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WO1997031016A2 - Nouveaux inhibiteurs des processus lies a la presence de sh2 - Google Patents

Nouveaux inhibiteurs des processus lies a la presence de sh2 Download PDF

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Publication number
WO1997031016A2
WO1997031016A2 PCT/US1997/002636 US9702636W WO9731016A2 WO 1997031016 A2 WO1997031016 A2 WO 1997031016A2 US 9702636 W US9702636 W US 9702636W WO 9731016 A2 WO9731016 A2 WO 9731016A2
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Prior art keywords
compound
compounds
fmoc
aryl
domain
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PCT/US1997/002636
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English (en)
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WO1997031016A3 (fr
Inventor
Berkley A. Lynch
Manfred Weigele
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Ariad Pharmaceuticals, Inc.
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Priority to AU22775/97A priority Critical patent/AU2277597A/en
Publication of WO1997031016A2 publication Critical patent/WO1997031016A2/fr
Publication of WO1997031016A3 publication Critical patent/WO1997031016A3/fr

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    • 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
    • C07K5/1016—Tetrapeptides with the first amino acid being neutral and aromatic or cycloaliphatic
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • cellular signal transduction the series of events leading from extracellular events to intracellular sequelae.
  • Numerous proteins that function as signal transducing molecules have been identified. These include receptor and non-receptor tyrosine kinases, phosphatases and other molecules with enzymatic or regulatory activities.
  • a common feature of many of these molecules is their capacity to associate specifically with other proteins to form a signaling complex that can alter cell activity.
  • PTKs protein tyrosine kinases
  • ISGF3alpha pi 13 ⁇ 91/84, Tensin, she; syk, zap, PTPase IC and PTPase 2; src and the src family of PTKs, abl and the abl farnily of PTKs, csk, tec; PLCgamma 1 and 2, GAP, p85alpha and p85 beta; vav, c-crk and GRB2; and nek.
  • PTKs protein tyrosine kinases
  • SH2 domains direct the association of specific proteins by binding selectively and with specificity to protein sequences containing phosphotyrosine. For example, upon binding of PDGF to the PDGF ⁇ -receptor, the receptor dimerizes and autophosphoiylates multiple tyrosine residues. This phosphorylation triggers the physical association of SH2-containing proteins such as c-src, PLC-gamma, PI3K and ras-GAP with the receptor, forming a signaling complex.
  • SH2-containing proteins such as c-src, PLC-gamma, PI3K and ras-GAP
  • This invention relates to compounds of formula:
  • A is H, Rl, -COR* or -CO- ORI where R is a substituted or unsubstituted alkyl, heteroalkyl, aryl or heteroaryl group and J is H or N ⁇ 2- L- forms of the compounds are currently preferred, although D- forms and racemic mixtures are also encompassed by this invention, as are N-alkylated derivatives thereof.
  • Alkyl as the term is used herein, is intended to include saturated and unsaturated, linear (straight-chain), branched, cyclic, and polycyciic aliphatic hydrocarbons, generally containing 1 - 10 contiguous aliphatic carbon atoms, which are optionally substituted with one or more functional groups selected from the group consisting of hydroxy, Ci-C ⁇ alkoxy, acyloxy, carbamoyl, amino, N-acylamino, keto, halo (chloro, bromo, fluoro or iodo), trihalomethyl, cyano, carboxyl, alkyl, cycloalkyl, aryl and heteroaryl, which functional groups may themselves (with the exception of hydroxy, halo and cyano groups) bear one or more of the foregoing functional groups.
  • functional groups selected from the group consisting of hydroxy, Ci-C ⁇ alkoxy, acyloxy, carbamoyl, amino, N-acylamino, keto,
  • alkyl, alkoxy and acyl groups contain 1-6 contiguous aliphatic carbon atoms, and may bear substituents.
  • Aryl as the term is used herein, is intended to include stable cyclic, heterocyclic, polycyciic, and polyheterocyclic unsaturated C3-C14 moieties (exemplified by but not limited to phenyl, biphenyl, naphthyl, pyridyl, furyl, thiophenyl, imidazoyl, pvri idinyl, and oxazoyl) which may be substituted with one to five functional groups selected from the group consisting of hydroxy, C1-C8 alkoxy, C3.-C8 branched or straight-chain alkyl, acyloxy, carbamoyl, amino, N-acylamino, nitro, halo, trifluoromethyl, cyano, and carboxyl, which functional groups may themselves (with the exception of hydroxy,
  • Heteroalkyl and heteroaryl refer to alkyl and aryl moieties respectively, which contain one or more of oxygen, sulfur, or nitrogen in place of one or more carbon atoms.
  • -CO-R 1 may also comprise a substituted or unsubstituted amino acid, itself containing an optional substituent A as previously defined (acetyl in the illustrations below) on its amino group, and an alkyl, aryl, arylalkyl, heteroaryl or heteroarylalkyl substituent on the alpha carbon, including for example:
  • Y is -CH2-, -O-, or -NH- (which may bear an aryl or alkyl substituent).
  • B is an aryl moiety or an alkyl moiety which may be linear, branched or cyclic (e.g. monocyclic, bicyclic or tricyclic), and in either case, may be further substituted. In some cases B preferably contains a hydrophobic substituent.
  • hydrophobic we mean a moiety comprising at least 3 contiguous carbon atoms.
  • Y-B may be an amino acid (including, among others, hydrophobic amino acids such as lie, Leu, Nle, Phe, Nal), a dipeptide (such as Glu-Thr, Asp-Val, Glu-Glu, Ser-Glu, or Asn-Glu), a tripeptide, a tetrapeptide or an oligopeptide, comprising one or more D- or L- amino acids (one or more of which may bear alkyl substituents on their alpha nitrogen atom(s)); or an alkyl or aryl ester, amide or carbamate derivative of any of the foregoing.
  • hydrophobic amino acids such as lie, Leu, Nle, Phe, Nal
  • a dipeptide such as Glu-Thr, Asp-Val, Glu-Glu, Ser-Glu, or Asn-Glu
  • a tripeptide such as Glu-Thr, Asp-Val, Glu-Glu, Ser-Glu, or Asn-Glu
  • a tripeptide such as Glu
  • B is a moiety containing about 6 to 50 atoms, with the proviso that in embodiments in which J is N ⁇ 2, where A is tBOC or triflouroacetyl, YB is not -OEt; where A is acetyl, YB is not -OEt, -OMe, or L-Phe; and where A is H, L- phenylalanyl- or N-cbz-L-phenylalanyl-, YB is not -OMe.
  • Illustrative Y-B substituents containing a monocyclic B moiety include the following:
  • Y-B substituents containing a bicyclic B moiety include the following:
  • R ⁇ is - , O or O2; ⁇ is as defined above; and R ⁇ is H, alkyl or aryl, preferably lower (i.e., 1-6 carbons) alkyl; and
  • R3 is as defined above and n and m are independently 1 or 2 (including individual stereoisomers thereof as well as mixtures of one or more of the stereoisomers).
  • R ⁇ is an aryl, arylalkyl or arylcycloalkyl group such as are illustrated above;
  • R ⁇ is an alkyl or aryl group and is preferably a Cl-6 aliphatic group such as methyl, ethyl, n-propyl, i-propyl, allyl and so forth;
  • R aa is H or an aryl or alkyl group and is preferably a side-chain of a naturally occurring amino acid (e.g., H (glycine), -CH3 (alanine), -CH2C6H5 (phenylalanine), and so forth); as well as esters, amides, carbamates and pharmaceutically acceptable salts thereof.
  • compounds of this invention which bind to a protein containing at least one SH2 domain with an affinity (i.e. an IC50 value, e.g. an IC50 BIAcore value) of at least about 200 ⁇ M, and more preferably at least about 100 ⁇ M, and more preferably at least about 50 ⁇ M as measured by any assay commonly used in the art Compounds with IC50 values below 10 ⁇ M are of particular interest. Compounds are also preferred which bind to one SH2 domain with an affinity at least an order of magnitude greater than with respect to a different SH2 domain, again, as may be determined by any conventional assay. Compounds of this invention interfere with intermolecular and/or intramolecular interactions which would otherwise be mediated by that SH2 domain.
  • an affinity i.e. an IC50 value, e.g. an IC50 BIAcore value
  • this invention further comprises a method for enhancing the binding of a compound of interest to an SH2 domain, where the compound which contains a YB moiety, which method comprises covalently attaching a moiety of formula L_(see below) to the compound of interest to produce a compound of formula M.
  • compounds of this invention may be prepared by condensing a tyrosine derivative, L, (or an activated form thereof) with the desired H-YB compound using conventional materials and methods such as are used routinely in peptide chemistry.
  • 3-Nitro- and 3,5-dinitro derivatives of the formula L are thus important intermediates, especially where A (in the structure depicted above) is other than H, acetyl, or N-acetyl-L-phenylalanyl in compounds in which J is nitro.
  • 3,5-Dinitrotyrosine intermediates are of particular interest, especially those that contain at least one phenyl substituent such as in the case of N- Fmoc-3,5-dinitro-L-tyrosine.
  • 3-Nitrotyrosine and 3,5-dimtrotyrosine may be prepared as described herein.
  • the underivatized L-amino acids and N-Fmoc-3-nitro-L- tyrosine may be obtained-rr m commercial sources. They may be modified by the addition of -R 1 , -COR* or -CO(ORl) using conventional methods and materials.
  • Preparation of representative compounds of this invention and intermediates useful for preparing them are detailed in the Experimental Examples which follow.
  • Compounds of this invention may be evaluated for binding activity with respect to one or more SH2 domains of interest, or with respect to proteins containing the SH2 domain(s), using various approaches, a number of which are well known in the art. For instance, compounds may be evaluated for activity as competitive inhibitors of the binding of an SH2 domain with a phosphorylated hgand thereto. See e.g. Pawson, US Patent No. 5,352,660 (4 October 1994). Compounds may be evaluated for binding to one or more SH2 domains of interest using surface plasmon resonance (BIAcore®) technology. See e.g., Panayotou et al, 1993, Molecular and Cellular Biology 13: 3567-3576.
  • BIOS® surface plasmon resonance
  • Compounds may further be evaluated for activity in inhibiting cellular or other biological events mediated by a pathway involving the SH2-based interaction of interest using a suitable cell-based assay or an animal model.
  • Cell-based assays and animal models suitable for evaluating inhibitory actvity of a compound with respect to a wide variety of cellular and other biological events are known in the art. New assays and models are regularly developed and reported in the scientific literature.
  • compounds which bind to an SH2 domain involved in the transduction of a signal leading to asthma or allergic episodes may be evaluated in a mast cell or basophil , degranulation assay.
  • the inhibitory activity of a compound of this invention with respect to cellular release of specific mediators such as histamine, leukotrienes, hormonal mediators and /or cytokines as well as its biological activity with respect to the levels of phosphatidylinositol hydrolysis or tyrosine phosphorylation can be characterized with conventional in vitro assays as an indication of biological activity.
  • mediators such as histamine, leukotrienes, hormonal mediators and /or cytokines
  • compounds Prior to in vivo models, compounds may also be tested in an ex-vivo assay for their ability to block antigen-stimulated contraction of sensitized guinea pig tracheal strip tissue. Activity in this assay has been shown to be useful in predicting the efficacy of potential anti-asthma drugs.
  • Numerous animal models of asthma have been developed and can be used (for reviews, see Larson, "Experimental Models of Reversible Airway Obstruction", in THE LUNG, Scientific Foundations, Crystal, West gt al (eds.), Raven Press, New York, pp. 953-965 (1991); Warner et a 1990, Am. Rev. Respir. Pis. 141:253-257).
  • mice used in animal models of asthma include mice, rats, guinea pigs, rabbits, dogs, sheep and primates. Other in vivo models available are described in Cross et al.. Lab Invest. 63:162-170 (1990)); and Koh, et al.. Science, 256:1210- 1213 (1992)).
  • compounds of this invention which bind to an SH2 domain involved in the transduction of a signal involved in the initiation, maintenance or spread of cancerous growth may be evaluated in relevant conventional in vitro and in vivo assays. See e.g., Ishii et al., J. Antibiot. XLII:1877-1878 (1989) (in vitro evaluation of cytotoxic /antitumor activity); Sun et al, US Patent 5,206,249 (issued 27 April 1993)(in vitro evaluation of growth inhibitory activity on cultured leukemia cells); and Sun et al, supra (xenograft models using various human tumor cell lines xenografted into mice, as well as various transgenic animal models).
  • Compounds of this invention may be used as biological reagents in assays as described herein for functional classification of an SH2 domain of a particular protein, particularly a newly discovered protein. Families or classes of SH2-bearing proteins may now be defined functionally, with respect to Ugand specificity.
  • compounds of this invention can be used to inhibit the occurrence of biological events resulting from molecular interactions mediated by an SH2 domain.
  • This invention thus provides a method and reagents for inhibiting (totally or partially) the interaction between a protein containing an SH2 domain and a natural Hgand thereto (i.e., a protein which normally binds in a cell to the SH2-bearing protein) or a biological activity mediated by such interaction.
  • an SH2 binding or blocking compound of this invention is combined or contacted with the SH2 dornain-containing protein, such as by introducing the compound into a cell in which the SH2-mediated interaction is to be inhibited.
  • the SH2 domain-bearing protein and its natural Hgand is inhibited as may be readily detected. Inhibiting such interactions can be useful in research aimed at better understanding the biology of SH2-mediated events.
  • SH2 binding or blocking agents would be useful, for example, in the diagnosis, prevention or treatment of conditions or diseases resulting from a cellular processes mediated by an SH2-based interaction.
  • a patient can be treated to prevent the occurence or progression of osteoporosis or to reverse its course by administering to the patient in need thereof an SH2 binding or blocking agent which selectively binds Src SH2.
  • SH2 binding or blocking agents may be useful therapeutically, including breast cancer where the SH2 domain-containing proteins Src, PLC ⁇ and Grb7 have been impUcated.
  • Other relevant conditions include prostate cancer, in which case targeting Grb2, PLC ⁇ , and PI3K, all of which contain SH2 domains, may be useful in treatment or prevention of the disease.
  • Inhibition of the interaction of Grb2 or Abl SH2 domains with Bcr-abl may be useful to treat chronic mylogenous leukemia (CML) or acute myelogenous leukemia (AML).
  • StUl other relevant appHcations of an SH2 inhibitor would be to prevent interferon-, growth factor-, or cytokine-mediated diseases (e.g. inflammatory diseases) by targeting the SH2 domains of STAT proteins.
  • Agents that block the SH2 domains of ZAP-70, which is beHeved to be involved in activation of T-cells, would be useful in the treatment of autoimmune diseases.
  • a compound that blocks one or both SH2 domains of ZAP-70 would also be useful as an immunosuppressant to prevent rejection of skin and organ transplants.
  • An SH2 binding or blocking agent of this invention can be formulated into a pharmaceutical composition containing a pharmaceutically acceptable carrier and/or other excipient(s) using conventional materials and means.
  • a composition can be administered to an animal, either human or non- human, for therapy of a disease or condition resulting from ceHular events involving an SH2-mediated molecular interaction. Administration of such composition may be by any conventional route (parenteral, oral, inhalation, and the like) using appropriate formulations as are weU known in this art.
  • the SH2 binding or blocking agent of this invention can be employed in admixture with conventional excipients, ie, pharmaceuticaHy acceptable organic or inorganic carrier substances suitable for parenteral administration.
  • a compound of this invention may be used in pharmaceutical compositions and methods for treatment or prevention in a mammal in need thereof.
  • Mammals include rodents such as mice, rats and guinea pigs as well as dogs, cats, horses, cattle, sheep, non-human primates and humans.
  • the preferred method of such treatment or prevention is by administering to a mammal an effective amount of the compound to prevent, aUeviate or cure said disease or disorder.
  • effective amounts can be readily determined by evaluating the compounds of this invention in conventional assays weU-known in the art, including assays described herein.
  • the invention provides methods of treating, preventing and/or alleviating the symptoms and /or severity of a disease or disorder referred to above by administration to a subject of a in an amount effective therefor.
  • the subject will be an animal, including but not limited to animals such as cows, pigs, chickens, etc., and is preferably a mammal, and most preferably human.
  • Various dehvery systems are known and can be used to administer the SH2 inhibitor, e.g., encapsulation in Hposomes, microparticles, microcapsules, etc.
  • One mode of deHveiy of interest is via pulmonary adrrdnistration, as detailed more fully infra.
  • the SH2 inhibitor may be adrninistered by any convenient route, for example by infusion or bolus injection, by absorption through epitheHal or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Administration can be systemic or local.
  • preferred routes of adrrdnistration are oral, nasal or via a bronchial aerosol or nebulizer.
  • the SH2 inhibitor locaUy may be desirable to administer to the area in need of treatment; this may be achieved by, for example, and not by way of limitation, local infusion during surgery, topical appHcation, by injection, by means of a catheter, by means of a suppository, or by means of a skin patch or implant, said implant being of a porous, non- porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers.
  • compositions comprise a therapeuticaUy (or prophylactically) effective amount of the SH2 inhibitor, and a pharmaceutically acceptable carrier or excipient.
  • a pharmaceutically acceptable carrier includes but is not limited to saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof.
  • the carrier and composition can be sterile. The formulation should suit the mode of administration.
  • the composition can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.
  • the composition can be a Hquid solution, suspension, emulsion, tablet, pill, capsule, sustained release formulation, or powder.
  • the composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides.
  • Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, ceUulose, magnesium carbonate, etc.
  • the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous adrriinistration to human beings.
  • compositions for intravenous administration are solutions in sterile isotonic aqueous buffer.
  • the composition may also include a solubilizing agent and a local anesthetic to ease pain at the side of the injection.
  • the ingredients are suppHed either separately or mixed together in unit dosage form, for example, as a lyophilized powder or water free concentrate in a hermeticaUy sealed container such as an ampoule or sachette indicating the quantity of active agent.
  • composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline.
  • an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to acLrrunistration.
  • Adrninistration to an individual of an effective amount of the SH2 inhibitor can also be accomplished topicaUy by administering the compound(s) directly to the affected area of the skin of the individual.
  • the SH2 inhibitor is administered or appHed in a composition including a pharmacologically acceptable topical carrier, such as a gel, an ointment, a lotion, or a cream, which includes, without limitation, such carriers as water, glycerol, alcohol, propylene glycol, fatty alcohols, triglycerides, fatty acid esters, or mineral oUs.
  • topical carriers include liquid petroleum, isopropyl pal itate, polyethylene glycol, ethanol (95%), polyoxyethyiene monolaurate (5%) in water, or sodium lauryl sulfate (5%) in water.
  • Other materials such as anti- oxidants, humectants, viscosity stabilizers, and similar agents may be added as necessary.
  • the SH2 inhibitor may be disposed within devices placed upon, in, or under the skin.
  • Such devices include patches, implants, and injections which release the compound into the skin, by either passive or active release mechanisms.
  • the effective dose of the SH2 inhibitor wiU typically be in the range of about 0.01 to about 50 mg/kgs, preferably about 0.1 to about 10 mg/kg of mammalian body weight, adrninistered in single or multiple doses.
  • the SH2 inhibitor may be adrninistered to patients in need of such treatment in a daily dose range of about 1 to about 2000 mg per patient
  • the amount of the SH2 inhibitor which wiU be effective in the treatment or prevention of a particular disorder or condition wiU depend on the nature of the disorder or condition, and can be deterrx ⁇ ned by standard clinical techniques.
  • in vitro or in vivo assays may optionaUy be employed to help identify optimal dosage ranges.
  • Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.
  • the precise dosage level of the SH2 inhibitor, as the active component(s), should be determined by the attending physician or other health care provider and wiU depend upon well known factors, including route of adrrdnistration, and the age, body weight, sex and general health of the individual; the nature, severity and clinical stage of the disease; and the use (or not) of concomitant therapies.
  • the invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention.
  • Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceutical or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.
  • the SH2 inhibitor is administered by pulmonary administration, e.g. via aerosolization.
  • This route of adrrdnistration may be particularly useful for treatment or prophylaxis of bronchial or pulmonary infection or tumors.
  • Pulmonary adrrdnistration can be accomplished, for example, using any of various deHvery devices known in the art (see e.g.,. Newman, S.P., 1984, in Aerosols and the Lung , Clarke and Davia (eds.), Butterworths, London, England, pp. 197-224; PCT PubHcation No. WO 92/16192 dated October 1, 1992; PCT Publication No. WO 91/08760 dated June 27, 1991; NTIS Patent
  • AppHcation 7-504-047 filed April 3, 1990 by Roosdorp and Crystal including but not limited to nebulizers, metered dose inhalers, and powder inhalers.
  • Various deHvery devices are commerciaUy avaUable and can be employed, e.g., Ultravent nebulizer (MaUinckrodt, Inc., St. Louis, Missouri); Acorn II nebulizer (Marquest Medical Products, Englewood, Colorado), Ventolin metered dose inhaler (Glaxo Inc., Research Triangle Park, North Carolina); Spinhaler powder inhaler (Fisons Corp., Bedford, Massachusetts) or Turbohaler (Astra).
  • Such devices typically entaU the ' use of formulations suitable for dispensing from such a device, in which a propellant material may be present.
  • Ultrasonic nebulizers tend to be more efficient than jet nebulizers in producing an aerosol of respirable size from a Hquid (Smith and Spino, "Pharmacokinetics of Drugs in Cystic Fibrosis," Consensus Conference, Clinical Outcomes for Evaluation of New CF Therapies, RockviUe, Maryland, December 10-11, 1992, Cystic Fibrosis Foundation).
  • a nebulizer may be used to produce aerosol particles, or any of various physiologicaUy acceptable inert gases may be used as an aerosolizing agent.
  • physiologicaUy acceptable surfactants e.g., glycerides
  • excipients e.g., lactose
  • carriers e.g., diluents
  • aU moisture-sensitive reactions were performed in heat-dried glassware with magnetic stirring under an atmosphere of dry 2.
  • Acetone, benzene, EtOAc, hexane, CH3OH, CH2O2, and THF were HPLC grade and used without purificatiorL Workup means drying the organic extracts over anhydrous MgS ⁇ 4, filtration under reduced pressure, and concentration on a rotary evaporator.
  • AU chromatographic purification was performed using flash chromatography according to StiU, W. C; Kahn, M.; Mitra, A. /. Org. Chem. 1978, 43, 2923, unless otherwise noted.
  • moieties may be readily deprotected by conventional means to yield a free amine group for covalent attachment to an N-A-3,5-dinitroty ⁇ osine moiety, where A s as defined above.
  • Other ring systems bearing free amino or hydroxyl groups which may be covalently attached to an N-A-3,5- di itrotyrosine moiety are known in the art.
  • Amino acids, dipeptides, tripeptides, oHgopeptides may be obtained by conventional means, in free or protected form, for incorporation into compounds of this invention.
  • R ⁇ is -OH and R ⁇ is -CH3 or -CH2 ⁇ (CO)CH3 may be prepared by known procedures or obtained from commercial sources. See e.g. the various references cited in the Merck Index for 7-aminocephalosporanic acid (Item 444) and Cephalosporin C (Item 1976). Related compounds can be prepared as previously described. See e.g. Teller et al, US Patent 3,926,984; Bohme et al, J. Org. Chem. (1973), 38(2), 230-6; and Ochiai et al, Tetrahedron Lett. (1972), (23), 2341-4.
  • Scheme 1 illustrates the synthesis of Z-L,L-BC "Me.
  • the other epimers are prepared in a similar manner using the combination of stereoisomers of N- cbz-glutamic acid and cysteine methyl ester corresponding to the stereochemistry of the desired product.
  • Any of Compounds 4(a) through 4(d) may be readily incorporated into the synthesis of other compounds of this invention through conventional deprotection and coupling steps.
  • the N-cbz group may be routinely removed by classical procedures with trifluoroacetic acid/thioanisole, and the methyl ester may be conveniently removed with Hthium hydroxide in methanol/water.
  • the oxazolidinone B-l which is formed from N-Cbz-L-glutarnic acid by heating with paraformaldehyde in benzene in the presence of catalytic amount of p-toluenesulfonic acid, is treated with oxalyl chloride foUowed by reduction of the acid chloride with n-Bu3SnH to give the aldehyde B-2.
  • Condensation of the aldehyde with L-cysteine methyl ester in pyridine over a 5-day period gives the bicyclic, hydroxymethyl derivative B-3.
  • the hydroxymethyl group is removed under mildly alkaline conditions to provide the Cbz-protected methyl ester 4(a) (Z-L,L-BC-OMe).
  • BusSnH (25.8 g, 1.0 eq) was added with stirring via a syringe pump at 0 °C uder N 2 over a 1.5 h period and the resulting solution was aUowed to warm from 0 °C to r. t. for 14 h.
  • the mixture was concentrated under reduced pressure and the oil was dissolved in 500 mL of CH3CN.
  • the CH3CN solution was washed with petroleum ether (5 x 200 mL) and then concentrated to give 24.5 g (100%) of the aldehyde as a yellowish oU that carried on without purification.
  • the monocycHc compounds XII can be synthesized from aldehydes IV and V. Reductive amination with a derivatized amino acid foUowing the method of Borch, R. F. et al /. Am. Chem. Soc. 1971, 93, 2897, foUowed by catalytic hydrogenation and cycHc amide formation can provide XII.
  • D-homoserine methyl ester XTH can be synthesized from D-homoserine by first forming the Cbz carbamate using standard conditions (Bodanszky, A et al The Practice of Peptide Synthesis (1984). p.14). Hydrolysis of the generated lactone, formation of the methyl ester (Wang, S.-S. et al /. Org. Chem. 1977, 42, 1286), and catalytic hydrogenation can provide D-homoserine methyl ester XUI. Coupling with aldehydes IV and V as previously described can afford spacer XIV.
  • N-Fmoc-3-nitro-L-tyrosine may be prepared as described above but substituting 3-nitro-L-tyrosine for 3,5-dinitro-L-tyrosine.
  • 3,5-Dirdtiotyrosine-YB compounds may be obtained by the following route:
  • n is a peptide comprising n amino acids which may be the same or different, where n is preferably 1-6 , a series of compounds of the formula:
  • synthesis was carried out in a manual peptide synthesis apparatus as foUows: 100 mgs of Fmoc-Rink amide resin (4-(2',4'-Dimethoxyphenyl- Fmoc-aminomethyl) phenoxy resin; Advanced Chemtech) with substitution levels of 0.3-0.6 mmole/g was added to a polypropylene chromatography column (PD-10, Pharmacia Inc.) with a porous frit above a leur tip, and with a siHcone cap. The column reaction vessels were fixed to a rotary shaker, and 5ml of dimethylacetamide (DMA) was added, and the vessels were shaken for 20 min. to swell the resin.
  • DMA dimethylacetamide
  • Amino acids (AA) used for this series of compounds included Fmoc- LeuOH, Fmoc-Thr(tBu)OH, Fmoc-Glu(tBu)OH, Fmoc-PheOH, Fmoc-D-PheOH, Fmoc-2-NalOH, Fmoc-1-NalOH, Fmoc-IleOH, Fmoc-AlaOH, Fmoc- Asp(tBu)OH, Fmoc-ValOH, Fmoc-GlyOH, and Fmoc-Asn(Trt)OH. Coupling
  • step 2 was initiated by adding solid Fmoc-amino acid to moist resin at the beginning of step 4, foUowed by adding NMP, NMM, and soHd TBTU.
  • any of a series of acids were coupled to the N-terrninus of the dnY containing peptide (step 4, table 1) using the methodology above for amino acids. Washes were done as in steps 5 and 6, table 1 and the synthesis was completed by washing the peptide-resin 3x with CH 2 C1 2 , and 3x with methanol (MeOH). Moist resins were desiccated overnight under vacuum with P 2 ⁇ 5 as a dessicant.
  • Dry resins (100-200 mg) were cleaved using a reaction mixture containing trifluoroacetic acid (TFA), ethane dithiol (EDT), and H 2 0, in a ratio of 90:5:5 respectively, for 2h, foUowed by filtration of the supernatant through a plug of glass wool, and a secondary cleavage of the resin using 90% TFA/H 0, for 30 min. Resin and supernatant were filtered and washed with two smaU portions of 90% TFA/H 0. The resin was discarded, and the supernatant, containing the cleaved, de ' protected peptide, was kept.
  • TFA trifluoroacetic acid
  • EDT ethane dithiol
  • TFA was evaporated from the peptide solution using a stream of dry N 2 gas, until a slurry remained. Approximately 4-5 ml of H 2 0 was added to the peptide slurry, and it was suspended with sonication in a water bath. This material was extracted 3 times with ice-cold diethyl ether. The ether layers were discarded, and remaining ether in the crude peptide solution was rotary evaporated under reduced pressure. The crude solutions were then lyophilized overnight on a Virtis lyophUizer, yielding a crude peptide powder. Purification and Characterization:
  • DMSO/H 2 0 required to achieve a clear solution.
  • concentrations of crude peptide solution was in the range of 40-100 mg/ml.
  • a series of sequential purification runs were performed, injecting 15-35ul of peptide solution per run (0.6-3.5 mg/injection). The central portion of the desired peak was coUected and lyophilized overnight on a Virtis lyophilizer.
  • the resultant solution is concentrated, generaUy to a thick residue, via short-path distiUation (40°/m vacuo), then diluted with EtOAc and washed successively with 5% aqueous citric acid, saturated aqueous NaHC ⁇ 3, H 2 0, and brine. Workup foUowed by chromatography in 50:1 (CH 2 Cl 2 /MeOH) yields the coupled product VLI.
  • the diastereomeric mixture l(f, g) was purified to individual components upon coupHng to H- ⁇ e-OMe ⁇ Cl, using conditions outlined in Scheme DI, to provide compounds VHI and DC.
  • Compound Vffl is converted to product l(i) as previously described.
  • Compound 1(1) may be synthesized as previously described utilizing H- ⁇ -Nal-OMe-HCl and N-Fmoc-3,5-dinitro-L-tyrosine.
  • the deriyatized amino acid H- ⁇ -Nal- Me HCl was prepared analogously to H-D-Cys-OMe-HCl starting with Boc- ⁇ -Nal-OH. Under the conditions of the reaction the Boc protecting group was removed.
  • Compound l(n) may be synthesized as previously described utilizing H- ⁇ -Nal-OMe-HCl and N-Fmoc-3-nitro-L-tyrosine.
  • the derivatized amino acid H- ⁇ -Nal-OMe ⁇ CI was prepared analogously to H-D-Cys-OMe-HCl starting with Boc- ⁇ -Nal-OH. Under the conditions of the reaction the Boc protecting group was removed.
  • Binding was measured by competition using surface plasmon resonance (Malmqvist, M. (1993) Current Opinions in Immunology 5, 282-286) as implemented in the BIAcore Biosensor (Pharmacia Biosensor, Piscataway, NJ).
  • SH2 proteins e.g. pp60 src , pp70 ZAP , or pp72 s y k , were pre-incubated with various concentrations of test compound and the ability of the test compound to competitively inhibit binding to a phophopeptide Hgand measured. Results were compared to binding measured in the absence of competitor and expressed as percent inhibition. IC50 values reflect the concentration of inhibitor required to reduce binding by 50%. Specifics of individual assays are described below.
  • HBS HEPES Buffered Saline
  • Tandem Syk Assay A pp72 s y k peptide Hgand corresponding to the ⁇ -chain ITAM of human Fc ⁇ RI
  • DGVY(P ⁇ 4)TGLSTRNQETY(P ⁇ 4)ETLK was synthesized as part of a larger peptide [Ac-CGGDGVY(P ⁇ 4)TGLSTRNQETY-(P ⁇ 4)ETLK-NH2] and used to generate a Syk-sensitive biosensor surface.
  • a Biosensor Chip CM5 was activated with 200 mM ethyl-3-(3- dimethylamino ⁇ ropyl)-carbodiimide hydrochloride (EDC)/ 50 mM
  • N-hydroxysuccinimide (NHS) to generate a surface reactive to primary amines; treated with ethylenediamine to generate a surface rich in primary amines; activated with m-maleimidobenzoyl-N- hydroxysuccinirnide ester (sulfo-MBS; 50 mM in 25 M aHCOs) to generate a surface reactive to free thiols; and the ITAM peptide immobilized through the N-terminal cysteine. Unreacted sites were blocked with ⁇ -mercaptoethanol and the chip cleaned of non ⁇ covalently bound ' peptide using 6 M guanidine hydrochloride. Assays were run in HBS using 20 nM pp72 s y k (l-265) ' +/- test inhibitor.
  • Tandem ZAP Assay A pp70 ZAP peptide Hgand corresponding to the ⁇ -chain ITAM-1 of the human T-cell receptor [NQLY(PO 4 )NELNIGRREEY(P04)DVLD] was synthesized as a part of a larger peptide [Ac-KGGNQLY(P0 4 )NELNIGRREEY-(P04)DVLD- NH ] and used to generate a ZAP-sensitive biosensor surface. SpecificaUy, a Biosensor Chip CM5 was activated with 200 mM EDC /
  • Src Assay A p60 src peptide Hgand corresponding to the hamster Middle-T antigen [QY(P04)EEEPI] was synthesized as a part of a larger peptide [Ac-KGGQY(P04)EEIPI-NH2] and used to generate a src-sensitive biosensor surface as described above for tandem ZAP. Assays were run in PIBS using 270 nM pp60 src (144-251) +/- test inhibitor.

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Abstract

L'invention se rapporte à des composés représentés par la formule (I) et à des sels pharmaceutiquement acceptables de ces composés. Dans la formule (I), A est H, R<1>, -CO-R<1> ou -CO-OR<1>, R<1> étant un groupe alkyle, hétéroalkyle, aryle ou hétéroaryle substitué ou non substitué et J étant H ou NO2. Bien que les formes L de ces composés soient actuellement préférées, l'invention englobe également des formes D et des mélanges racémiques.
PCT/US1997/002636 1996-02-23 1997-02-21 Nouveaux inhibiteurs des processus lies a la presence de sh2 WO1997031016A2 (fr)

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AU22775/97A AU2277597A (en) 1996-02-23 1997-02-21 New inhibitors of sh2-mediated processes

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Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999024442A1 (fr) * 1997-11-12 1999-05-20 Ariad Pharmaceuticals, Inc. Nouveaux inhibiteurs de transduction de signal, compositions les contenant
WO1999031066A1 (fr) * 1997-12-18 1999-06-24 Boehringer Ingelheim Pharmaceuticals, Inc. Pyridones utilisees comme inhibiteurs du domaine sh2 de la famille src
WO1999047529A1 (fr) * 1998-03-18 1999-09-23 Ariad Pharmaceuticals, Inc. Inhibiteurs de transduction de signaux heterocycliques et compositions les contenant
WO2000027802A1 (fr) * 1998-11-12 2000-05-18 Ariad Pharmaceuticals, Inc. Inhibiteurs de transduction de signaux bicycliques, compositions les contenant et utilisation de ces dernieres
US6156784A (en) * 1997-12-18 2000-12-05 Boehringer Ingelheim Pharmaceuticals, Inc. Compounds useful as phosphotyrosine mimics
US6730671B2 (en) 1999-03-02 2004-05-04 Boehringer Ingelheim Pharmaceuticals, Inc. Compounds useful as reversible inhibitors of cathespin S
US6756372B2 (en) 1999-09-13 2004-06-29 Boehringer Ingelheim Pharmaceuticals, Inc. Compounds useful as reversible inhibitors of cysteine proteases
WO2006059164A2 (fr) 2004-12-02 2006-06-08 Prosidion Limited Amides d'acide pyrrolopyridine-2-carboxylique
US7105307B2 (en) 1997-08-30 2006-09-12 Cyclacel, Ltd. Compositions and methods for screening for modulators of enzymatic activity
EP1800673A2 (fr) * 2005-12-23 2007-06-27 Canadian Blood Services Nitrophényles et composés associés et thimérosal pour l'inhibition de la destruction cellulaire ou tissulaire liée à l'immunité
US7405210B2 (en) 2003-05-21 2008-07-29 Osi Pharmaceuticals, Inc. Pyrrolopyridine-2-carboxylic acid amide inhibitors of glycogen phosphorylase
US7884112B2 (en) 2004-03-08 2011-02-08 Stuart Edward Bradley Pyrrolopyridine-2-carboxylic acid hydrazides
US10717703B2 (en) 2017-08-21 2020-07-21 Celgene Corporation Processes for the preparation of (S)-tert-butyl 4,5-diamino-5-oxopentanoate

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU4972096A (en) * 1995-02-01 1996-08-21 Affymax Technologies N.V. Peptides and compounds that bind to sh2 domains

Cited By (27)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7105307B2 (en) 1997-08-30 2006-09-12 Cyclacel, Ltd. Compositions and methods for screening for modulators of enzymatic activity
US6576766B1 (en) 1997-11-12 2003-06-10 Ariad Pharmaceuticals, Inc. Signal transduction inhibitors, compositions containing them
WO1999024442A1 (fr) * 1997-11-12 1999-05-20 Ariad Pharmaceuticals, Inc. Nouveaux inhibiteurs de transduction de signal, compositions les contenant
WO1999031066A1 (fr) * 1997-12-18 1999-06-24 Boehringer Ingelheim Pharmaceuticals, Inc. Pyridones utilisees comme inhibiteurs du domaine sh2 de la famille src
US6054470A (en) * 1997-12-18 2000-04-25 Boehringer Ingelheim Pharmaceuticals, Inc. Src family SH2 domain inhibitors
US6156784A (en) * 1997-12-18 2000-12-05 Boehringer Ingelheim Pharmaceuticals, Inc. Compounds useful as phosphotyrosine mimics
JP2003514762A (ja) * 1997-12-18 2003-04-22 ベーリンガー インゲルハイム ファーマシューティカルズ インコーポレイテッド Ｓｒｃファミリーｓｈ２ドメインインヒビターとしてのピリドン
WO1999047529A1 (fr) * 1998-03-18 1999-09-23 Ariad Pharmaceuticals, Inc. Inhibiteurs de transduction de signaux heterocycliques et compositions les contenant
WO2000027802A1 (fr) * 1998-11-12 2000-05-18 Ariad Pharmaceuticals, Inc. Inhibiteurs de transduction de signaux bicycliques, compositions les contenant et utilisation de ces dernieres
US6573295B2 (en) 1998-11-12 2003-06-03 Ariad Pharmaceuticals, Inc. Bicyclic signal transduction inhibitors, compositions containing them & uses thereof
US6482852B2 (en) 1998-11-12 2002-11-19 Ariad Pharmaceuticals, Inc. Bicyclic signal transduction inhibitors, compositions containing them & uses thereof
US6730671B2 (en) 1999-03-02 2004-05-04 Boehringer Ingelheim Pharmaceuticals, Inc. Compounds useful as reversible inhibitors of cathespin S
US6756372B2 (en) 1999-09-13 2004-06-29 Boehringer Ingelheim Pharmaceuticals, Inc. Compounds useful as reversible inhibitors of cysteine proteases
US6982272B2 (en) 1999-09-13 2006-01-03 Boehringer Ingelheim Pharmaceuticals, Inc. Compounds useful as reversible inhibitors of cysteine proteases
US7056915B2 (en) 1999-09-13 2006-06-06 Boehringer Ingelheim Pharmaceuticals, Inc. Compounds useful as reversible inhibitors of cysteine proteases
US7279472B2 (en) 1999-09-13 2007-10-09 Boehringer Ingelheim Pharmaceuticals Inc. Compounds useful as reversible inhibitors of cysteine proteases
US7265132B2 (en) 1999-09-13 2007-09-04 Boehringer Ingelheim Pharmaceuticals Inc. Compounds useful as reversible inhibitors of cysteine proteases
US6858623B2 (en) 2000-09-08 2005-02-22 Boehringer Ingelheim Pharmaceuticals, Inc. Compounds useful as reversible inhibitors of cysteine proteases
US7405210B2 (en) 2003-05-21 2008-07-29 Osi Pharmaceuticals, Inc. Pyrrolopyridine-2-carboxylic acid amide inhibitors of glycogen phosphorylase
US8158622B2 (en) 2003-05-21 2012-04-17 Prosidion Limited Pyrrolopyridine-2-carboxylic acid amide inhibitors of glycogen phosphorylase
US7884112B2 (en) 2004-03-08 2011-02-08 Stuart Edward Bradley Pyrrolopyridine-2-carboxylic acid hydrazides
WO2006059164A2 (fr) 2004-12-02 2006-06-08 Prosidion Limited Amides d'acide pyrrolopyridine-2-carboxylique
EP1800673A3 (fr) * 2005-12-23 2007-08-15 Canadian Blood Services Nitrophényles et composés associés et thimérosal pour l'inhibition de la destruction cellulaire ou tissulaire liée à l'immunité
EP1800673A2 (fr) * 2005-12-23 2007-06-27 Canadian Blood Services Nitrophényles et composés associés et thimérosal pour l'inhibition de la destruction cellulaire ou tissulaire liée à l'immunité
US10717703B2 (en) 2017-08-21 2020-07-21 Celgene Corporation Processes for the preparation of (S)-tert-butyl 4,5-diamino-5-oxopentanoate
US11505522B2 (en) 2017-08-21 2022-11-22 Celgene Corporation Processes for the preparation of (S)-tert-butyl 4,5- diamino-5-oxopentanoate
US11912644B2 (en) 2017-08-21 2024-02-27 Celgene Corporation Processes for the preparation of (S)-tert-butyl 4,5-diamino-5-oxopentanoate

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AU2277597A (en) 1997-09-10

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