WO1997007131A1 - Oligopeptides acyles divers - Google Patents

Oligopeptides acyles divers Download PDF

Info

Publication number
WO1997007131A1
WO1997007131A1 PCT/EP1996/003479 EP9603479W WO9707131A1 WO 1997007131 A1 WO1997007131 A1 WO 1997007131A1 EP 9603479 W EP9603479 W EP 9603479W WO 9707131 A1 WO9707131 A1 WO 9707131A1
Authority
WO
WIPO (PCT)
Prior art keywords
tyr
cys
asn
seq
glu
Prior art date
Application number
PCT/EP1996/003479
Other languages
English (en)
Inventor
Carlos Garcia-Echeverria
Brigitte Gay
Pascal Furet
Original Assignee
Novartis Ag
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Novartis Ag filed Critical Novartis Ag
Priority to AU68709/96A priority Critical patent/AU6870996A/en
Publication of WO1997007131A1 publication Critical patent/WO1997007131A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/02—Linear peptides containing at least one abnormal peptide link
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Definitions

  • the present invention relates to a new class of pharmaceutically active compounds comprising an acylated peptide structure, processes for the preparation of said compounds, pharmaceutical preparations comprising said compounds, the compounds for the use in the therapeutic (including prophylactic) or diagnostic treatment of the animal or especially human body, and the use of said compounds for the therapeutic or diagnostic treatment of the animal or especially human body or for the manufacture of pharmaceutical preparations.
  • Cancer and proliferative disorders affect a very large population and are one of the leading causes for death of human beings.
  • the process of signal transduction is responsible for relaying extracellular messages, e.g. chemical messages in the form of growth factors, hormones and neurotransmitters, via receptors, e.g. cell-surface receptors, to the interior of the cell.
  • extracellular messages e.g. chemical messages in the form of growth factors, hormones and neurotransmitters
  • receptors e.g. cell-surface receptors
  • a central feature of these biochemical communication processes is the working of protein-tyrosine kinases. These enzymes, for example found as either transmembrane growth factor receptors or as cytosolic or nuclear non-receptor proteins, catalyze the phosphorylation of specific tyrosine residues.
  • Examples for this class of enzymes include, but are not limited to, the PDGF receptor, the FGF receptor, the HGF receptor, members of the EGF receptor family such as the EGF receptor, erb-B2, erb-B3 and erb-B4, the src kinase family, Fak kinase and the Jak kinase family.
  • the tyrosine-phosphorylated proteins are involved in the regulation of a range of metabolic processes, ranging from proliferation and growth to differentiation and metabolism control.
  • Protein-tyrosine phosphorylation is known to be involved in modulating the activity of some special target enzymes as well as in initial and/or integral control of specific complex networks involved in signal transduction via various proteins containing a specific amino acid sequence called a Src Homology Region or SH2 domain (for review see Proc. Natl. Acad. Sci. USA 90, 5891 (1990)).
  • a malfunction in protein-tyrosine phosphorylation through tyrosine kinase overexpression or deregulation is causative for many oncogenic, degenerative and (hyper-) proliferative disorders such as cancer, inflammation, autoimmune disease, hyperproliferative skin disorders, such as psoriasis, and allergy/asthma.
  • Proteins comprising SH2 and/or SH3 domains that are effective in cellular signalling and transformation include, but are not limited to, the following: Src, Lck, Fps, ras GTPase- activating protein (GAP), phospholipase C, phosphoinositol-3 (PI-3) kinase, Fyn, Lyk, Fgr, Fes, ZAP-70, Sem-5, p85, SHPTP1 , SHPTP2, corkscrew, Syk, Lyn, Yes, Hck, Dsrc, Tec, Atk/Bpk, Itk/Tsk, Arg, Csk, tensin, Vav, Emt, Grb2, BCR-Abl, She, Nek, Crk, CrkL, Syp, Blk, 113TF, 91 TF, Tyk2, JAK1 , and JAK2, especially Src, phoshoiipase C, phosphoinositol-3 (
  • Grb2 protein a 26 kilodalton protein comprised of a single SH2 and two SH3 domains
  • the Grb2 SH2 domain binds to specific tyrosine phosphorylated sequences, e.g. in receptor tyrosine kinases, while the Grb2 SH3 domains bind to proline-rich sequences present in the Sos exchange factor.
  • SH2 domains are serving as recognition motifs for specific tyrosine-phosphorylated peptide sequences. Short, conserved motifs, primarily 3 to 6 amino acids on the carboxy-terminal side of a phosphotyrosine residue, carry the sequence-specific infor-mation for SH2- recognition. This concept has been supported by the mapping of separate sites for binding of SH2 domains from different signalling molecules on various receptors [see, e.g., Cell 69, 413 (1992); Proc. Natl. Acad. Sci. USA 89, 678 (1992); Mol. Cell. Biol. 12, 991 (1992); EMBO J. U, 1365 (1992); EMBO J. H* 559 (1992); EMBO J.
  • tyrosine 317 is the major site for SHC tyrosine phosphorylation and is the sole high-affinity binding site for Grb2 SH2.
  • Mutant SHC proteins with substitution of tyrosine 317 by phenylalanine loose the capacity to be highly phosphorylated on tyrosine upon growth factor activation, to bind Grb2 and to induce neoplastic transformation [see Oncogene 9, 2827 (1994)].
  • a FGR receptor with a point mutation at tyrosine 766 does not bind phospholipase C- ⁇ (an SH2-containing protein). It abolishes phosphatidylinositol turnover and calcium flux but not mitogenesis [see Nature 358, 678 (1992)].
  • EGFR Epidermal Growth Factor Receptor
  • PD 153035 rapidly suppressed autophosphorylation of the EGF receptor at low concentrations in human epidermoid carcinoma cells and selectively blocked EGF-mediated cellular processes including mitogenesis, early gene expression and oncogenic transformation [see Science 265, 1093 (1994)].
  • tyrosine kinase inhibitors RS-13022 and 14620 supressed EGF-stimulated proliferation of HER-14 cells (transfected NIH 3T3 cells) and MH-85 cells in vitro.
  • the MH-85 tumor is a human squamous cell carcinoma associated with three paraneoplastic syndromes: hypercalcemia, leukocytosis and cachexia.
  • the well-characterized cells show overexpression of endogenous EGF receptor tyrosine kinase and are dependent on the EGF receptor signal transduction pathway for growth in vitro and in nude mice.
  • the compounds suppressed the growth of MH-85 tumors in nude mice as well as the expression of the paraneoplastic syndromes. An increase in life span of 75% was observed for RG-13022- treated tumor bearing mice [see Cancer Res. 51, 4430 (1991)]
  • 4,5-Dianilinophthaiimides inhibit the growth of human tumor cells that overexpress EGFR or HER2-ErbB2 and exhibit good antitumor activity in mice in which these tumors are grown as xenografts (see Buchdunger et al., Proc. Natl. Acad. Sci USA 91, 2334 (1994) and Trinks et al., J. Med. Chem. 37, 1015 (1994)).
  • Anilinoquinazolines also represent a class of compounds which exhibit promising anti ⁇ cancer activity. They were shown to inhibit the EGF-stimulated growth of human KB nasopharyngeal cells in vitro at concentrations of 1 -10 ⁇ M. By these results it can be shown that the inhibition of regulatory pathways by way of inhibition of protein tyrosine kinases results in therapeutically useful effects. It is therefore reasonable that inhibition at the level of interaction of protein tyrosine kinases with other proteins, such as those with SH2 domains, will result in similar therapeutic usefulness.
  • the effect is to inhibit the association of SH2 containing (e.g. regulatory) proteins with a protein tyrosine kinase in order to inhibit downstream signalling through one or more specifically targeted effector proteins.
  • the compounds of the present invention show very favourable and valuable characteristics for pharmaceutical application, especially with regard to the therapeutic (including, in a broader sense, prophylactic) and/or diagnostic treatment of diseases that depend on the downstream signal transduction pathways, especially those mediated by an interaction of a protein comprising a SH2 domain with a tyrosine phosphorylated protein, such as a phosphorylated tyrosine protein kinase; proteins comprising one or more SH2 domains that are effective in cellular signalling and transformation include, but are not limited to, the following: Src, Lck, Fps, ras GTPase- activating protein (GAP), phospholipase C, phosphoinositol-3 (PI-3) kinase, Fyn, Lyk, Fgr, Fes, ZAP-70, Sem-5, p85, SHPTP1 , SHPTP2, corkscrew, Syk, Lyn, Yes, Hck
  • the new peptides of this invention preferably show selective inhibition of the binding of SH2-comprising proteins, such as Grb2, to phosphorylated proteins, especially activated growth factor receptor tyrosine kinases like EGF receptor tyrosine protein kinase, or She.
  • SH2-comprising proteins such as Grb2
  • phosphorylated proteins especially activated growth factor receptor tyrosine kinases like EGF receptor tyrosine protein kinase, or She.
  • the compounds of formula I disrupt the interaction between the SH2-comprising protein and the phosphoprotein, such as protein tyrosine kinase, and thus blocks the ability of the tyrosine protein kinases to initiate regulatory events depending on the SH2-comprising proteins, thus resulting in inhibition of specific downstream signal transduction pathways utilized in some hyperproliferative diseases, such as tumor diseases and psoriasis and the other diseases mentioned above and below, by uncoupling of the respective protein tyrosine kinase(s) from the respective SH2-containing effector protein.
  • the phosphoprotein such as protein tyrosine kinase
  • One feature of the present invention is the positive effect of the moieties X as defined below on the inhibitory action of the compounds of the present invention on the interaction of a broad variety of phosphoproteins, especially phosphotyrosine-comprising proteins, to SH2-comprising proteins (e.g. those mentioned below in the definition of the bivalent radical -(AA) m -).
  • moieties X are even able to allow for large sequence variability in the peptide derivatives of formula I.
  • the invention preferably relates to a an acylated peptide, namely a compound of the formula I,
  • X is arylcarbonyl, arylsulfonyl, cycloalkylcarbonyl, cycloalkanesulfonyl, heterocyclylcarbonyl or heterocyclylsulfonyl;
  • A is absent or the bivalent radical of a natural or unnatural amio acid
  • B is the bivalent radical of a natural amino acid
  • PTI is the bivalent radical of phosphotyrosine or a phosphotyrosine mimic
  • AA stands for a bivalent radical of a natural or unnatural amino acid
  • Y is hydroxy, a C-terminal protecting group or a primary, secondary or tertiary amino group
  • the term "Iower” defines a moiety with up to and including maximally 7, especially up to and including maximally 4, carbon atoms, said moiety being branched or straight-chained.
  • Lower alkyl for example, is methyl, ethyl, n-propyl, sec-propyl, n-butyl, isobutyl, sec-butyl, tert- butyl, n-pentyl, n-hexyl or n-heptyl.
  • the compounds of formula I with one or more centers of asymmetry may be present in the form of isomeric mixtures or pure isomers; for example, a compound of formula I with one center of asymmetry may be present in the form of a pure enantiomer or a mixture of enantiomers, e.g. a racemate, while a compound of formula I with two or more centers of asymmetry may be present in the form of a pure isomer (enantiomer) or in the form of diastereomeric mixtures, e.g. mixtures of epimers.
  • pure isomers of compounds of formula I are preferred over isomeric mixtures.
  • m is preferably 2 to 6, most preferably 2, 3 or 4.
  • Aryl has preferably from 6 to 14 ring carbon atoms, such as in phenyl (which is especially preferred), naphthyl (which is preferred), indenyl, indanyl, anthryl, phenanthryl, acenaphthyl or fluorenyl, and may be unsubstituted or preferably mono- to tri-substituted, especially by amino, mono- or di-lower alkylamino, Iower alkanoyiamino, such as acetylamino, amino- lower alkyl, mono- or di-loweralkylamino-lower alkyl, iower alkanoylamino-lower alkyl, hydroxy, Iower alkoxy, such as methoxy, carboxy, lower-alkoxycarbonyl, such as methoxycarbonyl, phenyl-, naphthyl- or fluorenyl-lower alkoxycarbonyl, such as benzyloxy ⁇
  • Cycloalkyl preferably has from 3 to 10 ring carbon atoms, preferably from 4 to 7 carbon atoms, and is unsubstituted or preferably mono- to tri-substituted, especially by amino, mono- or di-lower alkylamino, Iower alkanoyiamino, such as acetylamino, amino-lower alkyl, mono- or di-loweralkylamino-lower alkyl, Iower alkanoylamino-lower alkyl, hydroxy, Iower alkoxy, such as methoxy, carboxy, lower-alkoxycarbonyl, such as methoxycarbonyl, phenyl-, naphthyl- or fluorenyl-lower alkoxycarbonyl, such as benzyloxycarbonyl, Iower alkanoyl, cyano, Iower alkyl, for example methyl, ethyl or propyl, halo-lower alky
  • Heterocyclyl is preferably a single or double ring system having from 3 to 10 ring atoms, is bonded preferebly via a carbon atom or also via a nitrogen atom and contains up to 3 hetero atoms selected from oxygen, sulfur, sulfur linked to 1 or 2 oxygen atoms and, most preferably, nitrogen; which in addition may also be fused with 1 or 2 phenyl radicals or with 1 or 2 cycloalkyl radicals, cycloalkyl preferably having from 5 to 7 ring atoms; and which may be unsaturated or partially or fully saturated, for example thienyl, furyl, pyrrolyl, imidazolyl, such as imidazole-4-yl, pyrazolyl, oxazolyl, thiazolyl, such as 4- or 5-thiazolyl, tetrazolyl, pyridyl, such as pyridin-3- or pyridin-4-yl, pyrazinyl, such as pyra
  • arylcarbonyl X the aryl moiety is preferably defined as above; more preferably, arylcarbonyl is selected from benzoyl, 1-naphthoyl, 2-naphthoyl and, even more preferably, from benzoyl substituted with amino; Iower alkylamino; amino-lower alkyl; hydroxy; Iower alkoxy; amino and hydroxy; amino and Iower alkoxy; carboxy; lower-alkoxycarbonyl; cyano; halogen, especially chloro; lower-alkylthio; or Iower alkylsulfinyl, and from 1- or 2-naphthoyl substituted with amino; especially from 4-aminobenzoyl, 3-aminobenzoyl (more preferred), 2-aminobe ⁇ zoyl (most preferred); 3,5-diaminobe ⁇ zoyl; 4-lower alkylamino-benzoyl, such as 4-methylamino-benz
  • the aryl moiety is preferably defined as above; more preferably, arylsulfonyl is 1-or 2-naphthalenesulfonyl which is substituted with amino or mono- or di-lower alkylamino, such as dimethylamino; especially 5-dimethylamino- naphthalene-1 -sulfonyl.
  • cycloalkylcarbonyl X cycloalkyl is preferably as defined above; more preferably, cycloalkylcarbonyl is C 3 -C 7 -, especially C 4 - C 5 - or C 6 -cycloalkylcarbonyl, such as cyclohexylcarbonyl, and is unsubstituted or substituted by amino; most preferably 1 -amino- cyclohexylcarbonyl or 1 -amino-cyclopentylcarbonyl.
  • cycloalkyl is preferably as defined above; more preferably, cycloalkanesulfonyl is CrC-7-cycloalkanesulfonyl which is unsubstituted or preferably substituted with amino.
  • heterocyclylcarbonyl the heterocyclyl moiety is preferably as defined above; more preferably, heterocyclylcarbonyl is selected from pyridylcarbonyl which is unsubstituted or substituted with amino, such as pyridin-4-yl- or pyridin-3-ylcarbonyl, or amino-pyridin-3-yl- carbonyl, such as 2- or 6-amino-pyridin-3-ylcarbonyl; pyrazinyl, such as pyrazin-2-yl, which is unsubstituted or substituted with amino, such as in 3-amino-pyrazin-2-ylcarbonyl, quinolinyl-carbonyl, such as quinoline-2-, quinoline-3-, quinoline-4- or quinoline-8-ylcarbonyl, isoquinolyl, such as isoquinoiine-1-yl, indolylcarbonyl, such as indole-5-yl-, indolyl
  • A is absent or the bivalent radical of a natural or unnatural amio acid as defined beiow for AA.
  • B is the bivalent radical of a natural amino acid as defined beiow for AA.
  • B is an amino acid other than the moiety of the formula wherein K and L are independently H or methyl and p is 1 or 2, such as histidine.
  • the bivalent radical -A-B- in formula I is an analogue of an SH2 domain binding site of a protein with phosphotyrosine of a mammal, especially a human, for example one of the binding sites mentioned in Songyang et al., Cell 72, 767-778 (1993), e.g.
  • A is especially the bivalent radical of the amino acid proline which may be present in the D-, (D,L)- or preferably L-form or most preferably absent (that is, X is directly bound to B).
  • B is more preferably a bivalent radical of an amino acid selected from proline, S-protected cysteine, especially S-acetamidomethyl-cysteine, asparagine, ⁇ -alanine; and, more preferably, glutamic acid, cysteine that forms a disulfide bond with a further cysteine AA, glycine, alanine, valine, leucine and isoleucine; any of which is present in the (D,L)- or preferably the D- or (most preferably) the L-form.
  • a bivalent radical of a phosphotyrosine mimic PTI is defined as any radical that is able to replace a phosphotyrosine radical which resembles, but is structurally different from the respective phosphotyrosine radical and which cannot lose its phosphono-group too easily due to hydrolysis.
  • such a mimic is selected from the respective bivalent radical (which is bound N-terminally via the imino group resulting from the ⁇ -amino group and C- terminally via the carbonyl group resulting from its ⁇ -carboxy group) of an amino acid selected from phosphonomethyl-phenylalanine, especially 4-phosphonomethyl- phenylalanine, phosphono-( ⁇ -fluoro)methyl-phenylalanine, especially 4-phosphono-( ⁇ - fluoro)methyl-phenylalanine, phosphono-( ⁇ , ⁇ -difiuoro)methyl-phenylalanine, especially 4- phosphono-( ⁇ , ⁇ -difluoro)methyl-phenylalanine, phosphono-( ⁇ -hydroxy)methyl- phenylalanine, especially 4-phosphono-( ⁇ -hydroxy)methyl-phenylalanine, O-sulfo-tyrosine, such as 4-(O-sulfo)tyrosine, di
  • ( (HOOC) 2 -CH 2 -O-phenylalanine), especially p-(dicarboxymethoxy)-phenylalanine; (less preferably) phosphonophenylalanine, such as 4-phosphonophenylalanine; aspartic acid, glutamic acid, phosphoserine and phosphothreonine, each of which is present in the (D,L)-, D- or preferably the L-form.
  • a bivalent radical of phosphotyrosine and especially a bivalent radical of phosphono-( ⁇ , ⁇ -difluoro)methyl-phenylalanine, especially 4-phosphono-( ⁇ , ⁇ - difluoro)methyl-phenylalanine is especially preferred.
  • AA stands for a natural or unnatural amino acid, and is preferably a bivalent radical of an ⁇ - or ⁇ -amino acid which is preferably bonded N-terminally by way of its ⁇ - or ⁇ -amino group and C-terminally by way of its carboxy group and is preferably selected from the group comprising a bivalent radical of a natural ⁇ - amino acid having the L-configuration, such as those normally occurring in proteins, or an epimer of such an amino acid, that is to say having the unnatural D-configuration, or a (D,L)-isomeric mixture thereof; or a homologue of such an amino acid, for example a ⁇ -amino acid or an ⁇ -amino acid wherein the amino acid side chain has been shortened by one or two methylene groups or lengthened to up to 10 carbon atoms, such as an ⁇ -amino alkanoic acid with 5 up to and including 10 carbon atoms in a linear chain, a substituted aromatic ( ⁇ -aryl or
  • the bivalent radical bonded via its ⁇ -amino and its ⁇ - or ⁇ -carbonyl group, of an amino acid selected from glycine (H-Gly-OH), alanine (H-Ala-OH), ⁇ -alanine (H- ⁇ Ala-OH), valine (H-Val-OH), norvaline ( ⁇ -aminovaleric acid), leucine (H-Leu-OH), isoleucine (H-lle-OH), norleucine ( ⁇ -aminohexanoic acid, H-Nle-OH), ⁇ -amino-n-decanoic acid, serine (H-Ser-OH), homoserine ( ⁇ -amino- ⁇ -hydroxybutyric acid), threonine (H-Thr-OH), methionine (H-Met-OH), cysteine (H-Cys-OH), S-acetylaminomethyl-cysteine (H-Cys(Acm)- OH
  • the bivalent radical -(AA) m - in formula I is an analogue of an SH2 domain binding site of a protein with phosphotyrosine of a mammal, especially a human, for example one of the binding sites mentioned in Songyang et al., Cell 72, 767-778 (1993), e.g.
  • the -Asn- in position 2 of the mentioned sequence following Tyr 1068 in EGFR is present as such, while the amino acids in the other positions may be replaced with one of the other amino acids mentioned above or (as far as the C-terminal amio acid(s) following the Asn are concerned) may be deleted.
  • -(AA) m - has one of the following meanings:
  • -(AA 1 )- is preferably selected from -lie-, -Cys(Acm)-, -Cys- (which preferably forms a disulfide bond with another -Cys- B) and -Glu-;
  • -(AA 2 )- is preferably selected from -Asn-, and also from -Glu- and -He-, most preferably -Asn-;
  • -(AA 3 )- is preferably selected from -Gin-, -Cys(Acm)-, -Cys- (which preferably forms a disulfide bond with another -Cys- B); -lie- and -Pro-; or
  • a C-terminal protecting group Y is preferably an esterifying group, thus leading to an esterified C-terminal carboxy group. More preferred is a Iower alkoxy group that is preferably branched in the 1 -position of the Iower alkoxy group or substituted in the 1 - or 2- position of the Iower alkoxy group by (one) suitable substituent(s).
  • a lower alkoxy group that is branched in the 1 -position of the Iower alkoxy group is, for example, tert-lower alkoxy, for example tert-butoxy.
  • a iower alkoxy group that is substituted in the 1 - or 2-position of the Iower alkoxy group by (one) suitable substituent(s) is, for example, arylmethoxy having one or two aryl radicals, wherein aryl is preferably phenyl that is unsubstituted or mono-, di- or tri-substituted, for example, by Iower alkyl, for example tert-lower alkyl, such as tert-butyl, Iower alkoxy, for example methoxy, hydroxy, halogen, for example chlorine, and/or by nitro, for example benzyloxy, benzyloxy substituted by the mentioned substituents, for example 4-nitro- benzyloxy or 4-methoxybenzyloxy, diphenylmethoxy or diphenylmethoxy substituted by the mentioned substituents, for example di(4-methoxyphenyl) methoxy; 1 -lower alkoxy-lower
  • a C-terminal protecting Y group can furthermore be an organic silyloxy group.
  • An organic silyloxy group is, for example, a tri-lower alkylsilyloxy group, for example trimethyisilyloxy.
  • the silicon atom of the silyloxy group can also be substituted by two Iower alkyl groups, for example methyl groups.
  • a C-terminal protecting group Y is preferably tert-lower alkoxy, for example tert-butoxy, benzyloxy, 4-nitrobenzyloxy, 9-fluorenylmethoxy or diphenylmethoxy.
  • a primary, secondary or tertiary amino group Y is preferably a free amino group, a mono- or disubstituted amino group the substituents of which are preferably selected from the group comprising lower alkyl, such as methyl, ethyl; isobutyl or 3-methylbutyl; octyl, such as 2- ethyl-hexyl; aryloxy-lower alkyl, especially halonaphthyloxy-lower alkyl, such as 2-(1-bromo- naphthalen-2-yloxy)-ethyl, or naphthyloxy-lower alkyl, such as 2-(naphthalen-2-yloxy or naphthalen
  • 2-(1-pyrroIidinyl)-ethyl pyridyl-lower alkyl, e.g. 2-(2-pyridyl)-ethyl, furyl-lower alkyl, e.g. 2-furylmethyl, morpholinyl-lower alkyl, e.g. 2-(4-morpholinyl)-ethyl, and indolyl-lower alkyl, e.g. 2-(3-indolyl)-ethyl; cycloalkyl, such as cyclohexyl; and cycloalkyl- lower alkyl, such as cyclohexylmethyl.
  • a disubstituted amino group may also be N-contai ⁇ ning heterocyclyl bonded via its nitrogen atom, such as e.g. 1 -pyrrolidinyl or 4-morpholi ⁇ yl.
  • a primary, secondary or tertiary amino group Y is a free amino group, a mono- or di-substituted amino group the substituents of which are preferably selected from the group comprising Iower alkyl, e.g. methyl or ethyl, aryl-lower alkyl, such as phenyl-lower alkyl, e.g. benzyl, and heterocyclyl-lower alkyl, such as pyrrolidinyl-lower alkyl, e.g. 2-(1- pyrrolidinyl)-ethyl, pyridyl-lower alkyl, e.g.
  • a disubstituted amino group may also be N-containing heterocyclyl bonded via its nitrogen atom, such as e.g. 1 -pyrrolidinyl or 4-morpholinyl.
  • Y is a primary, secondary or tertiary amino group as defined above, most preferably amino (-NH 2 ).
  • the proviso that two or more sulfhydryl groups belonging to any amino acid A, B or AA are present as such or may form intramolecular disulfide bonds means that the respective peptide compounds of for-mula are present as cyclic compounds. It is to be interpreted so that, if an odd-numbered number of cysteine moieties is present, at least one sulfhydryl group per molecule is not part of a disulfide bond. Preferably, up to two cysteine moieties are present in a molecule of formula I; if two cysteine moieties are present, they may form an intramolecular disulfide bond, thus leading to pure constitution isomers.
  • Salts of compounds of formula I are especially acid addition salts, salts with bases or, where several salt-forming groups are present, can also be mixed salts or internal salts.
  • Salts are especially pharmaceutically acceptable salts of compounds of formula I.
  • Such salts are formed, for example, from compounds of formula I having an acid group, for example a carboxy group, a sulfo group, or a phosphoryl group substituted by one or two hydroxy groups, and are, for example, salts thereof with suitable bases, such as non-toxic metal salts derived from metals of groups la, lb, lla and lib of the Periodic Table of the Elements, especially suitable alkali metal salts, for example lithium, sodium or potassium salts, or alkaline earth metal salts, for example magnesium or calcium salts, also zinc salts or ammonium salts, as well as salts formed with organic amines, such as unsubstituted or hydroxy-substituted mono-, di- or tri-alkylamines, especially mono-, di- or tri-lower alkyl ⁇ amines, or with quaternary ammonium compounds, for example with N-methyl-N-ethyl- amine, diethylamine, triethylamine, mono
  • the compounds of formula I having a basic group, for example an amino group can form acid addition salts, for example with inorganic acids, for example hydrohalic acids, such as hydrochloric acid, sulfuric acid or phosphoric acid, or with organic carboxylic, sulfonic, sulfo or phospho acids or N-substituted sulfamic acids, for example acetic acid, propionic acid, glycolic acid, succinic acid, maleic acid, hydroxymaieic acid, methylmaleic acid, fumaric acid, malic acid, tartaric acid, gluconic acid, glucaric acid, glucuronic acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, salicylic acid, 4-aminosalicylic acid, 2-phenoxybenzoic acid, 2- acetoxybenzoic acid, embonic acid, nicotinic acid or isonicotinic acid, as well as with amino acids, for example the ⁇ -amino acids mentioned
  • the compounds of the invention have useful, in particular pharmacologically useful, properties.
  • the ability to inhibit the interaction between the SH2 domain of Grb2 and phosphorylated EGFR can be shown by the following type of assay:
  • the following procedure is used for the screening of inhibitors with regard to the interaction between Grb2 - for example, full-length (sequence: see Lowenstein et al., Cell 70, 431-42 (1992)) or SH2 alone - and phosphorylated EGFR (full-length cytoplasmic tyrosine kinase or a fusion pro ⁇ duct obtained from Maltose Binding Protein and the carboxy-terminal part of the EGF re ⁇ ceptor ("tail" EGFR-MBP fusion protein)) (for EGFR sequence, see Nature 309, 418-425 (1984); for purified recombinant intracellular domain (ICD), see Eur. J. Biochem.
  • full-length sequence: see Lowenstein et al., Cell 70, 431-42 (1992)
  • SH2 alone - and phosphorylated EGFR full-length cytoplasmic tyrosine kinase or a fusion pro ⁇ duct obtained from Maltose Bind
  • the EGFR-MBP fusion protein is made as follows: Oligonucleotides flanking the entire carboxy-terminal half (nucleotides 3112 to 3816) of the EGFR and containing engi ⁇ neered EcoRI-Hindlll restriction sites are used to amplify the appropriate DNA fragment by PCR. The amplified DNA fragment is recombined with purified EcoRI-Hindlll-digested pMALc2 vector (New England Biolabs, Inc., Beverly, USA) downstream from and in the same reading frame as the malE gene, which encodes maltose-binding protein (MBP). The vector containing the fused gene is transformed in E.
  • Coli and the fusion protein is expres ⁇ sed from the Pt ac promoter.
  • a crude cell extract is prepared and passed over a column of amylose resin.
  • the fusion protein is then eluted with neutral buffer, containing maltose. Aliquots are frozen in liquid nitrogen and stored at -70 °C).
  • Wells of polystyrene microtiter plates are coated overnight at 4 °C in incubation buffer (20 mM Tris pH 7.5) with phosphorylated EGFR or 'lair EGFR-MBP fusion protein (phosphorylation conditions: 0.5 mg/ml of purified recombinant EGFR-ICD, or EGFR-MBP (+0.03mg/ml EGFR-ICD) is phosphorylated by the addition of 10 mM MnCI 2 , 10 mM MgCI 2 , 40 ⁇ M ATP in 20 mM Tris buffer pH 7.5 for 45 min).
  • Grb2-SH2-GST [obtainable, e.g., from Santa Cruz Biotech, California, USA, or as follows: a cDNA clone encoding human Grb2 SH2 domain (e.g., aa 45-164) is amplified by polymerase chain reaction (PCR), using nucleotides with appropriate linkers, e.g. with BamHl (5').3' EcoRI linkers; the purified (e.g. BamHl -EcoRI) fragments from PCR products are then subcloned in-frame into the appropriate (e.g.
  • PCR polymerase chain reaction
  • This type of assays is not limited to the EGF receptor - it can also be used analogously with erb-B2 or other protein tyrosine kinases. Furthermore, it is possible to use other SH2 domains instead of Grb2 SH2.
  • the interactions of the SH2-comprising proteins and phosphotyrosine comprising proteins mentioned above in the definition of a bivalent radical -(AA) m - in formula I as an analogue of an SH2 domain binding site of a protein with phosphotyrosine of a mammal can be tested.
  • the compounds of the invention due to their ability to uncouple a phosphorylated protein, especially a protein tyrosine kinase, e.g. EGF receptor, from a respective SH2 containing protein, e.g. the SH2-containing Grb2, are able to inhibit subsequent cellular signal trans ⁇ duction pathways important for diseases such as viral, inflammatory, allergic, auto-immune, cardiovascular and especially proliferative diseases, such as for malignant hyperprolifera ⁇ tive diseases, e.g. tumor diseases, preferably breast cancer, chronic myelogenous leukemia (CML), thyroid carcinoma and osteosarcoma, or for hyperproliferation of epithelial cells, e.g. psoriasis, are appropriate for the treatment and prophylaxis of said diseases.
  • a phosphorylated protein especially a protein tyrosine kinase, e.g. EGF receptor
  • a respective SH2 containing protein e.g. the SH2-containing Grb2
  • the compounds of the present invention are useful for the treatment of diseases that respond to inhibition of the interaction of (a) protein(s) comprising (an) SH2 domain(s) and a phosphoprotein, preferably a protein tyrosine kinase or a modified version thereof, more preferably of Grb2 SH2 with EGFR or modified derivatives thereof.
  • modified version or “modified derivative” means mainly a derivative that is causative or active in the establishment of diseases, e.g. truncated versions, virus derived analogues, etc.
  • the treatment can also, e.g. in the case of hematopoietic cell proliferative disorders, such as leukemias, be used in conjunction with autologous bone marrow transplantation and chemotherapy techniques.
  • hematopoietic cell proliferative disorders such as leukemias
  • an aliquot of bone marrow cells (even one cell or some single cells, which may be treated by microinjection of a compound of the invention as described above) are obtained from a patient, e.g. from the pelvis.
  • the cells are then cultured in the presence of a compound of formula I (which may also be applied by microinjection) which is able to disrupt the protein tyrosine kinase/SH2-comprising protein interaction.
  • the compounds of formula I can also be bound covalently to chromatographic materials, thus making it possible to produce chromatographic materials for the affinity purification of natural or recombinant SH2-domains or SH2-comprising proteins from the cells of living organisms.
  • a compound of formula I with an appropriate free functional group e.g. -NH 2 , -SH, -OH and/or -COOH
  • an appropriate free functional group e.g. -NH 2 , -SH, -OH and/or -COOH
  • activated or activatable matrices appropriate for chromatography e.g.
  • cyanogen bromide activated matrices epoxy-activated matrices, nitrophenyl chloroformate and N-hydroxysuccinimde chloroformate.
  • polyacrylhydrazido agarose oxirane acrylic beads, bromoacetyl-cellulose, epichlorohydrin- activated matrices, tresyl-chlordie-activated agarose, vinylsulfone-activated agarose, and the like.
  • Preferred activated or activatable coupling gels for affinity chromatography include but are not limited to a) for coupling of compounds of formula I with an -NH 2 group employed for binding: cyanogen bromide activated Sepharose 4B; ECH Sepharose 4B (carbodiimide coupling method used most often in analogy to process for preparation of compounds of formula I as described below); or activated CH Sepharose 4B; b) for coupling of compounds of formula I with an -NH 2 and/or an -SH group: Tresyl- activated Sepharose 4B; c) for coupling of compounds of formula I with an -NH 2 ,-OH and/or -SH group: epoxy- activated Sepharose 6B; and d) for coupling of compounds of formula I with a -COOH group: EAH Sepharose 4B (carbodiimide method for coupling most often used in analogy to process for preparation of compounds of formula I as described below).
  • Sepharose stands for agarose derived chromatographic materials and is a trademark from Pharmacia, Uppsala, Sweden, from where the mentioned gels are available.
  • n 2 to 6, more preferably 2 , 3 or 4;
  • X is selected from
  • C-rCr-cycloalkylcarbonyi such as cyclohexylcarbo ⁇ yl, which is unsubstituted or substituted by amino; most preferably 1 -amino-cyclohexylcarbonyl or 1-amino- cyclopentylcarbonyl ;
  • pyridylcarbonyl which is unsubstituted or substituted with amino, such as pyridin-4-yl- or pyridin-3-ylcarbonyl, or amino-pyridin-3-yl-carbonyl, such as 2- or 6-amino-pyridin-3- ylcarbonyl; pyrazinyl, such as pyrazin-2-yl, which is unsubstituted or substituted with amino, such as in 3-amino-pyrazin-2-ylcarbonyl, quinolinyl-carbonyl, such as quinoline-2-, quinoline-3-, quinoline-4- or quinoline-8-ylcarbonyl, isoquinolyl, such as isoquinoiine-1-yl, indolylcarbonyl, such as indole-5-yl-, indolyl-3-yl- or indole-2-yl-carbo ⁇ yl; and indolinyl- carbonyl
  • A is absent or the bivalent radical of a natural or u ⁇ atural amino acid, preferably selected from glycine, alanine, ⁇ -alanine, valine, norvaline, leucine, isoleucine, norleucine, ⁇ -amino- n-decanoic acid, serine, homoserine, threonine, methionine, cysteine, which can form a disulfide bridge with another cysteine B or AA; S-protected cysteine, such as S- acetylaminomethyl-cysteine, proline, trans-3- and trans-4-hydroxyproline, phenylalanine, tyrosine, 4-aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine, 4-carboxy- phenylaianine, ⁇ -phenylserine, phenylglycine, ⁇ -naphthylalanine, cyclohex
  • B is the bivalent radical of a natural or unnatural amino acid as defined above for A, preferably being selected from proline or especially S-acetamidomethyl-cysteine, cysteine which may form a disulfide bond with another cysteine A or AA, glutamic acid, aspartic acid, glycine, alanine, valine, leucine, isoleucine and ⁇ -alanine, it being possible for each of the mentioned amino acids (with the exception of glycine or any other amino acid without asymmetric carbon atom) to be in the D-, L- or (D,L)-form, preferably in the L- or in the D- form;most preferably, B is selected from glutamic acid, S-acetamidomethyl-cysteine, cysteine which together with another cysteine AA forms a disulfide bond; glycine, alanine and isoleucine, it being possible for each of the mentioned amino acids (with the exception of glycine or
  • PTI is a bivalent radical of phosphotyrosine (in the D-, L- or less preferably the (D.L)-form) or of a phosphotyrosine mimic in the form of a bivalent radical (which is bound N-terminally via the imino group resulting from the ⁇ -amino group and C-terminally via the carbonyl group resulting from its ⁇ -carboxy group) of an amino acid selected from phosphonomethyl- phenylalanine, especially 4-phosphono-methyl-phenylalanine, phosphono-( ⁇ -fluoro)methyl- phenylaianine, especially 4-phosphono-( ⁇ -fluoro)methyl-phenylalanine, phosphono-( ⁇ , ⁇ - difluoro)methyl-phenylalanine, especially 4-phosphono-( ⁇ , ⁇ -difluoro)methyl-phenylalanine, phosphono-( ⁇ -hydroxy)methyl-phenylalanine, especially 4-phosphono-( ⁇
  • -(AA 1 )- is preferably selected from -He-, -Cys(Acm)-, -Cys- (which is present as such or preferably forms a disulfide bond with another -Cys- B), -Ac 5 c-, -Ac 6 c- and -Glu-;
  • -(AA 2 )- is preferably selected from -Asn-, and also from -Glu- and -lie-, most preferably -Asn-;
  • - (AA 3 )- is preferably selected from -Gin-, -Cys(Acm)-, -Cys- (which is present as such or preferably forms a disulfide bond with another -Cys- B); -lie- and -Pro-; and -(AA 4 )- wherein -(AA 1 )- is preferably selected from -He-, -Cys(Acm)-, -Cys-
  • -(AA 1 )- is preferably selected from -He-, -Cys(Acm)-, -Cys- (which is present as such or preferably forms a disulfide bond with another -Cys- B) and -Glu-;
  • -(AA 2 )- is preferably selected from -Asn-, and also from -Glu- and -He-, most preferably -Asn-;
  • -(AA 3 )- is preferably selected from -Gin-, -Cys(Acm)-, -Cys- (which is present as such or preferably forms a disulfide bond with another -Cys- B); -lie- and -Pro-; or
  • Y is a free amino group (preferrred), a mono- or disubstituted amino group the substituents of which are preferably selected from the group comprising Iower alkyl, e.g. methyl or ethyl, phenyl-lower alkyl, e.g. benzyl, pyrrolidinyl-lower alkyl, e.g. 2-(1-pyrrolidinyl)-ethyl, pyridyl- lower alkyl, e.g. 2-(2-pyridyl)-ethyl, furyl-lower alkyl, e.g. 2-furylmethyl, morpholinyl-lower alkyl, e.g.
  • X is selected from 4-aminobenzoyl, 3-aminobenzoyl (more preferred), 2-aminobenzoyl (most preferred); 3,5-diaminobenzoyl; 2-hydroxybenzoyl, 4-iower alkoxy-, such as 4- methoxybenzoyl, and 3-amino-2-naphthoyl;
  • A is absent or the bivalent amino acid radical of proline which is present in the (D,L)-, D- or preferably L-form; preferably, A is absent;
  • B is selected from glutamic acid, S-acetamidomethyl-cysteine, cysteine which together with another cysteine AA forms a disulfide bond; glycine, alanine and isoleucine, it being possible for each of the mentioned amino acids (with the exception of glycine or any other amino acid without asymmetric carbon atom) to be in the D-, L- or (D,L)-form, preferably in the L- or in the D-form;
  • PTI is a bivalent radical of phosphotyrosine (in the D-, L- (most preferred) or (less preferably) the (D,L)-form) or of a phosphotyrosine mimic of the phosphono-( ⁇ , ⁇ - dif luoro) methyl-phenylaianine type, especially 4-phosphono-( ⁇ , ⁇ -difluoro)methyl- phenylalanine
  • -(AA 1 )- is selected from -He-, -Cys(Acm)-, -Cys- (which is present as such or preferably forms a disulfide bond with another -Cys- B) and -Giu-;
  • -(AA 2 )- is selected from -Asn-, and also from -Glu- and -lie-, most preferably -Asn-;
  • -(AA 3 )- is selected from -Gin-, - Cys(Acm)-, and -Cys-; and -(AA 4 )- is selected from -Cys(Acm)- and -Cys- (which is present as such or preferably forms a disulfide bond with another -Cys- B);
  • -(AA 1 )- is selected from -He-, -Cys(Acm)-, -Cys- (which is present as such or preferably forms a disulfide bond with another -Cys- B) and -Glu-;
  • -(AA 2 )- is selected from -Asn- and -He-, most preferably -Asn-;
  • -(AA 3 )- is selected from -Gin-, -Cys(Acm)- and -Cys- (which is present as such or preferably forms a disulfide bond with another -Cys- B); or
  • Y is amino (-NH 2 )
  • a compound mentioned in the examples or a (preferably pharmaceutically acceptable) salt thereof.
  • the compounds of the present invention can be synthesized according to known procedures, especially by a process comprising reacting a fragment of a compound of formula I, which has a free carboxy group or a reactive derivative thereof, or, in the case of the introduction of X, a free carboxy or sulfo group, or a reactive derivative thereof, with a complementary fragment that has an amino group with at least one free hydrogen atom, or with a reactive derivative thereof, with formation of an amide bond; in the mentioned fragments free functional groups with the exception of those that participate in the reaction if required being present in protected form; and removing any protecting groups present;
  • the compounds of the present invention preferably can be readily prepared according to well-established, standard liquid or, preferably, solid-phase peptide synthesis methods, general descriptions of which are broadly available (see, for example, in J.M. Stewart and J.D. Young, Solid Phase Peptide Synthesis, 2nd edition, Pierce Chemical Company, Rockford, Illinois (1984), in M. Bodanzsky and A. Bodanzsky, The Practice of Peptide Synthesis, Springer Verlag, New York (1984); and Applied Biosystems 430A Users Manual, ABI Inc., Foster City, California), or they may be prepared in solution, by the liquid phase method or by any combination of solid-phase, liquid phase and solution chemistry, e.g. by first completing the respective peptide portion and then, if desired and appropriate, after removal of any protecting groups being present, by introduction of the residue X by reaction of the respective carbonic or sulfonic acid or a reactive derivative thereof.
  • a fragment with a free carboxy or sulfonic group can be an amino acid (if required, in suitably protected form) or a di- or other appropriate oligopeptide or also (in the case of the introduction of the N-terminal X of compounds of formula I with acylated or sulfonylated terminal amino group) the acylating carbonic or sulfonic acid.
  • Reactive derivatives of carbonic or sulfonic acids are preferably reactive esters, reactive anhydrides or reactive cyclic amides. Reactive carbonic acid or reactive sulfonic acid derivatives can also be formed in situ.
  • a reactive derivative of an "amino group with at least one free hydrogen” is preferably derivatized by the reaction with a phosphite, such as diethyl-chlorophosphite, 1 ,2- phenylene-chlorophosphite, ethyl-dichlorophosphite, ethylene-chlorophosphite or tetraethyl- pyrophosphite; or is present in the form of a carbamic acid chloride wherein the amino group participating in the reaction is subtituted by halocarbonyl, such as chlorocarbonyl.
  • halocarbonyl such as chlorocarbonyl.
  • free amino is used instead of a reactive derivative.
  • reaction steps required e.g. for the synthesis of amide or sulfonamide bonds usually depend on the type of activation of the carboxylic or sulfo group participating in the reaction.
  • the reactions normally run in the presence of a condensing agent or, when activating the carboxylic or sulfonic acids in the form of anhydrides, of an agent that binds the carboxylic or sulfonic acid formed.
  • chaotropic agents such as LiF in N-methyipyrrolidin-2-one.
  • the reactions are especially carried out in a temperature range from -30 to +150 °C, preferably from +10 to +70 °C, and, most preferably, from +20 to +50 °C, if appropriate, in an inert gas atmosphere, e.g. under nitrogen or argon.
  • unreacted amino groups can be acylated after a reaction cycle, e.g. by acetylation of unreacted amino groups with an excess of acetic anhydride/pyri- dine/DMA (1 :1 :8), thus facilitating later purification of the final product.
  • a suitably protected amino acid as a ligand is attached via its carboxyl group (- COOH) to a derivatized, insoluble polymeric support, e.g. a cross-linked polystyrene or polyamide resin, such as a 4-(2',4'-dimethoxyphenyl-[hydroxy- or amino-]methyl)-phenyoxy - polystyrene resin (the polymer is, e.g., a copolymer of styrene with 1% divinylbenzene, 100- 200 mesh) or a PAL-PEG-PS (synonym: PAL-PEG-MBHA-PS) resin (PAL stands for a trisalkoxy, especially trismethoxy, benzylamide linker; PEG for polyethyleneglycol; and MBHAfor 4-methylbenzhydryiamine - in this type of resin, polystyrene (PS) supports uniformly incorporate a derivatized polyethylene glycol (
  • Synthesis proceeds in a stepwise, cyclical fashion by successively removing the NH 2 protecting group of the amino group to be reacted next and then coupling an activated fragment (e.g. an amino acid, di-, tri- or oligopeptide or the carboxylic acid or sulfonic acid of formula II,
  • an activated fragment e.g. an amino acid, di-, tri- or oligopeptide or the carboxylic acid or sulfonic acid of formula II
  • activation of the COOH group of the amino acid to be reacted or ( in the case of the introduction of X) the carboxyl or sulfo group of the acid of of formula II to be attached by the condensation reaction is effected
  • a carbodiimide e.g. dicyclohexylcarbodiimide (DCC), N-ethyl-N'-(3-dimethyl- aminopropyl)-carbodiimide, N,N'-diethylcarbodiimide or N,N'-diisopropylcarbodiimide (DICD); with a carbonyl compound such as carbonyldiimidazole; with 1 ,2-oxazolium compounds such as 2-ethyl-5-phenyl-1 ,2-oxazolium-3'-sulfonate and 2-tert-butyl-5-methyiisoxazolium perchlorate; with acylamino compounds such as 2-ethoxy-1 -ethoxycarbonyl-1 ,2-dihydroqui- noline; with N-[(dimethylamino)-1 H-1 ,2,3-triazolo[4,5-b]pyridin-1 -yl
  • DCC di
  • symmetric anhydride obtainable, for example, by condensation of the corresponding acid in the presence of a carbodiimide or 1 -diethylaminopropyne; symmetric anhydrides method
  • an asymmetric anhydride such as the respective carbonic or sulfonic acid bromide, chloride or fluoride (obtainable, for example, by treatment of the respective carbonic or sulfonic acid with thionyl-, phosphopenta- or oxalyl-fluoride, -chloride or -bromide; acid halide (e.g. chloride) method), or
  • an "active ester” e.g. an amino- or amido ester, such as a N-hydroxy- benzotriazole (HOBT) or N-hydroxysuccinimide ester, or an aryl ester, such as a penta ⁇ fluorophenyl, 4-nitrophenyl or 2,4,5-trichlorophenyl ester (obtainable by treatment of the respective acid with a phenyl with the appropriate substituents, such as 4-nitrophenol or 2,4,5-trichlorophenol, and the like);
  • an active ester e.g. an amino- or amido ester, such as a N-hydroxy- benzotriazole (HOBT) or N-hydroxysuccinimide ester, or an aryl ester, such as a penta ⁇ fluorophenyl, 4-nitrophenyl or 2,4,5-trichlorophenyl ester (obtainable by treatment of the respective acid with a phenyl with the appropriate substituents, such as 4-nitrophenol or 2,4,5-trich
  • Useful acid binding agents that can be employed in the condensation reactions are, for example, alkaline metals, carbonates or bicarbonates, such as sodium or potassium carbonate or bicarbonate (if appropriate, together with a sulfate), or organic bases such as sterically hindered organic nitrogen bases, for example tri-lower alkylamines, such as N,N- diisopropyl-N-ethylamine, pyridine or N-methyl-pyrrolidin-2-one, which can be used alone or in any appropriate combination.
  • alkaline metals carbonates or bicarbonates, such as sodium or potassium carbonate or bicarbonate (if appropriate, together with a sulfate)
  • organic bases such as sterically hindered organic nitrogen bases, for example tri-lower alkylamines, such as N,N- diisopropyl-N-ethylamine, pyridine or N-methyl-pyrrolidin-2-one, which can be used alone or in any appropriate combination.
  • Reactive groups in the monomers of ligands or in the resin-bound or free intermediates resulting from one or more coupling steps can be protected by third groups as protecting groups that are customarily used in peptide synthesis.
  • third groups protecting groups that are customarily used in peptide synthesis. Examples of protecting groups, their introduction and their removal are, for example, described in standard works such as "Protective groups in Organic Chemistry", Plenum Press, London, New York 1973; “Methoden der organischen Chemie”, Houben-Weyl, 4. edition, Vol. 15/1 , Georg-Thieme Verlag, Stuttgart 1974; Th. W.
  • protecting groups comprises also resins used for solid phase synthesis, preferably those specifically mentioned above and below.
  • hydroxy protecting groups are acyl radicals, such as tert-lower alkoxycarbonyl radicals, for example tert-butoxycarbonyl, etherifying groups, such as tert-lower alkyl groups, for example t-butyl, or silyl- or tin radicals, such as tert-butyl-dimethylsilyl or the tri-n- butyltin radical.
  • acyl radicals such as tert-lower alkoxycarbonyl radicals, for example tert-butoxycarbonyl
  • etherifying groups such as tert-lower alkyl groups, for example t-butyl
  • silyl- or tin radicals such as tert-butyl-dimethylsilyl or the tri-n- butyltin radical.
  • Carboxy groups can be protected by groups as defined above for the C-terminal protecting groups Y, preferably by esterifying groups selected from those of the tert-butyl type, from benzyl, from trimethylsilylethyl and from 2-triphenylsilyl groups, or they can be protected as Iower alkenyl esters, such as allylic esters. .
  • Amino or guanidino (e.g. in H-Arg-OH) groups can be protected by removable acyl groups or by arylmethyl, etherified mercapto, 2-acyl-lower alk-1 -enyl, a silyl group or an organic sulfonyl group or tin amino protecting groups; tert-butoxycarbonyl, allyloxycarbonyl, benzyl ⁇ oxycarbonyl, 4-nitrobenzyloxycarbonyl, 2-chlorobenzyloxycarbonyl, 2-bromobenzyloxy- carbonyl, diphenyimethoxycarbonyl, nitrophenylsulfenyl, 2,2,2-trichloroethoxycarbonyl, 2,2,5,7,8-pentamethylchroman-6-sulfonyl (PMC - very preferred), 2,2,4,6,7-pentamethyl- dihydrobenzofuran-5-sulfonyl (Pbf) or 4-methoxy-2,3,
  • Carbamide groups (for example, in the side chains of asparagine and glutamine) can be protected at the nitrogen atom by arylmethyl groups, preferably triphenylmethyl (trityl) or analogues thereof with one or more Iower alkoxy, such as methoxy, and/or Iower alkyl, such as methyl, substituents in one or more phenyl rings.
  • arylmethyl groups preferably triphenylmethyl (trityl) or analogues thereof with one or more Iower alkoxy, such as methoxy, and/or Iower alkyl, such as methyl, substituents in one or more phenyl rings.
  • Imino groups (e.g. in imidazole) can be protected by 2,4-dinitrophenyl, trityl, tert-butoxy ⁇ carbonyl or p-toluenesulfonyl, or (e.g. in indole) by formyl or tert-butoxycarbonyl.
  • Mercapto groups can be protected, e.g., by acetamidomethyl, by trityl or by p-methyl benzyl.
  • Phospho groups can be protected in the form of their diesters, especially in the form of their di-lower alkyi esters, such as the di-methyl ester, the di-ethyl ester or the di-tert-butylester.
  • a large number of methods of removing protective groups in the final products or any inter ⁇ mediates are known in the art and comprise, inter alia, ⁇ -elimination, solvolysis, hydrolysis, alcoholysis, acidolysis, photolysis, enzymatical removal, treatment with a base or reduction.
  • the protective groups are usually removed after the complete synthesis of the resin-bound molecule by conventional methods of peptide chemistry, conveniently by treatment with 95 % trifluoroacetic acid (Fmoc-chemistry).
  • strong nucleophiles such as dimethylsulfide and/or 2-ethanedithiol, may be additionally added to capture the generated compounds resulting from the protecting groups, e.g. in a combination such as trimethyl- silyltrifluoro-methansulfonate/dimethylsulfide/trifluoroacetic acid/ethandithiol/m-cresol.
  • the two preferred methods of solid phase peptide synthesis are the Boc and the Fmoc methods, which are named with reference to their use of the tert-butoxycarbonyl (Boc) or 9- fluore ⁇ ylmethyloxycarbonyl (Fmoc) group, respectively, to protect the ⁇ -NH 2 or ⁇ -NHR 3 of the amino acid residue to be coupled (see J. M. Stewart, J. D. Young, Solid-Phase Peptide Synthesis, 2n edn., Pierce, Rockford, Illinois (1984) or G. Barany, R.B. Merrifield, Solid- phase Peptide Synthesis, in: The Peptides, Vol. 2 (E. Gross, J.
  • TFA trifluoroacetic acid
  • Preferred third groups as protecting groups are relatively stable in weak acid, e.g. TFA. Most can be cleaved by strong acids such as hydrofiuric acid (HF) or trifluoromethanesulfonic acid.
  • HF hydrofiuric acid
  • a small number of side chain groups e.g. 2,4-dinitrophenyl protected imino in the histidyl side chain
  • the product is typically cleaved from the resin and simultaneously deprotected by HF treatment at low temperature (e.g. around 0 °C).
  • the Fmoc-group can be cleaved off preferably in the presence of a mild nitrogen base, preferably piperidine, in an inert solvent, preferably dimethyl acetamide, thereby allowing the use of side-chain protecting groups which are labile to milder treatment, e.g. TFA.
  • a mild nitrogen base preferably piperidine
  • an inert solvent preferably dimethyl acetamide
  • an acid labile ether resin such as HMP-resin (p-hydroxymethylphenoxymethyl polystyrene), 4-(2',4'-dimethoxyphenyl-hydroxymethyl)-phenoxy-polystyrene (Rink-resin), or a resin with a benzyloxy- or alkyloxy linker (see Wang, J. Amer. Chem. Soc.
  • Iower alkyl-mercapto groups such as methylthio
  • sulfinyl groups e.g. iower alkyl sulfinyl, such as methylsulfinyl
  • organic or preferably inorganic peroxides such as hydrogen peroxide
  • reaction of lower alkylthio compounds with hydrogen peroxide in concentrations from 2 to 30 volume-% at preferred temperatures from 0 to 50 °C, especially around room temperature leads to the respective iower alkyl sulfinyl compounds.
  • esterified carboxy groups such as Iower alkoxycarbonyl groups
  • conditions for hydrolysis for example hydrolysis in the presence of a base, e.g. a hydroxide of an alkaline metal, such as sodium hydroxide, under conditions known in the art, e.g. in an aqueous solvent at preferred temperatures between 0 and 50 °C, preferably at room temperature.
  • disulfide bridges preferably intramolecular
  • an aqueous-organic solvent mixture such as acetic acid in water
  • Salts of compounds of formula I having at least one salt-forming group may be prepared in a manner known per se.
  • salts of compounds of formula I having acid groups may be formed, for example, by treating the compounds with metal compounds, such as alkali metal salts of suitable organic carboxylic acids, e.g. the sodium salt of 2-ethylhexanoic acid, with organic alkaii metal or alkaline earth metal compounds, such as the corres ⁇ ponding hydroxides, carbonates or hydrogen carbonates, such as sodium or potassium hydroxide, carbonate or hydrogen carbonate, with corresponding calcium compounds or with ammonia or a suitable organic amine, stoichiometric amounts or only a small excess of the salt-forming agent preferably being used.
  • metal compounds such as alkali metal salts of suitable organic carboxylic acids, e.g. the sodium salt of 2-ethylhexanoic acid
  • organic alkaii metal or alkaline earth metal compounds such as the corres ⁇ ponding hydroxides,
  • Acid addition salts of compounds of formula I are obtained in customary manner, e.g. by treating the compounds with an acid or a suitable anion exchange reagent.
  • Internal salts of compounds of formula I containing acid and basic salt-forming groups, e.g. a free carboxy group and a free amino group, may be formed, e.g. by the neutralisation of salts, such as acid addition salts, to the isoelectric point, e.g. with weak bases, or by treatment with ion exchangers.
  • Salts can be converted in customary manner into the free compounds; metal and ammonium salts can be converted, for example, by treatment with suitable acids, and acid addition salts, for example, by treatment with a suitable basic agent.
  • Mixtures of isomers obtainable according to the invention can be separated in a manner known p-er se into the individual isomers; diastereoisomers can be separated, for example, by partitioning between polyphasic solvent mixtures, recrystallisation and/or chromato ⁇ graphic separation, for example over silica gel or by e.g. medium pressure liquid chromato ⁇ graphy over a reversed phase column, and racemates can be separated, for example, by the formation of salts with optically pure salt-forming reagents and separation of the mixture of diastereoisomers so obtainable, for example by means of fractional crystallisation, or by chromatography over optically active column materials.
  • the present invention relates also to novel starting materials and/or intermediates and to processes for their preparation.
  • the starting materials used and the reaction conditions selected are preferably those that result in the compounds described as being preferred.
  • starting materials are known, can be prepared according to processes known ⁇ er se, especially in analogy to methods given in the Examples, and/or are commercially available.
  • D-, (D,L)- or L- amino acids, unnatural amino acids, di-, tri- or oligopeptides, derivatized and/or preloaded resins the ancillary reagents and solvents required for either Boc or Fmoc peptide synthesis are commercially available from various suppliers or can be prepared readily according to standard procedures.
  • di- or other oiigopeptoids can be prepared readily according to standard procedures.
  • automated peptide synthesizers with optimized, preprogrammed Boc and Fmoc synthesis cycles are available from numerous sources.
  • the starting materials for the phosphotyrosine mimics and the respective protected derivatives can be synthesized according to methods known in the art; (e.g., for phosphono- methyl-phenylalanine, especially 4-phosphonomethyl-phenylalanine, see Synthesis 1991 , 1019, Tetrahedron Lett. 32(43), 6061 (1991), Tetrahedron Lett. 33(9), 1193 (1992) and SynLett 1994. 233-254; for phosphono-( ⁇ -fluoro)methyl-phe ⁇ ylalanine, especially 4-phos- phono-( ⁇ -fluoro)methyl-phe ⁇ ylalanine, see J. Chem. Soc, Perkin Trans.
  • 014.024.114 presented at the 109 th American Chemical Society Meetin, April 2-6 (1995) in Anaheim, California; and for phosphono-phenylalanine, such as 4-phosphonophenylalanine, see Tetrahedron 46, 7793-7802 (1990)).
  • protecting groups in starting materials the reaction of which is to be avoided can be protected by suitable protect ⁇ ing groups (conventional protecting groups) which are customarily used in the synthesis of peptide compounds, and also in the synthesis of cephalosporins and penicillins as well as nucleic acid derivatives and sugars.
  • protect ⁇ ing groups conventional protecting groups
  • These protecting groups may already be present in the precursors and are intended to protect the functional groups in question against undesired secondary reactions, such as acylation, etherification, esterification, oxidation, solvolysis, etc.
  • the protecting groups can additionally cause the reactions to proceed selectively, for example stereoselectively.
  • the protecting groups can be so selected that more than one such group can be removed simultaneously, for example by acidolysis, such as by treatment with trifluoroacetic acid, or with hydrogen and a hydrogenation catalyst, such as a palladium-on-carbon catalyst.
  • the groups can also be so selected that they cannot all be removed simultaneously, but rather in a desired sequence, the corresponding intermediates being obtained.
  • any reference hereinbefore and hereinafter to a free compound or a salt thereof is to be understood as meaning also the corresponding salt or free compound, respectively, where appropriate and expedient.
  • All the above-mentioned process steps can be carried out under reaction conditions that are known p_er se, preferably those mentioned specifically, in the absence or, customarily, in the presence of solvents or diluents, preferably solvents or diluents that are inert towards the reagents used and are solvents therefor, in the absence or presence of catalysts, condensation agents or neutralising agents, for example ion exchangers, such as cation exchangers, e.g.
  • mixtures of isomers that are formed can be separated into the individual isomers, for example diastereoisomers or enantiomers, or into any desired mix ⁇ tures of isomers, for example racemates or mixtures of diastereoisomers, for example ana ⁇ logously to the methods described under "Additional process steps”.
  • solvents from which those solvents that are suitable for any particular reaction may be selected include, for example, water, esters, such as Iower alkyl-lower alkanoates, for example ethyl acetate, ethers, such as aliphatic ethers, for example diethyl ether, or cyclic ethers, for example tetrahydrofuran, liquid aromatic hydrocarbons, such as benzene or toluene, alcohols, such as methanol, ethanol or 1- or 2-propanol, nitriles, such as aceto ⁇ nitrile, halogenated hydrocarbons, such as methylene chloride, acid amides, such as dimethylformamide, bases, such as heterocyclic nitrogen bases, for example pyridine, carboxylic acid anhydrides, such as Iower alkanoic acid anhydrides, for example acetic anhydride, cyclic, linear or branched hydrocarbons, such as cyclohexane, hexan
  • Such solvent mixtures may also be used in working up, for example by chromatography or partitioning.
  • the compounds, including their salts may also be obtained in the form of hydrates, or their crystals may, for example, include the solvent used for crystallisation. Different crystalline forms may be present.
  • protected starting materials may be used in all process steps and the protecting groups may be removed at suitable stages of the reaction.
  • the invention relates also to those forms of the process in which a compound obtainable as intermediate at any stage of the process is used as starting material and the remaining process steps are carried out, or in which a starting material is formed under the reaction conditions or is used in the form of a derivative, for example in protected form or in the form of a salt, or a compound obtainable by the process according to the invention is produced under the process conditions and processed further in sjtu.
  • a starting material is formed under the reaction conditions or is used in the form of a derivative, for example in protected form or in the form of a salt, or a compound obtainable by the process according to the invention is produced under the process conditions and processed further in sjtu.
  • reaction conditions that are analogous to those mentioned in the Examples.
  • the invention relates also to pharmaceutical compositions comprising compounds of formula I, to their use in the therapeutic (including prophylactic) treatment of the diseases mentioned above, to the compounds for said use and to the preparation of pharmaceutical preparations.
  • the pharmacologically acceptable compounds of the present invention may be used, for example, for the preparation of pharmaceutical compositions that comprise an effective amount of the active ingredient together or in admixture with a significant amount of inorganic or organic, solid or liquid, pharmaceutically acceptable carriers.
  • compositions according to the invention are those for enteral, such as nasal, rectal or oral, or parenteral, such as intramuscular or intravenous, administration to warm-blooded animals (humans and animals), that comprise an effective dose of the pharmacologically active ingredient, alone or together with a significant amount of a pharmaceutically acceptable carrier.
  • enteral such as nasal, rectal or oral
  • parenteral such as intramuscular or intravenous, administration to warm-blooded animals (humans and animals)
  • the dose of the active ingredient depends on the species of warm-blooded animal, the body weight, the age and the individual condition, individual pharmacokinetic data, the disease to be treated and the mode of administration.
  • the invention relates also to a method of treating diseases that respond to inhibition of the interaction of proteins comprising SH2 domains and phosphoproteins, especially the phos ⁇ phorylated protein tyrosine kinases or modified versions thereof; preferably of Grb2 SH2 with a phospho-protein containing a -Tyr(P0 3 H 2 )-X-Asn- motif, such as phosphorylated EGFR protein tyrosine kinase or modified derivatives thereof, but also other phospho- proteins such as SHC or modified derivatives thereof; which comprises administering a prophylactically or especially therapeutically effective amount of a compound of formula I according to the invention, especially to a warm-blooded animal, for example a human, that, on account of one of the mentioned diseases, requires such treatment.
  • a prophylactically or especially therapeutically effective amount of a compound of formula I according to the invention especially to a warm-blooded animal, for example a human, that, on account of one of the mentioned diseases
  • the dose to be administered to warm-blooded animals is from approximately 3 mg to approximately 30 g, preferably from approximately 10 mg to approximately 1.5 g, for example approximately from 100 mg to 1000 mg per person per day, divided preferably into 1 to 3 single doses which may, for example, be of the same size. Usually, children receive half of the adult dose.
  • the pharmaceutical compositions comprise from approximately 1 % to approximately 95%, preferably from approximately 20 % to approximately 90%, active ingredient.
  • Pharma ⁇ ceutical compositions according to the invention may be, for example, in unit dose form, such as in the form of ampoules, vials, suppositories, dragees, tablets or capsules.
  • compositions of the present invention are prepared in a manner known per se, for example by means of conventional dissolving, lyophilising, mixing, granulating or confectioning processes.
  • Solutions of the active ingredient, and also suspensions, and especially isotonic aqueous solutions or suspensions are preferably used, it being possible, for example in the case of lyophilised compositions that comprise the active ingredient alone or together with a carrier, for example mannitol, for such solutions or suspensions to be produced prior to use.
  • the pharmaceutical compositions may be sterilised and/or may comprise excipients, for example preservatives, stabilisers, wetting and/or emulsifying agents, solubilisers, salts for regulating the osmotic pressure and/or buffers, and are prepared in a manner known perse, for example by means of conventional dissolving or lyophilising processes.
  • the said solutions or suspensions may comprise viscosity-increasing substances, such as sodium carboxy ⁇ methylcellulose, carboxymethylcellulose, dextran, polyvinylpyrrolidone or gelatin.
  • Suspensions in oil comprise as the oil component the vegetable, synthetic or semi-synthetic oils customary for injection purposes.
  • liquid fatty acid esters that contain as the acid component a long-chained fatty acid having from 8 to 22, especially from 12 to 22, carbon atoms, for example lauric acid, tridecylic acid, myristic acid, pentadecyiic acid, palmitic acid, margaric acid, stearic acid, arachidic acid, behenic acid or corresponding unsaturated acids, for example oleic acid, elaidic acid, erucic acid, brasidic acid or linoleic acid, if desired with the addition of antioxidants, for example vitamin E, ⁇ -carotene or 3,5-di-tert-butyl-4-hydroxytoluene.
  • the alcohol component of those fatty acid esters has a maximum of 6 carbon atoms and is a mono- or poly-hydroxy, for example a mono-, di- or tri-hydroxy, alcohol, for example methanol, ethanol, propanol, butanol or pentanol or the isomers thereof, but especially glycol and glycerol.
  • fatty acid esters are therefore to be mentioned: ethyl oleate, isopropyl myristate, isopropyl palmitate, "Labrafil M 2375” (polyoxyethylene glycerol trioleate, Gattefosse, Paris), "Miglyol 812” (triglyceride of saturated fatty acids with a chain length of C 8 to C ⁇ 2 , H ⁇ ls AG, Germany), but especially vegetable oils, such as cottonseed oil, almond oil, olive oil, castor oil, sesame oil, soybean oil and more especially groundnut oil.
  • vegetable oils such as cottonseed oil, almond oil, olive oil, castor oil, sesame oil, soybean oil and more especially groundnut oil.
  • the injection compositions are prepared in customary manner under sterile conditions; the same applies also to introducing the compositions into ampoules or vials and sealing the containers.
  • compositions for oral administration can be obtained by combining the active ingredient with solid carriers, if desired granulating a resulting mixture, and process ⁇ ing the mixture, if desired or necessary, after the addition of appropriate excipients, into tablets, dragee cores or capsules. It is also possible for them to be incorporated into plastics carriers that allow the active ingredients to diffuse or be released in measured amounts.
  • Suitable carriers are especially fillers, such as sugars, for example lactose, saccharose, mannitol or sorbitol, cellulose preparations and/or calcium phosphates, for example tricalcium phosphate or calcium hydrogen phosphate, and binders, such as starch pastes using for example corn, wheat, rice or potato starch, gelatin, tragacanth, methylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose and/or polyvinylpyrrolidone, and/or, if desired, disintegrators, such as the above-mentioned starches, also carboxy ⁇ methyl starch, crosslinked polyvinylpyrrolidone, agar, alginic acid or a salt thereof, such as sodium alginate.
  • fillers such as sugars, for example lactose, saccharose, mannitol or sorbitol, cellulose preparations and/or calcium phosphates, for example tricalcium phosphate or calcium hydrogen phosphate
  • Excipients are especially flow conditioners and lubricants, for example silicic acid, talc, stearic acid or salts thereof, such as magnesium or calcium stearate, and/or polyethylene glycol.
  • Dragee cores are provided with suitable, optionally enteric, coatings, there being used, inter alia, concentrated sugar solutions which may comprise gum arabic, talc, polyvinylpyrrolidone, polyethylene glycol and/or titanium dioxide, or coating solutions in suitable organic solvents, or, for the preparation of enteric coatings, solutions of suitable cellulose preparations, such as ethyicelluiose phthalate or hydroxypropylmethylcellulose phthalate.
  • Capsules are dry-filled capsules made of gelatin and soft sealed capsules made of gelatin and a plasticiser, such as glycerol or sorbitol.
  • the dry-filled capsules may comprise the active ingredient in the form of granules, for example with fillers, such as lactose, binders, such as starches, and/or glidants, such as talc or magnesium stearate, and if desired with stabilisers.
  • the active ingredient is preferably dissolved or suspended in suitable oily excipients, such as fatty oils, paraffin oil or liquid polyethylene glycols, it being possible also for stabilisers and/or antibacterial agents to be added.
  • suitable oily excipients such as fatty oils, paraffin oil or liquid polyethylene glycols, it being possible also for stabilisers and/or antibacterial agents to be added.
  • Dyes or pigments may be added to the tablets or dragee coatings or the capsule casings, for example for identification purposes or to indicate different dose
  • the peptide is synthesized on a Milligen 9050 automated peptide synthesizer (continuous flow; Millipore, Bedford, MA, USA), starting with an Fmoc-PAL-PEG-PS resin (see Albericio, F. et al, J. Org. Chem., 55 (1990) 3730-3743) for establishing the C-terminal carboxamide, and using chemical protocols based on the fluorenylmethoxycarbonyl chemistry (see E. Atherton and R.C. Sheppard, in Solid-Phase Peptide Synthesis-A Practical Approach, ed.: D. Rickwood and B.D. Hames, IRL Press at Oxford University Press, Oxford, 1989).
  • Fmoc-amino acids (3 equiv.) are coupled using their 2,4,5-trichlorophenyl esters (single coupling) with minimum reaction times of 30 min (see 9050 Plus PepSynthesizer, User's Guide, Millipore Corporation, Bedford, MA, 1992). Double coupling with 2-(2-pyridon- 1-yl)-1 ,1,3,3-tetramethyiuroniumtetrafluoroborate (TPTU) is carried out for glutamic acid.
  • TPTU 2-(2-pyridon- 1-yl)-1 ,1,3,3-tetramethyiuroniumtetrafluoroborate
  • N ⁇ -Fmoc-Tyr(P ⁇ 3H2)-OH 3 equiv.; see Ottinger, E.A, et al., Biochemistry 32, 4354 (1993)
  • benzotriazole-1 -yl-oxy-tris- (dimethylamino)-phosphonium hexafiuorophosphate/N-hydroxybe ⁇ zotriazole (1 :1 , 3 equiv.; first coupling) and N-[(dimethyiamino)-1 H-1 ,2,3-triazolo[4,5-b]pyridin-1 -ylmethylene]-N- methylmethanaminium hexafluorophosphate N-oxide (3 equiv.; second coupling) in the presence of diisopropylamine (6 equiv.).
  • 2-amino benzoic acid (Fluka, Buchs, Switzerland) is incorporated with benzotriazole-1 -yl-oxy-tris-(dimethylamino)-phosphoniumhexafiuoro- phosphate/N-hydroxybenzotriazole (double coupling).
  • Amino acid side chains are protected with the following groups: tert-butyl for glutamic acid; and trityl for asparagine and glutamine.
  • the completed peptide resin is simultaneosly deprotected and cleaved by treatment with trifluoroacetic acid:H2 ⁇ (95:5, v/v) for 3 h at room temperature.
  • the filtrate from the cleavage reaction is precipitated in diisopropyl ether/petroleum ether (1 :1 , v/v, 0 °C), and the precipitate collected by filtration.
  • the crude peptide is purified by medium- pressure liquid chromatography using a C-
  • the 2-aminobe ⁇ zoic acid acid building block is used in unprotected form.
  • the starting material is obtained as follows:
  • N ⁇ -Fmoc-Cys(Acm)-OH is from Calbiochem-Novabiochem (Laufelfingen, Switzerland).
  • the 2-aminobenzoic acid acid building block is used in unprotected form.
  • Title compound: Mass spectral analysis (negative-ion mode): 954.4 (calc. 954.0, C38H54N10O13S2P1), tR 6.24 min (HPLC System A).
  • 4-Methoxybenzoic acid is from Fluka, Buchs, Switzerland. Titie compound:
  • Example 8 2-Amino-nicotinoyl-Glu-Tyr(P ⁇ 3H 2 )-lie-Asn-Gln-NH2 (TFA salt)
  • the 2-aminobenzoic acid acid building block is used in unprotected form.
  • Title compound is used in unprotected form.
  • Example 1 2-Aminobenzoyl-Pro-Tyr(P03H 2 )-lle-Asn-Gln-NH2 (TFA salt)
  • the 2-aminobenzoic acid acid building block is used in unprotected form.
  • Title compound is used in unprotected form.
  • the 2-aminobenzoic acid acid building block is used in unprotected form.
  • Example 14 2-Aminobenzoyl-Cys(Acm)-Tyr(P ⁇ 3H2)-Cys(Acm)-Asn-Gln-NH2 (TFA salt) (SEQ ID NO: 14)
  • the 2-aminobenzoic acid acid building block is used in unprotected form. N ⁇ -Fmoc-D-
  • Cys(Acm)-OH is from Novabiochem, Laufelfingen, Switzerland. Title compound:
  • the 2-aminobenzoic acid acid building block is used in unprotected form.
  • Title compound is used in unprotected form.
  • the 2-aminobenzoic acid acid building block is used in unprotected form.
  • Title compound is used in unprotected form.
  • the 2-aminobenzoic acid acid building block is used in unprotected form.
  • Title compound is used in unprotected form.
  • Example 19 2-Aminobenzoyl- ⁇ Ala-Glu-Tyr(P ⁇ 3H2)-He-Asn-Gln-NH 2 (TFA salt) (SEQ ID NO: 19)
  • the 2-aminobenzoic acid acid building block is used in unprotected form.
  • Title compound: Mass spectral analysis (negative-ion mode): 805.0 (calc. 804.8, C34H47N9O12P1). t 6.20 min (HPLC System A).
  • the 2-aminobenzoic acid acid building block is used in unprotected form.
  • Title compound is used in unprotected form.
  • the 2-aminobenzoic acid acid building block is used in unprotected form.
  • Title compound is used in unprotected form.
  • the 2-aminobenzoic acid acid building block is used in unprotected form.
  • Title compound is used in unprotected form.
  • the peptide is synthesized manually on a 4-(2',4'-dimethoxyphenyl-aminomethyl)-phenoxy- resin (Novabiochem, Laufelfingen, Switzerland), employing the fluorenylmethoxycarbonyl strategy.
  • Fmoc-removal is with piperidine/dimethylacetamide (1 :4, v/v; 6 x 2 min), followed by washing with methanol (3 x 1 min), ⁇ /-methylpyrrolidin-2-one (2 x 1 min), methanol (3 x 1 min), and ⁇ /-methylpyrrolidin-2-one (3 x 2 min).
  • Coupling is achieved by first dissolving the Fmoc-amino acid (2 equiv.), diisopropylethylamine (2.2 equiv.), and the 2-(2-pyridon-1-yl)- 1 ,1 ,3,3-tetramethyluroniumtetrafluoroborate reagent (2 equiv.) in ⁇ /-methylpyrrolidin-2-one, then waiting 3 min for preactivation, adding the mixture to the resin, and finally shaking for at least 45 min.
  • Amino acid side chains are protected with the following groups: tert-butyl for glutamic acid; and trityl for asparagine and glutamine.
  • N ⁇ -Fmoc- Tyr(P ⁇ 3H2)-OH 3 equiv.
  • benzotriazole-l-yl-oxy-tris-(dimethylamino)- phosphoniumhexafluorophosphate/N-hydroxybenzotriazole (1 :1 ; 3 equiv.; first coupling) in the presence of diisopropylethylamine (7 equiv.) and N-[(dimethylamino)-1 H-1 ,2,3-triazolo- [4,5-b]pyridin-1 -ylmethylene]-N-methylmethanaminium hexafluorophosphate N-oxide (3 equiv., second coupling) in the presence of diisopropylethylamine (7 equiv.).
  • 3-(N-tert-But- oxycarbonyl-amino) benzoic acid (3 equiv.) is coupled with BOP/HOBt (1 :1 ; 3 equiv.; first coupling) in the presence of diisopropylethylamine (6 equiv.) and TPTU/HOBt (1 :1 ; 3 equiv: second coupling.) in the presence of diisopropylamine (6 equiv.).
  • the complete peptide resin obtained after the last coupling step is simultaneously deprotected and cleaved by treatment with trifluoroacetic acid/H2 ⁇ (95:5, v/v) for 3 h at room temperature.
  • the starting material is obtained as follows:
  • the starting material is obtained as follows:
  • Example 25 lsoquinoline-1 -ylcarbonyl-Glu-Tyr(P ⁇ 3H2)-He-Asn-Gln-NH2
  • the 2-aminobenzoic acid acid building block is used in unprotected form.
  • Title compound is used in unprotected form.
  • the unprotected (+/-)-lndoiine-2-ylcarboxyiic acid used is from Fluka, Buchs, Switzerland.
  • JExai ⁇ i ⁇ le_33 lndole-3-ylcarbonyl-Glu-Tyr(P ⁇ 3H2)-He-Asn-Gln-NH 2
  • the peptide derivative is synthesized manually using the 4-(2',4'-dimethoxyphenyl- aminomethyl)-phenoxy-resin Novabiochem, Laufelfingen, Switzerland), employing a procedure analogous to Example 23.
  • Incorporation of N ⁇ -Fmoc-[4-(0-diethyl)-phosphono- (difiuoromethyl)]-L-phenylalanine (synthesis see Tetrahedron Lett. 34(22), 3543 (1993)) to the protected peptide resin is carried out with BOP/HOBt (1 :1 ; 3 equiv.) in the presence of diisopropylethylamine (7 equiv.), with 3 h reaction time.
  • the protected peptide resin is treated with 1 M trimethylsilyltrifluoromethane sulfonate-2 M dimethylsulfide-trifluoroacetic acid (500 ⁇ l to 0.005 mmol of NH2)-ethane- dithiol (100 ⁇ l)-m-cresol (25 ⁇ l) 30 min at 4 °C and 3.5 h at room temperature (see Tetrahedron Lett. 34(44), 7039 (1993)).
  • the filtrate from the cleavage reaction is precipi ⁇ tated in diisopropyl ether-petroleum ether (1 :1 , v/v, 0 °C), and the precipitate collected by filtration.
  • Example 36 3-Aminobenzoy!-Glu-F2Pmp-lle-Asn-Gln-NH2 (TFA salt) (SEQ ID NO: 36)
  • Example 23 a The amino group of the 3-aminobenzoic acid building block is protected with the tert- butoxycarbonyl group as described in Example 23 a).
  • Example 37 The compound is prepared using a procedure analogous to Example 37, starting from the title compound of Example 13.
  • Title compound: Mass spectral analysis (negative-ion mode): 938.7 (calc. 938.0, C37H50N10O13S2P1), tR 6.27 min (HPLC System A).
  • Example 14 The compound is prepared using a procedure analogous to Example 37, starting from the title compound of Example 14.
  • Title compound: Mass spectral analysis (negative-ion mode): 825.5 (calc. 824.8, C31 H39N9O12S2P1), tR 6.32 min (HPLC System: Same column as for HPLC System A, using a linear gradient over 10 min of acetonitrile/0.09 % TFA and H 2 O/0.1 % TFA from 1 :49 to 3:7, flow rate 2.0 ml/min, detection at 215 nm).
  • Example 41 2-Hvdroxv-benzovl-Glu-TvrfP ⁇ 3H; -lle-Asn-Gln-NHp (SEQ ID NO: 41)
  • the peptide is synthesized on a Millipore 9050 Pius peptide synthesizer in analogy to the method described in Example 1.
  • the hydroxyl group of the 2-hydroxybenzoic acid (Fluka, Buchs, Switzerland) is not protected- Double coupling with BOP/HOBt (1 :1 , 3.0 equiv.).
  • Title compound Mass spectral analysis (negative-ion mode): 863.5 (calc.
  • the starting material is obtained as follows:
  • the titie compound is obtained in analogy to Examples 23.
  • Fmoc-1-amino-1 -cyclohexane- carboxylic acid (2 equiv.) is coupled with BOP/HOBt (1 :1 , 2 equiv.; first coupling) and HATU
  • a sterile-filtered aqueous solution, with 20 % cyclodextrins as solubilisers, of one of the compounds of formula I mentioned in the preceding Examples (e.g. Example 1) as active ingredient, is so mixed under aseptic conditions, with heating, with a sterile gelatine solution containing phenol as preservative, that 1.0ml of solution has the following composition:
  • Example 46 Sterile dry substance for miection:
  • Example 48 Film-coated tablets
  • a mixture of one of the compounds of formula I mentioned in the preceding Examples (e.g. Example 1) as active ingredient, 50 g of corn starch and the colloidal silica is processed with a starch paste, made from 250 g of corn starch and 2.2 kg of demineralised water, to form a moist mass. This is forced through a sieve having a mesh size of 3 mm and dried at 45° for 30min in a fluidised bed drier.

Abstract

L'invention concerne un composé de la formule (I). Dans cette formule, m est compris entre 2 et 15, X est un arylcarbonyle, arylsulfonyle, cycloalkylcarbonyle, cycloalcanesulfonyle, hétérocyclylcarbonyle ou hétérocyclylsulfonyle; A est absent ou il s'agit d'un radical bivalent d'un acide aminé naturel ou synthétique, B est un radical bivalent d'un acide aminé naturel, PTI est un radical bivalent de la phosphotyrosine ou d'une substance mimétique de la phosphotyrosine, AA est un radical bivalent d'un acide aminé naturel ou synthétique, et Y est un hydroxy, un groupe protégeant l'extrémité C-terminale ou un groupe amine primaire, secondaire ou tertiaire, à condition que deux groupes sulfydryle ou davantage appartenant à un quelconque des acides aminés A, B ou AA soient présents en tant que tels ou puissent former des liaisons disulfure intramoléculaires. L'invention concerne également les sels de ce composé. Ce composé et ses sels sont utiles pour traiter des maladies réagissant à l'inhibition de l'interaction entre une ou des protéines comprenant un ou des domaines SH2, et une protéine tyrosine kinase, ou une version modifiée d'une telle kinase.
PCT/EP1996/003479 1995-08-17 1996-08-06 Oligopeptides acyles divers WO1997007131A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU68709/96A AU6870996A (en) 1995-08-17 1996-08-06 Acylated oligopeptides containing phosphotyrosine as inhibitors of protein tyrosine kinases

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GBGB9516842.3A GB9516842D0 (en) 1995-08-17 1995-08-17 Various acylated oligopeptides
GB9516842.3 1995-08-17

Publications (1)

Publication Number Publication Date
WO1997007131A1 true WO1997007131A1 (fr) 1997-02-27

Family

ID=10779357

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP1996/003479 WO1997007131A1 (fr) 1995-08-17 1996-08-06 Oligopeptides acyles divers

Country Status (4)

Country Link
AU (1) AU6870996A (fr)
GB (1) GB9516842D0 (fr)
WO (1) WO1997007131A1 (fr)
ZA (1) ZA966968B (fr)

Cited By (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997030079A1 (fr) * 1996-02-15 1997-08-21 Pharmacia & Upjohn S.P.A. Peptides antagonistes de la mitogenese et de la motogenese cellulaires et leur utilisation therapeutique
WO1997042501A1 (fr) * 1996-05-07 1997-11-13 Boehringer Ingelheim Pharmaceuticals, Inc. Mimetiques de phosphotyrosine, procedes d'identification et d'utilisation de ces derniers
EP0871436A1 (fr) * 1995-06-30 1998-10-21 Smithkline Beecham Corporation UTILISATION DE COMPOSES SPECIFIQUES DU DOMAINE Stat 6 SH2 POUR LE TRAITEMENT DES REACTIONS ALLERGIQUES
US7125875B2 (en) 1999-04-15 2006-10-24 Bristol-Myers Squibb Company Cyclic protein tyrosine kinase inhibitors
EP2258371A1 (fr) 2001-08-10 2010-12-08 Novartis AG Utilisation d'inhibiteurs c-Src seuls ou en combinaison avec STI571 pour le traitement de leucemies
WO2011029915A1 (fr) 2009-09-10 2011-03-17 Novartis Ag Dérivés éthers d'hétéroaryles bicycliques
EP2308855A1 (fr) 2002-03-15 2011-04-13 Novartis AG Dérivés de 2,4-Diaminopyrimidine
WO2011064211A1 (fr) 2009-11-25 2011-06-03 Novartis Ag Dérivés hétérocycliques d'hétéroaryles bicycliques à 6 cycles benzéniques accolés de benzène contenant de l'oxygène
WO2011157793A1 (fr) 2010-06-17 2011-12-22 Novartis Ag Dérivés de 1,3‑dihydro-benzoimidazol-2-ylidène-amine à substitution pipéridinyle
WO2011157787A1 (fr) 2010-06-17 2011-12-22 Novartis Ag Dérivés de 1,3‑dihydro-benzoimidazol-2-ylidène-amine à substitution biphényle
EP2409969A1 (fr) 2004-06-24 2012-01-25 Novartis AG Dérivés d'urée de pyrimidine en tant qu'inhibiteurs de kinase
WO2012120469A1 (fr) 2011-03-08 2012-09-13 Novartis Ag Composés hétéroaryliques bicycliques de type fluorophényle
US8313729B2 (en) 2007-03-01 2012-11-20 Medibeacon, LLC Integrated photoactive small molecules and uses thereof
WO2016016894A1 (fr) 2014-07-30 2016-02-04 Yeda Research And Development Co. Ltd. Milieux pour la culture de cellules souches pluripotentes
WO2019119486A1 (fr) 2017-12-21 2019-06-27 中国科学院合肥物质科学研究院 Classe d'inhibiteurs de kinase dérivés de pyrimidine
WO2020152686A1 (fr) 2019-01-23 2020-07-30 Yeda Research And Development Co. Ltd. Milieux de culture pour cellules souches pluripotentes
WO2021097256A1 (fr) 2019-11-14 2021-05-20 Cohbar, Inc. Peptides antagonistes de cxcr4

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994007913A1 (fr) * 1992-09-25 1994-04-14 Warner-Lambert Company Antagonistes peptidiques de la fixation du sh2 et leurs usages therapeutiques
WO1994008600A1 (fr) * 1992-10-09 1994-04-28 Joslin Diabetes Center Inhibition de molecules transductrices de signaux

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994007913A1 (fr) * 1992-09-25 1994-04-14 Warner-Lambert Company Antagonistes peptidiques de la fixation du sh2 et leurs usages therapeutiques
WO1994008600A1 (fr) * 1992-10-09 1994-04-28 Joslin Diabetes Center Inhibition de molecules transductrices de signaux

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
BOBKO, MARK ET AL: "CD45 protein tyrosine phosphatase: determination of minimal peptide length for substrate recognition and synthesis of some tyrosine-based electrophiles as potential active-site directed irreversible inhibitors", BIOORG. MED. CHEM. LETT. (1995), 5(4), 353-6 CODEN: BMCLE8;ISSN: 0960-894X, 1995, XP000615949 *
CHALUPNY, N. JAN ET AL: "Specific binding of Fyn and phosphatidylinositol 3-kinase to the B cell surface glycoprotein CD19 through their src homology 2 domains", EUR. J. IMMUNOL. (1995), 25(10), 2978-84 CODEN: EJIMAF;ISSN: 0014-2980, October 1995 (1995-10-01), XP000615951 *
COWBURN, DAVID ET AL: "Enhanced affinities and specificities of consolidated ligands for the Src homology (SH) 3 and SH2 domains of Abelson protein-tyrosine kinase", J. BIOL. CHEM. (1995), 270(45), 26738-41 CODEN: JBCHA3;ISSN: 0021-9258, 10 November 1995 (1995-11-10), XP002023105 *
VAN DER GEER, PETER ET AL: "A conserved amino-terminal Shc domain binds to phosphotyrosine motifs in activated receptors and phosphopeptides", CURR. BIOL. (1995), 5(4), 404-12 CODEN: CUBLE2;ISSN: 0960-9822, 1 April 1995 (1995-04-01), XP000615948 *
ZVELEBIL, MARKETA J. J. M. ET AL: "Prediction and analysis of SH2 domain-phosphopeptide interactions", PROTEIN ENG. (1995), 8(6), 527-33 CODEN: PRENE9;ISSN: 0269-2139, June 1995 (1995-06-01), XP002023104 *

Cited By (22)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0871436A1 (fr) * 1995-06-30 1998-10-21 Smithkline Beecham Corporation UTILISATION DE COMPOSES SPECIFIQUES DU DOMAINE Stat 6 SH2 POUR LE TRAITEMENT DES REACTIONS ALLERGIQUES
EP0871436A4 (fr) * 1995-06-30 1999-10-20 Smithkline Beecham Corp UTILISATION DE COMPOSES SPECIFIQUES DU DOMAINE Stat 6 SH2 POUR LE TRAITEMENT DES REACTIONS ALLERGIQUES
WO1997030079A1 (fr) * 1996-02-15 1997-08-21 Pharmacia & Upjohn S.P.A. Peptides antagonistes de la mitogenese et de la motogenese cellulaires et leur utilisation therapeutique
WO1997042501A1 (fr) * 1996-05-07 1997-11-13 Boehringer Ingelheim Pharmaceuticals, Inc. Mimetiques de phosphotyrosine, procedes d'identification et d'utilisation de ces derniers
US7125875B2 (en) 1999-04-15 2006-10-24 Bristol-Myers Squibb Company Cyclic protein tyrosine kinase inhibitors
US8268837B2 (en) 2001-08-10 2012-09-18 Novartis Ag Use of c-Src inhibitors alone or in combination with STI571 for the treatment of leukaemia
US8119649B2 (en) 2001-08-10 2012-02-21 Novartis Ag Use of c-Src inhibitors alone or in combination with STI571 for the treatment of leukaemia
EP2258371A1 (fr) 2001-08-10 2010-12-08 Novartis AG Utilisation d'inhibiteurs c-Src seuls ou en combinaison avec STI571 pour le traitement de leucemies
EP2308855A1 (fr) 2002-03-15 2011-04-13 Novartis AG Dérivés de 2,4-Diaminopyrimidine
EP2409969A1 (fr) 2004-06-24 2012-01-25 Novartis AG Dérivés d'urée de pyrimidine en tant qu'inhibiteurs de kinase
EP2418205A1 (fr) 2004-06-24 2012-02-15 Novartis AG Dérivés d'urée de pyrimidine en tant qu'inhibiteurs de kinase
US8313729B2 (en) 2007-03-01 2012-11-20 Medibeacon, LLC Integrated photoactive small molecules and uses thereof
WO2011029915A1 (fr) 2009-09-10 2011-03-17 Novartis Ag Dérivés éthers d'hétéroaryles bicycliques
WO2011064211A1 (fr) 2009-11-25 2011-06-03 Novartis Ag Dérivés hétérocycliques d'hétéroaryles bicycliques à 6 cycles benzéniques accolés de benzène contenant de l'oxygène
WO2011157793A1 (fr) 2010-06-17 2011-12-22 Novartis Ag Dérivés de 1,3‑dihydro-benzoimidazol-2-ylidène-amine à substitution pipéridinyle
WO2011157787A1 (fr) 2010-06-17 2011-12-22 Novartis Ag Dérivés de 1,3‑dihydro-benzoimidazol-2-ylidène-amine à substitution biphényle
WO2012120469A1 (fr) 2011-03-08 2012-09-13 Novartis Ag Composés hétéroaryliques bicycliques de type fluorophényle
WO2016016894A1 (fr) 2014-07-30 2016-02-04 Yeda Research And Development Co. Ltd. Milieux pour la culture de cellules souches pluripotentes
WO2019119486A1 (fr) 2017-12-21 2019-06-27 中国科学院合肥物质科学研究院 Classe d'inhibiteurs de kinase dérivés de pyrimidine
US11602534B2 (en) 2017-12-21 2023-03-14 Hefei Institutes Of Physical Science, Chinese Academy Of Sciences Pyrimidine derivative kinase inhibitors
WO2020152686A1 (fr) 2019-01-23 2020-07-30 Yeda Research And Development Co. Ltd. Milieux de culture pour cellules souches pluripotentes
WO2021097256A1 (fr) 2019-11-14 2021-05-20 Cohbar, Inc. Peptides antagonistes de cxcr4

Also Published As

Publication number Publication date
AU6870996A (en) 1997-03-12
GB9516842D0 (en) 1995-10-18
ZA966968B (en) 1997-02-17

Similar Documents

Publication Publication Date Title
EP0846127A1 (fr) Derives oligopeptidiques acyles presentant une activite inhibant des signaux cellulaires
WO1997007131A1 (fr) Oligopeptides acyles divers
US5200397A (en) Use of peptide analogs of thrombospondin for the inhibition of angiogenic activity
US5506208A (en) Method for using synthetic analogs of thrombospondin for inhibiting angiogenesis activity
US5268358A (en) PDGF receptor blocking peptides
US5426100A (en) Piptide fragments and analogs of thrombospondin
JP2003500417A (ja) Ｆａｐ活性化抗腫瘍性化合物
JP2001500365A (ja) Ｐ５３とｍｄｍ２の間の相互作用の阻害剤
EP1069137A1 (fr) Peptides ayant une activité de type thrombospondine et leur utilisation thérapeutique
US4002740A (en) Tridecapeptide compositions and methods
US6180362B1 (en) Peptides which inhibit ras protein activity, their preparation and use
US5654277A (en) Peptides having thrombospondin-like activity and their therapeutic use
NZ231750A (en) Retroviral protease inhibitors and pharmaceutical compositions
JPH0565230A (ja) レトロウイルスプロテアーゼ阻害剤
US6339062B1 (en) Retroinverso polypeptides that mimic or inhibit thrombospondin activity
US5840692A (en) Peptide fragments and analogs of thrombospondin
US6307090B1 (en) Acylated oligopeptide derivatives having cell signal inhibiting activity
EP1044012B1 (fr) Inhibition de l'angiogenese au moyen d'analogues de peptide du domaine 5 de la kininogene de poids moleculaire eleve
US20070042962A1 (en) Peptide dependent upregulation of telomerase expression
JPH11504658A (ja) 細胞マイトジェネシス及びモトジェネシスのペプチドアンタゴニスト及びその治療的使用
EP0832110A1 (fr) Composes antiviraux de type peptidique
US6239110B1 (en) Synthetic analogs of thrombospondin and therapeutic use thereof
JPS6327360B2 (fr)
US5648461A (en) Synthetic analogs of thrombospondin and therapeutic use thereof
JPH07316191A (ja) アシルオキシヘキサノン酸誘導体

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AL AU BB BG BR CA CN CU CZ EE GE HU IL IS JP KP KR LK LR LT LV MG MK MN MX NO NZ PL RO SG SI SK TR TT UA US UZ VN AM AZ BY KG KZ MD RU TJ TM

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): KE LS MW SD SZ UG AT BE CH DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN ML MR NE SN TD TG

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application
122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: CA