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WO1998027065A1 - Modulateurs de proteines possedant des unites de reconnaissance de la phosphotyrosine - Google Patents

Modulateurs de proteines possedant des unites de reconnaissance de la phosphotyrosine Download PDF

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Publication number
WO1998027065A1
WO1998027065A1 PCT/US1996/020508 US9620508W WO9827065A1 WO 1998027065 A1 WO1998027065 A1 WO 1998027065A1 US 9620508 W US9620508 W US 9620508W WO 9827065 A1 WO9827065 A1 WO 9827065A1
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WIPO (PCT)
Prior art keywords
compound
solvates
prodrugs
esters
alkyl
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PCT/US1996/020508
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English (en)
Inventor
Adnan Mjalli
Sepehr Sarshar
Xiaodong Cao
Farid Bakir
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Ontogen Corporation
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Priority to US08/543,630 priority Critical patent/US5770620A/en
Priority to EP96940489A priority patent/EP0833629A4/fr
Application filed by Ontogen Corporation filed Critical Ontogen Corporation
Priority to JP52765098A priority patent/JP2001506997A/ja
Priority to AU15667/97A priority patent/AU740425B2/en
Priority to CA002275610A priority patent/CA2275610A1/fr
Priority to PCT/US1996/020508 priority patent/WO1998027065A1/fr
Priority to EP96945409A priority patent/EP0946518A1/fr
Priority to US08/766,114 priority patent/US5753687A/en
Publication of WO1998027065A1 publication Critical patent/WO1998027065A1/fr

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Definitions

  • the present invention relates to novel protein tyrosine phosphatase modulating compounds, to methods for their preparation, to compositions comprising the compounds, to their use for treatment of human and animal disorders, to their use for purification of proteins or glycoproteins, and to their use in diagnosis.
  • the invention relates to modulation of the activity of molecules with phosphotyrosine recognition units, including protein tyrosine phosphatases (PTPases) and proteins with Src- homology-2 domains, in in vitro systems, microorganisms, eukaryoic cells, whole animals and human beings.
  • PTPases protein tyrosine phosphatases
  • Reversible phosphorylation of proteins is a prevalent biological mechanism for modulation of enzymatic activity in living organisms. Tonks et al., J. Biol. Chem., 263(14):6722-3 (1988).
  • PK protein kinase
  • PP protein phosphatase
  • PK's/PP's the protein serine/threonine kinases and protein serine/threonine phosphatases - have been shown to play critical roles in the regulation of metabolism. See generally, Cohen, Trends Biochem. Sci., 77:408-413
  • the protein tyrosine kinases/phosphatases comprise a second, distinct family of PK/PP enzymes of significant interest, and have been implicated in the control of normal and neoplastic cell growth and proliferation. See Fisher et al., Science,
  • PTK Protein tyrosine kinase
  • PTPases Protein tyrosine phosphatases
  • R- PTPases are generally grouped into two categories: those which have both an extracellular domain and an intracellular catalytic domain, the receptor PTPases (R- PTPases); and those which are entirely intracellular. For R-PTPases much effort has been directed at determining the function of the extracellular domain. Most of the R- PTPases contain extracellular domains which are structurally similar to domains found in known adhesion molecules; these domains include fibronectin type III repeats, immunoglobulin domains, and cadherin extracellular repeats. See generally Brady-Kalnay and Tonks, Curr. Opin. Cell. Biol. 7:650-657 (1995); Streuli, Curr.
  • Adherens junctions contain, among others, adhesion receptors termed cadherins which mediate cell-cell contact through homophilic binding; the cadherins associate with ⁇ -, ⁇ -, and ⁇ - catenins, intracellular proteins which interact with cortical actin. Association between cadherins and catenins serves to stabilize the adherens junction and to strengthen cell- cell contact. See generally Cowin, Proc. Natl Acad. Sci. 97: 10759-10761 (1994). Association of cadherin with ⁇ -catenin is decreased by tyrosine phosphorylation of ⁇ - catenin [Kinch et al, J. Cell.
  • PTPK and PTP ⁇ mediate cellular aggregation through homophilic binding [Brady-
  • the neuronal PTP ⁇ (which has also been called R-PTP ⁇ ) binds to contactin, a neuronal cell recognition molecule; binding of PTP ⁇ to contactin increases cell adhesion and neurite outgrowth. Peles et al, Cell 52:251-260 (1995).
  • PTP ⁇ (also known as phosphacan) binds the extracellular matrix protein tenascin [Barnea et al. J. Biol. Chem. 269: 14349-14352 (1994)], and the neural cell adhesion molecules N-CAM and Ng-CAM [Maurel et al, Proc. Natl. Acad. Sci. 97:2512-2516 (1994)].
  • tenascin binds the extracellular matrix protein tenascin [Barnea et al. J. Biol. Chem. 269: 14349-14352 (1994)]
  • the neural cell adhesion molecules N-CAM and Ng-CAM Maurel et al, Proc. Natl. Acad. Sci. 97:2512-2516 (1994)].
  • PTP ⁇ As the expression of PTP ⁇ is restricted to radial glial cells in the developing central nervous system, which are though to form barriers to neuronal migration during embryogenesis, it is
  • mice loss of SHPl function (the motheaten and viable motheaten phenotypes) causes multiple hematopoietic defects resulting in immunodeficiency and severe autoimmunity; culminating in lethality by 2-3 weeks or 2-3 months depending on the severity of SHPl deficiency.
  • these mice have reduced numbers of hematopoietic cells, suggesting defects in development and maturation, those cells which survive and enter the periphery are characterized by hyper-responsiveness to growth factors and antigen. This observation suggested a role for SHPl in negative regulation of hematopoietic signaling events.
  • EpoR erythropoietin receptor
  • cytokine receptor family which also includes the receptors for interleukins 2, 3, 4, 5, 6, 7; granulocyte-macrophage colony stimulating factor, and macrophage colony stimulating factor.
  • SHPl associates via its SH2 domains with tyrosine-phosphorylated EpoR, causing dephosphorylation and inactivation of the EpoR-associated Janus kinase 2 and termination of the cellular response to erythropoietin. Klingmuller et al, Cell 50:729-738 (1995).
  • Mutation of the tyrosine on the EpoR to which SHPl binds results in enhanced cell proliferation to erythropoietin in vitro [Klingmuller, supra].
  • mutation of the EpoR resulting in loss of association with SHPl causes autosomal dominant benign erythrocytosis, which is characterized by increased numbers of erythrocytes in the periphery and increased hematocrit. de la Chapelle et al, Proc. Natl. Acad. Sci.
  • SHPl also appears to be a negative regulator of the cellular response to colony stimulating factor- 1 (CSF-1, a major macrophage mitogenic cytokine), as cells from viable motheaten and motheaten mice, which have reduced or absent SHPl function, are hyper-responsive to CSF-1 in vitro.
  • CSF-1 colony stimulating factor- 1
  • Reduced SHPl expression also results in increased cellular response to interleukin 3 [Yi et al, Mol. Cell. Biol. 75:7577-7586 (1993)].
  • PTPases appear to play a homologous role in the insulin signaling pathway.
  • Treatment of adipocytes with the PTPase inhibitor vanadate results in increased tyrosine phosphorylation and tyrosine kinase activity of the insulin receptor (InsR), and enhances or mimics the cellular effects of insulin including increased glucose transport.
  • InsR insulin receptor
  • CD45 is abundantly expressed on the cell surface of all nucleated hematopoietic cells, in several alternative splice variants. T and B lymphocytes which lack CD45 expression are incapable of responding normally to antigen, suggesting that CD45 is required for antigen receptor signaling.
  • CD45 genetically engineered mice which lack expression of CD45 exhibit severe defects in T lymphocyte development and maturation, indicating an additional role for CD45 in thymopoiesis.
  • the major substrates for CD45 appear to be members of the Src family of PTK's, particularly
  • Lck and Fyn whose kinase activity is both positively and negatively regulated by tyrosine phosphorylation.
  • Lck and Fyn isolated from CD45-deficient cells are hyperphosphorylated on negative regulatory tyrosine residues, and their PTK activity is reduced.
  • CD45 can dephosphorylate and activate purified Lck and Fyn in vitro, these data suggest that CD45 maintains the activity of Lck and Fyn in vivo through dephosphorylation of these negative regulatory tyrosines and that this is an important mechanism for maintaining lymphocyte homeostasis.
  • a second PTPase which is now believed to play an important positive role in signal transduction is the intracellular, SH2 -domain-containing SHP2 (which has also been called SHPTP-2, SHPTP-3, syp, PTP2c, and PTP-1D [Adachi, et al., supra ⁇ ).
  • SHP2 associates, via its SH2 domains, with the receptor for platelet- derived growth factor (PDGF-R), the receptor for epidermal growth factor (EGF-R), with the insulin receptor, and with the predominant substrate of the InsR, insulin receptor substrate 1 (IRS1). Bennett, et al, Proc. Natl. Acad. Sci. 97:7335-7339
  • SHP2 PTPase activity is required for cellular response to EGF and insulin, as competitive expression of inactive forms of SHP2 results in diminished signaling events and reduced cellular responses to EGF and insulin. Milarski and Saltiel, J.
  • PTP-IB overexpression has been correlated with breast and ovarian cancers [Weiner et al., J. Natl. Cancer Inst., 55:372-8 (1994); Weiner et al., Am J. Obstet. Gynecol, 770:1177-883 (1994)], and thus agents which modulate
  • PTP-IB activity would be helpful in elucidating the role of PTP-IB in these conditions and for the development of effective therapeutics against these disease states.
  • the important role of CD45 in hematopoietic development and T lymphocyte function likewise indicates a therapeutic utility for PTPase inhibitors in conditions that are associated with autoimmune disease, and as a prophylaxis for transplant rejection.
  • the antibiotic suramin which also appears to possess anti-neoplastic indications, has recently been shown to be a potent, irreversible, non-competitive inhibitor of CD45. See Ghosh and Miller, Biochem. Biophys. Res. Comm. 194:36-44 (1993).
  • the PTPase Yop2b is an essential virulence determinant in the pathogenic bacterium Yersinia, responsible for bubonic plague. Bliska et al, Proc. Natl. Acad Sci. USA, 55:1187-91 (1991), and thus an antimicrobial indication exists for PTPase inhibitor compounds, as well.
  • PTPases have been implicated in diabetic conditions.
  • PTPase inhibitors vanadium derivatives
  • metal-containing PTPase inhibitors act in a fairly non-specific fashion and act with similar potencies against all PTPase enzymes.
  • certain organic phosphotyrosine mimetics are reportedly capable of competitively inhibiting PTPase molecules when such mimetics are incorporated into polypeptide artificial PTPase substrates of 6-11 amino acid residues.
  • a "natural" (phosphorylated tyrosine) PTPase substrate which may be depicted by the Formula:
  • hexapeptide inhibitors nonetheless possess drawbacks for PTPase modulation in vivo. More particularly, the hexapeptide inhibitors described by Burke et al. are sufficiently large and anionic to potentially inhibit efficient migration across cell membranes, for interaction with the catalytic domains of transmembrane and intracellular PTPase enzymes which lie within a cell membrane. A need exists for small, organic-molecule based PTPase inhibitors having fewer anionic moieties, to facilitate migration across cell membranes.
  • the invention provides compounds and derivatives thereof useful for modulating, and especially inhibiting, the phosphatase activity of one or more protein tyrosine phosphatase (PTPase) and/or dual specificity phosphatase enzymes.
  • PTPase protein tyrosine phosphatase
  • the inventions further provides salts, esters, prodrugs, solvates, and the like of the compounds, and compositions comprising these compounds.
  • derivatives means: aryl acrylic acids with structure depicted in Formula (Al) having substitution (with, e.g., hydrogen, hydroxy, halo, amino, carboxy, nitro, cyano, methoxy, etc.) at one or more atoms of the aryl ring.
  • substitution with, e.g., hydrogen, hydroxy, halo, amino, carboxy, nitro, cyano, methoxy, etc.
  • Al having substitution at the alkene carbons with, e.g., an electron withdrawing group (e.g., Cl, F, Br, CF 3 , phenyl) or an electron donating group (e.g., CH 3 , alkoxy).
  • an electron withdrawing group e.g., Cl, F, Br, CF 3 , phenyl
  • an electron donating group e.g., CH 3 , alkoxy
  • attachment signifies a stable covalent bond, certain preferred points of attachment being apparent to those skilled in the art.
  • halogen or halo include fluorine, chlorine, bromine, and iodine.
  • alkyl includes Ci-C ⁇ straight chain saturated and C 2 -C ⁇ unsaturated aliphatic hydrocarbon groups, C[-Cn branched saturated and C 2 -C ⁇ unsaturated aliphatic hydrocarbon groups, C -C 8 cyclic saturated and C 5 -C 8 unsaturated aliphatic hydrocarbon groups, and C]-Cn straight chain or branched saturated and C 2 -C ⁇ straight chain or branched unsaturated aliphatic hydrocarbon groups substituted with C 3 -C 8 cyclic saturated and unsaturated aliphatic hydrocarbon groups having the specified number of carbon atoms.
  • this definition shall include but is not limited to methyl (Me), ethyl (Et), propyl (Pr), butyl (Bu), pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, ethenyl, propenyl, butenyl, penentyl, hexenyl, heptenyl, octenyl, nonenyl, decenyl, undecenyl, isopropyl (i-Pr), isobutyl (i-Bu), tert-butyl (t-Bu), sec-butyl (s-Bu), isopentyl, neopentyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclopentenyl, cyclohexeny
  • substituted alkyl represents an alkyl group as defined above wherein the substitutents are independently selected from halo, cyano, nitro, trihalomethyl, carbamoyl, C 0- ⁇ alkyloxy, arylC 0- nalkyloxy, C 0 - nalkylthio, arylC 0- iialkylthio, C 0- nalkylamino, arylC 0- nalkylamino, di(arylC 0- ⁇ alkyl)amino, .
  • alkyloxy (e.g. methoxy, ethoxy, propyloxy, allyloxy, cyclohexyloxy) represents an alkyl group as defined above having the indicated number of carbon atoms attached through an oxygen bridge.
  • alkyloxyalkyl represents an alkyloxy group attached through an alkyl group as defined above having the indicated number of carbon atoms.
  • alkylthio (e.g. methylthio, ethylthio, propylthio, cyclohexenylthio and the like) represents an alkyl group as defined above having the indicated number of carbon atoms attached through a sulfur bridge.
  • alkylthioalkyl represents an alkylthio group attached through an alkyl group as defined above having the indicated number of carbon atoms.
  • alkylamino (e.g. methylamino, diethylamino, butylamino, N- propyl-N-hexylamino, (2-cyclopentyl)propylamino, hexenylamino, pyrrolidinyl, piperidinyl and the like) represents one or two alkyl groups as defined above having the indicated number of carbon atoms attached through an amine bridge.
  • the two alkyl groups maybe taken together with the nitrogen to which they are attached forming a cyclic system containing 3 to 11 carbon atoms with at least one C Cnalkyl, arylCo-C ⁇ alkyl substituent.
  • alkylaminoalkyl represents an alkylamino group attached through an alkyl group as defined above having the indicated number of carbon atoms.
  • alkylcarbonyl (e.g. cyclooctylcarbonyl, pentylcarbonyl, 3- hexenylcarbonyl) represents an alkyl group as defined above having the indicated number of carbon atoms attached through a carbonyl group.
  • alkylcarbonylalkyl represents an alkylcarbonyl group attached through an alkyl group as defined above having the indicated number of carbon atoms.
  • alkylcarboxy (e.g. heptylcarboxy, cyclopropylcarboxy, 3- pentenylcarboxy) represents an alkylcarbonyl group as defined above wherein the carbonyl is in turn attached through an oxygen.
  • alkylcarboxy alkyl represents an alkylcarboxy group attached through an alkyl group as defined above having the indicated number of carbon atoms.
  • alkylcarbonylamino (e.g. hexylcarbonylamino, cyclopentylcarbonyl-aminomethyl, methylcarbonylaminophenyl) represents an alkylcarbonyl group as defined above wherein the carbonyl is in turn attached through the nitrogen atom of an amino group.
  • the nitrogen group may itself be substituted with an alkyl or aryl group.
  • alkylcarbonylaminoalkyl represents an alkylcarbonylamino group attached through an alkyl group as defined above having the indicated number of carbon atoms.
  • the nitrogen group may itself be substituted with an alkyl or aryl group.
  • aryl represents an unsubstituted, mono-, di- or trisubstituted monocyclic, polycyclic, biaryl and heterocyclic aromatic groups covalently attached at any ring position capable of forming a stable covalent bond, certain preferred points of attachment being apparent to those skilled in the art (e.g., 3-indolyl, 4- imidazolyl).
  • the aryl substituents are independently selected from the group consisting of halo, nitro, cyano, trihalomethyl, hydroxypyronyl, Cj. ⁇ alkyl, arylC
  • iialkyl C 0- ⁇ alkyloxyC 0- ⁇ alkyl, arylC 0- ⁇ alkyloxyC 0- ⁇ alkyl, C 0- ⁇ alkylthioC 0- ⁇ alkyl, arylC 0- ⁇ ⁇ alkylthioCo- 1 ⁇ alkyl, C 0-1 ⁇ alkylaminoC 0- ⁇ l ⁇ lkyl, arylC 0- ⁇ alky laminoC 0- 11 alkyl, d ⁇ arylC ⁇ alky ⁇ aminoCo-nalky 1 , Ci_nalkylcarbonylCo.i l alkyl, a ry ICj.
  • nalkylcarbonylCo.ii alkyl Ci.nalkylcarboxyCo-iialkyl, arylC ⁇ nalkylcarboxyCo. nalkyl, Ci.nalkylcarbonylaminoCo.ii alkyl, arylCi.nalkylcarbonylaminoCo.ii alkyl, - C 0 . ⁇ alkylCOOR 4 , -C 0 . ⁇ alkylCONR 5 R 6 wherein ⁇ , R 5 and R ⁇ are independently selected from hydrogen, C Cn alkyl, arylC 0 -C n alkyl, or R 5 and R ⁇ are taken together with the nitrogen to which they are attached forming a cyclic system containing 3 to 8 carbon atoms with at least one CpCj j alkyl, arylC 0 -C ⁇ j alkyl substituent.
  • aryl includes but is not limited to phenyl, biphenyl, naphthyl, dihydronaphthyl, tetrahydronaphthyl, indenyl, indanyl, azulenyl, anthryl, phenanthryl, fluorenyl, pyrenyl, thienyl, benzothienyl, isobenzothienyl, 2,3-dihydrobenzothienyl, furyl, pyranyl, benzofuranyl, isobenzofuranyl, 2,3-dihydrobenzofuranyl, pyrrolyl, indolyl, isoindolyl, indolizinyl, indazolyl, imidazolyl, benzimidazolyl, pyridyl, pyrazinyl, pyradazinyl, pyrimidinyl, triazinyl, quinolyl, isoquinolyl,
  • arylalkyl e.g. (4-hydroxyphenyl)ethyl, (2-aminonaphthyl)hexenyl, pyridylcyclopentyl
  • arylcarbonyl e.g. 2-thiophenylcarbonyl, 3 - methoxyanthrylcarbonyl, oxazolylcarbonyl
  • arylalkylcarbonyl e.g. (2,3-dimethoxyphenyl)propylcarbonyl, (2- chloronaphthyl)pentenylcarbonyl, imidazolylcyclopentylcarbonyl
  • alkyl group is in turn attached through a carbonyl.
  • signal transduction is a collective term used to define all cellular processes that follow the activation of a given cell or tissue.
  • Examples of signal transduction include but are not in any way limited to cellular events that are induced by polypeptide hormones and growth factors (e.g. insulin, insulin-like growth factors I and II, growth hormone, epidermal growth factor, platelet-derived growth factor), cytokines (e.g. interleukines), extracellular matrix components, and cell-cell interactions.
  • Phosphotyrosine recognition units/tyrosine phosphate recognition units/phosphotyrosine recognition units are defined as areas or domains of proteins or glycoproteins that have affinity for molecules containing phosphorylated tyrosine residues (pTyr).
  • Examples of pTyr recognition units include but are not in any way limited to: PTPases, SH2 domains and PTB domains.
  • PTPases are defined as enzymes with the capacity to dephosphorylate pTyr- containing proteins or glycoproteins.
  • Examples of PTPases include but are not in any way limited to: intracellular PTPases (e.g. PTP-IB, TC-PTP, PTP-1C, PTP-1D,PTP- Dl, PTP-D2), receptor-type PTPases (e.g. PTP ⁇ , PTP ⁇ , PTP ⁇ , PTP ⁇ , CD45, PTPK, PTP ⁇ ), dual specificity phosphatases (e.g. VH1, VHR, cdc25) and other PTPases such as LAR, SHP-1, SHP-2, PTP-1H, PTPMEGI, PTP-PEST, PTP ⁇ , PTPS31, IA-2 and
  • Modulation of cellular processes is defined as the capacity of compounds of the invention to 1) either increase or decrease ongoing, normal or abnormal, signal transduction, 2) initiate normal signal transduction, and 3) initiate abnormal signal transduction.
  • Modulation of pTyr-mediated signal transduction/modulation of the activity of molecules with pTyr recognition units is defined as the capacity of compounds of the invention to 1) increase or decrease the activity of proteins or glycoproteins with pTyr recognition units (e.g. PTPases, SH2 domains or PTB domains) or to 2) decrease or increase the association of a pTyr-containing molecule with a protein or glycoprotein with pTyr recognition units either via a direct action on the pTyr recognition site or via an indirect mechanism.
  • proteins or glycoproteins with pTyr recognition units e.g. PTPases, SH2 domains or PTB domains
  • Examples of modulation of pTyr-mediated signal transduction/modulation of the activity of molecules with pTyr recognition units are: a) inhibition of PTPase activity leading to either increased or decreased signal transduction of ongoing cellular processes; b) inhibition of PTPase activity leading to initiation of normal or abnormal cellular activity; c) stimulation of PTPase activity leading to either increased or decreased signal transduction of ongoing cellular processes; d) stimulation of PTPase activity leading to initiation of normal or abnormal cellular activity; e) inhibition of binding of SH2 domains or PTB domains to proteins or glycoproteins with pTyr leading to increase or decrease of ongoing cellular processes; f) inhibition of binding of SH2 domains or PTB domains to proteins or glycoproteins with pTyr leading to initiation of normal or abnormal cellular activity.
  • a subject is defined as any mammalian species, including humans.
  • R and R are independently selected from the group consisting of hydrogen, halo, cyano, nitro, trihalomethyl, alkyl, arylalkyl,
  • R'" is selected from the group consisting of hydrogen, alkyl, substituted alkyl, aryl, arylalkyl
  • Y is selected from hydrogen or
  • R , R , R and X are defined as above in Formula (Al), and wherein the remaining of Ri, R 2 and R 3 are independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, aryl, arylalkyl.
  • a class of preferred PTPase activity- modulating compounds have the general structural Formula depicted in (A3):
  • R , R , R and X are defined as above in Formula (Al), and wherein the remaining of R 1 ? R 2 and R 3 are independently selected from the group consisting ofhydrogen, alkyl, substituted alkyl, alkylcarbonyl, substituted alkylcarbonyl, aryl, arylalkyl, arylcarbonyl, arylalkylcarbonyl.
  • a class of preferred PTPase activity- modulating compounds have the general structural Formula depicted in (A4):
  • R , R , R and X are defined as above in Formula (Al), and wherein the remaining of Rj, R 2 is defined as above in Formula (A2).
  • a class of preferred PTPase activity- modulating compounds have the general structural Formula depicted in (A5):
  • R , R , R and X are defined as above in Formula (Al), and wherein the remaining of Rj . and R 2 is defined as above in Formula (A2).
  • a class of preferred PTPase activity- modulating compounds have the general structural Formula depicted in (A6):
  • R 2 (A6) wherein at least one of R R 2 , R 3 and R 4 substituents has the general structure depicted in Formula (B)
  • R , R , R and X are defined as above in Formula (Al), and wherein the remaining of Ri, R 2 , R 3 and » have the same definition as Rj, R 2 and R 3 in Formula (A2), with the proviso that when R 3 and R 4 are selected from substituted phenyl or substituted furyl then the phenyl and furyl substituents exclude hydroxy, halo, trifluoromethyl, C]- alkyl, C ⁇ -6 alkyloxy, C ⁇ -6 alkylthio, amino, C ⁇ alkylamino, di(C ⁇ . 6 alkyl)amino, pheny!C ⁇ -6 alkylamino and di(phenylC 1-6 alkyl)amino.
  • a class of preferred PTPase activity- modulating compounds have the general structural Formula depicted in (A6):
  • R 4 is selected from -COR 5 , -COOR 6 , -CONR 7 R 8 wherein R 5 thru R 8 are selected from hydrogen, alkyl, substituted alkyl, aryl, arylalkyl, or R 7 and R 8 are taken together with the nitrogen to which they are attached forming a cyclic system containing 3 to 8 carbon atoms with at least one alkyl, aryl, arylalkyl substituent, and wherein at least one of Rj, R 2 , and R 3 substituents has the general structure depicted in Formula (B)
  • R , R , R and X are defined as above in Formula (Al), and wherein the remaining of R b R 2 and R 3 are defined as above in Formula (A2).
  • a class of preferred PTPase activity- modulating compounds have the general structural Formula depicted in (A6):
  • a class of preferred PTPase activity- modulating compounds have the general structural Formula depicted in (A7):
  • R 2 is selected from -COR 5 , -COOR 6 , -CONR 7 R 8 wherein R 5 thru R 8 are defined as above in (6) and wherein at least one of R] and R 3 substituents has the general structure depicted in Formula (B)
  • R , R , R and X are defined as above in Formula (Al), and wherein the remaining of R) and R are defined as above in Formula (A2).
  • R , R , R and X are defined as above in Formula (Al), and wherein the remaining of Ri and R 2 is defined as above in Formula (A2), and wherein m is an integer between 0 and 4 and each R 3 is independently selected from the group consisting of halo, nitro, cyano, trihalomethyl, hydroxypyronyl, alkyl, arylalkyl, C 0- nalkyloxyCo-ii alkyl, arylC 0- ⁇ alkyloxyC 0- ⁇ alkyl, Co - ⁇ alkylthioCo-nalkyl, arylC 0 .
  • iialkylthioCo-nalkyl C 0 .nalkylaminoCo-n alkyl, arylCo .nalkylaminoC 0- ⁇ alkyl, i jalkylcarbonylC 0- ⁇ ⁇ alkyl, Cj.i i alkylcarboxy C 0- ⁇ l alkyl, ary l .i ⁇ alkylcarboxy C 0- l ialkyl, C ⁇ nalkylcarbonylaminoCo-n alkyl, arylC ⁇ .nalkylcarbonylaminoC 0 . ⁇ alkyl, -
  • C 0- ⁇ alkylCOOR , -Co.ualkylCONR 5 R 6 wherein j, R 5 and Rg are independently selected from hydrogen, CpCn alkyl, ary lC 0 -C ⁇ alkyl, or R 5 and R(, are taken together with the nitrogen to which they are attached forming a cyclic system containing 3 to 8 carbon atoms with at least one CpCnalkyl, arylC 0 -Cn alkyl substituent.
  • R] is selected from -COR 5 , -COOR 6 , -CONR 7 R 8 wherein R 5 thru R 8 are defined as above in (6) and wherein R 2 has the general structure depicted in Formula (B)
  • a class of preferred PTPase activity- modulating compounds have the general structural Formula depicted in (A9):
  • a class of preferred PTPase activity- modulating compounds have the general structural Formula depicted in (A9):
  • R ⁇ or R 2 is selected from -COR 5 , -COORg, -CONR 7 R 8 wherein R 5 thru R 8 are defined as in (6) and wherein the remainder of Rj and R 2 is defined as above in (9), and wherein m is an integer between 0 and 3 and each R 3 is defined as above in (9).
  • a class of preferred PTPase activity- modulating compounds have the general structural Formula depicted in (A10):
  • R , R , R and X are defined as above in Formula (Al), and wherein the remaining of Ri, R 2 is defined as above in Formula (A2), and wherein R 3 , j, R 5 , Rg are independently selected from hydrogen, alkyl, substituted alkyl, alkylcarbonyl, substituted alkylcarbonyl, aryl, arylalkyl, arylcarbonyl, arylalkylcarbonyl.
  • a class of preferred PTPase activity - modulating compounds have the general structural Formula depicted in (Al 1):
  • R , R , R and X are defined as above in Formula (Al), and wherein the remaining of R ls R 2 and R 3 are defined as above in Formula (A2).
  • compositions of the invention include compositions comprising compounds as defined above in structural formula (Al), (A2), (A3), (A4), (A5), (A6),
  • novel compounds which modulate the activity of PTPase or other molecules with pTyr recognition unit(s) as well as previously known aryl acrylic acid compounds which modulate the activity of PTPase or other molecules with pTyr recognition unit(s).
  • compositions comprising PTPase modulating compounds of the invention suitable for administration to a mammalian host.
  • the compounds of the invention act as inhibitors of PTPases, e.g. protein tyrosine phosphatases involved in the regulation of tyrosine kinase signaling pathways.
  • PTPases e.g. protein tyrosine phosphatases involved in the regulation of tyrosine kinase signaling pathways.
  • Preferred embodiments include modulation of receptor- tyrosine kinase signaling pathways via interaction with regulatory PTPases, e.g.
  • Another preferred embodiments of the invention is modulation of non-receptor tyrosine kinase signaling through modulation of regulatory PTPases, e.g. modulation of members of the Src kinase family.
  • One type of preferred embodiments of the invention relates to modulation of the activity of PTPases that negatively regulate signal transduction pathways.
  • Another type of preferred embodiments of the inventions relate to modulation of the activity of PTPases that positively regulate signale transduction pathways.
  • compounds of the inventions act as modulators of the active site of PTPases.
  • the compounds of the invention modulate the activity of PTPases via interaction with structures positioned outside the active sites of the enzymes, preferably SH2 domains.
  • Further preferred embodiments include modulation of signal transduction pathways via binding of the compounds of the invention to SH2 domains or PTB domains of non-PTPase signaling molecules.
  • Other preferred embodiments include use of the compounds of the invention for modulation of cell-cell interactions as well as cell-matrix interactions.
  • the compounds of the invention may be used as therapeutics to inhibit PTPases involved in the regulation of the insulin recptor tyrosine kinase signaling pathway in patients with type I diabetes, type II diabetes, impaired glucose tolerance, insuline resistance and obesity.
  • Further preferred embodiments include use of the compounds of the invention for treatment of disorders with general or specific dysfunction of PTPase activity, e.g. proliferative disorders including neoplastic diseases and psoriosis.
  • the compounds of the invention may be used in pharmaceutical preparations for treatment of osteoporosis.
  • Preferred embodiments of the invention further include use of compounds of the invention in pharmaceutical preparations to increase the secretion or action of growth hormone and its analogs or somatomedins including IGf-I and IGF-2 by modulating the activity of PTPases or other signal transduction molecules with affinity for phosphotyrosine involved controlling or inducing the action of these hormones or any regulating molecule.
  • compounds of the invention can be administered for purposes of stimulating the release of growth hormone from the pituitary or increase its action on target tissues thereby leading to similar effects or uses as growth hormone itself.
  • the uses of growth hormone maybe summarized as follows: stimulation of growth hormone release in the elderly; prevention of catabolic side effects of glucocorticoids; treatment of osteoporosis, stimulation of the immune system; treatment of retardation, accelaration of wound healing; accelerating bone fracture repair; treatment of growth retardation; treating renal failure or insufficiency resulting in growth retardation; treatment of physiological short stature including growth hormone deficient children and short stature associated with chronic illness; treatment of obesity and growth retardation associated with obesity; treating growth retardation associated with the Pader-Willi syndrom and Turner's syndrom; accelerating the recovery and reducing hospitalization of burn patients; treatment of intrauterine growth retardation, skeletal dysplasia, hypercortisolism and Cushings syndrome; induction of pulsatile growth hormone release; replacement of growth hormone in stressed patients; treatment of osteochondro-dysplasis, Noonans syndrome, schizophrenia, depressions, Alzheimer's disease, delayed wound healing and psychosocial deprivation; treatment of pulmonary dysfunction and ventilator
  • the compounds of the invention may be used in pharmaceutical preparations for treatment of various disorders of the immune system, either as stimulant or suppresor of normal or perturbed immune functions, including autoimmune reactions. Further embodiments of the invention for treatment of allergic reactions, e.g. asthma, dermal reactions, conjunctivitis.
  • allergic reactions e.g. asthma, dermal reactions, conjunctivitis.
  • compounds of the invention may be used in pharmaceutical preparations for prevention or induction of platelet aggregation.
  • compounds of the invention may be used in pharmaceutical preparations for treatment of infectious disorders.
  • the compounds of the invention may be used for treatment of infectious disorders caused by Yersinia and other bacteria as well as disorders caused by viruses or other microorganisms.
  • Compounds of the invention may additionally be used for treatment or prevention of diseases in animals, including commercially important animals.
  • Also included in the present invention is a process for isolation of PTPases via affinity purification procedures based on the use of immobilized compounds of the invention using procedures well-known to those skilled in the art.
  • the invention is further directed to a method for detecting the presence of PTPases in cell or in a subject comprising
  • the invention further relates to analysis and identification of the specific functions of certain PTPases by modulating their activity by using compounds of the invention in cellular assay systems or in whole animals.
  • the invention further provides methods for making compounds (Al), (A2), (A3), (A4), (A5), (A6), (A7), (A8), (A9), (AlO), (Al l) of the present invention having PTPase-modulatory/inhibitory activity.
  • compounds of the invention are synthesized in a multi-component combinatorial array, which permits rapid synthesis of numerous, structurally related compounds for subsequent evaluation.
  • the acrylic acid moiety of a compound is protected during synthesis by, e.g., esterification with a tert-butyl protecting group.
  • a preferred method of making compounds of the invention comprises use of a protected acrylic acid reagent and removal of the protective group by, e.g., treatment of a precursor ester compound with acid.
  • a method includes further esterifying or salifying the acrylic acid product thereby obtained.
  • the compounds of formula (Al), (A2), (A3), (A4), (A5), (A6), (A7), (A8), (A9), (AlO), (Al 1) may be prepared by procedures known to those skilled in the art from known compounds or readily preparable intermediates. General synthetic procedures and examples are as follow:
  • tert-butyl esters were converted to their corresponding carboxylic acids via treatment with a solution of 50% trifluoroacetic acid in dichloromethane for 1 hour at 23°C. The solvent was removed in vacuo and the residue was azeotroped with toluene or acetonitrile to yield the corresponding carboxylic acid.
  • reaction may be carried out neat or in a solvent such as dimethylformamide (DMF), tetrahydrofuran (THF), or toluene, in the presence of a catalyst (e.g. Pd(OAc) 2 , Pd(PPh 3 ) 4 , Pd 2 dba 3 ), a ligand (e.g. Ph 3 P, Ph 3 As, (o-tolyl) 3 P) and a base (e.g. K 2 CO 3 , CsCC» 3 , Et 3 N) at temperatures ranging from 23°C to 130°C, for 1 to 60 hours.
  • a catalyst e.g. Pd(OAc) 2 , Pd(PPh 3 ) 4 , Pd 2 dba 3
  • a ligand e.g. Ph 3 P, Ph 3 As, (o-tolyl) 3 P
  • a base e.g. K 2 CO 3 , CsCC» 3 , Et 3 N
  • the reaction mixture was heated at 100°C for 12 hours, cooled to 23 °C and the solvent was removed in vacuo. Ethyl acetate was added and the organic layer was washed with water and dried over sodium sulfate. The solvent was removed and the residue (mixture of dibromobenzil, mono and bis-tert-butylacrylate benzil) was recrystallized from hot 30% dichloromethane in hexane. The solid which crashed out (mixture of dibromobenzil and mono-tert-butylacrylate benzil) was filtered off and treated with 20% trifluoroacetic acid in dichloromethane.
  • These reactions may be carried out on functionalized cross linked polystyrene polymers such as Merrifield resin, Wang resin, Rink resin, TentagelTM resin, in a solvent such as dimethylformamide (DMF), tetrahydrofuran (THF), or toluene, in the presence of a catalyst (e.g. Pd(OAc) 2 , Pd(PPh 3 ) 4 , Pd 2 dba 3 ), a ligand (e.g. Ph 3 P, Ph 3 As, (o-tolyl) 3 P) and a base (e.g. K 2 CO 3 , CsCO 3 , Et 3 N) at temperatures ranging from 23°C to 130°C, for 1 to 60 hours.
  • a catalyst e.g. Pd(OAc) 2 , Pd(PPh 3 ) 4 , Pd 2 dba 3
  • a ligand e.g. Ph 3 P, Ph 3 As, (o-tolyl) 3 P
  • the resin was filtered and thoroughly washed with dichloromethane (500mL), methanol (500mL), dimethylformamide (500mL), dichloromethane (500mL) and methanol (500mL) and dried in vacuo (O.lmmHg) for 24 hours.
  • the coupling was repeated and resin 15 was filtered, washed and dried as above, and used directly in the next step.
  • the resin was filtered hot and washed thoroughly with hot dimethylformamide (500mL), hot acetic acid (500mL), methanol (500mL), dichloromethane (500mL), dimethylformamide (500mL), dichloromethane
  • the resin was filtered hot and washed thoroughly with hot dimethylformamide (500mL), hot acetic acid (500mL), methanol (500mL), dichloromethane (500mL), dimethylformamide (500mL), dichloromethane (500mL) and methanol (500mL) and dried in vacuo (O.lmmHg) for 24 hours.
  • the linker was cleaved from the resin with a solution of 20% trifluoroacetic acid in dichloromethane for 20min at ambient temperature.
  • ⁇ NMR for diacid linker 400 MHz, dg-DMSO) ⁇ 6.7 (d, 2H), 7.6 (d, 2H), 7.9 (s, 8H).
  • the resin was filtered hot and washed thoroughly with hot dimethylformamide (50mL), water (50mL), 10% sodium bicarbonate (50mL), 10% aqueous acetic acid (50mL), water (50mL), methanol (50mL), dichloromethane (50mL), methanol (50mL), dichloromethane (50mL) and dried in vacuo (O.lmmHg) for 24 hours.
  • the linker was cleaved from the resin with a solution of 20% trifluoroacetic acid in dichloromethane for 20min at ambient temperature.
  • ⁇ NMR for diacid linker 400 MHz, dg-DMSO) ⁇ 6.7 (d, 2H), 7.6 (d, 2H), 7.9 (s, 8H).
  • Resin 18 was treated with a 1.0M solution of oxalyl chloride in dichloromethane in the presence of a catalytic amount of dimethylformamide for 1 hour and filtered. The resin was subsequently treated with a dichloromethane solution containing the alcohol (ROH), pyridine and 4-dimethylaminopyridine for 20 hours at
  • Resin 18 was treated with a 1.0M solution of oxalyl chloride in dichloromethane in the presence of a catalytic amount of dimethylformamide for 1 hour and filtered. The resin was subsequently treated with a dichloromethane solution containing the aromatic amine (ArN(Rj)H), pyridine and 4-dimethylaminopyridine for 20 hours at 23°C to yield the monoamide resin 20.
  • Resin 18 was treated with a dichloromethane solution containing the amine (R]R 2 NH), EDCI and 4-dimethylamino ⁇ yridine for 20 hours at 23 °C to yield the monoamide resin 21.
  • reaction may be carried out in a solvent or combination of solvents such as tetrahydrofuran (THF), dichloromethane (CH 2 C1 2 ), in the presence of a catalyst (e.g. TiCl 3 ), and a base (e.g. pyridine) at temperatures ranging from -78°C to 23°C, for 1 to 60 hours.
  • solvents such as tetrahydrofuran (THF), dichloromethane (CH 2 C1 2 )
  • a catalyst e.g. TiCl 3
  • a base e.g. pyridine
  • the first step in this reaction may be carried out in a solvent such as tetrahydrofuran (THF), dichloromethane (CH 2 C1 2 ), in the presence of diisopropyl carbodiimide (DIC) and a base (e.g. 4-dimethylaminopyridine) at temperatures ranging from 0°C to 23 °C, for 1 to 60 hours.
  • the second step in this reaction may be carried out in a solvent such as dichloromethane (CH 2 C1 2 ), in the presence of an oxidizing reagent (e.g. tetrapropylammonium perruthenate (VII) (TPAP)) and activated 4A molecular sieves at temperatures ranging from 0°C to 23°C, for 1 to 60 hours.
  • a solvent such as tetrahydrofuran (THF), dichloromethane (CH 2 C1 2 )
  • DIC diisopropyl carbodiimide
  • a base e
  • Hydroxyester 24 (1 equiv) was oxidized to ketoester 26 at 23°C in CH 2 C1 2 , in the presence of catalytic amount of TPAP (0.1 equiv), N-methylmorpholine oxide (2 equiv) and 4A activated powdered molecular sieves (500mg/mol of substrate).
  • ⁇ NMR of 26 400MHz, CDC1 3 ) ⁇ 1.55 (s, 18H), 3.8 (s, 3H), 6.25 (d, IH), 6.29 (d, IH), 6.9 (d, 2H), 7.0 (s, IH), 7.5 (m, 10H), 7.95 (d, IH), 8.02 (d, IH).
  • the first step in this reaction may be carried out in a solvent or a combination of solvents such as tetrahydrofuran (THF), dichloromethane (CH 2 C1 2 ), in the presence of a catalyst (e.g. TiCl 3 ), and a base (e.g. pyridine) at temperatures ranging from - 78°C to 23°C, for 1 to 60 hours.
  • the second step in this reaction may be carried out in a solvent such as dichloromethane (CH 2 C1 2 ), in the presence of an oxidizing reagent (e.g. tetrapropylammonium perruthenate (VII) (TPAP)) and activated 4A molecular sieves at temperatures ranging from 0°C to 23 °C, for 1 to 60 hours.
  • a solvent or a combination of solvents such as tetrahydrofuran (THF), dichloromethane (CH 2 C1 2 ), in the presence of a catalyst (e.g.
  • reaction may be carried out in a solvent or a combination of solvents such as dichloromethane (CH 2 C1 2 ), chloroform (CHC1 3 ), methanol (MeOH), tetrahydrofuran (THF) or acetonitrile (CH 3 CN), in the presence or absence of a catalyst (e.g. ZnCl 2 , MgBr 2 ) at temperatures ranging from -78°C to 80°C, for 1 to 60 hours.
  • a catalyst e.g. ZnCl 2 , MgBr 2
  • reaction may be carried out in a solvent or combination of solvents such as tetrahydrofuran (THF), dichloromethane (CH 2 C1 2 ), in the presence of a catalyst (e.g. TiCl 3 ), and a base (e.g. pyridine) at temperatures ranging from -78°C to 23 °C, for 1 to 60 hours.
  • solvents such as tetrahydrofuran (THF), dichloromethane (CH 2 C1 2 )
  • a catalyst e.g. TiCl 3
  • a base e.g. pyridine
  • This reaction may be carried out on functionalized cross linked polystyrene polymers such as Merrifield resin, Wang resin, Rink resin, TentagelTM resin, in a solvent such as acetic acid (AcOH) at temperatures ranging from 23 °C to 120°C, for 1 to 60 hours.
  • a solvent such as acetic acid (AcOH) at temperatures ranging from 23 °C to 120°C, for 1 to 60 hours.
  • the product maybe released from the polymer using conditions known to those skilled in the art.
  • R l s R 2 , R 3 and 4 are defined as above in formula (A6).
  • the first step in this reaction may be carried out on functionalized cross linked polystyrene resins such as Merrifield resin, Wang resin, Rink resin, TentagelTM resin, in a solvent or a combination of solvents such as dichloromethane (CH 2 C1 2 ), chloroform (CHC1 ), methanol (MeOH), tetrahydrofuran (THF) or acetonitrile (CH 3 CN), in the presence or absence of a catalyst (e.g. ZnCl 2 , MgBr 2 ) at temperatures ranging from -78°C to 80°C, for 1 to 60 hours.
  • the second step in this reaction may be carried out in a solvent such as acetic acid (AcOH) at temperatures ranging from 23°C to 120°C, for 1 to 60 hours.
  • the first step in this reaction reaction may be carried out in a solvent or a combination of solvents such as dichloromethane (CH 2 C1 2 ), chloroform (CHC1 3 ), methanol (MeOH), tetrahydrofuran (THF), acetonitrile (CH 3 CN), in the presence or absence of a catalyst (e.g. ZnCl 2 , MgBr 2 ) at temperatures ranging from -78°C to 80°C, for 1 to 60 hours.
  • a catalyst e.g. ZnCl 2 , MgBr 2
  • the second step in this reaction may be carried out in a solvent such as acetic acid (AcOH) at temperatures ranging from 23 °C to 120°C, for 1 to 60 hours.
  • the first step in this reaction reaction may be carried out in a solvent or a combination of solvents such as dichloromethane (CH 2 C1 2 ), chloroform (CHC1 3 ), methanol (MeOH), tetrahydrofuran (THF), acetonitrile (CH 3 CN), in the presence or absence of a catalyst (e.g. ZnCl , MgBr 2 ) at temperatures ranging from -78°C to 80°C, for 1 to 60 hours.
  • a catalyst e.g. ZnCl , MgBr 2
  • the second step in this reaction may be carried out in a solvent such as acetic acid (AcOH) at temperatures ranging from 23 °C to 120°C, for 1 to 60 hours.
  • reaction may be carried out in a solvent or a combination of solvents such as dioxane or acetic acid (AcOH) at temperatures ranging from 23 °C to 120°C, for 1 to 60 hours.
  • solvents such as dioxane or acetic acid (AcOH)
  • the first step in this sequence of reactions may be carried out in a solvent such as tetrahydrofuran (THF), dichloromethane (CH 2 C1 2 ), in the presence of a base (e.g. 4,4-dimethylaminopyridine, triethylamine, triisopropylamine) and a sulfonyl chloride (e.g. tosyl chloride, mesyl chloride), at temperatures ranging from -20°C to 23°C, for 1 to 60 hours.
  • the second step in this sequence of reactions may be carried out in a solvent such as dichloromethane (CH 2 C1 2 ), in the presence of an oxidizing reagent (e.g.
  • TPAP tetrapropylammonium perruthenate
  • VI tetrapropylammonium perruthenate
  • the third step in this sequence of reactions may be carried out in a solvent such as acetic acid, toluene, dioxane at temperatures ranging from 0°C to 120°C, for 1 to 60 hours.
  • a human placental cDNA library was synthesized in a 50 ul reaction containing 1 ug human placental poly(A) + mRNA (Clontech, Palo Alto, CA), 4 ul random hexamer primers, 8 ul of lOmM dNTPs (Pharmacia, Piscataway NJ), lul (200 U/ul) Moloney murine leukemia virus reverse transcriptase (Gibco-BRL, Canada), 0.5 ul (26 U/ul) RNAsin (Promega, Madison WI), and 12 ul 5x buffer (Gibco-BRL). The synthesis reaction was incubated at 37 ° C for one hour and then heat inactivated at 95 ° C for five minutes.
  • a PTP-IB cDNA was amplified, using polymerase chain reaction (PCR), from the cDNAs synthesized as described above. More particularly, based on the published sequence of PTB- IB, two PCR primers were synthesized to amplify a portion of the PTP-IB coding sequence known to encode a 321 amino acid fragment containing the PTP-IB catalytic domain and having PTPase activity. See Hoppe et al., Eur. J. Biochem., 225. 1069-77 (1994); Barford, D., et al, J. Molec. Biol, 239:726-730 (1994); Chernoff et al. , Proc. Natl. Acad. Sci. USA, 57:2735-2739 (1990); Charbonneau et al. Proc Natl. Acad. Sci. USA, ⁇ °6 ' ;5252-5256 (1989).
  • the primers had the following respective sequences:
  • the first primer which hybridizes to the non-coding strand, corresponds to the 5' portion of the PTP-IB coding sequence and encodes a BamH I restriction site upstream of the initiation codon, to facilitate cloning.
  • the second primer which hybridizes to the coding strand, corresponds to the 3' portion of the PTB- IB fragment of interest, and encodes a stop codon and an EcoR I restriction site downstream from the stop codon.
  • a 100 ⁇ l PCR reaction mixture containing approx. 1 ug of the human placental cDNA library, 0.2 mM of each dNTP, 30 uM of each primer, lx Amplitaq DNA polymerase buffer (Perkin-Elmer, Norwalk CT), and 5 units Amplitaq DNA polymerase (Perkin-Elmer) was denatured at 94 ° C for 5 minutes and then subjected to 25 cycles of amplification as follows: 1) 94 ° C denaturation for 1 minute; 2) 55 ° C annealing for 1 minute; and 3) 72 ° C primer extension for 1 minute.
  • the PCR reaction product (992 bp) was digested with BamH I and EcoR I (New England Biolabs, Beverly MA) to yield a 975 bp product encoding the 321 amino acid PTP-IB protein fragment, and having "sticky ends" to facilitate cloning.
  • B Production of a PTP-IB expression vector.
  • the 975 bp PTP-IB partial cDNA was purified by agarose gel electrophoresis and ligated into a BamH HEcoR I-digested pGEX-3X plasmid vector (Pharmacia, Piscataway, NJ).
  • the pGEX vector is designed to produce a fusion of glutathione-S- transferase (GST) to a protein encoded by another DNA fragment inserted into the vector's cloning site.
  • GST glutathione-S- transferase
  • E. coli strain DH5 ⁇ (Gibco-BRL) was transformed with plasmid pGEX-3X-
  • PTP-1B following the supplier's transformation protocol and grown at 37 ° C with vigorous shaking in Luria-Bertani broth supplemented with 100 ug/ml ampicillin.
  • O.D. 600 of 0.7-1 production of the GST/PTP-IB fusion protein was induced with 0.1 mM IPTG (Isopropyl b-D-Thiogalactoside). After 3 additional hours of culturing at 37 C, the bacteria were pelleted by centrifugation.
  • the bacterial pellet was resuspended in lOx (w/v) lysis buffer consisting of 12.5 mM HEPES, 2 mM EDTA, pH 7.0, 15 mM b-mercaptoethanol (bME) and 1 mM PMSF.
  • the lysate was sonicated (on ice) until slight clearing was observed (approx. three min.) and then centrifuged at 10,000 revolutions per minute (RPM) for 10 min.
  • the supernatant was diluted 1 :4 with buffer A (25 mM HEPES, pH 7.0, and 15 mM bME).
  • Fractions containing PTPase activity were pooled, diluted 1 :4 with NET buffer (20 mM Tris, pH 8.8, 100 mM NaCl, 1 mM EDTA and 15 mM bME), and loaded onto a 10 ml GST-Sepharose 4B column (Pharmacia). After loading, the column was washed first with 3 bed volumes of NET buffer + 1%> NP40 (Sigma Chemical Co., St. Louis, MO), then with NET buffer until O.D. at 280 nm was basal. The GST/PTP-IB fusion protein was eluted from the column using 10 mM glutathione in 33 mM Tris, pH 8.0.
  • the GST/PTP- IB-containing fractions from the GST-Sepharose 4B purification were pooled, concentrated into a final storage buffer (0.2 M NaCl, 25 mM HEPES, 1 mM EDTA, and 5 mM DTT, pH 7.0) using a 1 ml Hi-Trap Q column (pre-packed, Pharmacia), and stored at -80 ° C (final concentration of 0.52 mg/ml).
  • the foregoing procedure yielded approximately 5mg of PTP-IB fusion protein per 500 ml of cultured cells, purified to substantial homogeneity as assessed by SDS- PAGE.
  • the protein concentration of the PTP-IB enzyme preparation was determined using the Bio-Rad Protein Assay kit (Bio-Rad, Hercules CA). An aliquot from each sample was taken and diluted to 2 mg protein/ml using activity assay buffer (100 mM Sodium Acetate, pH 6.0, 1 mM EDTA, 0.1% TX-100 (International Biotechnologies, Inc.) and 15 mM bME) to form a PTP-IB stock solution.
  • activity assay buffer 100 mM Sodium Acetate, pH 6.0, 1 mM EDTA, 0.1% TX-100 (International Biotechnologies, Inc.) and 15 mM bME
  • a 100 ul reaction mixture was prepared containing 10 ul of the PTP-IB stock solution, 10 ul of 9 mM p-nitrophenylphosphate ((pNPP), Sigma Chemical Co., St. Louis MO), and 80 ul of activity assay buffer (100 mM sodium acetate, pH 6.0, 1 mM EDTA, 0.1%) Triton X-100, 15 mM bME). Reactions were mixed gently and incubated at 37 ° C for 60 minutes. Enzymatic cleavage of phosphate from pNPP (a tyrosine phosphate analog) is marked by a colorimetric change in this substrate. See, e.g., Imbert et al, Biochem J., 297. 163-173 (1994); Ghosh and Miller, Biochem. Biophys. Res. Comm., 194:36-44 (1993); Zanke et al, Eur. J. Immunol, 22:235-39 (1992).
  • a human cDNA library was synthesized from RNA isolated from the human Jurkat cell line, as described above for PTP-IB
  • CD45 cDNA was amplified, using polymerase chain reaction (PCR), from the cDNAs synthesized above. Two PCR primers were synthesized to amplify the coding sequence of CD45. The primers had the following respective sequences:
  • CD45 (5') (SEQ ID NO: 3)
  • CD45 (3') (SEQ ID NO: 4)
  • the first primer corresponds to the 5' portion of the CD45 coding sequence and encodes a Sma I restriction site upstream of the initiation codon, to facilitate cloning.
  • the second primer corresponds to the 3' portion of the CD45 sequence, and encodes a stop codon and a Sma I restriction site downstream from the stop codon.
  • the PCR reaction product (2127 bp) was digested with Sma I (New England Biolabs, Beverly MA) to yield a 2110 bp product.
  • the pET24C plasmid vector (Novagen, Inc., Madison WI) was digested with the BamH I restriction enzyme, and the "sticky" ends were filled using T4 DNA polymerase according to the manufacturer's instructions (New England Biolabs, Beverly MA); the resulting plasmid DNA was ligated to Sma /-digested CD45 PCR product.
  • the pET24C vector is designed to produce high levels of the protein encoded by cDNA inserted into the vector's cloning site (CD45), in bacterial hosts. Complete sequencing of the insert of the resultant plasmid, designated pET24C-CD45, confirmed the identity of the CD45 cDNA, and insertion in the proper orientation and reading frame.
  • E. coli strain DH5 (Gibco-BRL) was transformed with pET24C-CD45 following the supplier's transformation protocol, plated onto Luria-Bertani agar plates supplemented with 30 ug/ml kanamycin and grown overnight at 37 ° C. A single bacterial colony was transferred into 50 mis Luria-Bertani broth supplemented with 30 ug/ml kanamycin and grown overnight with vigorous shaking. This overnight culture was split into two equal parts, and added to 2L Luria-Bertani broth supplemented with 50 ug/ml kanamycin. When the cultures reached an O.D.
  • the bacterial pellet (approximately 5 grams) was resuspended in lOx (w/v) lysis buffer consisting of 12.5 mM HEPES, 2 mM EDTA, pH 7.0, 15 mM bME and 1 mM PMSF. The lysate was sonicated (on ice) until slight clearing was observed (approx. three min.) and then centrifuged at 10,000 revolutions per minute (RPM) for 10 min. The supernatant was filtered through 1mm Wattman filter paper, and 9.7 grams (i.e., 194 grams/L) of ammonium sulfate were added to the solution on ice to precipitate soluble proteins.
  • lOx (w/v) lysis buffer consisting of 12.5 mM HEPES, 2 mM EDTA, pH 7.0, 15 mM bME and 1 mM PMSF.
  • the lysate was sonicated (on ice) until slight clearing was observed (approx
  • the lysate was spun at 10,000 RPM for 30 min. at 4 C; supernatant was removed, and an additional 7.6 grams (i.e., 151 grams/L) of ammonium sulfate were added.
  • the resulting pellet was resuspended in 3 mis of buffer B (33 mM imidazole-HCl pH 8.0, 2mM EDTA, 10 mM bME, 0.002% PMSF) and stored on ice.
  • the spin supernatant with ammonium sulfate was spun again at 10,000 RPM for 30 mins at 4 C.
  • the resulting pellet from the second centrifugation was resuspended in 2 mis of buffer B.
  • the two pellet solutions were pooled and dialyzed overnight against buffer B.
  • the CD45-containing fractions from the MonoQ column purification were pooled and stored at 4 C.
  • CD45 enzymatic activity of samples was assayed in microtiter plates as follows.
  • a 100 ul reaction mixture was prepared containing 10 ul of the CD45 stock solution, 10 ul of 9.3 mM p-nitrophenylphosphate ((pNPP), Sigma Chemical Co., St. Louis MO), and 80 ul of activity assay buffer (100 mM sodium acetate, pH 6.0, 1 mM EDTA, 0.1%) Triton X-100, 15 mM bME). Reactions were mixed gently and incubated at 37 ° C for 60 minutes. Reactions were stopped by addition of 10 ul of a 0.5 M NaOH/50% EtOH solution. To determine the enzymatic activity, absorbance readings of the reactions were measured at 405 nm using a Molecular Devices Thermomax Plate Reader (Menlo Park CA). In vitro PTPase Inhibition Assay
  • the ability of the compounds of the present invention, such as the cinnamic acid derivative compounds of Example 2, to inhibit the PTPase activity of PTP-IB, CD45, PTP-IC, and PTP ⁇ was determined using modifications of the PTP-IB and CD45 activity assays described in Examples 3 and 4.
  • 0.001 mmol of the cinnamic acid derivative (or other PTPase inhibitor compound) was dissolved in 100 ul of DMSO to create a 10 mM stock solution.
  • the 10 mM stock solution was used to add varying concentrations (100 uM, 33 uM, 10 uM, 3 uM, 1 uM, 0.3 uM, 0.1 uM, 0.03 uM, 0.01 uM or 0.003 uM) of the inhibitor compound to a series of otherwise identical PTPase activity assay reactions (100 ul final volume in microtiter wells).
  • each 100 ul reaction contained 10 ul PTPase enzyme stock solution (final phosphatase concentration of approximately 20 ng/well), 70 ul activity assay buffer, 10 ul pNPP stock solution (final pNPP concentration of .9 mM for PTP-IB assay, 0.93 mM for CD45 assay, 0.5 mM for PTP ⁇ assay, and 8 mM for PTP-IC assay), and 10 ul of the diluted inhibitor compound in DMSO.
  • Assay buffers contained: for CD45 and PTP-IB assays, 100 mM sodium acetate at pH 6.0, 1 mM EDTA, 0.1% Triton X-100, and 15 mM bME; for PTP-IC assays, 100 mM sodium acetate at pH 5.5, 0.1% BSA, and 15 mM bME; for PTP ⁇ assays, 100 mM sodium acetate at pH 5.25, 0.1 % BSA, and 15 mM bME. Purified phosphatase was added to the reaction mixtures to begin the reactions; the reactions were incubated at 37C for 60 min. (for PTP-IB and CD45 assays) or at 27 C for 60 min.
  • the concentration of inhibitor compound required to inhibit 50%> of the PTPase activity was determined as follows. First, absorbance readings from the negative control reactions were treated as a baseline and subtracted from the absorbance readings of the experimental reactions. Then, for each reaction, a percent inhibition was calculated using the following formula:
  • IC50 concentration was calculated from a best-fit computer analysis of the calculated percent inhibition for the various dilutions of the compound.
  • Inhibitor compounds having an IC50 less than 10 uM (and optimally less than 5uM) for a particular PTPase were scored as highly effective inhibitors of that PTPase enzyme, and are preferred inhibitors of the present invention.
  • the compounds of the present invention have asymmetric centers and may occur as racemates, racemic mixtures, and as individual enantiomers or diastereoisomers, with all isomeric forms being included in the present invention as well as mixtures thereof.
  • compositions of Formula (Al) thru (Al l) where a basic or acidic group is present in the structure are also included within the scope of this invention.
  • an acidic substituent such as - COOH 5 there can be formed the ammonium, sodium, potassium, calcium salt, and the like, for use as the dosage form.
  • a basic group such as amino or a basic heteroaryl radical, such as pyridyl
  • an acidic salt such as hydrochloride, hydrobromide, acetate, maleate, pamoate, methanesulfonate, p-toluenesulfonate, and the like, can be used as the dosage form.
  • esters can be employed, e.g., methyl, tert-butyl, pivaloyloxymethyl, and the like, and those esters known in the art for modifying solubility or hydrolysis characteristics for use as sustained release or prodrug formulations.
  • solvates may form solvates with water or common organic solvents. Such solvates are encompassed within the scope of the invention.
  • terapéuticaally effective amount shall mean that amount of drug or pharmaceutical agent that will elicit the biological or medical response of a tissue, system, animal, or human that is being sought by a researcher, veterinarian, medical doctor or other clinician.
  • a daily dose of about 0.5mg/Kg to lOOmg/Kg body weight in divided doses is suggested to treat PTPase related diseases. Such dosage has to be individualized by the clinician.
  • the present invention also has the objective of providing suitable topical, oral, and parenteral pharmaceutical formulations for use in the novel methods of treatment of the present invention.
  • the compounds of the present invention may be administered orally as tablets, aqueous or oily suspensions, lozenges, troches, powders, granules, emulsions, capsules, syrups or elixirs.
  • the composition for oral use may contain one or more agents selected from the group of sweetening agents, flavouring agents, colouring agents and preserving agents in order to produce pharmaceutically elegant and palatable preparations.
  • the tablets contain the acting ingredient in admixture with non-toxic pharmaceutically acceptable excipients which are suitable for the manufacture of tablets.
  • excipients may be, for example, (1) inert diluents , such as calcium carbonate, lactose, calcium phosphate or sodium phosphate; (2) granulating and disintegrating agents, such as corn starch or alginic acid; (3) binding agents, such as starch, gelatin or acacia; and (4) lubricating agents, such as magnesium stearate, stearic acid or talc.
  • inert diluents such as calcium carbonate, lactose, calcium phosphate or sodium phosphate
  • granulating and disintegrating agents such as corn starch or alginic acid
  • binding agents such as starch, gelatin or acacia
  • lubricating agents such as magnesium stearate, stearic acid or talc.
  • These tablets may be uncoated or coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period.
  • a time delay material such as g
  • Formulations for oral use may be in the form of hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin. They may also be in the form of soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, such as peanut oil, liquid paraffin or olive oil.
  • an inert solid diluent for example, calcium carbonate, calcium phosphate or kaolin.
  • the active ingredient is mixed with water or an oil medium, such as peanut oil, liquid paraffin or olive oil.
  • Aqueous suspensions normally contain the active materials in admixture with excipients suitable for the manufacture of aqueous suspension.
  • expicients may be: (1) suspending agent such as sodium carboxymethyl cellulose, methyl cellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia;
  • dispersing or wetting agents which may be (a) naturally occurring phosphatide such as lecithin; (b) a condensation product of an alkylene oxide with a fatty acid, for example, polyoxyethylene stearate; (c) a condensation product of ethylene oxide with a long chain aliphatic alcohol, for example, heptadecaethylenoxycetanol; (d) a condensation product of ethylene oxide with a partial ester derived from a fatty acid and hexitol such as polyoxyethylene sorbitol monooleate, or (e) a condensation product of ethylene oxide with a partial ester derived from fatty acids and hexitol anhydrides, for example polyoxyethylene sorbitan monooleate.
  • phosphatide such as lecithin
  • a condensation product of an alkylene oxide with a fatty acid for example, polyoxyethylene stearate
  • a condensation product of ethylene oxide with a long chain aliphatic alcohol for
  • the pharmaceutical compositions may be in the form of a sterile injectable aqueous or oleagenous suspension.
  • This suspension may be formulated according to known methods using those suitable dispersing or wetting agents and suspending agents which have been mentioned above.
  • the sterile injectable preparation may also a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example, as a solution in 1,3-butanediol.
  • the acceptable vehicles and solvents that may be employed are water, Ringer's solution, and isotonic sodium chloride solution.
  • sterile, fixed oils are conventionally employed as a solvent or suspending medium.
  • any bland fixed oil may be employed including synthetic mono- or diglycerides.
  • fatty acids such as oleic acid find use in the preparation of injectables.
  • Compounds of Formula (Al) thru (Al 1) may also be administered in the form of suppositories for rectal administration of the drug.
  • These compositions can be prepared by mixing the drug with a suitable non-irritating excipient which is solid at ordinary temperature but liquid at the rectal temperature and will therefore melt in the rectum to release the drug.
  • suitable non-irritating excipient which is solid at ordinary temperature but liquid at the rectal temperature and will therefore melt in the rectum to release the drug.
  • Such materials are cocoa butter and polyethylene glycols.
  • the compounds of the present invention may also be administered in the form of liposome delivery systems, such as small unilamellar vesicles, large unilamellar vesicles, and multilamellar vesicles.
  • Liposomes can be formed from a variety of phospholipids, such as cholesterol, stearylamine, or phosphatidylcholines.

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Abstract

La présente invention concerne de nouveaux composés modulant la protéine tyrosine phosphatase et correspondant à la structure générale illustrée dans la formule (A1) Y-X-C(R')=C(R'')COOR''', des procédés de préparation de ces composés et des compositions contenant ces composés. L'invention se rapporte en outre à l'utilisation de ces composés dans le traitement de troubles chez l'être humain et chez l'animal, dans la purification de protéines ou de glycoprotéines, et dans des diagnostics. L'invention se rapporte enfin à la modulation de l'activité de molécules possédant des unités de reconnaissance de la phosphotyrosine, y compris les protéines tyrosine phosphatases (PTPases) et les protéines possédant des domaines Src-homologie-2, dans des systèmes in vitro, des micro-organismes et des cellules eucaryotes, ainsi que chez des animaux et des êtres humains dans leur ensemble. R' et R'' sont choisis indépendamment l'un de l'autre dans le groupe comprenant un hydrogène, un halo, cyano, nitro, trihalométhyle, alkyle, arylalkyle. R''' est choisi dans le groupe comprenant un hydrogène, un alkyle, alkyle substitué, aryle, arylalkyle. X est un aryle. Y est choisi entre un hydrogène ou α où (*) indique un point éventuel de rattachement à X.
PCT/US1996/020508 1995-06-19 1996-12-16 Modulateurs de proteines possedant des unites de reconnaissance de la phosphotyrosine WO1998027065A1 (fr)

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US08/543,630 US5770620A (en) 1995-06-19 1995-10-16 Aryl acrylic acid derivatives useful as protein tyrosine phosphatase inhibitors
EP96940489A EP0833629A4 (fr) 1995-06-19 1996-06-19 Derives d'acide aryl-acrylique convenant comme inhibiteurs de proteine-tyrosine-phosphatase
JP52765098A JP2001506997A (ja) 1996-12-16 1996-12-16 ホスホチロシン認識ユニットを有する蛋白質の修飾物質
AU15667/97A AU740425B2 (en) 1996-12-16 1996-12-16 Modulators of proteins with phosphotyrosine recognition units
CA002275610A CA2275610A1 (fr) 1996-12-16 1996-12-16 Modulateurs de proteines possedant des unites de reconnaissance de la phosphotyrosine
PCT/US1996/020508 WO1998027065A1 (fr) 1996-12-16 1996-12-16 Modulateurs de proteines possedant des unites de reconnaissance de la phosphotyrosine
EP96945409A EP0946518A1 (fr) 1996-12-16 1996-12-16 Modulateurs de proteines possedant des unites de reconnaissance de la phosphotyrosine
US08/766,114 US5753687A (en) 1995-06-19 1996-12-16 Modulators of proteins with phosphotryrosine recognition units

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US6498182B2 (en) 2000-09-26 2002-12-24 Biovitrum Ab Compounds
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WO2003037332A1 (fr) * 2001-10-12 2003-05-08 Bayer Pharmaceuticals Corporation Heterocycles utiles pour le traitement de l'obesite
WO2003040107A1 (fr) * 2001-09-24 2003-05-15 Bayer Pharmaceuticals Corporation Preparation et utilisation de derives d'imidazole dans le traitement de l'obesite
US6613903B2 (en) 2000-07-07 2003-09-02 Novo Nordisk A/S Modulators of protein tyrosine phosphatases (PTPases)
WO2004016086A3 (fr) * 2002-08-19 2004-04-29 Lorus Therapeutics Inc Imidazoles 2,4,5-trisubstitues et utilisation de ceux-ci comme agents anti-microbiens
WO2004071447A2 (fr) * 2003-02-12 2004-08-26 Transtech Pharma Inc. Utilisation de derives d'azoles substitues en tant qu'agents therapeutiques
US7141596B2 (en) 2003-10-08 2006-11-28 Incyte Corporation Inhibitors of proteins that bind phosphorylated molecules
CN101810608A (zh) * 2009-02-24 2010-08-25 中国人民解放军第二军医大学 一种靶向于人磷脂酰乙醇胺结合蛋白4的抗肿瘤小分子化合物
US8148392B2 (en) 2005-05-25 2012-04-03 Lorus Therapeutics Inc. 2-indolyl imidazo [4,5-d] phenanthroline derivatives and their use in the treatment of cancer
US8969372B2 (en) 2003-11-14 2015-03-03 Aptose Boisciences Inc. Aryl imidazoles and their use as anti-cancer agents
US9309247B2 (en) 2013-03-20 2016-04-12 Lorus Therapeutics Inc. 2-substituted imidazo[4,5-D]phenanthroline derivatives and their use in the treatment of cancer
US11104957B2 (en) 2013-10-04 2021-08-31 Aptose Biosciences, Inc. Compositions and methods for treating cancers
US11149047B2 (en) 2017-10-30 2021-10-19 Aptose Biosciences, Inc. Aryl imidazoles for treatment of cancer
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US6613903B2 (en) 2000-07-07 2003-09-02 Novo Nordisk A/S Modulators of protein tyrosine phosphatases (PTPases)
US6498182B2 (en) 2000-09-26 2002-12-24 Biovitrum Ab Compounds
WO2003040107A1 (fr) * 2001-09-24 2003-05-15 Bayer Pharmaceuticals Corporation Preparation et utilisation de derives d'imidazole dans le traitement de l'obesite
US6960601B2 (en) 2001-09-24 2005-11-01 Bayer Pharmaceuticals Corporation Preparation and use of imidazole derivatives for treatment of obesity
DE10150172A1 (de) * 2001-10-11 2003-04-30 Morphochem Ag Neue Verbindungen, die Protein Tyrosin Phosphatase 1B (PTP-1B) inhibieren
WO2003037332A1 (fr) * 2001-10-12 2003-05-08 Bayer Pharmaceuticals Corporation Heterocycles utiles pour le traitement de l'obesite
US7884120B2 (en) 2002-08-19 2011-02-08 Lorus Therapeutics Inc. 2,4,5-trisubstituted imidazoles and their use as anti-microbial agents
WO2004016086A3 (fr) * 2002-08-19 2004-04-29 Lorus Therapeutics Inc Imidazoles 2,4,5-trisubstitues et utilisation de ceux-ci comme agents anti-microbiens
US8987305B2 (en) 2002-08-19 2015-03-24 Aptose Biosciences Inc. 2,4,5-trisubstituted imidazoles and their use as anti-microbial agents
US20130177632A1 (en) * 2002-08-19 2013-07-11 Raed H. Al-Qawasmeh 2,4,5-trisubstituted imidazoles and their use as anti-microbial agents
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US8394815B2 (en) 2002-08-19 2013-03-12 Lorus Therapeutics Inc. 2,4,5-trisubstituted imidazoles and their use as anti-microbial agents
WO2004071447A2 (fr) * 2003-02-12 2004-08-26 Transtech Pharma Inc. Utilisation de derives d'azoles substitues en tant qu'agents therapeutiques
WO2004071447A3 (fr) * 2003-02-12 2004-12-23 Transtech Pharma Inc Utilisation de derives d'azoles substitues en tant qu'agents therapeutiques
US7141596B2 (en) 2003-10-08 2006-11-28 Incyte Corporation Inhibitors of proteins that bind phosphorylated molecules
US10080739B2 (en) 2003-11-14 2018-09-25 Aptose Biosciences Inc. Aryl imidazoles and their use as anti-cancer agents
US8969372B2 (en) 2003-11-14 2015-03-03 Aptose Boisciences Inc. Aryl imidazoles and their use as anti-cancer agents
US8148392B2 (en) 2005-05-25 2012-04-03 Lorus Therapeutics Inc. 2-indolyl imidazo [4,5-d] phenanthroline derivatives and their use in the treatment of cancer
CN101810608A (zh) * 2009-02-24 2010-08-25 中国人民解放军第二军医大学 一种靶向于人磷脂酰乙醇胺结合蛋白4的抗肿瘤小分子化合物
US9309247B2 (en) 2013-03-20 2016-04-12 Lorus Therapeutics Inc. 2-substituted imidazo[4,5-D]phenanthroline derivatives and their use in the treatment of cancer
US11104957B2 (en) 2013-10-04 2021-08-31 Aptose Biosciences, Inc. Compositions and methods for treating cancers
US11149047B2 (en) 2017-10-30 2021-10-19 Aptose Biosciences, Inc. Aryl imidazoles for treatment of cancer
KR20220064450A (ko) * 2020-11-11 2022-05-19 주식회사 엘지화학 신규한 화합물 및 이를 이용한 유기발광 소자
WO2022102992A1 (fr) * 2020-11-11 2022-05-19 주식회사 엘지화학 Nouveau composé et dispositif électroluminescent organique le comprenant
CN116018338A (zh) * 2020-11-11 2023-04-25 株式会社Lg化学 新型化合物及包含其的有机发光器件
KR102623893B1 (ko) 2020-11-11 2024-01-11 주식회사 엘지화학 신규한 화합물 및 이를 이용한 유기발광 소자

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AU740425B2 (en) 2001-11-01
JP2001506997A (ja) 2001-05-29

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