WO2007121453A2 - Agents chélateurs de 2-hydroxy-1-oxo-1,2-dihydro-isoquinoline - Google Patents

Agents chélateurs de 2-hydroxy-1-oxo-1,2-dihydro-isoquinoline Download PDF

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WO2007121453A2
WO2007121453A2 PCT/US2007/066814 US2007066814W WO2007121453A2 WO 2007121453 A2 WO2007121453 A2 WO 2007121453A2 US 2007066814 W US2007066814 W US 2007066814W WO 2007121453 A2 WO2007121453 A2 WO 2007121453A2
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substituted
unsubstituted
complex
independently selected
hopo
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PCT/US2007/066814
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WO2007121453A3 (fr
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Kenneth N. Raymond
Michael Seitz
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The Regents Of The University Of California
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D217/00—Heterocyclic compounds containing isoquinoline or hydrogenated isoquinoline ring systems
    • C07D217/22—Heterocyclic compounds containing isoquinoline or hydrogenated isoquinoline ring systems with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to carbon atoms of the nitrogen-containing ring
    • C07D217/24—Oxygen atoms
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/0002—General or multifunctional contrast agents, e.g. chelated agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
    • A61K49/08—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by the carrier
    • A61K49/085—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by the carrier conjugated systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
    • A61K49/08—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by the carrier
    • A61K49/10—Organic compounds
    • A61K49/12—Macromolecular compounds
    • A61K49/126—Linear polymers, e.g. dextran, inulin, PEG
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/0474—Organic compounds complexes or complex-forming compounds, i.e. wherein a radioactive metal (e.g. 111In3+) is complexed or chelated by, e.g. a N2S2, N3S, NS3, N4 chelating group
    • A61K51/0478—Organic compounds complexes or complex-forming compounds, i.e. wherein a radioactive metal (e.g. 111In3+) is complexed or chelated by, e.g. a N2S2, N3S, NS3, N4 chelating group complexes from non-cyclic ligands, e.g. EDTA, MAG3
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/06—Macromolecular compounds, carriers being organic macromolecular compounds, i.e. organic oligomeric, polymeric, dendrimeric molecules
    • A61K51/065—Macromolecular compounds, carriers being organic macromolecular compounds, i.e. organic oligomeric, polymeric, dendrimeric molecules conjugates with carriers being macromolecules
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D217/00—Heterocyclic compounds containing isoquinoline or hydrogenated isoquinoline ring systems
    • C07D217/22—Heterocyclic compounds containing isoquinoline or hydrogenated isoquinoline ring systems with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to carbon atoms of the nitrogen-containing ring
    • C07D217/26—Carbon atoms having three bonds to hetero atoms with at the most one bond to halogen

Definitions

  • Chelators based on the hydroxamic acid functional group are known to be excellent binding motifs for metal cations.
  • ligands based on l-hydroxy-6-oxo-l ,6-dihydro- pyridine-2-carboxylic acid (1,2-HOPO) have been successfully used for a wide variety of applications like luminescence (Moore, E.G.; Xu, J.; Jocher, C. J.; Werner, E. J., Raymond K.N., manuscript submitted), MRI (Xu et al., Inorg. Chem., 43: 5492 (2004)), actinide sequestering (Gorden, et al., Chem.
  • Metal chelates and chelating agents are useful in diagnostic and therapeutic applications as well as for in vitro applications.
  • the present invention is directed to a new class of water-soluble chelating agents.
  • the invention further provides metal chelates, paramagnetic or otherwise, as well as methods of using these chelates.
  • the complexes of the invention may be used as contrast agents in medical imaging modalities, such as magnetic resonance imaging (MRI).
  • MRI magnetic resonance imaging
  • the paramagnetic chelates of the invention have unexpectedly high water exchange rates, and correspondingly high proton relaxation rates, making them highly effective MRI contrast agents.
  • the synthetic pathways to the chelates of the invention provide for the facile incorporation of subunits that modify one or more properties of the chelates.
  • chelates that include water-soluble groups, targeting groups, chelates conjugated to diverse macromolecules and the like.
  • the present invention provides a new class of highly water soluble Gd(III) complexes that also exhibit excellent stability and resistance to dissociation in vivo.
  • the Gd(III) complexes have surprisingly rapid water exchange kinetics, giving the compounds potent relaxivity.
  • the invention provides complexes that are water soluble, powerful, and non-toxic proton relaxation agents.
  • the present invention also provides a class of luminescent probes that are based on metal (e.g., lanthanide, such as terbium and europium) chelates, which are formed between the metal ion and a novel class of ligands. These complexes exhibit high chemical stability as well as high quantum yields of lanthanide ion luminescence in aqueous media without the need for secondary activating agents, such as by micelles or fluoride.
  • metal e.g., lanthanide, such as terbium and europium
  • Preferred ligands incorporate 2-hydroxy-l -oxo 1 ,2-dihydroisochinoline carboxylic acid (HOIQO) moieties in their structure and are characterized by surprisingly high chemical and kinetic stability as well as high water-solubility. These characteristics distinguish them from known ligands.
  • HOIQO 2-hydroxy-l -oxo 1 ,2-dihydroisochinoline carboxylic acid
  • the fluorophores of the invention can be used with other fluorophores or quenchers as components of energy transfer probes.
  • Many fluorescent labels are useful in combination with the complexes of the invention and many such labels are available from commercial sources, such as SIGMA (Saint Louis) or Invitrogen, that are known to those of skill in the art.
  • SIGMA Saint Louis
  • Invitrogen commercial sources
  • those of skill in the art will recognize how to select an appropriate fluorophore for a particular application and, if it is not readily available, will be able to synthesize the necessary fluorophore de novo or synthetically modify commercially available fluorescent compounds to arrive at the desired fluorescent label.
  • fluorescent proteins include, for example, green fluorescent proteins of cnidarians (Ward et al., Photochem. Photobiol. 1982, 35:803-808; Levine et al.. Camp. Biochem. Physiol. 1982, 72B:77 85), yellow fluorescent protein from Vibrio fischeri strain (Baldwin et al., Biochemistry 1990, 29:5509 15), Peridinin-chlorophyll from the dinoflagellate Symbiodinium sp.
  • green fluorescent proteins of cnidarians Ward et al., Photochem. Photobiol. 1982, 35:803-808; Levine et al.. Camp. Biochem. Physiol. 1982, 72B:77 85
  • yellow fluorescent protein from Vibrio fischeri strain Baldwin et al., Biochemistry 1990, 29:5509 15
  • Peridinin-chlorophyll from the dinoflagellate Symbiodinium sp.
  • phycobiliproteins from marine cyanobacteria such as Synechococcus, e.g., phycoerythrin and phycocyanin (Wilbanks et al., J. Biol. Chem. 1993, 268: 1226 35), and the like.
  • the compounds of the invention can be used as probes, as tools for separating particular ions from other solutes, as probes in microscopy, enzymology, clinical chemistry, molecular biology and medicine.
  • the compounds of the invention are also useful as therapeutic agents and as diagnostic agents in imaging methods.
  • the compounds of the invention are useful as components of optical amplifiers of light, waveguides and the like.
  • the compounds of the invention can be incorporated into inks and dyes, such as those used in the printing of currency and other instruments.
  • the compounds of the invention show luminescence after exciting them in any manner known in the art, including, for example, with light or electrochemical energy (see, for example, Kulmala et al, Analytica Chimica Acta 1999, 386: 1).
  • the luminescence can, in the case of chiral compounds of the invention, be circularly polarized (see, for example, Riehl et al, Chem. Rev. 1986, 86: 1).
  • the present invention provides a compound having a structure according to Formula (I):
  • Z is a member selected from substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, N, NR 9a , O, S and N-L 3 -N.
  • R 9a is a member selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl; and
  • each Q is a member independently selected from H, a protecting group, a negative charge and a positively charged counterion.
  • p is an integer selected from 1 to 7, with the proviso that when Z is NR a , O or S, then p is preferably 1 ; and with the further provido that when Z is N, then p is preferably 2. In one exemplary embodiment, in which Z is N-L -N, p is preferably 3.
  • Y 1 and each Y 2 are independently selected from -C(O)-, -C(O)NR 33 -, - C(O)O-, -C(O)S-, and -C(O)CR l la R 12a , wherein R 3a , R l l a and R l2a are members independently selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, and substituted or unsubstituted heterocycloalkyl.
  • Each L 1 , L 2 and L 3 are linker groups, which are members independently selected from a bond, substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl and substituted or unsubstituted heterocycloalkyl.
  • nl and n2 are integers independently selected from 0 to 4.
  • each R l a and R 2a is a member independently selected from an aryl group substituent.
  • each R la and R 2a is a member independently selected from H, halogen, cyano, substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, OR > 5 5 a d , o SrR> 6a , x N ⁇ Rr> 7 / d d rR> H ⁇ ) , SO 3 Q 1 , S(O) 2 R 33 , S(O) 2 Cl, S(O) 2 NR /d R 8d , NO 2 , NR 73 C(O)R 3 ", NR 7d S(O) 2 R Sd , C
  • Each R 5a , R 6a , R 7a and R 8a is a member independently selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl and substituted or unsubstituted heteroaryl.
  • the compound of the invention is complexed to a metal ion.
  • the compound of the invention has a structure according to Formula (II):
  • p is a member selected from 1 to 3.
  • nl and n2 are each 1.
  • the compound of the invention has a structure according to Formula (III):
  • each R , 2d is a member independently selected from H, a solubilizing group and an amino group.
  • the solubilizing group is a substituent, which is ionizable and increases the water solubility of the compound and its complex.
  • the solubilizing group is a member selected from a sulfonic acid group and a moiety comprising a tertiary amine.
  • the compound of the invention has a structure, which is a member selected from Formula (IV), Formula (V) and Formula (VI):
  • L 1 , L 2 , L 3 , L 4 and L 3 are linker groups, which are members independently selected from substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl and substituted or unsubstituted heterocycloalkyl.
  • L 1 , L 2 , L 3 , L 4 and L 5 are members independently selected from substituted or unsubstituted Ci to Cio alkyl.
  • L 1 , L 2 , L 4 and L 5 are members independently selected from substituted or unsubstituted ethylene.
  • the compound of the invention has a structure according to Formula (VII), Formula (VIII) and Formula (IX):
  • each R , 2a is independently selected.
  • the compound of the invention has a structure according to Formula (X) or Formula (XI):
  • each R , 2a a is independently selected.
  • each R , 2a a is a member independently selected from H and a sulfonic acid group.
  • L 3 has the structure:
  • R 30 and R 31 are members independently selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl and substituted or unsubstituted heterocycloalkyl, wherein R 30 and R 31 are optionally joined to form a 3- to 7-membered ring.
  • L 3 is a member selected from ethylene and
  • M is a metal ion.
  • Y 1 is a member selected from -C(O), -C(O)NR 33 -, -C(O)O-, -C(O)S-, and -C(O)CR l la R 12a , wherein R 3a , R l la and R l2a are members independently selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl and substituted or unsubstituted heterocycloalkyl; nl is an integer selected from O to 4; and R 40 is a linker to a backbone moiety.
  • R la and R 2a are members independently selected from an aryl group substituent.
  • R la and R 2a are members independently selected from H, halogen, cyano, substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, OR 5 ⁇ SR 6a , NR 7a R 8a , SO 3 Q 1 , S(O) 2 R 58 , S(O) 2 Cl, S(O) 2 NR 7a R 8a , NO 2 , NR 7a C(O)R 5a , NR 7a S(O) 2 R 5a , C(O)R 5a , C(O)OR 5a , C(O)SR 3a and a linker to a
  • the backbone moiety is a member selected from a polyamine (e.g., TREN), a tertiary amine, a polyether, an ether, a thioether and combinations thereof.
  • composition comprising a compound of the invention and a pharmaceutically acceptable carrier.
  • complex of the invention has a structure according to Formula (XII):
  • the complex of the invention has a structure according to Formula (XIII):
  • Z is a member selected from substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, N, NR 9a , O S, and N-L 3 -N, wherein R 9a is a member selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl.
  • p is an integer selected from 1 to 7, with the proviso that when Z is NR d , O or S, then p is preferably 1 , and with the further proviso that when Z is N, then p is preferably 1. In another embodiment, wherein Z is N-L -N, p is preferably 3.
  • Y 1 and each Y 2 are members independently selected from -C(O)-, -C(O)NR 3 ", -C(O)O-, -C(O)S-, and -C(O)CR 1 la R 12a , wherein R 3a , R l la and R 12 " are members independently selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl and substituted or unsubstituted heterocycloalkyl.
  • Each L 1 , L 2 and L 3 are linker groups, which are members independently selected from a bond, substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl and substituted or unsubstituted heterocycloalkyl.
  • nl and n2 are integers independently selected from 0 to 4.
  • Each R la and each R 2d is a member independently selected from H, halogen, cyano, substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, OR 5 ", SR 6a , NR 7d R 8a , SO 3 Q 1 , S(O) 2 R 53 , S(O) 2 Cl, S(O) 2 NR 7d R 8a , NR 7a S(O) 2 R 5a , C(O)R 5a , C(O)OR 5a , C(O)SR 5 " and a linker to a functional moiety, wherein Q 1 is a member selected from H, a negative charge and a positively charged counterion; each R 3d , R 6d , cyan
  • the comlex of the invention is a luminescent complex.
  • the complex of the invention has a structure according to Formula (XIV):
  • nl and n2 are each 1.
  • the complex of the invention has a structure according to Formula (XV):
  • L 1 and L 2 are members independently selected from substituted or unsubstituted Ci-Ci 0 alkyl.
  • the complex of the invention has a structure according to the formula:
  • L has the structure:
  • R 30 and R 31 are members independently selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl and substituted or unsubstituted heterocycloalkyl, wherein R 30 and R 31 are optionally joined to for a 3- to 7-membered ring.
  • L 3 is a member selected from ethylene and
  • Z is N and p is 2.
  • Z is a member selected from O, S and C(O) and p is 1.
  • each R a is a member independently selected from H, a solubilizing group and an amino group.
  • the solubilizing group is a member selected from a sulfonic acid group and a moiety comprising a tertiary amine.
  • composition comprising a complex of the invention and a pharmaceutically acceptable carrier.
  • the method includes administering to the patient an amount of a compound of the invention sufficient to provide contrast enhancement, and acquiring a contrast enhanced MR image.
  • composition comprising a luminescent complex of the invention and an analyte.
  • the invention also provides a method of detecting the presence of an analyte in a sample.
  • the method includes: (i) contacting the sample and a composition comprising a luminescent complex of the invention; (ii) exciting the complex; and (iii) detecting luminescense from the complex.
  • the invention further provides a method of removing a metal ion from a sample.
  • the method includes: (i) contacting the sample and a compound of the invention thereby forming a complex between the metal ion and the compound; and (ii) removing the complex from the sample.
  • the invention also provides selective structural modifications of the ligand scaffold result in in vivo residence times that are enhanced relevant to analogous compounds that are not modified. Other modifications are provided that generate greater specificity in the in vivo distribution of the modified Gd 3+ complexes.
  • FIG. 1 is a collection of poly(amino-carboxylate) ligands used for chelating Gd(III).
  • FIG. 2 is a diagram of a Gd(III) complex showing inner and outer-sphere water molecules, and the parameters that affect inner-sphere relaxivity.
  • FIG. 3 is a graphical presentation comparing relaxivity data for compounds of the invention and Gd(III)-polyaminocarboxylates.
  • FIG. 4 is a graphical presentation comparing water exchange rates for compounds of the invention and Gd(III)-polyaminocarboxylates.
  • FIG. 5 is a species distribution diagram for the Gd 3 VTREN-I-Me-HOPO system, calculated for 1 ⁇ M Gd and 10 ⁇ M ligand.
  • the ligand is represented as "L" in the diagram.
  • FIG. 6 is a graphical display of the selectivity of TREN- 1 -Me-HOPO for Gd 3+ over Ca2 + and Zn 2+ as compared with DTPA and DTPA-BMA.
  • FIG. 7 displays the structure of a representative Gd(III)-HOPO chelate and provides a graphical comparison the the NMRD profile of this compound with those of representative Gd(III)-polyaminocarboxylates.
  • FIG. 8 is an exemplary synthetic route to compounds of the invention having derivatized scaffolds with alkylene moieties of different lengths.
  • FIG. 9 is a graphical representation of the variation in ⁇ m with PEG length.
  • FIG. 10 is a graphical display of the relaxivities of various complexes of the invention.
  • FIG. 11 provides exemplary structures that are known to interact with human serum albumin.
  • FIG. 12 displays structures of exemplary protein binding chelates of the invention.
  • FIG. 13 is a graphical representation of the variation in the affinity of complexes of the invention for human serum albumin as the hydrophobicity/hydrophilicity of the complex is varied indicating that the invention provides a method to systematically vary the degree and strength of binding of a complex to a serum protein.
  • FIG. 14 is a graphical representation of the variation in the relatxation of complexes of the invention bound to human serum albumin as the hydrophobicity/hydrophilicity of the complex is varied indicating that the invention provides a method to systematically vary the relaxivity of the complex based on the degree and strength of binding of a complex to a serum protein.
  • FIG. 15 displays structures of motifs of protein binding complexes of the invention.
  • FIG. 17 is a graphical display of the 17 O NMR transverse relaxation rates as a function of temperature (273-355 K), measured at 2.1 T (90 MHz proton Larmor frequency) for 21 mM aqueous solutions of the complexes:
  • FIG. 19 displays the 1 H longitudinal relaxation rate of H 2 O as a function of HSA concentration.
  • Experiment involved titration of a 0.25 mmol/L solution of the 47 with HSA at 20 MHz, 25 0 C and pH-6.5.
  • the relatively weak interaction results in large standard errors.
  • FIG. 20 displays the temperature dependence of the paramagnetic contribution (R 2 ) to the transverse ' 7 O water relaxation rate of 55 (left) and 58 (right).
  • FIG. 21 displays the water proton longitudinal relaxation rate of a solution of complex as a function of HSA concentration: 55 (top left), 58 (top right), 48 (bottom)
  • FIG. 22 displays the 24 h post injection biodistribution of I S3 Gd[TREN-bis(HOPO)- (TAM-DME)].
  • FIG. 23 is a steady-state luminescence spectrum of a europium complex of compound 40.
  • the present invention provides chelating agents that are useful for many purposes including, but not limited to, contrast agents for imaging modalities (e.g., MRI, CT), metal ion decorporation (e.g., iron, actinides), and the like.
  • the chelating agents are also of use for binding radioisotopes utilized in nuclear medicine, gamma camera scintigraphy, and other medical applications.
  • the chelating agents form surprisingly stable, highly water-soluble complexes with metal ions.
  • HOPO refers to hydroxypyridonate
  • HOPY refers to hydroxypyrimidinone
  • TAM refers to dihydroxyterephthalamide
  • Analyte means any compound or molecule of interest for which a diagnostic test is performed, such as a biopolymer or a small molecular bioactive material.
  • An analyte can be, for example, a protein, peptide, carbohydrate, polysaccharide, glycoprotein, hormone, receptor, antigen, antibody, virus, substrate, metabolite, transition state analog, cofactor, inhibitor, drug, dye, nutrient, growth factor, lipid etc., without limitation.
  • energy transfer refers to the process by which the light emission of a luminescent group is altered by a luminescence-modifying group.
  • the luminescence-modifying group is a quenching group then the light emission from the luminescent group is attenuated (quenched).
  • Energy transfer mechanisms include luminescence resonance energy transfer by dipole-dipole interaction (e.g., in longer range energy transfer) or electron transfer (e.g., across shorter distances).
  • Energy transfer pair is used to refer to a group of molecules that participate in energy transfer. Such complexes may comprise, for example, two luminescent groups, which may be different from one-another and one quenching group, two quenching groups and one luminescent group, or multiple luminescent groups and multiple quenching groups. In cases where there are multiple luminescent groups and/or multiple quenching groups, the individual groups may be different from one another. Typically, one of the molecules acts as a luminescent group, and another acts as a luminescence-modifying group.
  • the preferred energy transfer pair of the invention comprises a luminescent group and a quenching group of the invention. There is no limitation on the identity of the individual members of the energy transfer pair in this application. All that is required is that the spectroscopic properties of the energy transfer pair as a whole change in some measurable way if the distance between the individual members is altered by some critical amount.
  • luminescence-modifying group refers to a molecule of the invention that can alter in any way the luminescence emission from a luminescent group.
  • a luminescence-modifying group generally accomplishes this through an energy transfer mechanism.
  • the luminescence emission can undergo a number of alterations, including, but not limited to, attenuation, complete quenching, enhancement, a shift in wavelength, a shift in polarity, and a change in luminescence lifetime.
  • a luminescence-modifying group is a fluorescence-modifying group.
  • Another exemplary luminescence-modifying group is a quenching group.
  • quenching group refers to any luminescence-modifying group of the invention that can attenuate at least partly the light emitted by a luminescent group. This attenuation is referred to herein as "quenching". Hence, excitation of the luminescent group in the presence of the quenching group leads to an emission signal that is less intense than expected, or even completely absent. Quenching typically occurs through energy transfer between the luminescent group and the quenching group.
  • FRET Fluorescence resonance energy transfer
  • FRET fluorescence resonance energy transfer
  • FRET fluorescence resonance energy transfer
  • FRET depends on energy transfer between the luminescent group and the luminescence-modifying group.
  • FRET also depends on the distance between the luminescence modifying group and the luminescent group.
  • Moiety refers to the radical of a molecule that is attached to another moiety.
  • targeting moiety is intended to mean any moiety attached to the complexes of the invention.
  • the targeting moiety can be a small molecule, which is intended to include both non-peptides and peptides.
  • the targeting group can also be a macromolecule, which includes saccharides, lectins, receptors, ligands for receptors, proteins such as BSA, antibodies, nucleic acids, solid supports and so forth.
  • the targeting group can also be a lipid as well as a polymer, such as a plastic surface, a poly-ethyleneglycol derivative and the like.
  • Reactive functional group refers to groups including, but not limited to, olefins, acetylenes, alcohols, phenols, ethers, oxides, halides, aldehydes, ketones, carboxylic acids, esters, amides, cyanates, isocyanates, thiocyanates, isothiocyanates, amines, hydrazines, hydrazones, hydrazides, diazo, diazonium, nitro, nitriles, mercaptans, sulfides, disulfides, sulfoxides, sulfones.
  • I i include those used to prepare bioconjugates, e.g., N-hydroxysuccinimide esters, maleimides and the like. Methods to prepare each of these functional groups are well known in the art and their application to or modification for a particular purpose is within the ability of one of skill in the art (see, for example, Sandler and Karo, eds. ORGANIC FUNCTIONAL GROUP PREPARATIONS, Academic Press, San Diego, 1989).
  • Non-covalent protein binding groups are moieties that interact with an intact or denatured polypeptide in an associative manner. The interaction may be either reversible or irreversible in a biological milieu.
  • the incorporation of a "non-covalent protein binding group" into a chelating agent or complex of the invention provides the agent or complex with the ability to interact with a polypeptide in a non-covalent manner.
  • Exemplary non-covalent interactions include hydrophobic-hydrophobic and electrostatic interactions.
  • non-covalent protein binding groups include anionic groups, e.g., phosphate, thiophosphate, phosphonate, carboxylate, boronate, sulfate, sulfone, thiosulfate, and thiosulfonate.
  • linking member refers to a covalent chemical bond that includes at least one heteroatom.
  • exemplary linking members include -C(O)NH-, -C(O)O-, -NH-, -S-, -O-, and the like.
  • targeting group is intended to mean a moiety that is: (1) able to actively direct the entity to which it is attached (e.g., contrast agent) to a target region, e.g., a tumor; or (2) is preferentially passively absorbed by or entrained within a target tissue, for example a tumor.
  • the targeting group can be a small molecule, which is intended to include both non- peptides and peptides.
  • the targeting group can also be a macromolecule, which includes, but is not limited to, saccharides, lectins, receptors, ligand for receptors, proteins such as BSA, antibodies, poly(ethers), dendrimers, poly(amino acids) and so forth.
  • cleavable group is intended to mean a moiety that allows for release of the chelate from the rest of the conjugate by cleaving a bond linking the chelate (or chelate linker arm construct) to the remainder of the conjugate. Such cleavage is either chemical in nature, or enzymatically mediated. Exemplary enzymatically cleavable groups include natural amino acids or peptide sequences that end with a natural amino acid. [0103] In addition to enzymatically cleavable sites, it is within the scope of the present invention to include one or more sites that are cleaved by the action of an agent other than an enzyme.
  • non-enzymatic cleavage agents include, but are not limited to, acids, bases, light (e.g., nitrobenzyl derivatives, phenacyl groups, benzoin esters), and heat.
  • Many cleaveable groups are known in the art. See, for example, Jung et al, Biochem. Biophys. Acta, 761 : 152-162 (1983); Joshi et al , J. Bi ⁇ l. Chem., 265: 14518-14525 (1990); Zarling et al, J. Immunol, 124: 913-920 (1980); Bouizar et al, Eur. J. Biochem., 155: 141-147 (1986); Park et al, J. Biol.
  • Certain compounds of the present invention can exist in unsolvated forms as well as solvated forms, including hydrated forms. In general, the solvated forms are equivalent to unsolvated forms and are encompassed 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.
  • Certain compounds of the present invention possess asymmetric carbon atoms (optical centers) or double bonds; the racemates, diastereomers, geometric isomers and individual isomers are encompassed within the scope of the present invention.
  • the compounds of the invention may be prepared as a single isomer (e.g., enantiomer, cis-trans, positional, diastereomer) or as a mixture of isomers.
  • the compounds are prepared as substantially a single isomer.
  • Methods of preparing substantially isomerically pure compounds are known in the art. For example, enantiomerically enriched mixtures and pure enantiomeric compounds can be prepared by using synthetic intermediates that are enantiomerically pure in combination with reactions that either leave the stereochemistry at a chiral center unchanged or result in its complete inversion. Alternatively, the final product or intermediates along the synthetic route can be resolved into a single stereoisomer.
  • the compounds of the present invention may also contain unnatural proportions of atomic isotopes at one or more of the atoms that constitute such compounds.
  • the compounds may be radiolabeled with radioactive isotopes, such as for example tritium ( 3 H), iodine-125 ( 123 I) or carbon-14 ( 14 C). All isotopic variations of the compounds of the present invention, whether radioactive or not, are intended to be encompassed within the scope of the present invention.
  • substituent groups are specified by their conventional chemical formulae, written from left to right, they equally encompass the chemically identical substituents, which would result from writing the structure from right to left, e.g., -CH 2 O- is intended to also recite -OCH 2 -.
  • alkyl by itself or as part of another substituent, means, unless otherwise stated, a straight or branched chain, or cyclic hydrocarbon radical, or combination thereof, which may be fully saturated, mono- or polyunsaturated and can include di- and multivalent radicals, having the number of carbon atoms designated (i.e. Ci-Ci 0 means one to ten carbons).
  • saturated hydrocarbon radicals include, but are not limited to, groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, cyclohexyl, (cyclohexyl)methyl, cyclopropylmethyl, homologs and isomers of, for example, n-pentyl, n- hexyl, n-heptyl, n-octyl, and the like.
  • An unsaturated alkyl group is one having one or more double bonds or triple bonds.
  • alkyl groups examples include, but are not limited to, vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl, 3-(l,4- pentadienyl), ethynyl, 1- and 3-propynyl, 3-butynyl, and the higher homologs and isomers.
  • alkyl unless otherwise noted, is also meant to include those derivatives of alkyl defined in more detail below, such as '"heteroalkyl.”
  • Alkyl groups that are limited to hydrocarbon groups are termed "homoalkyl".
  • alkylene' by itself or as part of another substituent means a divalent radical derived from an alkane, as exemplified, but not limited, by -CH 2 CHiCH 2 CH 2 -, and further includes those groups described below as “heteroalkylene.”
  • an alkyl (or alkylene) group will have from 1 to 24 carbon atoms, with those groups having 10 or fewer carbon atoms being preferred in the present invention.
  • a “lower alkyl” or “lower alkylene” is a shorter chain alkyl or alkylene group, generally having eight or fewer carbon atoms.
  • alkoxy alkylamino and “alkylthio” (or thioalkoxy) are used in their conventional sense, and refer to those alkyl groups attached to the remainder of the molecule via an oxygen atom, an amino group, or a sulfur atom, respectively.
  • heteroalkyl by itself or in combination with another term, means, unless otherwise stated, a stable straight or branched chain, or cyclic hydrocarbon radical, or combinations thereof, consisting of the stated number of carbon atoms and at least one heteroatom selected from the group consisting of O, N, Si and S, and wherein the nitrogen and sulfur atoms may optionally be oxidized and the nitrogen heteroatom may optionally be quaternized.
  • the heteroatom(s) O, N and S and Si may be placed at any interior position of the heteroalkyl group or at the position at which the alkyl group is attached to the remainder of the molecule.
  • heteroalkylene by itself or as part of another substituent means a divalent radical derived from heteroalkyl, as exemplified, but not limited by, -CH 2 - CH 2 -S-CH 2 -CH 2 - and -CH 2 -S-CH 2 -CH 2 -NH-CH 2 -.
  • heteroatoms can also occupy either or both of the chain termini (e.g., alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenediamino, and the like). Still further, for alkylene and heteroalkylene linking groups, no orientation of the linking group is implied by the direction in which the formula of the linking group is written. For example, the formula -C(O) 2 R " - represents both -C(O) 2 R'- and -R 5 C(O) 2 -.
  • cycloalkyl and “heterocycloalkyl”, by themselves or in combination with other terms, represent, unless otherwise stated, cyclic versions of “alkyl” and “heteroalkyl”, respectively. Additionally, for heterocycloalkyl, a heteroatom can occupy the position at which the heterocycle is attached to the remainder of the molecule. Examples of cycloalkyl include, but are not limited to, cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3- cyclohexenyl, cycloheptyl, and the like.
  • heterocycloalkyl examples include, but are not limited to, 1 -(1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4- morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl, 1 -piperazinyl, 2-piperazinyl, and the like.
  • halo or halogen
  • haloalkyl by themselves or as part of another substituent, mean, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom.
  • terms such as “haloalkyl,” are meant to include monohaloalkyl and polyhaloalkyl.
  • halo(Ci-C 4 )alkyl is mean to include, but not be limited to, trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, and the like.
  • aryl means, unless otherwise stated, a polyunsaturated, aromatic, substituent that can be a single ring or multiple rings (preferably from 1 to 3 rings), which are fused together or linked covalently.
  • heteroaryl refers to aryl groups (or rings) that contain from one to four heteroatoms selected from N, O, and S, wherein the nitrogen and sulfur atoms are optionally oxidized, and the nitrogen atom(s) are optionally quaternized.
  • a heteroaryl group can be attached to the remainder of the molecule through a heteroatom.
  • Non-limiting examples of aryl and heteroaryl groups include phenyl, 1-naphthyl, 2-naphthyl, 4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl, 2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4- isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl, 2-thienyl, 3- thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl, 1-isoquino
  • aryl when used in combination with other terms (e.g., aryloxy, arylthioxy, arylalkyl) includes both aryl and heteroaryl rings as defined above.
  • arylalkyl is meant to include those radicals in which an aryl group is attached to an alkyl group (e.g., benzyl, phenethyl, pyridylmethyl and the like) including those alkyl groups in which a carbon atom (e.g., a methylene group) has been replaced by, for example, an oxygen atom (e.g., phenoxymethyl, 2-pyridyloxymethyl, 3-(l- naphthyloxy)propyl, and the like).
  • alkyl group e.g., benzyl, phenethyl, pyridylmethyl and the like
  • an oxygen atom e.g., phenoxymethyl, 2-pyridyloxymethyl, 3-(l- naphthyloxy
  • R', R", R'" and R" each preferably independently refer to hydrogen, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, e.g., aryl substituted with 1-3 halogens, substituted or unsubstituted alkyl, alkoxy or thioalkoxy groups, or arylalkyl groups.
  • each of the R groups is independently selected as are each R', R", R'" and R"" groups when more than one of these groups is present.
  • R' and R" are attached to the same nitrogen atom, they can be combined with the nitrogen atom to form a 5-, 6-, or 7-membered ring.
  • -NR'R is meant to include, but not be limited to, 1 -pyrrolidinyl and 4-morpholinyl.
  • alkyl is meant to include groups including carbon atoms bound to groups other than hydrogen groups, such as haloalkyl (e.g., -CF 3 and -CH 2 CF 3 ) and acyl (e.g., -C(O)CH 3 , -C(O)CF 3 , -C(O)CH 2 OCH 3 , and the like).
  • substituents for the aryl and heteroaryl groups are generically referred to as "aryl group substituents.”
  • Two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be replaced with a substituent of the formula -T-C(O)-(CRR' ) q -U-, wherein T and U are independently -NR-, -0-, -CRR'- or a single bond, and q is an integer of from O to 3.
  • two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be replaced with a substituent of the formula -A-(CH 2 ) r -B-, wherein A and B are independently -CRR"-, -O-, -NR-, -S-, -S(O)-, -S(O) 2 -, -S(O) 2 NR"- or a single bond, and r is an integer of from 1 to 4.
  • One of the single bonds of the new ring so formed may optionally be replaced with a double bond.
  • two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be replaced with a substituent of the formula - (CRR') s -X-(CR"R'")d-, where s and d are independently integers of from O to 3, and X is -O- , -NR'-, -S-, -S(O)-, -S(O) 2 -, or -S(O) 2 NR'-.
  • the substituents R, R', R" and R'" are preferably independently selected from hydrogen or substituted or unsubstituted (C]-C6)alkyl.
  • heteroatom is meant to include oxygen (O), nitrogen (N), sulfur (S) and silicon (Si).
  • a "MRI signal generating moiety” is a species that affects Tl or T2 of water molecules in a sample or subject undergoing a MRI experiment.
  • the broken line as a component of a chemical structure implies that the position of the bond may be varied, such as within a ring structure, or that the bond may be either present or absent, such that the principles of chemical valency are obeyed.
  • the image intensity in MRI is the result of a complex interplay of numerous factors. These include the longitudinal (Ti) and transverse (T 2 ) proton relaxation times, proton density of the imaged tissues and instrumental parameters (such as magnetic field strength) (Aime, S. B. et al., Coord. Chem. Rev. 1999, 185-6, 321). Contrast agents (complexes of paramagnetic ions) decrease the relaxation times of nearby water protons by dipolar interactions, resulting in enhanced signal intensity of the tissue containing the agent (Lauffer, R. B., Chem. Rev. 1987, 87, 901).
  • Gd(III) The high magnetic moment and relatively long electronic relaxation time of Gd(III) make it ideally suited as the active component in MRI contrast agents (Banci, L. B. et al., C. Nuclear and Electronic Relaxation; VCH: Weinheim, 1991 ). Images that are acquired with the aid of a Gd(III) contrast agent are preferably visualized as "T ⁇ -weighted images" since the percentage change in ⁇ IT ⁇ in tissue is much greater than that in l/7 2 (Caravan, P. E. et al., Chem. Rev. 1999, 99, 2293).
  • the present invention relates to novel MRI contrast agents and compositions of novel MRI contrasting agents with faster water exchange rates (i.e. with ⁇ m and ⁇ r preferably in the low ns regime) and desirable in vivo persistence.
  • novel agents facilitate diagnosis of physiological abnormalities in specific regions of the body over longer time periods than are currently possible.
  • gadolinium as a representative paramagnetic ion that is coordinated by the chelating agents of the invention
  • the chelating agents of the invention are appropriate for complexing many metal ions.
  • ions of, e.g., transition metals, and lanthanides other than gadolinium include the ions of Dy, Fe, Mn, Pu, and U.
  • a metal chelate to be useful as a contrast enhancing agent in MRI, the agent must satisfy several requirements. Broadly stated the three most important requirements are stability, relaxivity and water solubility, each of which must high values.
  • the inventors have recognized that ligand systems that have only oxygen donor atoms provide complexes of Gd(III) that exhibit excellent stability, have high relaxivity and are highly water soluble. Each of the essential characteristics of a useful complex between Gd(III) and a ligand with only oxygen donor atoms is discussed below.
  • MRI contrast enhancing agent be as highly water soluble as possible.
  • the water solubility of MRI contrast agents is of particular importance because the agents are administered in multigram dosages to the subject of the imaging experiment.
  • a highly water-soluble agent requires a lower injection volume for administration. Lower injection volume correlates with ease of agent administration and decreased patient discomfort.
  • the complex preferably has a water solubility of at least about 15 mM and. more preferably, at least about 20 mM.
  • the present invention provides complexes between Gd(III) and ligands with only oxygen donors that meet and/or exceed this requirement.
  • the invention provides Gd(III) complexes as described above, having water solubilities of from about 50 mM to about 300 mM and, preferably from about 100 mM to about 1 M.
  • the compounds known in the art are relatively water insoluble.
  • TREN-MOE-3,2-HOPO has a water solubility at pH 7.4 of about 1 mM.
  • a compound of the present invention such as the Gd(III) complex of TREN-HOPY has a relaxivity of at least about 100 mM.
  • the present invention provides poly(ether) conjugates of homopodal and heteropodal ligands having water solubilities that are dramatically improved over compounds reported to date. Exemplary solubilities of compounds of the invention and known compounds are compared below in Table 1.
  • HOPO l-Me-3,2-HOPO or 6-Me-3,2-HOPO * minimum solubility in H 2 O (0.01 M HEPES).
  • the potency of a MRI contrast agent is generally given in terms of the magnitude of its relaxivity.
  • the relaxivity, r, , of a MRI contrast agent, as used herein, refers to the amount of increase in ⁇ IT ⁇ signal intensity that occurs per millimolar of Gd(III).
  • the relaxivity can be sub-divided into inner sphere and outer sphere contributions (Fig. 2) (Lauffer, R. B., Chem. Rev. 1987, 57, 901).
  • a mechanism underlying the enhanced relaxivity of the complexes of the invention is the near ideal time-scale of the water exchange kinetics of the complexes.
  • the inventors have recognized that for a complex formed between a Gd(III) ion and a ligand with only oxygen donor atoms to be a candidate MR imaging agent candidate, the metal ion of the complex preferably has a water exchange rate of at least about 10 x 10 sec " (FIG. 3).
  • the relaxivity of the complexes of the invention is typically at least two-fold greater than that of agents based upon the polyaminocarboxylate motif (FIG. 3).
  • Another factor that enhances the relaxivity of the complexes of the invention relative to the polyaminocarboxylates is the nearly ideal time-scale of the water exchange kinetics of the present complexes (FIG. 4).
  • TREN-I-Me-HOPO Another advantageous feature of TREN-I-Me-HOPO for clinical applications is its neutrality, which reduces osmolality effects in vivo, therefore lessening the discomfort of patients upon its intravenous administration (Lauffer, R.B., Chem. Rev. 1987, 87, 901).
  • the relaxivity in water of the complexes of the invention is preferably greater than about 5 mM ⁇ 's ⁇ ⁇ more preferably greater than about 6 mM ' V.
  • a presently preferred range of relaxivities of compounds of the invention is from about 6 mM ⁇ s " to about 15 mM " s " .
  • a complex between Gd(III) and an oxygen donor ligand will preferably form an aqueous solution that has a pGd of at least about 18 when the aqueous solution is pH about 7.4 and includes about 10 ⁇ M of ligand and about 1 ⁇ M of Gd. See, Equation 4, infra.
  • TREN-I-Me-HOPO exhibits enhanced selectivity (FIG. 6) for Gd 3 t over the physiological metal ions Ca 2+ and Zn 2+ (Xu, J. et al., J. Am. Chem. Soc.
  • the present invention provides a complex between a gadolinium (III) ion and an organic ligand comprising only oxygen donor atoms coordinating the gadolinium (III) ion.
  • the complexed gadolinium ion has a water exchange rate of at least about 10 x 10 6 sec " '.
  • the solubility in water of the complex is at least about 15, preferably, at least about 20 mM.
  • the present invention provides a complex between a gadolinium (III) ion and an organic ligand.
  • the ligand includes a structure according to Formula I:
  • R 1 , R 2 , and R 3 are independently selected from a linking member, an aryl group substituent, H, substituted or unsubstituted alkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroalkyl, hydroxy, carboxy, amide, ester, and amino groups, with the proviso that when A is nitrogen, R 1 is other than amino, and with the further proviso that when E is nitrogen, R 3 is not present.
  • R 4 is a linking member, alkyl group substituent, H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, hydroxy, carboxy, amide, or an ester group.
  • A, E and Z are independently selected from carbon and nitrogen.
  • the gadolinium complex has a solubility in water of at least about 15 mM, more preferably, at least about 20 mM.
  • R 1 and R 4 represent members independently selected from a linking member, H, substituted or unsubstituted alkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroalkyl, hydroxy, carboxy, amide, or an ester groups.
  • R 2 and R 3 are independently selected from a linking member, an aryl substituent, H, substituted or unsubstituted alkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroalkyl, hydroxy, carboxy, amide, ester, or an amino group.
  • R 1 and R 2 are independently C1-C 4 substituted or unsubstituted alkyl, e.g., methyl.
  • R 1 and R 2 are independently selected from methyl and H; and R 3 and R 4 are H.
  • R is selected from substituted or unsubstituted aryl and substituted or unsubstituted (alkyl)aryl; and R 2 is substituted or unsubstituted C 1 -C 4 alkyl.
  • the moiety according to Formula I has the structure: in which R 1 and R 4 are independently selected from a linking member, H, substituted or unsubstituted alkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroalkyl, hydroxy, carboxy, amide, and ester groups.
  • R represents a linking member, an aryl group substituent, H, substituted or unsubstituted alkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroalkyl, hydroxy, carboxy, amide, ester, and amino groups.
  • R 1 , R 2 and R 4 represent members independently selected from H and substituted or unsubstituted C 1 -C 4 alkyl. In a further embodiment, at least one of R 1 and R 2 is methyl.
  • R 5 is substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, or substituted or unsubstituted aryl.
  • R 5 is optionally substituted with one or more organic ligands that are also complexed to the gadolinium (III) ion. The identity of the remaining radicals is substantially as discussed above for Formula I.
  • R 3 is exemplary of a moiety referred to herein as a "scaffold.”
  • the scaffold is the backbone that tethers together two or more chelating units to form the ligands of the invention.
  • the scaffold is optionally further substituted with a reactive functional group.
  • the functional groups can be used to attach the ligand to another species, e.g., a targeting moiety, polymer, etc.
  • a further exemplary complex of the invention has a structure according to Formula II:
  • a first R 1 on a first chelating ring structure may be methyl; a second R 1 , which is located on a second ring is ethyl; and a third R 1 , which is located on a third ring may be benzyl.
  • the invention provides a complex wherein the structure according to Formula II is:
  • the invention provides a complex in which the structure according to Formula II is:
  • the invention provides a complex in which the structure according to Formula II is:
  • R 1 is methyl or substituted or unsubstituted benzyl.
  • the benzyl is substituted, in an exemplary embodiment, it is substituted with an alkoxy group.
  • at least one member selected from R 1 and R 2 is substituted or unsubstituted C 1 -C 4 alkyl, e.g., as methyl.
  • at least one R is selected from methyl and polyether.
  • the symbol q represents an integer from 1 to 2.
  • the invention provides a complex wherein the structure according to Formula II is; in which the radicals are each independently selected and their identities are substantially as discussed above.
  • the symbol q represents an integer from 1 to 2.
  • N-methyl-hydroxypyridinone precursor 1 is carboxylated to form the corresponding acid 2.
  • the hydroxyl group of the acid is protected as the benzyl adduct 3, which is converted to the corresponding thiazolide 4.
  • the thiazolide is combined with the TREN backbone, forming 5, which is deprotected by catalytic hydrogenation to afford 6.
  • the gadolinium complex 7, is formed by combining the ligand with a source of gadolinium ion in the presence of a base.
  • the method set forth above is general for the preparation of ligands based upon other backbone motifs. Moreover, the method is useful for preparing ligands in which the chelating units within a ligand have a structure other than the parent HOPO chelating unit. Furthermore, the N-methyl precursor is readily replaced with its unsubstituted analogue, or by analogues that are N-substituted with a moiety other than a methyl group, such as a substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl or a substituted or unsubstituted aryl moiety.
  • the protected hydroxypyridinone carboxylic acid ester 8 is a versatile intermediate for the preparation of variously substituted chelating units. Thus, in one route, 8 is benzylated at each available site, affording compound 10. In an alternate route, the endocyclic amine is left unbenzylated, providing compound 9.
  • the ester of compound 9 is hydrolyzed to the corresponding carboxylic acid 11, which is converted to thiazolide 12.
  • the thiazolide is conjugated with the amine backbone to yield 13, which is subsequently debenzylated to compound 14.
  • the Gd(III) complex 15 is formed by standard methods.
  • compound 10 is coupled with the amine backbone, debenzylated to yield the hydroxyl moiety and gadolinium chelate 18 is formed.
  • the route also provides for forming heteropodal ligands, such as 19, and ligands in which the endocyclic nitrogen is functionalized with groups other than benzyl, e.g,. 20.
  • [0162] is a representative embodiment of a ligand that is N-substituted with a moiety other than a methyl group
  • the ligand of the invention includes a HOPO chelating moiety, which is functionalized at the endocyclic nitrogen of the pyridinone moiety with a linker or other group, e.g., (Johnson, A. R. et al., Inorg. Chem. 2000, 39, 2652- 2660).
  • the MOE derivative is more than 10-times more water soluble than the parent complex.
  • TREN backbone 21 is contacted with protected thiazolide 22 to produce disubstituted TREN 24 with a single free primary amine group.
  • Compound 24 is contacted with the protected di-thiazolide TAM derivative 23, yielding activated heteropodal ligand 25 which can be subsequently reacted with an amine to form the functionalized heteropodal ligand 27.
  • the protected di-thiazolide 23 is functionalized with an amine, producing amide 26, prior to its reaction with the disubstituted TREN derivative 24, producing heteropodal ligand 27.
  • TREN-HOPO-TAM compounds of the invention are readily synthesized using either of the routes of Scheme 3.
  • the use of benzyl (Bn) protecting groups on TAM is generally preferable to the methyl groups previously reported (Cohen, S. M. et al., Inorg. Chem. 2000, 39, 4339), since the deprotection conditions are less severe, making the method amenable to use with a greater range of primary amines (RNH 2 ). Optimization of the reaction conditions for a particular amine (RNH 2 ) is well within the abilities of one of skill in the art.
  • R and R' are typically H, a substituted or unsubstituted alkyl, or substituted or unsubstituted heteroalkyl group.
  • R and /or R' are groups that include an ether or a reactive functional group.
  • the invention provides a compound according to Formula I, wherein at least one of R , R , R and R is a linker arm, including a reactive functional group that allows the compound to be tethered to another species.
  • at least one of R 1 , R 2 , R 3 and R 4 is a moiety that alters a property, e.g., water solubility, molecular weight, rotational correlation time, complex stability, etc., of the parent compound to which it is affixed.
  • R 1 , R 2 , R 3 and R 4 has the structure:
  • Z 1 is a member selected from H, CH 2 , OR 10 , SR 10 , NHR 10 , OCOR 1 1 , OC(O)NHR 1 1 , NHC(O)OR 10 , OS(O) 2 OR 10 , and C(O)R 1 '.
  • R 10 represents H, substituted or unsubstituted alkyl, or substituted or unsubstituted heteroalkyl.
  • R is a member selected from H, OR 12 , NR 12 NH 2 , SH, C(O)R 12 , NR 12 H . substituted or unsubstituted alkyl and substituted or unsubstituted heteroalkyl.
  • R is a member selected from H, substituted or unsubstituted alkyl and substituted or unsubstituted alkyl.
  • the symbol X represents a member selected from CH 2 , O, S and NR , wherein R is a member selected from H, substituted or unsubstituted alkyl and substituted or unsubstituted heteroalkyl; and the symbols j and k represent members independently selected from the group consisting of integers from 1 to 20.
  • Other linking moieties useful with the compounds of the invention will be apparent to those of skill in the art.
  • At least one of R 1 , R 2 , R 3 and R 4 is an ether or a polyether, preferably a member selected from ethylene glycol, and ethylene glycol oligomers, having a molecular weight of from about 60 daltons to about 10,000 daltons, and more preferably of from about 100 daltons to about 1,000 daltons.
  • polyether-based substituents include, but are not limited to, the following structures:
  • b is preferably a number from 1 to 100, inclusive.
  • Other functionalized polyethers are known to those of skill in the art, and many are commercially available from, for example, Shearwater Polymers, Inc. (Alabama).
  • the linker includes a reactive functional group for conjugating the compound to another molecule or to a surface.
  • a reactive functional group for conjugating the compound to another molecule or to a surface.
  • Representative useful reactive groups are discussed in greater detail in succeeding sections. Additional information on useful reactive groups is known to those of skill in the art. See, for example, Hermanson, BIOCONJUGATE TECHNIQUES, Academic Press, San Diego, 1996.
  • the moiety attached to at least one of R 1 , R 2 , R 3 and R 4 combines characteristics of one or more of the above-recited groups.
  • one preferred linker group combines both the attributes of a polyether and a reactive group:
  • linker groups include, but are not limited to, moieties such as sugars (e.g., polyol with reactive hydroxyl), amino acids, amino alcohols, carboxy alcohols, amino thiols, and the like.
  • the linkers of use in the compounds of the invention can also include a cleaveable group.
  • the cleaveable group is interposed between the signal- generating moiety, i.e., the complex, and a targeting agent or macromolecular backbone.
  • the invention also provides complexes based upon a new heterocyclic ligand system, hydrogen bond stabilized, bidentate oxo-hydroxy donor ligands.
  • TREN-HOPY The pK- d of TREN-HOPY is 6.37, close to that of TREN-I-Me- HOPO (6.12), indicating that the HOPY subunit is slightly more basic.
  • the pGd of [Gd(TREN-HOPY)(H 2 O) 2 ] is an acceptable 18.0.
  • the selectivity of Gd(III)TREN-HOPY over physiological cations Ca(II) and Zn(II) is similar to that of TREN- 1 -Me-HOPO.
  • the short water residence time of 2 ns, at 298 K, is comparable to members of the HOPO and heteropodand series and indicates that the relaxivity of the HOPY complexes is not limited by slow water exchange kinetics.
  • the rotational correlation time of [Gd(TREN- HOPY)(H 2 O) 2 ] is about 50% higher than that for the DOTA and DTPA Gd(III) complexes.
  • the relaxivity of a chelate such as [Gd(TREN-
  • HOPY (H 2 O) 2
  • HOPY (H 2 O) 2
  • Compound 32 is a useful intermediate in the preparation of multidentate HOPY- containing ligands where the electronic and structural influences of an amide group are not desired.
  • Oxidation of 32 using bleach with TEMPO catalyst under phase transfer conditions provided analytically pure 33 after careful acidification of the crude reaction extracts.
  • the present invention provides a synthetic route of HOPY-containing chelates, which does not require chromatography in preparing the key ligand precursor, HOPY carboxylic acid, 33.
  • a ligand can include only HOPY chelating moieties attached to a selected scaffold.
  • the HOPY subunits are combined with HOPO and/or TAM chelating units on a selected backbone to provide an array of mixed ligands.
  • the HOPY subunits are optionally derivatized with one or more linker arms or reactive functional groups.
  • the complexes of the invention are exemplified by embodiments in which one or more heterocyclic complexing group is attached to a linear, polyfunctional scaffold, forming a chelating agent with the correct geometry to complex a metal ion.
  • scaffolds of use in the complexes and chelating agents of the invention are exemplified by the use of TREN.
  • the focus on the TREN scaffold is for clarity of illustration only and should not be interpreted as limiting the scope of the invention to a genus of chelating agents and complexes having a TREN backbone.
  • Those of skill in the art will appreciate that a wide array of scaffold structures can be used as scaffold moieties in the compounds of the invention.
  • scaffolds of use in the present invention can be linear, cyclic, saturated or unsaturated species.
  • Some exemplary scaffold moieties are set forth below: , idicates poml of attachment R ⁇ H, alkoxy, aryloxy, ether, etc to chelating groups
  • the scaffolds can include reactive functional groups, infra, in addition to those that are used to form the link between the scaffold and the chelating heterocyclic rings.
  • the water solubility of the complexes of the invention is enhanced by the functionalization of the scaffold with an appropriate group.
  • synthetic methodologies were developed to enhance the solubility of TREN- 1 -Me- HOPO by the functionalization of the TREN cap to form homochiral tris(2-hydroxymethyl)- TREN-I-Me-HOPO (Hajela, S. B. et al., J. Am. Chem. Soc. 2000, 122, 1 1228).
  • the invention provides a Gd(III) complex of [Gd(SerTREN-l -Me-HOPO)(H 2 O) 2 ] (FIG. 7).
  • the complex has a solubility in water of ca. 15 mM (25 0 C, pH 7), a proton relaxivity (n) of 9.0 mM " 's " ' (at 25 0 C, 20 MHz), which is significantly higher than those of the current mono-aquo MRI contrast agents.
  • the water residence lifetime (x m ) is extremely short (14 ns) and is at least two orders of magnitude higher than commercial MRI agents; this water exchange rate is close to optimal for slowly rotating MRI contrast agents.
  • the substituent on the scaffold can be substantially any group including, but not limited to, reactive functional groups, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl or substituted or unsubstituted aryl groups.
  • the ligand backbone includes an ether or poly(ether), preferably an ethylene glycol oligomer.
  • a synthetic scheme to an exemplary ether derivatized backbone is set forth in Scheme 7:
  • HOIQO Hydro-Oxo-IsoQuinolinOne
  • 1 ,2-HOIQO derivatives (general formula in Scheme A) can be synthesized easily from homophthalic acid according to literature precedence (Chatterjea, J.N. et al., J lnd C hem Soc , 59: 707-709 (1982)).
  • the synthetic transformations only involve non-hazardous standard chemistry, easily performed on large scale. Protection, coupling to a polyamine backbone under standard conditions, and deprotection yields multidentate ligands in high yields (exemplified for a tris(2-aminoethyl)amine backbone in Scheme B).
  • R la , R 2a , R 3a and R 4a can be members independently selected from H, halogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, OR 3a , SR a , and NR 7a R 8d .
  • R 1a , R 6a , R 7a and R 8 " can be members independently selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl.
  • nl is an integer selected from 0 to 4.
  • R la and R 2a are members independently selected from halogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, OR 5a , SR 6a and NR 7a R 8a .
  • R 3a and R 4a can be members independently selected from any polyamine backbone, substituted or unsubstituted alkyl and substituted or unsubstituted aryl.
  • R la and R 2a along with the atoms to which they are attached, can be optionally joined to form a 4- to 8- membered ring.
  • R 3a and R a along with the atoms to which they are attached, can be optionally joined to form a 4- to 8- membered ring.
  • the invention provides a complex between a lanthanide or iron ion and an organic ligand, said ligand comprising a structure according to the following formula:
  • R l a , R 2a , R 3a and R 4a are members independently selected from H, halogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, OR 5a , SR 6a , and NR 7a R 8a .
  • R 5a , R 6a , R 7a and R 8a are members independently selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl.
  • the symbol nl is an integer selected from 0 to 4.
  • R la and R 2a are members independently selected from halogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, OR 5a , SR 6a and NR 7a R 8a .
  • R 3a and R a are members independently selected from a polyamine backbone, substituted or unsubstituted alkyl and substituted or unsubstituted aryl.
  • the invention provides a pharmaceutical formulation comprising an HOIQO chelate and an pharmaceutically acceptable carrier.
  • Scheme B Representative synthesis of a 1,2-HOIQO containing multidentate ligand with a polyamine backbone.
  • Scheme C Representative synthesis of a 1 ,2-HOIQO containing multidentate ligand with a polyamine backbone.
  • HOIQOs such as 1,2-HOIQO based scaffolds, offer the possibility for the introduction of substituents at the benzene ring. This can be achieved (among other reactions) by electrophilic aromatic substitutions yielding functional building blocks as exemplified in Scheme D.
  • Multidentate ligands with the 1 ,2-HOIQO chelator attached to polyamine backbones form highly stable metal complexes, as exemplified by the cerium(III) complex with ligand 5a (Scheme F).
  • the complex of the invention are formed between a compound of the invention and a metal ion.
  • the metal is selected to allow in vivo and in vitro imaging. Imaging techniques may be selected from MRI, luminescence detection, radioisotope labeling and the like.
  • the metal ion can have any charge.
  • the metal is M + .
  • the metal ion can be any metal ion, which forms a complex with the compounds of the invention.
  • the metal is a member selected from a group 3 (e.g., La), group 4 (e.g., Zr), group 5 (e.g., V), group 6 (e.g., Cr), group 7 (e.g., Mn), group 8 (e.g., Fe), group 9 (e.g., Co), group 10 (e.g., Ni), group 11 (e.g., Cu), or group 12 (e.g., Zn) element.
  • the metal is a lanthanide.
  • Exemplary lanthanides include La, Ce, Pr, Nd, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb and Lu.
  • the metal is an actinide.
  • Exemplary actinides include Ac, Th, Pa, U, Np, Pu, Am, Cm, Bk, Cf, Es, Fm, Md, No and Lr.
  • the invention provides complexes formed between at least one metal ion and a compound of the invention.
  • the metal is a member selected from the lanthanide group.
  • Exemplary lanthanides include Neodynium (Nd), Samarium (Sm), Europium (Eu), Terbium (Tb), Dysprosium (Dy) and Ytterbium (Yb), of which Europium and Terbium are preferred.
  • the complexes of the invention are luminescent.
  • the luminescent compounds of the invention can be used with a wide range of energy donor and acceptor molecules to construct luminescence energy transfer pairs, e.g., fluorescence energy transfer (FET) probes.
  • FET fluorescence energy transfer
  • Fluorophores useful in conjunction with the complexes of the invention are known to those of skill in the art. See, for example, Cardullo et al, Proc. Natl. Acad. Sci. USA 85: 8790-8794 (1988); Dexter, D.L., J. of Chemical Physics 21 : 836- 850 (1953); Hochstrasser et al, Biophysical Chemistry 45: 133-141 (1992); Selvin, P., Methods in Enzymology 246: 300-334 (1995); Steinberg, I.
  • DBITC 4-dimethylaminophenylazophenyl-4 '-isothiocyanate
  • FAM 5-carboxyfluorescein
  • Reactive Red 4 (CibacronTM Brilliant Red 3B-A) rhodamine and derivatives 6-carboxy-X-rhodamine (ROX)
  • 6-carboxyrhodamine (R6G) lissamine rhodamine B sulfonyl chloride rhodamine (Rhod) rhodamine B rhodamine 123 rhodamine X isothiocyanate sulforhodamine B sulforhodamine 101 sulfonyl chloride derivative of sulforhodamine 101 (Texas Red) N ,N ,N ' ,N ' -tetramethy 1-6-carboxyrhodam me (T AM RA) tetramethyl rhodamine tetramethyl rhodamine isothiocyanate (TRlTC) riboflavin rosolic acid lanthanide chelate derivatives
  • the literature also includes references providing exhaustive lists of fluorescent and chromogenic molecules and their relevant optical properties, for choosing reporter-quencher pairs (see, for example, Berlman, HANDBOOK OF FLUORESCENCE SPECTRA or AROMATIC MOLECULES, 2nd Edition (Academic Press, New York, 1971 ); Griffiths, COLOUR AND CONSTITUTION OF ORGANIC MOLECULES (Academic Press, New York, 1976); Bishop, Ed., INDICATORS (Pergamon Press, Oxford, 1972), Haugland, HANDBOOK OF FLUORESCENT PROBES AND RESEARCH CHEMICALS (Molecular Probes, Eugene, 1992) Pringsheim, FLUORESCENCE AND PHOSPHORESCENCE (Interscience Publishers, New York, 1949); and the like. Further, there is extensive guidance in the literature for de ⁇ vatizing reporter and quencher molecules for covalent attachment via readily available reactive groups that can be added to a molecule.
  • an absorbance band of the acceptor substantially overlap a fluorescence emission band of the donor.
  • the donor (fluorophore) is a component of a probe that utilizes fluorescence resonance energy transfer (FRET)
  • the donor fluorescent moiety and the quencher (acceptor) of the invention are preferably selected so that the donor and acceptor moieties exhibit fluorescence resonance energy transfer when the donor moiety is excited.
  • FRET fluorescence resonance energy transfer
  • the efficiency of FRET between the donor and acceptor moieties is at least 10%, more preferably at least 50% and even more preferably at least 80%. The efficiency of FRET can easily be empirically tested using the methods both described herein and known in the art.
  • the efficiency of FRET between the donor-acceptor pair can also be adjusted by changing ability of the donor and acceptor to dimerize or closely associate. If the donor and acceptor moieties are known or determined to closely associate, an increase or decrease in association can be promoted by adjusting the length of a linker moiety, or of the probe itself, between the two fluorescent entities.
  • the ability of donor-acceptor pair to associate can be increased or decreased by tuning the hydrophobic or ionic interactions, or the steric repulsions in the probe construct.
  • intramolecular interactions responsible for the association of the donor-acceptor pair can be enhanced or attenuated.
  • the association between the donor-acceptor pair can be increased by, for example, utilizing a donor bearing an overall negative charge and an acceptor with an overall positive charge.
  • a ligand molecule e.g., biotin
  • the ligand then binds to another molecules (e.g., streptavidin) molecule, which is either inherently detectable or covalently bound to a signal system, such as a fluorescent compound of the invention, or an enzyme that produces a fluorescent compound by conversion of a non-fluorescent compound.
  • a signal system such as a fluorescent compound of the invention, or an enzyme that produces a fluorescent compound by conversion of a non-fluorescent compound.
  • useful enzymes of interest as labels include, for example, hydrolases, particularly phosphatases, esterases and glycosidases, or oxidotases, particularly peroxidases.
  • Fluorescent compounds include fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, etc., as discussed above.
  • fluorescent labels can be detected by exciting the fluorophore with the appropriate wavelength of light and detecting the resulting fluorescence.
  • the fluorescence can be detected visually, by means of photographic film, by the use of electronic detectors such as charge coupled devices (CCDs) or photomultipliers and the like.
  • enzymatic labels may be detected by providing the appropriate substrates for the enzyme and detecting the resulting reaction product.
  • the complexes of the invention are tethered to other species by means of bonds formed between a reactive functional group on the ligand or a linker attached to the ligand, and a reactive functional group of complementary reactivity on the other species.
  • a reactive functional group on the ligand or a linker attached to the ligand and a reactive functional group of complementary reactivity on the other species.
  • the focus exemplifies selected embodiments of the invention from which, others are readily inferred by one of skill in the art. No limitation of the invention is implied, by focusing the discussion on the representative embodiments.
  • Exemplary ligands and complexes of the invention bear a reactive functional group, which is generally located on the scaffold or on a substituted or unsubstituted alkyl or heteroalkyl chain attached to the scaffold or a chelating moiety, allowing their facile attachment to another species.
  • a convenient location for the reactive group is the terminal position of the chain.
  • Reactive groups and classes of reactions useful in practicing the present invention are generally those that are well known in the art of bioconjugate chemistry.
  • Currently favored classes of reactions available with reactive ligand analogues are those proceeding under relatively mild conditions. These include, but are not limited to nucleophilic substitutions (e.g., reactions of amines and alcohols with acyl halides, active esters), electrophilic substitutions (e.g., enamine reactions) and additions to carbon-carbon and carbon-heteroatom multiple bonds (e.g., Michael reaction, Diels-Alder addition).
  • Exemplary reaction types include the reaction of carboxyl groups and various derivatives thereof including, but not limited to, N-hydroxysuccinimide esters, N- hydroxybenzotriazole esters, acid halides, acyl imidazoles, thioesters, p-nitrophenyl esters, alkyl, alkenyl, alkynyl and aromatic esters.
  • Hydroxyl groups can be converted to esters, ethers, aldehydes, etc.
  • Haloalkyl groups are converted to new species by reaction with, for example, an amine, a carboxylate anion, thiol anion, carbanion, or an alkoxide ion.
  • Dienophile (e.g., maleimide) groups participate in Diels-Alder.
  • Aldehyde or ketone groups can be converted to imines, hydrazones, semicarbazones or oximes, or via such mechanisms as Grignard addition or alkyllithium addition.
  • Sulfonyl halides react readily with amines, for example, to form sulfonamides.
  • Amine or sulfhydryl groups are, for example, acylated, alkylated or oxidized.
  • Alkenes can be converted to an array of new species using cycloadditions, acylation, Michael addition, etc. Epoxides react readily with amines and hydroxyl compounds.
  • the reactive functional groups can be chosen such that they do not participate in, or interfere with, the reactions necessary to assemble the reactive ligand analogue.
  • a reactive functional group can be protected from participating in the reaction by the presence of a protecting group.
  • a protecting group For examples of useful protecting groups, see Greene et al., PROTECTIVE GROUPS IN ORGANIC SYNTHESIS, John Wiley & Sons, New York, 1991.
  • the linkage between the ligand and the targeting (or other) agent, and optionally, the linker group at least one of the chemical functionalities will be activated.
  • the chemical functionalities including hydroxy, amino, and carboxy groups
  • a hydroxyl group of the ligand (or targeting agent) can be activated through treatment with phosgene to form the corresponding chloroformate, or p- nitrophenylchloroformate to form the corresponding carbonate.
  • the invention makes use of a targeting agent that includes a carboxyl functionality.
  • Carboxyl groups may be activated by, for example, conversion to the corresponding acyl halide or active ester. This reaction may be performed under a variety of conditions as illustrated in March, supra pp. 388-89.
  • the acyl halide is prepared through the reaction of the carboxyl-containing group with oxalyl chloride. The activated agent is combined with a ligand or ligand-linker arm combination to form a conjugate of the invention.
  • carboxyl-containing targeting agents is merely illustrative, and that agents having many other functional groups can be conjugated to the ligands of the invention. Macromolecular complexes
  • the invention provides a Gd(III) complex of the invention that is macromolecular, i.e., MW > 1000 D.
  • a macromolecular complex of the invention is formed by covalently conjugating a complex to a macromolecule via a reactive functional group.
  • the macromolecular complex is formed by a non-covalent interaction between a complex and a macromolecule, e.g, a serum protein.
  • poly(ethylene glycol) adducts of homopodal and heteropodal ligands and chelates are described by reference to poly(ethylene glycol) adducts of homopodal and heteropodal ligands and chelates.
  • biomolecules include polypeptides (e.g., antibodies, enzymes, receptors, antigens); polysaccharides (e.g., starches, inulin, dextran); lectins, non-peptide antigens and the like.
  • Exemplary synthetic polymers include poly(acrylic acid), poly(lysine), poly(glutamic acid), poly(ethylene imine), etc.
  • the bond formed between reactive functional groups of the macromolecule and that of the complex attaches the ligand to the macromolecule essentially irreversibly via a "stable bond" between the components.
  • a “stable bond”, as used herein, is a bond, which maintains its chemical integrity over a wide range of conditions ⁇ e.g., amide, carbamate, carbon-carbon, ether, etc.).
  • the macromolecule and the complex are linked by a "cleaveable bond”.
  • a “cleaveable bond”, as used herein, is a bond that undergoes scission under selected conditions. Cleaveable bonds include, but are not limited to, disulfide, imine, carbonate and ester bonds.
  • the reactive functional group can be located at one or more positions of the scaffold and/or one or more positions on the chelating ring structures.
  • the present invention also provides conjugates between the chelating agents and complexes of the invention and linear, branched and cyclic polymers, e.g., polysaccharides, poly(amino acids), cyclodextrins, synthetic polymers, etc.
  • linear, branched and cyclic polymers e.g., polysaccharides, poly(amino acids), cyclodextrins, synthetic polymers, etc.
  • a promising route to the optimization of proton relaxivity involves increasing the rotational correlation time, ⁇ r , by increasing the molecular weight of the complex, resulting in slower rotation of the molecule in solution. This has been achieved in a number of ways.
  • the present invention provides conjugates between oxygen donor ligands and saccharides, e.g., polysaccharides.
  • the invention provides a conjugate between an oxygen donor chelate and inulin.
  • Inulin is a naturally occurring polysaccharide which has been previously investigated as a carrier for Gd(IlI) chelates (using DTPA and DOTA derivatives) (Rongved, P. K., J. Carbohydr. Res. 1991, 214, 315; Corsi, D. M. V. E. et al., Chem. Eur. J. 2001, 7, 64).
  • inulin can be described as a mixture of linear ⁇ -(2 ⁇ l )-linked ⁇ -D- fructofuranosyl chains with a ⁇ -D-glucopyranosyl unit at the terminal end.
  • Inulin is commercially available in a variety of molecular weights and the degree of polymerization varies from 10 to 30, resulting in a molecular weight distribution of 1500 to 5000 Da.
  • the high hydrophilicity, pH stability, low solution viscosity and biocompatability of inulin should ensure that the conjugated-MRI agent has favorable pharmacological properties.
  • the inulin is attached to a TREN-HOPO scaffold.
  • the bond is formed using an activated intermediate of a carboxylic acid derivative that is attached to the endocyclic nitrogen of a HOPY moiety.
  • Other modes of conjugation are well within the skill of those of skill in the art.
  • similar molecules are readily synthesized using HOPO-TAM moieties in which the amide moiety of the TAM moiety is activated, and subsequently conjugated to inulin.
  • Scheme 8 An exemplary synthetic scheme to attach TREN-6-Me-H0P0 to the backbone ol inulin is shown in Scheme 8
  • the flexibility of the polymer chain allows for the preparation ol derivatives that have high degrees ot substitution, facilitating a higher rigidity in the Gd(III) complex
  • Also included in the present invention is a method of preparing a chelating agent having a polymeric backbone and at least one functionality to which a chelating hgand of the invention is bonded
  • suitable polymers include, but are not limited to, poly(styrene-divinylbenzene), agarose (manufactured by Bio-Rad Corp , Richmond, CA, under the name "Affi-Gel”), and polyacrylamide
  • the method of the invention is not limited by the identity of the backbone species, and that numerous amine-, hydroxyl- and sulfhydryl-containing compounds are useful as backbones
  • the present invention provides a metal chelate as set forth above, which is attached to a dend ⁇ mer via a reactive functional group Similar to the polymeric group discussed above, the dend ⁇ mer has at least two reactive functional groups In one embodiment, one or more fully assembled hgand is attached to the dend ⁇ mer Alternatively, the the chelate is formed directly on the dend ⁇ mer
  • dend ⁇ mers In contrast to linear polymers, dend ⁇ mers have a relatively rigid structure and the overall tumbling of the molecule contributes significantly to the rotational correlation time of the Gd-H vector 1 he high monodispersity, minimal shape variation and uniform surface chemistry of Gd(III) chelate dend ⁇ mers are also key features important in their potential application as MRI agents ( Krause, W et al , Top Curr C hem 2000, 210, 261) Although the rotational correlation time increases with higher generation dend ⁇ mers, the increase in proton relaxivity eventually reaches a plateau, an effect which is attributed to the slow water exchange ( ⁇ m ) inherent to the Gd(III) D 1 PA- and DOT A-based systems ( 1 oth, E et al , Chem Eur J 1996, 2, 1607) Hence, the reported relaxivity values for dend ⁇ mer conjugates range from 1 1 to 36 mM 's ' per Gd(IlI) center, depending on the nature of the chelate and the dend
  • Gd(III)-chelate-dendrimer conjugates have enhanced relaxivity due to their spherical rigid structure (which results in a slower ⁇ r ). According to the Enhanced Permeation and Retention Effect, high generation dendrimers of high molecular weight should be preferentially uptaken by tumor cells (Narayanan, V. V. et al., Macromolecules 2000, 33, 3944; Wiener, E. C. et al., J. Am. Chem. Soc. 1996, 118, 111 A; Maeda, H. et al., J. Controlled Release 2000, 75, 271 ; Malik, N. et al., J. Controlled Release 2000, 65, 133).
  • a water-soluble and bio-adapted polyester (polypropionate) class of dendrimer contrast agents has been designed to provide favorable pharmacokinetic properties. See, for example, Monopropionate (polypropionate) class of dendrimer contrast agents. See, for example, Monopropionate (polypropionate) class of dendrimer contrast agents. See, for example, Monopropionate (polypropionate) class of dendrimer contrast agents. See, for example, Monopropionate) of the invention.
  • the termini of the dendrimers are conjugated to an oxygen donor ligand of the invention, e.g., TREN-I -Me-HOPO-TAM.
  • the dendrimers are readily functionalized with thiaz-activated oxygen donor ligands, such as activated TREN-I-Me-HOPO-TAM ligands before complexation with Gd 3+ (Dong, Q. H. et al., Invest. Radiol. 1998, 33, 699).
  • the invention provides a complex, which includes a structure according to Formula I in which at least one of R 1 , R 2 , R 3 , and R 4 comprise a moiety derived from poly(ethyleneglycol).
  • the invention provides derivatives of TREN- l-Me-3,2-HOPO-TAM, such as Gd-TREN-Ms-(I -Me-HOPO)-(TAM- PEG-2000) (46); and Gd-TREN-bis-(l-Me-HOPO)-(TAM-PEG-5000) (47).
  • a complex having a PEG of average molecular weight 450 was also prepared.
  • the optimal value for ⁇ m depends on several variables, especially the field strength of the MRI scanner instrument.
  • the incorporation of PEG chains into HOPO- TAM ligands provides a method for the optimization of a MRI contrast agent to a particular field strength.
  • the method of the invention provides for the optimization of additional parameters relevant to a MRI contrast agent.
  • the relaxivity of the core complexes is enhanced by their substitution with PEG (FIG. 10).
  • the present invention provides compounds in which the PEG moiety is conjugated to positions other than the amide of a TAM group of a TREN-bis-(l -Me-HOPO)-(TAM).
  • the PEG moiety may be tethered to the endocyclic nitrogen or another position of the pyridinone moiety, another chelating moiety, the scaffold, or a combination of these positions.
  • PEG Polyethylene glycol
  • the PEG group is preferred for two reasons. First, the high water-solubility associated with PEG chains increases the rather low solubility of the parent complexes. Second, although it has been previously found that rapid internal motions within a PEG chain result in only a modest increase in ⁇ r (Toth, E. et al., Magn. Reson. Chem. 1998, 36, S 125), it has also been demonstrated that PEG chains can bind to HSA across a wide pH range (Azegami, S. T. et al., Langmuir 1999, 15, 940).
  • Methods of enhancing the specificity of agents include, but are not limited to, attaching the signal generating portion of the agent (e.g., an oxygen donor ligand complex with Gd(III) to a species that actively directs the conjugate to a selected tissue.
  • agent e.g., an oxygen donor ligand complex with Gd(III)
  • exemplary active agents are antibodies and ligands for biologically relevant receptors.
  • Another approach to improving the biodistribution of an agent relies on preparing an agent that interacts passively (non-specifically) with a biologically relevant species, e.g, a serum protein.
  • Yet another strategy exploits the enhanced permeability and uptake mechanism in which a damaged or diseased tissue preferentially uptakes a macromolecular agent.
  • the present invention provides an agent that binds to serum proteins in vivo, thus, affording a blood pool contrast enhancing agent.
  • a second advantage accrues from the interaction between the oxygen donor-metal complexes of the invention and a serum protein; the rotational correlation time of the complex increases, leading to an increase in the relaxivity of the complex (Lauffer, R. B., Magn. Reson. Med. 1991, 22, 339).
  • the complex MS-325 forms a noncovalent adduct with the blood protein human serum albumin (HSA) (Parmalee, D. J. W. et al., Invest. Radiol. 1997, 32, 741 ; Lauffer, R. B. P. et al., Radiology 1998, 207, 529).
  • HSA human serum albumin
  • the relaxivity of the resulting HSA adduct is 42.0 mM " 1 S " 1 , which is nearly seven times greater than the in vitro relaxivity of the free complex in water (6.6 mM ' V).
  • MS-325 is currently in phase II and phase III clinical trials for imaging the cardiovascular system. Complexes have also been designed to target other macromolecules.
  • Gd-BOPTA was designed to target hepatocytes in order to facilitate hepatobiliary imaging (Cavanga, F. M. et al., Invest. Radiol. 1997, 32, 780).
  • This MRI contrast agent has a relaxivity of 4.4 mM ' V in water, 6.9 mM “ 's " ' in rat plasma and 30 mM ' V in rat hepatocytes.
  • the present invention provides slowly tumbling Gd(III) complexes with faster water exchange rates (i.e. with ⁇ m and ⁇ r preferably in the low ns regime).
  • the invention also provides MRI contrast agents with desirable in vivo persistence, which facilitate diagnosis of physiological abnormalities in specific regions of the body over longer periods than currently possible.
  • ⁇ r , and ⁇ m other essential characteristics (such as stability and water-solubility) have not be worsened. Therefore, the new MRI contrast agents, which are the subject of this invention, contain the requisite structural features that result in images with better morphological and functional information (Comblin, V. et al., Coord. Chem. Rev. 1999, 185-186, 451 ).
  • the complexes of the invention include the covalent attachment of substituents to the oxygen donor ligand core, which facilitate non-covalent interactions with endogenous biomolecules.
  • substituents to the oxygen donor ligand core
  • the binding of a slowly rotating macromolecule to the contrast agent allows for longer rotational correlation times in vivo, which could dramatically increase the relaxivity.
  • the present invention is exemplified by reference to blood pool contrast agents.
  • the residence of the complexes of the invention in the blood pool is influenced by a number of structural features.
  • the molecular size (weight) of the complexes of the invention is readily increased to a value that prevents their rapid elimination by glomerular filtration.
  • the increase in molecule size can be achieved by attaching a macromolecular moiety to the ligand.
  • an array of ligands may be attached to a macromolecular carrier, such as a poly(peptide), poly(saccharide), or a dendrimer or other synthetic polymer.
  • a macromolecular carrier such as a poly(peptide), poly(saccharide), or a dendrimer or other synthetic polymer.
  • the resulting construct is biocompatible, substantially non- immunogenic, water-soluble and has an acceptable relaxivity.
  • the attachment can be through a stable linkage or a linkage that is cleaved under biologically relevant conditions.
  • Cleaveable linkages include a cleaveable group as discussed herein.
  • Strategies for preparing biodegradable contrast agents are known in the art. See, for example, US Pat.6,312,664.
  • Another strategy for enhancing the blood pool residence time of a chelate of the invention relies on the non-covalent, reversible interaction between the chelate and a component of the blood, for example, a protein.
  • HSA Human serum albumin
  • Binding of the MRI contrast agent to HSA serves three purposes. First, it targets the complex to the blood pool allowing selective enhancement of arteries and veins during MR angiographic evaluations. Second, protein binding slows down the molecular tumbling time of the complex and should provide a 5- to 10-fold increase in relaxation enhancement as compared to the parent complex (Caravan, P. et al., Inorg. Chem. 2001, 40, 2170). Finally, albumin binding increases the half-life of the drug in vivo, which allows the radiologist time to image multiple body regions and to employ pulse sequences which give high resolution images.
  • HSA possesses numerous binding sites of different levels of hydrophobicity and hydrophilicity, hence, different affinities for small molecules (FIG. 11).
  • the warfarin binding site (in subdomain IIA of HSA) is one such cavity and a recent study (Zaton, A. M. L. V., J. P. Chem.-Bi ⁇ Int. 2000, 124, 1) has shown that 4-hydroxycoumarin (CMN) has a very high affinity for this site in comparison with structural analogues and uracils (pyrimidine derivatives).
  • CCN 4-hydroxycoumarin
  • TREN-6-Me-HOPO The synthetic scheme the attachment of CMN to TREN-6-Me-HOPO is shown in Scheme 9.
  • Scheme 7 Synthesis of a potential blood-pool MRI contrast agent. presence of formaldehyde to form the protected coumarin-HOPO adduct. The adduct is subsequently reacted with an amine backbone as discussed herein and, optionally, with another chelating moiety, such as a HOPO substituted on the endocyclic nitrogen with an acetic acid residue. The benzyl protecting groups are optionally removed after the synthesis of the ligand is complete.
  • HSA-binding ligands of the invention include a hydrophilic moiety and a hydrophobic group, e.g., a benzyl group (FIG.12).
  • the hydrophilic group is PEG.
  • the hydrophilic group is a species such as a carboxylic acid.
  • Presently preferred hydrophobic moieties include substituted or unsubstituted benzyl groups.
  • Representative ligands according to this motif include:
  • Scheme 1C [0264] In Scheme 10, the ethyl ester (10) is hydrolyzed to the corresponding acid 49, which is converted to thiazolide 50. Two thiazolide units are coupled to the TREN backbone, affording a ligand having a free amine group 51 to which is coupled a protected PEG-TAM derivative 52. The resulting heteropodal ligand 53 is debenzylated, liberating the phenolic hydroxl moieties of the TAM subunit. The resulting compound 54 is metallated to provide Gd(III) chelate 55.
  • Scheme 12 sets forth the synthesis of thiaz-TAM-(L)Phen-TRI, which is used to form oxygen donor ligands of the invention.
  • N-protected phenylalanine 59 is activated as the thiazolide 60, which is coupled to an amino-PEG, affording PEGylated phenylalanine 61.
  • the BOC protecting group is then cleaved yielding the amine 62.
  • Compound 62 is coupled to di-thiaz-TAM 23, to provide compound 63, having both hydrophobic and hydrophilic characteristics.
  • Compound 63 is coupled to a ligand scaffold (such as 24) as discussed above.
  • the Gd complexes of the invention interact with HSA. It is within the scope of the invention to vary the substituents on the oxygen donor ligand to adjust the water solubility of the complex. Furthermore, the linker between the protein binding moiety and the Gd chelate can be adjusted to tune the degree of protein-ligand interaction.
  • the invention also provides complexes that are targeted to the liver and/or other components of the reticuloendothelial system.
  • the complex includes a hydrophobic moiety or other group known to be preferentially taken up by the liver or other components of the reticuloendothelial system.
  • a direct route to liver-specific MRI agents is to take advantage of the known biodistribution of cholesterol.
  • Cholesterol is a biomolecule that is synthesized in the liver and metabolized in the liver and bile glands.
  • a cholesterol derivative e.g., cholic acid (CHOL) is attached to the TREN-HOPO-TAM scaffold (Scheme 13).
  • This attachment enables specific in vivo localization of the conjugated-MRI agent to tissue of the reticuloendothelial system (Anelli, P. L. et al., Bioconj. Chem. 1999, 10, 137; Anelli, P. L. et al., Acta Radiologica 1997, 55, 15) resulting in hepatobiliary contrast enhancement.
  • cholic acid is activated and converted to the ethylamine derivative 64 by treating the activated intermediate with ethylenediamine.
  • Intermediate 64 is combined with an active thiazolide derivative of a ligand of the invention 65, to form the corresponding cholesterol derivatized ligand 66.
  • the invention provides liver-selective complexes that include both a poly(ether) and a hydrophobic moiety, such as a cholesterol derivative.
  • a representative synthetic route is provided in Scheme 14.
  • a ligand functionalized with a poly(ether) terminating with an active amine 67 is combined with activated cholesterol thiazolide 68 to provide the bifunctionalized ligand, bearing both a poly(ether) and a cholesterol moiety 69.
  • the benzyl protecting groups are removed from the phenolic oxygens of the TAM moiety, affording ligand 70.
  • the present invention also provides agents that are selective for tumors.
  • the invention provides a gadolinium chelate conjugated to a sapphyrin.
  • Sapphyrins which are expanded porphyrins, have been studied extensively for use in medicinal applications as anion binders and photosensitizers and have also been studied in cancer models to target tumors of pancreatic adenocarcinoma (constituting more than 75% of all pancreatic cancer).
  • pancreas specificity of porphyrins (cheap and readily available for screening) and expanded porphyrins, and the fast water exchange rate of the chelates of the invention can be combined to give promising pancreas-selective and fast water exchanging Gd-TREN-HOPO-porphyrin agents.
  • Pancreatic cancer most often occurs in the form of pancreatic adenocarcinoma.
  • Very recent and exciting research shows that a water-soluble derivative of sapphyrin accumulates specifically in human pancreatic adenocarcinoma when transplanted into and grown in mice (Ferucci. J. T. Annals of Oncology 10, Suppl. 4: S 18 (1999)).
  • thin cross-sections of tissue were analyzed by UV and resonance Raman spectroscopy for the sapphyrin, which was found to be present in the tumor in large quantities at all post-injection times.
  • pancreatic tumor selectivity is hundreds of times greater than that for the liver, kidney or muscle tissues in the mouse model.
  • a C 2 symmetric sapphyrin is disubstituted with amine pendants.
  • the addition of sapphyrin derivative 71 (R Me) and two equivalents of 23 form a disubstituted derivative 72 which is then be treated with 2 eq. of 24 in known amine coupling to form 73, which preserves the integrity of the [TREN-HOPO-TAM] geometry.
  • Ligand 73 is further deprotected and GdCl ⁇ is combined with 73 to form 74.
  • the present invention also provides water soluble sapphyrin-Gd(III) complexes.
  • the sapphyrin is derivatized with a polyethylene glycol moiety, to counter the lipophilicy of the sapphyrin.
  • the PEG pendant of 75 can be tethered at the hydroxypyridinoate nitrogen as shown in Scheme 16. Chlorinated 76 is then added directly to 12 at the HOPO ring nitrogen forming monosubstituted 77.
  • Derivative 77 can be further combined with 24 and be incorporated in the second step of Scheme 15.
  • the TREN is functionalized with PEG in a manner that is not detrimental to the stability of the complex.
  • one or more of the three TREN arms is functionalized by Tf- protection of the amine nitrogen with the concomitant labilizing of the vicinal proton for substitution by sapphyrin with alcohol pendant arms (Scheme 17).
  • An exemplary complex has a Gd to sapphyrin ratio of 4: 1.
  • TREN commercially available
  • the resulting product is reduced to form 81.
  • This tetra-TREN can be flexibly used to prepare compounds of the invention.
  • the compounds of the invention are further conjugated to quinacrine or secretin to further enhance pancreas affinity due to their individual molecular recognition, and pancreatic tumor selectivity.
  • quinacrine, 82 has an affinity for acidic phospholipids and the enzyme pancreatic phospholipase A2.
  • the acridine group also serves as a fluorescent marker, providing a means of detecting the compound in tissues.
  • Compound 82 which is commercially available can be Suzuki coupled with a borane functionalized propeneamine (commercially available) and then undergo amide coupling with 23 to yield the tethered quinacrine-TAM fragment (Scheme 18).
  • 84 is utilized in place of 23 (Scheme 15, Step 1) in conjunction with tetra-TREN 81 in place of reagent 24 (0.25 eq.) to prepare a Gd multiconjugate.
  • Secretin (commercially available) can also be conjugated at its N terminus.
  • Secretin a 27-amino acid polypeptide, is received in vivo on its carboxylate end at pancreatic parenchymal cells.
  • the N-terminus may be selectively iodinated in reasonable yield prior to substitution at the 6-Me-HOPO nitrogen.
  • the present invention generally utilizes art-recognized methods to characterize the new ligands and their metal complexes.
  • the following sections provide exemplary methods of characterizing the compounds of the invention. The methods set forth below are intended to illustrate useful techniques for characterizing the compositions of the invention, but should not be construed as limiting the methods of us in characterizing the compositions of the invention.
  • Methods of determining stability constants include, but are not limited to those set forth in, Johnson, A. R. et al., Inorg. Chem. 2000, 39, 2652-2660; and Cohen, S. M. X. et al., Inorg. Chem. 2000, 39, 5747.
  • the Bjerrum method can be used for metal complex stability measurements (pH titrations of ligand and metal + ligand). Competition titrations with DTPA can be performed to determine the stability of very stable complexes where direct pH titration methods are inappropriate. Spectrophotometric techniques can be used to monitor metal-ligand complexation reactions, which give rise to changes in the Vis/UV spectra relative to the parent metal and ligand species. With digitally-recording automated spectrophotometeric titrators, factor analysis of the Vis/UV spectra readily determined the species in solution, their individual spectra and the equilibrium constants which interrelate them.
  • the concentration of the saturated solution was then determined. This corresponds to the minimum solubility of the complex in H 2 O (pH 7, 25 0 C, 0.1 M). The solubility of the complexes were observed to increase at lower salt concentration.
  • the spectrophotometric data is refined using a model.
  • the model includes four components: LIl/, GdL, GdLH + and GdLH 2 2+ .
  • This speciation is similar to the solution behavior of Tren-MOE-3,2-HOPO, although the parent complex, Gd(Tren-Me-3,2-HOPO), was not formerly reported as possessing a diprotonated species. (Johnson et al., Inorg. Chem., 39:2652 (2000))
  • LogK.ML values reflect the metal-ligand affinity for deprotonated ligand in the reaction, M + L - ML.
  • the pM value is one way to make allowance for the competition for the ligand by protons in real solutions, and thus gives a more complete picture of the effectiveness of the ligand in chelating the metal.
  • a pM value can be calculated for many conditions, but it is generally preferable to calculate it for conditions relevant to biological considerations.
  • Potentiometric titrations of the ligand and/or complex with zinc and calcium ions can also be performed.
  • the potentiometric titrations provide a comparison to the high selectivity for Gd(III) afforded by the TrenHOPO system. (Xu et al., J. Am. Chem. Soc, 117:7245 (1995))
  • solutions of a 1 : 1 ratio of metal ion to ligand are titrated over a pH range of 2.4-1 1.
  • low concentrations (0.25 mM) are used to avoid precipitation in the pH region of 5.5-6.5 for both the Ca 2+ and Zn 2+ systems.
  • the longitudinal water proton relaxation rate at 20 MHz are measured (typically with a NMR spectrometer operating at 0.5 T) with a reproducibility of the T ⁇ data to ⁇ 0.5%.
  • Equipment for the control of temperature (with an accuracy of ⁇ 0.1 0 C) during these measurements are employed.
  • the mean residence water lifetime, ⁇ m is generally determined by measuring the transverse 17 O NMR relaxation rate (R 2/ ;) at various temperatures.
  • the variable-temperature 17 O NMR measurement is performed using spectrometers which operate at various magnetic field strengths (2.1 and 9.4 T are typically used) equipped with a 5 mm probe. A D 2 O external lock and solutions containing 2.6% of the 17 O isotope are used.
  • the observed transverse relaxation rates are calculated from the signal width at half-height. Details of the instrumentation, experimental methods, and data analysis are reported elsewhere and incorporated by reference herein (Cohen, S. M. X. et al., Inorg. Chem. 2000, 39, 5747; Aime, S. B. et al., J. Biol. Inorg. Chem. 1997, 2, 470; Aime. S. B. et al., Magn. Reson. Chem. 1998, 36, S200).
  • the Gd(III) complexes of the HOPO and HOPY systems set forth herein are promising candidates for MRI contrast agents since they possess high thermodynamic stability and high relaxivity due to the presence of two coordinated water molecules characterized by a fast rate of .
  • the higher values are at least about two times higher than that of the currently used contrast agents based on polyaminocarboxylate.
  • relaxometry Measuring the relaxation rates of an abundant nucleus in a large magnetic field range is called relaxometry.
  • a relaxometry profile is a plot of nuclear magnetic relaxation rates, usually ⁇ /T ⁇ , as a function of the Larmor frequency or the magnetic field on a logarithmic scale (see Fig. 7). This plot is also called a Nuclear Magnetic Relaxation
  • NMRD Dispersion
  • Nuclear magnetic resonance dispersion (NMRD) profiles can be used to determine the values of the parameters that contribute to the relaxivity of a Gd(III) complex (e.g. q and ⁇ r ).
  • the methods involved measuring the magnetic field strength (Larmor frequency) dependence of the solvent proton longitudinal relaxation rate in the presence of a Gd(III) complex.
  • the proton ⁇ IT ⁇ NMRD profiles were typically measured on a field-cycling relaxometer over a continuum of magnetic field strengths.
  • the relaxometer generally operates under computer control with an absolute uncertainty in ⁇ IT ⁇ of ⁇ 1%. Details of the instrumentation and data acquisition procedure are reported elsewhere and are incorporated by reference herein. (Aime, S. B. et al., J. Biol. Inorg. Chem.
  • the number of water molecules (q) bound to the metal ion is assessed by luminescence decay kinetics.
  • the metal ions are directly excited with a powerful Nd-YAG laser and the decay rates are recorded.
  • the method relies upon the preparation of the terbium(III) complexes of the new ligands.
  • the europopium (III) complexes of the new ligands are synthesized.
  • the NMRD profiles of complexes of the invention are in the fast exchange regime, a condition that precludes the assessment of the water exchange rate from the analysis of the magnetic field dependence of the proton relaxivities.
  • the value of X M a crucial parameter for the evaluation of the efficiency of a contrast agent, can be independently obtained by a variable temperature, proton decoupled 17 O NMR measurement of the water nuclear transverse relaxation rate (R 2 ) using a well-established (Powell et al., J. Am. Chem. Soc, 118:9333 (1996); Aime et al., Ace. Chem.
  • the R 2 values are dominated by the scalar relaxation mechanism which depends on k cx and its temperature dependence ( ⁇ H M ), the electronic relaxation rate and its temperature dependence ( ⁇ , ⁇ y, ⁇ Hv) and the hyperfine coupling constant AJh.
  • Biomolecule affinity measurements are generally measured by art-recognized methods. For example, the non-covalent interaction between HSA and the metal complexes containing hydrophobic groups have been thoroughly investigated using the well-established proton relaxation enhancement (PRE) method (Caravan, P. E. et al., Chem. Rev. 1999, 99, 2293) that allows both the binding parameters (K 3 ) and the relaxivity enhancement of the (Metal complex)-HSA adduct to be determined. In this method, the water proton relaxation rates of solutions containing the metal complex and increasing concentrations of the serum protein are measured.
  • PRE proton relaxation enhancement
  • An exemplary assay is performed with competitor probes (such as warfarin and ibuprofen), which elucidate the binding sites of the protein with which the MRI agents interact.
  • the measurements are typically performed at pH 7.4 (in phosphate buffer) and 298 K, which are close to physiological conditions.
  • the emissive properties of Eu(III) and/or Tb(IlI) 7 are used to reflect the rates of emissive decay in distinct sites that the metal ion occupies.
  • luminescence titration of the Eu(III) and Tb(III) complexes of the ligands with HSA is a good method for determining biomolecule affinity (Feig, A. L. P. et al., Chem. & Biol, 1999, 6, 801 ; Chaudhuri, D. H. et al., Biochem. 1997, 36, 9674; Cronce, D. T. H. et al., Biochem. 1992, 31, 7963.
  • the in vivo biodistribution of the chelating agents in tumor-induced rats is determined to assess tumor uptake of the agents.
  • An exemplary assay involves radiotracer studies using 153 Gd. The rodent is induced with the tumor of choice and the tumor is allowed to grow over several weeks to a diameter suitable for biodistribution experiments. The rodents are then transferred to a normal diet for an appropriate period of time. The radioactive MRI agent is then injected and the urine and feces collected and analyzed for counts periodically. The animal is subsequently sacrificed and the organs removed for radioactive analyses.
  • the biodistribution of the MRI agent as a percentage of the injected dose (% ID) per organ and as a %ID/g of tissue weight is then calculated. This is then compared with the distribution of the MRI agent in a control group (healthy rodents of the same cell line).
  • % ID percentage of the injected dose
  • %ID/g percentage of tissue weight
  • a study on the in vivo biodistribution of the chelating agents enables the identification of the localized distribution of the MRI agents in the presence and absence of tumors.
  • the compounds of the present invention can be prepared and administered in a wide variety of oral, parenteral and topical dosage forms.
  • the compounds of the present invention can be administered by injection, that is, intravenously, intramuscularly, intracutaneously, subcutaneously, intraduodenally, or intraperitoneally.
  • the compounds described herein can be administered by inhalation, for example, intranasally.
  • the compounds of the present invention can be administered transdermally.
  • the present invention also provides pharmaceutical compositions comprising a pharmaceutically acceptable carrier or excipient and one or more compounds of the invention.
  • pharmaceutically acceptable carriers can be either solid or liquid.
  • Solid form preparations include powders, tablets, pills, capsules, cachets, suppositories, and dispersible granules.
  • a solid carrier can be one or more substances, which may also act as diluents, flavoring agents, binders, preservatives, tablet disintegrating agents, or an encapsulating material.
  • the carrier is a finely divided solid, which is in a mixture with the finely divided active component.
  • the active component is mixed with the carrier having the necessary binding properties in suitable proportions and compacted in the shape and size desired.
  • the powders and tablets preferably contain from 5% or 10% to 70% of the active compound.
  • Suitable carriers are magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth, methylcellulose. sodium carboxymethylcellulose, a low melting wax, cocoa butter, and the like.
  • the term "preparation" is intended to include the formulation of the active compound with encapsulating material as a carrier providing a capsule in which the active component with or without other carriers, is surrounded by a carrier, which is thus in association with it.
  • cachets and lozenges are included. Tablets, powders, capsules, pills, cachets, and lozenges can be used as solid dosage forms suitable for oral administration.
  • a low melting wax such as a mixture of fatty acid glycerides or cocoa butter
  • the active component is dispersed homogeneously therein, as by stirring.
  • the molten homogeneous mixture is then poured into convenient sized molds, allowed to cool, and thereby to solidify.
  • Liquid form preparations include solutions, suspensions, and emulsions, for example, water or water/propylene glycol solutions.
  • liquid preparations can be formulated in solution in aqueous polyethylene glycol solution.
  • Aqueous solutions suitable for oral use can be prepared by dissolving the active component in water and adding suitable colorants, flavors, stabilizers, and thickening agents as desired.
  • Aqueous suspensions suitable for oral use can be made by dispersing the finely divided active component in water with viscous material, such as natural or synthetic gums, resins, methylcellulose, sodium carboxymefhylcellulose, and other well-known suspending agents.
  • solid form preparations which are intended to be converted, shortly before use, to liquid form preparations for oral administration.
  • liquid forms include solutions, suspensions, and emulsions.
  • These preparations may contain, in addition to the active component, colorants, flavors, stabilizers, buffers, artificial and natural sweeteners, dispersants, thickeners, solubilizing agents, and the like.
  • the pharmaceutical preparation is preferably in unit dosage form.
  • the preparation is subdivided into unit doses containing appropriate quantities of the active component.
  • the unit dosage form can be a packaged preparation, the package containing discrete quantities of preparation, such as packeted tablets, capsules, and powders in vials or ampoules.
  • the unit dosage form can be a capsule, tablet, cachet, or lozenge itself, or it can be the appropriate number of any of these in packaged form.
  • the quantity of active component in a unit dose preparation may be varied or adjusted from 0.1 mg to 1O g, more typically 1.0 mg to 1 g, most typically 10 mg to 500 mg, according to the particular application and the potency of the active component.
  • the composition can, if desired, also contain other compatible therapeutic or diagnostic agents.
  • MRI contrast agents are typically administered at a dosage of 0.1 - 0.3 mmol/kg patient in 0.5 M solutions (Caravan, P. E. et al., Chem. Rev. 1999, 99, 2293).
  • the improved properties of the present agents allows their administration in amounts lower than art- recognized aminocarboxylate-based contrast agents. Adjusting the dose to achieve maximal efficacy in humans based on the methods described above and other methods as are well-known in the art is well within the capabilities of the ordinarily skilled artisan.
  • the invention provides a method for performing a contrast enhanced imaging study on a subject.
  • the method includes administering a metal complex of the invention to the subject and acquiring an image of the subject.
  • the complexes of the invention are of use in a range of diagnostic imaging modalities including, but not limited to, MRI, X-ray and CT.
  • the invention also provides a method for tuning a physical property of a metal complex of the invention.
  • Exemplary physical properties that are tuned by the method of the invention include, but are not limited to, water exchange rate, rotational correlation time, in vivo residence time, relaxivity and water solubility.
  • the method includes preparing a parent complex that may or may not have a poly(ether) as a component of the ligand and measuring the physical property of the complex. If the measured property is less than ideal, the property is adjusted by preparing an analogue of the ligand that includes a poly(ether), or that has a poly(ether) of a size and/or structure different from that of the first ligand.
  • the inventors have recognized that, using the iterative method provided herein, it is possible to tune and refine a range of physical properties of the chelates of the invention.
  • Also provided by the present invention is a method for treating a patient for metal ion overload.
  • the method includes administering to a patient in need of such treatment an amount of a compound prepared by the method of the invention.
  • the amount of compound administered is effective to reduce the metal ion load in the patient. It is well within the abilities of one of skill in the art to ascertain an appropriate dosage and treatment regimen for a particular patient.
  • treating the patient with a compound prepared by a method of the invention results in a greater amount of metal ion being removed from the patient than is removed upon treating the patient with an identical dose of the same compound prepared by a previously known method.
  • the dicyclohexylurea (DCU) was removed by filtration, and the solvents removed. The remaining residue was dissolved in CH 2 Cl 2 , filtered and purified by a gradient flash silica column (2-6% CH 3 OH in CH 2 Cl 2 ). The solvents were removed from the appropriate fractions by rotary evaporation, and the residue recrystallized from acetone, yielding yellow crystals (3.78 g, 56 %).
  • K 2 CO 3 (10.5 g, 75.4 mmol) was added to a suspension of 6-methyl-3- hydroxy-2(lH)-pyridinone-4-carboxylic acid ethyl ester (5.20 g, 26.4 mmol) in dry DMF (10 ml) under an atmosphere of N 2 .
  • Benzyl chloride (6.96 g, 55.0 mmol) was added and the reaction stirred at 65 0 C for 3Oh, the progress of the reaction was monitored by TLC.
  • TREN-l-Me-3,2-HOPO-TAM-PEG-5000 (Formula 47A)
  • Benzyl protected TREN- l-Me-3,2-HOPO-TAM-Thiazolide (Formula 25 in Scheme 3)
  • a solution of TREN(I -Me-HOPO) 2 (3.00 g, 4.77 mmol) in CH 2 Cl 2
  • PEG-5000 monoamine (1.85 g, 0.370 mmol) was added to a solution of benzyl protected TREN- 1-Me-HOPO-TAM-thiazolide (0.4 g, 0.377 mmol) in dry CH 2 Cl 2 (5 ml). The resulting solution was left stirring for several days at 313K under a nitrogen atmosphere. After 4 days, a few drops of Net3 and a catalytic amount of dimethylaminopyridine (DMAP) were added.
  • DMAP dimethylaminopyridine
  • the first yellow band was unreacted BnTAMdiThiaz, and was saved for future use.
  • the second yellow band was the desired product: the solvents were removed to yield a thick yellow oil, which solidified on standing over 4h (Yield: 1.08 g, 87 %).
  • TAM-thiazolide-PEG-2000 (1.0 g, 0.42 mmol) was added to a solution Of TREN(I-Me-HOPO) 2 (0.341 g, 0.54 mmol) in CH 2 Cl 2 (20 ml). The resulting yellow solution was stirred under an atmosphere of N 2 until, after 2 days, the solution turned colorless. The product was purified by a gradient flash silica column (2-8% CH 3 OH in CH 2 Cl 2 ). The solvents were removed to yield a white solid (Yield: 0.91 g, 85%).
  • GdCl 3 -OH 2 O (0.043 g, 0.135 mmol) was added to a solution of TREN- l-Me-3,2-HOPO-TAM-PEG-2000 (0.35 g, 0.135 mmol) in H 2 O (10 ml). An excess of aqueous ammonia was added and the solution was heated to reflux for 2h. The solution was filtered and the solvents removed from the filtrate by rotary evaporation. The product was purified by elution down a Sephadex LH-20 column, after which the solution was added to a rapidly stirring solution Of Et 2 O (200 ml) to afford a pale yellow precipitate, which was filtered off and dried under vacuum.
  • Imidazole (0.058 g, 0.85mmol) was added to a solution of GdCl 3 -OH 2 O (0.060 g, 0.16 mmol) and TREN-HOPO-TAM-O 2 -NH 2 (0.150 g, 0.169 mmol) in H 2 O (10 ml). The resulting pale yellow solution was heated under reflux for 2 h. The solvent was removed to yield a yellow residue which was suspended in -PrOH, sonicated and filtered three times. The resulting light brown solid was dried under vacuum. (Yield: 0.1 17 g, 73%). Anal.
  • TAM-thiaz-ethanolamine (1.50 g, 2.81 mmol) in CH 2 Cl 2 (100 ml) was added dropwise to a rapidly stirring solution of tris(2-aminoethylamine) (TREN, 0.229 g, 1.57 mmol) in CH 2 Cl 2 (100 ml). Small extra portions of l-Bn-6-Me-3,2- HOPO-thiazolide were also added as deemed necessary by TLC.
  • the reaction mixture was purified by a gradient flash silica column (2-10% CH 3 OH and 1% Et 3 N in CH 2 Cl 2 ). The solvents were then removed to yield a colorless oil. (Yield: 0.63 g, 34 % relative to TREN).
  • l-Me-HOPO-3,2-thiazolide (Formula 4, Scheme 1) (3.00 g, 8.334 mmol) was dissolved in CH 2 CH 2 (50 ml) and i-PrOH (50 ml) and added to a solution of NaOH (0.350 g, 8.60 mmol) and glycine (0.626 g, 8.334 mmol) in water (3 ml). The yellow mixture was then stirred for 24h, during which time it turned colorless, and TLC indicated that the reaction was complete. The reaction mixture was purified by a gradient flash silica column (2-10% CH 3 OH in CH 2 CI 2 ).
  • N-hydroxysuccinimide (0.534 g, 4.64 mmol) was added to a solution of GIy-I -Me-HOPO (1.220 g, 3.87 mmol) in dry THF (60 ml) under a nitrogen atmosphere. After stirring for 20min, dicyclohexylcarbodiimide (DCC, 0.956, 4.64mmol) and dimethylaminopyridine (DMAP, 0.044 g, 0.46 mmol) were added. After 9h, a white precipitate of dicyclohexylurea (DCU) had formed and the formation of the NHS-activated ester was judged to be complete by TLC.
  • DCC dicyclohexylcarbodiimide
  • DMAP dimethylaminopyridine
  • TREN (0.141 g, 0.967 mmol, 0.25equiv.) was added. The solvent was evaporated and the residue taken up in a 1.0M aqueous solution of HCl (50ml). The suspension was filtered and the filtrate washed with CH 2 Cl 2 (2 x 50 ml). The combined organic fractions were then back-extracted once with 1.0 M HCl (50 ml). 1OM NaOH was added drop- wise to the combined aqueous fractions until the pH reached 1 1. The aqueous fractions were then extracted with CH 2 Cl 2 (10 x 50ml) and EtOAc (3 x 50ml). The organic extracts were combined, dried (Na 2 SO 4 ) and evaporated to dryness.
  • TREN-GIy- l-Me-3,2-HOPO-Bn (0.650 g, 0.624 mmol) was dissolved in a mixture of MeOH (325 ml) and EtOH (325 ml) and added to a slurry of 5% Pd on C (0.65 g) in EtOH (65 ml). The reaction was stirred under an atmosphere of hydrogen for 6h. The solution was filtered and the solvent removed by evaporation to afford a white solid (Yield: 0.296 g, 62%). This solid was found to be hygroscopic, and was therefore stored in a vacuum desiccator.
  • the fit is relatively poor, which may reflect a large degree of extra relaxivity due to the PEG chain organizing a network of hydrogen-bonded water molecules in the proximity of the metal center.
  • This "second-sphere" relaxivity cannot be accounted for quantitatively and will most likely be reflected in the values for X R or the zero-field value of the electronic relaxation time, xso being longer than in reality.
  • T M is an increase in T M as the PEG chain is lengthened, with values of 8 ⁇ 1, 19 ⁇ 2 ns and 31 ⁇ 2 ns for Gd-TREN-I- Me-3,2-HOPO-TAM and Gd-TREN- l-Me-3,2-HOPO-TAM-PEG-2000, Gd-TREN-I-Me- 3,2-HOPO-TAM-PEG-5000 respectively.
  • the optimal value for T M depends on several variables, in particular the field strength of the MRI scanner machine. Previous reports have suggested optimal ⁇ M values of a few tens of nanoseconds.
  • 35A (15.5 g, 0.1 mol) and anhydrous potassium carbonate (27.6 g, 0.2 mol) was mixed with benzyl chloride (15.2 g, 0.12 mol) in methanol (250 mL). The mixture was refluxed for 16 h, filtered, and the filtrate was evaporated to dryness. The residue was dissolved in water (50 mL) and acidified with 6 N HCl to pH 2. The resulting white precipitate was isolated by filtration, washed with cold water, and dried in vacuum to yield 22.3 g (91 %) of 36A, mp 176-177 0 C. Anal. Calc'd.
  • THPO-ethyl glycolate 34.Og, 0.195mol in ether ( 18OmL) was stirred with Na shot (2.24g, 0.0974mol) for 20hrs under a N 2 atmosphere, resulting in a yellow solution.
  • NaH (0.54 eq.) can be used in place of Na, but usually requires the addition of 2- 5% m/m EtOH to promote the reaction.
  • the ether was removed and the residue covered with abs. ethanol (2OmL).
  • An acetamidine solution was prepared from acetamidine.HCl (9.32g, 0.0989mol), which was stirred for 2hr in sodium ethoxide in ethanol (13OmL, by addition of 2.36g of Na).
  • (+)FABMS m/z 325 +.
  • 1 H NMR (CDCl 3 , 300Mhz): ⁇ 1.4 - 2.0 (m, 12H, 3,4,5-THP-CH 2 ), 2.42 (s, 3H, 2- CH 3 ), 3.50-356 (m, 211, 6-THP-CH 2 ), 3.86 -3.96 (m, 2H, 6-THP-CH 2 ), 4.44- 4.81(4 d's, 2H, CH 2 -OTHP), 4.79 (br m, IH, THP-CH), 5.83 (br m, I H, THP-CH), 1 1.44 (br s, IH, NH).
  • Method B A Hydrogen chloride/dioxane solution (5OmL, 4M) was dripped into a solution of the protected pyrimidine 30 (30. Ig, 89mmol) in EtOH (2OmL). A white solid rapidly precipitated and, after standing for a few hours at room temperature, the solid was filtered and washed with dioxane (3x1 OmL) and diethyl ether (2x1 OmL). Drying in vacuo overnight at 40 0 C gave 16.08g (88.8%) of 31»HC1. This absorbs l eq of H 2 O upon standing exposed in air. Mp: >200 0 C (dec). (+)FABMS: m/z 171 (MH] + .
  • PEG550-phthalimide (52B) [0396] PEG550-C1 (10.40 g, 18.0 mmol) and potassium phthalimide (10.74 g, 58.0 mmol) were dissolved in dry DMF (75 ml) and heated at 130 0 C for 42 h. The reaction mixture was filtered and the filtrate evaporated. The resulting yellow paste was partitioned between 1 : 1 CH 2 C1 2 :H 2 O (100 ml x 4), the organic component extracted and dried in MgSO4. Evaporation Of CH 2 Cl 2 yielded crude product which was purified twice by flash column chromatography (silica, eluent: CH 2 Cl 2 with increasing gradient of MeOH from 0 - 10%).
  • TREN-bis(HOPO-Bn)-(TAM-PEG550) (Formula 54, Scheme 10) [0400] TREN-bis(HOPO-Bn 2 )-(Bn 2 -TAM-PEG550) (53, 0.36 g, 0.22 mmol) was dissolved in acetic acid (10 ml) and then 12N HCl (10 ml) added to the solution. The solution was stirred in the dark at room temperature for 2 days. The solvents were evaporated yielding a yellow crystalline solid. MeOH (10 ml) was added and the solvent evaporated (x 3).
  • the isotopic abundance of the clusters are characteristic of mononuclear Gd-containing species.
  • the peaks are separated by 44 m/z units in the range 1284 - 1636 m/z, corresponding to 7 - 15 (CH 2 CH 2 O) units.
  • Table 3 shows some of the relaxivity properties of selected Gd complexes of the
  • ⁇ m The value of ⁇ m , was independently obtained by a variable temperature (VT) 17 O NMR measurement of the water nuclear transverse relaxation rate (R 2 ).
  • the VT 17 O NMR for 55 and 58 are shown in FIG. 20.
  • the data were measured at 2.1 T (90 MHz for the proton and 12 MHz for 17 O) and pH ⁇ 7.
  • the curve was analyzed in terms of the well established equations of Swift-Connick, rearranged in a form suitable for Gd. As an initial estimate of the values of the structural and dynamic parameters, those previously found for 47 were used in fitting the profile of 55.
  • a solution of 0.100 M KCl was prepared from 99.99% KCl (Fisher Scientific) and was used to maintain constant ionic strength during all titrations.
  • Carbonate-free 0.1 M KOH was prepared from Baker Dilut-It analytic concentrated KOH and was standardized against potassium hydrogen phthalate to a phenolphthalein endpoint.
  • Gadolinium(III) and zinc(II) solutions, each -0.100 M in metal ion, dissolved in -0.100 M HCl were prepared from anhydrous 99.99% chloride salts (Alpha). The metal ion content was checked by EDTA titration with Xylenol Orange as indicator using sodium acetate buffer.
  • the proton concentration of the standard solutions was checked by titration of a known volume of metal ion solution and a slight excess of EDTA (-1.005 eq.) to the equivalence point. (Harris et al., J. Am. Chem. Soc, 101:2722 (1979)). For all titrations, the observed pH was measured as - log[H + ].
  • the glass electrode was calibrated in hydrogen ion concentration units by titrating 2.000 mL of standardized HCl diluted in 50.00 mL of 0.100 M KCl, with 4.200 mL of standardized KOH. The calibration titration data were analyzed by a nonlinear least-squares program. (Martell, A. E.; Motekaitis, R. M, Determination and Use of Stability Constants; VCH: New York (1988)).
  • Potentiometric pH Titrations were performed using an automated apparatus consisting of a Accumet pH meter (models 925, 825MP or 15), a pH electrode (Orion Ross semi-micro combination, Cole Parmer semi-micro combination or Corning high performance combination electrodes), an autoburet (Metrohm 665 Dosimat or 702 SM Titrino) fitted with a 5 mL piston exchange unit and a jacketed Ar swept titration cell maintained at 25.0 0 C by a Lauda K-2/R or Neslab RTE-1 1 1 constant temperature circulating bath.
  • the electronic systems were integrated for automated collection with an IBM PC clone.
  • NMRD 1/Ti nuclear magnetic relaxation dispersion
  • the sample for the NMRD profile in blood serum was prepared by dissolving a 1 mol L " solution of the Gd(III) complex in a lyophilized serum of human origin (Seronorm I M , Nycomed) from controlled voluntary blood donors of Scandinavian blood banks.
  • Variable-temperature 17 O NMR measurements were recorded on a JEOL EX-400 (9.4 T) spectrometer, equipped with a 5 mm probe, by using D 2 O for external lock of the magnetic field. Experimental settings were: spectral width 10000 Hz, pulse width 7 ⁇ s, acquisition time 10 ms, 1000 scans and no sample spinning. The solution used contained ' 7 O enriched water (2.6%, Yeda, Israel). The observed transverse relaxation rates (R° 2obs ) were calculated from the linewidth of the resonance at half height.
  • NMR spectra were recorded on Bruker AMX 300, AMX 400 or DRX 500 spectrometers. Chemical shifts ( ⁇ ) are reported in ppm referenced to residual protio-solvent resonances. Melting points were obtained on a Buchi apparatus and are uncorrected. Electronic absorption spectra were recorded on a HP 8450A or HP 8452A UV-Vis diode array spectrophotometer with 1 cm quartz cells. Elemental analyses were performed by the Analytical Services Laboratory, College of Chemistry, University of California, Berkeley, CA. Mass spectra (F AB+ and El) were obtained by the Mass Spectrometry Laboratory at the College of Chemistry, University of California, Berkeley, CA.
  • the HOIQO chelators are attached to a TREN backbone.
  • these HOIQO chelators can be attached to other backbones described herein.
  • the biphasic system was cooled in an ice-bath and the pH was adjusted to 2 with 4 M HCl.
  • the resulting, slightly yellow solid was collected, washed with ice-cold water and Et 2 O, and dried in vacuo overnight at 6O 0 C.
  • the slightly yellow, solid product 2a was obtained as the hemihydrate (1 1.13 g, 91%).
  • the benzyl protected ligand 4a (880 mg, 900 ⁇ mol, 1.0 eq.) was dissolved in a mixture of 20 mL cone. HCl and 20 mL glacial acetic acid. The yellow solution was stirred at ambient temperature for 7 d and at 50 0 C for an additional 18 h. The mixture was filtered and the volatiles were removed under reduced pressure at room temperature. The red-brown residue was dissolved in 20 mL MeOH and the solvent was evaporated again. A minimum amount of MeOH, required to dissolve everything, was added. The yellow solution was filtered through a plug of cotton and treated with Et 2 O to precipitate the deprotected ligand. The precipitate was collected, washed with Et 2 O, and dried in vacuo. The product 5a was obtained as a light-pink solid (540 mg, 74%).
  • This acid chloride can be coupled with all suitable backbone moieties of the invention.
  • the backbone moiety is an amine and coupling can be accomplished by amide coupling procedures under standard conditions, such as those described herein (e.g., in Example 34 and 37).
  • the TREN backbone may be coupled to one or more of the above described sulfonic acid-derivatized monomers.
  • the resulting amides can be deprotected under aqueous acidic conditions to yield the water- soluble HOIQO chelators.
  • the benzyl protected ligand (630 mg, 470 ⁇ mol, 1.0 eq.) was dissolved in a mixture of 20 mL cone. HCl and 20 mL glacial acetic acid. The solution was stirred at ambient temperature for 1 13 h and at 50 0 C for additional 20 h. The mixture was filtered and the volatiles were removed under reduced pressure at room temperature. The colorless residue was suspended in 20 mL MeOH and stirred for 30 min. The solid was collected on a B ⁇ chner- funnel, washed with MeOH and dried in vacuo at 40 0 C for 15 h. The product was obtained as a pink solid (423 mg, 85%).
  • Cerium (Ce) Complex [0447] Starting with CeCl 3 * 6 H 2 O (9.6 mg, 25.6 ⁇ mol, 1.0 eq.), tren-tris(l,2-H0IQ0) * HCl * 2 H 2 O * MeOH (21.0 mg, 25.6 ⁇ mol, 1.0 eq.), pyridine (3 drops) in 5 mL MeOH (HPLC-grade) gave 12 mg (52%) complex.

Abstract

L'invention concerne une nouvelle catégorie de ligands polydentés fortement chélatants à base d'une structure d'acide 2-hydroxy-1-oxo-1,2-dihydro-isoquinoline-3-carboxylique (ci-après 1,2-HOIQO) en combinaison avec des squelettes de polyamines. Le groupe 1,2-HOIQO bidenté extrêmement stable peut être synthétisé par une méthodologie de synthèse standard sur une grande échelle. Une caractéristique avantageuse est la possibilité d'introduire une grande plage de substituants sur le noyau benzénique par des transformations standards (comme des substitutions aromatiques électrophiles). Cette caractéristique permet, par exemple, la mise au point de propriétés chimiques, photophysiques et de solubilité, mais aussi la fixation de groupes fonctionnels, utiles pour des applications de détection et d'imagerie (par exemple de l'ADN, des protéines, des anticorps, des fluorophores, etc.). La combinaison des chélateurs de 1,2-HOIQO à des squelettes de polyamines produit des ligands très fortement liants pour une variété de métaux, y compris les métaux de transition de la première rangée, les lanthanides, les actinides, etc. On s'attend à ce que les complexes métalliques de ce type soient utiles dans de nombreuses applications (par exemple, la luminescence, l'IRM, la séquestration des actinides, un traitement par chélation des métaux, le marquage par radio-isotopes métalliques, etc.).
PCT/US2007/066814 2006-04-17 2007-04-17 Agents chélateurs de 2-hydroxy-1-oxo-1,2-dihydro-isoquinoline WO2007121453A2 (fr)

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