SEQ ID NO: 2) which binds to nickel-containing resins and was used for purification purposes. Spacer (SEQ ID NO: 3) was utilized to design a BamHI restriction site at the indicated position of the construct.
  • the term -ek- refers to a recognition sequence (SEQ ID NO: 4) for the enterokinase protease which provides for the optional removal of the tags from the SH2 domain, thus producing an SH2 domain that contains no extraneous amino acids.
  • SH2 domains which contain no extraneous amino acids are preferable to tagged protein for crystallography studies.
  • SH2 refers to the SH2 domains of different proteins.
  • each DETl-DET2-spacer-ek-SH2 was designed such that the indicated restriction sites (BamHI and Xbal) flank the spacer- ek-SH2 region, thereby allowing different spacer-ek-SH2 contracts to be readily substimted into any one of the vectors described in Procedures 2, 3, 5 or 6 below to create a DETl-DET2-spacer-ek-SH2 tagged protein.
  • the DNA sequence encoding each DETl-DET2-spacer-ek-SH2 construct was also designed such that the entire tagged SH2 domain can be moved as an Ndel-Xbal fragment into any expression vector containing an Ndel site at an appropriate distance downstream of E.
  • the vector used in the instant experiments are the
  • This vector is a derivative ofthe series of vectors described in Shatzman, A, Gross, M, and Rosenberg, M, 1990, "Expression using vectors with phage lambda regulatory sequences", In: Current Protocols in Molecular Biology (F.A. Ausubel et al , eds.), pp. 16.3.1-16.3.11, Greene Publishing and Wiley-Interscience, N.Y. (hereinafter F.A.
  • Procedure 1 Cloning and Expression of chicken src SH2 domain containing tags DET1 and DET2 (DETl-DET2-spacer-SH2).
  • a DNA sequence encoding the tagged protein DETl-DET2-spacer-SH2 was PCR amplified from a cDNA clone containing the chicken src gene (p5H; Levy et al 1986. Proc. Natl. Acad. Sci. USA 83.4228) by methods well known to those skilled in the art by using the following primers:
  • the underlined sites are an Ndel recognition site (5') and a BamHI recognition site (3').
  • the underlined region is an Xbal recognition site.
  • the PCR product was digested with Ndel and Xbal, followed by isolation of the digested fragment on an agarose gel.
  • the fragment was ligated into Ndel-Xbal- digested pEAlKnRBS3 vector (Bergsma et al, supra- that had been agarose gel purified as a 6.5 kbp fragment.
  • the ligation reaction was used to transform R coli MM294c-T (F.A. Ausubel et al., supra).
  • a plasmid containing an insertion of the correct fragment was identified and confirmed by DNA sequencing.
  • the resultant plasmid encodes DETl-DET2-spacer-SH2 under the control of the phage lamda P L promoter and regulatory system.
  • Plasmid DNA was purified from MM294cI + and used to transform R coli strain ARl 20. In this host strain, expression of the phage promoter can be induced by addition of nalidixic acid to the growing culture as described in F.A. Ausubel et al, supra. Nalidixic acid induction of AR120 containing this plasmid, followed by analysis of the cellular proteins on an SDS- polyacrylamide gel stained with Coomassie Blue (F.A.
  • Procedure 2 Cloning, expression and purification of human src SH2 domain containing tags and an enterokinase proteolytic cleavage site (DETl-DET2-spacer- ek-src SH2).
  • a DNA sequence encoding protein ek-src SH2 was PCR amplified from a cDNA clone containing the human src gene (c-src SH2 DNA sequence identical to that described in Takeya,T. and Hanafusa, H, 1983 Cell 32:881-890) using the following primers:
  • the underlined site is a BamHI recognition site.
  • the underlined region is an Xbal recognition site.
  • the PCR product was digested with BamHI and Xbal, followed by isolation of the digested fragment on an agarose gel.
  • the fragment was ligated into BamHI- Xbal-digested expression vector containing the tagged chicken src gene DET1- DET2-spacer-SH2 described in Procedure 1 above.
  • the BamHI site is located between the coding regions for DET2 and SH2, and the Xbal site is located after the 3' end of the SH2 coding region.
  • the ligation reaction was used to transform ] c ⁇ ji MM294cI + .
  • Procedure 3 Cloning and expression of human lck SH2 domain containing tags and an enterokinase proteolytic cleavage site (DETl-DET2-spacer-ek-lck SH2).
  • a DNA sequence encoding protein ek-lck SH2 was PCR amplified from a cDNA clone containing the human lck gene (Genbank accession number M36881) using the following primers:
  • the underlined site is a BamHI recognition site.
  • the underlined region is an Xbal recognition site.
  • the PCR product was digested with BamHI and Xbal, followed by isolation of the digested fragment on an agarose gel.
  • the fragment was ligated into BamHI- Xbal-digested expression vector containing the tagged chicken src gene DET1- DET2-spacer-SH2 described in Procedure 1 above.
  • the BamHI site is located in between the coding regions for DET2 and SH2, and the Xbal site is located after the 3' end of the SH2 coding region.
  • the ligation reaction was used to transform E.
  • Procedure 4 Cloning and expression of human hep SH2 domain containing tags and an enterokinase proteolytic cleavage site (DETl-DET2-spacer-ek-hcp SH2).
  • a DNA sequence encoding protein ek-hep SH2 (hep SH2 DNA sequence identical to that described in Shen, S-H. Nature (1991) 352: 736-739) was reverse transcriptase-PCR amplified from human fetal liver RNA.
  • RNA isolation used Tri- Reagent (Molecular Research Center Inc.) and the Reverse Transcriptase system (GIBCO-BRL) according to the manufacture's instructions. PCR was carried out using the following primers:
  • the underlined site is a Bglll recognition site.
  • the underlined region is an Xbal recognition site.
  • the PCR product was digested with Bglll and Xbal, followed by isolation of the digested fragment on an agarose gel.
  • the fragment was ligated into BamHI- Xbal-digested expression vector containing the tagged human src gene DET1- DET2-spacer-ek-src SH2 described in Procedure 2 above.
  • the BamHI site is located in between the coding regions for DET2 and ek
  • the Xbal site is located after the 3' end ofthe SH2 coding region.
  • the ek-hcp SH2 sequence replaced the ek-src SH2 sequence in the above vector.
  • the ligation reaction was used to transform R coli MM294cI + .
  • the construct containing DET1- DET2-spacer-ek-hcp SH2 was confirmed by DNA sequencing (SEQ ID NO: 7) and used to transform R £ ⁇ ii GI698 (Invitrogen Corporation, San Diego, CA). Induction of the phage lambda promoter was induced by addition of tryptophan to the culture medium to 10 mg/ml, per the manufacture's instructions. A Coomassie-Blue- stained, Western-blot-positive induced protein band with an apparent molecular weight of 15,000 was observed after tryptophan induction of cells growing at 30° C. Cells were lysed at neutral pH by sonication in the presance of lysozyme.
  • the insoluble pellet was solubilized with 8 M urea in Tris buffer pH 8 and bound onto a Ni NTA column.
  • the resin was washed with equilibration buffer (Tris buffer pH 8 containing 0.5 M NaCl, 8 M urea and 5 mM BME) and the same buffer containing 15 mM imidazole.
  • the protein was refolded on the column during the removal of urea in the presence of 5 mM BME and the purified refolded protein eluted with 300 mM imidazole in Tris buffer pH 8.
  • the SH2 domain purified in this fashion, was found to bind with high affinity in a specific, saturable fashion to the appropriate pY peptide in the "Binding Assays" described below, demonstrating that the tag did not interfere with function and that the protein was refolded successfully.
  • This expressed fusion protein, DETl-DET2-spacer-ek-hcp SH2 was utilized in the "Binding Assays” described below in order to determine the specificity of compounds to selectively inhibit the human hep SH2 domain.
  • Procedure 5 Cloning, expression and purification of human Stat 6 SH2 domain containing tags and an enterokinase proteolytic cleavage site (DETl-DET2-spacer- ek-Stat 6 SH2).
  • a DNA sequence encoding protein ek-Stat 6-SH2 was PCR amplified from a cDNA clone containing the human Stat 6 gene (Stat 6 SH2 DNA sequence identical to that described in Science 265. (1994) 1701) using the following primers:
  • the underlined site is a BamHI recognition site.
  • the underlined region is an Xbal recognition site.
  • the PCR product was digested with BamHI and Xbal, followed by isolation of the digested fragment on an agarose gel.
  • the fragment was ligated with the vector fragment derived from BamHI-Xbal digestion of the expression vector containing the tagged human src gene DETl-DET2-spacer-ek-src SH2 described in Procedure 2 above.
  • the BamHI site is located between the coding regions for DET2 and SH2
  • the Xbal site is located after the 3' end of the SH2 coding region.
  • the ligation reaction was used to transform R coli MM294cI+.
  • the construct DETl-DET2-spacer-ek-Stat 6 SH2 was confirmed by DNA sequencing (SEQ ID NO: 29) and induced in R coli strain GI698 (Invitrogen Co ⁇ oration, San Diego, CA). Induction of the phage lambda promoter was induced by addition of tryptophan to the culture medium to 10 mg/ml, per the manufacture's instructions, also as described in procedure 4. A Coomassie-Blue-stained, Western-blot-positive induced protein band with an apparent molecular weight of 15,000 was observed after tryptophan induction of cells growing at 27° C.
  • Procedure 6 Cloning, expression and purification of human Stat 5 SH2 domain containing tags and an enterokinase proteolytic cleavage site (DETl-DET2-spacer- ek-Stat 5 SH2).
  • Restriction sites for BamHI and Xbal are underlined in the sequence above.
  • the ek-Stat 5-SH2 gene sequence was digested with BamHI and Xbal, followed by isolation of the digested fragment on an agarose gel.
  • the fragment was ligated with the vector fragment derived from BamHI-Xbal digestion of the expression vector containing the tagged human src gene DETl-DET2-spacer-ek-src SH2 described in Procedure 2 above.
  • the BamHI site is located between the coding regions for DET2 and SH2, and the Xbal site is located after the 3' end of the SH2 coding region.
  • the ligation reaction was used to transform R coli MM294cI+.
  • the construct DETl-DET2-spacer-ek-Stat 5 SH2 was confirmed by DNA sequencing (SEQ ID NO: 32) and induced in R coli strain GI698 (Invitrogen Co ⁇ oration, San Diego, CA). Induction of the phage lambda promoter was induced by addition of tryptophan to the culmre medium to 10 mg/ml, per the manufacture's instructions, also as described in procedure 4. A Coomassie-Blue-stained, Western- blot-positive induced protein band with an apparent molecular weight of 15,000 was observed after tryptophan induction of cells growing at 27° C.
  • Fusion proteins having the strucmre GST-X-SH2 are prepared as described in the GST gene fusion kit system available from Pharmacia (New Jersey).
  • GST is the tagging sequence glutathione s-transferase epitope (SEQ ID NO: 8) for fyn, Grb2 and SH-PTP2 and is the tagging sequence glutathione s-transferase epitope (SEQ ID NO: 9) for p85.
  • SH2 refers to the SH2 domains of fyn, Grb2, p85 and SH-PTP2 which were expressed and purified using glutathione Sepharose 4B (Pharmacia) according to "Current Protocols in Molecular Biology", ed.
  • X is an appropriate linker, preferably of 6 to 21 base pairs, used to keep the SH2 construct in frame and complement cloning. As such, the sequence of X is not critical. One skilled in the art can readily construct the appropriate linker.
  • the DNA sequence encoding each GST-X-SH2 fusion protein was designed such that the indicated restriction sites (BamHI and EcoRI) flank the SH2 region.
  • the vector used in the instant experiments was the R coli expression vector ⁇ GEX-2T (Pharmacia) for fyn, Grb2 and SH-PTP2, and pGEX-3X (Pharmacia) for p85. Each of these vectors result in SH2 constructs having additional C-terminal amino acids as described below.
  • the sequence encoding the SH2 domain of human fyn (amino acids 143- 252) (Yamamoto, T. et al. Proc. Natl. Acad. Sci. USA 83. 5459-5463 (1986)) was cloned into the BamHI and EcoRI sites of the expression vector pGEX-2T.
  • the SH2 domain including the additional C-terminal amino acids leucine-threonine- asparagine-serine-serine (SEQ ID NO: 10) was cloned by PCR techniques known to those skilled in the art to yield the expressed fusion protein GST-X-fyn. This expressed fusion protein was then utilized in the "Binding Assays" described below in order to determine the specificity of compounds to selectively inhibit the human fyn SH2 domain.
  • Human p85 SH2 domain The sequence encoding the SH2 domain of human p85 (amino acids 321-440) (Skolnik, E. et al., CeU 65, 83-90 (1991)) was cloned into the BamHI and EcoRI sites of the expression vector pGEX-3X.
  • the SH2 domain including the additional C-terminal amino acids asparagine-serine-serine (SEQ ID NO: 1 l) was cloned by PCR techniques known to those skilled in the art to yield the expressed fusion protein GST-X-p85. This expressed fusion protein was then utilized in the "Binding Assays" described below in order to determine the specificity of compounds to selectively inhibit the human p85 SH2 domain.
  • Human SH-PTP2 SH2 domain The sequence encoding the SH2 domain of human SH-PTP2 (amino acids l-106))(Bastien, L. et al., Biochem. Biophys. Res. Commun. 196. 124-133 (1993)) was cloned into the BamHI and EcoRI sites of the expression vector pGEX-2T.
  • the SH2 domain including the additional C-terminal amino acids glutamine-phenylalanine-isoleucine-valine-threonine-aspartate (SEQ ID NO: 12) was cloned by PCR techniques known to those skilled in the art to yield the expressed fusion protein GST-X-SH-PTP2. This expressed fusion protein was then utilized in the "Binding Assays" described below in order to determine the specificity of compounds to selectively inhibit the human SH-PTP2 SH2 domain.
  • Human Grb2 SH2 domain The sequence encoding the SH2 domain of human Grb2 (amino acids 58-159) (Lowenstein, E. et al., Ceil 70, 431-442 (1992)) was cloned into the BamHI and EcoRI sites of the expression vector pGEX-2T.
  • the SH2 domain including the additional C-terminal amino acids isoleucine-histidine- arginine-aspartate (SEQ ED NO: 25) was cloned by PCR techniques known to those skilled in the art to yield the expressed fusion protein GST-X-Grb2.
  • a six nucleotide linker was used and resulted in the amino acids glycine and serine between the GST and SH2 domain. This expressed fusion protein was then utilized in the "Binding Assays" described below in order to determine the specificity of compounds to selectively inhibit the human Grb2 SH2 domain.
  • Binding Assays The potency of compounds at the SH2 domains is determined based on the ability of such compounds to selectively inhibit such SH2 domain from binding to its respective specific pY peptide.
  • the binding assays for the SH2 domains and pY peptides are performed in an ELISA-based 96 well plate assay.
  • hydrophilic Durapore® pore size 0.65um Cat. No. MADVN6550
  • 2 ul 50% suspension
  • Protein-G Sepharose available from Pharmacia of N.J. Cat. No.
  • TBS-T 4°C
  • 90 ul of TBS-T is then added to each well.
  • Specific pY biotinylated peptides are diluted to a concentration of 1.0 uM in TBS-T (these peptides can be obtained from Bachem Bioscience of Pennsylvania, Genosys Biotechnologies of Texas and California Peptide Research of California). 10 ul is aliquoted per well to yield a final concentration of 0.1 uM (approx. the K for each SH2 domain peptide pair) and a final volume of 100 ul.
  • the assay plates are incubated until equilibrium binding was attained (3 hr at 4°C with shaking).
  • the assay plates are washed 2 X per well TBS-T (4°C), then 100 ul of SABC (Strepavidin biotinylated horseradish peroxidase complex, available from the Zymed co ⁇ oration of California cat. no. 93-0043, 1 drop reagent A (streptavidin) and 1 drop of reagent B (AH-biotin conjugated-horseradish peroxidase) per 10 ml of TBS- T, incubated at 37°C for 30 minutes, then cooled to 4°C) is added per well, then incubated at 4°C for 30-60 minutes. The plates are then washed 4 X with TBS-T (4°C) (250 ul/well)/wash).
  • SABC Stepavidin biotinylated horseradish peroxidase complex, available from the Zymed co ⁇ oration of California cat. no. 93-0043
  • 1 drop reagent A streptavidin
  • KD (uM)KD value for the specific pY and biotinylated peptide for each SH2 domain
  • IC 50 is the concentration of inhibitor at which the response or signal is inhibited by
  • KD is the dissociation constant for a ligand in a receptor/ligand interaction, normally equaling the concentration of ligand which is at 1/2 Vmax on a saturation binding curve>
  • the pY peptide ligands used in the above Binding Assays are as follows:
  • Biotinylated pY peptide ligand containing an aminocaproic acid (Aca) linker used for p85 SH2 Asp-Gly-Gly-pTyr-Met-Asp-Met-Ser-Lys-Asp-Glu (SEQ ID NO: 14)
  • Biotinylated pY peptide ligand containing an aminocaproic acid (Aca) linker used for SH-PTP2 SH2
  • Tables I and II illustrate the cross reactivity of SH2 antagonists at the indicated SH2 domains. From the results disclosed in these tables compounds which have binding affinities/inhibitory concentrations which are greater than fifty-fold higher at the Stat 6 SH2 domain than the binding affinities/inhibitory concentrations at other SH2 domains can be readily identified.
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • FRAGMENT TYPE internal
  • ORIGINAL SOURCE
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • FRAGMENT TYPE internal
  • ORIGINAL SOURCE
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • FRAGMENT TYPE internal
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • FRAGMENT TYPE internal
  • ORIGINAL SOURCE
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • FRAGMENT TYPE internal
  • ORIGINAL SOURCE
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • FRAGMENT TYPE internal
  • ORIGINAL SOURCE
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • FRAGMENT TYPE (vi) ORIGINAL SOURCE:
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • FRAGMENT TYPE (vi) ORIGINAL SOURCE:
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • FRAGMENT TYPE
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • FRAGMENT TYPE (vi) ORIGINAL SOURCE:
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • FRAGMENT TYPE internal
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • FRAGMENT TYPE internal
  • ORIGINAL SOURCE
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • FRAGMENT TYPE internal
  • ORIGINAL SOURCE

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Abstract

L'invention porte sur une méthode de traitement de réactions allergiques chez un sujet consistant à lui administrer une quantité à effet thérapeutique d'un composé qui se fixe au domaine humain Stat 6 SH2 avec une affinité plus de 50 fois supérieure à celle avec laquelle il se fixe au domaine humain Stat 5 SH2, et une affinité plus de 50 fois inférieure à celle avec laquelle il se fixe aux domaines humains hcp SH2, Grb2 SH2, SH-PTP2 SH2 et p85 SH2, et qui se fixe aux domaines humains src SH2, lck SH2 et fyn SH2 avec une affinité plus de 50 fois inférieure à celle avec laquelle il se fixe au domaine Stat 6 SH2.
EP96924322A 1995-06-30 1996-06-28 UTILISATION DE COMPOSES SPECIFIQUES DU DOMAINE Stat 6 SH2 POUR LE TRAITEMENT DES REACTIONS ALLERGIQUES Withdrawn EP0871436A4 (fr)

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US49735795A 1995-06-30 1995-06-30
US59871696A 1996-02-08 1996-02-08
US598716 1996-02-08
PCT/US1996/011074 WO1997002023A1 (fr) 1995-06-30 1996-06-28 UTILISATION DE COMPOSES SPECIFIQUES DU DOMAINE Stat 6 SH2 POUR LE TRAITEMENT DES REACTIONS ALLERGIQUES

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KR100481631B1 (ko) * 1996-12-28 2005-08-17 주식회사 엘지생활건강 세안화장료조성물
GB9705212D0 (en) * 1997-03-13 1997-04-30 Ciba Geigy Ag Chemical compounds
AU9123898A (en) * 1997-08-27 1999-03-16 Government Of The United States Of America, As Represented By The Secretary Of The Department Of Health And Human Services, The Attenuated and dominant negative variant cdnas of stat6: stat6b and stat6c
KR100503659B1 (ko) * 1997-12-26 2005-11-16 주식회사 엘지생활건강 전기 면도기용 화장료 조성물
KR100444900B1 (ko) * 2002-02-05 2004-08-21 주식회사 태평양 화장비누 및 그의 제조방법
AU2004224191A1 (en) 2003-03-24 2004-10-07 F. Hoffmann-La Roche Ag Non-nucleoside reverse transcriptase inhibitors
CN1934092A (zh) 2004-03-23 2007-03-21 弗·哈夫曼-拉罗切有限公司 非核苷逆转录酶抑制剂
DK1919878T3 (da) 2005-07-21 2010-10-25 Hoffmann La Roche Pyridazinonderivater som thyroidhormonreceptoragonister
US8076334B2 (en) 2007-09-20 2011-12-13 Hoffmann-La Roche Inc. Prodrugs of thyroid hormone analogs
LT3037104T (lt) * 2009-10-20 2020-09-10 Abbvie Inc. Anti-il-13 antikūnų izoliavimas ir gryninimas, naudojant afininę baltymo a chromatografiją
CN103360337B (zh) * 2013-07-11 2016-04-27 浙江海蓝化工集团有限公司 一种2-巯基-5-甲基-1,3,4-噻二唑的制备方法

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US5721266A (en) * 1993-05-28 1998-02-24 Warner-Lambert Company Substituted imidazolinyl-imidazolines as antagonists of SH-2 binding and therapeutic uses thereof

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CA2054602C (fr) * 1991-10-31 2003-04-22 Anthony Pawson Methode de dosage d'une substance influant sur un systeme de regulation d'un ligand phosphoryle-sh2
WO1994007913A1 (fr) * 1992-09-25 1994-04-14 Warner-Lambert Company Antagonistes peptidiques de la fixation du sh2 et leurs usages therapeutiques

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US5721266A (en) * 1993-05-28 1998-02-24 Warner-Lambert Company Substituted imidazolinyl-imidazolines as antagonists of SH-2 binding and therapeutic uses thereof
WO1997007131A1 (fr) * 1995-08-17 1997-02-27 Novartis Ag Oligopeptides acyles divers

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Title
See also references of WO9702023A1 *

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