US20050020810A1 - Peptides which target tumor and endothelial cells, compositions and uses thereof - Google Patents

Peptides which target tumor and endothelial cells, compositions and uses thereof Download PDF

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Publication number
US20050020810A1
US20050020810A1 US10/722,843 US72284303A US2005020810A1 US 20050020810 A1 US20050020810 A1 US 20050020810A1 US 72284303 A US72284303 A US 72284303A US 2005020810 A1 US2005020810 A1 US 2005020810A1
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compound
hydrogen
substituted
acyl
alkyl
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US10/722,843
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Robert Ternansky
Amy Allan
Patricia Gladstone
Won Yoon
Graham Parry
Fernando Donate
Andrew Mazar
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Tactic Pharma LLC
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Individual
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Assigned to ATTENUON LLC reassignment ATTENUON LLC ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: DONATE, FERNANDO, PARRY, GRAHAM, TERNANSKY, ROBERT J., MAZAR, ANDREW, ALLAN, AMY L., GLADSTONE, PATRICIA L., YOON, WON HYUNG
Publication of US20050020810A1 publication Critical patent/US20050020810A1/en
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/08—Peptides having 5 to 11 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates generally to peptide analogs of Ac—PHSCN—NH 2 which target tumor and endothelial cells and have anti-tumor, anti-angiogenic and anti-metasastic activity, methods of making these peptides, compositions thereof and methods of using these peptides and pharmaceutical compositions thereof to treat, prevent and detect diseases characterized by tumor growth, metastasis and angiogenesis.
  • the peptide analogs may serve, inter alia, as carriers of radioactivity, PET-active compounds, toxins, fluorescent molecules and PEG molecules.
  • Integrins are heterodimeric, transmembrane proteins that are involved in cell adhesion, motility and survival (Geiger et al., Nat. Rev. Mol. Cell Biol 2001, 2(11):793-805). Integrin ligands comprise the extracellular matrix (ECM) and basement membranes and include collagen, laminin, fibronectin, vitronectin, and fibrinogen (Bokel, Dev. Cell 2002, 3(3):311-21; Stupack et al., J Cell Sci 2002,115: 3729-38; Bornstein et al., Curr Opin Cell Biol 2002, 14(5): 608-16).
  • ECM extracellular matrix
  • basement membranes include collagen, laminin, fibronectin, vitronectin, and fibrinogen (Bokel, Dev. Cell 2002, 3(3):311-21; Stupack et al., J Cell Sci 2002,115: 3729-38; Bornstein et al., Curr Opin Cell Biol 2002, 14(5)
  • the integrin ⁇ 5 ⁇ 1 is normally not expressed in quiescent endothelial cells, but is upregulated during angiogenesis (Kim et al., Am J Pathol 2000, 156(4):1345-62).
  • ⁇ 5 ⁇ 1 is a receptor for fibronectin, an abundant plasma protein that may also be associated with the extracellular matrix (Labat-Robert, Semin Cancer Biol 2002, 12(3): 187-95).
  • Fibronectin interacts with the ⁇ 5 ⁇ 1 integrin through several epitopes with the major adhesive interaction mediated by the RGD sequence within the 10 th type III repeat.
  • the major adhesive interaction mediates cell signaling events and can be potentiated by a second epitope located in the ninth Type III repeat called the synergy region (i.e., PHSRN) (Akiyama et al., Cancer Metastasis Rev 1995, 14(3): 173-89).
  • Antagonists of ⁇ 5 ⁇ 1 were able to inhibit tumor angiogenesis and cause tumor regression which demonstrates the therapeutic potential of targeting the integrin ⁇ 5 ⁇ 1 (Kim et al., supra).
  • the integrin ⁇ 5 ⁇ 1 also appears to be important in the survival and metastasis of tumor cells (O'Brien et al., Exp Cell Res 1996, 224(1): 208-13; Ruoslahti, Invasion Metastasis 1994-95;14(1-6):87-9724-26); Kemperman et al., Invasion Metastasis 1994-95,14(1-6): 98-108; Tani et al., Br J Cancer 2003, 88(2): 327-33).
  • the integrin ⁇ v ⁇ 3 is also a therapeutic target for the inhibition of tumor angiogenesis since inhibitors of ⁇ v ⁇ 3 (e.g., monoclonal antibodies, cyclic RGD peptides and small non-peptidic organic compounds) are efficacious in multiple pre-clinical models of cancer progression (Kumar, Curr Drug Targets 2003, 4(2): 123-31; Varner et al., Important Adv Oncol 1996, 69-87; Brooks, J Clin Invest 1995, 96(4): 1815-22).
  • inhibitors of ⁇ v ⁇ 3 e.g., monoclonal antibodies, cyclic RGD peptides and small non-peptidic organic compounds
  • ⁇ v ⁇ 3 is not normally expressed on epithelial cells, it is up-regulated on tumor cells, leading to tumor cell adhesion, migration and invasion (Metzner et al., J Invest Dermatol 1996, 107(4): 597-602).
  • ⁇ v ⁇ 3 integrin has been implicated in melanoma progression and metastasis (Nip et al., Cancer Metastasis Rev 1995, 14(3): 241-52) and ⁇ v ⁇ 3 expression has been documented in a variety of tumor cell types including breast, prostate, pancreas, kidney, and glioma (Felding-Habermann et al., Proc Natl Acad Sci USA 2001, 98(4): 1853-8; Platten et al., Biochem Biophys Res Commun 2000, 268(2): 607-11; Lohr et al., Pancreas 1996, 12(3): 248-59; Rabb et al., Am J Nephrol 1996, 16(5): 402-8; Cooper et al., Neoplasia 2002, 4(3): 191-4). Further, the expression of ⁇ v ⁇ 3 has also been associated with metastasis to bone (Pecheur et al., FASEB J 2002,
  • Ac—PHSCN—NH 2 is derived from the synergy sequence of fibronectin and has been shown to target activated ⁇ 5 ⁇ 1 and ⁇ V ⁇ 3 integrins on cell surfaces (Livant, U.S. Pat. No. 6,001,965; Livant, U.S. Pat. No. 6,472,369; Livant et al., Cancer Res 2000, 60(2): 309-20).
  • Ac—PHSCN—NH 2 completely inhibits DU145 invasion and metastasis of MatLyLu cells in a rat model (Livant et al., supra) and in combination with 5-FU infusion improved survival in a CT26 colon cancer model (Stoeltzing et al., Int J Cancer 2003, 104(4): 496-503). Accordingly, Ac—PHSCN—NH 2 targets tumors and the blood vessels which nourish tumor cells as shown by visualization of a Ac—PHSCN—NH 2 derivative.
  • the peptide analogs will serve, inter alia, as carriers of radioactivity for imaging and radiotherapy, PET-active compounds for PET-imaging, toxins for targeted delivery of cellular toxins, fluorescent molecules for visualization and PEG molecules for improvement of pharmacokinetic parameters.
  • the present invention satisfies these and other needs by providing peptide analogs of Ac—PHSCN—NH 2 which target tumor and endothelial cells and have anti-tumor, anti-angiogenic and anti-metasastic activity, methods of making these peptides, compositions thereof and methods of using these peptides and pharmaceutical compositions thereof to treat, prevent and detect diseases characterized by tumor growth, metastasis and angiogenesis.
  • the peptide analogs may serve, inter alia, as carriers of radioactivity, PET-active compounds, toxins, fluorescent molecules and PEG molecules.
  • the present invention provides a compound of Formula (I):
  • the present invention provides pharmaceutical compositions of compounds of the invention.
  • the pharmaceutical compositions generally comprise one or more compounds of the invention or pharmaceutically acceptable salts, hydrates or solvates thereof and a pharmaceutically acceptable diluent, carrier, excipient and adjuvant.
  • diluent, carrier, excipient and adjuvant will depend upon, among other factors, the desired mode of administration.
  • the present invention provides methods for treating or preventing diseases or disorders such as cancer.
  • the methods generally involve administering to a patient in need of such treatment or prevention a therapeutically effective amount of a compound of the invention and/or a pharmaceutical composition thereof.
  • the present invention provides methods for detecting diseases or disorders such as cancer.
  • the methods generally involve administering to a patient in need of such treatment or prevention a diagnostically effective amount of a compound of the invention and/or a pharmaceutical composition thereof.
  • Compounds of the invention refers to compounds encompassed by structural formulae (I), (II), (III), (IV) and (V) disclosed herein and includes any specific compounds within that generic formula whose structure is disclosed herein.
  • the compounds of the invention may be identified either by their chemical structure and/or chemical name. When the chemical structure and chemical name conflict, the chemical structure is determinative of the identity of the compound.
  • the compounds of the invention may contain one or more chiral centers and/or double bonds and therefore, may exist as stereoisomers, such as double-bond isomers (i.e., geometric isomers), enantiomers or diastereomers.
  • the chemical structures depicted herein encompass all possible enantiomers and stereoisomers of the illustrated compounds including the stereoisomerically pure form (e.g., geometrically pure, enantiomerically pure or diastereomerically pure) and enantiomeric and stereoisomeric mixtures.
  • the compounds of the invention may also exist in several tautomeric forms. Accordingly, the chemical structures depicted herein encompass all possible tautomeric forms of the illustrated compounds.
  • the compounds of the invention also include isotopically labeled compounds where one or more atoms have an atomic mass different from the atomic mass conventionally found in nature.
  • isotopes examples include, but are not limited to, 2 H, 3 H, 13C, 14 C, 15 N, 17 O, 18 O, etc.
  • Compounds of the invention may exist in unsolvated forms as well as solvated forms, including hydrated forms and as N-oxides. In general, the hydrated, solvated and N-oxide forms are within the scope of the present invention.
  • Certain compounds of the present invention may exist in multiple crystalline or amorphous forms. In general, all physical forms are equivalent for the uses contemplated by the present invention and are intended to be within the scope of the present invention. Further, it should be understood, when partial structures of the compounds of the invention are illustrated, that brackets indicate the point of attachment of the partial structure to the rest of the molecule.
  • Alkyl by itself or as part of another substituent refers to a saturated or unsaturated, branched, straight-chain or cyclic monovalent hydrocarbon radical derived by the removal of one hydrogen atom from a single carbon atom of a parent alkane, alkene or alkyne.
  • Typical alkyl groups include, but are not limited to, methyl; ethyls such as ethanyl, ethenyl, ethynyl; propyls such as propan-1-yl, propan-2-yl, cyclopropan-1-yl, prop-1-en-1-yl, prop-1-en-2-yl, prop-2-en-1-yl (allyl), cycloprop-1-en-1-yl; cycloprop-2-en-1-yl, prop-1-yn-1-yl, prop-2-yn-1-yl, etc.; butyls such as butan-1-yl, butan-2-yl, 2-methyl-propan-1-yl, 2-methyl-propan-2-yl, cyclobutan-1-yl, but-1-en-1-yl, but-1-en-2-yl, 2-methyl-prop-1-en-1-yl, but-2-en-2-yl, buta-1,
  • alkyl is specifically intended to include groups having any degree or level of saturation, i.e., groups having exclusively single carbon-carbon bonds, groups having one or more double carbon-carbon bonds, groups having one or more triple carbon-carbon bonds and groups having mixtures of single, double and triple carbon-carbon bonds. Where a specific level of saturation is intended, the expressions “alkanyl,” “alkenyl,” and “alkynyl” are used.
  • an alkyl group comprises from 1 to 20 carbon atoms, more preferably, from 1 to 10 carbon atoms.
  • (C 1 -C 6 ) alkyl for example, refers to an alkyl group containing from 1 to 6 carbon atoms.
  • Alkanyl by itself or as part of another substituent refers to a saturated branched, straight-chain or cyclic alkyl radical derived by the removal of one hydrogen atom from a single carbon atom of a parent alkane.
  • Typical alkanyl groups include, but are not limited to, methanyl; ethanyl; propanyls such as propan-1-yl, propan-2-yl (isopropyl), cyclopropan-1-yl, etc.; butanyls such as butan-1-yl, butan-2-yl (sec-butyl), 2-methyl-propan-1-yl (isobutyl), 2-methyl-propan-2-yl (t-butyl), cyclobutan-1-yl, etc.; and the like.
  • Alkenyl by itself or as part of another substituent refers to an unsaturated branched, straight-chain or cyclic alkyl radical having at least one carbon-carbon double bond derived by the removal of one hydrogen atom from a single carbon atom of a parent alkene.
  • the group may be in either the cis or trans conformation about the double bond(s).
  • Typical alkenyl groups include, but are not limited to, ethenyl; propenyls such as prop-1-en-1-yl, prop-1-en-2-yl, prop-2-en-1-yl (allyl), prop-2-en-2-yl, cycloprop-1-en-1-yl; cycloprop-2-en-1-yl; butenyls such as but-1-en-1-yl, but-1-en-2-yl, 2-methyl-prop-1-en-1-yl, but-2-en-1-yl, but-2-en-1-yl, but-2-en-2-yl, buta-1,3-dien-1-yl, buta-1,3-dien-2-yl, cyclobut-1-en-1-yl, cyclobut-1-en-3-yl, cyclobuta-1,3-dien-1-yl, etc.; and the like.
  • Alkynyl by itself or as part of another substituent refers to an unsaturated branched, straight-chain or cyclic alkyl radical having at least one carbon-carbon triple bond derived by the removal of one hydrogen atom from a single carbon atom of a parent alkyne.
  • Typical alkynyl groups include, but are not limited to, ethynyl; propynyls such as prop-1-yn-1-yl, prop-2-yn-1-yl, etc.; butynyls such as but-1-yn-1-yl, but-1-yn-3-yl, but-3-yn-1-yl, etc.; and the like.
  • “Acyl” by itself or as part of another substituent refers to a radical —C(O)R 30 , where R 30 is hydrogen, alkyl, cycloalkyl, cycloheteroalkyl, aryl, arylalkyl, heteroalkyl, heteroaryl, heteroarylalkyl as defined herein.
  • Representative examples include, but are not limited to, formyl, acetyl, cyclohexylcarbonyl, cyclohexylmethylcarbonyl, benzoyl, benzylcarbonyl and the like.
  • “Acyl Chelate” by itself or as part of another substituent refers to a radical —C(O)R 31 , where R 31 is alkyl, cycloalkyl, aryl as defined herein substituted with a chelating group which binds an appropriate metal.
  • R 32 is a chelating group such as, for example, DOTA, TETA, a polyamino carboxylate (e.g., NODAGA, EDTA, tricine, —C(O)CH 2 -DTPA, etc.) and where the chelating group is bound to metals such as positron emitting labels (e.g., 18 F, 45 Ti, 44 Sc, 55 Co, 61 Cu, 64 Cu, 66 Ga, 68 Ga, 75 Br, 76 Br, 86 Y, 110 In, 124 I, 89 Zr, 99 Tc), radionuclides (e.g., 137 Cs, 60 Co, 131 I, 123 I, 192 Ir, 90 Y, 67 Ga, 99 Tc, 123 I, 125 I, 131 I, 111 In, 97 Ru, 67 Cu, 68 Ga, 72 As, 89 Zr, 90 Y, 201 Tl, etc.)
  • positron emitting labels e.g., 18 F,
  • Alkoxy by itself or as part of another substituent refers to a radical —OR 31 where R 33 represents an alkyl or cycloalkyl group as defined herein. Representative examples include, but are not limited to, methoxy, ethoxy, propoxy, butoxy, cyclohexyloxy and the like.
  • Aryl by itself or as part of another substituent refers to a monovalent aromatic hydrocarbon radical derived by the removal of one hydrogen atom from a single carbon atom of a parent aromatic ring system.
  • Typical aryl groups include, but are not limited to, groups derived from aceanthrylene, acenaphthylene, acephenanthrylene, anthracene, azulene, benzene, chrysene, coronene, fluoranthene, fluorene, hexacene, hexaphene, hexalene, as-indacene, s-indacene, indane, indene, naphthalene, octacene, octaphene, octalene, ovalene, penta-2,4-diene, pentacene, pentalene, pentaphene, perylene, phenalene, phenanthrene, picen
  • Arylalkyl by itself or as part of another substituent refers to an acyclic alkyl radical in which one of the hydrogen atoms bonded to a carbon atom, typically a terminal or sp 3 carbon atom, is replaced with an aryl group.
  • Typical arylalkyl groups include, but are not limited to, benzyl, 2-phenylethan-1-yl, 2-phenylethen-1-yl, naphthylmethyl, 2-naphthylethan-1-yl, 2-naphthylethen-1-yl, naphthobenzyl, 2-naphthophenylethan-1-yl and the like.
  • an arylalkyl group is (C 6 -C 30 ) arylalkyl, e.g., the alkanyl, alkenyl or alkynyl moiety of the arylalkyl group is (C 1 -C 10 ) and the aryl moiety is (C 6 -C 20 ), more preferably, an arylalkyl group is (C 6 -C 20 ) arylalkyl, e.g., the alkanyl, alkenyl or alkynyl moiety of the arylalkyl group is (C 1 -C 8 ) and the aryl moiety is (C 6 -C 12 ).
  • Cycloalkyl by itself or as part of another substituent refers to a saturated or unsaturated cyclic alkyl radical. Where a specific level of saturation is intended, the nomenclature “cycloalkanyl” or “cycloalkenyl” is used.
  • Typical cycloalkyl groups include, but are not limited to, groups derived from cyclopropane, cyclobutane, cyclopentane, cyclohexane and the like.
  • the cycloalkyl group is (C 3 -C 10 ) cycloalkyl, more preferably (C 3 -C 7 ) cycloalkyl.
  • “Cycloheteroalkyl” by itself or as part of another substituent refers to a saturated or unsaturated cyclic alkyl radical in which one or more carbon atoms (and any associated hydrogen atoms) are independently replaced with the same or different heteroatom.
  • Typical heteroatoms to replace the carbon atom(s) include, but are not limited to, N, P, O, S, Si, etc. Where a specific level of saturation is intended, the nomenclature “cycloheteroalkanyl” or “cycloheteroalkenyl” is used.
  • Typical cycloheteroalkyl groups include, but are not limited to, groups derived from epoxides, azirines, thiiranes, imidazolidine, morpholine, piperazine, piperidine, pyrazolidine, pyrrolidine, quinuclidine, and the like.
  • Diagnostically effective amount means the amount of a compound that, when administered to a patient for detection of a disease, is sufficient to detect the disease.
  • the “diagnostically effective amount” will vary depending on the compound, the disease and its severity and the age, weight, etc., of the patient to be treated.
  • Heteroalkyl, Heteroalkanyl, Heteroalkeniyl and Heteroalkvnyl by themselves or as part of another substituent refer to alkyl, alkanyl, alkenyl and alkynyl groups, respectively, in which one or more of the carbon atoms (and any associated hydrogen atoms) are independently replaced with the same or different heteroatomic groups.
  • Typical heteroatomic groups which can be included in these groups include, but are not limited to, —O—, —S—, —O—O—, —S—S—, —O—S—, —NR 34 R 35 , — ⁇ N—N ⁇ —, —N ⁇ N—, —N ⁇ N—, —N ⁇ N—NR 36 R 37 , —PR 38 —, —P(O) 2 —, —POR 39 —, —O—P(O) 2 —, —SO—, —SO 2 —, —SnR 40 R 41 — and the like, where R 34 , R 35 , R 36 , R 37 , R 38 , R 39 , R 40 and R 41 are independently hydrogen, alkyl, substituted alkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, cycloalkyl, substituted cycloalkyl, cycloheteroalkyl, substitute
  • Heteroaryl by itself or as part of another substituent refers to a monovalent heteroaromatic radical derived by the removal of one hydrogen atom from a single atom of a parent heteroaromatic ring system.
  • Typical heteroaryl groups include, but are not limited to, groups derived from acridine, arsindole, carbazole, ⁇ -carboline, chromane, chromene, cinnoline, furan, imidazole, indazole, indole, indoline, indolizine, isobenzofuran, isochromene, isoindole, isoindoline, isoquinoline, isothiazole, isoxazole, naphthyridine, oxadiazole, oxazole, perimidine, phenanthridine, phenanthroline, phenazine, phthalazine, pteridine, purine, pyran, pyrazine,
  • the heteroaryl group is from 5-20 membered heteroaryl, more preferably from 5-10 membered heteroaryl.
  • Preferred heteroaryl groups are those derived from thiophene, pyrrole, benzothiophene, benzofuran, indole, pyridine, quinoline, imidazole, oxazole and pyrazine
  • Heteroarylalkyl by itself or as part of another substituent refers to an acyclic alkyl radical in which one of the hydrogen atoms bonded to a carbon atom, typically a terminal or sp 3 carbon atom, is replaced with a heteroaryl group. Where specific alkyl moieties are intended, the nomenclature heteroarylalkanyl, heteroarylalkenyl and/or heterorylalkynyl is used.
  • the heteroarylalkyl group is a 6-30 membered heteroarylalkyl, e.g., the alkanyl, alkenyl or alkynyl moiety of the heteroarylalkyl is 1-10 membered and the heteroaryl moiety is a 5-20-membered heteroaryl, more preferably, 6-20 membered heteroarylalkyl, e.g., the alkanyl, alkenyl or alkynyl moiety of the heteroarylalkyl is 1-8 membered and the heteroaryl moiety is a 5-12-membered heteroaryl.
  • “Imino” by itself or as part of another substituent refers to a radical —C ⁇ NR 42 , where R 42 is hydrogen, alkyl, cycloalkyl, cycloheteroalkyl, aryl, arylalkyl, heteroalkyl, heteroaryl, heteroarylalkyl as defined herein.
  • Parent aromatic Ring System refers to an unsaturated cyclic or polycyclic ring system having a conjugated ⁇ electron system. Specifically included within the definition of “parent aromatic ring system” are fused ring systems in which one or more of the rings are aromatic and one or more of the rings are saturated or unsaturated, such as, for example, fluorene, indane, indene, phenalene, etc.
  • Typical parent aromatic ring systems include, but are not limited to, aceanthrylene, acenaphthylene, acephenanthrylene, anthracene, azulene, benzene, chrysene, coronene, fluoranthene, fluorene, hexacene, hexaphene, hexalene, as-indacene, s-indacene, indane, indene, naphthalene, octacene, octaphene, octalene, ovalene, penta-2,4-diene, pentacene, pentalene, pentaphene, perylene, phenalene, phenanthrene, picene, pleiadene, pyrene, pyranthrene, rubicene, triphenylene, trinaphthalene and the like.
  • Parent Heteroaromatic Ring System refers to a parent aromatic ring system in which one or more carbon atoms (and any associated hydrogen atoms) are independently replaced with the same or different heteroatom. Typical heteroatoms to replace the carbon atoms include, but are not limited to, N, P, O, S, Si, etc. Specifically included within the definition of “parent heteroaromatic ring systems” are fused ring systems in which one or more of the rings are aromatic and one or more of the rings are saturated or unsaturated, such as, for example, arsindole, benzodioxan, benzofuran, chromane, chromene, indole, indoline, xanthene, etc.
  • Typical parent heteroaromatic ring systems include, but are not limited to, arsindole, carbazole, ⁇ -carboline, chromane, chromene, cinnoline, furan, imidazole, indazole, indole, indoline, indolizine, isobenzofuran, isochromene, isoindole, isoindoline, isoquinoline, isothiazole, isoxazole, naphthyridine, oxadiazole, oxazole, perimidine, phenanthridine, phenanthroline, phenazine, phthalazine, pteridine, purine, pyran, pyrazine, pyrazole, pyridazine, pyridine, pyrimidine, pyrrole, pyrrolizine, quinazoline, quinoline, quinolizine, quinoxaline, tetrazole, thi
  • Patient includes humans.
  • human and “patient” are used interchangeably herein.
  • “Pharmaceutically acceptable salt” refers to a salt of a compound of the invention, which is pharmaceutically acceptable and possesses the desired pharmacological activity of the parent compound.
  • Such salts include: (1) acid addition salts, formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; or formed with organic acids such as acetic acid, propionic acid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, 3-(4-hydroxybenzoyl) benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethane-disulfonic acid, 2-hydroxyethanesulfonic acid, benzen
  • “Pharmaceutically acceptable vehicle” refers to a diluent, adjuvant, excipient or carrier with which a compound of the invention is administered.
  • Preventing refers to a reduction in risk of acquiring a disease or disorder (i.e., causing at least one of the clinical symptoms of the disease not to develop in a patient that may be exposed to or predisposed to the disease but does not yet experience or display symptoms of the disease).
  • “Substituted” refers to a group in which one or more hydrogen atoms are independently replaced with the same or different substituent(s).
  • Typical substituents include, but are not limited to, —M, —R 60 , —O ⁇ , ⁇ O, —OR 60 , —SR 60 , —S ⁇ , ⁇ S, —NR 60 R 61 , ⁇ NR 60 , —CF 3 , —CN, —OCN, —SCN, —NO, —NO 2 , ⁇ N 2 , —N 3 , —S(O) 2 O; —S(O) 2 OH, —S(O) 2 R 60 , —OS(O 2 )O ⁇ , —OS(O) 2 R 60 , —P(O)(O ⁇ ) 2 , —P(O)(OR 60 )(O ⁇ ), —OP(O)(OR 60 )(OR 61 ),
  • substituents include —M, —R 60 , ⁇ O, —OR 60 , —SR 60 , —S ⁇ , ⁇ S, —NR 60 R 61 , ⁇ NR 60 , —CF 3 , —CN, —OCN, —SCN, —NO, —NO 2 , ⁇ N 2 , —N 3 , —S(O) 2 R 60 , —OS(O 2 )O ⁇ , —OS(O) 2 R 60 , —P(O)(O ⁇ ) 2 , —P(O)(OR 60 )(O ⁇ ), —OP(O)(OR 60 )(OR 61 ), —C(O)R 60 , —C(S)R 60 , —C(O)OR 60 , —C(O)NR 60 R 61 ,—C(O)O ⁇ , —NR 62 C(O)NR 60 R 61 ,
  • Treating” or “treatment” of any disease or disorder refers, in one embodiment, to ameliorating the disease or disorder (i.e., arresting or reducing the development of the disease or at least one of the clinical symptoms thereof). In another embodiment “treating” or “treatment” refers to ameliorating at least one physical parameter, which may not be discernible by the patient. In yet another embodiment, “treating” or “treatment” refers to inhibiting the disease or disorder, either physically, (e.g., stabilization of a discernible symptom), physiologically, (e.g., stabilization of a physical parameter), or both. In yet another embodiment, “treating” or “treatment” refers to delaying the onset of the disease or disorder.
  • “Therapeutically effective amount” means the amount of a compound that, when administered to a patient for treating a disease, is sufficient to effect such treatment for the disease.
  • the “therapeutically effective amount” will vary depending on the compound, the disease and its severity and the age, weight, etc., of the patient to be treated.
  • the present invention satisfies these and other needs by providing peptide analogs of Ac—PHSCN—NH 2 which target tumor and endothelial cells and have anti-tumor, anti-angiogenic and anti-metasastic activity, methods of making these peptides, compositions thereof and methods of using these peptides and pharmaceutical compositions thereof to treat, prevent and detect diseases characterized by tumor growth, metastasis and angiogenesis.
  • the peptide analogs may serve, inter alia, as carriers of radioactivity, PET-active compounds, toxins, fluorescent molecules and PEG molecules.
  • the present invention provides a compound of Formula (I):
  • R 1 is not acetyl when R 4 and R 5 are hydrogen.
  • p and q are independently an integer between and including 1 and 50. In another embodiment, p and q are independently an integer between and including 1 and 25. In still another embodiment, p and q are independently an integer between and including 1 and 10. In still another embodiment, p and q are independently an integer between and including 1 and 5. In still another embodiment, p and q are independently an integer between and including 1 and 3.
  • p and q are independently an integer between and including 0 and 50. In another embodiment, p and q are independently an integer between and including 0 and 25. In still another embodiment, p and q are independently an integer between and including 0 and 10. In still another embodiment, p and q are independently an integer between and including 0 and 5. In still another embodiment, p and q are independently an integer between and including 0 and 3.
  • s is 0 and r is 1. In still another embodiment, s is 1 and r is 0. In still another embodiment, at least one of s and r is not 0.
  • R 1 is acyl, substituted acyl, acyl chelate, imino or substituted imino.
  • R 2 is C 1 -C 6 alkyl with at least one hydrogen atom replaced by a substituent selected from the group consisting of —NR 6 R 7 , —OR 8 and —CO 2 R 9 .
  • R 6 , R 7 , R 8 , R 9 , R 10 , R 11 and R 12 are independently selected from the group consisting of hydrogen, acyl, substituted acyl, acyl chelate, imino or substituted imino.
  • X 1 is —NHCH 2 CO— or —NHCH(CH 3 )CO—.
  • X 2 is
  • l is 1. In still another embodiment, n is 1 or 2. In still another embodiment, m is 1 or 2.
  • R 3 is C 1 -C 6 alkyl with at least one hydrogen atom replaced by a substituent selected from the group consisting of —NR 15 R 16 , —OR 17 and —CO 2 R 18 .
  • R 15 , R 16 , R 17 , R 18 , R 19 , R 20 and R 21 are independently selected from the group consisting of hydrogen, acyl, substituted acyl, acyl chelate, imino or substituted imino.
  • compounds of the invention has the structure of Formula (II):
  • R 1 is
  • r 0, R 1 is acetyl, R 4 is hydrogen, R 14 is hydrogen, methyl or acetyl and R 5 is
  • r 0, R 4 and R 5 are hydrogen, R 14 is hydrogen, methyl or acetyl and R 1 is
  • r 0, R 4 and R 5 are hydrogen, R 14 is hydrogen, methyl or acetyl and R 1 is
  • r 0, R 4 and R 5 are hydrogen, R 14 is hydrogen, methyl or acetyl and R 1 is
  • r 0, R 4 and R 5 are hydrogen, R 14 is hydrogen, methyl or acetyl and R 1 is and M is Cu, Ga, 111 In or 90 Y.
  • r 0, R 4 and R 5 are hydrogen, R 14 is hydrogen, methyl or acetyl and R 1 is and M is Cu.
  • r 0, R 4 and R 5 are hydrogen, R 14 is hydrogen, methyl or acetyl and R 1 is and M is 111 In, 90 Y or Ga.
  • r 0, R 4 and R 5 are hydrogen, R 14 is hydrogen, methyl or acetyl, R 1 is and M is 111 In, 67 Ga or 68 Ga.
  • r 0, R 4 and R 5 are hydrogen, R 14 is hydrogen, methyl or acetyl and R 1 is
  • r 0, R 4 and R 5 are hydrogen, R 14 is hydrogen, methyl or acetyl and R 1 is
  • r 0, R 4 and R 5 are hydrogen, R 14 is hydrogen, methyl or acetyl and R 1 is
  • r 0, R 4 and R 5 are hydrogen, R 14 is hydrogen, methyl or acetyl and R 1 is
  • r 0, R 4 and R 5 are hydrogen, R 14 is hydrogen, methyl or acetyl and R 1 is
  • r 0, R 4 and R 5 are hydrogen, R 14 is hydrogen, methyl or acetyl and R 1 is
  • r 0, R 4 and R 5 are hydrogen, R 14 is hydrogen, methyl or acetyl and R 1 is
  • r 0, R 4 and R 5 are hydrogen, R 14 is hydrogen, methyl or acetyl and R 1 is
  • r 0, R 4 and R 5 are hydrogen, R 4 is hydrogen, methyl or acetyl and R 1 is
  • R 1 is acyl or substituted acyl
  • R 2 is C 1 -C 4 alkyl with at least one hydrogen atom replaced by a substituent selected from the group consisting of —NR 6 R 7 , aryl and substituted aryl
  • R 6 and R 7 are independently selected from the group consisting of hydrogen, acyl and substituted acyl
  • X 1 is —NH(CH 2 ) h CO—
  • X 2 is
  • X 4 is
  • X 5 is R 13 is hydrogen, acyl, substituted acyl, alkyl or substituted alkyl.
  • X 6 is X 7 is —NH(CH 2 ) e CO—
  • R 3 is C 1 -C 4 alkyl with at least one hydrogen atom replaced by a substituent selected from the group consisting of —NR 15 R 16 , aryl and substituted aryl
  • R 15 and R 16 are independently selected from the group consisting of hydrogen, acyl and substituted acyl and R 4 and R 5 are hydrogen.
  • s is 0 and r is 1
  • k is 1
  • R 1 is acetyl
  • R 13 is hydrogen
  • e is 1
  • R 3 is —(CH 2 ) 4 NH 2 —.
  • q is 2, 4 or 6.
  • s is 0 and r is 1, k is 1, R 1 is acetyl, R 13 is hydrogen, e is 2, 4 or 6 and R 3 is —CH 2 ) 4 NHCO(CH 2 ) 2 —Ph—(4—OH).
  • q is 1.
  • s is 0 and r is 1, k is 1, R 1 is acetyl, R 13 is hydrogen, e is 2, 4 or 6 and R 3 is —CH 2 —Ph—(4—OH).
  • q is 1.
  • s is 0 and r is 1, k is 1, R 1 is acetyl, R 13 is methyl, e is 1 and R 3 is —(CH 2 ) 4 NH 2 .
  • q is 2.
  • s is 1 and r is 0, j is 1, R 1 is acetyl, R 13 is hydrogen, h is 1 and R 2 is —CH 2 —Ph—(4—OH).
  • p is 2, 4 or 6.
  • s is 1 and r is 0, j is 1, R 1 is acetyl, R 13 is hydrogen, h is 2, 4, or 6 and R 2 is —CH 2 —Ph—(4—OH).
  • p is 1.
  • s is 1 and r is 0, j is 0, R 1 is —CO(CH 2 ) 2 —Ph—(4—OH), R 13 is hydrogen and h is 1.
  • p is 2, 4 or 6.
  • s is 1 and r is 0, j is 0, R 1 is —O(CH 2 ) 2 —Ph—(4—OH), R 13 is hydrogen and h is 2, 4 or 6.
  • p is 1.
  • s is 0 and r is 0, R 1 is —(CH 2 ) 2 —Ph—(4—OH) and R 13 is hydrogen.
  • s is 0 and r is 0, R 1 is —COPh—(4—F) and R 13 is hydrogen.
  • s is 0 and r is 1, k is 1, R 1 is acetyl, R 13 is methyl or hydrogen, e is 1 and R 3 is —CH 2 ) 4 NHCOPh—(4—F).
  • q is 2.
  • s is 0 and r is 1, k is 1, R 1 is acetyl, R 13 is hydrogen, e is 1 and R 3 is —(CH 2 ) 4 NH—8-[4 40 —fluorobenzylamino]suberoyl or —(CH 2 ) 4 NHCOCH 2 F.
  • q is 2.
  • s is 1 and r is 0, j is 0, R 1 is 8-[4′-fluorobenzylamino]suberoyl or —COCH 2 F, R 13 is hydrogen and h is 2.
  • p is 1.
  • s is 0 and r is 1, k is 1, R 1 is acetyl, R 13 is hydrogen and R 3 is —CH 2 Ph—(3-I, 4-OH) or —CH 2 Ph—(3,5-diI, 4-OH).
  • q is 0.
  • q is 1 and e is 2.
  • q is 1 and e is 1.
  • s is 1 and r is 0, j is 1, R 1 is acetyl, R 13 is hydrogen and R 2 is —CH 2 Ph—(3-I, 4-OH) or —CH 2 Ph—(3,5-diI, 4-OH).
  • p is 0.
  • s is 0 and r is 0, R 1 is —CO(CH 2 ) 2 Ph (4-OH, 3, 5 di-I) and R 13 is hydrogen.
  • s is 1 and r is 0, j is 0, R 1 is —CO(CH 2 ) 2 Ph (4-OH, 3, 5 di-I), h is 2 and R 13 is hydrogen.
  • p is 1.
  • s is 1 and r is 0, j is 1, R 1 is acetyl, R 2 is —CH 2 —Ph (4-OH, 3, 5 di-I), h is 2 and R 13 is hydrogen.
  • p is 1.
  • s is 0 and r is 1, R 3 is —(CH 2 ) 4 NHCO(CH 2 ) 2 —Ph (4-OH, 3, 5 di-I), e is 1 and R 13 is hydrogen.
  • q is 2.
  • R 1 is acyl chelate
  • R 2 , R 6 , R 7 , X 1 , X 2 , X 4 , X 5 , R 13 , X 6 , X 7 , R 3 , R 15 , R 16 , R 4 and R 5 are as defined in the first embodiment.
  • s is 1 and r is 0, j is 0, R 1 is DOTA-In, h is 2 and R 13 is hydrogen.
  • p is 1.
  • s is 0 and r is 0, R 1 is DPTA or DPTA-In and R 13 is hydrogen.
  • the present invention provides a compound of Formula (III):
  • R 20 is acyl, substituted acyl, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, imino, substituted imino or a diagnostic agent;
  • R 21 is C 1 -C 6 alkyl with at least one hydrogen atom replaced by a substituent selected from the group consisting of —NHR 22 ;
  • R 22 is hydrogen, acyl, substituted acyl, alkyl, substituted alkyl or a diagnostic agent
  • R 2 , X 1 , X 2 , X 3 , X 4 , X 5 , X 6 , X 7 , R 4 and R 5 are as defined in structural formula (I);
  • R 20 and R 22 are diagnostic agents.
  • R 2 , X 1 , X 2 , X 3 , X 4 , X 5 , X 6 , X 7 , R 4 and R 5 are as defined in the first preferred embodiment.
  • R 20 is a fluorescent agent.
  • R 20 is ⁇ fraction (5/6) ⁇ carboxy fluorescein, s is 1, r is 0, j is 0, e is 2 and p is 1.
  • R 22 is a fluorescent agent.
  • R 21 is (CH 2 ) 4 NH—
  • R 22 is— ⁇ fraction (5/6) ⁇ carboxy fluorescein, s is 0, r is 1, k is 1, e is 1 and q is 2.
  • R 21 is (CH 2 )4NH—
  • R 22 is biotin
  • s is 0, r is 1, k is 1, e is 1 and q is 2.
  • the present invention provides a compound of Formula (IV):
  • R 23 is acyl, substituted acyl, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, imino, substituted imino or a pegylating agent;
  • R 24 is C 1 -C 6 alkyl with at least one hydrogen atom replaced by a substituent selected from the group consisting of —NHR 28 wherein R 28 is hydrogen, acyl, substituted acyl, alkyl substituted alkyl or a pegylating agent; and
  • R 2 , X 1 , X 2 , X 3 , X 4 , X 5 , X 6 , X 7 , R 4 and R 5 are as defined in structural formula (I);
  • R 23 or R 28 is a pegylating agent.
  • R 2 , X 1 , X 2 , X 3 , X 4 , X 5 , X 6 , X 7 , R 4 and R 5 are as defined in the first preferred embodiment.
  • R 23 is m-dPEG, s is 1, r is 0, j is 0, h is 2 and p is 1.
  • the present invention provides a compound of Formula (V):
  • R 29 is C 1 -C 6 alkyl with at least one hydrogen atom replace by —NHR 32 ;
  • R 30 is acyl, substituted acyl, alkyl, substituted alkyl or a therapeutic agent.
  • R 31 is hydrogen, alkyl, substituted alkyl or a therapeutic agent
  • R 32 is hydrogen, acyl substituted acyl, alkyl, substituted alkyl or a therapeutic agent
  • R 2 , X 1 , X 2 , X 3 , X 4 , X 5 , X 6 , X 7 and R 4 and R 5 are as defined structural formula (I);
  • R 30 , R 31 and R 32 is a therapeutic agent.
  • R 2 , X 1 , X 2 , X 3 , X 4 , X 5 , X 6 , X 7 and R 4 are as defined in the first preferred embodiment
  • R 13 is methyl or acetyl
  • s is 0, r is
  • R 30 is acetyl
  • R 31 is a therapeutic agent
  • the therapeutic agent is doxorubicin.
  • R 13 is methyl or hydrogen
  • s is 0, r is 1
  • k is 1
  • e 1, q is 2
  • R 30 is acetyl
  • R 31 is hydrogen
  • R 29 is —CH 2 ) 4 NHR 32 .
  • R 32 is —CO(CH 2 ) 3 -doxorubicin or protoporphyrin.
  • variations of compounds invention includes, for example, the D-amino acids of the naturally occurring amino acids, ⁇ -alanine, 3-aminopropionic acid, 2,3 diaminopropionic acid, 4-aminobutyric acid, etc., sarcosine, orthinine, N-methyl glycine, citrulline, t-butyl alanine, homoarginine, etc. are within the scope of the present invention
  • One or amide bonds in the compounds of the invention may be optionally replaced by isosteres such as —CH 2 —NH—, —CH 2 —S—, —CH 2 —S(O)—, CH 2 —S(O) 2 —, —COCH 2 — —CH ⁇ CH—, CH(OH)CH 2 which are well known in the art (see, e.g., Spatola, “Chemistry and Biochemistry of Amino Acids, Peptides and Proteins,” B. Weinstein, (eds.), Marcel Dekker, New York, 1983; Spatola et al., Life Sci. 1986, 38:1243-1249; Almquist et al., J. Med. Chem.
  • the peptides of the invention may also contain peptide mimetics such as those described in Olson et al., J. Med. Chem. 1993, 36:3039 and Chorev et aL, Science 1979, 204:1210.
  • Covalent modifications of the compounds of the invention are within the scope of the current invention and may improve the solubility, absorption, biological half life, etc. Such modifications may be effected by selective reaction of specific amino acid residues with organic reagents. For example, histidine residues may be selectively reacted with diethylpyrocarbonate at pH 5.5-7 and p-bromophenacyl bromide at pH 6.0. Residues containing free amino groups may be selectively reacted with carboxylic acid anhydrides, imidoesters, pyridoxal phosphate, trinitrobenzenesulfonic acid, O-methylisourea, 2,4 pentanedione, glyoxylate, etc.
  • Arginyl residues may be selectively reacted with phenylglyoxal, and various diones. Glutaminyl and asparaginyl residues may be deaminated under mildly acidic conditions to provide the corresponding glutamyl and aspartyl residues. Proline and lysine may be selectively hydroxylated while serine and threonine residues may be selectively phosphorylated. The ⁇ -amino groups of histidine and lysine may be selectively methylated (Creighton, Proteins: Structure and Molecule Properties , W.H. Freeman & Co., San Francisco, pp. 79-86 (1983)).
  • bi-fnctional cross-linking agents e.g., 1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde, N-hydroxy-succinimide esters, esters of 4-azidosalicylic acid, homobifunctional imidoesters (e.g., disuccinimidyl esters such as 3,3′- dithiobis(succinimidylpropionate)), bifinctional maleimides (e.g., bis-N-maleimido-1,8-octane, etc.) may be used to link compounds with water-insoluble support matrices or other macromolecular carriers.
  • bi-fnctional cross-linking agents e.g., 1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde, N-hydroxy-succinimide esters, esters of 4-azidosalicylic acid, homobifunctional imidoesters
  • Photoactivatable agents such as methyl-3-[(p-azidophenyl) dithio]propioimidate may also be used to attach compounds with water-insoluble support matrices.
  • compounds may be directly reacted with reactive water-insoluble matrices (e.g., cyanogen bromide-activated carbohydrates).
  • the present invention also includes longer peptides comprised of repeating units of the amino acid sequences of the compounds of the invention.
  • the repeating unit of such a multimer is the amino acid sequence of a compound where a, b, x, y, and z are 1.
  • the repeating unit is the amino acid sequence of a compound of invention where only one of a, b, x, y, and z is 0 and the rest are 1.
  • a multimer may be comprised of either the same or different combinations of repeating units comprised of amino acid sequences of compounds of structural formula (I).
  • Such multimeric peptides can be made by either by chemical synthesis or by recombinant DNA techniques, followed by chemical modification of the cysteine residues.
  • the synthetic multimers Preferably, have 2 to 12 repeats, more preferably, 2 to 8 repeats of the core peptide sequence. Accordingly, the total number of amino acids in the multimer should not exceed about 110 residues (or the equivalents, when including linkers or spacers).
  • a preferred multimer has the formula P 1 n . where P 1 is a pentapeptide, n is 2 to 8.
  • a multimer has the formula (P 1 -X m ) n -P 2 where P 1 and P 2 are pentapeptides.
  • P 1 and P 2 may be the same or different and each P 1 may represent a different pentapeptide derivative of structural formula (I).
  • the multimer may be optionally functionalized at both the N- and C-termini.
  • Compounds of the invention may be modified by the covalent attachment of any type of molecule as long as the modification does not prevent or inhibit biological function (i.e., inhibition or prevention of angiogenesis, cell invasion, cell proliferation, etc.).
  • a compound of the invention may be modified by glycosylation, acetylation, pegylation, phosphorylation, amidation, proteolytic cleavage, linkage to cellular ligand or protein, etc.
  • compounds of the invention are conjugated to a therapeutic agent or a diagnostic agent either directly or through a linking moiety.
  • the linking moiety is first attached to a diagnostic or therapeutic agent to form a linking moiety intermediate which is then further attached to a compound of structural formula (I).
  • the linking moiety can also be first attached to a compound of the invention to form a linking moiety intermediate which can then be attached to a diagnostic agent or therapeutic agent.
  • a linking moiety will include a linker and a linking group for conjugating a therapeutic agent or diagnostic agent to a peptide.
  • the nature of the linker will depend upon the particular application and the type of conjugation desired as the linker may be hydrophilic or hydrophobic, long or short, rigid or flexible.
  • the linker may be optionally substituted with one ore more linking groups which may be either the same or different, accordingly providing polyvalent linking moieties which are capable of conjugating multiple therapeutic agents or diagnostic agents with a antibody.
  • linkers comprised of stable bonds suitable for spacing linking groups from the amino nitro compound are known in the art, and include by way of example and not limitation, alkyl, heteroalkyl, acyclic heteroatomic bridges, aryl, arylaryl, arylalkyl, heteroaryl, heteroaryl-heteroaryl, substituted heteroaryl-heteroaryl, heteroarylalkyl, heteroaryl-heteroalkyl and the like.
  • the linker may include single, double, triple or aromatic carbon-carbon bonds, nitrogen-nitrogen bonds, carbon-nitrogen, carbon-oxygen bonds and/or carbon-sulfur bonds.
  • finctionalities such as carbonyls, ethers, thioethers, carboxarnides, sulfonamides, ureas, urethanes, hydrazines, etc. may be included in a linker.
  • linker may be rigid polyunsaturated alkyl or an aryl, biaryl, heteroaryl, etc.
  • the linker may be a flexible peptide such as Gly-Gly-Gly or a flexible saturated alkanyl or heteroalkanyl.
  • Hydrophilic linkers may be, for example, polyalcohols or polyethers such as polyalkyleneglycols.
  • Hydrophobic linkers may be, for example, alkyls or aryls.
  • a linking group is capable of mediating formation of a covalent bond with complementary reactive functionality of, for example, peptide to provide the therapeutic agent or diagnostic agent conjugated to the peptide.
  • the linking group may be any reactive functional group known to those of skill in the art that will react with common chemical groups found in peptides (e.g., amino, sulfhydryl, hydroxyl, carboxylate, imidizaloyl, guandinium, amide, etc.).
  • the linking group may be, for example, a photochemically activated group, an electrochemically activated group, a free radical donor, a free radical acceptor, a nucleophilic group or an electrophilic group.
  • the linking group may be —NHR 1 , —NH 2 , —OH, —SH, halogen, —CHO, —R 1 CO, —SO 2 H, —PO 2 H, —N 3 , —CN, —CO 2 H, —SO 3 H, —PO 3 H, —PO 2 (O R 1 )H, —CO 2 R 1 , —SO 3 R 1 or —PO(OR 1 ) 2 where R 1 is alkyl.
  • the linking group is —NHR 1 , —NH 2 , —OH, —SH, —CHO, —CO 2 H, R 1 CO—, halogen and —CO 2 R 1 .
  • linker and the linking group include, for example, compounds where the linker is —(CH 2 ) n —, n is an integer between 1 and 8, the linking group is —NH 2 , —OH, —CO 2 H, and —CO 2 R 1 and the corresponding analogues where any suitable hydrogen is substituted.
  • Other embodiments of the linking moiety include any amino acid, which may be, for example, a D or L amino acid.
  • the linking moiety may be a dipeptide, a tripeptide or a tetrapeptide comprised of any combination of amino acids.
  • the polarity of the peptide bond in these peptides may be either C—N or N—C.
  • Therapeutic agents and diagnostic agents may be linked to peptides directly using a variety of conventional reactions known to the skilled artisan.
  • condensation reagents e.g., carbodiimides, carbonyldiimidazoles, etc.
  • residues such as glutamic acid, aspartic acid and the C-terminal carboxyl group of a compound of structural formula (I).
  • diagnostic agents and therapeutic agents containing a linker and linking group may be attached to the amino group of lysine, the carboxylic acid groups of glutamic acid and aspartic acid, the sulfhydryl group of cysteine, the hydroxyl groups of threonine and serine and the various moieties of aromatic amino acids of peptides using conventional approaches known to the skilled artisan.
  • selection of an appropriate strategy for conjugating diagnostic agents or therapeutic agents to a peptide either directly or through a linker and linking group is well within the ambit of the skilled artisan.
  • Therapeutic agents which can be conjugated to peptides include, but are not limited to, radionuclides, porphyrins and porphyrin derivatives for photodynamic therapy (e.g., protoporphyrin, benzoporphyrin derivative monoacid A, tin-etio purpurin, meta-tetrahydroxyphenylchlorin, HPD, photofrin, protoporhyrin IX, Pc4, mono aspartyl chlorin e 6 , for others see T. Hassan et al, “PhotoDynamic Therapy of Cancer” in Cancer medicine, fiflh edition, R. C. Blast et al., Ed., B. C. Decker Inc, Canada, 2000, p.
  • photodynamic therapy e.g., protoporphyrin, benzoporphyrin derivative monoacid A, tin-etio purpurin, meta-tetrahydroxyphenylchlorin, HPD, photofrin, pro
  • protein toxins e.g., ricin, Pseudomonas exotoxin, diptheria toxin, saporin, pokeweed antiviral protein, bouganin, etc.
  • cytotoxic cancer agents e.g., camptothecins (e.g., 9-nitrocamptothecin (9NC), 9-aminocamptothecin (9AC), 10-aminocamptothecin, 9-chlorocamptothecin, 10,11-methylendioxycamptothecin, irinothecin, aromatic camptothecin esters, alkyl camptothecin esters, topotecan, (1S,9S)-1-amino-9-ethyl-5-fluoro-2,3-dihydro-9-hydroxy4-methyl-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1 ,2-b]quinoline-10,13(9H,15H)-d
  • the therapeutic agent is a cytotoxic cancer agent, such as, for example, a taxane, a camptothecin, an epithilone or a anthracycline.
  • the therapeutic agent is doxorubicin.
  • the therapeutic agent is a radionuclide.
  • peglyating agents include, but are not limited to, a-methoxy-w-carboxy-PEG 2K & 5K1, a-methoxy-w-N-succinimidylglutarate-PEG 2K & 5K1, a-methoxy-w-glutarate-PEG 2K, 5K, 20K, 30K2, a-methoxy-w-GGGglutarate-PEG 2K & 5K1, mPEG-Succinimidyl propionate 2K, 5K, 20K, 30K2 and m-PEG-ButyrALD 2K, 5K, 20K, 30K2 (for other peglyating agents see Li et al., Biomacromolecules, 2003, 4, 1055.1067). Common pegylating agents are also available from commercial supplies such as Nektar Therapeutics, San Carlos, Calif. Methods for attachment of various PEG groups
  • diagnostically labeled means that a peptide has an attached diagnostically detectable label.
  • labels include but are not limited to, radioactive isotopes, paramagnetic isotopes, compounds which can be imaged by positron emission tomography (PET), fluorescent or colored compounds, compounds which can be imaged by magnetic resonance, chemiluminescent compounds, bioluminescent compounds, etc.
  • Suitable detectable labels include, but are not limited to, radioactive, fluorescent, fluorogenic or chromogenic labels.
  • Useful radiolabels (radionuclides) which are detected simply by gamma counter, scintillation counter or autoradiography include, but are not limited to, 3 H, 125 I, 131 I, 35 S and 14 C.
  • the metals are preferably detectable metal atoms, including radionuclides, and are complexed to proteins and other molecules (See, e.g., U.S. Pat. Nos. 5,627,286, 5,618,513, 5,567,408, 5,443,816 and 5,561,220).
  • Common fluorescent labels include, but are not limited to, fluorescein, rhodamine, dansyl, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine (Haugland, Handbook of Fluorescent Probes and Research Chemicals , Sixth Ed., Molecular Probes, Eugene, Or., 1996) may be used to label compounds of structural formula (I). Fluorescein, fluorescein derivatives and fluorescein-like molecules such as Oregon GreenTM and its derivatives, Rhodamine GreenTM and Rhodol GreenTM, are coupled to amine groups using the isothiocyanate, succinimidyl ester or dichlorotriazinyl-reactive groups.
  • fluorophores may also be coupled to thiols using maleimide, iodoacetamide, and aziridine-reactive groups.
  • the long wavelength rhodamines which are basically Rhodamine GreenTM derivatives with substituents on the nitrogens are preferred labeling reagents. This group includes the tetramethylrhodamines, X-rhodamines and Texas RedTM derivatives.
  • Other preferred fluorophores are those excited by ultraviolet light. Examples include, but are not limited to, cascade blue, coumarin derivatives, naphthalenes (of which dansyl chloride is a member), pyrenes and pyridyloxazole derivatives.
  • Inorganic materials such as semiconductor nanocrystals (Bruchez, et al., 1998, Science 281:2013-2016) and quantum dots, e.g., zinc-sulfide-capped Cd selenide (Chan, et al., Science 1998, 281:2016-2018) may also be used as diagnostic labels.
  • Peptides can also be labeled with fluorescence-emitting metals such as 152 Eu or others of the lanthanide series. These metals can be attached to compounds of structural formula (I) through acyl chelating groups such as
  • DTPA diethylenetriaminepentaacetic acid
  • EDTA ethylene-diamine-tetraacetic acid
  • Radionuclides may be attached to peptides either directly or indirectly using an acyl chelating group such as DTPA and EDTA for in vivo diagnosis.
  • an acyl chelating group such as DTPA and EDTA
  • the chemistry of chelation is well known in the art and varying ranges of chelating agent to peptide may be used to provide the labeled peptide.
  • the labeled peptide must retain the biological activity of the native peptide.
  • radionuclide having diagnostic or therapeutic value can be used as the radiolabel in the present invention.
  • the radionuclide is a y -emitting or beta -emitting radionuclide, for example, one selected from the lanthanide or actinide series of the elements.
  • Positron-emitting radionuclides e.g 68 Ga or 64 Cu, may also be used.
  • Suitable gamma -emitting radionuclides include those which are useful in diagnostic imaging applications.
  • the gamma -emitting radionuclides preferably have a half-life of from 1 hour to 40 days, preferably from 12 hours to 3 days. Examples of suitable gamma -emitting radionuclides include 67 Ga, 111 In, 99m Tc, 169Yb and 186 Re. Most preferably, the radionuclide is 99m Tc.
  • radionuclides are 67 Cu, 67 Ga, 68 Ga, 72 As, 89 Zr, 90 Y, 97 Ru, 99 Tc, 111 In, 123 I, 125 I, 131 I, 169 Yb, 186 Re, and 201 Tl. Though limited work have been done with positron-emitting radiometals as labels, certain proteins, such as transferrin and human serum albumin, have been labeled with 68 Ga.
  • a number of metals (not radioisotopes) useful for magnetic resonance imaging include gadolinium, manganese, copper, iron, gold and europium. Gadolinium is most preferred.
  • the amount of labeled peptide needed for detectability in diagnostic use will vary depending on considerations such as age, condition, sex, and extent of disease in the patient, contraindications, if any, and other variables, and is to be adjusted by the individual physician or diagnostician. Dosage can vary from 0.01 mg/kg to 100 mg/kg.
  • Peptides may also be detected by coupling to a phosphorescent or a chemiluminescent compound, as is well known to the skilled artisan.
  • Preferred chemiluminescent compounds include but are not limited to, luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester.
  • bioluminescent compounds may be used to detect antibodies and/or conjugates thereof and include, but are not limited to, luciferin, luciferase and aequorin.
  • Colorimetric detection based on chromogenic compounds which have, or result in, chromophores with high extinction coefficients may also be used to detect compounds of structural formula (I).
  • the compounds of the invention may be obtained via conventional synthetic methods.
  • Starting materials useful for preparing compounds of the invention and intermediates thereof are commercially available or can be prepared by well-known synthetic methods.
  • Peptides may be prepared using solid-phase synthesis such as that generally described by Merrifield, J. Amer. Chem. Soc. 1963, 85:2149-54 using automated equipment, which may be purchased from chemical suppliers (e.g., Applied Biosystems, Foster City, Calif.) or manual equipment.
  • Solid-phase peptide synthesis may be initiated from the C-terminus of the peptide by coupling a protected ⁇ -amino acid (either Boc or FMOC protected), to a suitable resin.
  • a starting material can be prepared by attaching an ⁇ -amino-protected amino acid by an ester linkage to a chloromethylated resin, hydroxymethyl resin, BHA resin, MBHA resin or a Rink resin.
  • transwells are coated with type I collagen (50 ⁇ g/mL) by adding 200 ⁇ L of the collagen solution per transwell, then incubating overnight at 37° C.
  • the transwells are assembled in a 24-well plate and a chemoattractant (e.g., FGF-2) is added to the bottom chamber in a total volume of 0.8 mL media.
  • ECs such as human umbilical vein endothelial cells (HUVEC), which have been detached from monolayer culture using typsin, are diluted to a final concentration of about 10 6 cells/niL with serum-free media and 0.2 mL of this cell suspension is added to the upper chamber of each transwell.
  • a chemoattractant e.g., FGF-2
  • Inhibitors to be tested may be added to both the upper and lower chambers and the migration is allowed to proceed for 5 hrs in a humidified atmosphere at 37° C.
  • the transwells are removed from the plate stained using DiffQuik®.
  • Cells which did not migrate are removed from the upper chamber by scraping with a cotton swab and the membranes are detached, mounted on slides, and counted under a high-power field (400x) to determine the number of cells migrated.
  • Matrigel® is a reconstituted basement membrane containing type IV collagen, laminin, heparan sulfate proteoglycans such as perlecan, which bind to and localize bFGF, vitronectin as well as transforming growth factor- ⁇ (TGF ⁇ ), urokinase-type plasminogen activator (uPA), tissue plasminogens activator (tpA) and the serpin known as plasminogen activator inhibitor type 1 (PAI- 1) (Chambers et al., Canc. Res. 1995, 55:1578-1585).
  • TGF ⁇ transforming growth factor- ⁇
  • uPA urokinase-type plasminogen activator
  • tpA tissue plasminogens activator
  • PAI- 1 plasminogen activator inhibitor type 1
  • Such assays employ transwell tissue culture inserts.
  • Invasive cells are defined as cells which are able to traverse through the Matrigel® and upper aspect of a polycarbonate membrane and adhere to the bottom of the membrane.
  • Transwells (Costar) containing polycarbonate membranes (8.0 ⁇ m pore size) are coated with Matrigel® (Collaborative Research), which has been diluted in sterile PBS to a final concentration of 75 ⁇ g/mL (60 ⁇ L of diluted Matrigel® per insert), and placed in the wells of a 24-well plate.
  • the membranes are dried overnight in a biological safety cabinet, then rehydrated by adding 100 ⁇ L of DMEM containing antibiotics for 1 hour on a shaker table.
  • the DMEM is removed from each insert by aspiration and 0.8 mL of DMEM/10% FBS/antibiotics is added to each well of the 24-well plate such that it surrounds the outside of the transwell (“lower chamber”).
  • Fresh DMEM/antibiotics 100 ⁇ L
  • human Glu-plasminogen 5 ⁇ g/mL
  • any inhibitors to be tested are added to the top, inside of the transwell (“upper chamber”).
  • the cells which are to be tested are trypsinized and resuspended in DMEM/antibiotics, then added to the top chamber of the transwell at a final concentration of 800,000 cells/mL.
  • the final volume of the upper chamber is adjusted to 200 ⁇ L.
  • the assembled plate is then incubated in a humid 5% CO 2 atmosphere for 72 hours. After incubation, the cells are fixed and stained using DiffQuik® (Giemsa stain) and the upper chamber is then scraped using a cotton swab to remove the Matrigel® and any cells which did not invade through the membrane.
  • the membranes are detached from the transwell using an X-acto® blade, mounted on slides using Permount® and cover-slips, then counted under a high-powered (400x) field. An average of the cells invaded is determined from 5-10 fields counted and plotted as a function of inhibitor concentration.
  • Endothelial cells for example, human umbilical vein endothelial cells (HUVEC) or human microvascular endothelial cells (HMVEC) which can be prepared or obtained commercially, are mixed at a concentration of 2 ⁇ 10 5 cells/mL with fibrinogen (5 mg/mL in phosphate buffered saline (PBS) in a 1:1 (v/v) ratio.
  • fibrinogen 5 mg/mL in phosphate buffered saline (PBS) in a 1:1 (v/v) ratio.
  • Thrombin is added (5 units/mL final concentration) and the mixture is immediately transferred to a 24-well plate (0.5 mL per well).
  • the fibrin gel is allowed to form and then VEGF and bFGF are added to the wells (each at 5 ng/mL final concentration) along with the test compound.
  • the cells are incubated at 37° C.
  • results are expressed as the average of 5 different wells for each concentration of compound.
  • cells typically, in the presence of angiogenic inhibitors, cells remain either rounded or form undifferentiated tubes (e.g. 0 or 1 branch). This assay is recognized in the art to be predictive of angiogenic (or anti-angiogenic) efficacy in vivo (Min et aL, Cancer Res. 1996, 56: 2428-2433).
  • endothelial cell tube formation is observed when endothelial cells are cultured on Matrigel® (Schnaper et aL, J. Cell. PhysioL 1995, 165: 107-118). Endothelial cells (1 ⁇ 10 4 cells/well) are transferred onto Matrigel®-coated 24-well plates and tube formation is quantitated after 48 hrs. Inhibitors are tested by adding them either at the same time as the endothelial cells or at various time points thereafter. Tube formation can also be stimulated by adding (a) angiogenic growth factors such as bFGF or VEGF, (b) differentiation stimulating agents (e.g., PMA) or (c) a combination of these.
  • angiogenic growth factors such as bFGF or VEGF
  • differentiation stimulating agents e.g., PMA
  • this assay models angiogenesis by presenting to the endothelial cells a particular type of basement membrane, namely the layer of matrix which migrating and differentiating endothelial cells might be expected to first encounter.
  • the matrix components found in Matrigel® (and in basement membranes in situ) or proteolytic products thereof may also be stimulatory for endothelial cell tube formation which makes this model complementary to the fibrin gel angiogenesis model previously described (Blood et aL, Biochim. Biophys. Acta 1990, 1032: 89-118; Odedrat al., Pharmac. Ther. 1991, 49: 111-124).
  • the ability of the compounds of the invention to inhibit the proliferation of EC's may be determined in a 96-well format.
  • Type I collagen (gelatin) is used to coat the wells of the plate (0.1-1 mg/mL in PBS, 0.1 mL per well for 30 minutes at room temperature). After washing the plate (3x w/PBS), 3-6,000 cells are plated per well and allowed to attach for 4 hrs (37° C./5% CO 2 ) in Endothelial Growth Medium (EGM; Clonetics ) or M199 media containing 0.1-2% FBS.
  • the media and any unattached cells are removed at the end of 4 hrs and fresh media containing bFGF (1-10 nglmL) or VEGF (1-10 ng/mL) is added to each well.
  • Compounds to be tested are added last and the plate is allowed to incubate (37° C./5% CO 2 ) for 24-48 hrs.
  • MTS Promega
  • the absorbance at 490 nm, which is proportional to the cell number, is then measured to determine the differences in proliferation between control wells and those containing test compounds.
  • a similar assay system can be set up with cultured adherent tumor cells. However, collagen may be omitted in this fonnat.
  • Tumor cells e.g., 3,000-10,000/well
  • Serum free medium is then added to the wells, and the cells are synchronized for 24 hrs.
  • Medium containing 10% FBS is then added to each well to stimulate proliferation.
  • Compounds to be tested are included in some of the wells. After 24 hrs, MTS is added to the plate and the assay developed and read as described above.
  • the anti-proliferative and cytotoxic effects of compounds of the invention may be determined for various cell types including tumor cells, ECs, fibroblasts and macrophages. This is especially useful when testing a compound of the invention which has been conjugated to a therapeutic moiety such as a radiotherapeutic or a toxin.
  • a conjugate of one of the compounds of the invention with Bolton-Hunter reagent which has been iodinated with 131 I would be expected to inhibit the proliferation of cells expressing an PHSCN binding site/receptor (most likely by inducing apoptosis).
  • Anti-proliferative effects would be expected against tumor cells and stimulated endothelial cells but, under some circumstances not quiescent endothelial cells or normal human dermal fibroblasts. Any anti-proliferative or cytotoxic effects observed in the normal cells may represent non-specific toxicity of the conjugate.
  • a typical assay would involve plating cells at a density of 5-10,000 cells per well in a 96-well plate.
  • the compound to be tested is added at a concentration I Ox the IC 50 measured in a binding assay (this will vary depending on the conjugate) and allowed to incubate with the cells for 30 minutes.
  • the cells are washed 3X with media, then fresh media containing [ 3 H]thymidine (1 ⁇ Ci/mL) is added to the cells and they are allowed to incubate at 37° C. in 5% CO 2 for 24 and 48 hours.
  • Cells are lysed at the various time points using 1 M NaOH and counts per well determined using a ⁇ -counter.
  • Proliferation may be measured non-radioactively using MTS reagent or CyQuant® to measure total cell number.
  • MTS reagent or CyQuant® for cytotoxicity assays (measuring cell lysis), a Promega 96-well cytotoxicity kit is used. If there is evidence of anti-proliferative activity, induction of apoptosis may be measured using TumorTACS (Genzyme).
  • the ability of the compounds of the invention to promote apoptosis of EC's may be determined by measuring activation of caspase-3.
  • Type I collagen (gelatin) is used to coat a P100 plate and 5 ⁇ 10 5 ECs are seeded in EGM containing 10% FBS. After 24 hours (at 37° C. in5% CO 2 ) the medium is replaced by EGM containing 2% FBS, 10 ng/ml bFGF and the desired test compound. The cells are harvested after 6 hours, cell lysates prepared in 1% Triton and assayed using the EnzChek®Caspase-3 Assay Kit #1 (Molecular Probes) according to the manufactures instructions.
  • Neovascularization is assessed at 5 and 7 days after implantation. On day 7, animals are anesthetized and infused with a dye such as colloidal carbon to stain the vessels. The animals are then euthanized, the corneas fixed with formalin, and the corneas flattened and photographed to assess the degree of neovascularization. Neovessels may be quantitated by imaging the total vessel area or length or simply by counting vessels.
  • This assay is performed essentially as described by Passaniti et al., 1992, Lab Invest. 67:519-528. Ice-cold Matrigel® (e.g., 500 ⁇ L) (Collaborative Biomedical Products, Inc., Bedford, Mass.) is mixed with heparin (e.g., 50 ⁇ g/ml), FGF-2 (e.g., 400 ng/ml) and the compound to be tested. In some assays, bFGF may be substituted with tumor cells as the angiogenic stimulus.
  • the Matrigel® mixture is injected subcutaneously into 4-8 week-old athymic nude mice at sites near the abdominal midline, preferably 3 injections per mouse.
  • the injected Matrigel® forms a palpable solid gel. Injection sites are chosen such that each animal receives a positive control plug (such as FGF-2+heparin), a negative control plug (e.g., buffer+heparin) and a plug that includes the compound being tested for its effect on angiogenesis, e.g., (FGF-2+heparin+compound). All treatments are preferably run in triplicate. Animals are sacrificed by cervical dislocation at about 7 days post injection or another time that may be optimal for observing angiogenesis. The mouse skin is detached along the abdominal midline, and the Matrigel® plugs are recovered and scanned immediately at high resolution. Plugs are then dispersed in water and incubated at 37° C. overnight.
  • a positive control plug such as FGF-2+heparin
  • a negative control plug e.g., buffer+heparin
  • All treatments are preferably run in triplicate. Animals are sacrificed by cervical dislocation at about 7 days post injection or another time that may be optimal for
  • Hemoglobin (Hb) levels are determined using Drabkin's solution (e.g., obtained from Sigma) according to the manufacturers' instructions.
  • the amount of Hb in the plug is an indirect measure of angiogenesis as it reflects the amount of blood in the sample.
  • animals may be injected prior to sacrifice with a 0.1 ml buffer (preferably PBS) containing a high molecular weight dextran to which is conjugated a fluorophore.
  • the amount of fluorescence in the dispersed plug determined fluorimetrically, also serves as a measure of angiogenesis in the plug.
  • Staining with mAb anti-CD31 (CD31 is “platelet-endothelial cell adhesion molecule or PECAM”) may also be used to confirm neovessel formation and microvessel density in the plugs.
  • This assay is performed essentially as described by Nguyen et al., Microvascular Res. 1994, 47:3140.
  • a mesh containing either angiogenic factors (bFGF) or tumor cells plus inhibitors is placed onto the CAM of an 8-day old chick embryo and the CAM observed for 3-9 days after implantation of the sample.
  • Angiogenesis is quantitated by determining the percentage of squares in the mesh which contain blood vessels.
  • tumor cells for example 1 ⁇ 5 ⁇ 10 6 cells of the 3LL Lewis lung carcinoma or the rat prostate cell line MatLyLu, are mixed with Matrigel® and then injected into the flank of a mouse following the protocol described in Sec. B., above.
  • a mass of tumor cells and a powerful angiogenic response can be observed in the plugs after about 5 to 7 days.
  • the anti-tumor and anti-angiogenic action of a compound in an actual tumor environment can be evaluated by including it in the plug.
  • Measurement is then made of tumor weight, Hb levels or fluorescence levels (of a dextran-fluorophore conjugate injected prior to sacrifice).
  • the plugs are first homogenize with a tissue homogenizer.
  • Nude mice are inoculated with MDA-MB-231 cells (human breast carcinoma) and MatrigelTM (1 ⁇ 10 6 cells in 0.2 mL) s.c. in the right flank of the animals.
  • the tumors are staged to 200 mm 3 and then treatment with a test composition is initiated (100 ⁇ g/animal/day given q.d. IP).
  • Tumor volumes are obtained every other day and the animals are sacrificed after 2 weeks of treatment.
  • the tumors are excised, weighed and paraffin embedded. Histological sections of the tumors are analyzed by H and E, anti-CD31, Ki-67, TUNEL, and CD68 staining.
  • the compounds of the invention are also tested for inhibition of late metastasis using an experimental metastasis model (Crowley et al., Proc. Natl. Acad. Sci. USA 1993, 90 5021-5025). Late metastasis involves the steps of attachment and extravasation of tumor cells, local invasion, seeding, proliferation and angiogenesis.
  • Human prostatic carcinoma cells PC-3) transfected with a reporter gene, preferably the green fluorescent protein (GFP) gene, but as an alternative with a gene encoding the enzymes chloramphenicol acetyl-transferase (CAT), luciferase or LacZ, are inoculated into nude mice.
  • a reporter gene preferably the green fluorescent protein (GFP) gene
  • This approach permits utilization of either of these markers (fluorescence detection of GFP or histochemical calorimetric detection of enzymatic activity) to follow the fate of these cells.
  • Cells are injected, preferably iv, and metastases identified after about 14 days, particularly in the lungs but also in regional lymph nodes, femurs and brain. This mimics the organ tropism of naturally occurring metastases in human prostate cancer.
  • GFP-expressing PC-3 cells (1 ⁇ 10 6 cells per mouse) are injected iv into the tail veins of nude (nu/nu) mice. Animals are treated with a test composition at 100 ⁇ g/animal/day given q.d. IP.
  • Single metastatic cells and foci are visualized and quantitated by fluorescence microscopy or light microscopic histochemistry or by grinding the tissue and quantitative colorimetric assay of the detectable label.
  • the rat syngeneic breast cancer system employs Mat BIII rat breast cancer cells (Xing et al., Int. J. Cancer 1996, 67:423429).
  • Tumor cells for example, about 10 6 suspended in 0.1 mL PBS, are inoculated into the mammary fat pads of female Fisher rats.
  • a 14-day Alza osmotic mini-pump is implanted intraperitoneally to dispense the test compound.
  • the compound is dissolved in PBS (e.g., 200 mM stock), sterile filtered and placed in the minipump to achieve a release rate of about 4 mg/kg/day.
  • Control animals receive vehicle (PBS) alone or a vehicle control peptide in the minipump.
  • This tumor line arose spontaneously as carcinoma of the lung in a C57BL/6 mouse (Malave et al., J. Nat'l. Canc. Inst. 1979, 62:83-88). It is propagated by passage in C57BL/6 mice by subcutaneous (sc) inoculation and is tested in semiallogeneic C57BL/6 ⁇ DBA/2 F 1 mice or in allogeneic C3H mice. Typically six animals per group for subcutaneously (sc) implant, or ten for intramuscular (im) implant are used. Tumor may be implanted sc as a 2-4 mm fragment, or im or sc as an inoculum of suspended cells of about 0.5-2 ⁇ 10 6 -cells.
  • Treatment begins 24 hours after implant or is delayed until a tumor of specified size (usually approximately 400 mg) can be palpated.
  • the test compound is administered ip daily for 11 days Animals are followed by weighing, palpation, and measurement of tumor size. Typical tumor weight in untreated control recipients on day 12 after im inoculation is 500-2500 mg. Typical median survival time is 18-28 days.
  • a positive control compound, for example cyclophosphamide at 20 mg/kg/injection per day on days 1-11 is used. Results computed include mean animal weight, tumor size, tumor weight, survival time. For confirmed therapeutic activity, the test composition should be tested in two multi-dose assays.
  • Test mice are male C57BL/6 mice, 2-3 months old. Following sc, im, or intra-footpad implantation, this tumor produces metastases, preferentially in the lungs.
  • Single-cell suspensions are prepared from solid tumors by treating minced tumor tissue with a solution of 0.3% trypsin. Cells are washed 3 times with PBS (pH 7.4) and suspended in PBS. Viability of the 3LL cells prepared in this way is generally about 95-99% (by trypan blue dye exclusion). Viable tumor cells (3 ⁇ 10 4 -5 ⁇ 10 6 ) suspended in 0.05 ml PBS are injected subcutaneously, either in the dorsal region or into one hind foot pad of C57BL/6 mice.
  • Visible tumors appear after 3-4 days after dorsal sc injection of 10 6 cells. The day of tumor appearance and the diameters of established tumors are measured by caliper every two days. The treatment is given as one to five doses of peptide or derivative, per week. In another embodiment, the peptide is delivered by osmotic minipump.
  • mice are randomized into two groups: (1) primary tumor is completely excised; or (2) sham surgery is performed and the tumor is left intact. Although tumors from 500-3000 mm 3 inhibit growth of metastases, 1500 mm 3 is the largest size primary tumor that can be safely resected with high survival and without local regrowth. After 21 days, all mice are sacrificed and autopsied.
  • Lungs are removed and weighed. Lungs are fixed in Bouin's solution and the number of visible metastases is recorded. The diameters of the metastases are also measured using a binocular stereoscope equipped with a micrometer-containing ocular under 8 ⁇ magnification. On the basis of the recorded diameters, it is possible to calculate the volume of each metastasis. To determine the total volume of metastases per lung, the mean number of visible metastases is multiplied by the mean volume of metastases. To further determine metastatic growth, it is possible to measure incorporation of 125 IdUrd into lung cells (Thakur et al., J. Lab. Clin. Med. 1977, 89:217-228).
  • mice Ten days following tumor amputation, 25 ⁇ g of fluorodeoxyuridine is inoculated into the peritoneums of tumor-bearing (and, if used, tumor-resected mice). After 30 min, mice are given 1 ⁇ Ci of 125 IdUrd (iododeoxyuridine). One day later, lungs and spleens are removed and weighed, and a degree of 125 IdUrd incorporation is measured using a gamma counter.
  • mice with footpad tumors when tumors reach about 8-10 mm in diameter, mice are randomized into two groups: (1) legs with tumors are amputated after ligation above the knee joints; or (2) mice are left intact as nonamputated tumor-bearing controls. (Amputation of a tumor-free leg in a tumor-bearing mouse has no known effect on subsequent metastasis, ruling out possible effects of anesthesia, stress or surgery). Mice are killed 10-14 days after amputation. Metastases are evaluated as described above.
  • This assay is a surrogate marker of biological activity. Briefly, DU145 cell are harvested by brief incubation with trypsin, the peptide derivative to be tested (competitor) and standard are added and the suspension is agitated at 4° C. for 2 hours. The cells are pelleted, supernatant aspirated, PBS added and the process is repeated three times (washes). HPR-Streptavidin is added, allowed to bind followed by a second series of wash steps. The appropriate HRP substrate is added and color is developed within the previously defined linear part of the reaction.
  • the assay is carried out in a 96 well assay and takes place for 48 hours. Briefly, HUVECs (Cascade Biologicals) are cultured overnight in M200 containing 2% FBS. The following day, 3,000 cells are plated in each well of a gelatin-coated 96-well plate. The cells are allowed to adhere and spread for 4-6 hours, at which time the medium is replaced with fresh medium containing 2% FBS, 1 ng/mL FGF-2, and varying concentrations of specific test compounds. Cells are then cultured for an additional 48 hours, and relative cell numbers in each well are determined using the Cell Titer Aqueous Cell Proliferation Assay (Promega). The result will be an IC 50 that can be compared to a standard compound. This is an assay that reflects biological function of the peptide derivatives against their target cells.
  • peptide 2, 4, 8 and 10 ⁇ Ci for biodistribution and 10, 50, 100, and 200 ⁇ Ci for Gamma scintigraphy
  • mice Four doses of peptide (2, 4, 8 and 10 ⁇ Ci for biodistribution and 10, 50, 100, and 200 ⁇ Ci for Gamma scintigraphy) are injected via tail vein into tumor (B16F10 melanoma cells) bearing (200-300 mm 3 ) C57Bl/6 mice.
  • the lowest and highest dosages of peptides are tested first and second.
  • the dosage of peptide derivative to be tested may be modified based on the initial results obtained.
  • 1251 labeled peptide at a dose determined by the method of section 4.4.17 will be injected via tail vein into tumor bearing mice (200-300 mm 3 tumor). Three mice per time point will be anesthetized and imaged at 0.5 h, 1 h, 2 h, 4 h, 6 h, 24 h, and 48 h which will determine the optimal time for imaging. As a control, unlabelled peptide at a 100-fold molar excess will be co-injected with the 125 I labeled peptide to demonstrate equivalency of both species.
  • nucleic acid is synonymous with “polynucleotide” and is intended to include a gene, a cDNA molecule, an mRNA molecule, as well as a fragment of any of these such as an oligonucleotide, and further, equivalents thereof (explained more fully below). Sizes of nucleic acids are stated either as kilobases (kb) or base pairs (bp).
  • Protein size is stated as molecular mass in kilodaltons (kDa) or as length (number of amino acid residues). Protein size is estimated from PAGE, from sequencing, from presumptive amino acid sequences based on the coding nucleic acid sequence or from published amino acid sequences.
  • DNA molecules encoding the amino acid sequence corresponding to the peptide multimers of the present invention, or active variants thereof, can be synthesized by the polymerase chain reaction (PCR) (see, for example, U.S. Pat. No. 4,683,202) using primers derived the sequence of the protein disclosed herein.
  • PCR polymerase chain reaction
  • These cDNA sequences can then be assembled into a eukaryotic or prokaryotic expression vector and the resulting vector can be used to direct the synthesis of the fusion polypeptide or its fragment or derivative by appropriate host cells, for example COS or CHO cells.
  • nucleic acid as used herein is intended to include such fragments or equivalents.
  • the nucleic acid sequences of this invention can be DNA or RNA.
  • Prokaryotic or eukaryotic host cells transformed or transfected to express the multimers are within the scope of the invention.
  • the peptide multimer may be expressed in bacterial cells such as E. coli, insect cells ( baculovirus ), yeast, or mammalian cells such as Chinese hamster ovary cells (CHO) or human cells (which are preferred for human therapeutic use of the transfected cells).
  • Other suitable host are known to those skilled in the art.
  • yeast S. cerevisiae examples include pYepSec1 (Baldari et al., 1987, EMBO J. 6:229-234), pMFa (Kurjan et al. 1982 Cell 30:933-943), pJRY88 (Schultz et al., 1987, Gene 54:113-123), and pYES2 (Invitrogen Corporation, San Diego, Calif.).
  • Baculovirus vectors available for expression of proteins in cultured insect cells include the pAc series (Smith et al., 1983, Mol. Cell Biol. 3:2156-2165) and the pVL series (Lucklow et al., (1989) Virology 170:31-39).
  • COS cells Gluzman 1981 Cell 23:175-182
  • pCDM 8 Gluzman 1981 Cell 23:175-182
  • CHO dhfr-negative CHO
  • vectors such as pMT2PC (Kaufman et al., 1987, EMBO J. 6:187-195) for stable amplification/expression in mammalian cells.
  • the NS0 myeloma cell line (a glutamine synthetase expression system.) is available from Celltech Ltd.
  • Suitable vectors containing the desired coding and control sequences employs standard ligation and restriction techniques which are well understood in the art. Isolated plasmids, DNA sequences, or synthesized oligonucleotides are cleaved, tailored, and re-ligated in the form desired. The DNA sequences which form the vectors are available from a number of sources. Backbone vectors and control systems are generally found on available “host” vectors which are used for the bulk of the sequences in construction. For the pertinent coding sequence, initial construction may be, and usually is, a matter of retrieving the appropriate sequences from cDNA or genomic DNA libraries.
  • the entire gene sequence for genes of sizeable length may be prepared by synthesizing individual overlapping complementary oligonucleotides and filling in single stranded nonoverlapping portions using DNA polymerase in the presence of the deoxyribonucleotide triphosphates.
  • This approach has been used successfully in the construction of several genes of known sequence. See, for example, Edge, Nature 1981, 292:756; Nambair et al., Science 1984, 223:1299; and Jay, J. Biol. Chem. 1984, 259:6311.
  • Synthetic oligonucleotides are prepared by either the phosphotriester method as described by references cited above or the phosphoramidite method as described by Beaucage et al., Tetrahedron Lett. 1981, 22:1859; and Matteucci et al., J. Am. Chem. Soc. 1981, 103:3185 and can be prepared using commercially available automated oligonucleotide synthesizers. Kinase treatment of single strands prior to annealing or for labeling is achieved using well-known methods.
  • the components of the desired vectors can be excised and ligated using standard restriction and ligation procedures.
  • Site-specific DNA cleavage is performed by treating with the suitable restriction enzyme (or enzymes) under conditions which are generally understood in the art, and the particulars of which are specified by the manufacturer of these commercially available restriction enzymes. See, e.g., New England Biolabs, Product Catalog.
  • size separation of the cleaved fragments may be performed by polyacrylamide gel or agarose gel electrophoresis using standard techniques. A general description of size separations is found in Meth. Enzymol. (1980) 65:499-560.
  • any of a number of methods are used to introduce mutations into the coding sequence to generate variants if these are to be produced recombinantly. These mutations include simple deletions or insertions, systematic deletions, insertions or substitutions of clusters of bases or substitutions of single bases. Modifications of the DNA sequence are created by site-directed mutagenesis, a well-known technique for which protocols and reagents are commercially available (Zoller et al., Nucleic Acids Res. 1982, 10:6487-6500 and Adelman et al., DNA 1983,2:183-193)). The isolated DNA is analyzed by restriction and/or sequenced by the dideoxy nucleotide method of Sanger, Proc. Natl. Acad. Sci. USA 1977, 74:5463) as further described by Messing, et al., Nucleic Acids Res. 1981, 9:309, or by the method of Maxam et al., Meth. Enzymol., supra.
  • Vector DNA can be introduced into mammalian cells via conventional techniques such as calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, or electroporation. Suitable methods for transforming host cells can be found in Sambrook et al. supra and other standard texts.
  • a proteolytic cleavage site is introduced at the junction of the reporter group and the target protein to enable separation of the target protein from the reporter group subsequent to purification of the fusion protein.
  • Proteolytic enzymes for such cleavage and their recognition sequences include Factor Xa, thrombin and enterokinase.
  • a compound of the invention and/or a pharmaceutical composition thereof is administered to a patient, preferably a human, suffering from cancer or form a disease characterized by angiogenesis.
  • the compounds of the invention and/or pharmaceutical compositions thereof may be used to treat or prevent cancer or undesired angiogenesis.
  • cancers include any vascularized tumor, preferably, a solid tumor, including but not limited to, carcinomas of the lung, breast, ovary, stomach, pancreas, larynx, esophagus, testes, liver, parotid, bilary tract, colon, rectum, cervix, uterus, endometrium, kidney, bladder, prostrate, thyroid, squamous cell carcinomas, adenocarcinomas, small cell carcinomas, melanomas, gliomas, neuroblastomas, sarcomas (e.g., angiosarcomas, chondrosarcomas )).
  • a solid tumor including but not limited to, carcinomas of the lung, breast, ovary, stomach, pancreas, larynx, esophagus, testes, liver, parotid, bilary tract, colon, rectum, cervix, uterus, endometrium, kidney, bladder, prostrate
  • Disease characterized by undesired angiogenesis include, but are not limited to, arthritis, diabetes, arteriosclerosis, arteriovenous, malformations, corneal graft neovascularization, delayed wound healing, diabetic retinopathy, age related macular degeneration, granulations, burns, hemophilic joints, rheumatoid arthritis, hypertrophic scars, neovascular glaucoma, nonunion fractures, Osier Weber Syndrome, psoriasis, pyogenic, granuloma, retrolental fibroplasia, pterygium, scleroderma, trachoma, vascular adhesions, ocular neovascularization, parasitic diseases, hypertrophy following surgery, inhibition of hair growth, macular degeneration (including both wet and dry type), rheumatoid arthritis and osteoarthritis.
  • Also contemplated are methods for treating a patient having a disease or condition associated with undesired cell migration, invasion or proliferation comprising administering to the subject an therapeutically effective amount of a compound of the invention and/or a pharmaceutical composition thereof.
  • the patient has a tumor, and angiogenesis inhibition results in reduction in size or growth rate of the tumor or destruction of the tumor.
  • the subject is a human.
  • diseases or conditions against which the above methods may be effective include primary growth of a solid tumor, leukemia or lymphoma, tumor invasion, metastasis or growth of tumor metastases; benign hyperplasia; atherosclerosis, myocardial angiogenesis; post-balloon angioplasty vascular restinosis, neointima formation following vascular trauma, vascular graft restinosis, coronary collateral formation, deep venous thrombosis, ischemic limb angiogenesis; telangiectasia, pyogenic granuloma, corneal disease, rubeosis, neovascular glaucoma, diabetic and other retinopathy, retrolental fibroplasias, diabetic neovascularization, macular degeneration, endometriosis, arthritis, fibrosis associated with a chronic inflammatory condition, traumatic spinal cord injury including ischemia, scarring or fibrosis, lung fibrosis, chemotherapy-induced fibrosis
  • a preferred disease or condition to be treated by the above methods are tumor growth, invasion or metastasis, particularly brain tumors.
  • brain tumors are astrocytoma, anaplastic astrocytoma, glioblastoma, glioblastoma multiformae, pilocytic astrocytoma, pleiomorphic xanthoastrocytoma, subependymal giant cell astrocytoma, fibrillary astrocytoma, gemistocytic astrocytoma, protoplasmic astrocytoma, oligodendroglioma, anaplastic oligodendroglioma, ependymoma, anaplastic ependymoma, myxopapillary ependymoma, subependymoma, mixed oligoastrocytoma and malignant oligoastrocytoma.
  • the above methods may also be used to treat uterine diseases such as endometriosis and pathogenic ocular neovascularization associated with, or a cause of, proliferative diabetic retinopathy, neovascular age-related macular degeneration, retinopathy of prematurity, sickle cell retinopathy or retinal vein occlusion.
  • uterine diseases such as endometriosis and pathogenic ocular neovascularization associated with, or a cause of, proliferative diabetic retinopathy, neovascular age-related macular degeneration, retinopathy of prematurity, sickle cell retinopathy or retinal vein occlusion.
  • compounds of the invention and/or pharmaceutical compositions thereof are administered to a patient, preferably a human, as a preventative measure against various diseases or disorders characterized by undesired angiogenesis including cancer.
  • the compounds of the invention and/or pharmaceutical compositions thereof may be administered as a preventative measure to a patient having a predisposition for a disease characterized by undesired angiogenesis.
  • the compounds and/or pharmaceutical compositions of the invention may be used for the prevention of one disease or disorder and concurrently treating another (e.g., preventing arthritis while treating cancer).
  • a compound of the invention and/or a pharmaceutical composition thereof is administered to a patient, preferably a human, in a diagnostically effective amount to detect or image a disease such as those listed in Section 5.6 above.
  • compounds of the invention and/or pharmaceutical compositions thereof may be used to detect or image diseases or conditions associated with undesired cell migration, invasion or proliferation such as those listed above in Section 5.6 by administering to a subject an diagnostically effective amount of a compound of the invention and/or a pharmaceutical composition thereof.
  • Compounds of the invention may be diagnostically labeled and used, for example, to detect cell migration, cell invasion and cell proliferation.
  • the disposition of a compound of the invention during and after binding may be followed in vitro or in vivo by using an appropriate method to detect the label.
  • Diagnostically labeled compounds may be utilized in vivo for diagnosis and prognosis, for example, to image occult metastatic foci or for other types of in situ evaluations.
  • compounds of the invention may include bound linker moieties, which are well known to those of skill in the art
  • In situ detection of the labeled compound may be accomplished by removing a histological specimen from a subject and examining it by microscopy under appropriate conditions to detect the label.
  • histological methods such as staining procedures
  • the type of detection instrument available is a major factor in selecting a radionuclide.
  • the radionuclide chosen must have a type of decay which is detectable by a particular instrument.
  • any conventional method for visualizing diagnostic imaging can be utilized in accordance with this invention.
  • Another factor in selecting a radionuclide for in vivo diagnosis is that its half-life be long enough so that the label is still detectable at the time of maximum uptake by the target tissue, but short enough so that deleterious irradiation of the host is minimized.
  • a radionuclide used for in vivo imaging does not emit particles, but produces a large number of photons in a 140-200 keV range, which may be readily detected by conventional gamma cameras.
  • In vivo imaging may be used to detect occult metastases which are not observable by other methods.
  • Compounds of the present invention may be used in diagnostic, prognostic or research procedures in conjunction with any appropriate cell, tissue, organ or biological sample of a desired animal species.
  • biological sample any fluid or other material derived from the body of a normal or diseased subject, such as blood, serum, plasma, lymph, urine, saliva, tears, cerebrospinal fluid, milk, amniotic fluid, bile, ascites fluid, pus and the like.
  • a organ or tissue extract and a culture fluid in which any cells or tissue preparation from the subject has been incubated is also included within the meaning of this term.
  • an effective amount means an amount sufficient to be detected using the appropriate detection system e.g., magnetic resonance imaging detector, gamma camera, etc.
  • the minimum detectable amount will depend on the ratio of labeled compound specifically bound to a tumor (signal) to the amount of labeled compound either bound non-specifically or found free in plasma or in extracellular fluid.
  • the amount of a composition to be administered depends on the precise compound selected, the disease or condition, the route of administration, and the judgment of the skilled imaging professional. Generally, the amount of a compound needed for detectability in diagnostic use will vary depending on considerations such as age, condition, sex, and extent of disease in the patient, contraindications, if any, and other variables, and is to be adjusted by the individual physician or diagnostician. Dosage can vary from 0.01 mg/kg to 100 mg/kg.
  • the compounds the invention and/or pharmaceutical compositions thereof may be advantageously used in human medicine. As previously described in Section 4.6 above, compounds of the invention and/or pharmaceutical compositions thereof are useful for the treatment or prevention of various diseases or disorders such as cancer.
  • compounds of the invention and/or pharmaceutical compositions thereof When used to treat or prevent the above disease or disorders, compounds of the invention and/or pharmaceutical compositions thereof may be administered or applied singly, or in combination with other agents.
  • the compounds of the invention and/or pharmaceutical compositions thereof may also be administered or applied singly, in combination with other pharmaceutically active agents (e.g., other anti-cancer agents, other anti-angiogenic agents such as chelators as zinc, penicillamine, thiomolybdate etc.), including other compounds of the invention.
  • the current invention provides methods of treatment and prophylaxis by administration to a patient of a therapeutically effective amount of a compound and/or pharmaceutical composition of the invention.
  • the patient may be an animal, is more preferably, a mammal and most preferably, a human.
  • the present compounds and/or pharmaceutical compositions of the invention may be administered orally.
  • the compounds and/or pharmaceutical compositions of the invention may also be administered by any other convenient route, for example, by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.). Administration can be systemic or local.
  • Various delivery systems e.g., encapsulation in liposomes, microparticles, microcapsules, capsules, etc.
  • Methods of administration include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, oral, sublingual, intranasal, intracerebral, intravaginal, transdermal, rectally, by inhalation, or topically, particularly to the ears, nose, eyes, or skin.
  • the preferred mode of administration is left to the discretion of the practitioner, and will depend in-part upon the site of the medical condition. In most instances, administration will result in the release of the compounds and/or pharmaceutical compositions of the invention into the bloodstream.
  • This may be achieved, for example, and not by way of limitation, by local infusion during surgery, topical application, e.g., in conjunction with a wound dressing after surgery, by injection, by means of a catheter, by means of a suppository, or by means of an implant, said implant being of a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers.
  • administration can be by direct injection at the site (or former site) of cancer or arthritis.
  • Intraventricular injection may be facilitated by an intraventricular catheter, for example, attached to a reservoir, such as an Ommaya reservoir.
  • a compound and/or pharmaceutical composition of the invention may also be administered directly to the lung by inhalation.
  • a compound and/or pharmaceutical composition of the invention may be conveniently delivered to the lung by a number of different devices.
  • a Metered Dose Inhaler (“MDI”), which utilizes canisters that contain a suitable low boiling propellant, (e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or any other suitable gas) may be used to deliver compounds of the invention directly to the lung.
  • a suitable low boiling propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or any other suitable gas
  • a Dry Powder Inhaler (“DPI”) device may be used to administer a compound and/or pharmaceutical composition of the invention to the lung.
  • DPI devices typically use a mechanism such as a burst of gas to create a cloud of dry powder inside a container, which may then be inhaled by the patient.
  • DPI devices are also well known in the art.
  • a popular variation is the multiple dose DPI (“MDDPI”) system, which allows for the delivery of more than one therapeutic dose.
  • MDDPI devices are available from companies such as AstraZeneca, GlaxoWellcome, IVAX, Schering Plough, SkyePharma and Vectura.
  • capsules and cartridges of gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of a compound of the invention and a suitable powder base such as lactose or starch for these systems.
  • liquid spray device Another type of device that may be used to deliver a compound and/or pharmaceutical composition of the invention to the lung is a liquid spray device supplied, for example, by Aradigm Corporation, Hayward, Calif.
  • Liquid spray systems use extremely small nozzle holes to aerosolize liquid drug formulations that may then be directly inhaled into the lung.
  • a nebulizer is used to deliver a compound and/or pharmaceutical composition of the invention to the lung.
  • Nebulizers create aerosols from liquid drug formulations by using, for example, ultrasonic energy to form fine particles that may be readily inhaled (see e.g., Verschoyle et al., British J. Cancer, 1999, 80, Suppl. 2, 96, which is herein incorporated by reference).
  • Examples of nebulizers include devices supplied by Batelle Pulmonary Therapeutics (Columbus, Ohio) (See, Armer et al., U.S. Pat. No.5,954,047; van der Linden et al., U.S. Pat. No.5,950,619; van der Linden et al., U.S. Pat. No. 5,970,974).
  • an electrohydrodynamic (“EHD”) aerosol device is used to deliver a compound and/or pharmaceutical composition of the invention to the lung.
  • EHD aerosol devices use electrical energy to aerosolize liquid drug solutions or suspensions (see e.g., Noakes et al., U.S. Pat. No. 4,765,539).
  • the electrochemical properties of the formulation may be important parameters to optimize when delivering a compound and/or pharmaceutical composition of the invention to the lung with an EHD aerosol device and such optimization is routinely performed by one of skill in the art.
  • EHD aerosol devices may more efficiently deliver drugs to the lung than existing pulmonary delivery technologies.
  • the compounds and/or pharmaceutical compositions of the invention can be delivered in a vesicle, in particular a liposome (see Langer, 1990, Science, 249:1527-1533; Treat et al., in “Liposomes in the Therapy of Infectious Disease and Cancer,” Lopez-Berestein and Fidler (eds.), Liss, New York, pp.353-365 (1989); see generally “Liposomes in the Therapy of Infectious Disease and Cancer,” Lopez-Berestein and Fidler (eds.), Liss, New York, pp.353-365 (1989)).
  • a liposome see generally “Liposomes in the Therapy of Infectious Disease and Cancer,” Lopez-Berestein and Fidler (eds.), Liss, New York, pp.353-365 (1989)).
  • the compounds and/or pharmaceutical compositions of the invention can be delivered via sustained release systems, preferably, oral sustained release systems.
  • a pump may be used (Langer, supra; Sefton, 1987, CRC Crit. Ref Biomed. Eng. 14:201; Saudek et al., 1989, N. Engl. J Med. 321:574).
  • polymeric materials can be used (see “Medical Applications of Controlled Release,” Langer and Wise (eds.), CRC Pres., Boca Raton, Fla. (1974); “Controlled Drug Bioavailability,” Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984); Langer t al., 1983, J Macromol. Sci. Rev. Macromol Chem. 23:61; Levy et al., 1985, Science 228: 190; During et al., 1989, Ann. Neurol. 25:351; Howard et al., 1989, J. Neurosurg. 71:105).
  • polymeric materials are used for oral sustained release delivery.
  • Preferred polymers include sodium carboxymethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose and hydroxyethylcellulose (most preferred, hydroxypropyl methylcellulose).
  • Other preferred cellulose ethers have been described (Alderman, Int. J. Pharm. Tech. & Prod. Mfr., 1984, 5(3) 1-9). Factors affecting drug release are well known to the skilled artisan and have been described in the art (Bamba et al., Int. J. Pharm., 1979, 2, 307).
  • enteric-coated preparations can be used for oral sustained release administration.
  • Preferred coating materials include polymers with a pH-dependent solubility (i.e., pH-controlled release), polymers with a slow or pH-dependent rate of swelling, dissolution or erosion (i.e., time-controlled release), polymers that are degraded by enzymes (i.e., enzyme-controlled release) and polymers that form firm layers that are destroyed by an increase in pressure (i.e., pressure-controlled release).
  • osmotic delivery systems are used for oral sustained release administration (Verma et al., Drug Dev. Ind. Pharm. 2000, 26:695-708).
  • OROSTM osmotic devices are used for oral sustained release delivery devices (Theeuwes et al., U.S. Pat. No. 3,845,770; Theeuwes et al., U.S. Pat. No.3,916,899).
  • a controlled-release system can be placed in proximity of the target of the compounds and/or pharmaceutical composition of the invention, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in “Medical Applications of Controlled Release,” supra, vol. 2, pp. 115-138 (1984)).
  • Other controlled-release systems discussed in Langer, 1990, Science 249:1527-1533 may also be used.
  • the present pharmaceutical compositions contain a therapeutically or diagnostically effective amount of one or more compounds of the invention, preferably, in purified form, together with a suitable amount of a pharmaceutically acceptable vehicle, so as to provide the form for proper administration to a patient.
  • a pharmaceutically acceptable vehicle When administered to a patient, the compounds of the invention and pharmaceutically acceptable vehicles are preferably sterile. Water is a preferred vehicle when the compounds of the invention are administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid vehicles, particularly for injectable solutions.
  • Suitable pharmaceutical vehicles also include excipients such as starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like.
  • excipients such as starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like.
  • the present pharmaceutical compositions can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.
  • auxiliary, stabilizing, thickening, lubricating and coloring agents may be used.
  • compositions comprising a compound of the invention may be manufactured by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.
  • Pharmaceutical compositions may be formulated in conventional manner using one or more physiologically acceptable carriers, diluents, excipients or auxiliaries, which facilitate processing of compounds of the invention into preparations which can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.
  • the present pharmaceutical compositions can take the form of solutions, suspensions, emulsion, tablets, pills, pellets, capsules, capsules containing liquids, powders, sustained-release formulations, suppositories, emulsions, aerosols, sprays, suspensions, or any other form suitable for use.
  • the pharmaceutically acceptable vehicle is a capsule (see e.g., Grosswald et al., U.S. Pat. No. 5,698,155).
  • suitable pharmaceutical vehicles have been described in the art (see Remington's Pharmaceutical Sciences, Philadelphia College of Pharmacy and Science, 19th Edition, 1995).
  • compounds of the invention may be formulated as solutions, gels, ointments, creams, suspensions, etc. as is well-known in the art.
  • Systemic formulations include those designed for administration by injection, e.g., subcutaneous, intravenous, intramuscular, intrathecal or intraperitoneal injection, as well as those designed for transdermal, transmucosal, oral or pulmonary administration.
  • Systemic formulations may be made in combination with a further active agent that improves mucociliary clearance of airway mucus or reduces mucous viscosity.
  • active agents include, but are not limited to, sodium channel blockers, antibiotics, N-acetyl cysteine, homocysteine and phospholipids.
  • the compounds of the invention are formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings.
  • compounds of the invention for intravenous administration are solutions in sterile isotonic aqueous buffer.
  • a compound of the invention may be formulated in aqueous solutions, preferably, in physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiological saline buffer.
  • the solution may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • the pharmaceutical compositions may also include a solubilizing agent.
  • Pharmaceutical compositions for intravenous administration may optionally include a local anesthetic such as lignocaine to ease pain at the site of the injection.
  • the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent.
  • a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent.
  • the compound of the invention When the compound of the invention is administered by infusion, it can be dispensed, for example, with an infusion bottle containing sterile pharmaceutical grade water or saline.
  • an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.
  • penetrants appropriate to the barrier to be permeated are used in the formulation.
  • penetrants are generally known in the art.
  • compositions for oral delivery may be in the form of tablets, lozenges, aqueous or oily suspensions, granules, powders, emulsions, capsules, syrups, or elixirs, for example.
  • Orally administered pharmaceutical compositions may contain one or more optionally agents, for example, sweetening agents such as fructose, aspartame or saccharin; flavoring agents such as peppermint, oil of wintergreen, or cherry coloring agents and preserving agents, to provide a pharmaceutically palatable preparation.
  • the compositions when in tablet or pill form, the compositions may be coated to delay disintegration and absorption in the gastrointestinal tract, thereby providing a sustained action over an extended period of time.
  • Selectively permeable membranes surrounding an osmotically active driving compound are also suitable for orally administered compounds of the invention.
  • fluid from the environment surrounding the capsule is imbibed by the driving compound, which swells to displace the agent or agent composition through an aperture.
  • delivery platforms can provide an essentially zero order delivery profile as opposed to the spiked profiles of immediate release formulations.
  • a time delay material such as glycerol monostearate or glycerol stearate may also be used.
  • Oral compositions can include standard vehicles such as mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Such vehicles are preferably of pharmaceutical grade.
  • suitable carriers, excipients or diluents include water, saline, alkyleneglycols (e.g., propylene glycol), polyalkylene glycols (e.g., polyethylene glycol) oils, alcohols, slightly acidic buffers between pH 4 and pH 6 (e.g., acetate, citrate, ascorbate at between about 5.0 mM to about 50.0 mM), etc.
  • alkyleneglycols e.g., propylene glycol
  • polyalkylene glycols e.g., polyethylene glycol
  • slightly acidic buffers between pH 4 and pH 6 e.g., acetate, citrate, ascorbate at between about 5.0 mM to about 50.0 mM
  • flavoring agents, preservatives, coloring agents, bile salts, acylcamitines and the like may be added.
  • the pharmaceutical compositions may take the form of tablets, lozenges, etc. formulated in conventional manner.
  • Liquid drug formulations suitable for use with nebulizers and liquid spray devices and EHD aerosol devices will typically include a compound of the invention with a pharmaceutically acceptable vehicle.
  • the pharmaceutically acceptable vehicle is a liquid such as alcohol, water, polyethylene glycol or a perfluorocarbon.
  • another material may be added to alter the aerosol properties of the solution or suspension of compounds of the invention.
  • this material is liquid such as an alcohol, glycol, polyglycol or a fatty acid.
  • Other methods of formulating liquid drug solutions or suspension suitable for use in aerosol devices are known to those of skill in the art (see, e.g., Biesalski, U.S. Pat. No. 5,112,598; Biesalski, U.S. Pat. No. 5,556,611).
  • a compound of the invention may also be formulated in rectal or vaginal pharmaceutical compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.
  • a compound of the invention may also be formulated as a depot preparation. Such long acting formulations may be administered by implantation (for example, subcutaneously or intramuscularly) or by intramuscular injection.
  • a compound of the invention may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
  • a compound of the invention when it is acidic, it may be included in any of the above-described formulations as the free acid or a pharmaceutically acceptable salt.
  • Pharmaceutically acceptable salts substantially retain the activity of the free acid, may be prepared by reaction with bases and tend to be more soluble in aqueous and other protic solvents than the corresponding free acid form.
  • a compound of the invention, or pharmaceutical compositions thereof will generally be used in an amount effective to achieve the intended purpose.
  • the amount of a compound of the invention that will be effective in the treatment, prevention or detection of a particular disorder or condition disclosed herein will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques known in the art as previously described. In addition, in vitro or in vivo assays may optionally be employed to help identify optimal dosage ranges.
  • the amount of a compound of the invention administered will, of course, be dependent on, among other factors, the subject being treated, the weight of the subject, the severity of the affliction, the manner of administration and the judgment of the prescribing physician.
  • the dosage may be delivered in a pharmaceutical composition by a single administration, by multiple applications or controlled release.
  • the compounds of the invention are delivered by oral sustained release administration.
  • the compounds of the invention are administered twice per day (more preferably, once per day). Dosing may be repeated intermittently, may be provided alone or in combination with other drugs and may continue as long as required for effective treatment of the disease state or disorder.
  • Suitable dosage ranges for oral administration are dependent on the potency of the drug, but are generally about 0.001 mg to about 200 mg of a compound of the invention per kilogram body weight. Dosage ranges may be readily determined by methods known to the artisan of ordinary skill.
  • Suitable dosage ranges for intravenous (i.v.) administration are about 0.01 mg to about 100 mg per kilogram body weight.
  • Suitable dosage ranges for intranasal administration are generally about 0.01 mg/kg body weight to about 1 mg/kg body weight.
  • Suppositories generally contain about 0.01 milligram to about 50 milligrams of a compound of the invention per kilogram body weight and comprise active ingredient in the range of about 0.5% to about 10% by weight.
  • Recommended dosages for intradermal, intramuscular, intraperitoneal, subcutaneous, epidural, sublingual or intracerebral administration are in the range of about 0.001 mg to about 200 mg per kilogram of body weight.
  • Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems. Such animal models and systems are well-known in the art.
  • the compounds of the invention are preferably assayed in vitro and in vivo, as described above, for the desired therapeutic, prophylactic or diagnostic activity, prior to use in humans.
  • in vitro assays can be used to determine whether administration of a specific compound of the invention or a combination of compounds of the invention is preferred for treating, preventing or diagnosing cancer.
  • the compounds of the invention may also be demonstrated to be effective and safe using animal model systems.
  • a therapeutically effective dose of a compound of the invention described herein will provide therapeutic benefit without causing substantial toxicity.
  • a diagnostically effective dose of a compound of the invention described herein will provide diagnostic benefit without causing substantial toxicity.
  • Toxicity of compounds of the invention may be determined using standard pharmaceutical procedures and may be readily ascertained by the skilled artisan.
  • the dose ratio between toxic and therapeutic effect is the therapeutic index.
  • a compound of the invention will preferably exhibit particularly high therapeutic indices in treating disease and disorders.
  • the dosage of a compound of the inventions described herein will preferably be within a range of circulating concentrations that include an effective therapeutic or diagnostic does dose with little or no toxicity.
  • the compounds and/or pharmaceutical compositions of the invention can be used in combination therapy with at least one other therapeutic agent.
  • the compound and/or pharmaceutical composition of the invention and the therapeutic agent can act additively or, more preferably, synergistically.
  • a compound and/or pharmaceutical composition of the invention is administered concurrently with the administration of another therapeutic agent, which may be part of the same pharmaceutical composition or a different pharmaceutical composition.
  • a pharmaceutical composition of the invention is administered prior or subsequent to administration of another therapeutic agent.
  • the compounds and/or pharmaceutical compositions of the invention can be used in combination therapy with other chemotherapeutic agents (e.g., alkylating agents (e.g., nitrogen mustards (e.g., cyclcphosphamide, ifosfamide, mechlorethamine, melphalen, chlorambucil, hexamethylmelamine, thiotepa), alkyl sulfonates (e.g., busulfan), nitrosoureas, triazines), antimetabolites (e.g., folic acid analogs, pyrimidine analogs (e.g., fluorouracil, floxuridine, cytosine arabinoside, etc.), purine analogs (e.g., mercaptopurine, thiogunaine, pentostatin, etc.), natural products (e.g., vinblastine, vincristine, etoposide, tertiposide, dact
  • alkylating agents
  • the current invention provides therapeutic kits comprising the compounds of the invention or pharmaceutical compositions of the invention.
  • the therapeutic kits may also contain other compounds (e.g., chemotherapeutic agents, natural products, hormones or antagonists, anti-angiogenesis agents or inhibitors, apoptosis-inducing agents or chelators) or pharmaceutical compositions of these other compounds.
  • Therapeutic kits may have a single containers which contains the compound of the invention or pharmaceutical compositions of the invention with or without other components (e.g., other compounds or pharmaceutical compositions of these other compounds) or may have distinct container for each component.
  • therapeutic kits of the invention include a compound and/or a pharmaceutical composition of the invention packaged for use in combination with the co-administration of a second compound (preferably, a chemotherapeutic agent, a natural product, a hormone or antagonist, a anti-angiogenesis agent or inhibitor, a apoptosis-inducing agent or a chelator) and/or a pharmaceutical composition thereof.
  • a second compound preferably, a chemotherapeutic agent, a natural product, a hormone or antagonist, a anti-angiogenesis agent or inhibitor, a apoptosis-inducing agent or a chelator
  • the components of the kit may be pre-complexed or each component may be in a separate distinct container prior to administration to a patient.
  • the components of the kit may be provided in one or more liquid solutions, preferably, an aqueous solution, more preferably, a sterile aqueous solution.
  • the components of the kit may also be provided as solids, which may be converted into liquids by addition of suitable solvents, which are preferably provided in another distinct container.
  • the container of a therapeutic kit may be a vial, test tube, flask, bottle, syringe, or any other means of enclosing a solid or liquid.
  • the kit will contain a second vial or other container, which allows for separate dosing.
  • the kit may also contain another container for a pharmaceutically acceptable liquid.
  • a therapeutic kit will contain apparatus (e.g., one or more needles, syringes, eye droppers, pipette, etc.), which enables administration of the components of the kit.
  • apparatus e.g., one or more needles, syringes, eye droppers, pipette, etc.
  • Rink Amide AM resin (Novabiochem) was treated with 20% piperidine in DMF (1 mL per 100 mg of resin) for three minutes with nitrogen agitation or vibration and the reaction mixture was filtered. This step was repeated an additional two times. The resin was washed three times with DMF, three times with methanol and three times with dichloromethane.
  • NMM N-Methylmorpholine
  • the resin was treated with 20% piperidine in DMF (3 ⁇ 3 minutes each) as described above to remove the Fmoc group on the first amino acid.
  • the next desired Fmoc protected, tritylated amino acid (3 eq), HBTU (3 eq), and HOBt (3 eq) were dissolved in DMF (1 mL per 100 mg of resin) and added to the above resin, followed by the addition of N-Methylmorpholine (NMM) (6 eq) and the mixture was agitated for 1 hour.
  • NMM N-Methylmorpholine
  • the reaction mixture was filtered and the resin was washed with DMF three times, MeOH three times and DCM three times. Subsequent amino acids were single coupled in a similar manner.
  • peptides containing an N-terminus acetyl the following commercially available amino acids, Ac-Pro-OH (3 eq) or Ac-Tyr-OH (3 eq) were used and coupled with HBTU (3 eq), HOBT (3 eq) and NMM (6 eq) for an hour.
  • the capping of the N-terminus was performed by coupling the terminal amine on the resin with the appropriate acid (3 eq), HBTU (3 eq), HOBT (3 eq) and NMM (6 eq).
  • the crude peptide was dissolved in a minimum amount of methanol and water or in a few drops of glacial acetic acid and water and was purified by preparative reverse phase HPLC (Beckman) with a Phenomenex Synergi hydro-RP C18 column (250 mm ⁇ 21.2 mm).
  • the peptide was eluted using a gradient from 3-100% B over 30 min with a flow rate of 20 mL/min, where solvent A was water containing 0.1% trifluoroacetic acid and solvent B was acetonitrile containing 0.1% trifluoroacetic acid. Detection was at 220 or 254 nm.
  • the resin was treated with 2% hydrazine in DMF (1 mL per 100 mg of resin) for three minutes with nitrogen agitation or vibration and the reaction mixture was filtered. This step was repeated an additional five times.
  • the resin was washed three times with DMF and three times with dichloromethane.
  • the desired carboxylic acid (4 eq), HBTU (4 eq) and HOBt (4 eq) were dissolved in DMF (1 mL per 100 mg of resin) and added to the above resin, followed by the addition of N-methylmorpholine (8 eq) and the mixture was agitated for 1 hour.
  • the reaction mixture was filtered and the resin was washed three times with DMF and three times with dichloromethane.
  • the resin was treated with TFA/TIS/water (95:2.5:2.5, 1 mL per 100 mg of resin) and agitated with nitrogen or vibration for 1 hour.
  • the reaction mixture was filtered, the resin was washed once with TFA/TIS/water, three times with dichloromethane and the filtrate was concentrated. The residue was triturated 4 times with diethyl ether.
  • Example 2 The procedure of Example 2 was used to cleave and purify the peptide and afforded 282 mg (46%) of a white solid, and as a mixture of two compounds in a ratio of 83:17: 1 H NMR (300 MHz, DMSO-d6) ⁇ 8.97 (s, 1H), 8.46-8.27 (m, 2H), 8.25-8.15 (m, 1H), 7.98-7.87 (m, 1H), 7.40-7.35 (m, 2H), 6.93 (s, 1H), 4.79-4.60 (m, 1H), 4.56-4.47 (m, 2H), 4.39-4.25 (m, 2H), 3.66-3.60 (m, 3H, overlapping with water peak), 3.22-3.12 (m, 2H), 3.05-2.94 (m, 1H), 2.91-2.82 (m, 1H), 2.68-2.41 (m, 3H, overlapping with DMSO peak), 2.07 (s, 3H), 2.00 (s, 3H), 1.91-1.
  • Triethylamine (17 ⁇ L, 0.12 mmol) was added to a solution of Ac-Pro-His-Ser-Cys(Me)-Asn-Gly-Gly-Lys-NH 2 (21 mg, 0.024 mmol) and 4-fluorobenzoyl succinimide (6.2 mg, 0.026 mmol) in 1 mL of DMF and the solution was stirred at room temperature for 3.5 hours.
  • This compound was prepared from Rink amide AM resin (0.0584 mmol) according to the procedures of Example 1 and 2, with the exceptions that the coupling of Fmoc-Cys(trt)-OH was performed using 3 equivalents of amino acid, 3 equivalents of HBTU, 3 equivalents of HOBt, and 6 equivalents of NMM, while the couplings of Fmoc-Ser(trt)-OH, Fmoc-His(trt)-OH, Fmoc-Pro-OH and 5-(&6-)carboxyfluorescein were performed using 2 equivalents of the acid, 2 equivalents of HBTU, 2 equivalents of HOBt, and 4 equivalents of NMM.
  • This compound was prepared from Ac-Pro-His(trt)-Ser(trt)-Cys(trt)-Asn(trt)-Gly-Gly-Lys(ivDde)-Rink amide AM resin (208 mg, loading of 0.32 mmol/g, 0.067 mmol) according to Procedure C.
  • This compound was prepared from Ac-Pro-His-Ser-Cys(Me)-Asn-OH (62 mg, 0.10 mmol) and doxorubicin hydrochloride (37 mg, 0.063 mmol) according to the procedure of Example 4.
  • This compound was prepared from Ac-Pro-His-Ser-Cys(Ac)-Asn-OH (52 mg, 0.081 mmol) and doxorubicin hydrochloride (33 mg, 0.057 mmol) according to the procedure of Example 3.
  • This compound was prepared from Ac-Pro-His(trt)-Ser(trt)-Cys(trt)-Asn(trt)-Gly-Gly-Lys(ivDde)-Rink amide AM resin (317 mg, loading of 0.32 mmol/g, 0.101 mmol) according to the procedure of Example 3.
  • This compound was prepared from Ac-Pro-His(trt)-Ser(trt)-Cys(trt)-Asn(trt)-Gly-Gly-Lys(ivDde)-Rink amide AM resin (312 mg, loading of 0.32 mmol/g, 0.0997 mmol) according to the procedure of Example 3.
  • the product (42.3 mg of crude material) was purified according to the procedure of Example 2 and afforded 3.9 mg (11%) of PLG-107 as a fluffy, white solid: ES MS m/z (M+H) + 1104.0.
  • This compound was prepared according to the procedures of Examples 1 and 2 with the following modification: capping of the N-terminus was carried out using N-succinimidyl-3-(4-hydroxyphenyl)propionate (4.0 eq) and TEA (12.0 eq). The title compound (43.0 mg, 38%) was isolated as a fine white powder.
  • the NMR data indicated a mixture of two species in a ratio of about 80:20: 1 H NMR (300 MHz, DMSO-d 6 ) ⁇ 9.12 (bs, 1 H),8.87-8.95 (m, 1 H), 8.09-8.39 (m, 4 H), 7.97 (app d, 1 H), 7.39 (s, 1 H), 7.33 (bs, 1 H), 6.99-7.09 (m, 4 H), 6.90-6.92 (2 H), 6.61-6.66 (m, 2 H), 5.09 (bs, 1 H), 4.59-4.77 (m, 2 H), 4.27-4.46 (m, 5 H), 3-57-3.70 (m, 3 H), 3.12-3.19 (m, 1 H), 2.97-3.02 (m, 1 H), 2.66-2.80 (m, 4 H), 2.37-2.45 (m, 2 H), 1.63-2.30 (m, 5 H); MS m/z (C 30 H 41 N 9 O 9 S+H) + 704.4; Anal. calcd
  • This compound was prepared according to the procedures of Examples 1 and 2 with the following modification: capping of the N-terminus was performed by doing a single coupling with 3-(4-hydroxyphenyl)propionic acid (3.0 eq). The title compound (23.9 mg, 23%) was isolated as a fine white powder.
  • This compound was prepared according to the procedures of Examples 1 and 2 with the following modification: only single couplings were performed with Fmoc- ⁇ -Ala-OH and Acetyl-Tyr(3,5-diiodo)-OH. The title compound was isolated as a fine white powder (55.0 mg, 51%).
  • the NMR data indicated a mixture of two species in a ratio of about 80:20: 1 H NMR (300 MHz, DMSO-d 6 ) ⁇ 9.35 (bs, 1 H), 8.96 (m, 1 H), 8.22-8.41 (m, 3 H), 7.97-8.14 (m, 4 H), 7.60-7.63 (m, 2 H), 7.39 (s, 1 H), 7.34 (bs, 1 H), 7.09 (app d, 2 H), 6.92 (s, 1 H), 4.60-4.78 (m, 2 H), 4.28-4.50 (m, 6 H), 3.59-3.71 (m, 2 H), 2.94-3.06 (m, 2 H), 2.72-2.83 (m, 3 H), 2.38-2.46 (m, 3 H), 1.76-2.09 (m, 7 H); MS m/z (C 35 H 47 I 2 N 11 O 11 S+H) + 1084.5; Anal. calcd for C 35 H 47 I 2 N 11 O 11 S: N, 14.22. Found: N
  • This compound was prepared according to the procedures of Examples 3 and 2 with the following modifications: the amide formation on lysine was carried out with 3-(4-hydroxy-3,4-diiodophenyl)propionic acid (4 eq), HBTU (4 eq), HOBT (4 eq) and NMM (8 eq). The title compound was isolated (16.2 mg, 13%) as a fine white powder.
  • This compound was prepared according to the procedures of Examples 1 and 2 with the following modification: capping of the N-terminus was carried out using 3-(4-hydroxy-3,5-diiodophenyl)propionic acid (4.0 eq), HBTU (4.0 eq), HOBT (4.0 eq) and NMM (8.0 eq). The title compound (23.3 mg, 32%) was isolated as a fine white powder.
  • This compound was prepared according to the procedures of Examples 2 and 3 with the following modifications: the amide formation on lysine was carried out with 5-(and 6)-carboxyfluorescein (3 eq), HBTU (3 eq), HOBT (3 eq) and NMM (6 eq). The title compound was isolated (10.5 mg, 18%) as a fine white powder.
  • This compound was prepared according to the procedures of Examples 1 and 2 with the following modification: capping of the N-terminus was carried out using 3-(4-hydroxy-3,5-diiodophenyl)propionic acid (3 eq), HBTU (3 eq), HOBT (3 eq) and NMM (6 eq). The title compound was isolated as a fine white powder (24.2 mg, 30%).
  • This compound was prepared according to the procedures of Examples 2 and 3 with the following modifications: the amide formation on lysine was carried out with 3-(4-hydroxyphenyl)propionic acid (4 eq), HBTU (4 eq), HOBT (4 eq) and NMM (8 eq). The title compound was isolated (16.2 mg, 13%) as a fine white powder.
  • This compound was prepared according to the procedures of Examples 2 and 3 with the following modification: capping of the N-terminus was carried out using 3-(4-hydroxy-3,5-diiodophenyl)propionic acid (3 eq.), HBTU (3 eq.), HOBT (3 eq.) and NMM (6 eq.). The title compound was isolated as a fine white powder (27.3 mg, 29%).
  • This compound was prepared according to the procedures of Examples 1 and 2 with the following modification: capping of the N-terminus was carried out using 3-(4-hydroxy-3,5-diiodophenyl)propionic acid (3 eq), HBTU (3 eq), HOBT (3 eq) and NMM (6 eq). The title compound was isolated as a fine white powder (17.1 mg, 16%).
  • the NMR data indicated a mixture of two species in a ratio of about 70:30: 1 H NMR (300 MHz, DMSO-d 6 ) ⁇ 9.11 (s, 1 H), 8.86, 8.92 (s, s, 1 H), 8.51, 8.05-8.16 (m, d, 6 H, J 8.5 Hz), 8.22-8.32 (m, 2 H) 7.87-7.98 (m, 2 H), 7.34-7.39 (m, 2 H), 7.11 (s, 1 H), 7.08 (s, 1 H), 6.98 (app d, 2 H), 6.92 (s, 1 H), 6.64 (app d, 2 H), 5.11 (bs, 1 H), 4.59-4.77 (m, 1 H), 4.31-4.50 (m, 4 H), 3.83-4.02 (m, 2 H), 3.71-3.79 (m, 9 H), 3.58-3.67 (m, 2 H), 3.48-3.53 (m, 2 H), 3.11-3.19 (m, 1 H), 2.91-3.04 (m
  • This compound was prepared according to the procedures of Examples 1 and 2 with the following modification: capping of the N-terminus was carried out using 3-(4-hydroxyphenyl)propionic acid (3 eq), HBTU (3 eq), HOBT (3 eq) and NMM (6 eq). The title compound was isolated as a fine white powder (47.4 mg, 57%).
  • the NMR data indicated a mixture of two species in a ratio of about 70:30: 1 H NMR (300 MHz, DMSO-d 6 ) ⁇ 8.98 (m, 1 H), 8.21-8.44 (m, 2 H), 7.98-8.14 (m, 2 H), 7.71-7.77 (m, 1 H), 7.40 (s, 1 H), 7.35 (m, 1 H), 7.09 (d, 2 H, J 9.8 Hz), 6.95-6.98 (m, 2 H), 6.92 (s, 1 H), 6.62-6.54 (m, 2 H), 4.62-4.79 (m, 4 H), 4.28-4.50 (m, 8 H), 3.47-3.70 (m, 4 H), 3.12-3.19 (m, 1 H), 2.90-3.03 (m, 3 H), 2.76-2.83 (m, 2 H), 2.65-2.70 (m, 2 H), 2.40-2.46 (m, 2 H), 2.25-2.30 (m, 3 H), 1.72-2.08 (m, 4 H), 1.14-1
  • the NMR data indicated a mixture of two species in a ratio of about 80:20: 1 H NMR (300 MHz, DMSO-d 6 ) ⁇ 8.97 (m, 1 H), 8.21-8.44 (m, 2 H), 8.06-8.14 (m, 1 H), 7.97-7.99 (m, 2 H), 7.85-7.87 (m, 1 H), 7.40 (s, 1 H), 7.35 (s, 1 H), 7.09 (d, 2 H, J 9.7 Hz), 6.89-7.00 (m, 2 H), 6.92 (s, 1 H), 6.61-6.64 (m, 2 H), 4.59-4.66 (m, 2 H), 4.25-4.50 (m, 6 H), 3.59-3.71 (m, 2 H), 3.48-3.55 (m, 2 H), 3.12-3.19 (m, 1 H), 2.95-3.10 (m, 3 H), 2.76-2.83 (m, 3 H), 2.57-2.65 (m, 1 H), 2.38-2.46 (m, 2 H), 2.24-2.27
  • the NMR data indicated a mixture of three species in a ratio of about 70:20:10 1 H NMR (300 MHz, D 2 O) ⁇ 8.69-8.90 (m, 1 H), 7.39-7.55 (m, 1 H), 4.53-4.64 (m, 3 H), 4.39-4.48 (m, 1 H), 3.88-3.96 (m, 5 H), 3.24-3.68 (m, 28 H), 3.00-3.02 (m, 3 H), 2.69-2.94 (m, 9 H), 2.24-2.43 (m, 2 H), 1.84-2.10 (m, 5 H); MS m/z (C 40 H 61 InN 14 O 15 S+H) + 11245.8; Anal. Calcd for C 40 H 61 InN 14 O 15 S: N, 17.43 Found: N, 12.42 (peptide content: 71%).
  • This compound was prepared according to the procedures of Examples 1 and 2 with the following modifications: capping of the N-terminus was carried out using 3-(4-hydroxyphenyl)propionic acid (3 eq), HBTU (3 eq), HOBT (3 eq) and NMM (6 eq). The title compound was isolated (61.1 mg, 53%) as a fine white powder.
  • This compound was prepared according to the procedures of Examples 1 and 2 with the following modification: capping of the N-terminus was carried out using 8-(4-fluorobenzylamino)suberic acid (3 eq), HBTU (3 eq), HOBT (3 eq) and NMM (6 eq). The title compound was isolated as a fine white powder (70.8 mg, 53%).
  • the NMR data indicated a mixture of two species in a ratio of about 70:30: 1 H NMR (300 MHz, DMSO-d 6 ) ⁇ 8.97 (s, 1 H), 8.22-8.28 (m, 3 H), 8.11-8.14 (m, 1 H), 7.96-7.99 and 8.40-8.43 (m, 1 H), 7.74-7.80 (m, 1 H), 7.34-7.39 (m, 2 H), 7.24-7.29 (m, 2 H), 7.08-7.16 (m, 4 H), 6.92 (bs, 1 H), 4.29-4.50 (m, 7 H), 4.22 (d, 2 H, J 5.9 Hz), 3.63-3.69 (m, 2 H), 3.44-3.52 (m, 2 H), 3.16-3.26 (m, 3 H), 2.97-3.02 (m, 1 H), 2.76-2.83 (m, 2 H), 2.38-2.49 (m, 3 H), 1.99-2.13 (m, 5 H), 1.72-1.89 (m, 3 H), 1.4
  • DTPA-PHSCN (20 mg, 0.018 mmol) was dissolved in 1 mL of 0.1M AcOH(aq) solution and InCl 3 (40 mg, 0.18 mmol) was dissolved in 2 mL of 0.02M HCl solution. The two solutions were combined and incubated for an hour at room temperature.
  • This compound was prepared according to procedures of Examples 2 and 3 with the following modifications: the 2% hydrazine treatment for the deprotection of ivDde on lysine was repeated 10 times and the amide formation on lysine was carried out with protoporphyrin IX (2 eq), PYBOP (2 eq), and NMM (6 eq). The title compound was isolated (7.6 mg, 5.49 ⁇ M, 8.6%) as a fine dark red powder.; MS m/z (C 67 H 85 N 17 O 14 S+H) + 1385.6.
  • This compound was prepared from Rink amide AM resin according to the procedures of Examples 1 and 2 sand was isolated as a white, fluffy solid; ES MS m/z (M+H) + 1066.5.

Abstract

The present invention relates generally to peptide analogs of Ac—PHSCN—NH2 which target tumor and endothelial cells and have anti-tumor, anti-angiogenic and anti-metasastic activity, methods of making these peptides, compositions thereof and methods of using these peptides and pharmaceutical compositions thereof to treat, prevent and detect diseases characterized by tumor growth, metastasis and angiogenesis. The peptide analogs may serve, inter alia, as carriers of radioactivity, PET-active compounds, toxins, fluorescent molecules and PEG molecules.

Description

  • This application claims the benefit under 35 U.S.C. § 119(e) from U.S. Provisional Application Ser. No. 60/429,174, filed Nov. 25, 2002 and U.S. Provisional Application Ser. No. 60/475,539, filed Jun. 2, 2003 which are herein incorporated by reference in their entirety.
    • 1. FIELD OF THE INVENTION
  • The present invention relates generally to peptide analogs of Ac—PHSCN—NH2 which target tumor and endothelial cells and have anti-tumor, anti-angiogenic and anti-metasastic activity, methods of making these peptides, compositions thereof and methods of using these peptides and pharmaceutical compositions thereof to treat, prevent and detect diseases characterized by tumor growth, metastasis and angiogenesis. The peptide analogs may serve, inter alia, as carriers of radioactivity, PET-active compounds, toxins, fluorescent molecules and PEG molecules.
    • 2. BACKGROUND OF THE INVENTION
  • Integrins are heterodimeric, transmembrane proteins that are involved in cell adhesion, motility and survival (Geiger et al., Nat. Rev. Mol. Cell Biol 2001, 2(11):793-805). Integrin ligands comprise the extracellular matrix (ECM) and basement membranes and include collagen, laminin, fibronectin, vitronectin, and fibrinogen (Bokel, Dev. Cell 2002, 3(3):311-21; Stupack et al., J Cell Sci 2002,115: 3729-38; Bornstein et al., Curr Opin Cell Biol 2002, 14(5): 608-16). Integrin expression and importantly, integrin activation state is altered in tumor tissue (Liu et al., Semin Oncol 2002, 29 (3 Suppl 11): 96-103; Hood et al., Nat. Rev. Cancer 2002 2(2): 91-100; Felding-Habermann, Clin Exp Metastasis 2003, 20(3): 203-13). Accordingly, several integrins, including αvβ3, αvβ5 and α5β1, have been recognized as validated therapeutic targets for the treatment and prevention of cancer (Kumar, Curr Drug Targets 2003, 4(2): 123-31; Kerr et al., Expert Opin Investig Drugs 2002, 11(12): 1765-74; Rust et al., J Biomed Biotechnol. 2002, 2(3): 124-130; Damiano, Curr Cancer Drug Targets 2002, 2(1): 3743; Tucker, Curr Opin Phannacol 2002, 2(4): 394-402) and receptor-targeted imaging methods for oncology (Herschman, Science 2003, 302(5645): 605-8); Aboagye et al., Invest New Drugs 2003, 21(2): 169-81; Van De Wiele et al., Eur J Nucl Med Mol Imaging 2002 29(5): 699-709; Glaser et al., Int J Oncol 2003, 22(2):253-67).
  • The integrin α5β1 is normally not expressed in quiescent endothelial cells, but is upregulated during angiogenesis (Kim et al., Am J Pathol 2000, 156(4):1345-62). α5β1 is a receptor for fibronectin, an abundant plasma protein that may also be associated with the extracellular matrix (Labat-Robert, Semin Cancer Biol 2002, 12(3): 187-95). Fibronectin interacts with the α5β1 integrin through several epitopes with the major adhesive interaction mediated by the RGD sequence within the 10th type III repeat. The major adhesive interaction mediates cell signaling events and can be potentiated by a second epitope located in the ninth Type III repeat called the synergy region (i.e., PHSRN) (Akiyama et al., Cancer Metastasis Rev 1995, 14(3): 173-89). Antagonists of α5β1 were able to inhibit tumor angiogenesis and cause tumor regression which demonstrates the therapeutic potential of targeting the integrin α5β1 (Kim et al., supra). The integrin α5β1 also appears to be important in the survival and metastasis of tumor cells (O'Brien et al., Exp Cell Res 1996, 224(1): 208-13; Ruoslahti, Invasion Metastasis 1994-95;14(1-6):87-9724-26); Kemperman et al., Invasion Metastasis 1994-95,14(1-6): 98-108; Tani et al., Br J Cancer 2003, 88(2): 327-33).
  • The integrin αvβ3 is also a therapeutic target for the inhibition of tumor angiogenesis since inhibitors of αvβ3 (e.g., monoclonal antibodies, cyclic RGD peptides and small non-peptidic organic compounds) are efficacious in multiple pre-clinical models of cancer progression (Kumar, Curr Drug Targets 2003, 4(2): 123-31; Varner et al., Important Adv Oncol 1996, 69-87; Brooks, J Clin Invest 1995, 96(4): 1815-22). Although αvβ3 is not normally expressed on epithelial cells, it is up-regulated on tumor cells, leading to tumor cell adhesion, migration and invasion (Metzner et al., J Invest Dermatol 1996, 107(4): 597-602). The αvβ3 integrin has been implicated in melanoma progression and metastasis (Nip et al., Cancer Metastasis Rev 1995, 14(3): 241-52) and αvβ3 expression has been documented in a variety of tumor cell types including breast, prostate, pancreas, kidney, and glioma (Felding-Habermann et al., Proc Natl Acad Sci USA 2001, 98(4): 1853-8; Platten et al., Biochem Biophys Res Commun 2000, 268(2): 607-11; Lohr et al., Pancreas 1996, 12(3): 248-59; Rabb et al., Am J Nephrol 1996, 16(5): 402-8; Cooper et al., Neoplasia 2002, 4(3): 191-4). Further, the expression of αvβ3 has also been associated with metastasis to bone (Pecheur et al., FASEB J 2002, 16(10): 1266-8.).
  • Ac—PHSCN—NH2 is derived from the synergy sequence of fibronectin and has been shown to target activated α5β1 and αVβ3 integrins on cell surfaces (Livant, U.S. Pat. No. 6,001,965; Livant, U.S. Pat. No. 6,472,369; Livant et al., Cancer Res 2000, 60(2): 309-20). Further, Ac—PHSCN—NH2 completely inhibits DU145 invasion and metastasis of MatLyLu cells in a rat model (Livant et al., supra) and in combination with 5-FU infusion improved survival in a CT26 colon cancer model (Stoeltzing et al., Int J Cancer 2003, 104(4): 496-503). Accordingly, Ac—PHSCN—NH2 targets tumors and the blood vessels which nourish tumor cells as shown by visualization of a Ac—PHSCN—NH2 derivative.
  • Thus, what is needed are novel peptide analogs of Ac—PHSCN—NH2 to fully explore the potential of Ac—PHSCN—NH2 derivatives in targeting tumors and the vasculature which nourishes tumor cells. Ideally, the peptide analogs will serve, inter alia, as carriers of radioactivity for imaging and radiotherapy, PET-active compounds for PET-imaging, toxins for targeted delivery of cellular toxins, fluorescent molecules for visualization and PEG molecules for improvement of pharmacokinetic parameters.
    • 3. SUMMARY OF THE INVENTION
  • The present invention satisfies these and other needs by providing peptide analogs of Ac—PHSCN—NH2 which target tumor and endothelial cells and have anti-tumor, anti-angiogenic and anti-metasastic activity, methods of making these peptides, compositions thereof and methods of using these peptides and pharmaceutical compositions thereof to treat, prevent and detect diseases characterized by tumor growth, metastasis and angiogenesis. The peptide analogs may serve, inter alia, as carriers of radioactivity, PET-active compounds, toxins, fluorescent molecules and PEG molecules.
  • In one aspect the present invention provides a compound of Formula (I):
    Figure US20050020810A1-20050127-C00001
      • or a pharmaceutically acceptable salt, solvate, hydrate or N-oxide thereof wherein:
      • j and k are independently 0 or 1;
      • p and q are independently an integer including and between 0 and 100;
      • r and s are independently 0 or 1;
      • R1 is acyl, substituted acyl, acyl chelate, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, imino or substituted imino;
      • R2 is C1-C6 alkyl with at least one hydrogen atom replaced by a substituent selected from the group consisting of —NR6R7, —OR8, —CO2R9, —S(O)zR10, —P(OR11)OR12, aryl and substituted aryl;
      • R6, R7, R8, R9, R10, R11 and R12 are independently selected from the group consisting of hydrogen, acyl, substituted acyl, acyl chelate, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, imino and substituted imino;
      • X1 is —NH(C═C)gCO—, —NH(CH2)hCO— or —NHCH(CH3)CO—;
      • g and h are independently 1, 2, 3, 4, 5 or 6
        Figure US20050020810A1-20050127-C00002
      • l is an integer from 1 to 4;
        Figure US20050020810A1-20050127-C00003
      • R13 is hydrogen, alkyl, substituted alkyl, acyl, substituted acyl, arylalkyl, substituted arylalkyl, aryl, substituted aryl or —S(O)xR14;
      • n is an integer from 1 to 5;
      • R14 is alkyl, substituted alkyl, acyl, substituted acyl, arylalkyl, substituted arylalkyl, aryl or substituted aryl;
      • y and x are independently 0, 1 or 2;
        Figure US20050020810A1-20050127-C00004
      • m is an integer from 1, 2, 3 or 4;
      • X7 is —NH(C═C)dCO—, —NH(CH2)eCO— or —NHCH(CH3)CO—;
      • d and e are independently 1, 2, 3, 4, 5 or 6;
      • R3 is C1-C6 alkyl with at least one hydrogen atom replace by a substituent selected from the group consisting of —NR15R16, —OR17, —CO2R18, —S(O)nR19, —P(OR20)OR21, aryl and substituted aryl;
      • R4 and R5 are independently hydrogen, alkyl or substituted alkyl; and
      • R15, R16, R17, R18, R19, R20 and R21 are independently selected from the group consisting of hydrogen, acyl, substituted acyl, acyl chelate, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, imino and substituted imino;
        with the proviso that R1 is not acetyl when R4 and R5 are hydrogen and r and s are 0.
  • In a second aspect, the present invention provides pharmaceutical compositions of compounds of the invention. The pharmaceutical compositions generally comprise one or more compounds of the invention or pharmaceutically acceptable salts, hydrates or solvates thereof and a pharmaceutically acceptable diluent, carrier, excipient and adjuvant. The choice of diluent, carrier, excipient and adjuvant will depend upon, among other factors, the desired mode of administration.
  • In a third aspect, the present invention provides methods for treating or preventing diseases or disorders such as cancer. The methods generally involve administering to a patient in need of such treatment or prevention a therapeutically effective amount of a compound of the invention and/or a pharmaceutical composition thereof.
  • In a fourth aspect, the present invention provides methods for detecting diseases or disorders such as cancer. The methods generally involve administering to a patient in need of such treatment or prevention a diagnostically effective amount of a compound of the invention and/or a pharmaceutical composition thereof.
    • 4. DETAILED DESCRIPTION OF THE INVENTION 4.1 Definitions
  • “Compounds of the invention” refers to compounds encompassed by structural formulae (I), (II), (III), (IV) and (V) disclosed herein and includes any specific compounds within that generic formula whose structure is disclosed herein. The compounds of the invention may be identified either by their chemical structure and/or chemical name. When the chemical structure and chemical name conflict, the chemical structure is determinative of the identity of the compound. The compounds of the invention may contain one or more chiral centers and/or double bonds and therefore, may exist as stereoisomers, such as double-bond isomers (i.e., geometric isomers), enantiomers or diastereomers. Accordingly, the chemical structures depicted herein encompass all possible enantiomers and stereoisomers of the illustrated compounds including the stereoisomerically pure form (e.g., geometrically pure, enantiomerically pure or diastereomerically pure) and enantiomeric and stereoisomeric mixtures. The compounds of the invention may also exist in several tautomeric forms. Accordingly, the chemical structures depicted herein encompass all possible tautomeric forms of the illustrated compounds. The compounds of the invention also include isotopically labeled compounds where one or more atoms have an atomic mass different from the atomic mass conventionally found in nature. Examples of isotopes that may be incorporated into the compounds of the invention include, but are not limited to, 2H, 3H, 13C, 14C, 15N, 17O, 18O, etc. Compounds of the invention may exist in unsolvated forms as well as solvated forms, including hydrated forms and as N-oxides. In general, the hydrated, solvated and N-oxide forms are within the scope of the present invention. Certain compounds of the present invention may exist in multiple crystalline or amorphous forms. In general, all physical forms are equivalent for the uses contemplated by the present invention and are intended to be within the scope of the present invention. Further, it should be understood, when partial structures of the compounds of the invention are illustrated, that brackets indicate the point of attachment of the partial structure to the rest of the molecule.
  • “Alkyl” by itself or as part of another substituent refers to a saturated or unsaturated, branched, straight-chain or cyclic monovalent hydrocarbon radical derived by the removal of one hydrogen atom from a single carbon atom of a parent alkane, alkene or alkyne. Typical alkyl groups include, but are not limited to, methyl; ethyls such as ethanyl, ethenyl, ethynyl; propyls such as propan-1-yl, propan-2-yl, cyclopropan-1-yl, prop-1-en-1-yl, prop-1-en-2-yl, prop-2-en-1-yl (allyl), cycloprop-1-en-1-yl; cycloprop-2-en-1-yl, prop-1-yn-1-yl, prop-2-yn-1-yl, etc.; butyls such as butan-1-yl, butan-2-yl, 2-methyl-propan-1-yl, 2-methyl-propan-2-yl, cyclobutan-1-yl, but-1-en-1-yl, but-1-en-2-yl, 2-methyl-prop-1-en-1-yl, but-2-en-1-yl, but-2-en-2-yl, buta-1,3-dien-1-yl, buta-1,3-dien-2-yl, cyclobut-1-en-1-yl, cyclobut-1-en-3-yl, cyclobuta-1,3-dien-1-yl, but-1-yn-1-yl, but-1-yn-3-yl, but-3-yn-1-yl, etc.; and the like.
  • The term “alkyl” is specifically intended to include groups having any degree or level of saturation, i.e., groups having exclusively single carbon-carbon bonds, groups having one or more double carbon-carbon bonds, groups having one or more triple carbon-carbon bonds and groups having mixtures of single, double and triple carbon-carbon bonds. Where a specific level of saturation is intended, the expressions “alkanyl,” “alkenyl,” and “alkynyl” are used. Preferably, an alkyl group comprises from 1 to 20 carbon atoms, more preferably, from 1 to 10 carbon atoms. (C1-C6) alkyl, for example, refers to an alkyl group containing from 1 to 6 carbon atoms.
  • “Alkanyl” by itself or as part of another substituent refers to a saturated branched, straight-chain or cyclic alkyl radical derived by the removal of one hydrogen atom from a single carbon atom of a parent alkane. Typical alkanyl groups include, but are not limited to, methanyl; ethanyl; propanyls such as propan-1-yl, propan-2-yl (isopropyl), cyclopropan-1-yl, etc.; butanyls such as butan-1-yl, butan-2-yl (sec-butyl), 2-methyl-propan-1-yl (isobutyl), 2-methyl-propan-2-yl (t-butyl), cyclobutan-1-yl, etc.; and the like.
  • “Alkenyl” by itself or as part of another substituent refers to an unsaturated branched, straight-chain or cyclic alkyl radical having at least one carbon-carbon double bond derived by the removal of one hydrogen atom from a single carbon atom of a parent alkene. The group may be in either the cis or trans conformation about the double bond(s). Typical alkenyl groups include, but are not limited to, ethenyl; propenyls such as prop-1-en-1-yl, prop-1-en-2-yl, prop-2-en-1-yl (allyl), prop-2-en-2-yl, cycloprop-1-en-1-yl; cycloprop-2-en-1-yl; butenyls such as but-1-en-1-yl, but-1-en-2-yl, 2-methyl-prop-1-en-1-yl, but-2-en-1-yl, but-2-en-1-yl, but-2-en-2-yl, buta-1,3-dien-1-yl, buta-1,3-dien-2-yl, cyclobut-1-en-1-yl, cyclobut-1-en-3-yl, cyclobuta-1,3-dien-1-yl, etc.; and the like.
  • “Alkynyl” by itself or as part of another substituent refers to an unsaturated branched, straight-chain or cyclic alkyl radical having at least one carbon-carbon triple bond derived by the removal of one hydrogen atom from a single carbon atom of a parent alkyne. Typical alkynyl groups include, but are not limited to, ethynyl; propynyls such as prop-1-yn-1-yl, prop-2-yn-1-yl, etc.; butynyls such as but-1-yn-1-yl, but-1-yn-3-yl, but-3-yn-1-yl, etc.; and the like.
  • “Acyl” by itself or as part of another substituent refers to a radical —C(O)R30, where R30 is hydrogen, alkyl, cycloalkyl, cycloheteroalkyl, aryl, arylalkyl, heteroalkyl, heteroaryl, heteroarylalkyl as defined herein. Representative examples include, but are not limited to, formyl, acetyl, cyclohexylcarbonyl, cyclohexylmethylcarbonyl, benzoyl, benzylcarbonyl and the like.
  • “Acyl Chelate” by itself or as part of another substituent refers to a radical —C(O)R31, where R31 is alkyl, cycloalkyl, aryl as defined herein substituted with a chelating group which binds an appropriate metal. Representative examples include —C(O)CH2CH2—R32 where R32 is a chelating group such as, for example, DOTA, TETA, a polyamino carboxylate (e.g., NODAGA, EDTA, tricine, —C(O)CH2-DTPA, etc.)
    Figure US20050020810A1-20050127-C00005
    and where the chelating group is bound to metals such as positron emitting labels (e.g., 18F, 45Ti, 44Sc, 55Co, 61Cu, 64Cu, 66Ga, 68Ga, 75Br, 76Br, 86Y, 110In, 124I, 89Zr, 99Tc), radionuclides (e.g., 137Cs, 60Co, 131I, 123I, 192Ir, 90Y, 67Ga, 99Tc, 123I, 125I, 131I, 111In, 97Ru, 67Cu, 68Ga, 72As, 89Zr, 90Y, 201Tl, etc.) or lanthanide metals.
  • “Alkoxy” by itself or as part of another substituent refers to a radical —OR31 where R33 represents an alkyl or cycloalkyl group as defined herein. Representative examples include, but are not limited to, methoxy, ethoxy, propoxy, butoxy, cyclohexyloxy and the like.
  • “Aryl” by itself or as part of another substituent refers to a monovalent aromatic hydrocarbon radical derived by the removal of one hydrogen atom from a single carbon atom of a parent aromatic ring system. Typical aryl groups include, but are not limited to, groups derived from aceanthrylene, acenaphthylene, acephenanthrylene, anthracene, azulene, benzene, chrysene, coronene, fluoranthene, fluorene, hexacene, hexaphene, hexalene, as-indacene, s-indacene, indane, indene, naphthalene, octacene, octaphene, octalene, ovalene, penta-2,4-diene, pentacene, pentalene, pentaphene, perylene, phenalene, phenanthrene, picene, pleiadene, pyrene, pyranthrene, rubicene, triphenylene, trinaphthalene and the like. Preferably, an aryl group comprises from 6 to 20 carbon atoms, more preferably from 6 to 12 carbon atoms.
  • “Arylalkyl” by itself or as part of another substituent refers to an acyclic alkyl radical in which one of the hydrogen atoms bonded to a carbon atom, typically a terminal or sp3 carbon atom, is replaced with an aryl group. Typical arylalkyl groups include, but are not limited to, benzyl, 2-phenylethan-1-yl, 2-phenylethen-1-yl, naphthylmethyl, 2-naphthylethan-1-yl, 2-naphthylethen-1-yl, naphthobenzyl, 2-naphthophenylethan-1-yl and the like. Where specific alkyl moieties are intended, the nomenclature arylalkanyl, arylalkenyl and/or arylalkynyl is used. Preferably, an arylalkyl group is (C6-C30) arylalkyl, e.g., the alkanyl, alkenyl or alkynyl moiety of the arylalkyl group is (C1-C10) and the aryl moiety is (C6-C20), more preferably, an arylalkyl group is (C6-C20) arylalkyl, e.g., the alkanyl, alkenyl or alkynyl moiety of the arylalkyl group is (C1-C8) and the aryl moiety is (C6-C12).
  • “Cycloalkyl” by itself or as part of another substituent refers to a saturated or unsaturated cyclic alkyl radical. Where a specific level of saturation is intended, the nomenclature “cycloalkanyl” or “cycloalkenyl” is used. Typical cycloalkyl groups include, but are not limited to, groups derived from cyclopropane, cyclobutane, cyclopentane, cyclohexane and the like. Preferably, the cycloalkyl group is (C3-C10) cycloalkyl, more preferably (C3-C7) cycloalkyl.
  • “Cycloheteroalkyl” by itself or as part of another substituent refers to a saturated or unsaturated cyclic alkyl radical in which one or more carbon atoms (and any associated hydrogen atoms) are independently replaced with the same or different heteroatom. Typical heteroatoms to replace the carbon atom(s) include, but are not limited to, N, P, O, S, Si, etc. Where a specific level of saturation is intended, the nomenclature “cycloheteroalkanyl” or “cycloheteroalkenyl” is used. Typical cycloheteroalkyl groups include, but are not limited to, groups derived from epoxides, azirines, thiiranes, imidazolidine, morpholine, piperazine, piperidine, pyrazolidine, pyrrolidine, quinuclidine, and the like.
  • “Diagnostically effective amount” means the amount of a compound that, when administered to a patient for detection of a disease, is sufficient to detect the disease. The “diagnostically effective amount” will vary depending on the compound, the disease and its severity and the age, weight, etc., of the patient to be treated.
  • “Heteroalkyl, Heteroalkanyl, Heteroalkeniyl and Heteroalkvnyl” by themselves or as part of another substituent refer to alkyl, alkanyl, alkenyl and alkynyl groups, respectively, in which one or more of the carbon atoms (and any associated hydrogen atoms) are independently replaced with the same or different heteroatomic groups. Typical heteroatomic groups which can be included in these groups include, but are not limited to, —O—, —S—, —O—O—, —S—S—, —O—S—, —NR34R35, —═N—N═—, —N═N—, —N═N—, —N═N—NR36R37, —PR38—, —P(O)2—, —POR39—, —O—P(O)2—, —SO—, —SO2—, —SnR40R41— and the like, where R34, R35, R36, R37, R38, R39, R40 and R41 are independently hydrogen, alkyl, substituted alkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, cycloalkyl, substituted cycloalkyl, cycloheteroalkyl, substituted cycloheteroalkyl, heteroalkyl, substituted heteroalkyl, heteroaryl, substituted heteroaryl, heteroarylalkyl or substituted heteroarylalkyl.
  • “Heteroaryl” by itself or as part of another substituent refers to a monovalent heteroaromatic radical derived by the removal of one hydrogen atom from a single atom of a parent heteroaromatic ring system. Typical heteroaryl groups include, but are not limited to, groups derived from acridine, arsindole, carbazole, β-carboline, chromane, chromene, cinnoline, furan, imidazole, indazole, indole, indoline, indolizine, isobenzofuran, isochromene, isoindole, isoindoline, isoquinoline, isothiazole, isoxazole, naphthyridine, oxadiazole, oxazole, perimidine, phenanthridine, phenanthroline, phenazine, phthalazine, pteridine, purine, pyran, pyrazine, pyrazole, pyridazine, pyridine, pyrimidine, pyrrole, pyrrolizine, quinazoline, quinoline, quinolizine, quinoxaline, tetrazole, thiadiazole, thiazole, thiophene, triazole, xanthene, and the like. Preferably, the heteroaryl group is from 5-20 membered heteroaryl, more preferably from 5-10 membered heteroaryl. Preferred heteroaryl groups are those derived from thiophene, pyrrole, benzothiophene, benzofuran, indole, pyridine, quinoline, imidazole, oxazole and pyrazine
  • “Heteroarylalkyl” by itself or as part of another substituent refers to an acyclic alkyl radical in which one of the hydrogen atoms bonded to a carbon atom, typically a terminal or sp3 carbon atom, is replaced with a heteroaryl group. Where specific alkyl moieties are intended, the nomenclature heteroarylalkanyl, heteroarylalkenyl and/or heterorylalkynyl is used. In preferred embodiments, the heteroarylalkyl group is a 6-30 membered heteroarylalkyl, e.g., the alkanyl, alkenyl or alkynyl moiety of the heteroarylalkyl is 1-10 membered and the heteroaryl moiety is a 5-20-membered heteroaryl, more preferably, 6-20 membered heteroarylalkyl, e.g., the alkanyl, alkenyl or alkynyl moiety of the heteroarylalkyl is 1-8 membered and the heteroaryl moiety is a 5-12-membered heteroaryl.
  • “Imino” by itself or as part of another substituent refers to a radical —C═NR42, where R42 is hydrogen, alkyl, cycloalkyl, cycloheteroalkyl, aryl, arylalkyl, heteroalkyl, heteroaryl, heteroarylalkyl as defined herein.
  • “Parent Aromatic Ring System” refers to an unsaturated cyclic or polycyclic ring system having a conjugated π electron system. Specifically included within the definition of “parent aromatic ring system” are fused ring systems in which one or more of the rings are aromatic and one or more of the rings are saturated or unsaturated, such as, for example, fluorene, indane, indene, phenalene, etc. Typical parent aromatic ring systems include, but are not limited to, aceanthrylene, acenaphthylene, acephenanthrylene, anthracene, azulene, benzene, chrysene, coronene, fluoranthene, fluorene, hexacene, hexaphene, hexalene, as-indacene, s-indacene, indane, indene, naphthalene, octacene, octaphene, octalene, ovalene, penta-2,4-diene, pentacene, pentalene, pentaphene, perylene, phenalene, phenanthrene, picene, pleiadene, pyrene, pyranthrene, rubicene, triphenylene, trinaphthalene and the like.
  • “Parent Heteroaromatic Ring System” refers to a parent aromatic ring system in which one or more carbon atoms (and any associated hydrogen atoms) are independently replaced with the same or different heteroatom. Typical heteroatoms to replace the carbon atoms include, but are not limited to, N, P, O, S, Si, etc. Specifically included within the definition of “parent heteroaromatic ring systems” are fused ring systems in which one or more of the rings are aromatic and one or more of the rings are saturated or unsaturated, such as, for example, arsindole, benzodioxan, benzofuran, chromane, chromene, indole, indoline, xanthene, etc. Typical parent heteroaromatic ring systems include, but are not limited to, arsindole, carbazole, β-carboline, chromane, chromene, cinnoline, furan, imidazole, indazole, indole, indoline, indolizine, isobenzofuran, isochromene, isoindole, isoindoline, isoquinoline, isothiazole, isoxazole, naphthyridine, oxadiazole, oxazole, perimidine, phenanthridine, phenanthroline, phenazine, phthalazine, pteridine, purine, pyran, pyrazine, pyrazole, pyridazine, pyridine, pyrimidine, pyrrole, pyrrolizine, quinazoline, quinoline, quinolizine, quinoxaline, tetrazole, thiadiazole, thiazole, thiophene, triazole, xanthene, and the like.
  • “Patient” includes humans. The terms “human” and “patient” are used interchangeably herein.
  • “Pharmaceutically acceptable salt” refers to a salt of a compound of the invention, which is pharmaceutically acceptable and possesses the desired pharmacological activity of the parent compound. Such salts include: (1) acid addition salts, formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; or formed with organic acids such as acetic acid, propionic acid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, 3-(4-hydroxybenzoyl) benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethane-disulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, 4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid, 4-toluenesulfonic acid, camphorsulfonic acid, 4-methylbicyclo[2.2.2]-oct-2-ene-1-carboxylic acid, glucoheptonic acid, 3-phenylpropionic acid, trimethylacetic acid, tertiary-butylacetic acid, lauryl sulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, muconic acid, and the like; or (2) salts formed when an acidic proton present in the parent compound is replaced by a metal ion, e.g., an alkali metal ion, an alkaline earth ion, or an aluminum ion; or coordinates with an organic base such as ethanolamine, diethanolamine, triethanolamine, N-methylglucamine and the like.
  • “Pharmaceutically acceptable vehicle” refers to a diluent, adjuvant, excipient or carrier with which a compound of the invention is administered.
  • “Preventing” or “prevention” refers to a reduction in risk of acquiring a disease or disorder (i.e., causing at least one of the clinical symptoms of the disease not to develop in a patient that may be exposed to or predisposed to the disease but does not yet experience or display symptoms of the disease).
  • “Substituted” refers to a group in which one or more hydrogen atoms are independently replaced with the same or different substituent(s). Typical substituents include, but are not limited to, —M, —R60, —O−, ═O, —OR60, —SR60, —S−, ═S, —NR60R61, ═NR60, —CF3, —CN, —OCN, —SCN, —NO, —NO2, ═N2, —N3, —S(O)2O; —S(O)2OH, —S(O)2R60, —OS(O2)O−, —OS(O)2R60, —P(O)(O−)2, —P(O)(OR60)(O−), —OP(O)(OR60)(OR61), —C(O)R60, —C(S)R60, —C(O)OR60, —C(O)NR60R61, —C(O)O—, —C(S)OR60, —NR62C(O)NR60R61, —NR62C(S)NR60R61, —NR62C(NR63)NR60R61 and —C(NR62)NR60R61 where M is independently a halogen; R60, R61, R62 and R63 are independently hydrogen, alkyl, substituted alkyl, alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloheteroalkyl, substituted cycloheteroalkyl, aryl, substituted aryl, heteroaryl or substituted heteroaryl, or optionally R60 and R61 together with the nitrogen atom to which they are bonded form a cycloheteroalkyl or substituted cycloheteroalkyl ring; and R64 and R65 are independently hydrogen, alkyl, substituted alkyl, aryl, cycloalkyl, substituted cycloalkyl, cycloheteroalkyl, substituted cycloheteroalkyl, aryl, substituted aryl, heteroaryl or substituted heteroaryl, or optionally R64 and R65 together with the nitrogen atom to which they are bonded form a cycloheteroalkyl or substituted cycloheteroalkyl ring. Preferably, substituents include —M, —R60, ═O, —OR60, —SR60, —S−, ═S, —NR60R61, ═NR60, —CF3, —CN, —OCN, —SCN, —NO, —NO2, ═N2, —N3, —S(O)2R60, —OS(O2)O−, —OS(O)2R60, —P(O)(O−)2, —P(O)(OR60)(O−), —OP(O)(OR60)(OR61), —C(O)R60, —C(S)R60, —C(O)OR60, —C(O)NR60R61,—C(O)O−, —NR62C(O)NR60R61, more preferably, —M, —R60, ═O, —OR60, —SR60, —NR60R61, —CF3, —CN, —NO2, —S(O)2R60, —P(O)(OR60)(O−), —OP(O)(OR60)(OR61), —C(O)R60, —C(O)OR60, —C(O)NR60R61, —C(O)O−, most preferably, —M, —R60, ═O, —OR60, —SR60, —NR60R61, —CF3, —CN, —NO2, —S(O)2R60, —OP(O)(OR60)(OR61), —C(O)R60, —C(O)OR60, —C(O)O−, where R60, R61 and R62 are as defined above.
  • “Treating” or “treatment” of any disease or disorder refers, in one embodiment, to ameliorating the disease or disorder (i.e., arresting or reducing the development of the disease or at least one of the clinical symptoms thereof). In another embodiment “treating” or “treatment” refers to ameliorating at least one physical parameter, which may not be discernible by the patient. In yet another embodiment, “treating” or “treatment” refers to inhibiting the disease or disorder, either physically, (e.g., stabilization of a discernible symptom), physiologically, (e.g., stabilization of a physical parameter), or both. In yet another embodiment, “treating” or “treatment” refers to delaying the onset of the disease or disorder.
  • “Therapeutically effective amount” means the amount of a compound that, when administered to a patient for treating a disease, is sufficient to effect such treatment for the disease. The “therapeutically effective amount” will vary depending on the compound, the disease and its severity and the age, weight, etc., of the patient to be treated.
  • Reference will now be made in detail to preferred embodiments of the invention. While the invention will be described in conjunction with the preferred embodiments, it will be understood that it is not intended to limit the invention to those preferred embodiments. To the contrary, it is intended to cover alternatives, modifications, and equivalents as may be included within the spirit and scope of the invention as defined by the appended claims.
    • 4.2 Compounds of the Invention
  • The present invention satisfies these and other needs by providing peptide analogs of Ac—PHSCN—NH2 which target tumor and endothelial cells and have anti-tumor, anti-angiogenic and anti-metasastic activity, methods of making these peptides, compositions thereof and methods of using these peptides and pharmaceutical compositions thereof to treat, prevent and detect diseases characterized by tumor growth, metastasis and angiogenesis. The peptide analogs may serve, inter alia, as carriers of radioactivity, PET-active compounds, toxins, fluorescent molecules and PEG molecules.
  • Accordingly, in one aspect, the present invention provides a compound of Formula (I):
    Figure US20050020810A1-20050127-C00006
      • or a pharmaceutically acceptable salt, solvate, hydrate or N-oxide thereof wherein:
      • j and k are independently 0 or 1;
      • p and q are independently an integer including and between 0 and 100;
      • r and s are independently 0 or 1;
      • R1 is acyl, substituted acyl, acyl chelate, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, imino or substituted imino;
      • R2 is C1-C6 alkyl with at least one hydrogen atom replaced by a substituent selected from the group consisting of —NR6R7, —OR8, —CO2R9, —S(O)zR10, —P(OR11)OR12, aryl and substituted aryl;
      • R6, R7, R8, R9, R10, R11 and R12 are independently selected from the group consisting of hydrogen, acyl, substituted acyl, acyl chelate, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, imino and substituted imino;
      • X1 is —NH(C═C)gCO—, —NH(CH2)hCO— or —NHCH(CH3)CO—;
      • g and h are independently 1, 2, 3, 4, 5 or 6
        Figure US20050020810A1-20050127-C00007
      • l is an integer from 1 to 4;
        Figure US20050020810A1-20050127-C00008
      • R13 is hydrogen, alkyl, substituted alkyl, acyl, substituted acyl, arylalkyl, substituted arylalkyl, aryl, substituted aryl or —S(O)xR14;
      • n is an integer from 1 to 5;
      • R14 is alkyl, substituted alkyl, acyl, substituted acyl, arylalkyl, substituted arylalkyl, aryl or substituted aryl;
      • y and x are independently 0, 1 or 2;
        Figure US20050020810A1-20050127-C00009
      • m is an integer from 1, 2, 3 or 4;
      • X7 is —NH(C═C)dCO—, —NH(CH2)eCO— or —NHCH(CH3)CO—;
      • d and e are independently 1, 2, 3, 4, 5 or 6;
      • R3 is C1-C6 alkyl with at least one hydrogen atom replace by a substituent selected from the group consisting of —NR15R16, —OR17, —CO2R18, —S(O)nR19, —P(OR20)OR21, aryl and substituted aryl;
      • R4 and R5 are independently hydrogen, alkyl or substituted alkyl; and
      • R15, R16, R17, R18, R19, R20 and R21 are independently selected from the group consisting of hydrogen, acyl, substituted acyl, acyl chelate, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, imino and substituted imino;
      • with the proviso that R1 is not acetyl when R4 and R5 are hydrogen and r and s are 0.
  • In one embodiment, R1 is not acetyl when R4 and R5 are hydrogen.
  • In one embodiment, p and q are independently an integer between and including 1 and 50. In another embodiment, p and q are independently an integer between and including 1 and 25. In still another embodiment, p and q are independently an integer between and including 1 and 10. In still another embodiment, p and q are independently an integer between and including 1 and 5. In still another embodiment, p and q are independently an integer between and including 1 and 3.
  • In one embodiment, p and q are independently an integer between and including 0 and 50. In another embodiment, p and q are independently an integer between and including 0 and 25. In still another embodiment, p and q are independently an integer between and including 0 and 10. In still another embodiment, p and q are independently an integer between and including 0 and 5. In still another embodiment, p and q are independently an integer between and including 0 and 3.
  • In still another embodiment, s is 0 and r is 1. In still another embodiment, s is 1 and r is 0. In still another embodiment, at least one of s and r is not 0.
  • In still another embodiment, R1 is acyl, substituted acyl, acyl chelate, imino or substituted imino. In still another embodiment, R2 is C1-C6 alkyl with at least one hydrogen atom replaced by a substituent selected from the group consisting of —NR6R7, —OR8 and —CO2R9. In still another embodiment, R6, R7, R8, R9, R10, R11 and R12 are independently selected from the group consisting of hydrogen, acyl, substituted acyl, acyl chelate, imino or substituted imino.
  • In still another embodiment, X1 is —NHCH2CO— or —NHCH(CH3)CO—. In still another embodiment, X2 is
    Figure US20050020810A1-20050127-C00010
  • In still another embodiment, l is 1. In still another embodiment, n is 1 or 2. In still another embodiment, m is 1 or 2.
  • In still another embodiment, R3 is C1-C6 alkyl with at least one hydrogen atom replaced by a substituent selected from the group consisting of —NR15R16, —OR17 and —CO2R18. In still another embodiment, R15, R16, R17, R18, R19, R20 and R21 are independently selected from the group consisting of hydrogen, acyl, substituted acyl, acyl chelate, imino or substituted imino.
  • In one embodiment, compounds of the invention has the structure of Formula (II):
    Figure US20050020810A1-20050127-C00011
      • wherein:
      • R1 is acyl, substituted acyl, acyl chelate, alkyl, substituted alkyl, imino or substituted imino;
      • R4 is hydrogen;
      • R5 is hydrogen, alkyl or substituted alkyl; and
      • R25 is hydrogen, acyl, substituted acyl, acyl chelate, alkyl, substituted alkyl, imino or substituted imino; and
      • q is 1, 2, 3, 4 or 5.
  • In one embodiment of a compound of Formulae (I) and (II), R1 is
    Figure US20050020810A1-20050127-C00012
      • and R14 is hydrogen, methyl or acetyl. In one specific embodiment, r=0 and R4 and R5 are hydrogen. In another specific embodiment, r=1, q is 2, R4, R5 and R25 are hydrogen.
  • In another embodiment of a compound of Formulae (I) and (II), r=1, q is 2, R14 is hydrogen, methyl or acetyl and R25 is
    Figure US20050020810A1-20050127-C00013
  • In another embodiment of a compound of Formulae (I) and (II), r=0, R1 is acetyl, R4 is hydrogen, R14 is hydrogen, methyl or acetyl and R5 is
    Figure US20050020810A1-20050127-C00014
  • In still another embodiment of a compound of Formulae (I) and (II), r=1, q is 2, R1 is acetyl, R4 and R25 are hydrogen, R14 is hydrogen, methyl or acetyl and R5 is
    Figure US20050020810A1-20050127-C00015
  • In still another embodiment of a compound of Formulae (I) and (II), r=0, R4 and R5 are hydrogen, R14 is hydrogen, methyl or acetyl and R1 is
    Figure US20050020810A1-20050127-C00016
  • In still another embodiment of a compound of Formulae (I) and (II), r=1, q=2, R1=acetyl, R4 and R5 are hydrogen, R14 is hydrogen, methyl or acetyl and R25 is
    Figure US20050020810A1-20050127-C00017
  • In still another embodiment of a compound of Formulae (I) and (II), r=0, R4 and R5 are hydrogen, R14 is hydrogen, methyl or acetyl and R1 is
    Figure US20050020810A1-20050127-C00018
  • In still another embodiment of a compound of Formulae (I) and (II), r=1, q=2, R4, R5 and R25 are hydrogen, R14 is hydrogen, methyl or acetyl and R1 is
    Figure US20050020810A1-20050127-C00019
  • In still another embodiment of a compound of Formulae (I) and (II), r=1, q=2, R1 is acetyl, R4 and R5 are hydrogen, R14 is hydrogen, methyl or acetyl and R25 is
    Figure US20050020810A1-20050127-C00020
  • In still another embodiment of a compound of Formulae (I) and (II), r=0, R4 and R5 are hydrogen, R14 is hydrogen, methyl or acetyl and R1 is
    Figure US20050020810A1-20050127-C00021
  • In still another embodiment of a compound of Formulae (I) and (II), r=1, q=2, R4, R5 and R25 are hydrogen, R14 is hydrogen, methyl or acetyl and R1 is
    Figure US20050020810A1-20050127-C00022
  • In still another embodiment of a compound of Formulae (I) and (II), r=1, q=2, R1 is acetyl, R4 and R5 are hydrogen, R14 is hydrogen, methyl or acetyl and R25 is
    Figure US20050020810A1-20050127-C00023
  • In still another embodiment of a compound of Formulae (I) and (II), r=0, R4 and R5 are hydrogen, R14 is hydrogen, methyl or acetyl and R1 is
    Figure US20050020810A1-20050127-C00024
    and M is Cu, Ga, 111In or 90Y.
  • In still another embodiment of a compound of Formulae (I) and (II), r=1, q=2, R4, R5 and R25 are hydrogen, R14 is hydrogen, methyl or acetyl, R1 is
    Figure US20050020810A1-20050127-C00025
    and M is Cu, Ga, 111In or 90Y.
  • In still another embodiment of a compound of Formulae (I) and (II), r=1, q=2, R1 is acetyl, R4 and R5 are hydrogen, R14 is hydrogen, methyl or acetyl, R25 is
    Figure US20050020810A1-20050127-C00026
    and M is Cu, Ga, 111In or 90Y.
  • In still another embodiment of a compound of Formulae (I) and (II), r=0, R4 and R5 are hydrogen, R14 is hydrogen, methyl or acetyl and R1 is
    Figure US20050020810A1-20050127-C00027
    and M is Cu.
  • In still another embodiment of a compound of Formulae (1) and (II), r=1, q=2, R4, R5 and R25 are hydrogen, R14 is hydrogen, methyl or acetyl, R1 is
    Figure US20050020810A1-20050127-C00028
    and M is Cu.
  • In still another embodiment of a compound of Formulae (I) and (II), r=1, q=2, R1 is acetyl, R4 and R5 are hydrogen, R14 is hydrogen, methyl or acetyl, R25 is
    Figure US20050020810A1-20050127-C00029
    and M is Cu.
  • In still another embodiment of a compound of Formulae (I) and (II), r=0, R4 and R5 are hydrogen, R14 is hydrogen, methyl or acetyl and R1 is
    Figure US20050020810A1-20050127-C00030
    and M is 111In, 90Y or Ga.
  • In still another embodiment of a compound of Formulae (I) and (II), r=1, q=2, R4, R5 and R25 are hydrogen, R14 is hydrogen, methyl or acetyl, R1 is
    Figure US20050020810A1-20050127-C00031
    and M is 111In, 90Y or Ga.
  • In still another embodiment of a compound of Formulae (I) and (II), r=1, q=2, R1 is acetyl, R4 and R5 are hydrogen, R14 is hydrogen, methyl or acetyl, R25 is
    Figure US20050020810A1-20050127-C00032
    and M is 111In, 90Y or Ga.
  • In still another embodiment of a compound of Formulae (I) and (II), r=0, R4 and R5 are hydrogen, R14 is hydrogen, methyl or acetyl, R1 is
    Figure US20050020810A1-20050127-C00033
    and M is 111In, 67Ga or 68 Ga.
  • In still another embodiment of a compound of Formulae (I) and (II), r=1, q=2, R4, R5 and R25 are hydrogen, R14 is hydrogen, methyl or acetyl, R1 is
    Figure US20050020810A1-20050127-C00034
    and M is 111In, 67Ga or 68Ga.
  • In still another embodiment of a compound of Formulae (I) and (II), r=1, q=2, R1 is acetyl, R4 and R5 are hydrogen, R14 is hydrogen, methyl or acetyl, R25 is
    Figure US20050020810A1-20050127-C00035
    and M is 111In, 67Ga or 68Ga.
  • In still another embodiment of a compound of Formulae (I) and (II), wherein r=0, R4 and R5 are hydrogen, R14 is hydrogen, methyl or acetyl, R1 is
    Figure US20050020810A1-20050127-C00036
    and M is Tc.
  • In still another embodiment of a compound of Formulae (I) and (II), r=1, q=2, R4, R5 and R25 are hydrogen, R14 is hydrogen, methyl or acetyl, R1 is
    Figure US20050020810A1-20050127-C00037
    and M is Tc.
  • In still another embodiment of a compound of Formulae (I) and (II), r=1, q=2, R1 is acetyl, R4 and R5 are hydrogen, R14 is hydrogen, methyl or acetyl, R25 is
    Figure US20050020810A1-20050127-C00038
    and M is Tc.
  • In still another embodiment of a compound of Formulae (I) and (II), r=0, R4 and R5 are hydrogen, R14 is hydrogen, methyl or acetyl and R1 is
    Figure US20050020810A1-20050127-C00039
  • In still another embodiment of a compound of Formulae (I) and (II), wherein r=1, q=2, R4, R5 and R25 are hydrogen, R14 is hydrogen, methyl or acetyl and R1 is
    Figure US20050020810A1-20050127-C00040
  • In still another embodiment of a compound of Formulae (I) and (II), wherein r=1, q=2, R1 is acetyl, R4 and R5 are hydrogen, R14 is hydrogen, methyl or acetyl and R25 is
    Figure US20050020810A1-20050127-C00041
  • In still another embodiment of a compound of Formulae (I) and (II), r=0, R4 and R5 are hydrogen, R14 is hydrogen, methyl or acetyl and R1 is
    Figure US20050020810A1-20050127-C00042
  • In still another embodiment of a compound of Formulae (I) and (II), r=1, q=2, R4, R5 and R25 are hydrogen, R14 is hydrogen, methyl or acetyl and R1 is
    Figure US20050020810A1-20050127-C00043
  • In still another embodiment of a compound of Formulae (I) and (II), r=1, q=2, R1 is acetyl, R4 and R5 are hydrogen, R14 is hydrogen, methyl or acetyl and R25 is
    Figure US20050020810A1-20050127-C00044
  • In still another embodiment of a compound of Formulae (I) and (II), r=0, R4 and R5 are hydrogen, R14 is hydrogen, methyl or acetyl and R1 is
    Figure US20050020810A1-20050127-C00045
  • In still another embodiment of a compound of Formulae (I) and (II), r=1, q=2, R4, R5 and R25 are hydrogen, R14 is hydrogen, methyl or acetyl and R1 is
    Figure US20050020810A1-20050127-C00046
  • In still another embodiment of a compound of Formulae (I) and (II), r=1, q=2, R1 is acetyl, R4 and R5 are hydrogen, R14 is hydrogen, methyl or acetyl and R25 is
    Figure US20050020810A1-20050127-C00047
  • In still another embodiment of a compound of Formulae (I) and (II), r=0, R4 and R5 are hydrogen, R14 is hydrogen, methyl or acetyl and R1 is
    Figure US20050020810A1-20050127-C00048
  • In still another embodiment of a compound of Formulae (I) and (II), r=1, q=2, R4, R5 and R25 are hydrogen, R14 is hydrogen, methyl or acetyl and R1 is
    Figure US20050020810A1-20050127-C00049
  • In still another embodiment of a compound of Formulae (I) and (II), r=1, q=2, R1 is acetyl, R4 and R5 are hydrogen, R14 is hydrogen, methyl or acetyl and R25 is
    Figure US20050020810A1-20050127-C00050
  • In still another embodiment of a compound of Formulae (I) and (II), r=0, R4 and R5 are hydrogen, R14 is hydrogen, methyl or acetyl and R1 is
    Figure US20050020810A1-20050127-C00051
  • In still another embodiment of a compound of Formulae (I) and (II), r=1, q=2, R4, R5 and R25 are hydrogen, R14 is hydrogen, methyl or acetyl and R1 is
    Figure US20050020810A1-20050127-C00052
  • In still another embodiment of a compound of Formulae (I) and (II), r=1, q=2, R1 is acetyl, R4 and R5 are hydrogen, R14 is hydrogen, methyl or acetyl and R25 is
    Figure US20050020810A1-20050127-C00053
  • In still another embodiment of a compound of Formulae (I) and (II), r=0, R4 and R5 are hydrogen, R14 is hydrogen, methyl or acetyl and R1 is
    Figure US20050020810A1-20050127-C00054
  • In still another embodiment of a compound of Formulae (I) and (II), r=1, q=2, R4, R5 and R25 are hydrogen, R14 is hydrogen, methyl or acetyl and R1 is
    Figure US20050020810A1-20050127-C00055
  • In still another embodiment of a compound of Formulae (I) and (II), r=1, q=2, R1 is acetyl, R4 and R5 are hydrogen, R14 is hydrogen, methyl or acetyl and R25 is
    Figure US20050020810A1-20050127-C00056
  • In still another embodiment of a compound of Formulae (I) and (II), wherein r=0, R4 and R5 are hydrogen, R14 is hydrogen, methyl or acetyl and R1 is
    Figure US20050020810A1-20050127-C00057
  • In still another embodiment of a compound of Formulae (I) and (II), wherein r=1, q=2, R4, R5 and R25 are hydrogen, R14 is hydrogen, methyl or acetyl and R1 is
    Figure US20050020810A1-20050127-C00058
  • In still another embodiment of a compound of Formulae (I) and (II), r=1, q=2, R1 is acetyl, R4 and R5 are hydrogen, R14 is hydrogen, methyl or acetyl and R25 is
    Figure US20050020810A1-20050127-C00059
  • In still another embodiment of a compound of Formulae (I) and (II), r=0, R4 and R5 are hydrogen, R14 is hydrogen, methyl or acetyl and R1 is
    Figure US20050020810A1-20050127-C00060
  • In still another embodiment of a compound of Formulae (I) and (II), r=1, q=2, R4, R5 and R25 are hydrogen, R14 is hydrogen, methyl or acetyl and R1 is
    Figure US20050020810A1-20050127-C00061
  • In still another embodiment of a compound of Formulae (I) and (II), r=1, q=2, R1 is acetyl, R4 and R5 are hydrogen, R14 is hydrogen, methyl or acetyl and R25 is
    Figure US20050020810A1-20050127-C00062
  • In still another embodiment of a compound of Formulae (I) and (II), r=0, R4 and R5 are hydrogen, R14 is hydrogen, methyl or acetyl and R1 is
    Figure US20050020810A1-20050127-C00063
  • In still another embodiment of a compound of Formulae (I) and (II), r=0, R4 and R5 are hydrogen, R4 is hydrogen, methyl or acetyl and R1 is
    Figure US20050020810A1-20050127-C00064
  • In still another embodiment of a compound of Formulae (I) and (II), r=1, q=2, R4, R5 and R25 are hydrogen, R14 is hydrogen, methyl or acetyl and R1 is
    Figure US20050020810A1-20050127-C00065
  • In still another embodiment of a compound of Formulae (I) and (II), r=1, q=2, R1 is acetyl, R4 and Rs are hydrogen, R14 is hydrogen, methyl or acetyl and R25 is
    Figure US20050020810A1-20050127-C00066
  • In still another embodiment of a compound of Formulae (I) and (II), r=1, q=2, R4, R5 and R25 are hydrogen, R14 is hydrogen, methyl or acetyl and R1 is
    Figure US20050020810A1-20050127-C00067
  • In still another embodiment of a compound of Formulae (I) and (II), r=1, q=2, R1 is acetyl, R4 and R5 are hydrogen, R14 is hydrogen, methyl or acetyl and R25 is
    Figure US20050020810A1-20050127-C00068
  • In a first preferred embodiment, R1 is acyl or substituted acyl, R2 is C1-C4 alkyl with at least one hydrogen atom replaced by a substituent selected from the group consisting of —NR6R7, aryl and substituted aryl, R6 and R7 are independently selected from the group consisting of hydrogen, acyl and substituted acyl, X1 is —NH(CH2)hCO—, X2 is
    Figure US20050020810A1-20050127-C00069
    X4 is
    Figure US20050020810A1-20050127-C00070
    X5 is
    Figure US20050020810A1-20050127-C00071
    R13 is hydrogen, acyl, substituted acyl, alkyl or substituted alkyl.
  • X6is
    Figure US20050020810A1-20050127-C00072
    X7 is —NH(CH2)eCO—, R3 is C1-C4 alkyl with at least one hydrogen atom replaced by a substituent selected from the group consisting of —NR15R16, aryl and substituted aryl, R15 and R16 are independently selected from the group consisting of hydrogen, acyl and substituted acyl and R4 and R5 are hydrogen. In one embodiment, s is 0 and r is 1, k is 1, R1 is acetyl, R13 is hydrogen, e is 1 and R3 is —(CH2)4NH2—. Preferably, q is 2, 4 or 6. In another embodiment, s is 0 and r is 1, k is 1, R1 is acetyl, R13 is hydrogen, e is 2, 4 or 6 and R3 is —CH2)4NHCO(CH2)2—Ph—(4—OH). Preferably, q is 1. In still another embodiment, s is 0 and r is 1, k is 1, R1 is acetyl, R13 is hydrogen, e is 2, 4 or 6 and R3 is —CH2—Ph—(4—OH). Preferably, q is 1. In still another embodiment, s is 0 and r is 1, k is 1, R1 is acetyl, R13 is methyl, e is 1 and R3 is —(CH2)4NH2. Preferably, q is 2. In still another embodiment, s is 1 and r is 0, j is 1, R1 is acetyl, R13 is hydrogen, h is 1 and R2 is —CH2—Ph—(4—OH). Preferably, p is 2, 4 or 6. In still another embodiment, s is 1 and r is 0, j is 1, R1 is acetyl, R13 is hydrogen, h is 2, 4, or 6 and R2 is —CH2—Ph—(4—OH). Preferably, p is 1. In still another embodiment, s is 1 and r is 0, j is 0, R1 is —CO(CH2)2—Ph—(4—OH), R13 is hydrogen and h is 1. Preferably, p is 2, 4 or 6. In still another embodiment, s is 1 and r is 0, j is 0, R1 is —O(CH2)2—Ph—(4—OH), R13 is hydrogen and h is 2, 4 or 6. Preferably, p is 1. In still another embodiment, s is 0 and r is 0, R1 is —(CH2)2—Ph—(4—OH) and R13 is hydrogen. In still another embodiment, s is 0 and r is 0, R1 is —COPh—(4—F) and R13 is hydrogen. In still another embodiment, s is 0 and r is 1, k is 1, R1 is acetyl, R13 is methyl or hydrogen, e is 1 and R3 is —CH2)4NHCOPh—(4—F). Preferably, q is 2. In still another embodiment, s is 0 and r is 1, k is 1, R1 is acetyl, R13 is hydrogen, e is 1 and R3 is —(CH2)4NH—8-[440 —fluorobenzylamino]suberoyl or —(CH2)4NHCOCH2F. Preferably, q is 2. In still another embodiment, s is 1 and r is 0, j is 0, R1 is 8-[4′-fluorobenzylamino]suberoyl or —COCH2F, R13 is hydrogen and h is 2. Preferably, p is 1. In still another embodiment, s is 0 and r is 1, k is 1, R1 is acetyl, R13 is hydrogen and R3 is —CH2Ph—(3-I, 4-OH) or —CH2Ph—(3,5-diI, 4-OH). Preferably, q is 0. Preferably, q is 1 and e is 2. Preferably, q is 1 and e is 1. In still another embodiment, s is 1 and r is 0, j is 1, R1 is acetyl, R13 is hydrogen and R2 is —CH2Ph—(3-I, 4-OH) or —CH2Ph—(3,5-diI, 4-OH). Preferably, p is 0. In still another embodiment, s is 0 and r is 0, R1 is —CO(CH2)2Ph (4-OH, 3, 5 di-I) and R13 is hydrogen. In still another embodiment, s is 1 and r is 0, j is 0, R1 is —CO(CH2)2Ph (4-OH, 3, 5 di-I), h is 2 and R13 is hydrogen. Poreferably, p is 1. In still another embodiment, s is 1 and r is 0, j is 1, R1 is acetyl, R2 is —CH2—Ph (4-OH, 3, 5 di-I), h is 2 and R13 is hydrogen. Preferably, p is 1. In still another embodiment, s is 0 and r is 1, R3 is —(CH2)4NHCO(CH2)2—Ph (4-OH, 3, 5 di-I), e is 1 and R13 is hydrogen. Preferably, q is 2.
  • In still another embodiment, R1 is acyl chelate, R2, R6, R7, X1, X2, X4, X5, R13, X6, X7, R3, R15, R16, R4 and R5 are as defined in the first embodiment. In one embodiment, s is 1 and r is 0, j is 0, R1 is DOTA-In, h is 2 and R13 is hydrogen. Preferably, p is 1. In another embodiment, s is 0 and r is 0, R1 is DPTA or DPTA-In and R13 is hydrogen.
  • In another aspect, the present invention provides a compound of Formula (III):
    Figure US20050020810A1-20050127-C00073
  • or a pharmaceutically acceptable salt, solvate, hydrate or N-oxide thereof wherein:
  • R20 is acyl, substituted acyl, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, imino, substituted imino or a diagnostic agent;
  • R21 is C1-C6 alkyl with at least one hydrogen atom replaced by a substituent selected from the group consisting of —NHR22;
  • R22 is hydrogen, acyl, substituted acyl, alkyl, substituted alkyl or a diagnostic agent; and
  • j, k, p, q, r, s, R2, X1, X2, X3, X4, X5, X6, X7, R4 and R5 are as defined in structural formula (I);
  • with the proviso that at least one of R20 and R22 is a diagnostic agent.
  • In one embodiment, R2, X1, X2, X3, X4, X5, X6, X7, R4 and R5 are as defined in the first preferred embodiment. In one embodiment, R20 is a fluorescent agent. Preferably, R20 is {fraction (5/6)} carboxy fluorescein, s is 1, r is 0, j is 0, e is 2 and p is 1. In another embodiment, R22 is a fluorescent agent. Preferably, R21 is (CH2)4NH—, R22 is—{fraction (5/6)} carboxy fluorescein, s is 0, r is 1, k is 1, e is 1 and q is 2. Preferably, R21 is (CH2)4NH—, R22 is biotin, s is 0, r is 1, k is 1, e is 1 and q is 2.
  • In anothere aspect, the present invention the present invention provides a compound of Formula (IV):
    Figure US20050020810A1-20050127-C00074
  • or a pharmaceutically acceptable salt, solvate, hydrate or N-oxide thereof wherein:
  • R23 is acyl, substituted acyl, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, imino, substituted imino or a pegylating agent;
  • R24 is C1-C6 alkyl with at least one hydrogen atom replaced by a substituent selected from the group consisting of —NHR28 wherein R28 is hydrogen, acyl, substituted acyl, alkyl substituted alkyl or a pegylating agent; and
  • j, k, p, q, r, s, R2, X1, X2, X3, X4, X5, X6, X7, R4 and R5 are as defined in structural formula (I);
  • with the proviso that at least one of R23 or R28 is a pegylating agent.
  • In one embodiment, R2, X1, X2, X3, X4, X5, X6, X7, R4 and R5 are as defined in the first preferred embodiment. Preferably, R23 is m-dPEG, s is 1, r is 0, j is 0, h is 2 and p is 1.
  • In still another aspect, the present invention provides a compound of Formula (V):
    Figure US20050020810A1-20050127-C00075
  • or a pharmaceutically acceptable salt, solvate, hydrate or N-oxide thereof wherein:
  • R29 is C1-C6 alkyl with at least one hydrogen atom replace by —NHR32;
  • R30 is acyl, substituted acyl, alkyl, substituted alkyl or a therapeutic agent.
  • R31 is hydrogen, alkyl, substituted alkyl or a therapeutic agent;
  • R32 is hydrogen, acyl substituted acyl, alkyl, substituted alkyl or a therapeutic agent; and;
  • j, k, p, q, r, s, R2, X1, X2, X3, X4, X5, X6, X7 and R4 and R5 are as defined structural formula (I);
  • with the proviso that at least one of R30, R31 and R32 is a therapeutic agent.
  • In one embodiment, R2, X1, X2, X3, X4, X5, X6, X7 and R4 are as defined in the first preferred embodiment Preferably, R13 is methyl or acetyl, s is 0, r is 0, R30 is acetyl and R31 is a therapeutic agent In one embodiment, the therapeutic agent is doxorubicin. In another embodiment, R13 is methyl or hydrogen, s is 0, r is 1, k is 1, e is 1, q is 2, R30 is acetyl, R31 is hydrogen, R29 is —CH2)4NHR32. Preferably, R32 is —CO(CH2)3-doxorubicin or protoporphyrin.
  • The use of unnatural amino acids is specifically contemplated in the present invention. Accordingly, variations of compounds invention includes, for example, the D-amino acids of the naturally occurring amino acids, β-alanine, 3-aminopropionic acid, 2,3 diaminopropionic acid, 4-aminobutyric acid, etc., sarcosine, orthinine, N-methyl glycine, citrulline, t-butyl alanine, homoarginine, etc. are within the scope of the present invention
  • One or amide bonds in the compounds of the invention may be optionally replaced by isosteres such as —CH2—NH—, —CH2—S—, —CH2—S(O)—, CH2—S(O)2—, —COCH2— —CH═CH—, CH(OH)CH2 which are well known in the art (see, e.g., Spatola, “Chemistry and Biochemistry of Amino Acids, Peptides and Proteins,” B. Weinstein, (eds.), Marcel Dekker, New York, 1983; Spatola et al., Life Sci. 1986, 38:1243-1249; Almquist et al., J. Med. Chem. 1980, 23:1392; Holladay et al., Tetrahedron Lett. 1983, 24:4401; Hruby, Life Sci. 1982, 4; 189:199; Jennings-White et al., Tetrahedron Lett. 1982, 23:2533; Hruby, Biopolymers 1993; 33:1073-1082; Wiley et aL, Med. Res. Rev. 1993 13:327-384; Moore et aL, Adv. in Phannacol 1995, 33:91-141; Giannis et aL, 1997, Adv. in Drug Res. 29:1-78). The peptides of the invention may also contain peptide mimetics such as those described in Olson et al., J. Med. Chem. 1993, 36:3039 and Chorev et aL, Science 1979, 204:1210.
  • Covalent modifications of the compounds of the invention are within the scope of the current invention and may improve the solubility, absorption, biological half life, etc. Such modifications may be effected by selective reaction of specific amino acid residues with organic reagents. For example, histidine residues may be selectively reacted with diethylpyrocarbonate at pH 5.5-7 and p-bromophenacyl bromide at pH 6.0. Residues containing free amino groups may be selectively reacted with carboxylic acid anhydrides, imidoesters, pyridoxal phosphate, trinitrobenzenesulfonic acid, O-methylisourea, 2,4 pentanedione, glyoxylate, etc. Arginyl residues may be selectively reacted with phenylglyoxal, and various diones. Glutaminyl and asparaginyl residues may be deaminated under mildly acidic conditions to provide the corresponding glutamyl and aspartyl residues. Proline and lysine may be selectively hydroxylated while serine and threonine residues may be selectively phosphorylated. The α-amino groups of histidine and lysine may be selectively methylated (Creighton, Proteins: Structure and Molecule Properties, W.H. Freeman & Co., San Francisco, pp. 79-86 (1983)).
  • Derivatization with bi-fnctional cross-linking agents (e.g., 1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde, N-hydroxy-succinimide esters, esters of 4-azidosalicylic acid, homobifunctional imidoesters (e.g., disuccinimidyl esters such as 3,3′- dithiobis(succinimidylpropionate)), bifinctional maleimides (e.g., bis-N-maleimido-1,8-octane, etc.) may be used to link compounds with water-insoluble support matrices or other macromolecular carriers. Photoactivatable agents such as methyl-3-[(p-azidophenyl) dithio]propioimidate may also be used to attach compounds with water-insoluble support matrices. Alternatively, compounds may be directly reacted with reactive water-insoluble matrices (e.g., cyanogen bromide-activated carbohydrates).
  • The present invention also includes longer peptides comprised of repeating units of the amino acid sequences of the compounds of the invention. In one embodiment, the repeating unit of such a multimer is the amino acid sequence of a compound where a, b, x, y, and z are 1. In another embodiment, the repeating unit is the amino acid sequence of a compound of invention where only one of a, b, x, y, and z is 0 and the rest are 1.
  • A multimer may be comprised of either the same or different combinations of repeating units comprised of amino acid sequences of compounds of structural formula (I). Such multimeric peptides can be made by either by chemical synthesis or by recombinant DNA techniques, followed by chemical modification of the cysteine residues. Preferably, the synthetic multimers have 2 to 12 repeats, more preferably, 2 to 8 repeats of the core peptide sequence. Accordingly, the total number of amino acids in the multimer should not exceed about 110 residues (or the equivalents, when including linkers or spacers).
  • A preferred multimer has the formula P1 n. where P1 is a pentapeptide, n is 2 to 8. In another embodiment, a multimer has the formula (P1-Xm)n-P2 where P1 and P2 are pentapeptides. P1 and P2 may be the same or different and each P1 may represent a different pentapeptide derivative of structural formula (I). X is C1-C5 alkyl, C1-C5 polyether containing up to 4 oxygen atoms or Glyz wherein, z=1-6, m=0 or 1 and n=1-7.
  • A preferred recombinantly produced peptide multimer has the formula: (P1-Glyz)n-P2 where P1 and P2 are pentapeptides which are the same or different and each P1 in the multimer may be a different pentapeptide, n=1-100 and z=0-6. The multimer may be optionally functionalized at both the N- and C-termini.
  • Compounds of the invention may be modified by the covalent attachment of any type of molecule as long as the modification does not prevent or inhibit biological function (i.e., inhibition or prevention of angiogenesis, cell invasion, cell proliferation, etc.). For example, a compound of the invention may be modified by glycosylation, acetylation, pegylation, phosphorylation, amidation, proteolytic cleavage, linkage to cellular ligand or protein, etc. Preferably, compounds of the invention are conjugated to a therapeutic agent or a diagnostic agent either directly or through a linking moiety.
  • Preferably, the linking moiety is first attached to a diagnostic or therapeutic agent to form a linking moiety intermediate which is then further attached to a compound of structural formula (I). As will be apparent to the skilled artisan, the linking moiety can also be first attached to a compound of the invention to form a linking moiety intermediate which can then be attached to a diagnostic agent or therapeutic agent.
  • Typically, a linking moiety will include a linker and a linking group for conjugating a therapeutic agent or diagnostic agent to a peptide. The nature of the linker will depend upon the particular application and the type of conjugation desired as the linker may be hydrophilic or hydrophobic, long or short, rigid or flexible. The linker may be optionally substituted with one ore more linking groups which may be either the same or different, accordingly providing polyvalent linking moieties which are capable of conjugating multiple therapeutic agents or diagnostic agents with a antibody.
  • A wide variety of linkers comprised of stable bonds suitable for spacing linking groups from the amino nitro compound are known in the art, and include by way of example and not limitation, alkyl, heteroalkyl, acyclic heteroatomic bridges, aryl, arylaryl, arylalkyl, heteroaryl, heteroaryl-heteroaryl, substituted heteroaryl-heteroaryl, heteroarylalkyl, heteroaryl-heteroalkyl and the like. Thus, the linker may include single, double, triple or aromatic carbon-carbon bonds, nitrogen-nitrogen bonds, carbon-nitrogen, carbon-oxygen bonds and/or carbon-sulfur bonds. Accordingly, finctionalities such as carbonyls, ethers, thioethers, carboxarnides, sulfonamides, ureas, urethanes, hydrazines, etc. may be included in a linker.
  • Choosing a suitable linker is within the capabilities of those of skill in the art. For example, where a rigid linker is desired, the linker may be rigid polyunsaturated alkyl or an aryl, biaryl, heteroaryl, etc. Where a flexible linker is desired, the linker may be a flexible peptide such as Gly-Gly-Gly or a flexible saturated alkanyl or heteroalkanyl. Hydrophilic linkers may be, for example, polyalcohols or polyethers such as polyalkyleneglycols. Hydrophobic linkers may be, for example, alkyls or aryls.
  • Preferably, a linking group is capable of mediating formation of a covalent bond with complementary reactive functionality of, for example, peptide to provide the therapeutic agent or diagnostic agent conjugated to the peptide. Accordingly, the linking group may be any reactive functional group known to those of skill in the art that will react with common chemical groups found in peptides (e.g., amino, sulfhydryl, hydroxyl, carboxylate, imidizaloyl, guandinium, amide, etc.). Accordingly, the linking group may be, for example, a photochemically activated group, an electrochemically activated group, a free radical donor, a free radical acceptor, a nucleophilic group or an electrophilic group. However, those of skill in the art will recognize that a variety of functional groups which are typically unreactive under certain reaction conditions can be activated to become reactive. Groups that can be activated to become reactive include, e.g., alcohols, carboxylic acids and esters, including salts thereof.
  • The linking group may be —NHR1, —NH2, —OH, —SH, halogen, —CHO, —R1CO, —SO2H, —PO2H, —N3, —CN, —CO2H, —SO3H, —PO3H, —PO2(O R1)H, —CO2R1, —SO3R1 or —PO(OR1)2 where R1 is alkyl. Preferably, the linking group is —NHR1, —NH2, —OH, —SH, —CHO, —CO2H, R1CO—, halogen and —CO2R1.
  • Some embodiments of the linker and the linking group include, for example, compounds where the linker is —(CH2)n—, n is an integer between 1 and 8, the linking group is —NH2, —OH, —CO2H, and —CO2R1 and the corresponding analogues where any suitable hydrogen is substituted. Other embodiments of the linking moiety include any amino acid, which may be, for example, a D or L amino acid. Thus, the linking moiety may be a dipeptide, a tripeptide or a tetrapeptide comprised of any combination of amino acids. The polarity of the peptide bond in these peptides may be either C—N or N—C.
  • Therapeutic agents and diagnostic agents may be linked to peptides directly using a variety of conventional reactions known to the skilled artisan. For example, condensation reagents (e.g., carbodiimides, carbonyldiimidazoles, etc.) may be used to form an amide bond linkage between an amino group of the therapeutic or diagnostic agent and the carboxylic acid groups of residues such as glutamic acid, aspartic acid and the C-terminal carboxyl group of a compound of structural formula (I).
  • Similar methods may be used to attach therapeutic agents and diagnostic agents containing a linker and linking group to compounds of structural formula (I). For example, diagnostic agents and therapeutic agents containing a linker and linking group may be attached to the amino group of lysine, the carboxylic acid groups of glutamic acid and aspartic acid, the sulfhydryl group of cysteine, the hydroxyl groups of threonine and serine and the various moieties of aromatic amino acids of peptides using conventional approaches known to the skilled artisan. In general, selection of an appropriate strategy for conjugating diagnostic agents or therapeutic agents to a peptide either directly or through a linker and linking group is well within the ambit of the skilled artisan.
  • Therapeutic agents which can be conjugated to peptides include, but are not limited to, radionuclides, porphyrins and porphyrin derivatives for photodynamic therapy (e.g., protoporphyrin, benzoporphyrin derivative monoacid A, tin-etio purpurin, meta-tetrahydroxyphenylchlorin, HPD, photofrin, protoporhyrin IX, Pc4, mono aspartyl chlorin e6, for others see T. Hassan et al, “PhotoDynamic Therapy of Cancer” in Cancer medicine, fiflh edition, R. C. Blast et al., Ed., B. C. Decker Inc, Canada, 2000, p. 489-502), protein toxins (e.g., ricin, Pseudomonas exotoxin, diptheria toxin, saporin, pokeweed antiviral protein, bouganin, etc.), cytotoxic cancer agents, camptothecins (e.g., 9-nitrocamptothecin (9NC), 9-aminocamptothecin (9AC), 10-aminocamptothecin, 9-chlorocamptothecin, 10,11-methylendioxycamptothecin, irinothecin, aromatic camptothecin esters, alkyl camptothecin esters, topotecan, (1S,9S)-1-amino-9-ethyl-5-fluoro-2,3-dihydro-9-hydroxy4-methyl-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1 ,2-b]quinoline-10,13(9H,15H)-dione methanesulfonate dihydrate (DX-8951f), 7-[(2-trimethyl-silyl)ethyl]-20(S)camptothecin (BNP1350), Rubitecan, Exatecan, Lurtotecan, Diflomotecan and other homocamptothecins, etc.), taxanes (e.g., taxol), epithilones, calicheamycins, hydroxy urea, cytarabine, cyclophosamide, ifosamide, nitrosureas, cisplatin, mitomycins maytansines, carboplatin, dacarbazine, procarbazine, etoposides, tenoposide, bleomycin, doxurobicin, 2-pyrrolinodoxurobicin, daunomycin, idarubican, daunorubicin, dactinomycin, plicamycin, mitoxantrone, asparginase, dihydroxy anthracine dione, mithrimycin, actinomycin D, 1-dehydrotestosterone, cytochlasins, vinblastine, vincristine, vinorelbine, paclitaxel, docetaxel, gramicidin D, glucocorticoids, anthracyclines, procaine, teracaine, lidocaine, propanolol, puromycin, methotrexate, 6-mercaptopurine, 6-thioguanine, mustard toxins, anthyrimycin, paclitaxel, alkylating agents (e.g., mechoremethamine, thioepa chlorambucil, melphalan, carmustine, loustine, cyclothosphamide, busulfan, dibromomannitol, streptozotocin, etc.) homologues and analogues thereof. Preferably, the therapeutic agent is a cytotoxic cancer agent, such as, for example, a taxane, a camptothecin, an epithilone or a anthracycline. In one embodiment, the therapeutic agent is doxorubicin. In another embodiment the therapeutic agent is a radionuclide.
  • Also within the current invention is conjugation of the compounds with various peglyating agents. Representative peglyating agents include, but are not limited to, a-methoxy-w-carboxy-PEG 2K & 5K1, a-methoxy-w-N-succinimidylglutarate-PEG 2K & 5K1, a-methoxy-w-glutarate-PEG 2K, 5K, 20K, 30K2, a-methoxy-w-GGGglutarate-PEG 2K & 5K1, mPEG-Succinimidyl propionate 2K, 5K, 20K, 30K2 and m-PEG-ButyrALD 2K, 5K, 20K, 30K2 (for other peglyating agents see Li et al., Biomacromolecules, 2003, 4, 1055.1067). Common pegylating agents are also available from commercial supplies such as Nektar Therapeutics, San Carlos, Calif. Methods for attachment of various PEG groups to peptides are numerous and are well known to the skilled artisan.
  • The term “diagnostically labeled” means that a peptide has an attached diagnostically detectable label. Many different labels exist in the art and methods of labeling are well known the skilled artisan. General classes of labels, which can be used in the present invention, include but are not limited to, radioactive isotopes, paramagnetic isotopes, compounds which can be imaged by positron emission tomography (PET), fluorescent or colored compounds, compounds which can be imaged by magnetic resonance, chemiluminescent compounds, bioluminescent compounds, etc. Suitable detectable labels include, but are not limited to, radioactive, fluorescent, fluorogenic or chromogenic labels. Useful radiolabels (radionuclides), which are detected simply by gamma counter, scintillation counter or autoradiography include, but are not limited to, 3H, 125I, 131I, 35S and 14C.
  • Methods and compositions for complexing metals to peptides are well known in the art. The metals are preferably detectable metal atoms, including radionuclides, and are complexed to proteins and other molecules (See, e.g., U.S. Pat. Nos. 5,627,286, 5,618,513, 5,567,408, 5,443,816 and 5,561,220).
  • Common fluorescent labels include, but are not limited to, fluorescein, rhodamine, dansyl, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine (Haugland, Handbook of Fluorescent Probes and Research Chemicals, Sixth Ed., Molecular Probes, Eugene, Or., 1996) may be used to label compounds of structural formula (I). Fluorescein, fluorescein derivatives and fluorescein-like molecules such as Oregon Green™ and its derivatives, Rhodamine Green™ and Rhodol Green™, are coupled to amine groups using the isothiocyanate, succinimidyl ester or dichlorotriazinyl-reactive groups. Similarly, fluorophores may also be coupled to thiols using maleimide, iodoacetamide, and aziridine-reactive groups. The long wavelength rhodamines, which are basically Rhodamine Green™ derivatives with substituents on the nitrogens are preferred labeling reagents. This group includes the tetramethylrhodamines, X-rhodamines and Texas Red™ derivatives. Other preferred fluorophores are those excited by ultraviolet light. Examples include, but are not limited to, cascade blue, coumarin derivatives, naphthalenes (of which dansyl chloride is a member), pyrenes and pyridyloxazole derivatives.
  • Inorganic materials such as semiconductor nanocrystals (Bruchez, et al., 1998, Science 281:2013-2016) and quantum dots, e.g., zinc-sulfide-capped Cd selenide (Chan, et al., Science 1998, 281:2016-2018) may also be used as diagnostic labels.
  • Peptides can also be labeled with fluorescence-emitting metals such as 152Eu or others of the lanthanide series. These metals can be attached to compounds of structural formula (I) through acyl chelating groups such as
  • diethylenetriaminepentaacetic acid (DTPA), ethylene-diamine-tetraacetic acid (EDTA), etc.
  • Radionuclides may be attached to peptides either directly or indirectly using an acyl chelating group such as DTPA and EDTA for in vivo diagnosis. The chemistry of chelation is well known in the art and varying ranges of chelating agent to peptide may be used to provide the labeled peptide. Of course, the labeled peptide must retain the biological activity of the native peptide.
  • Any radionuclide having diagnostic or therapeutic value can be used as the radiolabel in the present invention. In a preferred embodiment, the radionuclide is a y -emitting or beta -emitting radionuclide, for example, one selected from the lanthanide or actinide series of the elements. Positron-emitting radionuclides, e.g 68Ga or 64Cu, may also be used. Suitable gamma -emitting radionuclides include those which are useful in diagnostic imaging applications. The gamma -emitting radionuclides preferably have a half-life of from 1 hour to 40 days, preferably from 12 hours to 3 days. Examples of suitable gamma -emitting radionuclides include 67Ga, 111In, 99mTc, 169Yb and 186Re. Most preferably, the radionuclide is 99mTc.
  • Examples of preferred radionuclides (ordered by atomic number) are 67Cu, 67Ga, 68Ga, 72As, 89Zr, 90Y, 97Ru, 99Tc, 111In, 123I, 125I, 131I, 169Yb, 186Re, and 201Tl. Though limited work have been done with positron-emitting radiometals as labels, certain proteins, such as transferrin and human serum albumin, have been labeled with 68Ga.
  • A number of metals (not radioisotopes) useful for magnetic resonance imaging include gadolinium, manganese, copper, iron, gold and europium. Gadolinium is most preferred. Generally, the amount of labeled peptide needed for detectability in diagnostic use will vary depending on considerations such as age, condition, sex, and extent of disease in the patient, contraindications, if any, and other variables, and is to be adjusted by the individual physician or diagnostician. Dosage can vary from 0.01 mg/kg to 100 mg/kg.
  • Peptides may also be detected by coupling to a phosphorescent or a chemiluminescent compound, as is well known to the skilled artisan. Preferred chemiluminescent compounds include but are not limited to, luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester. Similarly, bioluminescent compounds may be used to detect antibodies and/or conjugates thereof and include, but are not limited to, luciferin, luciferase and aequorin.
  • Colorimetric detection, based on chromogenic compounds which have, or result in, chromophores with high extinction coefficients may also be used to detect compounds of structural formula (I).
    • 4.3 Synthesis
  • The compounds of the invention may be obtained via conventional synthetic methods. Starting materials useful for preparing compounds of the invention and intermediates thereof are commercially available or can be prepared by well-known synthetic methods.
  • Peptides may be prepared using solid-phase synthesis such as that generally described by Merrifield, J. Amer. Chem. Soc. 1963, 85:2149-54 using automated equipment, which may be purchased from chemical suppliers (e.g., Applied Biosystems, Foster City, Calif.) or manual equipment. Solid-phase peptide synthesis may be initiated from the C-terminus of the peptide by coupling a protected α-amino acid (either Boc or FMOC protected), to a suitable resin. Such a starting material can be prepared by attaching an α-amino-protected amino acid by an ester linkage to a chloromethylated resin, hydroxymethyl resin, BHA resin, MBHA resin or a Rink resin. Such methods, well-known in the art, are disclosed, for example, in U.S. Pat. No. 5,994,309. Alternatively, compounds of the invention may be made by solution phase synthesis using protected α-amino acids (see e g., Bodanszky, “Methods of Peptide Synthesis,” Springer Verlag, New York, 1984). As is apparent to those of skill in the art, unnatural amino acids can be easily employed in the above standard methods of chemical synthesis and may be made by conventional methods know to those of skill in the art.
  • The skilled artisan will appreciate that two general synthetic strategies exist for synthesis of compounds of the invention. Compounds with sulfur containing amino acids may be synthesized either directly by incorporation of the appropriate sulfur containing amino acid into a standard method of chemical synthesis as described above or indirectly by selective functionalization of an appropriate thiol containing peptide precursor and, if necessary, selective oxidation of the resultant thioether containing peptide. Methods for selectively finctionalizing free thiols (e.g., selective alkylation, acylation, disulfide formation, etc.) in the presence of diverse organic functionality are well known to the skilled artisan as are methods of oxidizing sulfides to sulfoxides (e.g., NaBO3, acetonitrile: water, NaIO4, acetonitrile: water, etc.) and sulfones (e.g., H2O2, HCO2H).
    • 4.4 Assays for Compounds of the Invention
  • Those of skill in the art will appreciate that the in vitro and in vivo assays usefull for measuring the activity of the compounds of the invention described herein are illustrative rather than comprehensive.
    • 4.4.1 Assay for Endothelial Cell Migration
  • For endothelial cell (EC) migration, transwells are coated with type I collagen (50 μg/mL) by adding 200 μL of the collagen solution per transwell, then incubating overnight at 37° C. The transwells are assembled in a 24-well plate and a chemoattractant (e.g., FGF-2) is added to the bottom chamber in a total volume of 0.8 mL media. ECs, such as human umbilical vein endothelial cells (HUVEC), which have been detached from monolayer culture using typsin, are diluted to a final concentration of about 106 cells/niL with serum-free media and 0.2 mL of this cell suspension is added to the upper chamber of each transwell. Inhibitors to be tested may be added to both the upper and lower chambers and the migration is allowed to proceed for 5 hrs in a humidified atmosphere at 37° C. The transwells are removed from the plate stained using DiffQuik®. Cells which did not migrate are removed from the upper chamber by scraping with a cotton swab and the membranes are detached, mounted on slides, and counted under a high-power field (400x) to determine the number of cells migrated.
    • 4.4.2 Biological Assay of Anti-Invasive Activity
  • The ability of cells such as ECs or tumor cells (e.g., PC-3 human prostatic carcinoma) cells to invade through a reconstituted basement membrane (Matrigel®) in an assay known as a Matrigel® invasion assay system has been described in detail in the art (Kleinman et al., Biochemistry 1986, 25: 312-318; Parish et al., 1992, Int. J. Cancer 52:378-383). Matrigel® is a reconstituted basement membrane containing type IV collagen, laminin, heparan sulfate proteoglycans such as perlecan, which bind to and localize bFGF, vitronectin as well as transforming growth factor-β (TGFβ), urokinase-type plasminogen activator (uPA), tissue plasminogens activator (tpA) and the serpin known as plasminogen activator inhibitor type 1 (PAI- 1) (Chambers et al., Canc. Res. 1995, 55:1578-1585). It is accepted in the art that results obtained in this assay for compounds which target extracellular receptors or enzymes are predictive of the efficacy of these cornpounds in vivo (Rabbani et al., Int. J. Cancer 1995, 63: 840-845).
  • Such assays employ transwell tissue culture inserts. Invasive cells are defined as cells which are able to traverse through the Matrigel® and upper aspect of a polycarbonate membrane and adhere to the bottom of the membrane. Transwells (Costar) containing polycarbonate membranes (8.0 μm pore size) are coated with Matrigel® (Collaborative Research), which has been diluted in sterile PBS to a final concentration of 75 μg/mL (60 μL of diluted Matrigel® per insert), and placed in the wells of a 24-well plate. The membranes are dried overnight in a biological safety cabinet, then rehydrated by adding 100 μL of DMEM containing antibiotics for 1 hour on a shaker table. The DMEM is removed from each insert by aspiration and 0.8 mL of DMEM/10% FBS/antibiotics is added to each well of the 24-well plate such that it surrounds the outside of the transwell (“lower chamber”). Fresh DMEM/antibiotics (100 μL), human Glu-plasminogen (5 μg/mL), and any inhibitors to be tested are added to the top, inside of the transwell (“upper chamber”). The cells which are to be tested are trypsinized and resuspended in DMEM/antibiotics, then added to the top chamber of the transwell at a final concentration of 800,000 cells/mL. The final volume of the upper chamber is adjusted to 200 μL. The assembled plate is then incubated in a humid 5% CO2 atmosphere for 72 hours. After incubation, the cells are fixed and stained using DiffQuik® (Giemsa stain) and the upper chamber is then scraped using a cotton swab to remove the Matrigel® and any cells which did not invade through the membrane. The membranes are detached from the transwell using an X-acto® blade, mounted on slides using Permount® and cover-slips, then counted under a high-powered (400x) field. An average of the cells invaded is determined from 5-10 fields counted and plotted as a function of inhibitor concentration.
    • 4.4.3 Tube-Formation Assays of Anti-Angiogenic Activity
  • Endothelial cells, for example, human umbilical vein endothelial cells (HUVEC) or human microvascular endothelial cells (HMVEC) which can be prepared or obtained commercially, are mixed at a concentration of 2×105 cells/mL with fibrinogen (5 mg/mL in phosphate buffered saline (PBS) in a 1:1 (v/v) ratio. Thrombin is added (5 units/mL final concentration) and the mixture is immediately transferred to a 24-well plate (0.5 mL per well). The fibrin gel is allowed to form and then VEGF and bFGF are added to the wells (each at 5 ng/mL final concentration) along with the test compound. The cells are incubated at 37° C. in 5% CO2 for 4 days at which time the cells in each well are counted and classified as either rounded, elongated with no branches, elongated with one branch, or elongated with 2 or more branches. Results are expressed as the average of 5 different wells for each concentration of compound. Typically, in the presence of angiogenic inhibitors, cells remain either rounded or form undifferentiated tubes (e.g. 0 or 1 branch). This assay is recognized in the art to be predictive of angiogenic (or anti-angiogenic) efficacy in vivo (Min et aL, Cancer Res. 1996, 56: 2428-2433).
  • In an alternate assay, endothelial cell tube formation is observed when endothelial cells are cultured on Matrigel® (Schnaper et aL, J. Cell. PhysioL 1995, 165: 107-118). Endothelial cells (1×104 cells/well) are transferred onto Matrigel®-coated 24-well plates and tube formation is quantitated after 48 hrs. Inhibitors are tested by adding them either at the same time as the endothelial cells or at various time points thereafter. Tube formation can also be stimulated by adding (a) angiogenic growth factors such as bFGF or VEGF, (b) differentiation stimulating agents (e.g., PMA) or (c) a combination of these.
  • While not wishing to be bound by theory, this assay models angiogenesis by presenting to the endothelial cells a particular type of basement membrane, namely the layer of matrix which migrating and differentiating endothelial cells might be expected to first encounter. In addition to bound growth factors, the matrix components found in Matrigel® (and in basement membranes in situ) or proteolytic products thereof may also be stimulatory for endothelial cell tube formation which makes this model complementary to the fibrin gel angiogenesis model previously described (Blood et aL, Biochim. Biophys. Acta 1990, 1032: 89-118; Odedrat al., Pharmac. Ther. 1991, 49: 111-124).
    • 4.4.4. Assays for Inhibition of Proliferation
  • The ability of the compounds of the invention to inhibit the proliferation of EC's may be determined in a 96-well format. Type I collagen (gelatin) is used to coat the wells of the plate (0.1-1 mg/mL in PBS, 0.1 mL per well for 30 minutes at room temperature). After washing the plate (3x w/PBS), 3-6,000 cells are plated per well and allowed to attach for 4 hrs (37° C./5% CO2) in Endothelial Growth Medium (EGM; Clonetics ) or M199 media containing 0.1-2% FBS. The media and any unattached cells are removed at the end of 4 hrs and fresh media containing bFGF (1-10 nglmL) or VEGF (1-10 ng/mL) is added to each well. Compounds to be tested are added last and the plate is allowed to incubate (37° C./5% CO2) for 24-48 hrs. MTS (Promega) is added to each well and allowed to incubate from 1-4 hrs. The absorbance at 490 nm, which is proportional to the cell number, is then measured to determine the differences in proliferation between control wells and those containing test compounds. A similar assay system can be set up with cultured adherent tumor cells. However, collagen may be omitted in this fonnat. Tumor cells (e.g., 3,000-10,000/well) are plated and allowed to attach overnight. Serum free medium is then added to the wells,, and the cells are synchronized for 24 hrs. Medium containing 10% FBS is then added to each well to stimulate proliferation. Compounds to be tested are included in some of the wells. After 24 hrs, MTS is added to the plate and the assay developed and read as described above.
    • 4.4.5 Assays of Cytotoxicity
  • The anti-proliferative and cytotoxic effects of compounds of the invention may be determined for various cell types including tumor cells, ECs, fibroblasts and macrophages. This is especially useful when testing a compound of the invention which has been conjugated to a therapeutic moiety such as a radiotherapeutic or a toxin. For example, a conjugate of one of the compounds of the invention with Bolton-Hunter reagent which has been iodinated with 131I would be expected to inhibit the proliferation of cells expressing an PHSCN binding site/receptor (most likely by inducing apoptosis). Anti-proliferative effects would be expected against tumor cells and stimulated endothelial cells but, under some circumstances not quiescent endothelial cells or normal human dermal fibroblasts. Any anti-proliferative or cytotoxic effects observed in the normal cells may represent non-specific toxicity of the conjugate.
  • A typical assay would involve plating cells at a density of 5-10,000 cells per well in a 96-well plate. The compound to be tested is added at a concentration I Ox the IC50 measured in a binding assay (this will vary depending on the conjugate) and allowed to incubate with the cells for 30 minutes. The cells are washed 3X with media, then fresh media containing [3H]thymidine (1 μCi/mL) is added to the cells and they are allowed to incubate at 37° C. in 5% CO2 for 24 and 48 hours. Cells are lysed at the various time points using 1 M NaOH and counts per well determined using a β-counter. Proliferation may be measured non-radioactively using MTS reagent or CyQuant® to measure total cell number. For cytotoxicity assays (measuring cell lysis), a Promega 96-well cytotoxicity kit is used. If there is evidence of anti-proliferative activity, induction of apoptosis may be measured using TumorTACS (Genzyme).
    • 4.4.6 Caspase-3 Activity
  • The ability of the compounds of the invention to promote apoptosis of EC's may be determined by measuring activation of caspase-3. Type I collagen (gelatin) is used to coat a P100 plate and 5×105 ECs are seeded in EGM containing 10% FBS. After 24 hours (at 37° C. in5% CO2) the medium is replaced by EGM containing 2% FBS, 10 ng/ml bFGF and the desired test compound. The cells are harvested after 6 hours, cell lysates prepared in 1% Triton and assayed using the EnzChek®Caspase-3 Assay Kit #1 (Molecular Probes) according to the manufactures instructions.
    • 4.4.7. Corneal Angiogenesis Model
  • The protocol used is essentially identical to that described by Volpert et aL, J. Clin. Invest. 1996, 98:671-679. Briefly, female Fischer rats (120-140 gms) are anesthetized and pellets (5 μl) comprised of Hydron®, bFGF (150 nM), and the compounds to be tested are implanted into tiny incisions made in the cornea 1.0-1.5 mm from the limbus. Neovascularization is assessed at 5 and 7 days after implantation. On day 7, animals are anesthetized and infused with a dye such as colloidal carbon to stain the vessels. The animals are then euthanized, the corneas fixed with formalin, and the corneas flattened and photographed to assess the degree of neovascularization. Neovessels may be quantitated by imaging the total vessel area or length or simply by counting vessels.
    • 4.4.8 Matrigel® Plug Assay
  • This assay is performed essentially as described by Passaniti et al., 1992, Lab Invest. 67:519-528. Ice-cold Matrigel® (e.g., 500 μL) (Collaborative Biomedical Products, Inc., Bedford, Mass.) is mixed with heparin (e.g., 50 μg/ml), FGF-2 (e.g., 400 ng/ml) and the compound to be tested. In some assays, bFGF may be substituted with tumor cells as the angiogenic stimulus. The Matrigel® mixture is injected subcutaneously into 4-8 week-old athymic nude mice at sites near the abdominal midline, preferably 3 injections per mouse. The injected Matrigel® forms a palpable solid gel. Injection sites are chosen such that each animal receives a positive control plug (such as FGF-2+heparin), a negative control plug (e.g., buffer+heparin) and a plug that includes the compound being tested for its effect on angiogenesis, e.g., (FGF-2+heparin+compound). All treatments are preferably run in triplicate. Animals are sacrificed by cervical dislocation at about 7 days post injection or another time that may be optimal for observing angiogenesis. The mouse skin is detached along the abdominal midline, and the Matrigel® plugs are recovered and scanned immediately at high resolution. Plugs are then dispersed in water and incubated at 37° C. overnight. Hemoglobin (Hb) levels are determined using Drabkin's solution (e.g., obtained from Sigma) according to the manufacturers' instructions. The amount of Hb in the plug is an indirect measure of angiogenesis as it reflects the amount of blood in the sample. In addition, or alternatively, animals may be injected prior to sacrifice with a 0.1 ml buffer (preferably PBS) containing a high molecular weight dextran to which is conjugated a fluorophore. The amount of fluorescence in the dispersed plug, determined fluorimetrically, also serves as a measure of angiogenesis in the plug. Staining with mAb anti-CD31 (CD31 is “platelet-endothelial cell adhesion molecule or PECAM”) may also be used to confirm neovessel formation and microvessel density in the plugs.
    • 4.4.9 Chick Chorioallantoic Membrane (CAM) Angiogenesis Assay
  • This assay is performed essentially as described by Nguyen et al., Microvascular Res. 1994, 47:3140. A mesh containing either angiogenic factors (bFGF) or tumor cells plus inhibitors is placed onto the CAM of an 8-day old chick embryo and the CAM observed for 3-9 days after implantation of the sample. Angiogenesis is quantitated by determining the percentage of squares in the mesh which contain blood vessels.
    • 4.4.10 In Vivo Assessment of Angiogenesis Inhibition and Anti-Tumor Effects Using the Matrigel® Plug Assay with Tumor Cells
  • In this assay, tumor cells, for example 1−5×106 cells of the 3LL Lewis lung carcinoma or the rat prostate cell line MatLyLu, are mixed with Matrigel® and then injected into the flank of a mouse following the protocol described in Sec. B., above. A mass of tumor cells and a powerful angiogenic response can be observed in the plugs after about 5 to 7 days. The anti-tumor and anti-angiogenic action of a compound in an actual tumor environment can be evaluated by including it in the plug. Measurement is then made of tumor weight, Hb levels or fluorescence levels (of a dextran-fluorophore conjugate injected prior to sacrifice). To measure Hb or fluorescence, the plugs are first homogenize with a tissue homogenizer.
    • 4.4.11 Xenograft Model of Subcutaneous (s.c.) Tumor Growth
  • Nude mice are inoculated with MDA-MB-231 cells (human breast carcinoma) and Matrigel™ (1×106 cells in 0.2 mL) s.c. in the right flank of the animals. The tumors are staged to 200 mm3 and then treatment with a test composition is initiated (100 μg/animal/day given q.d. IP). Tumor volumes are obtained every other day and the animals are sacrificed after 2 weeks of treatment. The tumors are excised, weighed and paraffin embedded. Histological sections of the tumors are analyzed by H and E, anti-CD31, Ki-67, TUNEL, and CD68 staining.
    • 4.4.12 Xenograft Model of Metastasis
  • The compounds of the invention are also tested for inhibition of late metastasis using an experimental metastasis model (Crowley et al., Proc. Natl. Acad. Sci. USA 1993, 90 5021-5025). Late metastasis involves the steps of attachment and extravasation of tumor cells, local invasion, seeding, proliferation and angiogenesis. Human prostatic carcinoma cells (PC-3) transfected with a reporter gene, preferably the green fluorescent protein (GFP) gene, but as an alternative with a gene encoding the enzymes chloramphenicol acetyl-transferase (CAT), luciferase or LacZ, are inoculated into nude mice. This approach permits utilization of either of these markers (fluorescence detection of GFP or histochemical calorimetric detection of enzymatic activity) to follow the fate of these cells. Cells are injected, preferably iv, and metastases identified after about 14 days, particularly in the lungs but also in regional lymph nodes, femurs and brain. This mimics the organ tropism of naturally occurring metastases in human prostate cancer. For example, GFP-expressing PC-3 cells (1×106 cells per mouse) are injected iv into the tail veins of nude (nu/nu) mice. Animals are treated with a test composition at 100 μg/animal/day given q.d. IP. Single metastatic cells and foci are visualized and quantitated by fluorescence microscopy or light microscopic histochemistry or by grinding the tissue and quantitative colorimetric assay of the detectable label.
    • 4.4.13. Inhibition of Spontaneous Metastasis In Vivo by PHSCN and Functional Derivatives
  • The rat syngeneic breast cancer system employs Mat BIII rat breast cancer cells (Xing et al., Int. J. Cancer 1996, 67:423429). Tumor cells, for example, about 106 suspended in 0.1 mL PBS, are inoculated into the mammary fat pads of female Fisher rats. At the time of inoculation, a 14-day Alza osmotic mini-pump is implanted intraperitoneally to dispense the test compound. The compound is dissolved in PBS (e.g., 200 mM stock), sterile filtered and placed in the minipump to achieve a release rate of about 4 mg/kg/day. Control animals receive vehicle (PBS) alone or a vehicle control peptide in the minipump. Animals are sacrificed at about day 14. In the rats treated with the compounds of the present invention, significant reductions in the size of the primary tumor and in the number of metastases in the spleen, lungs, liver, kidney and lymph nodes (enumerated as discrete foci) may be observed. Histological and immunohistochemical analysis reveal increased necrosis and signs of apoptosis in tumors in treated animals. Large necrotic areas are seen in tumor regions lacking neovascularization. Human or rabbit PHSCN and their derivatives to which 131I is conjugated (either 1 or 2 I atoms per molecule of peptide) are effective radiotherapeutics and are found to be at least two-fold more potent than the unconjugated polypeptides. In contrast, treatment with control peptides fails to cause a significant change in tumor size or metastasis.
    • 4.4.14. 3LL Lewis Lung Carcinoma: Primary Tumor Growth
  • This tumor line arose spontaneously as carcinoma of the lung in a C57BL/6 mouse (Malave et al., J. Nat'l. Canc. Inst. 1979, 62:83-88). It is propagated by passage in C57BL/6 mice by subcutaneous (sc) inoculation and is tested in semiallogeneic C57BL/6×DBA/2 F1 mice or in allogeneic C3H mice. Typically six animals per group for subcutaneously (sc) implant, or ten for intramuscular (im) implant are used. Tumor may be implanted sc as a 2-4 mm fragment, or im or sc as an inoculum of suspended cells of about 0.5-2×106-cells. Treatment begins 24 hours after implant or is delayed until a tumor of specified size (usually approximately 400 mg) can be palpated. The test compound is administered ip daily for 11 days Animals are followed by weighing, palpation, and measurement of tumor size. Typical tumor weight in untreated control recipients on day 12 after im inoculation is 500-2500 mg. Typical median survival time is 18-28 days. A positive control compound, for example cyclophosphamide at 20 mg/kg/injection per day on days 1-11 is used. Results computed include mean animal weight, tumor size, tumor weight, survival time. For confirmed therapeutic activity, the test composition should be tested in two multi-dose assays.
    • 4.4.15 3LL Lewis Lung Carcinoma: Primary Growth and Metastasis Model
  • This assay is well known in the art (Gorelik et al., J. Nat'l. Canc. Inst. 1980, 65:1257-1264; Gorelik et al., Rec. Results Canc. Res. 1980, 75:20-28; Isakov et al., Invasion Metas. 2:12-32 (1982); Talmadge et al., J. Nat'l. Canc. Inst. 1982, 69:975-980; Hilgard et al., Br. J. Cancer 1977, 35:78-86). Test mice are male C57BL/6 mice, 2-3 months old. Following sc, im, or intra-footpad implantation, this tumor produces metastases, preferentially in the lungs. With some lines of the tumor, the primary tumor exerts anti-metastatic effects and must first be excised before study of the metastatic phase (see also U.S. Pat. No. 5,639,725). Single-cell suspensions are prepared from solid tumors by treating minced tumor tissue with a solution of 0.3% trypsin. Cells are washed 3 times with PBS (pH 7.4) and suspended in PBS. Viability of the 3LL cells prepared in this way is generally about 95-99% (by trypan blue dye exclusion). Viable tumor cells (3×104-5×106) suspended in 0.05 ml PBS are injected subcutaneously, either in the dorsal region or into one hind foot pad of C57BL/6 mice. Visible tumors appear after 3-4 days after dorsal sc injection of 106 cells. The day of tumor appearance and the diameters of established tumors are measured by caliper every two days. The treatment is given as one to five doses of peptide or derivative, per week. In another embodiment, the peptide is delivered by osmotic minipump.
  • In experiments involving tumor excision of dorsal tumors, when tumors reach about 1500 mm3 in size, mice are randomized into two groups: (1) primary tumor is completely excised; or (2) sham surgery is performed and the tumor is left intact. Although tumors from 500-3000 mm3 inhibit growth of metastases, 1500 mm3 is the largest size primary tumor that can be safely resected with high survival and without local regrowth. After 21 days, all mice are sacrificed and autopsied.
  • Lungs are removed and weighed. Lungs are fixed in Bouin's solution and the number of visible metastases is recorded. The diameters of the metastases are also measured using a binocular stereoscope equipped with a micrometer-containing ocular under 8× magnification. On the basis of the recorded diameters, it is possible to calculate the volume of each metastasis. To determine the total volume of metastases per lung, the mean number of visible metastases is multiplied by the mean volume of metastases. To further determine metastatic growth, it is possible to measure incorporation of 125IdUrd into lung cells (Thakur et al., J. Lab. Clin. Med. 1977, 89:217-228). Ten days following tumor amputation, 25 μg of fluorodeoxyuridine is inoculated into the peritoneums of tumor-bearing (and, if used, tumor-resected mice). After 30 min, mice are given 1 μCi of 125IdUrd (iododeoxyuridine). One day later, lungs and spleens are removed and weighed, and a degree of 125IdUrd incorporation is measured using a gamma counter.
  • In mice with footpad tumors, when tumors reach about 8-10 mm in diameter, mice are randomized into two groups: (1) legs with tumors are amputated after ligation above the knee joints; or (2) mice are left intact as nonamputated tumor-bearing controls. (Amputation of a tumor-free leg in a tumor-bearing mouse has no known effect on subsequent metastasis, ruling out possible effects of anesthesia, stress or surgery). Mice are killed 10-14 days after amputation. Metastases are evaluated as described above.
  • Statistics: Values representing the incidence of metastases and their growth in the lungs of tumor-bearing mice are not normally distributed. Therefore, non-parametric statistics such as the Mann-Whitney U-Test may be used for analysis. Study of this model by Gorelik et al. (1980, supra) showed that the size of the tumor cell inoculum determined the extent of metastatic growth. The rate of metastasis in the lungs of operated mice was different from primary tumor-bearing mice. Thus in the lungs of mice in which the primary tumor had been induced by inoculation of larger doses of 3LL cells (1-5×106) followed by surgical removal, the number of metastases was lower than that in nonoperated tumor-bearing mice, though the volume of metastases was higher than in the nonoperated controls. Using 125IdUrd incorporation as a measure of lung metastasis, no significant differences were found between the lungs of tumor-excised mice and tumor-bearing mice originally inoculated with 106 3LL cells. Amputation of tumors produced following inoculation of 105 tumor cells dramatically accelerated metastatic growth. These results were in accord with the survival of mice after excision of local tumors. The phenomenon of acceleration of metastatic growth following excision of local tumors had been repeatedly observed (for example, see U. S. Pat. No. 5,639,725). These observations have implications for the prognosis of patients who undergo cancer surgery.
    • 4.4.15 Competition Binding Assay with DU145 Prostate Cancer Cells
  • These can be carried at 6 concentrations of peptide to determine an IC50. This assay is a surrogate marker of biological activity. Briefly, DU145 cell are harvested by brief incubation with trypsin, the peptide derivative to be tested (competitor) and standard are added and the suspension is agitated at 4° C. for 2 hours. The cells are pelleted, supernatant aspirated, PBS added and the process is repeated three times (washes). HPR-Streptavidin is added, allowed to bind followed by a second series of wash steps. The appropriate HRP substrate is added and color is developed within the previously defined linear part of the reaction.
    • 4.4.16 Human Endothelial Cell (HUVEC) Proliferation Assay
  • The assay is carried out in a 96 well assay and takes place for 48 hours. Briefly, HUVECs (Cascade Biologicals) are cultured overnight in M200 containing 2% FBS. The following day, 3,000 cells are plated in each well of a gelatin-coated 96-well plate. The cells are allowed to adhere and spread for 4-6 hours, at which time the medium is replaced with fresh medium containing 2% FBS, 1 ng/mL FGF-2, and varying concentrations of specific test compounds. Cells are then cultured for an additional 48 hours, and relative cell numbers in each well are determined using the Cell Titer Aqueous Cell Proliferation Assay (Promega). The result will be an IC50 that can be compared to a standard compound. This is an assay that reflects biological function of the peptide derivatives against their target cells.
    • 4.4.17 Dosing Range and Kinetics
  • Four doses of peptide (2, 4, 8 and 10 μCi for biodistribution and 10, 50, 100, and 200 μCi for Gamma scintigraphy) are injected via tail vein into tumor (B16F10 melanoma cells) bearing (200-300 mm3) C57Bl/6 mice. The lowest and highest dosages of peptides are tested first and second. Thus, it is possible that the dosage of peptide derivative to be tested may be modified based on the initial results obtained.
  • (Group I, Dosimetry 3 animals/dose) Animals will be sacrificed after 2 hours after dosing with 125I labeled peptides. Tumors (theoretical high counts) and hearts (theoretical low counts) will then be removed and radioactivity counted.
  • (Group 2 Gamma scintigraphy 3 animals/dose) Animals are anesthetized and imaged (gamma scintigraphy) 2 hours after injection of 125I radiolabeled peptide A peptide dosage is chosen based on these results.
    • 4.4.18 Imaging Feasability
  • 1251 labeled peptide at a dose determined by the method of section 4.4.17 will be injected via tail vein into tumor bearing mice (200-300 mm3 tumor). Three mice per time point will be anesthetized and imaged at 0.5 h, 1 h, 2 h, 4 h, 6 h, 24 h, and 48 h which will determine the optimal time for imaging. As a control, unlabelled peptide at a 100-fold molar excess will be co-injected with the 125I labeled peptide to demonstrate equivalency of both species.
    • 4.5 Recombinant DNA Methods
  • General methods of molecular biology have been amply described in the art (Sambrook, et al., Molecular Cloning: A Laboratory Manual, 2nd (or later) Edition, Cold Spring Harbor Press, Cold Spring Harbor, N.Y., 1989; Ausube et al., Current Protocols in Molecular Biology, Vol. 2, Wiley-Interscience, New York, (current edition); Kriegler, Gene Transfer and Expression: A Laboratory Manual (1990); Glover, D M, editor, DNA Cloning: A Practical Approach, vol. I & II, IRL Press, 1985; Alberts et al., Molecular Biology of the Cell, 2nd (or later) Ed., Garland Publishing, Inc., New York, N.Y. (1989); Watson et al., Recombinant DNA, 2nd (or later) Ed., Scientific American Books, New York, 1992; and Old et al., Principles of Gene Manipulation: An Introduction to Genetic Engineering, 2nd (or later) Ed., University of California Press, Berkeley, Calif. (1981)).
  • Unless otherwise indicated, a particular nucleic acid sequence is intended to encompasses conservative substitution variants thereof (e.g., degenerate codon substitutions) and a complementary sequence. The term “nucleic acid” is synonymous with “polynucleotide” and is intended to include a gene, a cDNA molecule, an mRNA molecule, as well as a fragment of any of these such as an oligonucleotide, and further, equivalents thereof (explained more fully below). Sizes of nucleic acids are stated either as kilobases (kb) or base pairs (bp). These are estimates derived from agarose or polyacrylamide gel electrophoresis (PAGE), from nucleic acid sequences which are determined by the user or published. Protein size is stated as molecular mass in kilodaltons (kDa) or as length (number of amino acid residues). Protein size is estimated from PAGE, from sequencing, from presumptive amino acid sequences based on the coding nucleic acid sequence or from published amino acid sequences.
  • Specifically, DNA molecules encoding the amino acid sequence corresponding to the peptide multimers of the present invention, or active variants thereof, can be synthesized by the polymerase chain reaction (PCR) (see, for example, U.S. Pat. No. 4,683,202) using primers derived the sequence of the protein disclosed herein. These cDNA sequences can then be assembled into a eukaryotic or prokaryotic expression vector and the resulting vector can be used to direct the synthesis of the fusion polypeptide or its fragment or derivative by appropriate host cells, for example COS or CHO cells.
  • The term “nucleic acid” as used herein is intended to include such fragments or equivalents. The nucleic acid sequences of this invention can be DNA or RNA. Prokaryotic or eukaryotic host cells transformed or transfected to express the multimers are within the scope of the invention. For example, the peptide multimer may be expressed in bacterial cells such as E. coli, insect cells (baculovirus), yeast, or mammalian cells such as Chinese hamster ovary cells (CHO) or human cells (which are preferred for human therapeutic use of the transfected cells). Other suitable host are known to those skilled in the art. Expression in eukaryotic cells leads to partial or complete glycosylation and/or formation of relevant inter- or intra-chain disulfide bonds of the recombinant polypeptide. Examples of vectors for expression in yeast S. cerevisiae include pYepSec1 (Baldari et al., 1987, EMBO J. 6:229-234), pMFa (Kurjan et al. 1982 Cell 30:933-943), pJRY88 (Schultz et al., 1987, Gene 54:113-123), and pYES2 (Invitrogen Corporation, San Diego, Calif.). Baculovirus vectors available for expression of proteins in cultured insect cells (SF 9 cells) include the pAc series (Smith et al., 1983, Mol. Cell Biol. 3:2156-2165) and the pVL series (Lucklow et al., (1989) Virology 170:31-39). Generally, COS cells (Gluzman 1981 Cell 23:175-182) are used in conjunction with such vectors as pCDM 8 (Aruffoet al., supra, for transient amplification/expression in mammalian cells, while CHO (dhfr-negative CHO) cells are used with vectors such as pMT2PC (Kaufman et al., 1987, EMBO J. 6:187-195) for stable amplification/expression in mammalian cells. The NS0 myeloma cell line (a glutamine synthetase expression system.) is available from Celltech Ltd.
  • Construction of suitable vectors containing the desired coding and control sequences employs standard ligation and restriction techniques which are well understood in the art. Isolated plasmids, DNA sequences, or synthesized oligonucleotides are cleaved, tailored, and re-ligated in the form desired. The DNA sequences which form the vectors are available from a number of sources. Backbone vectors and control systems are generally found on available “host” vectors which are used for the bulk of the sequences in construction. For the pertinent coding sequence, initial construction may be, and usually is, a matter of retrieving the appropriate sequences from cDNA or genomic DNA libraries. However, once the sequence is disclosed it is possible to synthesize the entire gene sequence in vitro starting from the individual nucleotide derivatives. The entire gene sequence for genes of sizeable length, e.g., 500-1000 bp may be prepared by synthesizing individual overlapping complementary oligonucleotides and filling in single stranded nonoverlapping portions using DNA polymerase in the presence of the deoxyribonucleotide triphosphates. This approach has been used successfully in the construction of several genes of known sequence. See, for example, Edge, Nature 1981, 292:756; Nambair et al., Science 1984, 223:1299; and Jay, J. Biol. Chem. 1984, 259:6311.
  • Synthetic oligonucleotides are prepared by either the phosphotriester method as described by references cited above or the phosphoramidite method as described by Beaucage et al., Tetrahedron Lett. 1981, 22:1859; and Matteucci et al., J. Am. Chem. Soc. 1981, 103:3185 and can be prepared using commercially available automated oligonucleotide synthesizers. Kinase treatment of single strands prior to annealing or for labeling is achieved using well-known methods.
  • Once the components of the desired vectors are thus available, they can be excised and ligated using standard restriction and ligation procedures. Site-specific DNA cleavage is performed by treating with the suitable restriction enzyme (or enzymes) under conditions which are generally understood in the art, and the particulars of which are specified by the manufacturer of these commercially available restriction enzymes. See, e.g., New England Biolabs, Product Catalog. If desired, size separation of the cleaved fragments may be performed by polyacrylamide gel or agarose gel electrophoresis using standard techniques. A general description of size separations is found in Meth. Enzymol. (1980) 65:499-560. Any of a number of methods are used to introduce mutations into the coding sequence to generate variants if these are to be produced recombinantly. These mutations include simple deletions or insertions, systematic deletions, insertions or substitutions of clusters of bases or substitutions of single bases. Modifications of the DNA sequence are created by site-directed mutagenesis, a well-known technique for which protocols and reagents are commercially available (Zoller et al., Nucleic Acids Res. 1982, 10:6487-6500 and Adelman et al., DNA 1983,2:183-193)). The isolated DNA is analyzed by restriction and/or sequenced by the dideoxy nucleotide method of Sanger, Proc. Natl. Acad. Sci. USA 1977, 74:5463) as further described by Messing, et al., Nucleic Acids Res. 1981, 9:309, or by the method of Maxam et al., Meth. Enzymol., supra.
  • Vector DNA can be introduced into mammalian cells via conventional techniques such as calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, or electroporation. Suitable methods for transforming host cells can be found in Sambrook et al. supra and other standard texts. In fusion expression vectors, a proteolytic cleavage site is introduced at the junction of the reporter group and the target protein to enable separation of the target protein from the reporter group subsequent to purification of the fusion protein. Proteolytic enzymes for such cleavage and their recognition sequences include Factor Xa, thrombin and enterokinase.
    • 4.6 Therapeutic Uses
  • In accordance with the invention, a compound of the invention and/or a pharmaceutical composition thereof is administered to a patient, preferably a human, suffering from cancer or form a disease characterized by angiogenesis. The compounds of the invention and/or pharmaceutical compositions thereof may be used to treat or prevent cancer or undesired angiogenesis.
  • Preferably, cancers include any vascularized tumor, preferably, a solid tumor, including but not limited to, carcinomas of the lung, breast, ovary, stomach, pancreas, larynx, esophagus, testes, liver, parotid, bilary tract, colon, rectum, cervix, uterus, endometrium, kidney, bladder, prostrate, thyroid, squamous cell carcinomas, adenocarcinomas, small cell carcinomas, melanomas, gliomas, neuroblastomas, sarcomas (e.g., angiosarcomas, chondrosarcomas)). Disease characterized by undesired angiogenesis include, but are not limited to, arthritis, diabetes, arteriosclerosis, arteriovenous, malformations, corneal graft neovascularization, delayed wound healing, diabetic retinopathy, age related macular degeneration, granulations, burns, hemophilic joints, rheumatoid arthritis, hypertrophic scars, neovascular glaucoma, nonunion fractures, Osier Weber Syndrome, psoriasis, pyogenic, granuloma, retrolental fibroplasia, pterygium, scleroderma, trachoma, vascular adhesions, ocular neovascularization, parasitic diseases, hypertrophy following surgery, inhibition of hair growth, macular degeneration (including both wet and dry type), rheumatoid arthritis and osteoarthritis.
  • Also contemplated are methods for treating a patient having a disease or condition associated with undesired cell migration, invasion or proliferation comprising administering to the subject an therapeutically effective amount of a compound of the invention and/or a pharmaceutical composition thereof. In the above methods, the patient has a tumor, and angiogenesis inhibition results in reduction in size or growth rate of the tumor or destruction of the tumor. Preferably, the subject is a human.
  • Other examples of diseases or conditions against which the above methods may be effective include primary growth of a solid tumor, leukemia or lymphoma, tumor invasion, metastasis or growth of tumor metastases; benign hyperplasia; atherosclerosis, myocardial angiogenesis; post-balloon angioplasty vascular restinosis, neointima formation following vascular trauma, vascular graft restinosis, coronary collateral formation, deep venous thrombosis, ischemic limb angiogenesis; telangiectasia, pyogenic granuloma, corneal disease, rubeosis, neovascular glaucoma, diabetic and other retinopathy, retrolental fibroplasias, diabetic neovascularization, macular degeneration, endometriosis, arthritis, fibrosis associated with a chronic inflammatory condition, traumatic spinal cord injury including ischemia, scarring or fibrosis, lung fibrosis, chemotherapy-induced fibrosis; wound healing with scarring and fibrosis, peptic ulcers, a bone fracture, keloids, or a disorder of vasculogenesis, hematopoiesis, ovulation, menstruation, pregnancy or placentation associated with pathogenic cell invasion or with angiogenesis.
  • A preferred disease or condition to be treated by the above methods are tumor growth, invasion or metastasis, particularly brain tumors. Examples of such brain tumors are astrocytoma, anaplastic astrocytoma, glioblastoma, glioblastoma multiformae, pilocytic astrocytoma, pleiomorphic xanthoastrocytoma, subependymal giant cell astrocytoma, fibrillary astrocytoma, gemistocytic astrocytoma, protoplasmic astrocytoma, oligodendroglioma, anaplastic oligodendroglioma, ependymoma, anaplastic ependymoma, myxopapillary ependymoma, subependymoma, mixed oligoastrocytoma and malignant oligoastrocytoma.
  • The above methods may also be used to treat uterine diseases such as endometriosis and pathogenic ocular neovascularization associated with, or a cause of, proliferative diabetic retinopathy, neovascular age-related macular degeneration, retinopathy of prematurity, sickle cell retinopathy or retinal vein occlusion.
  • Further, in certain embodiments, compounds of the invention and/or pharmaceutical compositions thereof are administered to a patient, preferably a human, as a preventative measure against various diseases or disorders characterized by undesired angiogenesis including cancer. Thus, the compounds of the invention and/or pharmaceutical compositions thereof may be administered as a preventative measure to a patient having a predisposition for a disease characterized by undesired angiogenesis. Accordingly, the compounds and/or pharmaceutical compositions of the invention may be used for the prevention of one disease or disorder and concurrently treating another (e.g., preventing arthritis while treating cancer).
  • The suitability of the compounds of the invention and/or pharmaceutical compositions thereof in treating or preventing various diseases or disorders such as cancer may be assayed by methods described herein and in the art. Accordingly, it is well with the capability of those of skill in the art to assay and use the compounds of the invention and/or pharmaceutical compositions thereof to treat or prevent diseases or disorders such as cancer.
    • 4.7 Diagnostic Uses and Methods
  • A compound of the invention and/or a pharmaceutical composition thereof is administered to a patient, preferably a human, in a diagnostically effective amount to detect or image a disease such as those listed in Section 5.6 above. Further, compounds of the invention and/or pharmaceutical compositions thereof may be used to detect or image diseases or conditions associated with undesired cell migration, invasion or proliferation such as those listed above in Section 5.6 by administering to a subject an diagnostically effective amount of a compound of the invention and/or a pharmaceutical composition thereof.
  • Compounds of the invention may be diagnostically labeled and used, for example, to detect cell migration, cell invasion and cell proliferation. The disposition of a compound of the invention during and after binding may be followed in vitro or in vivo by using an appropriate method to detect the label. Diagnostically labeled compounds may be utilized in vivo for diagnosis and prognosis, for example, to image occult metastatic foci or for other types of in situ evaluations. For diagnostic applications, compounds of the invention may include bound linker moieties, which are well known to those of skill in the art
  • In situ detection of the labeled compound may be accomplished by removing a histological specimen from a subject and examining it by microscopy under appropriate conditions to detect the label. Those of ordinary skill will readily perceive that any of a wide variety of histological methods (such as staining procedures) can be modified in order to achieve such in situ detection.
  • For diagnostic in vivo radioimaging, the type of detection instrument available is a major factor in selecting a radionuclide. The radionuclide chosen must have a type of decay which is detectable by a particular instrument. In general, any conventional method for visualizing diagnostic imaging can be utilized in accordance with this invention. Another factor in selecting a radionuclide for in vivo diagnosis is that its half-life be long enough so that the label is still detectable at the time of maximum uptake by the target tissue, but short enough so that deleterious irradiation of the host is minimized. In one preferred embodiment, a radionuclide used for in vivo imaging does not emit particles, but produces a large number of photons in a 140-200 keV range, which may be readily detected by conventional gamma cameras.
  • In vivo imaging may be used to detect occult metastases which are not observable by other methods. Compounds of the present invention may be used in diagnostic, prognostic or research procedures in conjunction with any appropriate cell, tissue, organ or biological sample of a desired animal species. By the term “biological sample” is intended any fluid or other material derived from the body of a normal or diseased subject, such as blood, serum, plasma, lymph, urine, saliva, tears, cerebrospinal fluid, milk, amniotic fluid, bile, ascites fluid, pus and the like. Also included within the meaning of this term is a organ or tissue extract and a culture fluid in which any cells or tissue preparation from the subject has been incubated.
  • Useful doses are defined as effective amount of a compound for the particular diagnostic measurement. Thus, an effective amount means an amount sufficient to be detected using the appropriate detection system e.g., magnetic resonance imaging detector, gamma camera, etc. The minimum detectable amount will depend on the ratio of labeled compound specifically bound to a tumor (signal) to the amount of labeled compound either bound non-specifically or found free in plasma or in extracellular fluid.
  • The amount of a composition to be administered depends on the precise compound selected, the disease or condition, the route of administration, and the judgment of the skilled imaging professional. Generally, the amount of a compound needed for detectability in diagnostic use will vary depending on considerations such as age, condition, sex, and extent of disease in the patient, contraindications, if any, and other variables, and is to be adjusted by the individual physician or diagnostician. Dosage can vary from 0.01 mg/kg to 100 mg/kg.
    • 4.8 Therapeutic/Prophylactic Administration
  • The compounds the invention and/or pharmaceutical compositions thereof may be advantageously used in human medicine. As previously described in Section 4.6 above, compounds of the invention and/or pharmaceutical compositions thereof are useful for the treatment or prevention of various diseases or disorders such as cancer.
  • When used to treat or prevent the above disease or disorders, compounds of the invention and/or pharmaceutical compositions thereof may be administered or applied singly, or in combination with other agents. The compounds of the invention and/or pharmaceutical compositions thereof may also be administered or applied singly, in combination with other pharmaceutically active agents (e.g., other anti-cancer agents, other anti-angiogenic agents such as chelators as zinc, penicillamine, thiomolybdate etc.), including other compounds of the invention.
  • The current invention provides methods of treatment and prophylaxis by administration to a patient of a therapeutically effective amount of a compound and/or pharmaceutical composition of the invention. The patient may be an animal, is more preferably, a mammal and most preferably, a human.
  • The present compounds and/or pharmaceutical compositions of the invention, may be administered orally. The compounds and/or pharmaceutical compositions of the invention may also be administered by any other convenient route, for example, by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.). Administration can be systemic or local. Various delivery systems (e.g., encapsulation in liposomes, microparticles, microcapsules, capsules, etc.) may be used to administer a compound and/or pharmaceutical composition of the invention. Methods of administration include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, oral, sublingual, intranasal, intracerebral, intravaginal, transdermal, rectally, by inhalation, or topically, particularly to the ears, nose, eyes, or skin. The preferred mode of administration is left to the discretion of the practitioner, and will depend in-part upon the site of the medical condition. In most instances, administration will result in the release of the compounds and/or pharmaceutical compositions of the invention into the bloodstream.
  • In specific embodiments, it may be desirable to administer one or more compounds and/or pharmaceutical composition of the invention locally to the area in need of treatment. This may be achieved, for example, and not by way of limitation, by local infusion during surgery, topical application, e.g., in conjunction with a wound dressing after surgery, by injection, by means of a catheter, by means of a suppository, or by means of an implant, said implant being of a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers. In one embodiment, administration can be by direct injection at the site (or former site) of cancer or arthritis.
  • In certain embodiments, it may be desirable to introduce one or more compounds and/or pharmaceutical compositions of the invention into the central nervous system by any suitable route, including intraventricular, intrathecal and epidural injection. Intraventricular injection may be facilitated by an intraventricular catheter, for example, attached to a reservoir, such as an Ommaya reservoir.
  • A compound and/or pharmaceutical composition of the invention may also be administered directly to the lung by inhalation. For administration by inhalation, a compound and/or pharmaceutical composition of the invention may be conveniently delivered to the lung by a number of different devices. For example, a Metered Dose Inhaler (“MDI”), which utilizes canisters that contain a suitable low boiling propellant, (e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or any other suitable gas) may be used to deliver compounds of the invention directly to the lung.
  • Alternatively, a Dry Powder Inhaler (“DPI”) device may be used to administer a compound and/or pharmaceutical composition of the invention to the lung. DPI devices typically use a mechanism such as a burst of gas to create a cloud of dry powder inside a container, which may then be inhaled by the patient. DPI devices are also well known in the art. A popular variation is the multiple dose DPI (“MDDPI”) system, which allows for the delivery of more than one therapeutic dose. MDDPI devices are available from companies such as AstraZeneca, GlaxoWellcome, IVAX, Schering Plough, SkyePharma and Vectura. For example, capsules and cartridges of gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of a compound of the invention and a suitable powder base such as lactose or starch for these systems.
  • Another type of device that may be used to deliver a compound and/or pharmaceutical composition of the invention to the lung is a liquid spray device supplied, for example, by Aradigm Corporation, Hayward, Calif. Liquid spray systems use extremely small nozzle holes to aerosolize liquid drug formulations that may then be directly inhaled into the lung.
  • In one embodiment, a nebulizer is used to deliver a compound and/or pharmaceutical composition of the invention to the lung. Nebulizers create aerosols from liquid drug formulations by using, for example, ultrasonic energy to form fine particles that may be readily inhaled (see e.g., Verschoyle et al., British J. Cancer, 1999, 80, Suppl. 2, 96, which is herein incorporated by reference). Examples of nebulizers include devices supplied by Batelle Pulmonary Therapeutics (Columbus, Ohio) (See, Armer et al., U.S. Pat. No.5,954,047; van der Linden et al., U.S. Pat. No.5,950,619; van der Linden et al., U.S. Pat. No. 5,970,974).
  • In another embodiment, an electrohydrodynamic (“EHD”) aerosol device is used to deliver a compound and/or pharmaceutical composition of the invention to the lung. EHD aerosol devices use electrical energy to aerosolize liquid drug solutions or suspensions (see e.g., Noakes et al., U.S. Pat. No. 4,765,539). The electrochemical properties of the formulation may be important parameters to optimize when delivering a compound and/or pharmaceutical composition of the invention to the lung with an EHD aerosol device and such optimization is routinely performed by one of skill in the art. EHD aerosol devices may more efficiently deliver drugs to the lung than existing pulmonary delivery technologies.
  • In another embodiment, the compounds and/or pharmaceutical compositions of the invention can be delivered in a vesicle, in particular a liposome (see Langer, 1990, Science, 249:1527-1533; Treat et al., in “Liposomes in the Therapy of Infectious Disease and Cancer,” Lopez-Berestein and Fidler (eds.), Liss, New York, pp.353-365 (1989); see generally “Liposomes in the Therapy of Infectious Disease and Cancer,” Lopez-Berestein and Fidler (eds.), Liss, New York, pp.353-365 (1989)).
  • In another embodiment, the compounds and/or pharmaceutical compositions of the invention can be delivered via sustained release systems, preferably, oral sustained release systems. In one embodiment, a pump may be used (Langer, supra; Sefton, 1987, CRC Crit. Ref Biomed. Eng. 14:201; Saudek et al., 1989, N. Engl. J Med. 321:574).
  • In another embodiment, polymeric materials can be used (see “Medical Applications of Controlled Release,” Langer and Wise (eds.), CRC Pres., Boca Raton, Fla. (1974); “Controlled Drug Bioavailability,” Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984); Langer t al., 1983, J Macromol. Sci. Rev. Macromol Chem. 23:61; Levy et al., 1985, Science 228: 190; During et al., 1989, Ann. Neurol. 25:351; Howard et al., 1989, J. Neurosurg. 71:105). In another embodiment, polymeric materials are used for oral sustained release delivery. Preferred polymers include sodium carboxymethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose and hydroxyethylcellulose (most preferred, hydroxypropyl methylcellulose). Other preferred cellulose ethers have been described (Alderman, Int. J. Pharm. Tech. & Prod. Mfr., 1984, 5(3) 1-9). Factors affecting drug release are well known to the skilled artisan and have been described in the art (Bamba et al., Int. J. Pharm., 1979, 2, 307).
  • In another embodiment, enteric-coated preparations can be used for oral sustained release administration. Preferred coating materials include polymers with a pH-dependent solubility (i.e., pH-controlled release), polymers with a slow or pH-dependent rate of swelling, dissolution or erosion (i.e., time-controlled release), polymers that are degraded by enzymes (i.e., enzyme-controlled release) and polymers that form firm layers that are destroyed by an increase in pressure (i.e., pressure-controlled release).
  • In still another embodiment, osmotic delivery systems are used for oral sustained release administration (Verma et al., Drug Dev. Ind. Pharm. 2000, 26:695-708). In still another embodiment, OROS™ osmotic devices are used for oral sustained release delivery devices (Theeuwes et al., U.S. Pat. No. 3,845,770; Theeuwes et al., U.S. Pat. No.3,916,899).
  • In yet another embodiment, a controlled-release system can be placed in proximity of the target of the compounds and/or pharmaceutical composition of the invention, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in “Medical Applications of Controlled Release,” supra, vol. 2, pp. 115-138 (1984)). Other controlled-release systems discussed in Langer, 1990, Science 249:1527-1533 may also be used.
    • 4.9 Pharmaceutical Compositions
  • The present pharmaceutical compositions contain a therapeutically or diagnostically effective amount of one or more compounds of the invention, preferably, in purified form, together with a suitable amount of a pharmaceutically acceptable vehicle, so as to provide the form for proper administration to a patient. When administered to a patient, the compounds of the invention and pharmaceutically acceptable vehicles are preferably sterile. Water is a preferred vehicle when the compounds of the invention are administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid vehicles, particularly for injectable solutions. Suitable pharmaceutical vehicles also include excipients such as starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The present pharmaceutical compositions, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. In addition, auxiliary, stabilizing, thickening, lubricating and coloring agents may be used.
  • Pharmaceutical compositions comprising a compound of the invention may be manufactured by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes. Pharmaceutical compositions may be formulated in conventional manner using one or more physiologically acceptable carriers, diluents, excipients or auxiliaries, which facilitate processing of compounds of the invention into preparations which can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.
  • The present pharmaceutical compositions can take the form of solutions, suspensions, emulsion, tablets, pills, pellets, capsules, capsules containing liquids, powders, sustained-release formulations, suppositories, emulsions, aerosols, sprays, suspensions, or any other form suitable for use. In one embodiment, the pharmaceutically acceptable vehicle is a capsule (see e.g., Grosswald et al., U.S. Pat. No. 5,698,155). Other examples of suitable pharmaceutical vehicles have been described in the art (see Remington's Pharmaceutical Sciences, Philadelphia College of Pharmacy and Science, 19th Edition, 1995).
  • For topical administration, compounds of the invention may be formulated as solutions, gels, ointments, creams, suspensions, etc. as is well-known in the art. Systemic formulations include those designed for administration by injection, e.g., subcutaneous, intravenous, intramuscular, intrathecal or intraperitoneal injection, as well as those designed for transdermal, transmucosal, oral or pulmonary administration. Systemic formulations may be made in combination with a further active agent that improves mucociliary clearance of airway mucus or reduces mucous viscosity. These active agents include, but are not limited to, sodium channel blockers, antibiotics, N-acetyl cysteine, homocysteine and phospholipids.
  • In one embodiment, the compounds of the invention are formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings. Typically, compounds of the invention for intravenous administration are solutions in sterile isotonic aqueous buffer. For injection, a compound of the invention may be formulated in aqueous solutions, preferably, in physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiological saline buffer. The solution may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. When necessary, the pharmaceutical compositions may also include a solubilizing agent. Pharmaceutical compositions for intravenous administration may optionally include a local anesthetic such as lignocaine to ease pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. When the compound of the invention is administered by infusion, it can be dispensed, for example, with an infusion bottle containing sterile pharmaceutical grade water or saline. When a compound of the invention is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.
  • For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
  • Pharmaceutical compositions for oral delivery may be in the form of tablets, lozenges, aqueous or oily suspensions, granules, powders, emulsions, capsules, syrups, or elixirs, for example. Orally administered pharmaceutical compositions may contain one or more optionally agents, for example, sweetening agents such as fructose, aspartame or saccharin; flavoring agents such as peppermint, oil of wintergreen, or cherry coloring agents and preserving agents, to provide a pharmaceutically palatable preparation. Moreover, when in tablet or pill form, the compositions may be coated to delay disintegration and absorption in the gastrointestinal tract, thereby providing a sustained action over an extended period of time. Selectively permeable membranes surrounding an osmotically active driving compound are also suitable for orally administered compounds of the invention. In these later platforms, fluid from the environment surrounding the capsule is imbibed by the driving compound, which swells to displace the agent or agent composition through an aperture. These delivery platforms can provide an essentially zero order delivery profile as opposed to the spiked profiles of immediate release formulations. A time delay material such as glycerol monostearate or glycerol stearate may also be used. Oral compositions can include standard vehicles such as mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Such vehicles are preferably of pharmaceutical grade.
  • For oral liquid preparations such as, for example, suspensions, elixirs and solutions, suitable carriers, excipients or diluents include water, saline, alkyleneglycols (e.g., propylene glycol), polyalkylene glycols (e.g., polyethylene glycol) oils, alcohols, slightly acidic buffers between pH 4 and pH 6 (e.g., acetate, citrate, ascorbate at between about 5.0 mM to about 50.0 mM), etc. Additionally, flavoring agents, preservatives, coloring agents, bile salts, acylcamitines and the like may be added.
  • For buccal administration, the pharmaceutical compositions may take the form of tablets, lozenges, etc. formulated in conventional manner.
  • Liquid drug formulations suitable for use with nebulizers and liquid spray devices and EHD aerosol devices will typically include a compound of the invention with a pharmaceutically acceptable vehicle. Preferably, the pharmaceutically acceptable vehicle is a liquid such as alcohol, water, polyethylene glycol or a perfluorocarbon. Optionally, another material may be added to alter the aerosol properties of the solution or suspension of compounds of the invention. Preferably, this material is liquid such as an alcohol, glycol, polyglycol or a fatty acid. Other methods of formulating liquid drug solutions or suspension suitable for use in aerosol devices are known to those of skill in the art (see, e.g., Biesalski, U.S. Pat. No. 5,112,598; Biesalski, U.S. Pat. No. 5,556,611).
  • A compound of the invention may also be formulated in rectal or vaginal pharmaceutical compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.
  • In addition to the formulations described previously, a compound of the invention may also be formulated as a depot preparation. Such long acting formulations may be administered by implantation (for example, subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, a compound of the invention may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
  • When a compound of the invention is acidic, it may be included in any of the above-described formulations as the free acid or a pharmaceutically acceptable salt. Pharmaceutically acceptable salts substantially retain the activity of the free acid, may be prepared by reaction with bases and tend to be more soluble in aqueous and other protic solvents than the corresponding free acid form.
    • 4.10 Doses
  • A compound of the invention, or pharmaceutical compositions thereof, will generally be used in an amount effective to achieve the intended purpose. For use to treat or prevent diseases or disorders uch as cancer the compounds of structural Formula (I) and/or pharmaceutical compositions thereof, are administered or applied in a therapeutically effective amount. For use to detect diseases or disorders such as cancer the compounds of structural Formula (I) and/or pharmaceutical compositions thereof, are administered or applied in a diagnostically effective amount.
  • The amount of a compound of the invention that will be effective in the treatment, prevention or detection of a particular disorder or condition disclosed herein will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques known in the art as previously described. In addition, in vitro or in vivo assays may optionally be employed to help identify optimal dosage ranges. The amount of a compound of the invention administered will, of course, be dependent on, among other factors, the subject being treated, the weight of the subject, the severity of the affliction, the manner of administration and the judgment of the prescribing physician.
  • For example, the dosage may be delivered in a pharmaceutical composition by a single administration, by multiple applications or controlled release. In one embodiment, the compounds of the invention are delivered by oral sustained release administration. Preferably, in this embodiment, the compounds of the invention are administered twice per day (more preferably, once per day). Dosing may be repeated intermittently, may be provided alone or in combination with other drugs and may continue as long as required for effective treatment of the disease state or disorder.
  • Suitable dosage ranges for oral administration are dependent on the potency of the drug, but are generally about 0.001 mg to about 200 mg of a compound of the invention per kilogram body weight. Dosage ranges may be readily determined by methods known to the artisan of ordinary skill.
  • Suitable dosage ranges for intravenous (i.v.) administration are about 0.01 mg to about 100 mg per kilogram body weight. Suitable dosage ranges for intranasal administration are generally about 0.01 mg/kg body weight to about 1 mg/kg body weight. Suppositories generally contain about 0.01 milligram to about 50 milligrams of a compound of the invention per kilogram body weight and comprise active ingredient in the range of about 0.5% to about 10% by weight. Recommended dosages for intradermal, intramuscular, intraperitoneal, subcutaneous, epidural, sublingual or intracerebral administration are in the range of about 0.001 mg to about 200 mg per kilogram of body weight. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems. Such animal models and systems are well-known in the art.
  • The compounds of the invention are preferably assayed in vitro and in vivo, as described above, for the desired therapeutic, prophylactic or diagnostic activity, prior to use in humans. For example, in vitro assays can be used to determine whether administration of a specific compound of the invention or a combination of compounds of the invention is preferred for treating, preventing or diagnosing cancer. The compounds of the invention may also be demonstrated to be effective and safe using animal model systems.
  • Preferably, a therapeutically effective dose of a compound of the invention described herein will provide therapeutic benefit without causing substantial toxicity. Similarly, a diagnostically effective dose of a compound of the invention described herein will provide diagnostic benefit without causing substantial toxicity. Toxicity of compounds of the invention may be determined using standard pharmaceutical procedures and may be readily ascertained by the skilled artisan. The dose ratio between toxic and therapeutic effect is the therapeutic index. A compound of the invention will preferably exhibit particularly high therapeutic indices in treating disease and disorders. The dosage of a compound of the inventions described herein will preferably be within a range of circulating concentrations that include an effective therapeutic or diagnostic does dose with little or no toxicity.
    • 4.11 Combination Therapy
  • In certain embodiments of the present invention, the compounds and/or pharmaceutical compositions of the invention can be used in combination therapy with at least one other therapeutic agent. The compound and/or pharmaceutical composition of the invention and the therapeutic agent can act additively or, more preferably, synergistically. In one embodiment, a compound and/or pharmaceutical composition of the invention is administered concurrently with the administration of another therapeutic agent, which may be part of the same pharmaceutical composition or a different pharmaceutical composition. In another embodiment, a pharmaceutical composition of the invention is administered prior or subsequent to administration of another therapeutic agent.
  • In particular, in one preferred embodiment, the compounds and/or pharmaceutical compositions of the invention can be used in combination therapy with other chemotherapeutic agents (e.g., alkylating agents (e.g., nitrogen mustards (e.g., cyclcphosphamide, ifosfamide, mechlorethamine, melphalen, chlorambucil, hexamethylmelamine, thiotepa), alkyl sulfonates (e.g., busulfan), nitrosoureas, triazines), antimetabolites (e.g., folic acid analogs, pyrimidine analogs (e.g., fluorouracil, floxuridine, cytosine arabinoside, etc.), purine analogs (e.g., mercaptopurine, thiogunaine, pentostatin, etc.), natural products (e.g., vinblastine, vincristine, etoposide, tertiposide, dactinomycin, daunorubicin, doxurubicin, bleomycin, mithrmycin, mitomycin C, L-asparaginase, interferon alpha), platinum coordination complexes (e.g., cis-platinum, carboplatin, etc.), mitoxantrone, hydroxyurea, procarbazine, hormones and antagonists (e.g., prednisone, hydroxyprogesterone caproate, medroxyprogesterone acetate, megestrol acetate, diethylstilbestrol, ethinyl estradiol, tamoxifen, testosterone propionate, fluoxymesterone, flutamide, leuprolide, etc.), anti-angiogenesis agents or inhibitors (e.g., angiostatin, retinoic acids and paclitaxel, estradiol derivatives, thiazolopyrimidine derivatives, etc.), apoptosis-inducing agents (e.g., antisense nucleotides that block oncogenes which inhibit apoptosis, tumor suppressors, TRAIL, TRAIL polypeptide, Fas-associated factor 1, interleukin-1β-converting enzyme, phosphotyrosine inhibitors, RXR retinoid receptor agonists, carbostyril derivatives, etc.) and chelators (penicillamine, zinc, trientine, etc.)).
    • 4.12 Therapeutic Kits
  • The current invention provides therapeutic kits comprising the compounds of the invention or pharmaceutical compositions of the invention. The therapeutic kits may also contain other compounds (e.g., chemotherapeutic agents, natural products, hormones or antagonists, anti-angiogenesis agents or inhibitors, apoptosis-inducing agents or chelators) or pharmaceutical compositions of these other compounds.
  • Therapeutic kits may have a single containers which contains the compound of the invention or pharmaceutical compositions of the invention with or without other components (e.g., other compounds or pharmaceutical compositions of these other compounds) or may have distinct container for each component. Preferably, therapeutic kits of the invention include a compound and/or a pharmaceutical composition of the invention packaged for use in combination with the co-administration of a second compound (preferably, a chemotherapeutic agent, a natural product, a hormone or antagonist, a anti-angiogenesis agent or inhibitor, a apoptosis-inducing agent or a chelator) and/or a pharmaceutical composition thereof. The components of the kit may be pre-complexed or each component may be in a separate distinct container prior to administration to a patient.
  • The components of the kit may be provided in one or more liquid solutions, preferably, an aqueous solution, more preferably, a sterile aqueous solution. The components of the kit may also be provided as solids, which may be converted into liquids by addition of suitable solvents, which are preferably provided in another distinct container.
  • The container of a therapeutic kit may be a vial, test tube, flask, bottle, syringe, or any other means of enclosing a solid or liquid. Usually, when there is more than one component, the kit will contain a second vial or other container, which allows for separate dosing. The kit may also contain another container for a pharmaceutically acceptable liquid.
  • Preferably, a therapeutic kit will contain apparatus (e.g., one or more needles, syringes, eye droppers, pipette, etc.), which enables administration of the components of the kit.
    • 5. EXAMPLES
  • The invention is further defined by reference to the following examples, which describe in detail, preparation of compounds of the invention and methods for assaying for biological activity. It will be apparent to those skilled in the art that many modifications, both to materials and methods, may be practiced without departing from the scope of the invention.
  • In the examples below, the following abbreviations have the following meanings. If an abbreviation is not defined, it has its generally accepted meaning.
      • AcCN=acetonitrile
      • Boc=tert-butyloxycarbonyl
      • CPM=counts per minute
      • DMF=N,N-dimethylformamide
      • DMSO=dimethylsulfoxide
      • Fmoc=9-fluorenylmethyloxycarbonyl
      • g=gram
      • h=hour
      • HBTU=O-Benzotriazole, N, N, N, N, tetramethyl uronium hexafluoro phosphate
      • HBSS=Hank's buffered saline solution
      • HOBT=N-hydroxybenxotriazole
      • HPLC=high pressure liquid chromatography
      • L=liter
      • LC/MS=liquid chromatography/mass spectroscopy
      • M=molar
      • min=minute
      • mL=milliliter
      • mmol=millimoles
      • NHS=N-hydroxysuccinimide
      • NMM=N-methyl morpholine
      • TFA=trifluoroacetic acid
      • TIS=triisopropylsilane
      • TLC=thin layer chromatography
      • μL=microliter
      • μM=micromolar
      • v/v=volume to volume
    5.1 Example 1 Standard Resin Bound Amino Acid Coupling
  • Rink Amide AM resin (Novabiochem) was treated with 20% piperidine in DMF (1 mL per 100 mg of resin) for three minutes with nitrogen agitation or vibration and the reaction mixture was filtered. This step was repeated an additional two times. The resin was washed three times with DMF, three times with methanol and three times with dichloromethane. The desired Fmoc protected, tritylated amino acid (BOC or iVDde was used for lysine and t-Bu used for tyrosine) (3 eq), HBTU (3 eq), and HOBt (3 eq) were dissolved in DMF (1 mL per 100 mg of resin) and added to the above resin, followed by the addition of N-Methylmorpholine (NMM) (6 eq) and the mixture was agitated for 1 hour. The reaction mixture was filtered and the resin was washed with DMF. This coupling step was repeated and then the resin was washed with DMF three times, MeOH three times and DCM three times. The resin was treated with 20% piperidine in DMF (3×3 minutes each) as described above to remove the Fmoc group on the first amino acid. The next desired Fmoc protected, tritylated amino acid (3 eq), HBTU (3 eq), and HOBt (3 eq) were dissolved in DMF (1 mL per 100 mg of resin) and added to the above resin, followed by the addition of N-Methylmorpholine (NMM) (6 eq) and the mixture was agitated for 1 hour. The reaction mixture was filtered and the resin was washed with DMF three times, MeOH three times and DCM three times. Subsequent amino acids were single coupled in a similar manner. For peptides containing an N-terminus acetyl, the following commercially available amino acids, Ac-Pro-OH (3 eq) or Ac-Tyr-OH (3 eq) were used and coupled with HBTU (3 eq), HOBT (3 eq) and NMM (6 eq) for an hour. For all other examples, the capping of the N-terminus was performed by coupling the terminal amine on the resin with the appropriate acid (3 eq), HBTU (3 eq), HOBT (3 eq) and NMM (6 eq). The fully assembled N-capped peptide on Rink Amide AM resin was treated with TFA/TIS/water (95:2.5:2.5, 1 mL per 100 mg of resin) and agitated with nitrogen or vibration for 1 hour. The reaction mixture was filtered, the resin was washed with TFA/TIS/water and with dichloromethane. The solvent was removed in vacuo and the resulting residue was triturated three times with ether.
    • 5.2 Example 2 Purification of Peptides
  • The crude peptide was dissolved in a minimum amount of methanol and water or in a few drops of glacial acetic acid and water and was purified by preparative reverse phase HPLC (Beckman) with a Phenomenex Synergi hydro-RP C18 column (250 mm×21.2 mm). The peptide was eluted using a gradient from 3-100% B over 30 min with a flow rate of 20 mL/min, where solvent A was water containing 0.1% trifluoroacetic acid and solvent B was acetonitrile containing 0.1% trifluoroacetic acid. Detection was at 220 or 254 nm. Fractions >95% pure by analytical HPLC analysis (Waters, Phenomenex hydro RP (250 mm×4.6 mm over 40 minutes) using gradient 3-100% B were combined, concentrated to a volume of about 2-4 ml by rotary evaporation, and lyophilized.
    • 5.3 Example 3 Cleavage of Peptides from Resin
  • The resin was treated with 2% hydrazine in DMF (1 mL per 100 mg of resin) for three minutes with nitrogen agitation or vibration and the reaction mixture was filtered. This step was repeated an additional five times. The resin was washed three times with DMF and three times with dichloromethane. The desired carboxylic acid (4 eq), HBTU (4 eq) and HOBt (4 eq) were dissolved in DMF (1 mL per 100 mg of resin) and added to the above resin, followed by the addition of N-methylmorpholine (8 eq) and the mixture was agitated for 1 hour. The reaction mixture was filtered and the resin was washed three times with DMF and three times with dichloromethane. The resin was treated with TFA/TIS/water (95:2.5:2.5, 1 mL per 100 mg of resin) and agitated with nitrogen or vibration for 1 hour. The reaction mixture was filtered, the resin was washed once with TFA/TIS/water, three times with dichloromethane and the filtrate was concentrated. The residue was triturated 4 times with diethyl ether.
    • 5.4 Example 4 Coupling of Peptides to Doxorubicin
  • The desired peptide (1 eq) and doxorubicin hydrochloride (0.6-0.7 eq) were dissolved in DMF (0.05 M) and then HOBt (1.2 eq) and diisopropylethylamine (4 eq) were added to the red solution. The reaction mixture was stirred at room temperature for 5 minutes, and EDAC (1.2 eq) was then added. The reaction mixture was stirred for 17 hours at room temperature and the solvent was removed in vacuo.
    • 5.5 Example 5 4-Fluorobenzoyl-Pro-His-Ser-Cys-Asn-NH2
  • This compound was prepared according to the procedure of Examples 1 and 2, with 4-fluorobenzoic acid being used in place of the amino acid. (10.4 mg, 19%) was isolated as a white, fluffy solid: 1H NMR (300 MHz, DMSO-d6) δ 8.94-8.84 (m, 1H), 8.53-7.96 (m, 4H), 7.66-7.58 (m, 2H), 7.44-7.21 (m, 4H), 7.09 (d, J=10.9 Hz, 2H), 6.92 (s, 1H), 5.09 (br s, 1H), 4.72-4.65 (m, 1H), 4.52-4.31 (m, 4H), 3.70-3.48 (m, 6H, overlapping with water peak), 3.07-2.72 (m, 4H), 2.22-2.08 (m, 1H), 1.93-1.75 (m, 3H); ES MS m/z (M+H)+ 678.4.
    • 5.6 Example 6 Ac-Pro-His-Ser-Cys(Me)-Asn-OH
  • Wang resin (397 mg, 1.00 mmol) was washed three times with dichloromethane, three times with methanol and three times with dichloromethane. Fmoc-Asn(trt)-OH (2.40 g, 4.02 mmol) and diisopropylcarbodiimide (0.625 mL, 4.01 mmol) were dissolved in 4 mL of dichloromethane and 3 mL of DMF at 0° C. and stirred for 20 minutes. The dichloromethane was removed in vacuo, 3 mL of DMF added to the solution and the solution added to the Wang resin from above in a sintered glass funnel. DMAP (49 mg, 0.40 mmol) in 1 mL DMF was added to the resin and the mixture was agitated with nitrogen for one hour. The mixture was filtered, the resin was washed three times with DMF and three times with dichloromethane, and the coupling procedure was repeated exactly as described above. The remainder of the amino acids were added using the procedure of Example 1, with the exception that the couplings of Fmoc-Ser(trt)-OH and Fmoc-His(trt)-OH were performed using 3 equivalents of amino acid, 3 equivalents of HBTU, 3 equivalents of HOBt, and 6 equivalents of NMM. The procedure of Example 2 was used to cleave and purify the peptide and afforded 282 mg (46%) of a white solid, and as a mixture of two compounds in a ratio of 83:17: 1H NMR (300 MHz, DMSO-d6) δ 8.97 (s, 1H), 8.46-8.27 (m, 2H), 8.25-8.15 (m, 1H), 7.98-7.87 (m, 1H), 7.40-7.35 (m, 2H), 6.93 (s, 1H), 4.79-4.60 (m, 1H), 4.56-4.47 (m, 2H), 4.39-4.25 (m, 2H), 3.66-3.60 (m, 3H, overlapping with water peak), 3.22-3.12 (m, 2H), 3.05-2.94 (m, 1H), 2.91-2.82 (m, 1H), 2.68-2.41 (m, 3H, overlapping with DMSO peak), 2.07 (s, 3H), 2.00 (s, 3H), 1.91-1.65 (m, 4H); ES MS m/z (M+H)+ 613.4.
    • 5.7 Example 7 Ac-Pro-His-Ser-Cys(Me)-Asn-Gly-Gly-Lys(4-fluorobenzoyl)-NH2
  • Triethylamine (17 μL, 0.12 mmol) was added to a solution of Ac-Pro-His-Ser-Cys(Me)-Asn-Gly-Gly-Lys-NH2 (21 mg, 0.024 mmol) and 4-fluorobenzoyl succinimide (6.2 mg, 0.026 mmol) in 1 mL of DMF and the solution was stirred at room temperature for 3.5 hours. The reaction mixture was concentrated and the residue was purified by preparative HPLC (3 to 100% acetonitrile-water over 30 minutes) to give 17 mg (71%) of a white fluffy solid: 1H NMR (300 MHz, DMSO-d6) δ 8.98 (s, 1H), 8.49-8.42 (m, 1H), 8.35-8.22 (m, 3H), 8.14-8.07 (m, 2H), 7.94-7.87 (m, 3H), 7.82 (d, J=8.0 Hz, 1H), 7.46 (s, 1H), 7.40-7.35 (m, 1H), 7.32-7.26 (m, 3H), 7.02 (s, 2H), 4.82-4.61 (m, 1H), 4.60-4.47 (m, 2H), 4.40-4.26 (m, 2H), 4.21-4.13 (m, 1H), 3.77-3.69 (m, 5H), 3.68-3.61 (m, 2H, overlapping with water peak), 3.27-3.14 (m, 5H, overlapping with water peak), 3.05-2.95 (m, 2H), 2.91-2.83 (m, 1H), 2.72-2.55 (m, 3H, overlapping with DMSO peak), 2.08 (s, 3H), 2.01 (s, 3H), 1.92-1.65 (m, 5H), 1.61-1.46 (m, 3H), 1.38-1.24 (m, 2H); ES MS m/z (M+H)+ 976.7.
    • 5.8 Example 8 5-(& 6-)Carboxyfluorescein-β-Ala-Pro-His-Ser-Cys-Asn-NH2
  • This compound was prepared from Rink amide AM resin (0.0584 mmol) according to the procedures of Example 1 and 2, with the exceptions that the coupling of Fmoc-Cys(trt)-OH was performed using 3 equivalents of amino acid, 3 equivalents of HBTU, 3 equivalents of HOBt, and 6 equivalents of NMM, while the couplings of Fmoc-Ser(trt)-OH, Fmoc-His(trt)-OH, Fmoc-Pro-OH and 5-(&6-)carboxyfluorescein were performed using 2 equivalents of the acid, 2 equivalents of HBTU, 2 equivalents of HOBt, and 4 equivalents of NMM. The title compound (27 mg, 46%) was isolated as a yellow, fluffy solid, and as a mixture of two compounds: 1H NMR (300 MHz, DMSO-d6)□10.38-10.04 (br s, 1H), 8.98-8.92 (m, 1H), 8.90-8.68 (m, 1H), 8.48-8.41 (m, 1H), 8.35-8.18 (m, 2H), 8.18-8.04 (m, 2H), 8.02-7.96 (m, 1H), 7.66 (s, 0.5H), 7.42-7.33 (m, 2.5H), 7.10 (d, J=7.3 Hz, 2H), 6.95-6.90 (m, 1H), 6.70 (s, 2H), 6.62-6.51 (m, 4H), 4.76-4.57 (m, 1H), 4.51-4.25 (m, 4H), 3.05-2.92 (m, 2H), 2.85-2.71 (m, 3H), 2.69-2.60 (m, 1H), 2.58-2.54 (m, 1H), 2.47-2.37 (m, 2H), 2.09-1.70 (m, 5H); ES MS m/z (M+H)+ 985.6; Anal. calcd for C45H48N10O14S: N, 14.22. Found: 10.54 (peptide content, 74%).
    • 5.9 Example 9 Ac-Pro-His-Ser-Cys-Asn-Gly-Gly-Lys(4-fluorobenzoyl)-NH2
  • This compound was prepared from Ac-Pro-His(trt)-Ser(trt)-Cys(trt)-Asn(trt)-Gly-Gly-Lys(ivDde)-Rink amide AM resin (208 mg, loading of 0.32 mmol/g, 0.067 mmol) according to Procedure C. The product was cleaved and purified according to Procedure B and afforded 28 mg (44%) of the title compound as a fluffy, white solid: 1H NMR (300 MHz, DMSO-d6) δ 8.96 (s, 1H), 8.48-8.40 (m, 1H), 8.30-8.18 (m, 2H), 8.16-8.00 (m, 2H), 7.97-7.79 (m, 4H), 7.46-7.33 (m, 2H), 7.32-7.24 (m, 4H), 7.11 (s, 0.5H), 7.01 (s, 2H), 6.94 (s, 0.5H), 4.79-4.11 (m, 7H), 3.78-3.60 (m, 8H, overlapping with water peak), 3.27-3.12 (m, 5H, overlapping with water peak), 3.04-2.93 (m, 1H), 2.89-2.70 (m, 2H), 2.64-2.44 (m, 2H, overlapping with DMSO peak), 2.40-2.32 (m, 1H), 2.09-1.97 (m, 3H), 1.91-1.66 (m, 5H), 1.58-1.45 (m, 3H), 1.37-1.23 (m, 2H); ES MS m/z (M+H)+ 962.8.
    • 5.10 Example 10 Ac-Tyr-Gly-Gly-Pro-His-Ser-Cys-Asn-NH2
  • This compound was prepared from Rink amide AM resin (0.100 mmol) according to the procedures of Examples 1 and 2. The title compound (54 mg, 61%) was isolated as a white, fluffy solid and as a mixture of two compounds in a ratio of 3:1: 1H NMR (300 MHz, DMSO-d6) δ 8.94 (s, 0.75H), 8.89 (s, 0.25H), 8.54-8.22 (m, 3H), 8.11 (t, J=8.7 Hz, 2H), 7.98 (d, J=7.0 Hz, 1H), 7.84-7.78 (m, 1H), 7.41-7.32 (m, 2H), 7.10 (d, J=8.6 Hz, 2H), 7.05-6.99 (m, 2H), 6.92 (s, 1H), 6.63 (d, J=8.2 Hz, 2H), 4.79-4.55 (m, 13H, overlapping with water peak), 4.49-4.30 (m, 7H, overlapping with water peak), 4.03-3.84 (m, 2H), 3.83-3.58 (m, 4H), 3.55-3.47 (m, 1H), 3.44-3.25 (m, 1H), 3.22-3.09 (m, 1H), 3.06-2.72 (m, 4H), 2.69-2.55 (m, 1H), 2.47-2.38 (m,2H), overlapping with DMSO peak), 2.09-1.96 (m, 1H), 1.93-1.82 (m, 2H), 1.77 (s, 4H); ES MS m/z (M+H)+ 875.7; Anal. calcd for C36H50N12O12S: N, 19.21. Found: 12.90 (peptide content, 67%).
    • 5.11 Example 11 Ac-Tyr-Gly-Gly-Gly-Gly-Pro-His-Ser-Cys-Asn-NH2
  • This compound was prepared from Rink amide AM resin (0.100 mmol) according to the procedures of Examples 1 and 2. The title compound (51 mg, 52%) was isolated as a white, fluffy solid, and as a mixture of two compounds in a ratio of 73:27: 1H NMR (300 MHz, DMSO-d6)□8.94 (s, 0.73H), 8.88 (s, 0.27H), 8.56-8.21 (m, 3H), 8.18-8.01 (m, 4H), 7.99-7.87 (m, 2H), 7.40 (s, 1H), 7.37-7.33 (m, 1H), 7.10 (d, J=8.4 Hz, 2H), 7.02 (d, J=8.5 Hz, 2H), 6.92 (s, 1H), 6.64 (d, J=8.5 Hz, 2H), 4.78-4.26 (m, 26H, overlapping with water peak), 4.03-3.85 (m, 3H, overlapping with water peak), 3.81-3.58 (m, 8H), 3.55-3.46 (m, 1H), 3.21-3.10 (m, 1H), 3.05-2.71 (m, 4H), 2.69-2.56 (m, 1H), 2.51-2.38 (m, 2H, overlapping with DMSO peak), 2.05-1.95 (m, 1H), 1.92-1.82 (m, 2H), 1.77 (s, 4H); ES MS m/z (M+H)+ 989.7; Anal. calcd for C40H56N14O14S: N, 19.83. Found: 14.52 (peptide content, 73%).
    • 5.12 Example 12 Ac-Tyr-Gly-Gly-Gly-Gly-Gly-Gly-Pro-His-Ser-Cys-Asn-NH2
  • This compound was prepared from Rink amide AM resin (0.100 mmol) according to the procedures of Examples 1 and 2. The title compound (37 mg, 33%) was isolated as a white, fluffy solid, and as a mixture of two compounds in a ratio of 63:37: 1H NMR (300 MHz, DMSO-d6) δ 8.94 (s, 0.63H), 8.88 (s, 0.37H), 8.55-8.20 (m, 3H), 8.17-7.86 (m, 7H), 7.40 (s, 1H), 7.37-7.32 (m, 1H), 7.10 (d, J=8.3 Hz, 2H), 7.02 (d, J=8.4 Hz, 2H), 6.92 (s, 1H), 6.63 (d, J=8.4 Hz, 2H), 4.76-4.59 (m, 3H, overlapping with water peak), 4.50-4.30 (m, 6H), 4.03-3.85 (m, 2H), 3.81-3.69 (m, 8H), 3.68-3.58 (m, 2H), 3.55-3.47 (m, 2H), 3.43-3.10 (m, 2H), 3.04-2.70 (m, 4H), 2.68-2.55 (m, 2H), 2.47-2.37 (m, 2H, overlapping with DMSO peak), 2.05-1.94 (m, 1H), 1.92-1.81 (m, 2H), 1.77 (s, 4H); ES MS m/z (M+H)+ 1103.8; Anal. calcd for C44H62N16O16S: N, 20.32. Found: 14.61 (peptide content, 72%).
    • 5.13 Example 13 Ac-Tyr-Gly-Gly-Gly-Gly-Gly-Gly-Pro-His-Ser-Cys-Asn-NH2
  • This compound was prepared from Rink amide AM resin (0.100 mmol) according to the procedures of Examples 1 and 2. The title compound (37 mg, 33%) was isolated as a white, fluffy solid, and as a mixture of two compounds in a ratio of 63:37: 1H NMR (300 MHz, DMSO-d6) δ 8.94 (s, 0.63H), 8.88 (s, 0.37H), 8.55-8.20 (m, 3H), 8.17-7.86 (m, 7H), 7.40 (s, 1H), 7.37-7.32 (m, 1H), 7.10 (d, J=8.3 Hz, 2H), 7.02 (d, J=8.4 Hz, 2H), 6.92 (s, 1H), 6.63 (d, J=8.4 Hz, 2H), 4.76-4.59 (m, 3H, overlapping with water peak), 4.50-4.30 (m, 6H), 4.03-3.85 (m, 2H), 3.81-3.69 (m, 8H), 3.68-3.58 (m, 2H), 3.55-3.47 (m, 2H), 3.43-3.10 (m, 2H), 3.04-2.70 (m, 4H), 2.68-2.55 (m, 2H), 2.47-2.37 (m, 2H, overlapping with DMSO peak), 2.05-1.94 (m, 1H), 1.92-1.81 (m, 2H), 1.77 (s, 4H); ES MS m/z (M+H)+ 1103.8; Anal. calcd for C44H62N16O16S: N, 20.32. Found: 14.61 (peptide content, 72%).
    • 5.14 Example 14 Ac-Tyr-NH—(CH2)4—CO-Pro-His-Ser-Cys-Asn-NH2
  • This compound was prepared from Rink amide AM resin (0.103 mmol) according to the procedures of Examples 1 and 2. The title compound (53 mg, 60%) was isolated as a white, fluffy solid, and as a mixture of two compounds in a ratio of 66:34: 1H NMR (300 MHz, DMSO-d6) δ 8.97 (s, 1H), 8.45-8.21 (m, 2H), 8.13 (d, J=7.9 Hz, 1H), 8.08-7.96 (m, 2H), 7.93-7.85 (m, 1H), 7.40 (s, 1H), 7.37-7.32 (m, 1H), 7.09 (d, J=10.0 Hz, 2H), 7.02-6.95 (m, 2H), 6.92 (s, 1H), 6.63 (d, J=8.5 Hz, 2H), 4.79-4.59 (m, 4H, overlapping with water peak), 4.51-4.27 (m, 7H, overlapping with water peak), 3.72-3.56 (m, 2H), 3.54-3.28 (m, 2H), 3.21-3.10 (m, 1H), 3.07-2.94 (m, 3H), 2.88-2.72 (m, 3H), 2.65-2.55 (m, 1H), 2.46-2.38 (m, 2H, overlapping with DMSO peak), 2.33-2.17 (m, 2H), 2.04-1.95 (m, 1H), 1.93-1.74 (m, 6H), 1.50-1.23 (m, 4H); ES MS m/z (M+H)+ 860.7; Anal. calcd for C37H53N11O11S: N, 17.92. Found: 12.40 (peptide content, 69%).
    • 5.15 Example 15 3-(4-Hydroxyphenyl)propionyl-NH—(CH2)4—CO-Pro-His-Ser-Cys-Asn-NH2
  • This compound was prepared from Rink amide AM resin (0.103 mmol) according to the procedures of Examples 1 and 2. The title compound (50 mg, 60%) was isolated as a white, fluffy solid and as a mixture of two compounds in a ratio of 7:3: 1H NMR (300 MHz, DMSO-d6) δ 8.97 (s, 1H), 8.47-8.21 (m, 2H), 8.12 (d, J=7.9 Hz, 1H), 8.09-7.92 (m, 1H), 7.81-7.73 (m, 1H), 7.40 (s, 1H), 7.37-7.32 (m, 1H), 7.09 (d, J=10.0 Hz, 2H), 6.99-6.89 (m, 3H), 6.64 (d, J=8.5 Hz, 2H), 4.78-4.59 (m, 2H), 4.51-4.27 (m, 6H, overlapping with water peak), 3.71-3.56 (m, 3H), 3.54-3.45 (m, 2H), 3.44-3.27 (m, 1H), 3.21-3.10 (m, 1H), 3.06-2.93 (m, 3H), 2.85-2.75 (m, 2H), 2.72-2.64 (m, 2H), 2.46-2.37 (m, 2H, overlapping with DMSO peak), 2.32-2.21 (m, 4H), 2.08-1.95 (m, 1H), 1.90-1.69 (m, 3H), 1.52-1.23 (m, 4H); ES MS m/z (M+H)+ 803.6; Anal. calcd for C35H50N10O10S: N, 17.45. Found: 12.86 (peptide content, 74%).
    • 5.16 Example 16 Ac-Pro-His-Ser-Cys(Me)-Asn-Dox
  • This compound was prepared from Ac-Pro-His-Ser-Cys(Me)-Asn-OH (62 mg, 0.10 mmol) and doxorubicin hydrochloride (37 mg, 0.063 mmol) according to the procedure of Example 4. The resulting residue was purified according to the procedure of Example 2 and afforded the title compound (28 mg, 39%) as a fluffy, orange solid: 1H NMR (300 MHz, DMSO-d6) δ 8.97-8.93 (m, 1H), 8.44-8.16 (m, 3H), 7.96-7.87 (m, 3H), 7.70-7.63 (m, 1H), 7.41-7.26 (m, 3H), 6.93-6.86 (m, 1H), 5.48-5.42 (m, 1H), 5.24-5.20 (m, 1H), 5.10-4.92 (m, 2H), 4.79-4.40 (m, 7H), 4.37-4.24 (m, 2H), 4.16-4.11 (m, 1H), 4.01-3.88 (m, 4H), 3.65-3.48 (m, 6H, overlap with water peak), 3.25-3.11 (m, 2H), 3.06-2.91 (m, 3H), 2.86-2.70 (m, 1H), 2.67-2.55 (m, 2H, overlapping with DMSO peak), 2.39-2.25 (m, 1H), 2.22-2.07 (m, 2H), 2.04 (s, 3H), 2.00 (s, 3H), 1.90-1.62 (m,4H), 1.52-1.41 (m, 1H), 1.12 (d, J=6.4 Hz, 3H); ES MS m/z (M+H)+ 1138.5.
    • 5.17 Example 17 Ac-Pro-His-Ser-Cys(Ac)-Asn-Dox
  • This compound was prepared from Ac-Pro-His-Ser-Cys(Ac)-Asn-OH (52 mg, 0.081 mmol) and doxorubicin hydrochloride (33 mg, 0.057 mmol) according to the procedure of Example 3. The resulting residue was purified according to the procedure of Example 2 and afforded the title compound (36 mg, 54%) as a fluffy, orange solid: 1H NMR (300 MHz, DMSO-d6) δ 8.95 (s, 1H), 8.44-8.10 (m, 3H), 7.96-7.85 (m, 2H), 7.69-7.62 (m, 1H), 7.40-7.28 (m, 2H), 6.90 (s, 1H), 5.46 (br s, 1H), 5.32-5.21 (m, 1H), 4.98-4.92 (m, 1H), 4.76-4.12 (m, 8H), 4.00-3.88 (m, 3H), 3.07-2.73 (m, 5H), 2.38-2.26 (m, 1H), 2.28 (s, 3H), 2.22-2.06 (m, 2H), 2.00 (s, 3H), 1.91-1.63 (m, 5H), 1.52-1.42 (m, 1H), 1.13 (d, J=6.4 Hz, 3H); ES MS m/z (M+H)+ 1166.6.
    • 5.18 Example 18 Ac-]Pro-]H[is-Ser-Cys(Me)-Asn-Gly-Gly-Lys(CO—(CH[2)3—CO-]Dox)-NH2
  • Ac-Pro-His-Ser-Cys(Me)-Asn-Gly-Gly-Lys-NH2 (74 mg, 0.087 mmol) and Fmoc-Dox-hemiglutarate (70% pure, 74 mg, 0.059 mmol) were dissolved in 3 mL of DMF at room temperature. HOBt (14 mg, 0.11 mmol) and diisopropylethylamine (60 μL, 0.34 mmol) were added to the red solution and the reaction mixture was stirred for 5 minutes. EDAC (21 mg, 0.11 mmol) was added to the reaction mixture and stirred for an additional 18 hours. The solvent was removed in vacuo and the resulting red oil was triturated three times with ethyl acetate to afford a red solid. The red solid was dissolved in 5 mL of DMF and 500 μL of piperidine. The reaction mixture was cooled to 0° C. in an ice bath and a mixture of 450 μL TFA, 1.05 mL pyridine and 3mL of DMF was added. After 5 minutes, the solvent was removed in vacuo and the residue was triturated once with ethyl acetate and once with diethyl ether to give a red solid. This product was purified according to the procedure of Example 2 and afforded the title compound (14 mg, 16%) as a fluffy, orange solid: ES MS m/z (M+H)+ 1493.8.
    • 5.19 Example 19 2-Fluoroacetyl-β-Ala-Pro-His-Ser-Cys-Asn-NH2
  • This compound was prepared from Rink amide AM resin (0.111 mmol) according to the procedures of Examples 1 and 2. The title compound (30 mg, 40%) was isolated as a white, fluffy solid: 1H NMR (300 MHz, DMSO-d6)□8.98-8.93 (m, 1H), 8.43-8.21 (m, 2H), 8.15-8.07 (m, 2H), 8.01-7.90 (m, 1H), 7.41-7.32 (M, 2H), 7.09 (d, J=9.9 Hz, 2H), 6.92 (s, 1H), 4.87-4.70 (m, 2H), 4.68-4.59 (m, 1H), 4.50-4.28 (m, 5H), 3.36-3.28 (m, 4H, overlapping with water peak), 3.22-3.10 (m, 2H), 3.05-2.91 (m, 1H), 2.89-2.72 (m, 3H), 2.46-2.36 (m, 3H, overlapping with DMSO peak), 2.09-1.95 (m, 1H), 1.90-1.55 (m, 4H); ES MS m/z (M+H)+687.5; Anal. calcd for C26H39FN10O9S: N, 20.40. Found: 14.89 (peptide content, 73%).
    • 5.20 Example 20 Ac-Pro-His-Ser-Cys-Asn-Gly-Gly-Lys(2-fluoroacetyl)-NH2
  • This compound was prepared from Ac-Pro-His(trt)-Ser(trt)-Cys(trt)-Asn(trt)-Gly-Gly-Lys(ivDde)-Rink amide AM resin (317 mg, loading of 0.32 mmol/g, 0.101 mmol) according to the procedure of Example 3. The product was purified according to the procedure of Example 2 and afforded 21 mg (23%) of the title compound as a fluffy, white solid: 1H NMR (300 MHz, DMSO-d6) δ 8.97 (s, 1H), 8.45-8.17 (m, 3H), 8.16-8.01 (m, 3H), 7.95 (d, J=7.2 Hz, 1H), 7.80 (d, J=8.1 Hz, 1H), 7.45 (s, 1H), 7.41-7.33 (m, 1H), 7.27 (s, 1H), 7.01 (s, 2H), 4.74 (d, J=47 Hz, 2H), 4.78-4.61 (m, 1H), 4.59-4.24 (m, 5H), 4.19-4.10 (m, 2H), 3.55-3.46 (m, 3H, overlapping with water peak), 3.38-2.94 (m, 6H), 2.87-2.70 (m, 2H), 2.65-2.42 (m, 2H, overlapping with DMSO peak), 2.36 (t, J=8.5 Hz, 1H), 2.00 (s, 3H), 1.90-1.61 (m, 5H), 1.56-1.36 (m, 3H), 1.31-1.15 (m, 2H); ES MS m/z (M+H)+ 900.8; Anal. calcd for C35H54FN13O12S: N, 20.23. Found: 14.55 (peptide content, 72%).
    • 5.21 Example 21 Ac-Pro-His-Ser-Cys-Asn-Gly-Gly-Lys(8-[(4′-fluorobenzyl)amino]suberoyl)-NH2
  • This compound was prepared from Ac-Pro-His(trt)-Ser(trt)-Cys(trt)-Asn(trt)-Gly-Gly-Lys(ivDde)-Rink amide AM resin (312 mg, loading of 0.32 mmol/g, 0.0997 mmol) according to the procedure of Example 3. The product (42.3 mg of crude material) was purified according to the procedure of Example 2 and afforded 3.9 mg (11%) of PLG-107 as a fluffy, white solid: ES MS m/z (M+H)+ 1104.0.
    • 5.22 Example 22 Ac-Pro-His-Ser-Cys-Asn-β-Ala-Lys(3-(4-hydroxyphenyl)propionyl)-NH2
  • Ac-Pro-His(trt)-Ser(trt)-Cys(trt)-Asn(trt)-β-Ala-Lys(ivDde) was prepared on Rink amide AM resin (0.100 mmol) according to the procedure of Example 1, but was not cleaved from the resin. The title compound was prepared from this resin bound peptide according to the procedure of Example 3. The crude product was purified according to the procedure of Example 2 and afforded 39 mg (41%) of the title compound as a fluffy, white solid: 1H NMR (300 MHz, DMSO-d6) δ 8.97 (s, 1H), 8.46-8.26 (m, 1H), 8.21 (d, J=7.7 Hz, 1H), 8.13 (d, J=7.7 Hz, 1H), 8.07-7.87 (m, 2H), 7.77-7.66 (m, 2H), 7.40 (s, 1H), 7.36-7.28 (m, 2H), 6.99-6.88 (m, 4H), 6.67-6.62 (m, 2H), 4.80-4.60 (m, 1H), 4.52-4.25 (m, 6H), 3.72-3.59 (m, 4H, overlapping with water peak), 3.56-3.44 (m, 2H), 3.38-3.12 (m, 4H), 3.05-2.93 (m, 3H), 2.87-2.75 (m, 2H), 2.71-2.64 (m, 2H), 2.45-2.35 (m, 2H), 2.31-2.23 (m, 4H), 2.00 (s, 3H), 1.91-1.58 (m, 5H), 1.54-1.41 (m, 1H), 1.39-1.17 (m, 4H); ES MS m/z (M+H)+ 945.8; Anal. calcd for C41H60N12O12S: N, 17.79. Found: 12.95 (peptide content, 73%).
    • 5.23 Example 23 Ac-Pro-His-Ser-Cys-Asn-NH—(CH2)4—CO-Lys(3-(4-hydroxyphenyl)propionyl)-NH2
  • Ac-Pro-His(trt)-Ser(trt)-Cys(trt)-Asn(trt)-NH—(CH2)4—CO-Lys(ivDde) was prepared on Rink amide AM resin (0.100 mmol) according to the procedure of Example 1, but was not cleaved from the resin. The title compound was prepared from this resin bound peptide according to the procedure of Example 3. The crude product was purified according to the procedure of Example 2 and afforded 34 mg (35%) of the title compound as a fluffy, white solid: 1H NMR (300 MHz, DMSO-d6) δ 8.97 (s, 1H), 8.45-8.19 (m, 2H), 8.13 (d, J=7.8 Hz, 1H), 8.04-7.92 (m, 1H), 7.81-7.71 (m, 2H), 7.66-7.59 (m, 1H), 7.40 (s, 1H), 7.35 (br s, 1H), 7.29 (br s, 1H), 6.96 (d, J=8.4 Hz, 2H), 6.93 (br s, 2H), 6.64 (d, J=8.4 Hz, 2H), 4.81-4.60 (m, 2H), 4.53-4.24 (m, 8H, overlapping with water peak), 3.72-3.60 (m, 3H, overlapping with water peak), 3.56-3.46 (m, 2H), 3.39-3.25 (m, 1H), 3.24-3.11 (m, 1H), 3.07-2.92 (m, 5H), 2.84-2.72 (m, 2H), 2.71-2.63 (m, 2H), 2.46-2.36 (m, 2H, overlapping with DMSO peak), 2.30-2.24 (m, 2H), 2.16-2.07 (m, 2H), 2.00 (s, 3H), 1.91-1.55 (m, 5H), 1.53-1.16 (m, 9H); ES MS m/z (M+H)+ 973.8; Anal. calcd for C43H64N12O12S: N, 17.27. Found: 11.42 (peptide content, 66%).
    • 5.24 Example 24 Ac-Pro-His-Ser-Cys-Asn-NH—(CH2)6—CO-Lys(3-(4-hydroxyphenyl)propionyl)-NH2
  • Ac-Pro-His(trt)-Ser(trt)-Cys(trt)-Asn(trt)-NH—(CH2)6—CO-Lys(ivDde) was prepared on Rink amide AM resin (0.100 mmol) according to the procedure of Example 1, but was not cleaved from the resin. The title compound was prepared from this resin bound peptide according to the procedure of Example 3. The crude product was purified according to the procedure of Example 2 and afforded 29 mg (29%) of the title compound as a fluffy, white solid: 1H NMR (300 MHz, DMSO-d6) δ 9.12 (s, 1H), 8.91-8.82 (m, 1H), 8.43-8.26 (m, 2H), 8.03-7.97 (m, 1H), 7.94-7.83 (m, 1H), 7.79-7.71 (m, 2H), 7.41-7.20 (m, 3H), 6.96 (d, J=8.5 Hz, 2H), 6.93-6.87 (m, 2H), 6.64 (d, J=8.5 Hz, 2H), 5.09 (br s, 1H), 4.80-4.57 (m, 1H), 4.48-4.39 (m, 1H), 4.36-4.20 (m, 3H), 4.19-4.09 (m, 1H), 3.69-3.53 (m, 4H), 3.04-2.88 (m, 7H), 2.73-2.63 (m, 3H), 2.45-2.33 (m, 3H, overlapping with DMSO peak), 2.29-2.23 (m, 3H), 2.12-2.05 (m, 3H), 1.99 (s, 3H), 1.91-1.56 (m, 5H), 1.51-1.12 (m, 13 H); ES MS m/z (M+H)+ 1002.1; Anal. calcd for C45H68N12O12S: N, 16.79. Found: 10.78 (peptide content, 64%).
    • 5.25 Example 25 Acetyl-Pro-His-Ser-Cys-Asn-Tyr(3-iodo)-NH2
  • This compound was prepared according to the procedures of Examples 1 and 2 giving the title compound (32.9 mg, 16%) as a fine white powder. The NMR data indicated a mixture of two species in a ratio of about 80:20: 1H NMR (300 MHz, DMSO-d6) δ 10.06 (s, 1H), 8.96 (m, 1 H), 8.14-8.43 (m, 3 H), 7.88-8.05 (m, 2 H), 7.50 (d, 1 H, J=1.8 Hz), 7.40 (s, 3 H), 7.09 (bs, 1 H), 6.98-7.03 (m, 2 H), 6.75 (d, 1 H, J=8.4 Hz), 5.06 (bs, 1 H), 4.60-4.74 (m, 1 H), 4.47-4.54 (m, 1 H), 4.37-4.46 (m, 1 H), 4.22-4.36 (m, 3 H), 3.57-3.69 (m, 2 H), 3.47-3.55 (m, 2 H), 3.11-3.21 (m, 1 H), 2.86-3.04 (m, 3 H), 2.59-2-78 (m, 3 H), 2.35-2.43 (m, 1 H), 2.26-2.31 (t, 1 H, J=7.8 Hz), 2.00 (s, 3 H), 1.69-1.94 (m, 4 H); MS m/z (C32H43IN10O10S+H)+ 887.4; Anal. calcd for C32H43IN10O10S: N, 15.80. Found: N, 12.30 (peptide content: 78%).
    • 5.26 Example 26 Acetyl-Pro-His-Ser-Cys-Asn-Tyr(3,5-diiodo)-NH2
  • This compound was prepared according to the procedures of Examples 1 and 2 giving the title compound (25.0 mg, 11%) as a fine white powder. The NMR data indicated a mixture of two species in a ratio of about 80:20: 1H NMR (300 MHz, DMSO-d6) δ 9.32 (bs, 1 H), 8.94-8.95 (m, 1 H), 8.14-8.43 (m, 3 H), 7.91-8.04 (m, 2 H), 7.58 (s, 2 H), 7.34-7.42 (m, 3 H), 7.12 (s, 1 H), 6.98 (s, 1 H), 5.06 (bs, 1 H) 4.60-4.79 (m 1, H), 4.39-4.53 (m, 2 H), 4.23-4.35 (3 H), 3.58-3.69 (m, 2 H), 3.47-3.54 (m, 2 H), 3.11-3.21 (m, 1 H), 2.56-3.07 (m, 7 H), 2.37-2.44 (m 1 H), 2.29 (t, 1 H, J=9.7 Hz), 2.00 (s, 3 H), 1.68-1.90 (m, 4 H); MS m/z (C32H42I2N10O10S+H)+ 1013.4; Anal. calcd for C32H42I2N10O10S: N, 13.83. Found: N, 11.26 (peptide content: 81%).
    • 5.27 Example 27 Acetyl-Pro-His-Ser-Cys(methyl)-Asn-Gly-Gly-Lys-NH2
  • This compound was prepared according to the procedures of Examples 1 and giving the title compound as (151.2 mg, 44%) as a fine white powder. The NMR data indicated a mixture of two species in a ratio of about 80:20: 1H NMR (300 MHz, DMSO-d6) δ 8.96-8.97 (m, 1 H), 8.24-8.46 (m, 3 H), 8.09 (app t, 2 H), 7.79-7.95 (m, 2 H), 7.65 (bs, 3 H), 7.45 (bs, 1 H), 7.34-7.39 (m, 1 H), 7.29 (bs, 1 H), 7.05 (bs, 1 H), 7.00 (bs, 1 H), 5.05 (bs, 1 H), 4.60-4.79 (m, 1 H), 4.47-4.58 (2 H), 4.25-4.40 (m, 2 H), (m, 2 H) 4.12-4.20 (m, 1 H) 3.69-3.79 (m, 4 H), 3.60-3.66 (m, 3 H), 3.12-3.22 (m, 2 H), 2.94-3.04 (m, 1 H), 2.56-2.89 (m, 6 H), 2.07 (s, 3 H), 2.00 (s, 3 H), 1.64-1.88 (m, 4 H), 1.46-1.58 (m, 3 H), 1.23-1.37 (m, 2 H); MS m/z (C34H55N13O11S+H)+ 854.7; Anal. calcd for C34H55N13O11S: N, 21.32. Found: N, 14.88 (peptide content: 70%).
    • 5.28 Example 28 3-(4-Hydroxyphenyl)propionyl-Pro-His-Ser-Cys-Asn-NH2
  • This compound was prepared according to the procedures of Examples 1 and 2 with the following modification: capping of the N-terminus was carried out using N-succinimidyl-3-(4-hydroxyphenyl)propionate (4.0 eq) and TEA (12.0 eq). The title compound (43.0 mg, 38%) was isolated as a fine white powder. The NMR data indicated a mixture of two species in a ratio of about 80:20: 1H NMR (300 MHz, DMSO-d6) δ 9.12 (bs, 1 H),8.87-8.95 (m, 1 H), 8.09-8.39 (m, 4 H), 7.97 (app d, 1 H), 7.39 (s, 1 H), 7.33 (bs, 1 H), 6.99-7.09 (m, 4 H), 6.90-6.92 (2 H), 6.61-6.66 (m, 2 H), 5.09 (bs, 1 H), 4.59-4.77 (m, 2 H), 4.27-4.46 (m, 5 H), 3-57-3.70 (m, 3 H), 3.12-3.19 (m, 1 H), 2.97-3.02 (m, 1 H), 2.66-2.80 (m, 4 H), 2.37-2.45 (m, 2 H), 1.63-2.30 (m, 5 H); MS m/z (C30H41N9O9S+H)+ 704.4; Anal. calcd for C30H41N9O9S: N, 17.91. Found: N, 13.88 (peptide content: 77%).
    • 5.29 Example 29 Acetyl-Tyr(3,5-diiodo)-Pro-His-Ser-Cys-Asn-NH2
  • This compound was prepared according to the procedures of Examples 1 and 2 giving the title compound (48.8 mg, 29%) as a fine white powder. The NMR data indicated a mixture of two species in a ratio of about 80:20: 1H NMR (300 MHz, MeOD) δ 8.79 (m, 1 H), 8.26-8.46 (m, 1 H), 7.61-7.65 (m, 2 H), 7.42-7.47 (m, 1 H), 4.70-4.76 (m, 1 H), 4.52-4.56 (m, 1 H), 4.38-4.45 (m, 2 H), 3.80-3.96 (m, 3 H), 3.59-3.67 (m, 1 H), 3.35-3.43 (m, 2 H), 3.17-3.27 (m, 1 H), 2.99-3.06 (m, 1 H), 2.93-2.95 (m, 2 H), 2.65-2.80 (m, 3 H), 1.99-2.25 (m, 3 H), 1.93-1.96 (m, 3 H); MS m/z (C32H42I2N10O10S+H)+ 1013.0; Anal. calcd for C32H42I2N10O10S: N, 13.83. Found: N, 11.33 (peptide content: 82%).
    • 5.30 Example 30 Acetyl-Pro-His-Ser-Cys-Asn-β-Ala-Tyr(3-iodo)-NH2
  • This compound was prepared according to the procedures of Examples 1 and 2 giving the title compound (8.1 mg, 6%) as a fine white powder. The NMR data indicated a mixture of two species in a ratio of about 80:20: 1H NMR (300 MHz, DMSO-d6) δ 10.07 (s, 1 H), 8.96 (m, 1 H), 8.11-8.44 (m, 3 H), 7.94-8.02 (m, 2 H), 7.68 (app t, 1 H), 7.54 (d, 1 H, J=2.1 Hz), 7.40-7.41 (m, 2 H), 7.34 (m, 1 H), 7.05 (m, 2 H), 6.90 (bs, 1 H), 6.75 (d, 1 H, J=8.4 Hz), 5.09 (bs, 1 H), 4.60-4.80 (m, 2 H), 4.26-4.51 (m, 4 H), 3.59-3.70 (m, 2 H), 3.47-3.54 (m, 2 H), 3.11-3.21 (m, 4 H), 2.72-3.03 (m, 5 H), 2.56-2.64 (m, 1 H), 2.36-2.43 (m, 2 H), 2.14-2.29 (m, 3 H), 2.00 (s, 3 H), 1.67-1.91 (m, 3 H); MS m/z (C35H48IN11O11S+H)+ 958.5; Anal. calcd for C35H48IN11O11S: N, 16.09. Found: N, 12.85 (peptide content: 80%).
    • 5.31 Example 31 3-(4-Hydroxy-3,5-diiodophenyl)propionyl-Pro-His-Ser-Cys-Asn-NH2
  • This compound was prepared according to the procedures of Examples 1 and 2 with the following modification: capping of the N-terminus was performed by doing a single coupling with 3-(4-hydroxyphenyl)propionic acid (3.0 eq). The title compound (23.9 mg, 23%) was isolated as a fine white powder. The NMR data indicated a mixture of two species in a ratio of about 80:20: 1H NMR (300 MHz, DMSO-d6); δ 9.31 (bs, 1 H), 8.93-8.97 (m, 1 H), 7.99-8.44 (m, 4 H), 7.62 (m, 2 H), 7.41-7.56 (m, 1 H), 7.34 (bs, 1 H), 7.09 (app d, 2 H), 6.92 (s, 1 H), 5.12 (bs, 1 H), 4.29-4.78 (m, 6 H), 3.59-3.72 (m, 3 H), 3.14-3.22 (m, 2 H), 2.96-3.03 (m, 2 H), 2.77-2.84 (m, 2 H), 2.65-2.71 (m, 2 H), 2.39-2.46 (m, 2 H), 1.56-2.15 (m, 5 H); MS m/z (C30H39I2N9O9S+H)+ 956.3; Anal. calcd for C30H39I2N9O9S: N, 13.19. Found: N, 10.84 (peptide content: 82%).
    • 5.32 Example 32 Acetyl-Tyr(3,5-diiodo)-β-Ala-Pro-His-Ser-Cys-Asn-NH2
  • This compound was prepared according to the procedures of Examples 1 and 2 with the following modification: only single couplings were performed with Fmoc-β-Ala-OH and Acetyl-Tyr(3,5-diiodo)-OH. The title compound was isolated as a fine white powder (55.0 mg, 51%). The NMR data indicated a mixture of two species in a ratio of about 80:20: 1H NMR (300 MHz, DMSO-d6) δ 9.35 (bs, 1 H), 8.96 (m, 1 H), 8.22-8.41 (m, 3 H), 7.97-8.14 (m, 4 H), 7.60-7.63 (m, 2 H), 7.39 (s, 1 H), 7.34 (bs, 1 H), 7.09 (app d, 2 H), 6.92 (s, 1 H), 4.60-4.78 (m, 2 H), 4.28-4.50 (m, 6 H), 3.59-3.71 (m, 2 H), 2.94-3.06 (m, 2 H), 2.72-2.83 (m, 3 H), 2.38-2.46 (m, 3 H), 1.76-2.09 (m, 7 H); MS m/z (C35H47I2N11O11S+H)+ 1084.5; Anal. calcd for C35H47I2N11O11S: N, 14.22. Found: N, 11.39 (peptide content: 80%).
    • 5.33 Example 33 Acetyl-Pro-His-Ser-Cys-Asn-β-Ala-Tyr(3,5-diiodo)-NH2
  • This compound was prepared according to the procedures of Examples 1 and 2 with the following modification: only single couplings were performed with every amino acid. The title compound was isolated as a fine white powder (14.5 mg, 9%). The NMR data indicated a mixture of two species in a ratio of about 80:20: 1H NMR (300 MHz, DMSO-d6) δ 9.35 (bs, 1 H), 8.96 (m, 1 H), 7.94-8.44 (m, 6 H), 7.68-7.72 (m, 1 H), 7.62 (s, 2 H), 7.46 (bs, 1 H), 7.40 (bs, 1 H), 7.34 (bs, 1 H), 7.06 (bs, 1 , H), 6.90 (bs, 1 H), 5.11 (bs, 1 H), 4.61-4.80 (m, 6 H), 3.57-3.71 (m, 2 H), 3.08-3.22 (m, 3 H), 2.94-3.07 (m, 2 H), 2.73-2.86 (m, 4 H), 2.36-2.43 (m, 2 H), 2.14-2.30 (m, 3 H), 2.00 (s, 3 H), 1.68-1.89 (m, 4 H); MS m/z (C35H47I2N11O11S)+ 1084.5; Anal. calcd for C35H47I2N11O11S: N, 14.22. Found: N, 11.64 (peptide content: 82%).
    • 5.34 Example 34 Acetyl-Tyr(3-iodo)-Pro-His-Ser-Cys-Asn-NH2
  • This compound was prepared according to the procedures of Example 2 with the following modifications: the final amino acid coupling was carried out with Ac-Tyr(3-iodo)-OH (4 eq), HBTU (4 eq), HOBT (4 eq) and NMM (8 eq). The title compound was isolated (7.7 mg, 16%) as a fine white powder. The NMR data indicated a mixture of two species in a ratio of about 75:25: 1H NMR (300 MHz, DMSO-d6) δ 10.10-10.17 (m, 1 H), 8.91-8.97 (m, 1 H), 8.25-8.52-m (2 H), 8.05-8.15 (m, 2 H), 7.58 (d, 1 H, J=2.0 Hz), 7.35-7.43 (m, 2 H), 7.09-7.11 (m, 3 H), 6.92 (bs, 1 H), 6.75-6.77 (m, 1 H), 5.11 (bs, 1 H), 4.55-4.69 (m, 2 H), 4.33-4.50 (m, 4 H), 3.49-3.77 (m, 4 H), 3.17 (s, 2 H), 2.69-2.85 (m, 4 H), 1.76-2.07 (m, 6 H); MS m/z (C32H43IN10O10S+H)+ 887.5; Anal. calcd for C32H43IN10O10S: N, 15.80. Found: N, 12.15 (peptide content: 77%).
    • 5.35 Example 35 Acetyl-Pro-His-Ser-Cys-Asn-Gly-Gly-Lys(3-(4-hydroxy-3,4-diiodophenyl) propionyl)-NH2
  • This compound was prepared according to the procedures of Examples 3 and 2 with the following modifications: the amide formation on lysine was carried out with 3-(4-hydroxy-3,4-diiodophenyl)propionic acid (4 eq), HBTU (4 eq), HOBT (4 eq) and NMM (8 eq). The title compound was isolated (16.2 mg, 13%) as a fine white powder. The NMR data indicated a mixture of two species in a ratio of about 75:25: 1H NMR (300 MHz, DMSO-d6) δ 9.31 (bs, 1 H), 8.96 (m, 1 H), 8.19-8.44 (m, 3 H), 8.01-8.14 (m, 2 H), 7.95 (m, 1 H), 7.76-7.82 (m, 2 H), 7.55 (s, 2 H), 7.45 (s, 1 H) 7.34, 7.39 (s, 1 H), 7.27 (s, 1 H), 7.01 (s, 2 H), 5.09 (bs, 1 H), 4.61-4.77 (m, 1 H), 4.51-4.57 (m, 1 H), 4.26-4.37 (m, 2 H), 4.10-4.17 (m, 1 H), 3.62-3.74 (m, 5 H), 3.47-3.52 (m, 2 H), 2.94-3.02 (m, 5 H), 2.73-2.83 (m, 3 H), 2.64-2.69 (m, 3 H), 2.26-2.39 (m, 4 H), 2.00 (s, 3 H), 1.61-1.89 (m, 4 H), 1.44-1.56 (m, 1 H), 1.18-1.39 (m, 5H); MS m/z (C42H59I2N13O13S+H)+ 1240.7; Anal. calcd for C42H59I2N13O13S: N, 14.69 Found: N, 12.28 (peptide content: 84%).
    • 5.36 Example 36 3-(4-Hydroxy-3,5-diiodophenyl)propionyl-β-Ala-Pro-His-Ser-Cys-Asn-NH2
  • This compound was prepared according to the procedures of Examples 1 and 2 with the following modification: capping of the N-terminus was carried out using 3-(4-hydroxy-3,5-diiodophenyl)propionic acid (4.0 eq), HBTU (4.0 eq), HOBT (4.0 eq) and NMM (8.0 eq). The title compound (23.3 mg, 32%) was isolated as a fine white powder. The NMR data indicated a mixture of two species in a ratio of about 80:20: 1H NMR (300 MHz, DMSO-d6) δ 9.32 (bs, 1 H), 8.95 (s, 1 H), 8.22-8.42 (m, 2 H), 8.11-8.13 app d, 1 H), 7.97-7.99 (app d, 1 H), 7.81-7.87 (m, 1 H), 7.56 (s, 2 H), 7.35-7.39 (m, 2 H), 7.08-7.11 (app d, 2 H), 6.92 (s, 1 H), 5.12 (bs, 1 H), 4.60-4.78 m(m, 1 H), 4.27-4.50 (m, 5 H), 3.59-3.70 (m, 3 H), 2.94-3.02 (m, 1 H), 2.76-2.83 (m, 2 H), 2.65-2.69 (m, 2 H), 2.28-2.46 (m, 6 H), 1.72-2.09 (m, 4 H); MS m/z (C33H44I2N10O10S+H)+ 1027.5; Anal. calcd for C33H44I2N10 O 10S: N, 13.64. Found: N, 10.85 (peptide content: 80%).
    • 5.37 Example 37 Acetyl-Pro-His-Ser-Cys-Asn-Gly-Gly-Lys(5- and 6-carboxyfluorescein)-NH2
  • This compound was prepared according to the procedures of Examples 2 and 3 with the following modifications: the amide formation on lysine was carried out with 5-(and 6)-carboxyfluorescein (3 eq), HBTU (3 eq), HOBT (3 eq) and NMM (6 eq). The title compound was isolated (10.5 mg, 18%) as a fine white powder. The NMR data indicated a mixture of isomers: 1H NMR (300 MHz, DMSO-d6) δ 10.16 (s, 1 H), 8.78-8.92 (m, 1 H), 8.44, 8.68 (m, m, 1 H), 8.05-8.28 (m, 3 H), 7.79-7.96 (m, 1 H), 7.45, 7.66 (s, s, 1 H), 7.25-7.37 (m, 2 H), 6.97-7.02 (m, 2 H), 6.69 (m, 1 H), 6.52-6.59 (m, 2 H), 5.08 (bs, 1 H), 4.06-4.80 (m, 8 H), 2.24-2.31, 1.99 (m, s, 3 H), 1.67-1.92 (m, 2 H), 1.23-1.59 (m, 3 H); MS m/z (C54H63N13O17S+H)+ 1198.7; Anal. calcd for C54H63N13O17S: N, 15.20. Found: N, 11.18 (peptide content: 73%).
    • 5.38 Example 38 (3-(4-Hydroxyphenyl)propionyl)-Gly-Gly-Pro-His-Ser-Cys-Asn-NH2
  • This compound was prepared according to the procedures of Examples 1 and 2 with the following modification: capping of the N-terminus was carried out using 3-(4-hydroxy-3,5-diiodophenyl)propionic acid (3 eq), HBTU (3 eq), HOBT (3 eq) and NMM (6 eq). The title compound was isolated as a fine white powder (24.2 mg, 30%). The NMR data indicated a mixture of two species in a ratio of about 80:20: 1H NMR (300 MHz, DMSO-d6) δ 9.17, 8.51 (bs, d, 1 H, J=8.2 Hz), 8.87-8.93 (m, 1 H), 8.10-8.31 (m, 3 H), 7.80-7.98 (m, 2 H), 7.35-7.38 (m, 2 H), 7.09 (d, 2 H, J=7.4 Hz), 6.96-6.99 (m, 2 H), 6.92 (s, 1 H), 6.63-6.66 (m, 2 H), 5.14 (bs, 1 H), 4.59-4.77 (m, 1 H), 4.30-4.50 (m, 4 H), 3.85-4.00 (m, 2 H), 3.59-3.75 (m, 5 H), 2.89-3.05 (m, 1 H), 2.66-2.83 (m, 4 H), 2.36-2.46 (m, 4 H), 1.97-2.08 (m, 1 H), 1.67-1.90 (m, 2 H); MS m/z (C34H47N11O11S+H)+ 818.6; Anal. Calcd for C34H47N11O11S: N, 18.84. Found: N, 14.36 (peptide content:76%).
    • 5.39 Example 39 Acetyl-Pro-His-Ser-Cys-Asn-Gly-Gly-Lys(3-(4-hydroxyphenyl)propionyl)-NH2
  • This compound was prepared according to the procedures of Examples 2 and 3 with the following modifications: the amide formation on lysine was carried out with 3-(4-hydroxyphenyl)propionic acid (4 eq), HBTU (4 eq), HOBT (4 eq) and NMM (8 eq). The title compound was isolated (16.2 mg, 13%) as a fine white powder. The NMR data indicated a mixture of two species in a ratio of about 75:25: 1H NMR (300 MHz, DMSO-d6) δ 8.97 (m, 1 H), 8.42-8.44, 8.19-8.30 (m, m, 3 H), 8.07-8.15 (m, 2 H), 7.94-8.04 (m, 1 H), 7.73-7.82 (m, 2 H), 7.45 (bs, 1 H), 7.35, 7.40 (bs, 1 H), 7.27 (m, 1 H), 6.95-7.01 (m, 4 H), 6.63-6.65 (m, 2 H), 4.72-4.79 (m, 1 H), 4.61-4.68 (m, 1 H), 4.51-4.58 (m, 1 H), 4.41-4.48 (m, 1 H), 4.26-4.39 (m, 2 H), 4.10-4.17 (m, 1 H), 3.61-3.79 (m, 6 H), 3.48-3.55 (m, 2 H), 3.12-3.21 (m, 1 H), 2.96-3.02 (m, 3 H), 2.61-2.86 (m, 4 H), 2.25-2.39 (m, 3 H), 2.00 (s, 3 H), 1.63-1.91 (m, 3 H), 1.44-1.56 (m, 1 H), 1.18-1.37 (m, 4 H); MS m/z (C42H61N13O13S+H)+ 988.8; Anal. calcd for C42H61N13O13S: N, 18.43. Found: N, 12.75 (peptide content: 69%).
    • 5.40 Example 40 (3-(4-Hydroxyphenyl)propionyl)-Gly-Gly-Gly-Gly-Pro-His-Ser-Cys-Asn-NH2
  • This compound was prepared according to the procedures of Examples 2 and 3 with the following modification: capping of the N-terminus was carried out using 3-(4-hydroxy-3,5-diiodophenyl)propionic acid (3 eq.), HBTU (3 eq.), HOBT (3 eq.) and NMM (6 eq.). The title compound was isolated as a fine white powder (27.3 mg, 29%). The NMR data indicated a mixture of two species in a ratio of about 80:20: 1H NMR (300 MHz, DMSO-d6) δ 8.87-8.92 (m, 1 H), 8.24, 8.53 (d, d, 1 H, J=7.4, 8.4 Hz), 8.31 (1 H, J=8.4 Hz), 8.08-8.19 (m, 4 H), 7.87-7.98 (m, 2 H), 7.39 (s, 1 H), 7.36 (s, 1 H), 7.10 (d, 1 H, J=6.5 Hz), 6.99 (app d, 2 H), 6.92 (s, 1 H), 6.65 (app d, 2 H), 4.59-4.77 (m, 1 H), 4.30-4.50 (m, 4 H), 3.86-4.02 (m, 2 H), 3.63-3.79 (m, 7 H), 2.92-3.04 (m, 3 H), 2.67-2.84 (m, 6 H), 2.36-2.46 (m, 5 H), 1.97-2.05 (m, 1 H), 1.82-1.90 (m, 2 H), 1.69-1.79 (m, 2 H); MS m/z (C38H53N13O13S+H)+ 932.6; Anal. Calcd for C38H53N13O13S: N, 19.54. Found: N, 12.89 (peptide content: 66%).
    • 5.41 Example 41 (3-(4-Hydroxyphenyl)propionyl)-Gly-Gly-Gly-Gly-Gly-Gly-Pro-His-Ser-Cys-Asn-NH2
  • This compound was prepared according to the procedures of Examples 1 and 2 with the following modification: capping of the N-terminus was carried out using 3-(4-hydroxy-3,5-diiodophenyl)propionic acid (3 eq), HBTU (3 eq), HOBT (3 eq) and NMM (6 eq). The title compound was isolated as a fine white powder (17.1 mg, 16%). The NMR data indicated a mixture of two species in a ratio of about 70:30: 1H NMR (300 MHz, DMSO-d6) δ 9.11 (s, 1 H), 8.86, 8.92 (s, s, 1 H), 8.51, 8.05-8.16 (m, d, 6 H, J=8.5 Hz), 8.22-8.32 (m, 2 H) 7.87-7.98 (m, 2 H), 7.34-7.39 (m, 2 H), 7.11 (s, 1 H), 7.08 (s, 1 H), 6.98 (app d, 2 H), 6.92 (s, 1 H), 6.64 (app d, 2 H), 5.11 (bs, 1 H), 4.59-4.77 (m, 1 H), 4.31-4.50 (m, 4 H), 3.83-4.02 (m, 2 H), 3.71-3.79 (m, 9 H), 3.58-3.67 (m, 2 H), 3.48-3.53 (m, 2 H), 3.11-3.19 (m, 1 H), 2.91-3.04 (m, 2 H), 2.76-2.83 (m, 2 H), 2.67-2.72 (m, 2 H), 2.35-2.46 (m, 4 H), 1.96-2.09 (m, 1 H), 1.90 (m, 4 H); MS m/z (C42H59N15O15S+H)+ 1046.8; Anal. Calcd for C42H59N15O15S: N, 20.08. Found: N, 15.84 (peptide content: 79%).
    • 5.42 Example 42 (3-(4-Hydroxyphenyl)propionyl)-Ahp-Pro-His-Ser-Cys-Asn-NH2
  • This compound was prepared according to the procedures of Examples 1 and 2 with the following modification: capping of the N-terminus was carried out using 3-(4-hydroxyphenyl)propionic acid (3 eq), HBTU (3 eq), HOBT (3 eq) and NMM (6 eq). The title compound was isolated as a fine white powder (47.4 mg, 57%). The NMR data indicated a mixture of two species in a ratio of about 70:30: 1H NMR (300 MHz, DMSO-d6) δ 8.98 (m, 1 H), 8.21-8.44 (m, 2 H), 7.98-8.14 (m, 2 H), 7.71-7.77 (m, 1 H), 7.40 (s, 1 H), 7.35 (m, 1 H), 7.09 (d, 2 H, J=9.8 Hz), 6.95-6.98 (m, 2 H), 6.92 (s, 1 H), 6.62-6.54 (m, 2 H), 4.62-4.79 (m, 4 H), 4.28-4.50 (m, 8 H), 3.47-3.70 (m, 4 H), 3.12-3.19 (m, 1 H), 2.90-3.03 (m, 3 H), 2.76-2.83 (m, 2 H), 2.65-2.70 (m, 2 H), 2.40-2.46 (m, 2 H), 2.25-2.30 (m, 3 H), 1.72-2.08 (m, 4 H), 1.14-1.50 (m, 7 H); MS m/z (C37H54N10O10S+H)+ 831.8; Anal. Calcd for C37H54N10O10S: N, 16.86. Found: N, 13.10 (peptide content: 78%).
    • 5.43 Example 43 Ac-Tyr-Ahp-Pro-His-Ser-Cys-Asn-NH2
  • This compound was prepared according to the procedures of Examples 1 and 2 with the following modifications: the final amino acid coupling was carried out with Ac-Tyr(3-iodo)-OH (3 eq), HBTU (3 eq), HOBT (3 eq) and NMM (6 eq). The title compound was isolated (40.9 mg, 46%) as a fine white powder. The NMR data indicated a mixture of two species in a ratio of about 80:20: 1H NMR (300 MHz, DMSO-d6) δ 8.97 (m, 1 H), 8.21-8.44 (m, 2 H), 8.06-8.14 (m, 1 H), 7.97-7.99 (m, 2 H), 7.85-7.87 (m, 1 H), 7.40 (s, 1 H), 7.35 (s, 1 H), 7.09 (d, 2 H, J=9.7 Hz), 6.89-7.00 (m, 2 H), 6.92 (s, 1 H), 6.61-6.64 (m, 2 H), 4.59-4.66 (m, 2 H), 4.25-4.50 (m, 6 H), 3.59-3.71 (m, 2 H), 3.48-3.55 (m, 2 H), 3.12-3.19 (m, 1 H), 2.95-3.10 (m, 3 H), 2.76-2.83 (m, 3 H), 2.57-2.65 (m, 1 H), 2.38-2.46 (m, 2 H), 2.24-2.27 (m, 2 H), 1.80-2.10 (m, 1 H), 1.84-1.89 (m, 2 H), 1.76 (s, 3 H), 1.45-1.49 (m, 2 H), 1.15-1.35 (m, 6 H); MS m/z (C39H57N11O11S+H)+ 888.8; Anal. Calcd for C39H57N11O11S: N, 17.35. Found: N, 13.81 (peptide content: 80%).
    • 5.44 Example 44 DOTA-In-β-Ala-Pro-His-Ser-Cys-Asn-NH2
  • This compound was prepared according to the procedures of Examples 1 and 2 with the following modifications: the final amide coupling was carried out with DOTA-tris(t-butyl ester) (3 eq), HBTU (3 eq), HOBT (3 eq) and NMM (6 eq). To the purified peptide-DOTA compound (22.0 mg, 0.022 mmol) in 0.1 M AcOH (aq.) (0.5 mL) was added Indium chloride (48.6 mg, 0.22 mmol) dissolved in 1.0 mL of 0.2 M HCl (aq.) and stirred at rt for 2 hours. The complex was purified according to the procedure of Example 2. The title compound was isolated (40.9 mg, 46%) as a fine white powder. The NMR data indicated a mixture of three species in a ratio of about 70:20:10 1H NMR (300 MHz, D2O) δ 8.69-8.90 (m, 1 H), 7.39-7.55 (m, 1 H), 4.53-4.64 (m, 3 H), 4.39-4.48 (m, 1 H), 3.88-3.96 (m, 5 H), 3.24-3.68 (m, 28 H), 3.00-3.02 (m, 3 H), 2.69-2.94 (m, 9 H), 2.24-2.43 (m, 2 H), 1.84-2.10 (m, 5 H); MS m/z (C40H61InN14O15S+H)+ 11245.8; Anal. Calcd for C40H61InN14O15S: N, 17.43 Found: N, 12.42 (peptide content: 71%).
    • 5.45 Example 45 Acetyl-Pro-His-Ser-Cys-Asn-Gly-Gly-Gly-Gly-Gly-Gly-Lys-NH2
  • This compound was prepared according to the procedures of Examples 1 and 2. The title compound was isolated (15.4 mg, 10%) as a fine white powder. The NMR data indicated a mixture of two species in a ratio of about 75:25: 1H NMR (300 MHz, DMSO-d6) δ 8.95-8.97 (m, 1 H), 8.43 and 8.02 (d, 1 H, J=7.6 Hz), 8.11-8.31 (m, 9 H), 7.84-7.86 (m, 1 H), 7.62 (bs, 3 H), 7.44 (bs 1 H), 7.34-7.39 (m, 1 H), 7.29 (s, 1 H), 7.05 (s, 1 H), 7.00 (s, 1 H), 4.51-4.79 (m, 3 H), 4.25-4.48 (m, 4 H), 4.12-4.19 (m, 2 H), 3.74 (m, 10 H), 3.11-3.21 (m, 2 H), 2.94-3.03 (m, 2 H), 2.73-2.82 (m, 5 H), 2.34-2.39 (m, 1 H), 2.00 (s, 3 H), 1.67-1.89 (m, 5 H), 1.46-1.57 (m, 3 H), 1.24-1.34 (m, 2 H); MS m/z (C41H65N17O15S+H)+ 1068.7; Anal. calcd for C41H65N17O15S: N, 22.29. Found: N, 15.33 (peptide content: 69%).
    • 5.46 Example 46 Acetyl-Pro-His-Ser-Cys-Asn-Ava-Tyr-NH2
  • This compound was prepared according to the procedures of Examples 1 and 2. The title compound was isolated (56.5 mg, 44%) as a fine white powder. The NMR data indicated a mixture of two species in a ratio of about 75:25: 1H NMR (300 MHz, DMSO-d6) δ 9.13 (bs, 1 H), 8.96-8.97 (m, 1 H), 8.20-8.30 (m, 2 H), 8.13 (d, 1 H, J=7.8), 7.96-8.04 (m, 1 H), 7.82 (d, 1 H, J=8.5 Hz), 7.57-7.61 (m, 1 H), 7.39 (s, 1 H), 7.34 (bs, 1 H), 6.93-7.01 (m, 4 H), 6.61-6.64 (m, 2 H), 4.62-4.80 (m, 1 H), 4.25-4.50 (m, 5 H), 3.15-3.20 (m, 1 H), 2.95-3.01 (m, 3 H), 2.75-2.87 (m, 4 H), 2.58-2.66 (m, 1 H), 2.36-2.49 (m, 2 H), 2.00-2.02 (m, 5 H), 1.69-1.88 (m, 3 H), 1.35-1.37 (m, 2 H), 1.25-1.28 (m, 2 H); MS m/z (C37H53N11O11S+H)+ 860.7; Anal. calcd for C37H53N11O11S: N, 17.92. Found: N, 14.34 (peptide content: 80%).
    • 5.47 Example 47 Acetyl-Pro-His-Ser-Cys-Asn-Ahp-Tyr-NH2
  • This compound was prepared according to the procedures of Examples 1 and 2. The title compound (49.5 mg, 37%) was isolated as a fine white powder. The NMR data indicated a mixture of two species in a ratio of about 80:20: 1H NMR (300 MHz, DMSO-d6) δ 9.12 (bs, 1 H), 8.97-8.98 (m, 1 H), 8.26-8.29 (m, 1 H), 8.42 and 8.21 (d, 1 H, J=7.6 Hz), 8.13 and 7.95 (d, 1 H, J=7.8 Hz), 7.81 (d, 1 H, J=8.5 Hz), 7.57-7.60 (m, 1 H), 7.39 (s, 1 H), 7.33 (bs, 2 H), 6.99-7.01 (app d, 2 H), 6.96 (s, 1 H), 6.93 (s, 1 H), 6.61-6.64 (app d, 2 H), 4.60-4.79 (m, 1 H), 4.26-4.53 (m, 5 H), 3.11-3.21 (m, 1 H), 2.94-3.04 (m, 3 H), 2.73-2.88 (m, 3 H), 2.58-2.65 (m, 1 H), 2.39-2.49 (m, 2 H), 2.00-2.04 (m, 5 H), 1.70-1.91 (m, 3 H), 1.28-1.40 (m, 4 H), 1.05-1.20 (m, 4 H); MS m/z (C39H57N11O11S+H)+ 888.8; Anal. calcd for C39H57N11O11S: N, 17.35. Found: N, 13.84 (peptide content: 80%).
    • 5.48 Example 48 3-(4-Hydroxyphenyl)propionyl-bAla-Pro-His-Ser-Cys-Asn-NH2
  • This compound was prepared according to the procedures of Examples 1 and 2 with the following modifications: capping of the N-terminus was carried out using 3-(4-hydroxyphenyl)propionic acid (3 eq), HBTU (3 eq), HOBT (3 eq) and NMM (6 eq). The title compound was isolated (61.1 mg, 53%) as a fine white powder. The NMR data indicated a mixture of two species in a ratio of about 75:25: 1H NMR (300 MHz, DMSO-d6) δ 8.97 (bs, 1 H), 8.22-8.29 (m, 2 H), 8.10-8.13 (m, 1 H), 7.97 and 8.41 (d, 1 H, J=7.2 Hz), 7.80 (app t, 1 H), 7.34-7.39 (m, 2 H), 7.08 (d, 2 H, J=9.8 Hz), 6.92-6.98 (m, 3 H), 6.63-6.66 (app d, 2 H), 4.60-4.77 (m, 1 H), 4.27-4.50 (m, 5 H), 3.65-3.71 (m, 3 H), 3.39-3.49 (m, 3 H), 3.11-3.29 (m, 4 H), 2.94-3.04 (m, 1 H), 2.76-2.83 (m, 2 H), 2.37-2.46 (m, 3 H), 2.26-2.31 (m, 2 H), 1.97-2.08 (m, 1 H), 1.72-1.87 (m, 3 H); MS m/z (C33H46N10O10S+H)+ 775.7; Anal. calcd for C33H46N10O10S: N, 18.08. Found: N, 12.76 (peptide content: 71%).
    • 5.49 Example 49 8-(4-Fluorobenzylamino)suberoyl-β-Ala-Pro-His-Ser-Cys-Asn-NH2
  • This compound was prepared according to the procedures of Examples 1 and 2 with the following modification: capping of the N-terminus was carried out using 8-(4-fluorobenzylamino)suberic acid (3 eq), HBTU (3 eq), HOBT (3 eq) and NMM (6 eq). The title compound was isolated as a fine white powder (70.8 mg, 53%). The NMR data indicated a mixture of two species in a ratio of about 70:30: 1H NMR (300 MHz, DMSO-d6) δ 8.97 (s, 1 H), 8.22-8.28 (m, 3 H), 8.11-8.14 (m, 1 H), 7.96-7.99 and 8.40-8.43 (m, 1 H), 7.74-7.80 (m, 1 H), 7.34-7.39 (m, 2 H), 7.24-7.29 (m, 2 H), 7.08-7.16 (m, 4 H), 6.92 (bs, 1 H), 4.29-4.50 (m, 7 H), 4.22 (d, 2 H, J=5.9 Hz), 3.63-3.69 (m, 2 H), 3.44-3.52 (m, 2 H), 3.16-3.26 (m, 3 H), 2.97-3.02 (m, 1 H), 2.76-2.83 (m, 2 H), 2.38-2.49 (m, 3 H), 1.99-2.13 (m, 5 H), 1.72-1.89 (m, 3 H), 1.42-1.54 (m, 4 H), 1.20-1.24 (m, 4 H); MS m/z (C34H47N11O11S+H)+ 818.6; Anal. Calcd for C39H56FN11O10S: N, 17.31. Found: N, 11.72 (peptide content: 68%).
    • 5.50 Example 50 m-dPEG-β-Ala-His-Ser-Cys-Asn-NH2
  • This compound was prepared according to the procedures of Examples 1 and 2 with the following modification: capping of the N-terminus was carried out using NHS-m-dPEG™ (Quanta Biodesign, 1.9 eq), and TEA (9 eq). The title compound was isolated as a fine white powder (38 mg, 21%): 1H NMR (300 MHz, D2O) δ 8.58 (d, 1 H, J=1.2 Hz), 7.28 (d, 1 H, J=0.8 Hz), 4.75-4.61 (m, 2 H), 4.53 (t, 1 H, J=6.2 Hz), 4.44 (t, 1 H, J=5.6 Hz), 4.34-4.29 (dd, 1 H, J=5.1 Hz, 3.6 Hz), 3.82 (t, 2 H, J=5.4 Hz), 3.71 (t, 2 H, J=6.1 Hz), 3.64-3.61 (m, 44 H), 3.58-3.54 (m, 4 H), 3.40 (t, 2 H, J=6.7 Hz), 3.63-3.69 (m, 2 H), 3.44-3.52 (m, 2 H), 3.16-3.26 (m, 3 H), 2.97-3.02 (m, 1 H), 3.32 (s, 3 H), 3.25 (d, 1 H, J=5.9 Hz), 3.18 (d, 1 H, J=8.4 Hz), 2.90 (d, 2 H, J=6.2 Hz), 2.83-2.56 (m, 3 H), 2.46 (t, 2 H, J=6.1 Hz), 2.25-2.15 (m, 1H), 1.92-1.87 (m, 1H), 1.83-1.76 (m, 1H); MS m/z (C50H88N10O21S+H)+ 1198.1.
    • 5.51 Example 51 DTPA Conjugation to PHSCN
  • A solution of resin (Rink Amide AM resin) bound PHSCN (143 mg, 0.36 mmol/g, 0.051 mmol), p-SCN-benzyl DTPA (66 mg, 0.103 mmol), NNM (120 μL, 2.1 mmol), in anhydrous DMF (2 mL) was agitated gently with nitrogen for 7 hours at room temperature, and the reaction mixture was filtered and washed with DMF once. This step was repeated twice more. The resin was washed three times with DMF, three times with methanol and three times with dichloromethane. The resulting resin bound compound was treated with TFA/TIS/water (95:2.5:2.5, 1 mL per 100 mg of resin) and agitated with nitrogen for 1 hour. The reaction mixture was filtered, the resin was washed once with TFA/TIS/water and three times with dichloromethane. The solvent was removed in vacuo and the resulting residue was triturated three times with ether. The resulting white powder was subjected to a prep HPLC purification according to the procedure of Example 2 to give 15.5 mg (0.014 mmol, 27.7%) of desired product: 1H NMR (300 MHz, D2O) δ 8.66 (s, 1H), 7.40-7.31 (m, 5H), 4.97-4.92 (dd, J=4.92, 5.31 Hz, 1H), 4.87-4.83 (m, 2H), 4.77-4.72 (m, 1H), 4.65-4.56 (m, 2H), 4.00-3.97 (m, 4H), 3.91-3.72 (m, 10H), 3.49-3.20 (m, 6H), 3.03-2.97 (m, 2H), 2.92-2.73 (m, 4H), 2.38 (m, 1H), 2.20 (m, 2H), 2.04-1.85 (m, 2H); MS m/z (C43H61N13O17S2+H)+ 1096.8.
    • 5.52 Example 52 In3+ chelation to DTPA-PHSCN
  • DTPA-PHSCN (20 mg, 0.018 mmol) was dissolved in 1 mL of 0.1M AcOH(aq) solution and InCl3 (40 mg, 0.18 mmol) was dissolved in 2 mL of 0.02M HCl solution. The two solutions were combined and incubated for an hour at room temperature. The solvent was evaporated under reduced pressure and the resulting white powder was subjected to a prep HPLC purification according to the procedure of Example 2 to give 7.1 mg (0.00588 mmol, 32.6%) of desired product: 1H NMR (300 MHz, D2O) δ 8.66 (s, 1H), 7.41-7.31 (m, 5H), 4.97-4.92 (dd, J=4.86, 5.46 Hz, 1H), 4.86-4.84 (m, 2H), 4.76-4.72 (dd, J=5.43, 2.88 Hz, 1H), 4.61-4.57 (m, 2H), 4.04 (d, J=17.22 Hz, 1H), 3.90 (d, J=5.82 Hz, 2H), 3.81-3.60 (m, 5H), 3.50-3.19 (m, 10H), 3.14-2.73 (m, 8H), 2.39 (m, 1H), 2.16 (m, 1H), 2.04-1.91 (m, 2H); MS m/z (C43H58InN13O17S2+H)+ 1208.8; Anal. calcd for C43H58InN13O17S2: N, 15.07. Found: N, 11.58 (peptide content: 76.8%).
    • 5.53 Example 53 Acetyl-Pro-His-Ser-Cys-Asn-Gly-Gly-Gly-Gly-Lys-NH2
  • This compound was prepared according to the procedures of Examples 1 and 2. The title compound was isolated (101 mg, 70%) as a fine white powder: 1H NMR (300 MHz, D2O) δ 8.57 (d, J=1.32 Hz, 1H), 7.26 (s, 1H), 4.73-4.72 (m, 2H), 4.52 (t, J=6.24, 1H), 4.43 (t, J=5.25 Hz,1H), 4.27 (m, 2H), 3.94-3.91(m, 8H), 3.81 (t, J=5.40 Hz, 2H), 3.57 (t, J=6.78 Hz, 1H), 3.31-3.24 (dd, J=15.57, 5.67 Hz, 1H), 1H), 3.18-3.09 (dd, J=15.6, 8.64Hz, 2H), 2.96-2.83 (m, 4H), 2.80-2.73 (m, 2H), 2.20-2.16 (m, 1H), 2.05 (s, 3H), 1.94-1.57 (m, 6H), 1.42-1.34 (m, 2H); MS m/z (C37H59N15O13S+H)+ 954.9.
    • 5.54 Example 54 Acetyl-Pro-His-Ser-Cys-Asn-Gly-Gly-Lys(Protoporphyrin)-NH2
  • This compound was prepared according to procedures of Examples 2 and 3 with the following modifications: the 2% hydrazine treatment for the deprotection of ivDde on lysine was repeated 10 times and the amide formation on lysine was carried out with protoporphyrin IX (2 eq), PYBOP (2 eq), and NMM (6 eq). The title compound was isolated (7.6 mg, 5.49 μM, 8.6%) as a fine dark red powder.; MS m/z (C67H85N17O14S+H)+ 1385.6.
    • 5.55 Example 55 Ac-Tyr-β-Ala-Pro-His-Ser-Cys-Asn-NH2
  • This compound was prepared from Rink amide AM resin according to the procedures of Examples 1 and 2 and was isolated as a white, fluffy solid; ES MS m/z (M+H)+ 832, (M+Na)+ 854.
    • 5.56 Example 56 Ac-Pro-His-Ser-Cys-Asn-β-Ala-Tyr-NH2
  • This compound was prepared from Rink amide AM resin according to the procedures of Examples 1 and 2 and was isolated as a white, fluffy solid; ES MS m/z (M+H)+ 832, (M+Na)+ 854.
    • 5.57 Example 57 Ac-Pro-His-Ser-Cys-Asn-Gly-Gly-Lys-NH2
  • This compound was prepared from Rink amide AM resin according to the procedures of Examples 1 and 2 and was isolated as a white, fluffy solid; ES MS m/z (M+H)+ 840
    • 5.58 Example 58 Ac-Pro-His-Ser-Cys-Asn-Gly-Gly-Lys(biotin)-NH2
  • This compound was prepared from Rink amide AM resin according to the procedures of Examples 1 and 2 sand was isolated as a white, fluffy solid; ES MS m/z (M+H)+ 1066.5.
  • Finally, it should be noted that there are alternative ways of implementing the present invention. Accordingly, the present embodiments are to be considered as illustrative and not restrictive, and the invention is not to be limited to the details given herein, but may be modified within the scope and equivalents of the appended claims. All publications and patents cited herein are incorporated by reference in their entirety.

Claims (74)

1. A compound of Formula (I):
Figure US20050020810A1-20050127-C00076
or a pharmaceutically acceptable salt, solvate, hydrate or N-oxide thereof wherein:
j and k are independently 0 or 1;
p and q are independently an integer including and between 0 and 100;
r and s are independently 0 or 1;
R1 is acyl, substituted acyl, acyl chelate, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, imino or substituted imino;
R2 is C1-C6 alkyl with at least one hydrogen atom replaced by a substituent selected from the group consisting of —NR6R7, —OR8, —CO2R9, —S(O)zR10, —P(OR11)OR12, aryl and substituted aryl;
R6, R7, R8, R9, R10, R11 and R12 are independently selected from the group consisting of hydrogen, acyl, substituted acyl, acyl chelate, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, imino and substituted imino;
X1 is —NH(C═C)gCO—, —NH(CH2)hCO— or —NHCH(CH3)CO—;
g and h are independently 1, 2, 3, 4, 5 or 6
Figure US20050020810A1-20050127-C00077
l is an integer from 1 to 4;
Figure US20050020810A1-20050127-C00078
R13 is hydrogen, alkyl, substituted alkyl, acyl, substituted acyl, arylalkyl, substituted arylalkyl, aryl, substituted aryl or —S(O)xR14;
n is an integer from 1 to 5;
R14 is alkyl, substituted alkyl, acyl, substituted acyl, arylalkyl, substituted arylalkyl, aryl or substituted aryl;
y and x are independently 0, 1 or 2;
Figure US20050020810A1-20050127-C00079
m is an integer from 1, 2, 3 or 4;
X7 is —NH(C═C)dCO—, —NH(CH2)eCO— or —NHCH(CH3)CO—;
d and e are independently 1, 2, 3, 4, 5 or 6;
R3 is C1-C6 alkyl with at least one hydrogen atom replace by a substituent selected from the group consisting of —NR15R16, —OR17, —CO2R18, —S(O)nR19, —P(OR20)OR21 aryl and substituted aryl;
R4 and R5 are independently hydrogen, alkyl or substituted alkyl; and
R15, R16, R17, R18, R19, R20 and R21 are independently selected from the group consisting of hydrogen, acyl, substituted acyl, acyl chelate, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, imino and substituted imino;
with the proviso that R1 is not acetyl when R4 and R5 are hydrogen and r and s are 0.
2. The compound of claim 1, wherein R1 is not acetyl when R4 and R5 are hydrogen.
3. The compound of claim 1, wherein at least one of r or s are 1.
4. The compound of claim 1 wherein s is 1 and r is 0.
5. The compound of claim 1, wherein s is 0 and r is 1.
6. The compound of claim 1, wherein R1 is acyl, substituted acyl, acyl chelate, imino or substituted imino.
7. The compound of claim 1, wherein R2 is C1-C6 alkyl with at least one hydrogen atom replaced by a substituent selected from the group consisting of —NR6R7, —OR8 and —CO2R9.
8. The compound of claim 7, wherein R6, R7, R8 and R9 are independently selected from the group consisting of hydrogen, acyl, substituted acyl, acyl chelate, imino or substituted imino.
9. The compound of claim 1, wherein X1 is —NH(CH2)hCO—.
10. The compound of claim 1, wherein R3 is C1-C6 alkyl with at least one hydrogen atom replaced by a substituent selected from the group consisting of —NR15R16, —OR17 and —CO2R18.
11. The compound of claim 1, wherein R15, R16, R17 and R18 are independently selected from the group consisting of hydrogen, acyl, substituted acyl, acyl chelate, imino or substituted imino.
12. The compound of claim 1, wherein:
R1 is acyl or substituted acyl;
R2 is C1-C4 alkyl with at least one hydrogen atom replaced by a substituent selected from the group consisting of, —NR6R7, aryl and substituted aryl;
R6 and R7 are independently selected from the group consisting of hydrogen, acyl and substituted acyl;
Figure US20050020810A1-20050127-C00080
R13 is hydrogen, acyl, substituted acyl, alkyl or substituted alkyl;
Figure US20050020810A1-20050127-C00081
R3 is C1-C4 alkyl with at least one hydrogen atom replaced by a substituent selected from the group consisting of —NR15R16, aryl and substituted aryl;
R15 and R16 are independently selected from the group consisting of hydrogen, acyl and substituted acyl; and
R4 and R5 are hydrogen.
13. The compound of claim 12, wherein:
s is 0 and r is 1;
k is 1;
R1 is acetyl;
R13 is hydrogen;
e is 1; and
R3 is —(CH2)4NH2.
14. The compound of claim 13, wherein q is 2, 4 or 6.
15. The compound of claim 12, wherein
s is 0 and r is 1;
k is 1;
R1 is acetyl;
R13 is hydrogen;
e is 2, 4 or 6; and
R3 is —(CH2)4NHCO(CH2)2-Ph-(4-OH).
16. The compound of claim 15, wherein q is 1.
17. The compound of claim 12, wherein:
s is 0 and r is 1;
k is 1;
R1 is acetyl;
R13 is hydrogen;
e is 2, 4 or 6 and
R3is —CH2-Ph-(4-OH).
18. The compound of claim 17, wherein q is 1;
19. The compound of claim 12, wherein
s is 0 and r is 1;
k is 1;
R1 is acetyl;
R13 is methyl;
e is 1; and
R3 is —CH2)4NH2.
20. The compound of claim 19, wherein q is 2.
21. The compound of claim 12, wherein:
s is 1 and r is 0;
j is 1;
R1 is acetyl;
R13 is hydrogen;
h is 1; and
R2 is —CH2-Ph-(4-OH).
22. The compound of claim 21, wherein p is 2, 4 or 6.
23. The compound of claim 12, wherein
s is 1 and r is 0;
j is 1;
R1 is acetyl;
R13 is hydrogen;
h is 2, 4, or 6; and
R2 is —CH2-Ph-(4-OH).
24. The compound of claim 23, wherein p is 1.
25. The compound of claim 12, wherein:
s is 1 and r is 0;
j is 0;
R1 is —CO(CH2)2-Ph-(4-OH).;
R13 is hydrogen; and
h is 1.
26. The compound of claim 25, wherein p is 2, 4 or 6.
27. The compound of claim 12, wherein:
s is 1 and r is 0;
j is 0;
R1 is —CO(CH2)2-Ph-(4-OH).;
R13 is hydrogen; and
h is 2, 4 or 6.
28. The compound of claim 27, wherein p is 1.
29. The compound of claim 12, wherein:
s is 0 and r is 0;
R1 is —CH2)2-Ph-(4-OH); and
R13 is hydrogen.
30. The compound of claim 12, wherein:
s is 0 and r is 0;
R1 is —COPh-(4-F); and
R13 is hydrogen.
31. The compound of claim 12, wherein:
s is 0 and r is 1;
k is 1;
R1 is acetyl;
R13 is methyl or hydrogen;
e is 1; and
R3 is —CH2)4NHCOPh-(4-F).
32. The compound of claim 31, wherein q is 2.
33. The compound of claim 12, wherein:
s is 0 and r is 1;
k is 1;
R1 is acetyl;
R13 is hydrogen;
e is 1; and
R3 is —(CH2)4NH-8-[4′-fluorobenzylamino]suberoyl or —(CH2)4NHCOCH2F
34. The compound of claim 33, wherein q is 2.
35. The compound of claim 12, wherein:
s is 1 and r is 0;
j is 0;
R1 is 8-[4′-fluorobenzylamino]suberoyl or —COCH2F;
R13 is hydrogen; and
h is 2.
36. The compound of claim 35, wherein p is 1.
37. The compound of claim 12, wherein
s is 0 and r is 1;
k is 1;
R1 is acetyl;
R13 is hydrogen; and
R3 is —CH2Ph-(3-I, 4-OH) or —CH2Ph-(3,5-diI, 4-OH).
38. The compound of claim 37, wherein q is 0.
39. The compound of claim 37, wherein q is 1 and e is 2.
40. The compound of claim 37, wherein q is 1 and e is 1.
41. The compound of claim 12, wherein
s is 1 and r is 0;
j is 1;
R1 is acetyl;
R13 is hydrogen; and
R2 is —CH2Ph-(3-I, 4-OH) or —CH2Ph-(3,5-diI, 4-OH).
42. The compound of claim 41, wherein p is 0.
43. The compound of claim 12, wherein
s is 0 and r is 0;
R1 is —CO(CH2)2Ph(4-OH, 3, 5 di-I); and
R13 is hydrogen.
44. The compound of claim 12, wherein
s is 1 and r is 0;
j is 0;
R1 is —O(CH2)2Ph(4-OH, 3, 5 di-I);
h is 2; and
R13 is hydrogen.
45. The compound of claim 44, wherein p is 1.
46. The compound of claim 12, wherein
s is 1 and r is 0;
j is 1;
R1 is acetyl;
R2 is —CH2-Ph(4-OH, 3, 5 di-I);
h is 2; and
R13 is hydrogen.
47. The compound of claim 46, wherein p is 1.
48. The compound of claim 12, wherein
s is 0 and r is 1;
R3 is —(CH2)4NHCO(CH2)2-Ph(4-OH, 3, 5 di-D);
e is 1; and
R13 is hydrogen.
49. The compound of claim 48, wherein q is 2.
50. The compound of claim 1, wherein:
R1 is acyl chelate;
R2, R6, R7, X1, X2, X4, X5, R13, X6, X7, R3, R15, R16, R4 and R5 are as defined in claim 12.
51. The compound of claim 50, wherein
s is 1 and r is 0;
j is 0;
R1 is DOTA-In;
h is 2; and
R13 is hydrogen.
52. The compound of claim 51, wherein p is 1.
53. The compound of claim 50, wherein
s is 0 and r is 0;
R1 is DPTA or DPTA-In; and
R13 is hydrogen.
54. A compound of Formula (III):
Figure US20050020810A1-20050127-C00082
or a pharmaceutically acceptable salt, solvate, hydrate or N-oxide thereof wherein:
R20 is acyl, substituted acyl, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, imino, substituted imino or a diagnostic agent;
R21 is C1-C6 alkyl with at least one hydrogen atom replaced by a substituent selected from the group consisting of —NHR22;
R22 is hydrogen, acyl, substituted acyl, alkyl, substituted alkyl or a diagnostic agent; and
j, k, p, q, r, s, R2, X1, X2, X3, X4, X5, X6, X7, R4 and R5 are as defined in claim 1;
with the proviso that at least one of R20 and R22 is a diagnostic agent.
55. The compound of claim 54, wherein R2, X1, X2, X3, X4, X5, X6, X7, R4 and R5 are as defined in claim 12.
56. The compound of claim 55, wherein R20 is a fluorescent agent.
57. The compound of claim 56, wherein R20 is ⅚ carboxy fluorescein, s is 1, r is 0, j is 0, e is 2 and p is 1.
58. The compound of claim 55 wherein R22 is a fluorescent agent.
59. The compound of claim 58, wherein R21 is (CH2)4NH—, R22 is −⅚ carboxy fluorescein, s is 0, r is 1, k is 1, e is 1 and q is 2.
60. The compound of claim 55, wherein R21 is (CH2)4NH—, R22 is biotin, s is 0, r is 1, k is 1, e is 1 and q is 2.
61. A compound of Formula (IV):
Figure US20050020810A1-20050127-C00083
or a pharmaceutically acceptable salt, solvate, hydrate or N-oxide thereof wherein:
R23 is acyl, substituted acyl, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, imino, substituted imino or a pegylating agent;
R24 is C1-C6 alkyl with at least one hydrogen atom replaced by a substituent selected from the group consisting of —NHR28 wherein R28 is hydrogen, acyl, substituted acyl, alkyl substituted alkyl or a pegylating agent; and
j, k, p, q, r, s, R2, X1, X2, X3, X4, X5, X6, X7, R4 and R5 are as defined in claim 1;
with the proviso that at least one of R23 or R28 is a pegylating agent.
62. The compound of claim 61, wherein R2, X1, X2, X3, X4, X5, X6, X7, R4 and R5 are as defined in claim 12.
63. The compound of claim 62 wherein R23 is m-dPEG, s is 1, r is 0, j is 0, h is 2 and p is 1.
64. A compound of Formula (V):
Figure US20050020810A1-20050127-C00084
or a pharmaceutically acceptable salt, solvate, hydrate or N-oxide thereof wherein:
R29 is C1-C6 alkyl with at least one hydrogen atom replace by —NHR32;
R30 is acyl, substituted acyl, alkyl, substituted alkyl or a therapeutic agent.
R31 is hydrogen, alkyl, substituted alkyl or a therapeutic agent;
R32 is hydrogen, acyl substituted acyl, alkyl, substituted alkyl or a therapeutic agent; and;
j, k, p, q, r, s, R2, X1, X2, X3, X4, X5, X6, X7 and R4 and R5 are as defined in claim 1;
with the proviso that at least one of R30, R31 and R32 is a therapeutic agent.
65. The compound of claim 64, wherein R2; X1, X2, X3, X4, X5, X6, X7 and R4 are as defined in claim 12.
66. The compound of claim 65, wherein R13 is methyl or acetyl, s is 0, r is 0, R30 is acetyl and R31 is a therapeutic agent.
67. The compound of claim 66, wherein the therapeutic agent is doxorubicin.
68. The compound of claim 65, wherein R13 is methyl or hydrogen, s is 0, r is 1, k is 1, e is 1, q is 2, R30 is acetyl, R31 is hydrogen, R29 is —(CH2)4NHR32.
69. The compound of claim 68, wherein the R32 is —CO(CH2)3-doxorubicin.
70. The compound of claim 68, wherein R32 is protoporphyrin.
71. A pharmaceutical composition comprising a compound of claim 1, and a pharmaceutically acceptable vehicle.
72. A method for treating or preventing cancer in a patient comprising administering to the patient in need of such treatment or prevention a therapeutically effective amount of a compound of claim 1.
73. A method for treating or preventing cancer in a patient comprising administering to the patient in need of such treatment or prevention a therapeutically effective amount of the pharmaceutical composition of claim 71.
74. A method for detecting cancer in a patient comprising administering to the patient in need of such detection a diagnostically effective amount of the pharmaceutical composition of claim 71.
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