Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
  • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
  • C07K5/10—Tetrapeptides
  • C07K5/1027—Tetrapeptides containing heteroatoms different from O, S, or N
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
  • C07K14/71—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
  • C07K5/02—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link
  • C07K5/0202—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link containing the structure -NH-X-X-C(=0)-, X being an optionally substituted carbon atom or a heteroatom, e.g. beta-amino acids
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
  • C07K5/02—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link
  • C07K5/0207—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link containing the structure -NH-(X)4-C(=0), e.g. 'isosters', replacing two amino acids
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
  • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
  • C07K5/06—Dipeptides
  • C07K5/06191—Dipeptides containing heteroatoms different from O, S, or N
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
  • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
  • C07K5/08—Tripeptides
  • C07K5/0827—Tripeptides containing heteroatoms different from O, S, or N
• • 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 pharmaceutically active compounds comprising a N-acyl 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.
• Signal transduction is the process of 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
• protein-tyrosine kinases At the heart of this biological communication are the protein-tyrosine kinases. These enzymes, found, for example, as either transmembrane growth factor receptors or as nuclear or cytosolic non-receptor proteins, catalyze the phosphorylation of specific tyrosine residues.
• This class of enzymes includes, but is 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 a range of metabolic processes, from proliferation and growth to differentiation. Protein-tyrosine phosphorylation is known to be involved in modulating the activity of some target enzymes as well as in generating specific complex networks involved in signal transduction via various proteins containing a specific amino acid sequence called
• - overexpression or deregulation is manifested by various oncogenic and (hyper-) proliferative disorders such as cancer, inflammation, autoimmune disease, hyperproliferative skin disorders, such as psoriasis, and allergy/asthma.
• SH2- and/or SH3-comprising proteins that play a role in cellular signaling and transforma ⁇ tion 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, phosholipase C, phosphoi
• a direct link has been established between activated receptor kinases and Ras with the finding that the mammalian Grb2 protein, a 26 kilodalton protein comprising a single SH2 and two SH3 domains, directly couples receptor tyrosine kinases to the Ras guanine nucleotide exchange factor Sos in mammals and also Drosophila.
• 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 represent 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 information 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. tg, 991 (1992); EMBO J. JJ . , 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)].
• An 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
• tyrosine 1068 is the binding site for Grb2 SH2 (see Buday et al., Cell 73, 611-620 (1993)).
• a phosphopeptide based on the surrounding sequence, Pro-Val-Pro-Glu-Tyr(PO 3 H 2 )-lle-Asn-Gln-Ser was shown to inhibit the interaction of phosphorylated EGFR and Grb2.
• PD 153035 rapidly suppressed autophosphoryla ⁇ tion 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-Dianilinophthalimides 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.
• 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.
• 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)n-).
• These moieties X are even able to allow for large sequence variability in the pep ⁇ tide derivatives of formula I.
• Many of the compounds of formula I show inhibition if n in for ⁇ mula I given below is 3, 2 and even 1 or 0. in addition, the C- and especially the N-terminal modification even allows that PTI in formula I given below is simply tyrosine without any phosphono group or an analogue thereof; even then very active compounds can be obtained.
• the invention relates to an acylated peptide, namely a compound of the formula I,
• X is arylcarbonyl, cycloalkylcarbonyl, tricycloalkylcarbonyl, arylsulfonyl, heterocyclylcarbonyl, heterocyclylsulfonyl, carbamoyl-lower alkanoyl, aryl-lower alkylcarbonyl, cycloalkyl-lower alkylcarbonyl, aryl-lower alkylsulfonyl, heterocyclyl-lower alkylcarbonyl, heterocyclyl-lower alkylsulfonyl with the proviso that in any of the lower alkyl radicals mentioned a methylene group may be replaced with oxa, aza or thia; heterocyclyl-lower alkenylcarbonyl or aryl- lower-alkenylcarbonyl; or, if Y is a secondary or tertiary amino group, is one of the morieties X mentioned above or lower
• PTI is the bivalent radical of tyrosine or (preferably) 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
• lower 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.
• lower alkenyl designates a residue with 2 to 7 carbon atoms, preferably with 2 to 4 carbon atoms, such as vinyl, allyl or 1 - or 2-butenyl.
• 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.
• a double bond in a compound of formula I may be present in the cis (Z) or trans (E) form.
• the respective compound may be present as a mixture of isomers or as a pure isomer.
• n is preferably 1 to 15, more preferably 1 to 4, even more preferably 1 to 3 and most preferably 2 or especially 3.
• Aryl has preferably from 6 to 14 ring carbon atoms, such as in phenyl (which is especially preferred), naphthyl (which is especially preferred), such as 1 -naphthyl or 2-naphthyl, indenyl, indanyl, anthryl, phenanthryl (which is especially preferred), acenaphthyl or fluorenyl (which is preferred), and may be unsubstituted or preferably mono- to tri ⁇ substituted, especially by amino, mono- or di-lower alkylamino, lower alkanoylamino, such as acetylamino, amino-lower alkyl, mono- or di-loweralkylamino-lower alkyl, lower alkanoyl- amino-lower alkyl, hydroxy, lower alkoxy, such as methoxy, carboxy, lower-alkoxycarbonyl, such as methoxycarbonyl, phenyl-, naphthyl- or flu
• 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, lower alkanoylamino, such as acetylamino, amino-lower alkyl, mono- or di-loweralkylamino-lower alkyl, lower alkanoylamino-lower alkyl, hydroxy, lower alkoxy, such as methoxy, carboxy, lower-alkoxycarbonyl, such as methoxycarbonyl, phenyl-, naphthyl- or fluorenyl-lower alkoxycarbonyl, such as benzyloxycarbonyl, lower alkanoyl, cyano, lower alkyl, for example methyl, ethyl or propyl, halo-lower alkyl, for example tri ⁇ fluoromethyl,
• Tricycloalkyl preferably has 8 to 16 carbon atoms and is, for example, tricyclo[5.2.1.0 2,6 ]dec- 8-yl or especially adamantyl, preferably 1 -adamantyl.
• 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, imid ⁇ azolyl, such as imidazole-4-yl, pyrazolyl, oxazolyl, thiazolyl, such as 4- or 5-thiazolyl, tetraz ⁇ olyl, pyridyl, such as pyridin-3- or pyridin-4-yl, pyrazinyl,
• a methylene group may be replaced with aza, thia or (preferably) oxa, preferably the oxa being bound to the respective aryl or heterocylyl or to the carbonyl group in formula I
• the lower alkyl or the lower alkyl wherein a methylene group may be replaced with oxa, aza or thia can be linear or branched and is preferably selected from methyl (forming methyle ⁇ ne), 1 , 2-ethyl (forming 1 ,2-ethylene), 1 , 1 -ethyl (forming methyl-methylene), methoxy whe ⁇ rein the methyl is bound to the aryl or heterocyclyl and
• radicals given in parenthesiws are to be regarded in the following way:
• the bond on the left of each radical is to be regarded to be directed to the N-terminus in formula I
• the bond on the right is to be regarded to be directed to the C-terminus of formula I.
• arylcarbonyl X the aryl moiety is preferably defined as above; more preferably, arylcar ⁇ bonyl is selected from benzoyi or naphthoyl and, even more preferably, from benzoyi sub ⁇ stituted with amino; lower alkylamino; amino-lower alkyl; hydroxy; lower alkoxy; amino and hydroxy; amino and lower alkoxy; carboxy; lower-alkoxycarbonyl; cyano; halogen, especially chloro; lower-alkylthio; or lower alkylsulfinyl; or (altematively or in addition to the group of moieties mentioned just before) selected from naphthoyl or hydroxy-naphthoyl, such as naphthalene-2-yl-carbonyl or 6-hydroxy-naphthalene-2-yl-carbonyl, and, less preferably, from fluorenylcarbonyl, such as fluoren-9-ylcarbonyl; especially
• cycloalkylcarbonyl X cycloalkyl is preferably as defined above; more preferably, cyclo ⁇ alkylcarbonyl is C3-C7-, especially C - C 5 - or C 6 -cycloalkylcarbonyl, such as cyclohexyl-car- bonyl, and is unsubstituted or substituted by amino or anneiated to a benzo ring; most pre ⁇ ferably cyclohexylcarbonyl, 1 ,2,3,4-tetrahydronaphthylcarbonyl, such as 1 ,2,3,4-tetrahydro- naphthyl-2-carbonyl, or especially 1 -amino-cyclohexylcarbonyl or 1-amino-cyclopentylcarbo- nyl. Cyclohexylcarbonyl and especially 1 ,2,3,4-tetrahydronaphthyl-2-carbonyl are most pre ⁇ fer
• Tricycloalkylcarbonyl tricycloalkyl preferably has 8 to 16 carbon atoms and is, for ex ⁇ ample, tricyclo[5.2.1.0 2,6 ]dec-8-yl or especially adamantyl, preferably 1 -adamantyl.
• 1- Adamantylcarbonyl is especially preferred.
• the aryl moiety is preferably defined as above; more pre ⁇ ferably, arylsulfonyl is 2-or 3-napthylsulfonyl which is substituted with amino or mono- or di- lower alkylamino, such as dimethylamino, especially 5-dimethylamino-naphthalenesulfonyl.
• heterocyclylcarbonyl the heterocyclyl moiety is preferably as defined above; more pre ⁇ ferably, heterocyclylcarbonyl is selected from pyridylcarbonyl which is unsubstituted or sub ⁇ stituted with amino, such as pyridin-4-yl- or pyridin-3-ylcarbonyl, or amino-pyridin-3-yl-car- bonyl, such as 2- or 6-amino-pyridin-3-ylcarbonyl, benzimidazolylcarbonyl, such as benz- imidazol-5-ylcarbonyl, quinolinyl-carbonyl, such as quinoline-2-, quinoline-3- or quinoline-6- ylcarbonyl, 2,3-dihydrobenzofuranylcarbonyl, such as 2,3-dihydrobenzofuran-5-ylcarbonyl, and indolylcarbonyl, such as indole-5-yl-, indo
• lower alkanoyl is especially propionyl.
• Preferred is 3- carbamoylpropionyl.
• aryl-lower alkylcarbonyl X with the proviso that in the lower alkyl radical mentioned a methylene group may be replaced with oxa, aza or thia (the latter two being less preferred than oxa), the aryl moiety is preferably as defined above for aryl (most preferably lower alkylaminophenyl, such as 2-, 3- or 4-acetylaminophenyl, or especially hydroxyphenyl, such as 3-hydroxyphenyl, or (in the sequence of growing preference) 2-amino-, 3,5-diamino-, 4- amino- or 3-aminophenyl); or (alternatively or in addition to the group of moieties mentioned just before) amino-lower alkylphenyl, such as aminomethyl-phenyl, or preferably 3,4- dihydroxyphenyl; and the lower alkyl or the lower alkyl wherein a methylene group may be replaced with oxa, aza or thia can be linear or
• cycloalkyl-lower alkylcarbonyl X cycloalkyl is preferably as defined above; more prefer ⁇ ably, cycloalkyl-lower alkylcarbonyl is C 3 -C 7 -, especially C 4 - C 5 - or C 6 -cycloalkyl-d-C -car- bonyl, such as 3-cyclohexylpropanoyl, and is unsubstituted or substituted by amino; most preferably 1 -amino-cyclohexylcarbonyl or 1-amino-cyclopentylcarbonyl; or (alternatively or in addition to the group of moieties mentioned just before) 3-(cyclohexyl)-propionyl.
• aryl-lower alkylsulfonyl X with the proviso that in the lower alkyl radical mentioned a methylene group may be replaced with oxa, aza or thia; aryl and the lower alkyl or lower alkyl wherein a methylene group is replaced with oxa, aza or thia are preferably as described above.
• heterocyclyl-lower alkylcarbonyl X with the proviso that in the lower alkyl radical mentio ⁇ ned a methylene group may be replaced with oxa, aza or thia;
• the heterocyclyl moiety is preferably as defined above, more preferably being selected from unsubstituted or amino- or lower alkyl-substituted thiazolyl, such as 4-methyl-thiazol-5-yl or 2-amino-thiazole-4-yl, indolyl, such as indole-3-yl, and benzimidazolyl, such as benzimidazol-5-yl;
• the lower alkyl or the lower alkyl wherein a methylene group may be replaced with oxa, aza or thia can be linear or branched and is preferably selected from methyl (forming methylene), 1 ,2- ethyl (forming 1 ,2-ethylene), 1 , 1 -eth
• 2-(thiazolyl)-ethoxycarbonyl such as 2-(4-methyl-thiazol-5-yl)-ethoxy- carbonyl
• unsubstituted or amino-substituted thiazolyl-lower alkylcarbonyl such as 2-amino- thiazole-5-ylacetyl
• indolyl-lower alkylcarbonyl such as indole-3-yl-acetyl, 3- (indole-3-yl)propionyl or 4-(indole-3-yl)butyroyl.
• a methylene group may be replaced with oxa, aza or thia, the heterocyclyl moiety and the lower alkyl or the lower alkyl radical wherein a methylene group is replaced with oxa, aza or thia are preferably as defined above, respectively.
• aryl is preferably as defined above, especially being selec ⁇ ted from phenyl which is substituted by 1 to 2 moieties independently selected from lower alkoxy, preferably methoxy, and especially hydroxy, while the lower alkenyl radical prefer ⁇ ably is linear and has one double bond and 2 to 7 carbon atoms, preferably being a 1 ,2- vinyl radical.
• heterocyclyl-lower alkenylcarbonyl X the heterocyclyl moiety is preferably as defined above, especially being selected from imidazolyl, such as 4-imidazolyl, and from indolyl, such as indole-3-yl, while the lower alkenyl radical preferably is linear and has one double bond and 2 to 7 carbon atoms, preferably being a 1 ,2-vinyl radical; more preferably, hete ⁇ rocyclyl-lower alkenylcarbonyl is selected from imidazolyl-lower alkenylcarbonyl, such as imidazole-4-yl-lower alkenylcarbonyl, especially imidazole-4-yl-acryloyl, and indolyl-lower alkenylcarbonyl, such as indole-3-ylacryloyl.
• carbamoyl-lower alkanoyl, cycloalkyl-carbonyl and aryl-lower alkylcarbonyl are especially preferred.
• Lower alkanoyl X which may be present only if Y is a secondary or tertiary amino group, is especially acetyl, but may also be pivaloyi. - 16
• Halo-lower alkanoyl preferably has one to three halogen substituents, preferably selected from chloro and fluoro, and is for example trifluoroacetyl or trichloroacetyl.
• Lower-alkoxycarbonyl X which may be present only if Y is a secondary or tertiary amino group, is especially methoxycarbonyl, ethoxycarbonyl or tert-butoxycarbonyl.
• Aryl-lower alkoxycarbonyl X which may be present only if Y is a secondary or tertiary amino group, has an aryl moiety as defined above, especially phenyl or fluorenyl, and is especially benzyloxycarbonyl or fluoren-9-ylmethoxycarbonyl.
• Cycloalkyl-lower alkoxycarbonyl X which may be present only if Y is a secondary or tertiary amino group, has a cycloalkyl moiety as defined above and is especially cyclohexyl-lower alkoxycyrbonyl, such as cyclohexylmethyl-oxycarbonyl.
• PTI is the bivalent radical of tyrosine (-Tyr-) or preferably a bivalent radical of phosphotyrosine or a phosphotyrosine mimic.
• 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 selec ⁇ ted from phosphonomethyl-phenylalanine, especially 4-phosphonomethyl-phenylalanine, phosphono-( ⁇ -fluoro)methyl-phenylalanine, especially 4-phosphono-( ⁇ -fluoro)methyl-phe- nylalanine, phosphono-( ⁇ , ⁇ -difluoro)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)
• 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, an unsubstituted or substituted aromatic (
• hetero atoms selected from oxygen, nitrogen, sulfur, and sulfur linked to 1 or 2 oxygen atoms, and may be unsaturated or partially or fully saturated, for example furyl, pyrrolyl, pyrrolidinyl, morpholinyl, pyridyl or indolyl, a cyclic ⁇ -amino-( ⁇ , ⁇ - lower alkylene)-carbonic acid; or an ⁇ -amino-[(C 6 -C 8 )-bicyclo]-carbonic acid; each being present in the L-, D- or (D,L)-configuration and in unprotected or amino-, carboxy- or sulfhydryl-protected form.
• 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), iso ⁇ leucine (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)-
• an amino acid
• the bivalent radical -(AA) n - 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.
• cylce gene 1 protein chain following Tyr 139 that is, -Asp-Glu- Asp-Asp-Tyr-
• Tyr 1092 that is, -lle-Asn-Gln-Ser-Val-
• Tyr 1138 that is, -Leu-Asn-Thr-Val- Gln-
• an analogue of the human SHC chain following Tyr 317 that is, -Val-Asn-Val-Gln- Asn
• an analogue of the human HGF receptor chain following Tyr 1374 that is, -Val-Asn- Val-Leu-Cys-
• an analogue of the human ErbB2 chain following Tyr 1139 that is, -Val-Asn- Gln-Pro-Asp-
• an analogue of the human ErbB3 that is, -Val-Asn- Gln-Pro-Asp-
• 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 concemed) may be deleted.
• -(AA) n - has one of the following meanings:
• a C-terminal protecting group Y is preferably an esterifying group, thus leading to an esterified C-terminal carboxy group. More preferred is a lower alkoxy group that is preferably branched in the 1 -position of the lower alkoxy group or substituted in the 1 - or 2- position of the lower alkoxy group by (one) suitable substituent(s).
• a lower alkoxy group that is branched in the 1 -position of the lower alkoxy group is, for example, tert-lower alkoxy, for example tert-butoxy.
• a lower alkoxy group that is substituted in the 1 - or 2-position of the lower 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 lower alkyl, for example tert-lower alkyl, such as tert-butyl, lower 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-iower alkoxy, for example meth
• 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 trimethylsilyloxy.
• the silicon atom of the silyloxy group can also be substituted by two lower alkyl groups, for example methyl groups.
• a C-terminal protecting group Y is preferably tert-lower alkoxy, for example tert-butyloxy, benzyloxy, 4-nitrobenzyloxy, 9-fluoreny I methoxy 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-1-yloxy)-ethyl; aryl-lower alkyl, such as phenyl-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.
• a primary, secondary or tertiary amino group Y is a free amino group, a mono- or disubstituted amino group the substituents of which are pre ⁇ ferably selected from the group comprising lower alkyl, e.g. methyl or ethyl, aryl-lower alkyl, such as phenyl-lower alkyl, e.g. benzyl, or 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 hetero ⁇ cyclyl 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 pre ⁇ ferably amino (-NH 2 ) or monosubstituted amino selected from lower alkylamino, such as methylamino, ethylamino; isobutylamino or 3-methylbutylamino; octylamino, such as 2-ethyl- hexyl-amino; aryloxy-lower alkylamino, especially halonaphthyloxy-lower alkylamino, such as 2-(1-bromo-naphthalen-2-yloxy)-ethylamino, or naphthyloxy-lower alkylamino, such as 2- (naphthalen-2-yloxy or naphthalen-1 -yloxy)-ethylamino; aryl-lower alkylamino, such as phenyl-lower alkylamino, e.g
• 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, Ib, 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, hydroxymaleic 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 here
• the compounds of the invention have useful, in particular pharmacologically useful, proper ⁇ ties.
• 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 recep ⁇ tor ("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 Binding
• the EGFR-MBP fusion protein is made as follows: Oligonucleotides flanking the en ⁇ tire carboxy-terminal half (nucleotides 3112 to 3816) of the EGFR and containing enginee ⁇ red EcoRI-Hindlll restriction sites are used to amplify the appropriate DNA fragment by PCR. The amplified DNA fragment is recombined with purified EcoRI-Hindl I I-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 P tac 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. Ali ⁇ quots are frozen in liquid nitrogen and stored at -70 °C).
• Wells of polystyrene microtiter plates are coated ovemight at 4 °C in incubation buffer (20 mM Tris pH 7.5) with phosphorylated EGFR or "tail" 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 BamHI (5').3' EcoRI linkers; the purified (e.g. BamHI -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. For example, the interactions of the SH2-comprising proteins and phosphotyrosine comprising proteins mentioned above in the definition of a bivalent radical -(AA) n - in formula I as an analogue of an SH2 domain binding site of a protein with phosphotyrosine of a mammal can be tested.
• the SH2-containing Grb2 are able to inhibit subsequent cellular signal trans ⁇ duction pathways important for diseases such as viral, inflammatory, allergic, autoimmune, 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.
• diseases such as viral, inflammatory, allergic, autoimmune, 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.
• 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 can be treated by microinjection of a compound of formula I 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-chloride-activated agarose, vinylsuifone-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.
• One preferred embodiment of the invention relates to a compound of formula I, wherein n is 1 to 15,
• X is arylcarbonyl, cycloalkylcarbonyl, arylsulfonyl, heterocyclylcarbonyl, heterocyclylsulfonyl; aryl-lower alkylcarbonyl, cycloalkyl-lower alkylcarbonyl, aryl-lower alkylsulfonyl, heterocyclyl- lower alkylcarbonyl, heterocyclyl-lower alkylsulfonyl with the proviso that in any of the lower alkyl radicals mentioned a methylene group may be replaced with oxa, aza or thia; or heterocyclyl-lower alkenylcarbonyl,
• 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
• n is 1 to 4, more preferably 1 to 3, most preferably 2 or especially 3;
• X is selected from
• benzoyi or, even more preferably, from benzoyi substituted with amino; lower alkylamino; amino-lower alkyl; hydroxy; lower alkoxy; amino and hydroxy; amino and lower alkoxy; carboxy; lower-alkoxycarbonyl; cyano; halogen, especially chloro; lower-alkylthio; or lower alkylsulfinyl; especially from 4-aminobenzoyl, 3-aminobenzoyl, 2-aminobenzoyl, 4- lower alkylamino-benzoyl, such as 4-methylamino-benzoyl, 4-(amino-lower alkyl)-benzoyl, such as 4-(methylamino)-benzoyl, 4-hydroxy-benzoyl, 4-lower alkoxy-, such as 4- methoxybenzoyl, 4-amino-2-hydroxy-benzoyl, 4-amino-3-lower alkoxy-benzoyl, such as 4- amino-3-me
• naphthoyl or hydroxy-naphthoyl such as naphthalene-2-yl-carbonyl or especially 6- hydroxy-naphthalene-2-yl-carbonyl, or (less preferably) fluorenylcarbonyl, such as fluoren-9- ylcarbonyl;
• chromenylcarbonyl such as 2H-chromen-3-ylcarbonyl, that is unsubstituted by 1 to 3 substitutents selected from oxo and hydroxy, especially 7,8-dihydroxy-2-oxo-2H- (benzopyran)-3-yl-carbonyl;
• phenyl-methoxycarbonyl wherein the phenyl residue is unsubstituted or substituted with amino in the (in growing preference) 2-, 3- and 5-, 4- or 3-position of the phenyl ring; ( ⁇ )-, (+) or (-)-1-(3-aminophenyl)ethyloxycarbonyl; hydroxybenzyloxy- or hydroxyphenyl-2- ethoxycarbonyl, such as 3-hydroxybenzyloxycarbonyl or 3-hydroxyphenyl-2-ethoxy- carbonyl; (with less preference) lower alkanoylamino-phenyloxymethylcarbonyl, such as 2-, 3- or especially 4-acetylaminophenyloxymethylcarbonyl, aminophenyloxymethylcarbonyl, such as 3- or 4-aminophenyloxycarbonyl; aminophenyl-lower alkylcarbonyl, such as 4- aminophenyl-acetyl or 3-(3-aminophenyi)-propion
• dihydroxyphenyl-lower alkylcarbonyl especially 3-(3,4-dihydroxyphenyl)-propionyl or 2-(3,4-dihydroxyphenyl)-acetyl;
• imidazolyl-lower alkenylcarbonyl such as imidazole-4-yl-lower alkenylcarbonyl, especially imidazole-4-yl-acryloyl, or indolyl-lower alkenylcarbonyl, such as indole-3-yl- acryloyl;
• cinnamoyl substituted by 1 to 2 moieties selected independently from methoxy and especially hydroxy, especially p-hydroxycinnamoyl, m-hydroxy-p-methoxy-cinnamoyl or preferably m,p-dihydroxy-cinnamoyl; and
• PTI is a bivalent radical of tyrosine or (preferably) 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-phenylalanine, especially 4-phosphono-( ⁇ - fluoro)methyl-phenylalanine, phosphono-( ⁇ , ⁇ -difluoro)methyl-phenylalanine, especially 4-
• Y is amino (-NH 2 ) or monosubstituted amino selected from lower alkylamino, such as methylamino, ethylamino; isobutylamino or 3-methylbutylamino; octylamino, such as 2-ethyl- hexyl-amino; aryloxy-lower alkylamino, especially halonaphthyloxy-lower alkylamino, such as 2-(1-bromo-naphthalen-2-yloxy)-ethylamino, or naphthyloxy-lower alkylamino, such as 2- (naphthalen-2-yloxy or naphthalen-1-yloxy)-ethylamino; aryl-lower alkylamino, such as phenyl-lower alkylamino, e.g.
• n is 1 to 4, more preferably 1 to 3, most preferably 2 or especially 3;
• X is selected from
• benzoyi or, even more preferably, from benzoyi substituted with amino; lower alkylamino; amino-lower alkyl; hydroxy; lower alkoxy; amino and hydroxy; amino and lower alkoxy; carboxy; lower-alkoxycarbonyl; cyanc; halogen, especially chloro; lower-alkylthio; or lower alkylsulfinyl; especially from 4-aminobenzoyl, 3-ami no benzoyi, 2-aminobenzoyl, 4- lower alkylamino-benzoyl, such as 4-methylamino-benzoyl, 4-(amino-lower alkyl)-benzoyl, such as 4-(methylamino)-benzoyl, 4-hydroxy-benzoyl, 4-lower alkoxy-, such as 4- methoxybenzoyl, 4-amino-2-hydroxy-benzoyl, 4-amino-3-lower alkoxy-benzoyl, such as 4- amino-3-meth
• 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, benzimidazolylcarbonyl, such as benzimidazol-5-ylcarbonyl, quinolinyl-carbonyl, such as quinoline-2-, quinoline-3- or quinoline-6-ylcarbonyl, 2,3-dihydrobenzofuranyl- carbonyl, such as 2,3-dihydrobenzofuran-5-ylcarbonyl, or indolylcarbonyl, such as indole-5- yl-, indole-3-yl- or indole-2-yl-carbonyl; most preferably from quinoline-6-ylcarbonyl and especially from indolyl; most
• phenyl-methoxycarbonyl wherein the phenyl residue is unsubstituted or substituted with amino in the (in growing preference) 2-, 3- and 5-, 4- or 3-position of the phenyl ring; ( ⁇ )-, (+) or (-)-1 -(3-aminophenyl)ethyloxycarbonyl; hydroxybenzyloxy- or hydroxyphenyl-2- ethoxycarbonyl, such as 3-hydroxybenzyloxycarbonyl or 3-hydroxyphenyl-2-ethoxy- carbonyl; (with less preference) lower alkanoylamino-phenyioxymethylcarbonyl, such as 2-, 3- or especially 4-acetylaminophenyloxymethylcarbonyl, aminophenyloxymethylcarbonyl, such as 3- or 4-aminophenyioxycarbonyl; aminophenyl-lower alkylcarbonyl, such as 4- aminophenyl-acetyl or 3-(3-aminophenyl)
• imidazolyl-lower alkenylcarbonyl such as imidazole-4-yl-lower alkenylcarbonyl, especially imidazole-4-yl-acryloyl, or indolyl-lower alkenylcarbonyl, such as indole-3-yl- acryloyl;
• 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- phenylalanine, especially 4-phosphono-( ⁇ -fiuoro)methyl-phenylalanine, phosphono-( ⁇ , ⁇ - difluoro)methyl-phenylalanine, especially 4-phosphono-( ⁇ , ⁇ -difluoro)methyl-phenylalanine, phosphono-( ⁇ -hydroxy)methyl-phenylalanine, especially 4-phosphono-(
• Y is a free amino group (preferred), a mono- or disubstituted amino group the substituents of which are preferably selected from the group comprising lower 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.
• n is 1 to 4, more preferably 1 to 3, most preferably 2 or especially 3;
• X is selected from
• naphthoyl or hydroxy-naphthoyl such as naphthalene-2-yl-carbonyl or especially 6- hydroxy-naphthalene-2-yl-carbonyl, or (less preferably) fluorenylcarbonyl, such as fluoren-9- ylcarbonyl;
• chromenylcarbonyl such as 2H-chromen-3-ylcarbonyl, that is unsubstituted by 1 to 3 substitutents selected from oxo and hydroxy, especially 7,8-dihydroxy-2-oxo-2H-
• PTI is a bivalent radical of tyrosine or (preferably) 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-phenylalanine, especially 4-phosphono-( ⁇ - fluoro)methyl-phenylalanine, phosphono-( ⁇ , ⁇ -difluoro)methyi-phenylalanine, especially 4- phosphono-( ⁇ , ⁇ -difluoro)methyl-phenylalanine, phosphono-( ⁇ -
• Y is amino (-NH 2 ) or monosubstituted amino selected from lower alkylamino, such as methylamino, ethylamino; isobutylamino or 3-methylbutylamino; octylamino, such as 2-ethyl- hexyl-amino; aryloxy-lower alkylamino, especially halonaphthyloxy-lower alkylamino, such as 2-(1-bromo-naphthalen-2-yloxy)-ethylamino, or naphthyloxy-lower alkylamino, such as 2- (naphthalen-2-yloxy or naphthalen-1 -yloxy)-ethylamino; aryl-lower alkylamino, such as phenyl-lower alkylamino, e.g.
• n is 1 , 2 or 3, especially 2 or 3;
• X is selected from 4-aminobenzoyl, 3-aminobenzoyl, 4-amino-2-hydroxy-benzoyl, 4-lower alkoxycarbonyl-benzoyl, such as 4-methoxycarbonyl-benzoyl, quinolinyl-carbonyl, such as quinoline-2-, quinoline-3- or quinoiine-6-ylcarbonyl, indolylcarbonyl, such as indole-5-yi-, indole-3-yl- or indole-2-yi-carbonyl; phenyl-methoxycarbonyl wherein the phenyl residue is unsubstituted or substituted with amino in the (in growing preference) 2-, 3- and 5-, 4- or 3- position of the phenyl ring; ( ⁇ )-, (+) or (-)-1-(3-aminophenyl)ethyloxycarbonyl; hydroxy-
• 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-( ⁇ , ⁇ - difluoro)methyl-phenylalanine or the phosphonomethyl-phenylalanine type, especially 4- phosphono-( ⁇ , ⁇ -difiuoro)methyl-phenylalanine
• Y is amino (-NH 2 ) or monosubstituted amino selected from lower alkylamino, such as methylamino, ethylamino; isobutylamino or 3-methylbutylamino; octylamino, such as 2-ethyl- hexyl-amino; aryloxy-lower alkylamino, especially halonaphthyloxy-lower alkylamino, such as 2-(1-bromo-naphthalen-2-yloxy)-ethylamino, or naphthyloxy-lower alkylamino, such as 2- (naphthalen-2-yloxy or naphthalen-1 -yloxy)-ethylamino; aryl-lower alkylamino, such as phenyl-lower alkylamino, e.g.
• n is 2 or 3;
• X is selected from 4-aminobenzoyl, 3-aminobenzoyl, 4-amino-2-hydroxy-benzoyl, 4-lower alkoxycarbonyl-benzoyl, such as 4-methoxycarbonyl-benzoyl, quinolinyl-carbonyl, such as quinoli ⁇ e-2-, quinoline-3- or quinoline-6-ylcarbonyl, indolylcarbonyl, such as indole-5-yl-, indole-3-yl- or indole-2-yl-carbonyl; phenyl-methoxycarbonyl wherein the phenyl residue is unsubstituted or substituted with amino in the (in growing preference) 2-, 3- and 5-, 4- or 3- position of the phenyl ring; ( ⁇ )-, (+) or (-)-1-(3-aminophenyl)ethyloxycarbonyl; hydroxy- benzyloxy-
• 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-( ⁇ , ⁇ - difluoro)methyl-phenylalanine type, especially 4-phosphono-( ⁇ , ⁇ -difluoro)methyl- phenylalanine
• Y is a free amino group (-NH 2 )
• n is 1 , 2 or 3, especially 2 or 3;
• X is selected from naphthoyl or hydroxy-naphthoyi, such as naphthalene-2-yl-carbonyl or especially 6-hydroxy-naphthalene-2-yl-carbonyl, cyclohexylcarbonyl, 1 ,2,3,4- tetrahydronaphthyl-2-carbonyl (preferred), adamantoyl, preferably 1-adamantoyl, 7,8- dihydroxy-2-oxo-2H-(benzopyran)-3-yl-carbonyl, carbamoyl-lower alkanoyl, especially 3- carbamoylpropionyl, dihydroxyphenyl-lower alkylcarbonyl, especially 3-(3,4- dihydroxyphenyl)-propionyl or 2-(3,4-dihydroxyphenyl)-acetyl, and cinnamoyi substituted by 1 to 2 moieties selected independently from methoxy and especially hydroxy, especially p-
• 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-( ⁇ , ⁇ - difluoro)methyl-phenylalanine or the phosphonomethyl-phenylalanine type, especially 4- phosphono-( ⁇ , ⁇ -difluoro)methyl-phenylalanine
• Y is monosubstituted amino selected from lower alkylamino, such as methylamino, ethylamino; isobutylamino or 3-methylbutylamino; octylamino, such as 2-ethyl-hexyl-amino; aryloxy-lower alkylamino, especially halonaphthyloxy-lower alkylamino, such as 2-(1 -bromo- naphthalen-2-yloxy)-ethylamino, or naphthyloxy-lower alkylamino, such as 2-(naphthalen-2- yloxy or naphthalen-1-yloxy)-ethylamino; aryl-lower alkylamino, such as phenyl-lower alkylamino, e.g.
• a compound mentioned in the examples or a (preferably pharmaceutically acceptable) salt thereof.
• a compound of formula I being selected from the following compounds: trans-3,4-dihydroxy-cinnamoyl-Tyr(P ⁇ 3H2)-He-NH2
• indole-5-ylcarbonyI-Tyr-lle-Asn-Gln-NH2 SEQ ID NO: 112
• a pharmaceutically acceptable salt thereof is also indole-5-ylcarbonyI-Tyr-lle-Asn-Gln-NH2 (SEQ ID NO: 112), or a 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 Veriag, 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-chiorophosphite, 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 chiorocarbonyl.
• halocarbonyl such as chiorocarbonyl.
• 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-methylpyrrolidin-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 MBHA for 4-methylbenzhydrylamine - 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
• X has the meanings given under formula I) to the deprotected NH 2 (e.g. ⁇ - or ⁇ -NH 2 ).
• 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. dicydohexylcarbodiimide (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-methylisoxazolium 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-ylm
• DCC
• an "active ester” e.g. an amino- or amido ester, such as a 1 -hydroxy ⁇ benzotriazole (HOBT) or N-hydroxysuccinimide ester, or an aryl ester, such as a penta- fluorophenyl, 4-nitrophenyl or 2,4,5-tetrachlorophenyl 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 1 -hydroxy ⁇ benzotriazole (HOBT) or N-hydroxysuccinimide ester, or an aryl ester, such as a penta- fluorophenyl, 4-nitrophenyl or 2,4,5-tetrachlorophenyl ester (obtainable by treatment of the respective acid with a phenyl with the appropriate substituents, such as 4-nitrophenol
• 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-o ⁇ e, 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-o ⁇ e, 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 Veriag, 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 lower 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, diphenylmethoxycarbonyl, 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,
• 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 lower alkoxy, such as methoxy, and/or lower alkyl, such as methyl, substituents in one or more phenyl rings.
• arylmethyl groups preferably triphenylmethyl (trityl) or analogues thereof with one or more lower alkoxy, such as methoxy, and/or lower 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-methylbenzyl.
• 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 dimethyl sulfide 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/dimethyisulfide/trifluoroacetic acid/ethanedithiol/m-cresol.
• Cleavage of phosphonate diester protecting groups is possible in appropriate solvents, such as acetonitrile, in the presence of tri-lower alkylsilylhalogenides, such as trimethylsilyl- iodide, and subsequent hydrolysis of the resulting tri-lower alkylsilyl-ester intermediate in the presence of an acid, especially a lower alkanoic acid, such as acetic acid, in aqueous solution.
• solvents such as acetonitrile
• Cleavage of lower-alkenoic esters of carboxy groups is preferably effected in a solution of a lower alkanoic acid, such as acetic acid, and a sterically hindered base, such as N-methylmorpholine, in an appropriate solvent, such as a halogenated hydrocarbon, especially chloroform, in the presence of tetrakis- (triphenylphosphin)palladium, preferably under inert gas, such as argon.
• 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- fluorenylmethyloxycarbonyl (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 hydrofluric acid (HF) or trifluoromethanesulfonic acid.
• HF hydrofluric acid
• a small number of side chain groups e.g. 2,4-dinitrophenyl protected imino in the histidyl side chain
• may require a separate deprotection step e.g. treatment with thiophenol or ammonolysis.
• 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.
• mercapto groups e.g. lower alkyl-mercapto groups, such as methylthio
• sulfinyl groups e.g. lower 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 lower alkyl sulfinyl compounds.
• esterified carboxy groups such as lower 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.
• 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 alkali 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 alkali 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 per se into the individual isomers; diastereoisomers and/or cis/trans-isomers can be separated, for example, by partitioning between poiyphasic solvent mixtures, recrystal ⁇ lisation and/or chromatographic separation, for example over silica gel or by e.g. medium pressure liquid chromatography 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 per 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 oligopeptoids 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-phenylalanine, especially 4-phos- phono-( ⁇ -fluoro)methyl-phenylalanine, 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 per 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 dissolve them, 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 lower 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 lower alkanoic acid anhydrides, for example acetic anhydride, cyclic, linear or branched hydrocarbons, such as cydohexane, hexane or
• 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(PO 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.
• 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 perse, 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 lyophiiising 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, pentadecylic 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 , Huls 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 ethylcellulose 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
• Boc tert-Butoxycarbonyl
• BOP benzotriazole- 1-yl- oxy-tris-(dimethylamino)-phosphoniumhexa-fluorophosphate
• DHph bivalent radical of D- homophenylalanine (Bachem, Bubendorf, Switzerland)
• Fmoc fluorenylmethoxy-carbonyl
• Fmoc-PAL-PEG-PS (Millipore, Bedford, USA): a resin used for peptide synthesis
• F2Pmp 4-phosphono(difluoromethyl)-L-phenyl-alanine
• HATU N-[(dimethylamino)-1 H-1 ,2,3- triazolo[4,5-b]pyridin-1-ylmethylene]-N-methylmethanaminiumhexafluorophosphat
• Example 1 3-Aminobenzyloxycarbonyl-Tyr(P ⁇ 3H2)-He-Asn-Gln-NH2 (TFA salt) (SEQ ID NO: 1)
• the peptide is synthesized manually on a 4-(2',4'-dimethoxyphenyl-aminomethyl)-phenoxy- resin (Fmoc-protected at the amino group, obtainable from Novabiochem, Laufelfingen, Switzerland; 0.47 mmol/g, 300 mg), employing the fluorenylmethoxycarbonyl (Fmoc) strategy (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 is removed 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 (TPTU, Senn Chemicals, Dielsdorf, Switzerland; 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.
• TPTU Senn Chemicals, Dielsdorf, Switzerland
• N ⁇ -Fmoc- Tyr(PO3H2)-OH (see Biochemistry 32, 4354 (1993)) (3 equiv.) is accomplished with benzotriazole-1-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-terf-butoxy-carbonyl-aminobenzyl-4- nitrophenyi-carbonate (3 equiv.) is coupled to the ⁇ /-terminal residue of the peptide resin in the presence of an equimolar amount of diisopropylethyl-amine in N-methylpyrrolidin-2-one during 17 h at room temperature.
• the complete peptide resin obtained after the final coupling reaction is simultaneously 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 is collected by filtration.
• the crude peptide is purified by medium-pressure liquid chromatography using a C-
• Mass spectral analysis (matrix-assisted laser-desoption ionization time-of-flight mass spectrometry, MALDI-TOF) reveales a molecular mass within 0.1 % of the expected values (negative-ion mode): 763.7 (calc.
• the starting materials are prepared as follows:
• the ether mixture is extracted with brine (1x50 ml) and then with water (7x 50 ml), dried over anhydrous Na2SO4 and evaporated in vacuo.
• the starting material is obtained as follows:
• the starting material is obtained as follows:
• the starting material is obtained as follows:
• Example 7 3-Aminobenzyloxycarbonyl-Tyr(P ⁇ 3H2)-Ac6C-Asn-Gln-NH2 (TFA salt) (SEQ ID NO: 7)
• the starting material is prepared as follows:
• the starting material is obtained as follows:
• the starting material is prepared as follows:
• Example 1 3-Aminobenzyloxycarbonyl-Tyr(P ⁇ 3H2)-Ac ⁇ C- ⁇ Ala-NH2 (TFA salt) (SEQ ID NO: 11)
• Example 12 3-Aminobenzyloxycarbonyl-Tyr(PO3H2)-Ac6C-Gly-NH2 (TFA salt) (SEQ ID NO: 12)
• aj ⁇ r j le_13 Benzyloxycarbonyl-Tyr(P ⁇ 3H2)-He-Asn-Gln-NH2
• Example 14 3,5-Diaminobenzyloxycarbonyl-Tyr(P ⁇ 3H2)-He-Asn-Gln-NH2 (TFA salt) (SEQ ID NO: 14)
• N-terminal group is coupled as 3,5-di-tert-butoxycarbonyl-diaminobenzyl-4-nitrophenyl- dicarbonate.
• the starting material is prepared as follows:
• Example 16 3-.N-Acetylamino)phenoxvacetyl-Tyr(PO3H2.-He-Asn-Gln-NH2
• the starting material is prepared as follows:
• Example 17 4-(N-Acetylamino)phenoxyacetyl-Tyr(PO3H2)-He-Asn-Gln-NH2 (TFA salt) (SEQ ID NO: 17)
• the starting material is obtained as follows:
• the starting material is obtained as follows:
• the starting material is obtained as follows: a) 4-(N-tert-Butoxycarbonylamino)phenoxy-acetic acid
• Example 20 3-Aminophenoxyacetvl-Tvr(P ⁇ 3H2--He-Asn-Gln-NH2 (TFA salt)
• the title compound is prepared using a procedure analogous to Example 19, starting from 3-(N-tert-butoxycarbonylamino)phenoxy-acetic acid.
• Mass spectral analysis (negative-ion mode): 764.0 (calc. 763.7, C32H44N8O12P1), -R 4.92 min (HPLC System A).
• the starting material is obtained as follows:
• the starting material is obtained as follows:
• Example 26 2-Amino-thiazol-4-yl-acetyl-Tyr(P ⁇ 3H )-lle-Asn-Gln-NH2 (TFA salt) (SEQ ID NO: 26)
• caj ⁇ iBle_3P 4-Methoxybenzoyl-Tyr(P ⁇ 3H2)-He-Asn-Gln-NH2
• the compound is prepared using a protocol analogous to Example 25, using 4-methoxy- benzoic acid (Fluka, Buchs, Switzerland).
• the compound is obtained by oxidation of the title compound of Example 31 with 5 % hydrogen peroxide at room temperature; the course of the reaction is monitored by HPLC (System A). After 45 min, the starting material has been converted to the sulfoxide deri ⁇ vative. The solution is then concentrated to dryness and lyophilized from 80 % acetic acid.
• Example 33 trans-(4-lmidazolyl-acryloyl)-Tyr(P0 3 H2)-He-Asn-Gln-NH2 (TFA salt) (SEQ ID NO: 33)
• Exarnr j le_36 Pyridin-4-carbonyl-Tyr(P ⁇ 3H2)-He-Asn-Gln-NH2 (SEQ ID NO: 36)
• the compound is prepared using a protocol analogous to Example 25, using pyridin-4- carbonic acid (Fluka, Buchs, Switzerland).
• Title compound Mass spectral analysis (nega ⁇ tive-ion mode): 719.9 (calc. 719.7, C3oH 4 oN 8 0-
• 1 P- j ), .R 4.21 min (HPLC System A).
• Example 37 4-Aminomethylbenzoyl-Tyr(P ⁇ 3H2)-He-Asn-Gln-NH2 (TFA salt) (SEQ ID NO: 37)
• the compound is prepared using a protocol analogous to Example 25, using 4-aminome- thyl-benzoic acid (Fluka, Buchs, Switzerland).
• Title compound Mass spectral analysis (ne ⁇ gative-ion mode): 747.9 (calc. 747.7, C32H44N8O11 P-
• ), tR 4.39 min (HPLC System A).
• Example 39 4-Aminophenylacetyl-Tyr(P ⁇ 3H2)-He-Asn-Gln-NH2 (TFA salt) (SEQ ID NO: 39)
• the starting material is prepared as follows:
• the compound is obtained by saponification of the title compound of Example 42 with sodium hydroxide as follows: 4-Methoxycarbonylbenzoyl-Tyr(P ⁇ 3H2)-He-Asn-Gln-NH2 (SEQ ID NO: 42) (27 mg, 35 ⁇ mol) is dissolved in 2.7 ml of water and 34.7 ⁇ l of a 1 N solu ⁇ tion of NaOH is added. The course of the reaction is followed by anayltical HPLC. After 2 h, a further 312 ⁇ l of 1 N NaOH are added.
• Example 44 4-Amino-2-chlorobenzoyl-Tyr(P ⁇ 3H2)-He-Asn-Gln-NH2 (TFA salt) (SEQ ID NO: 44)
• Example 45 6-Amino-nicotinoyl-Tyr(P ⁇ 3H2)-He-Asn-Gln-NH2 (TFA salt) (SEQ ID NO: 45)
• the compound is prepared using a protocol analogous to Example 16, using 6-amino-nicoti- nic acid (Fluka, Buchs, Switzerland).
• Title compound Mass spectral analysis (negative-ion mode): 734.7 (calc. 734.7, C30H41 NgO- j 1 P-
• Example 47 3-(3-Aminophenyl)propionoyl-Tyr(P ⁇ 3H2)-He-Asn-Gin-NH2 (TFA salt) (SEQ ID NO: 47)
• the starting material is prepared as follows: a) 4-(tert-Butoxycarbonylamino)benzoic acid
• Example 49 trans-(3-lndolvl-acrvlovn-Tvr(PO3H2l-lle-Asn-Gln-NH (SEQ ID NO: 49) trans-3-lndolyl-acrylic acid (Fluka, Buchs, Switzerland) is coupled with BOP/HOBt (1 :1 , 3 equiv.; first coupling) and N-[(dimethylamino)-1H-1 ,2,3-triazolo[4,5-b]pyridin-1-ylmethylene]- N-methylmethanaminium hexafluorophosphate N-oxide (3 equiv.; second coupling) in the presence of diisopropylethylamine (6 equiv.).
• Title compound Mass spectral analysis (negative-ion mode): 783.6 (calc. 783.8, C35H44N8O11 P-
• ), tR 4.32 min (HPLC System A).
• Example 52 4-Aminobenzoyl-Tyr(P0 3 H 2 )-Gly-Asn-Gln-NH2 (TFA salt) (SEQ ID NO: 52)
• Example 60 4-Aminobenzoyl-Tyr(P03H 2 )-lle-Gln-Gln-NH2 (TFA salt)
• Example 61 lndole-5-vlcarbonyl-Tyr(P03H ⁇ -lle-Asn-Gln-NH2 (SEQ ID NO: 61)
• the peptide is synthesized manually on a 4-(2',4'-dimethoxyphenyl-aminomethyl)-phenoxy resin, employing a procedure analogous to Example 1.
• the N-terminal group is incorporated as N-terf-butoxycarbonyl-indole-5-ylcarboxylic acid (3 equiv.) using benzotriazole- 1yl-N-oxy- tris-(dimethylamino)-phosphonium hexafluorophosphate/N-hydroxybenzotriazole (1:1 ; 3 equiv.) in the presence of diisopropylethylamine (7 equiv.) in N-methylpyrrolidin-2-one.
• the complete peptide resin obtained after the final coupling is simultaneosly deprotected- cleaved by treatment with trifluoro-acetic acid/ethanedithiol/H2 ⁇ (76:20:4, v/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.
• Decarboxylation of the resulting N-carboxy of the N-terminal indole ring is carried out in 0.1 M acetic acid (1 h at room temperature; see White, P, in Smith, J.A., Rivier, J.E.
• the starting material is prepared as follows:
• Example 64 3-Aminobenzyloxycarbonyl- -F2Pmp-lle-Asn-Gln-NH2 (TFA salt) (SEQ ID NO: 64)
• the peptide is synthesized manually on a 4-(2',4'-dimethoxyphenyl-aminomethyl)-phenoxy resin, employing a procedure analogous to Example 1. Incorporation of N ⁇ -Fmoc-[4-(0- diethyl)-phosphono(difluoromethyl)]-L-phenylalanine (for synthesis see Tetrahedron Lett.
• the peptide resin is treated with 1 M trimethylsilyltrifluoro- methane sulfonate-2 M dimethylsulfide-trifluoroacetic acid (500 ml to 0.005 mmol of NH2)- ethanedithiol (100 ml)-m-cresol (25 ml) 30 min at 4 °C and 3.5 h at room temperature (see Tetrahedron Lett. 34(44), 7093 (1993).
• 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 as described in Example 1.
• the crude title compound in Boc-protected form is obtained by reaction of H- .-F2Pmp-lle-Asn-Gln-NH2 (1 equiv.) with 3-tert-butoxycarbonyl-aminobenzyl- 4-nitrophenyl-carbonate (see Example 1 b)) (3 equiv.) in the presence of diisopropylethylamine (3 equiv.).
• the crude product is purified by medium-pressure liquid chromatography as described in Example 1.
• the peptide is synthesized manually on a 4-(2',4'-dimethoxyphenyl-aminomethyl)-phenoxy resin, employing a procedure analogous to Example 1. Incorporation of N ⁇ -Fmoc-[4-(O-di- ethyl)-phosphono(difluoromethyl)]-L-phenylalanine (reference: see Example 64) to the pro ⁇ tected peptide resin is carried out as in Example 64.
• the peptide resin is treated with 1 M trimethylsilyltrifiuoro-methane sulfonate-2 M dimethylsulfide-trifluoroacetic acid (500 ml to 0.005 mmol of NH2)-ethanedithiol (100 m ⁇ )-m- cresol (25 ml) 30 min at 4 °C and 3.5 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 preci ⁇ pitate collected by filtration.
• Example 66 4-Aminobenzoyl- Tyr(P0 3 H 2 )-Phe-Asn-Gln-NH2 (TFA Salt) (SEQ ID NO: 66)
• 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-tetramethyluronium tetrafluoroborate 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. Asparagine and glutamine side chains are protected with the trityl group.
• N ⁇ -Fmoc-Tyr(P0 3 H2)-OH 3 equiv.
• benzo- tri izole-1-yl-oxy-tris-dimethylamino)-phosphoniumhexafluorophosphate/N-hydroxybenzo- triazole (1 :1 ; 3 equiv.; first coupling) 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.).
• trans-3,4-Dihydroxy-cinnamic acid (2 equiv.; Fluka, Buchs, Switzerland) is incorporated (double coupling) with benzotri- azole-1-yl-oxy-tris-dimethylamino)-phosphoniumhexafluorophosphate/N-hydroxybenzo- triazole (1 :1 ; 3 equiv.) in the presence of diisopropylethylamine (4 equiv.).
• the complete peptide resin obtained after the last coupling step is simultaneosly deprotected and cleaved by treatment with trifluoroacetic acid/H 2 0 (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-pres ⁇ sure liquid chromatography using a C ⁇ 8 -column (Merck LICHROPREP RP-18, 15-25 ⁇ m bead diameter, reversed phase material based on Cis'derivatized silicagel, Merck, Darm ⁇ stadt, FRG; column length 46 cm, diameter 3.6 cm; flow rate 53.3 ml/min; detection at 215 nm) eluted with an acetonitrile-water gradient containing 0.1 % of TFA.
• trans-3-Hydroxv-cinnamoyl-Tvr(PO.'-;H?)-He-Asn-Gln-NH9 SEQ ID NO: 68
• trans-3-Hydroxy-cinnamic acid is from Fluka (Buchs, Switzerland) and is used without side- chain protection.
• tR 6.04 (HPLC system A).
• trans-3-Hydroxy-4-methoxv-cinnamoyl-Tvr(P03Hp)-lle-Asn-Gln-NHp SEQ ID NO: 69
• trans-3-Hydroxy-4-methoxy-cinnamic acid is from Janssen (Olen, Belgium) and is used without side-chain protection.
• Title compound Mass-spectral analysis (negative-ion mode): 790.1 (calc. 790.7, C34H45N7O13P1).
• tR 5.77 (HPLC system A).
• Example 70 trans-3.4-Dihvdroxy-cinnamovl-Tvr(POqHp)-lle-NHp (SEQ ID NO: 70) trans-3,4-Dihydroxy-cinnamic acid (3 equiv.) is incorporated with benzotriazole-1-yl-oxy-tris- dimethylamino)-phosphoniumhexafluorophosphate/N-hydroxybenzotriazole (1:1 ; 3 equiv.; first coupling) 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.).
• Title compound Mass-spectral analysis (negative-ion mode): 534.4 (calc. 534.5, C24H29N3O
• Example 71 trans-3.4-Dihvdroxv-cinnamoyl-Tvr(P ⁇ 3H.>)-AcfiC-NH;> (SEQ ID NO: 71) trans-3,4-Dihydroxy-cinnamic acid (3 equiv.) is incorporated with benzotriazole-1 -yl-oxy-tris- dimethylamino)-phosphoniumhexafluorophosphate/N-hydroxybenzotriazole (1 :1 ; 3 equiv.; first coupling) 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.).
• the starting material is prepared as follows: a) Fmoc-1 -amino-cvclohexanecarboxylic acid
• the title compound is synthesized starting from 1 -amino-cyclohexanecarboxylic acid
• Example 76 3-(3,4-Dihvdroxvphenvl)-propionvl-Tvr(PQ.-:Hp)-lle-Asn-NHp (SEQ ID NO: 76)
• 3-(3,4-Dihydroxyphenyl)-propionic acid is from Fluka (Buchs, Switzerland) and is incorpora ⁇ ted with benzotriazole- 1 -yi-oxy-tris-dimethylamino)-phosphoniumhexafluorophosphate/N-hy- droxybenzotriazole (1 :1 ; 3 equiv.; first coupling) 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.).
• Example 77 3.4-Dihydroxvphenvl-acetvl-Tvr(PO..Hp)-lle-Asn-NH ⁇ (SEQ ID NO: 77)
• 3,4-Dihydroxyphenyl-acetic acid is from Fluka, Buchs, Switzerland and is incorporated with benzotriazole-1-yl-oxy-tris-dimethylamino)-phosphoniumhexafluorophosphate/N-hydroxy- benzotriazole (1 :1 ; 3 equiv.; first coupling) 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.).
• Example 80 trans-3,4-Dihvdroxv-cinnamovl-Tyr(PO..Hp)-Ac7C-Asn-NHp (SEQ ID NO: 80)
• the starting material is prepared as follows: a) Fmoc-1-amino-cvcloheptanecarboxylic acid
• Fmoc-2-amino-2-norbornanecarboxyiic acid (2 equiv.) is incorporated with benzotriazole-1- yl-oxy-tris-dimethylamino)-phosphoniumhexafluorophosphate/N-hydroxybenzotriazole (1 :1;
• the starting material is prepared as follows: a) Fmoc-2-amino-2-norbornanecarboxylic acid
• the title compound is synthesized starting from 2-amino-2-norbornanecarboxylic acid
• Fmoc-2-amino-2-norbornanecarboxylic acid (2 equiv.) is incorporated with benzotriazole-1- yl-oxy-tris-dimethylamino)-phosphoniumhexafluorophosphate/N-hydroxybenzotriazole (1 :1 ;
• Example 83 trans-3,4-Dihydroxy-cinnamoyl-Tyr(P ⁇ 3H2)-Nbo-NH2 (epimer 2) (SEQ ID NO: 83)
• Fmoc-2-amino-2-norbornanecarboxylic acid (2 equiv.) is incorporated with benzotriazole-1- yl-oxy-tris-dimethylamino)-phosphoniumhexafluorophosphate/N-hydroxybenzotriazole (1 :1 ; 2 equiv., first coupling) and N-[(dimethylamino)-1 H-1 ,2,3-triazolo[4,5-b]pyridin-1 -ylmethyle- ne]-N-methylmethanaminium hexafluorophosphate N-oxide (2 equiv.; second coupling) in the presence of diisopropylethylamine (6 equiv.).
• Title compound: Mass-spectral analysis (negative-ion mode): 558.7 (calc. 558.5, C26H29N3O9P1). tR 6.21 (HPLC system A).
• trans-4-Hydroxy-cinnamoyl-Tyr(PO:-.Hg)-lle-Asn-NHp SEQ ID NO: 84
• trans-4-Hydroxy-cinnamic acid is from Fluka, Buchs, Switzerland and is incorporated (2 equiv.; double coupling) with benzotriazole-1-yl-oxy-tris-dimethylamino)-phosphoniumhexa- fluorophosphate/N-hydroxybenzotriazole (1 :1; 2 equiv.) in the presence of diisopropylethyl ⁇ amine (4 equiv.).
• 6-Hydroxy-2-naphthoic acid is from Lancaster, France, France and is incorporated (2 equiv.; double coupling) with benzotriazole-1-yl-oxy-tris-dimethylamino)-phosphoniumhexa- fluorophosphate/N-hydroxybenzotriazole (1 :1 ; 2 equiv.) in the presence of diisopropylethyl ⁇ amine (4 equiv.).
• Example 87 trans-3.4-Dihydroxv-cinnamoyl-Tyr(P ⁇ 3Hp)-Glu-NHp (SEQ ID NO: 87)
• the side chain of the glutamic acid building block is protected with the tert-butyl group.
• N -Fmoc-Z homophenylalanine (2 equiv.; double coupling) is incorporated with 2-(2-pyri- don-1-yl)-1 ,1 ,3,3-tetramethyluronium-tetrafluoroborate (2 equiv.) in the presence of diiso ⁇ propylethylamine (2.2 equiv.).
• the side chain of the glutamic acid building block is protected with the tert-butyl group.
• the starting material is prepared as follows: a) N ⁇ -Fmoc-D-homophenylalanine
• N ⁇ -Fmoc-D-homophenylalanine (2 equiv.) is incorporated with 2-(2-pyridon-1-yl)-1 ,1 ,3,3- tetramethyluroniumtetrafluoroborate (2 equiv.) in the presence of diisopropylethylamine (2.2 equiv.).
• t R 7.04 (HPLC system A).
• Example 90 trans-3.4-Dihydroxv-cinnamovl-Tvr(POr.Hp)-Ac5C-NHp (SEQ ID NO: 90)
• Fmoc-1-amino-cyclopentanecarboxylic acid (2 equiv.) is incorporated with benzotriazole- 1 - yl-oxy-tris-dimethylamino)-phosphoniumhexafluorophosphate/N-hydroxybenzotriazole (1 :1 ; 2 equiv.; first coupling) and N-[(dimethylamino)-1 H-1 ,2,3-triazolo[4,5-b]pyridin-1-ylmethyle- ne]-N-methylmethanaminium hexafluorophosphate N-oxide (2 equiv.; second coupling) in the presence of diisopropylethylamine (6 equiv.).
• 2-Naphthoic acid is from Fluka, Buchs, Switzerland, and is incorporated (2 equiv.) with ben- zotriazole-1-yl-oxy-tris-dimethylamino)-phosphoniumhexafluorophosphate/N-hydroxybenzo- triazole (1 :1 ; 2 equiv.; double coupling) in the presence of diisopropylethylamine (4 equiv.).
• 1-Adamantoic acid is from Fluka, Buchs, Switzerland, and is incorporated (2 equiv.) with benzotriazole-1-yl-oxy-tris-dimethylamino)-phosphoniumhexafluorophosphate/N-hydroxy- benzotriazole (1 :1 ; 2 equiv.; double coupling) in the presence of diisopropylethylamine (4 equiv.).
• Cyclohexanoic acid is from Fluka, Buchs, Switzerland, and is incorporated (2 equiv.) with benzotriazole-1-yl-oxy-tris-dimethylamino)-phosphoniumhexafluorophosphate/N-hydroxy- benzotriazole (1 :1 ; 2 equiv.; double coupling) in the presence of diisopropylethylamine (4 equiv.).
• 3-Cyclohexyl-propionic acid is from Fluka, Buchs, Switzerland, and is incorporated (2 equiv.) with benzotriazole-1-yl-oxy-tris-dimethylamino)-phosphoniumhexafluorophosphate/N-hydro- xybenzotriazole (1 :1 ; 2 equiv.; double coupling) in the presence of diisopropylethylamine (4 equiv.).
• Example 96 1 ,2,3,4-Tetrahydro-2-naphthoyl-Tyr(PO3H2)-He-NH2 (SEQ ID NO: 96)
• 1 ,2,3,4-Tetrahydro-2-naphthoic acid is from Fluka, Buchs, Switzerland, and is incorporated (2 equiv.) with benzotriazole-1-yl-oxy-tris-dimethylamino)-phosphoniumhexafluorophos- phate/N-hydroxybenzotriazole (1 :1 ; 2 equiv.; double coupling) in the presence of diisopro ⁇ pylethylamine (4 equiv.).
• Cyclohexanoic acid is from Fluka, Buchs, Switzerland, and is incorporated (2 equiv.) with benzotriazole-1-yl-oxy-tris-dimethylamino)-phosphoniumhexafluorophosphate/N-hydroxy- benzotriazole (1 :1 ; 2 equiv.; double coupling) in the presence of diisopropylethylamine (4 equiv.).
• 1 -Adamantoic acid is from Fluka, Buchs, Switzerland and is incorporated (2 equiv.) with benzotriazole-1-yl-oxy-tris-dimethylamino)-phosphoniumhexafluorophosphate/N-hydroxy- benzotriazole (1 :1 ; 2 equiv.; double coupling) in the presence of diisopropylethylamine (4 equiv.).
• 4-Acetamino-benzoic acid is from Fluka, Buchs, Switzerland and is incorporated (2 equiv.) with benzotriazole-1-yl-oxy-tris-dimethylamino)-phosphoniumhexafluorophosphate/N-hydro- xybenzotriazole (1 :1 ; 2 equiv.; double coupling) in the presence of diisopropylethylamine (4 equiv.).
• Succinamic acid is from Aldrich, Buchs, Switzerland, and is incorporated (2 equiv.) with ben- zotriazole-1-yl-oxy-tris-dimethylamino)-phosphoniumhexafluorophosphate/N-hydroxybenzo- triazole (1 :1 ; 2 equiv.; double coupling) in the presence of diisopropylethylamine (4 equiv.).
• Example 102 1 ,2,3,4-Tetrahydro-2-naphthoyl-Tyr(PO3H2)-He-Asn-NH2
• Example 103 1 ,2,3,4-Tetrahydro-2-naphthoyl-Tyr(P ⁇ 3H2)-lle-lle-Pro-NH2 (SEQ ID NO: 103)
• 1 ,2,3,4-Tetrahydro-2-naphthoic acid is from Fluka, Buchs, Switzerland, and is incorporated (2 equiv.) with benzotriazole-1 -yl-oxy-tris-dimethylamino)-phosphoniumhexafluorophospha- te/N-hydroxybenzotriazole (1 :1 ; 2 equiv.; double coupling) in the presence of diisopropyl ⁇ ethylamine (4 equiv.).
• the starting material is prepared as follows: a) Fmoc-L-homo-phenylalanine
• Example 105 trans-3.4-Dihydroxy-cinnamovl-Tyr(P03Hp)-lle-Asn-NH2 (SEQ ID NO: 105)
• 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 caboxamide, and using chemical protocols based on the fluorenylmethoxycarbonyl chemistry (see E. Atherton and R.C. Sheppard, in Solid-Phase Peptide Synthesis- A Practical Approach, eds.: D. Rickwood and B.D. Hames, IRL Press at Oxford University Press, Oxford, 1989).
• N ⁇ -Fmoc- Tyr(P0 3 H 2 )-OH (3 equiv.) and trans-3,4-dihydroxy-cinnamic acid are accomplished with benzotriazole-1-yl-oxy-tris-dimethylamino)-phosphoniumhexafluorophosphate/N-hydroxy- benzotriazole (1 :1 ; 3 equiv., first coupling) 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 (6 equiv.).
• the complete peptide resin obtained after the last coupling step is simultaneously deprotected and cleaved by treatment with trifluoroacetic acid/H 2 O (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 as described in -Example 1.
• Title compound: Mass-spectral analysis (negative-ion mode): 648.3 (calc. 648.6, C28H35N5O11 P1). tR 5.88 (HPLC system A).
• Fmoc- ⁇ -alanine is from Novabiochem (Laufelfingen, Switzerland) and is incorporated (3 equiv.) with benzotriazole-1 -yl-oxy-tris-dimethylamino)-phosphoniumhexafluorophospha- te/N-hydroxybenzotriazole (1 :1 ; 3 equiv.) in the presence of diisopropylethylamine (6 equiv.).
• the peptide is synthesized manually on a 4-(2',4'-dimethoxyphenyl-aminomethyl)-phenoxy resin, employing a procedure analogous to Example 67. Incorporation of N ⁇ -Fmoc-[4-(O- diethyl)-phophono(difluoromethyl)]-L-phenylalanine (for synthesis see Tetrahedron Lett. 34
• the complete peptide resin obtained after the last coupling step is deprotected and cleaved via a two step process.
• the peptide is removed from the solid-support and partially deprotected by treatment with trifluoroacetic acid/H 2 O (95:5, v/v) for 3 h at room temperature.
• the cleavage of the phosphonate diester protecting groups is carried out as follows: The crude compound is suspended in MeCN (1 ml) and trimethylsilyl-iodide (1 ml) is added dropwise to the suspension. The course of the reaction is followed by analytical HPLC. After 60 min complete reaction is achieved.
• Example 117 3-Aminobenzyloxycarbonyl-Tyr(P ⁇ 3H2)-Gln-Asn-NH2 (TFA salt) (SEQ ID NO: 117)
• the Fmoc group is removed and the peptide resin is cleaved with trifluoroacetic acid/water (95:5, v/v, v/v) for 3 h at room temperature.
• the filtrate is precipitated in diisopropyl ether- petroleum ether (1 :1 , v/v) at 0 °C, and the precipitate is collected by filtration.
• the com ⁇ pound (68 mg) is dissolved in acetonitrile (2 ml) and trimethylsilyl iodide (1.7 ml; Fluka, Buchs, Switzerland) is added. The course of the reaction is monitored by analytical HPLC using a reversed-phase column.
• Example 121 Acetyl-Tyr(PO3H )-lle-Asn-NH-(3-naphthalen-1 -yl-propyi) (SEQ ID NO: 121)
• the peptide is synthesized manually on a 4-(2',4',-dimethoxyphenyl-Fmoc-aminomethyl)- phenoxyacetamido-norleucyl-MBHA resin (commercially available from Novabiochem, Laufelfingen, Switzerland, 0.55 mmol/g), employing the Fmoc strategy (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 is removed with piperidine/ dimethylacetamide (1 :4, v/v; 6 x 2 min) followed by washing with isopropa ⁇ nol (3 x 1 min), dimethylacetamide (2 x 1 min), isopropanol (3 x1 min) and dimethylacet ⁇ amide (2 x 1 min).
• Coupling is achieved by first dissolving the Fmoc-amino acid (3 equiv.) , diisopropylethyl amin (3.3 equiv.), and the 2-(2-pyridon-1 -yl)-1 ,1 ,3,3-tetramethyluroniumte- trafluoroborate reagent (TPTU, commercially available from Senn Chemicals, Dielsdorf, Switzerland; 3 equiv.) in N-methylpyrrolidin-2-one, then waiting 3 min for preactivation, ad ⁇ ding the mixture to the resin, and finally shaking for at least 45 min.
• TPTU 2-(2-pyridon-1 -yl)-1 ,1 ,3,3-tetramethyluroniumte- trafluoroborate reagent
• N ⁇ -Fmoc-aspartic acid ⁇ -allylic ester (Novabiochem, Laufelfingen, Switzerland) is coupled through its side chain to the resin.
• the incorporation of N ⁇ -Fmoc-Tyr(PO 3 H)-OH (see Biochemistry 32, 4354 (1993) (3 equiv.) is accomplished with BOP/ HOBt (1 :1 ; 2 equiv.; first coupling) in the presence of diisopropylethylamine (6 equiv.) and N-[(dimethylamino)-1 H-1 ,2,3-triazolo[4,5-b]pyridin-1- ylmethylene]-N-methylmethanaminium hexafluorophosphate N-oxide (HATU, 2 equiv., second coupling) in the presence of diisopropylethylamine (6 equiv.).
• Acetylation is perfor ⁇ med with acetic anhydride and pyridine in dimethylacetamide (1 :1 :8 v/v) for 2 min followed by washing with dimethylacetamide and dichloromethane.
• the dried peptide resin is re-suspended in a degassed solution of acetic acid and N-methylmorpholin in chloroform (2:1 :37 v/v) followed by addition of tetrakis- (triphenyl ⁇ phosphine)- palladium(O) (0.8 equiv.) under an argon atmosphere (see F. Albericio, G. Barany, G.B. Fields, D. Hudson, S.A.
• the resin is washed with chlo ⁇ roform (3 x 1 min) , dimethylformamide (3 x 1min), a solution of sodium diethyldithiocarba- mate (0.05M) containing 0.5% diisopropylethylamine in dimethylformamide (2 x 1 min), di ⁇ methylformamide (2 x 1 min) and dichloromethane (3 x1 min) and dried.
• the crude peptide is purified by medium-pressure liquid chromato ⁇ graphy using a C 18 reversed phase column (Merck ⁇ LICHROPREP RP-18, 15-25 ⁇ m bead diameter, reversed phase HPLC column material based on C-
• a C 18 reversed phase column Merck ⁇ LICHROPREP RP-18, 15-25 ⁇ m bead diameter, reversed phase HPLC column material based on C-
• the starting material is obtained as follows:
• the free base is dissolved in 40 ml of ethanol and treated with 10 ml of a 10% solution of hydrogen chloride in ethanol (approximately 0.03 mol). The solution is concentrated and the product precipitated by addition of ether. The product is collected by filtration, yielding the title compound: Melting point 154-156°C.
• the starting material is obtained as follows: a) 3-(2-Naphthyl)-acrylonitril
• Exarnrjle_124 Acetyl-Tyr(P ⁇ 3H2)-He-Asn-NH-(3-phenanthren-9-yl-propyl) (SEQ ID NO: 124)
• the starting material is obtained as follows: a) 3-(Phenanthren-9-yl)-propylamine
• the title compound is synthesized starting with a Grignard reaction between 9- bromophenanthrene (Fluka, Buchs, Switzerland) and ethylene oxide (Fluka, Buchs,
• Example 125 Acetvl-Tvr(P ⁇ 3H2.-He-Asn-NH-[2-(1 -bromo-naphthalen-2-yloxv)-ethvll (SEQ ID NO: 125)
• the starting material is obtained as follows: a) (1-Bromo-naphthalen-2-yloxy)-acetonitrile
• Example 126 Acetvl-Tvr(P ⁇ 3Hp)-lle-Asn-NH-.3.3-diphenvl-propyl)
• 3,3-Diphenylpropylamine is from Aldrich, Buchs, Switzerland.
• Example 127 Acetvl-Tv POg ⁇ -He-Asn-NH-Q-phenvl-propvl)
• 3-Phenylpropylamine is from Fluka, Buchs, Switzerland.
• 2,2-Diphenylethylamine is from Aldrich, Buchs, Switzerland.
• 2-(4-Chloro-phenyl)-ethylamine is from Fluka, Buchs, Switzerland.
• Benzylamine is from Fluka, Buchs, Switzerland.
• Isobutylamine is from Fluka, Buchs, Switzerland.
• Cyclohexylamine is from Fluka, Buchs, Switzerland.
• Aminomethyl-cyclohexane is from Fluka, Buchs, Switzerland.
• Example 137 Acetyl-Tyr(P ⁇ 3H2)-Ac6C-Asn-NH-(3-naphthalen-1 -yl-propyl)
• Example 138 Acetvl-Tvr(P ⁇ 3H2.-Acgc-Asn-NH-(3-naphthalen-2-vl-propyl)
• Example 140 3-Aminobenzvloxvcarbonvl-Tvr(P ⁇ 3H2. -He-Asn-NH-(3-naphthalen-1 -vl- propyl)
• the peptide is synthesized manually on a 4-(2',4',-dimethoxyphenyi-Fmoc-aminomethyl)- phenoxyacetamido-norleucyl-MBHA resin (commercially available from Novabiochem, Lau ⁇ felfingen, Switzerland, 0.55 mmol/g), employing the Fmoc strategy (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 is removed with piperidine/ dimethylacetamide (1 :4, v/v; 6 x 2 min) followed by washing with isopropa ⁇ nol (3 x 1 min), dimethylacetamide (2 x 1 min), isopropanol (3 x1 min) and dimethylacet ⁇ amide (2 x 1 min).
• Coupling is achieved by first dissolving the Fmoc-amino acid (3 equiv.) , diisopropylethylamine (3.3 equiv.), and the 2-(2-pyridon-1-yl)-1 ,1 ,3,3-tetramethyluroniumte- trafluoroborate reagent (TPTU, commercially available Senn chemicals, Dielsdorf, Switzer ⁇ land; 3 equiv.) in N-methylpyrrolidin-2-one, then waiting 3 min for preactivation, adding the mixture to the resin, and finally shaking for at least 45 min.
• TPTU 2-(2-pyridon-1-yl)-1 ,1 ,3,3-tetramethyluroniumte- trafluoroborate reagent
• N ⁇ -Fmoc-asparagine- ⁇ -allylic ester (Novabiochem, Laufelfingen, Switzerland) is coupled through its side chain to the resin.
• the incorporation of N ⁇ -Fmoc-Tyr(P0 3 H 2 )-OH (see Biochemistry 32, 4354 (1993) (3 equiv.) is accomplished with BOP/ HOBt (1 :1 ; 2 equiv.; first coupling) in the presence of diisopropylethylamine (6 equiv.) and N-[(dimethylamino)-1 H-1 ,2,3-triazolo[4,5-b]pyridin-1- ylmethylene]-N-methylmethanaminium hexafluorophosphate N-oxide (HATU, 2 equiv., se ⁇ cond coupling) in the presence of diisopropylethylamine (6 equiv.).
• 3-N-tert-Butoxy-carbo- nyl-aminobenzyl-4-nitrophenyl-carbonate (3 equiv.) is coupled to the N-terminal residue of the peptide in the presence of an equimolar amount of diisopropylethylamine in N-methyl- pyrrolidin-2-one during 17h at room temperature.
• the crude peptide is purified by medium-pres ⁇ sure liquid chromatography using a C-J S reversed phase column (Merck ® LICHROPREP RP- 18, 15-25 ⁇ m bead diameter, reversed phase HPLC column material based on Ci 8-deri- vatized silicagel, Merck, Darmstadt, FRG; column length 46 cm, diameter 3.6 cm; flow rate 53.3 ml/min; detection at 215nm), eluted with an acetonitrile-water gradient containing 0.1% of TFA.
• a C-J S reversed phase column Merck ® LICHROPREP RP- 18, 15-25 ⁇ m bead diameter, reversed phase HPLC column material based on Ci 8-deri- vatized silicagel, Merck, Darmstadt, FRG; column length 46 cm, diameter 3.6 cm; flow rate 53.3 ml/min; detection at 215nm
• Mass spectral analysis matrix-assisted laser-de- sorption ionisation time-of-flight mass spectrometry, MALDI-TOF and electro-spray ionisa- tion mass spectroscopy, ESI reveals a molecular mass within 0.1% of the expected values (negative- ion mode: 803.0 [M-H]+, (C40H49N6O10P. calc. 804.85).
• Example 141 S-Aminobenzvloxvcarbonvl-Tvr OgHp) -He-Asn-NH-r3-(2-hydroxy- naphthalen-1 -yl)-propyl]
• Example 142 3-Aminobenzvloxvcarbonvl-Tvr(P ⁇ 3Hp)-lle-Asn-NH-(3-naphthalen-2-yl- propyl) (SEQ ID NO: 142)
• Example 144 3-Aminobenzvloxvcarbonvl-Tvr(P ⁇ 3H2.-He-Asn-NH-f2-(1 -bromo-naphthalen-
• Example 149 3-Aminobenzyloxvcarbonyl-Tyr(P ⁇ 3H2)-Acgc-Asn-NH-[3-(2-hydroxy- naphthaien-1 -yl)-propyl]
• 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 151 Sterile dry substance for injection:
• Example 152 Nasal spray:
• Example 153 Film-coated tablets
• active ingredient 1000 g corn starch 680 g colloidal silica 200 g magnesium stearate 20 g stearic acid 50 g sodium carboxymethyl starch 250 g water quantum satis
• 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.
• the dry granules are pressed through a sieve having a mesh size of 1 mm, mixed with a pre-sieved mixture (1 mm sieve) of 330 g of corn starch, the magnesium stearate, the stearic acid and the sodium carboxymethyl starch, and compressed to form slightly biconvex tablets.

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