WO2007022307A2

WO2007022307A2 - Ligands for metal ions and methods for making and using the same

Info

Publication number: WO2007022307A2
Application number: PCT/US2006/032043
Authority: WO
Inventors: Robert D. Hancock
Original assignee: University Of North Carolina At Wilmington
Priority date: 2005-08-16
Filing date: 2006-08-16
Publication date: 2007-02-22
Also published as: CA2619832A1; WO2007022307A3; MX2008002233A

Abstract

Ligands for metal ions are disclosed. Also disclosed are uses for the ligands and/or for coordination complexes containing the ligands. Representative uses include uses as imaging and/or contrast enhancement agents, as therapeutic agents, as fluorescent ligands for monitoring metal ions in biological systems, and as metal ion extractants.

Description

TITLE

LIGANDS FOR METAL IONS AND METHODS FOR MAKING AND USING THE SAME

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of and priority to U.S. Provisional Patent Application Serial No.60/708,637, filed August 16, 2005, the disclosure of which is incorporated herein by reference in its entirety.

TECHNICAL FIELD

Ligands for metal ions and their representative uses as an imaging and/or contrast enhancement agent, a therapeutic agent, a fluorescent ligand for monitoring metal ions in biological systems, and a metal ion extractant.

ABBREVIATIONS

BAPTA = 1 ,2-bis-2-aminophenoxyethane-

Λ/,Λ/,Λ/',Λ/'-tetraacetic acid

Ca = calcium

CHEF = chelation-enhanced fluorescence

CN. = coordination number

Cs = cesium

¹³⁷Cs = cesium

DMF = dimethylformamide

DMSO = dimethylsulfoxide

DPADA = dipyridinoacridinediacetate

DTPA = diethylenetriaminepentaacetate

EDTA = ethylenediaminetetraacetate

EtOAc = ethyl acetate

EtOH = ethanol

Fe = iron g = grams

Gd = gadolinium

Kd = dissociation constant kg = kilograms

La = lanthanum

M = molar

Mg = magnesium ml_ = milliliters mM = millimolar

Mn = manganese

MRI = magnetic resonance imaging

PDA = 1 ,10-phenanthroline-2,9-dicarboxylate

Pu = plutonium

Sr = strontium

UV = ultraviolet

Zn = zinc

BACKGROUND

The field of coordination chemistry, the study of compounds formed between metal ions and other neutral or negatively charged species, was pioneered by Alfred Werner, who won the Nobel Prize in 1913 for his coordination theory of transition metal-amine complexes. Since that time, the field has grown to include the development of a wide range of coordination complexes and their uses as analytical and bioanalytical tools, medical diagnostics, anti-cancer drugs, chelation therapy agents, and biomimetic models for the study of enzymes.

Key aspects in many of these analytical, bioanalytical and medical uses of coordination complexes are the selectivity of the chelating agent for the ion of interest and the solubility properties of the chelating agent and/or the coordination complex. For example, the use of chelating agents to detect and quantify Ca²⁺ in a living cell involves the selective recognition of Ca²⁺ in the presence of a number of other ion species, such as Mg²⁺, Mn²⁺, Zn²⁺, and Fe³⁺. Such agents also must possess the proper solubility to permeate cell membranes. Given the myriad uses of coordination complexes, an ability to fine-tune such properties to fit a specific use is an ever-present need in the art.

SUMMARY

The presently disclosed subject matter provides a family of metal-chelating ligands comprising a rigid fused-ring heteroaromatic backbone, wherein the size, Lewis base properties, and geometry of the metal binding site are tunable for different metal ions based on their size and coordination chemistries.

In some embodiments, the presently disclosed subject matter describes metal chelating ligands of Formula (I):

(Rn)_n,

D₁ -Ar -D, (I)

wherein Ar is an aromatic moiety, which in some embodiments, comprises at least two pyridine rings, and can be selected from the group consisting of:

Di and D₂ are independently selected from the group consisting Of-CO₂H, a carboxylate salt, an alkyl ester of -CO₂H, an acyloxymethyl ester of -CO₂H, -CH₂CO₂H, an acyloxymethyl ester Of-CH₂CO₂H, a salt Of-CH₂CO₂ ^", an alkyl ester of -CH₂CO₂H, -C(=O)NH₂, -CH₂C(=O)NH₂, phosphate, methyl phosphate, phosphonate, methyl phosphonate, amino, alkylamino, dialkylamino, an amine salt, an amino-substituted alkyl, the salt of an amino-substituted alkyl, hydroxyl, alkylhydroxyl, alkoxyl, mercapto, and alkylmercapto; m is an integer corresponding to the number of sites on a given Ar that can bear an additional substituent (or the number of H atoms on the Ar portion of the Di-Ar-D₂ molecule); each n is an integer between 1 and m; and each R is independently selected from the group consisting of H, alkyl, substituted alkyl, branched alkyl, perfluoroalkyl, halo, nitro, cyano, amino, thio, alkylamino, dialkylamino, alkylthio, alkoxyl, sulfate, carboxylate, carbamoyl, alkylcarbamoyl, dialkylcarbamoyl, acyloxyl, acylamino, aroylamino, phosphate, substituted sulfonate, sulfonic acid, aryl, substituted aryl, aralkyloxyl, aryloxyl, arylthio, alkoxycarbonyl, and cycloalkyl; wherein any two R groups on adjacent carbons optionally can form an additional fused ring or ring system or wherein any two R groups substituted on the same carbon can together be (=0); subject to the proviso that when Ar is:

Di and D₂ are not one Of-CO₂H, a carboxylate salt, an alkyl ester Of-CO₂H, an acyloxymethyl ester Of -CO₂H, and C(=O)NH₂. In some embodiments, the presently disclosed subject matter also describes the metal complexes formed between ligands of Formula (I) and alkali and alkaline metal ions, transition metal ions, lanthanide metal ions, and actinide metal ions. In some embodiments, the presently disclosed subject matter also describes the conjugates formed between ligands of Formula (I) or metal complexes of ligands of Formula (I) and carriers, substrates and targeting moieties. In some embodiments, the presently disclosed subject matter provides compounds for use in medical diagnostics, such as, but not limited to, metal complexes for magnetic resonance imaging (MRI), complexes of radioisotopes, such as, but not limited to, ¹⁸F, ^99mTc, ¹¹¹In, ⁶⁷Ga, ²¹²Bi and ⁶⁴Cu, for use in positron emission tomography (PET), radioscintigraphy, and the like. In some embodiments, the presently disclosed subject matter provides compounds for use in biological research, such as, but not limited to, compounds for detecting and/or quantifying metal ions in biological samples.

In some embodiments, the presently disclosed subject matter provides ligands for complexing and recovering metal ions through ion exchange or solvent extraction. In some embodiments, the metal ions are economically valuable metal ions from mining and/or other operations.

In some embodiments, the presently disclosed subject matter provides ligands for complexing radioactive metal ions, such as, but not limited to, ligands for reprocessing nuclear waste and/or for decontaminating environmental samples contaminated by nuclear waste or nuclear fall-out. In some embodiments, the presently disclosed subject matter provides ligands for chelation therapy to treat radiation sickness or other metal intoxication, such as poisoning with mercury, lead, cadmium and the like.

Accordingly, it is an object of the presently disclosed subject matter to provide a metal-chelating ligand and uses thereof. It is another object of the presently disclosed subject matter to provide a metal complex for use in medical diagnostics, such as an imaging and/contrast enhancement agent for magnetic resonance imaging (MRI), positron emission tomography (PET), radioscintigraphy, and the like. It is another object of the presently disclosed subject matter to provide a metal complexing ligand for detecting and/or quantifying metal ions in biological samples. It is another object of the presently disclosed subject matter to provide a ligand for complexing radioactive or other toxic metal ions. It is another object of the presently disclosed subject matter to provide a ligand for chelation therapy to treat metal intoxication.

Certain objects of the presently disclosed subject matter having been stated hereinabove, which are addressed in whole or in part by the presently disclosed subject matter, other objects and aspects will become evident as the description proceeds when taken in connection with the accompanying Examples as best described herein below.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1A is a space-filling molecular model of the complex between 1 ,10- phenanthroline-2,9-dicarboxylate (PDA) and a calcium cation (Ca²⁺). As indicated by the arrows, the small, light grey colored, hemispherical atoms are hydrogen. The medium grey, medium-sized atoms are carbons. The two wedge-shaped, dark grey atoms are nitrogens. The four hemispherically shaped, dark grey atoms are the oxygen atoms of the PDA carboxylate groups. The large, circular atom is a Ca²⁺ atom. The Ca²⁺ atom is of a size where contact with the oxygen and nitrogen donor atoms can occur.

Figure 1 B is a space-filling molecular model of the complex between PDA and a magnesium cation (Mg²⁺). As indicated by the arrows, the small, light grey, hemispherical atoms are hydrogens. The medium grey, medium-sized atoms are carbons. The two wedge-shaped, dark grey atoms are nitrogen atoms. The four hemispherically shaped, dark grey atoms are the oxygen atoms of the PDA carboxylate groups. The circular atom is a Mg²⁺ atom. As depicted, the Mg²⁺ atom is too small to make contact with the four donor atoms.

Figure 2A is a ball-and-stick molecular model of a gadolinium (Gd³⁺) PDA complex, showing the five coordinated water molecules. The small, light grey colored atoms are hydrogens. The small, medium grey colored atoms are carbons. The dark grey atoms are oxygens or nitrogens. The large, light grey atom is a Gd³⁺ atom. There are five water molecules occupying binding sites on the Gd³⁺ atom. Figure 2B is a ball-and-stick molecular model of a Gd³⁺ dipyridinoacridinediacetate (DPADA) complex, showing four coordinated water molecules. The small, light grey colored atoms are hydrogens. The small, medium grey colored atoms are carbons. The dark grey atoms are oxygens or nitrogens. The large, light grey atom is a Gd³⁺ atom. There are four water molecules occupying binding sites on the Gd3+ atom.

Figure 3 is a fluorescence image showing the presence of Ca²⁺ in a rat neuron as detected by the ligand (1-[2-(5-carboxyoxal-2-yl)-6-aminobenzofuran-5- oxyl]-2-(2'-amino-5'-methylphenoxy)ethane-Λ/,Λ/,Λ/',Λ/-tetraacetic acid (Fura-2).

DETAILED DESCRIPTION

The presently disclosed subject matter will now be described more fully hereinafter with reference to the accompanying Examples, in which representative embodiments are shown. The presently disclosed subject matter can, however, be embodied in different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the embodiments to those skilled in the art.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this presently described subject matter belongs. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.

Throughout the specification and claims, a given chemical formula or name shall encompass all optical and stereoisomers, as well as racemic mixtures where such isomers and mixtures exist.

L Definitions

As used herein, the term "ligand" refers generally to a species, such as a molecule or ion, which interacts, e.g., binds, in some way with another species.

More particularly, a "ligand" refers to a molecule or ion that binds a metal ion in solution to form a "coordination complex." See Martell, A. E., and Hancock, R. P., Metal Complexes in Aqueous Solutions, Plenum: New York (1996), which is incorporated herein by reference in its entirety.

The term "chelating agent" refers to a molecule or molecular ion having two or more unshared electron pairs available for donation to a metal ion. In some embodiments, the metal ion is coordinated by two or more electron pairs to the chelating agent. The terms "bidentate chelating agent," "tridentate chelating agent," "tetradentate chelating agent," and "pentadentate chelating agent" refer to chelating agents having two, three, four, and five electron pairs, respectively, available for simultaneous donation to a metal ion coordinated by the chelating agent. In some embodiments, the electron pairs of a chelating agent form coordinate bonds with a single metal ion. In some embodiments, the electron pairs of a chelating agent form coordinate bonds with more than one metal ion, with a variety of binding modes being possible. The term "donor group" refers to a moiety having an unshared pair of electrons available for donation to a metal ion. In some embodiments, the presently disclosed ligands optionally can have a donor group that is part of a ring structure, such a nitrogen atom of a heteroaromatic ring structure, or a "non-ring" donor group associated with a substituent group attached to a ring structure. The term "coordination" refers to an interaction in which one multi-electron pair donor coordinately bonds, i.e., is "coordinated," to one metal ion.

The term "coordinate bond" refers to an interaction between an electron pair donor and a coordination site on a metal ion resulting in an attractive force between the electron pair donor and the metal ion. The use of this term is not intended to be limiting, in so much as certain coordinate bonds also can be classified as have more or less covalent character (if not entirely covalent character) depending on the characteristics of the metal ion and the electron pair donor.

The term "coordination site" refers to a point on a metal ion that can accept an electron pair donated, for example, by a chelating agent. The term "free coordination site" refers to a coordination site on a metal ion that is either vacant or is occupied by a species that is weakly donating, such that the weakly donating species can be displaced by another species.

The term "coordination number" refers to the number of coordination sites on a metal ion that are occupied by donor atoms from one or more ligands.

The term "coordination geometry" refers to the manner in which coordination sites and free coordination sites are spatially arranged around a metal ion. Examples of coordination geometry include, but are not limited to, octahedral, square planar, trigonal, trigonal bipyramidal, and the like. The term "complex" refers to a compound formed by the union of one or more electron-rich and electron-poor molecules or atoms capable of independent existence with one or more electronically poor molecules or atoms, each of which also is capable of independent existence.

A "coordination complex" is one type of complex, in which there is a coordinate bond between a metal ion and an electron pair donor. A "transition metal complex" is a coordination complex in which the metal ion is a transition metal ion. The term "coordination complex" as used herein also is taken to include complexes formed between alkali or alkaline metal ions and electron pair donors and complexes formed between metal ions of the rare earth elements (e.g., the lanthanides and actinides) and electron pair donors.

In some embodiments, a coordination complex can be understood to comprise (i) one or more metal ions, which, in some embodiments, may or may not be the same atom, have the same charge, coordination number or coordination geometry, and the like; and (ii) one or more ligands or chelating agents that form coordinate bonds with the one or more metal ions.

In general, the terms "compound," "composition," "agent," and the like as provided herein include complexes, coordination complexes, or the ligands and chelating agents that form complexes or coordination complexes.

In some embodiments, the "metal complex" is charged. That is the metal ion and the ligand or chelating agent, in the aggregate, are not neutral. In such embodiments, the metal complex typically is associated with one or more counterions to form a neutral compound. Depending on how the term "coordination complex" is used, such counterions, in some embodiments, are considered part of the coordination complex and, in some embodiments, such counterions are not considered part of the coordination complex. Counterions generally do not form coordinate bonds to the metal ion, although they can be associated, often in the solid state, with the metal ion or ligand that make up the coordination complex. Examples of counterions include, but are not limited to, monoanions, such as nitrate, chloride, tetrafluoroborate, hexafluorophosphate, and mono-carboxylates having the general formula RC(=O)O^', and dianions, such as sulfate.

Additionally, a coordination complex can be solvated. The term "solvated" refers to the association of a coordination complex and another molecule, wherein in some embodiments, the other molecule is a solvent, which, in some embodiments, is water. The term "heteroatom" refers to an atom of any element other than carbon or hydrogen. Examples of heteroatoms, include, but are not limited to, nitrogen, oxygen, boron, phosphorous, sulfur, and selenium.

It will be understood that the terms "substituted" or "substituted with" and grammatical variations thereof include the implicit proviso that such substitution is in accordance with the permitted valence of the substituted atom and the substituent, and that the substitution results in a stable compound, e.g., a compound that does not spontaneously undergo transformation, such as by rearrangement, cyclization, elimination, or other reaction.

Further, the term "substituted" and grammatical variations thereof is meant to include all permissible substituents of organic compounds. Permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic substituents of organic compounds. The permissible substituents can be one or more and the same or different for appropriate organic compounds. For example, a heteroatom, such as nitrogen, can have hydrogen substituents and/or any permissible substituents of organic compounds as described herein that satisfy the valences of the heteroatom. The term "imaging agent" refers to a composition capable of generating a detectable image upon binding with a target and includes contrast agents for Magnetic Resonance Imaging (MRI).

The term "radiopharmaceutical" refers to a radioactive compound used in therapy or in diagnosis, such as positron emission tomography (PET) and radioscintigraphy. In some embodiments, such radiopharmaceutical compounds comprise radioisotopes including, but not limited to, ^99mTc, ¹¹¹In, ⁶⁷Ga, ²¹²Bi and

⁶⁴Cu.

The term "radionuclide" refers to an isotope of artificial or natural origin that exhibits radioactivity. Radionuclides can serve, for example, as agents, e.g., a contrast enhancement or imaging agent, in nuclear medicine.

The terms "subject," "patient," and "host" are used interchangeably and can refer to either a human or non-human animal to which a compound of the presently disclosed subject matter is administered. The terms "parenteral administration" and "administered parenterally" refer to modes of administration other than enteral and topical administration, in some embodiments, by injection, and includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intra- articulare, subcapsular, subarachnoid, intraspinal, and intrasternal, and infusion.

As used herein the term "alkyl" refers to Ci_₂o inclusive, linear {i.e., "straight- chain"), branched, or cyclic, saturated or at least partially and in some cases fully unsaturated {i.e., alkenyl and alkynyl) hydrocarbon chains, including for example, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, terf-butyl, pentyl, hexyl, octyl, ethenyl, propenyl, butenyl, pentenyl, hexenyl, octenyl, butadienyl, propynyl, butynyl, pentynyl, hexynyl, heptynyl, and allenyl groups. "Branched" refers to an alkyl group in which a lower alkyl group, such as methyl, ethyl or propyl, is attached to a linear alkyl chain. "Lower alkyl" refers to an alkyl group having 1 to about 8 carbon atoms {i.e., a Ci_-8 alkyl), e.g., 1 , 2, 3, 4, 5, 6, 7, or 8 carbon atoms. "Higher alkyl" refers to an alkyl group having about 10 to about 20 carbon atoms, e.g., 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbon atoms. In certain embodiments, "alkyl" refers, in particular, to C_1-8 straight-chain alkyls. In other embodiments, "alkyl" refers, in particular, to Ci_-8 branched-chain alkyls.

Alkyl groups can optionally be substituted (a "substituted alkyl") with one or more alkyl group substituents, which can be the same or different. The term "alkyl group substituent" includes but is not limited to alkyl, substituted alkyl, halo, nitro, cyano, hydroxyl, thio, amino, alkylamino, dialkylamino, aryl, arylamino, acyl, aryloxyl, alkoxyl, alkylthio, arylthio, aralkyloxyl, aralkylthio, carboxyl, alkoxycarbonyl, acyloxycarbonyl, carbamoyl, alkylcarbamoyl, dialkylcarbamoyl, and cycloalkyl. "Alkyl group substituent" also can include a sulfate or phosphate group.

Thus, as used herein, the term "substituted alkyl" includes alkyl groups, as defined herein, in which one or more atoms or functional groups of the alkyl group are replaced with one or more atoms or functional groups, including for example, alkyl, substituted alkyl, halogen (e.g. ,-CH₂X, -CHX₂, and -CX₃, wherein X is a halogen selected from the group consisting of Cl, Br., F, and I), aryl, substituted aryl, alkoxyl, hydroxyl, nitro, amino, alkylamino, dialkylamino, sulfate, and mercapto.

The term "perfluoroalkyl" as used herein to describe an alkyl group substituent refers to an alkyl group in which every hydrogen (H) atom has been replaced with a fluorine (F) atom.

The term "aryl" is used herein to refer to an aromatic substituent that can be a single aromatic ring, or multiple aromatic rings that are fused together, linked covalently, or linked to a common group, such as, but not limited to, a methylene or ethylene moiety. The common linking group also can be a carbonyl, as in benzophenone, or oxygen, as in diphenylether, or nitrogen, as in diphenylamine.

The term "aryl" specifically encompasses heterocyclic aromatic compounds. The aromatic ring(s) can comprise phenyl, naphthyl, biphenyl, diphenylether, diphenylamine and benzophenone, among others. In particular embodiments, the term "aryl" means a cyclic aromatic comprising about 5 to about 24 carbon atoms, e.g., 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, or 24 carbon atoms, and including 5- and 6-membered hydrocarbon and heterocyclic aromatic rings and fused or linked systems made up of 5- and 6-membered hydrocarbon and heterocyclic aromatic rings.

The aryl group can be optionally substituted (a "substituted aryl") with one or more aryl group substituents, which can be the same or different, wherein "aryl group substituent" includes alkyl, substituted alkyl, aryl, substituted aryl, aralkyl, hydroxyl, cyano, alkoxyl, aryloxyl, aralkyloxyl, carboxyl, acyl, halo, nitro, alkoxycarbonyl, aryloxycarbonyl, aralkoxycarbonyl, acyloxyl, acylamino, aroylamino, carbamoyl, alkylcarbamoyl, dialkylcarbamoyl, arylthio, alkylthio, alkylene, and -NR¹R", wherein R¹ and R" can each be independently hydrogen, alkyl, substituted alkyl, aryl, substituted aryl, and aralkyl.

Thus, as used herein, the term "substituted aryl" includes aryl groups, as defined herein, in which one or more atoms or functional groups of the aryl group are replaced with another atom or functional group, including for example, alkyl, substituted alkyl, halogen, aryl, substituted aryl, alkoxyl, hydroxyl, nitro, amino, alkylamino, dialkylamino, sulfate, and mercapto.

Specific examples of aryl groups include, but are not limited to, cyclopentadienyl, phenyl, furan, thiophene, pyrrole, pyran, pyridine, imidazole, benzimidazole, isothiazole, isoxazole, pyrazole, pyrazine, triazine, pyrimidine, quinoline, isoquinoline, indole, carbazole, and the like. As used herein, the term "aza" refers to a heterocyclic ring structure containing at least one nitrogen atom. Specific examples of aza groups include, but are not limited to, pyrrolidine, piperidine, quinuclidine, pyridine, pyrrole, indole, purine, pyridazine, pyrimidine, and pyrazine.

A fused-ring structure represented by a formula such as:

can be substituted by "n" number of "R" groups, wherein "n" is an integer from 0 to the number of carbon atoms available for substitution on the ring. When the integer "n" is 0, the structure is not substituted by an "R" group. When "n" is an integer equal to or greater than one, each "R" group is substituted on a carbon of the ring, thereby replacing a hydrogen atom that would be bonded to that carbon in the absence of the "R" group. Each "R" group, if more than one, is substituted on an available carbon on the ring structure rather than on another "R" group. In the case of a fused ring cyclic system as provided immediately hereinabove, the "R" group can be substituted on any otherwise unsubstituted carbon atom through the fused system. Thus, in the case of the preceding example, the fused ring structure is substituted at carbons 2 and 9 by substituents X and Y, respectively. Thus, in cases where "n" is one, the "R" substituent can be bound to any available carbon on the ring structure not occupied by another designated substituent, e.g., carbon 2 substituted by X and carbon 9 substituted by Y.

When a named atom of an aromatic ring or a heterocyclic aromatic ring is defined as being "absent," the named atom is replaced by a direct bond. When the linking group or spacer group is defined as being absent, the linking group or spacer group is replaced by a direct bond.

"Alkylene" refers to a straight or branched bivalent aliphatic hydrocarbon group having from 1 to about 20 carbon atoms, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbon atoms. The alkylene group can be straight, branched or cyclic. The alkylene group also can be optionally unsaturated and/or substituted with one or more "alkyl group substituents." There can be optionally inserted along the alkylene group one or more oxygen, sulfur or substituted or unsubstituted nitrogen atoms (also referred to herein as

"alkylaminoalkyl"), wherein the nitrogen substituent is alkyl as previously described.

Exemplary alkylene groups include methylene (-CH₂-); ethylene (-CH₂-CH₂-); propylene (-(CH₂)H; cyclohexylene (-C₆H₁₀-); -CH=CH-CH=CH-; -CH=CH-

CH₂-; -(CH₂)_q-N(R)-(CH₂)ι-, wherein each of q and r is independently an integer from 0 to about 20, e.g., 0, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17,

18, 19, or 20, and R is hydrogen or lower alkyl; methylenedioxyl (-0-CH₂-O-); and ethylenedioxyl (-0-(CH₂J₂-O-). An alkylene group can have about 2 to about 3 carbon atoms and can further have 6-20 carbons.

As used herein, the term "acyl" refers to an organic acid group wherein the -OH of the carboxyl group has been replaced with another substituent (i.e., as represented by RC(=O) — , wherein R is an alkyl or an aryl group as defined herein). As such, the term "acyl" specifically includes arylacyl groups, such as a phenacyl group. Specific examples of acyl groups include acetyl and benzoyl.

"Cyclic" and "cycloalkyl" refer to a non-aromatic mono- or multicyclic ring system of about 3 to about 10 carbon atoms, e.g., 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms. The cycloalkyl group can be optionally partially unsaturated. The cycloalkyl group also can be optionally substituted with an alkyl group substituent as defined herein. There can be optionally inserted into the cyclic alkyl chain one or more oxygen, sulfur or substituted or unsubstituted nitrogen atoms, wherein the nitrogen substituent is hydrogen, alkyl, substituted alkyl, aryl, or substituted aryl, thus providing a heterocyclic group. Representative monocyclic cycloalkyl rings include cyclopentyl, cyclohexyl, and cycloheptyl. Multicyclic cycloalkyl rings include adamantyl, octahydronaphthyl, decalin, camphor, camphane, and noradamantyl. "Alkoxyl" or "alkoxyalkyl" refer to an alkyl-O- or alkyl-O-alkyl- group respectively, wherein alkyl is as previously described. The term "alkoxyl" as used herein can refer to C₁-₂₀ inclusive, linear, branched, or cyclic, saturated or unsaturated oxo-hydrocarbon chains, including, for example, methoxyl, ethoxyl, propoxyl, isopropoxyl, butoxyl, f-butoxyl, and pentoxyl.

"Aryloxyl" refers to an aryl-O- group wherein the aryl group is as previously described, including a substituted aryl. The term "aryloxyl" as used herein can refer to phenyloxyl or napthyloxyl, and alkyl, substituted alkyl, halo, or alkoxyl substituted phenyloxyl or napthyloxyl.

"Aralkyl" refers to an aryl— alkyl— group wherein aryl and alkyl are as previously described, and include substituted aryl and substituted alkyl. Exemplary aralkyl groups include benzyl, phenylethyl, and naphthylmethyl.

"Aralkyloxyl" refers to an aralkyl-O- group wherein the aralkyl group is as previously described. An exemplary aralkyloxyl group is benzyloxyl.

"Dialkylamino" refers to an -NRR¹ group wherein each of R and R' is independently an alkyl group and/or a substituted alkyl group as previously described. Exemplary dialkylamino groups include ethylmethylamino, dimethylamino, diethylamino, and diisopropylamino.

Since the term "alkylamino" might be taken to mean a substituent such as - NHR, the term "amino-substituted alkyl" will be used to avoid confusion when the substituent -alkyl-NRR' is intended. Thus an exemplary amino-substituted alkyl group is -CH₂NH₂.

"Alkoxycarbonyl" refers to an alkyl-O-C(=O)- group. Exemplary alkoxycarbonyl groups include methoxycarbonyl, ethoxycarbonyl, butyloxycarbonyl, and f-butyloxycarbonyl.

"Aryloxycarbonyl" refers to an aryl-O-C(=O)- group. Exemplary aryloxycarbonyl groups include phenoxy- and naphthoxy-carbonyl.

"Aralkoxycarbonyl" refers to an aralkyl-O-C(=O)- group. An exemplary aralkoxycarbonyl group is benzyloxycarbonyl.

An "alkyl ester" of a carboxylic acid refers to the -C(=O)-OR group wherein R is alkyl as previously described. Thus, an alkyl ester can refer to, for example, a methyl ester, an ethyl ester, a propyl ester, an isopropyl ester, a butyl ester, a sec- butyl ester, a tert-butyl ester, and the like. The term "acyloxymethyl ester of a carboxylic acid" refers to the -C(=O)-O- CH₂-O-C(=O)-R group, wherein R is an alkyl, substituted alkyl, aryl or substituted aryl group as previously described.

"Carbamoyl" refers to an H₂N-C(=O)- group. "Alkylcarbamoyl" refers to a R'RN-C(=O)- group wherein one of R and R¹ is hydrogen and the other of R and R¹ is alkyl and/or substituted alkyl as previously described.

"Dialkylcarbamoyl" refers to a R'RN-C(=O)- group wherein each of R and R¹ is independently alkyl and/or substituted alkyl as previously described. "Acyloxyl" refers to an acyl-O- group wherein acyl is as previously described.

"Acylamino" refers to an acyl-NH- group wherein acyl is as previously described.

"Aroyl" refers to an aryl-C(=O)- group. "Aroylamino" refers to an aroyl-NH- group wherein aroyl is as previously described.

The term "amino" refers to the -NH₂ group, as well as to alkylamino groups (-NHR, wherein R is alkyl) and dialkylamino groups, as described previously. "Amino" can also refer to nitrogen groups substituted with one or two aryl groups. The term "carbonyl" refers to the -(C=O)- group.

The term "carboxyl" refers to the -C(=O)OH group.

The term "carboxylate" refers to the anion of a carboyl group (i.e., a - C(=O)O^" group).

The terms "halo", "halide", or "halogen" as used herein refer to fluoro, chloro, bromo, and iodo groups.

The terms "hydroxyl" or "hydroxyl" refer to the -OH group.

The term "hydroxyalkyl" refers to an alkyl group substituted with an -OH group, such as a -CH₂OH group.

The term "mercapto" refers to the -SH group. The term "alkylmercapto" refers to an alkyl group substituted at any suitable position by an -SH group, such as a -CH₂SH group. The term "oxo" refers to a compound described previously herein wherein a carbon atom is replaced by an oxygen atom.

The term "nitro" refers to the -NO₂ group.

The term "cyano" or "nitrile" refers to the -CΞN group. The term "thio" refers to a compound described previously herein wherein a carbon or oxygen atom is replaced by a sulfur atom.

The term "sulfate" refers to the -SO₄ group.

The term "sulfonate" refers to the -SO₃ group.

The term "phosphate" refers to the -PO₄ group. The term phosphate includes fully protonated, partially protonated, and non-protonated derivatives of phosphoric acid. Thus, phosphates include hydrogen phosphates (i.e., -O-

P(=O)(OH)(O^') groups), dihydrogen phosphates, (i.e. ,-O-P(=O)(OH)₂ groups) and phosphate dianions (i.e., -O-P(=O)(O^")₂ groups). The number of protons can vary depending on the pH conditions. The term phosphate also includes phosphate salts, wherein one or more suitable cationic group, such as, for example, an ammonium group or sodium ion, is associated with a negative charge or charges on the phosphate group. The term "methyl phosphate" refers to a phosphate group substituent attached to the structure being substituted via a methylene linker. Thus, methyl phosphate refers to groups including: the -CH₂-O- P(=O)(OH)₂ group, the -CH₂-O-P(=O)(O^")₂ group, and -CH₂-O-P(=O)(OH)(O^").

The term "phosphonate" refers to the -PO₃ group. The term phosphonate refers to fully protonated, partially protonated and non-protonated phosphonates. Thus, phosphonates include hydrogen phosphonates (i.e.,-P(=O)(OH)(O^") groups); dihydrogen phosphonates (i.e.,-P(=O)(OH)₂ groups); and phosphonate dianions (i.e., -P(=O)(O^")₂ groups). The number of protons associated with the phosphonate group can vary depending on pH. The term phosphonate also includes phosphonate salts, wherein one or more suitable cationic group, such as, for example, an ammonium group or sodium ion, is associated with a negative charge or charges on the phosphate group. The term "methyl phosphonate" refers to a phosphonate group substituent attached to the structure being substituted through a methylene linker. Thus, methyl phosphate refers to groups including: the -CH₂-P(=O)(OH)₂ group, the -CH₂-P(=O)(O^')₂ group, and the -CH₂- P(=O)(OH)(O^") group.

An "alkyl ester of a phosphate" refers to a phosphate as described above wherein either or both of the protonate-able oxygens is substituted with an alkyl group, wherein alkyl is as defined hereinabove. Thus, an alkyl ester of a phosphate is a -O-P(=O)(OR)(OR') group, wherein R and R' are independently selected from H and alkyl, so long as at least one of R and R' is alkyl. When both R and R' are alkyl, they can be the same alkyl group (i.e., both R and R' can be methyl) or different alkyl groups (i.e., R can be methyl and R' can be ethyl). An "alkyl ester of a phosphonate" refers to a -P(=O)(OR)(OR') group, wherein R and R' are independently selected from H and alkyl so long as at least one of R and R¹ is alkyl. When both R and R' are alkyl, they can be the same alkyl group (i.e., both R and R' can be methyl) or different alkyl groups (i.e., R can be methyl and R' can be ethyl). When the term "independently selected" is used, the substituents being referred to (e.g., R groups, such as groups R₁ and R₂, or groups X and Y), can be identical or different. For example, both Ri and R₂ can be substituted alkyls, or Ri can be hydrogen and R₂ can be a substituted alkyl, and the like.

A named "R", "D," or "Ar" group will generally have the structure that is recognized in the art as corresponding to a group having that name, unless specified otherwise herein. Forthe purposes of illustration, certain representative "R," "X," "Y", and "A" groups as set forth above are defined below. These definitions are intended to supplement and illustrate, not preclude, the definitions that would be apparent to one of ordinary skill in the art upon review of the present disclosure.

As used herein, the variable "M", when used in a chemical formula, represents a metal atom.

]L General Considerations 1 ,10-Phenanthroline is an Λ/-heterocyclic chelating agent with a rigid planar structure that has been used in all aspects of coordination chemistry, as well as in the design of metalloenzyme models, self-assembling systems and in metal complexes with anti-cancer activity. See Moghimi, A., et al., Inorg. Chem., 42, 1616-1624, (2003); Bretonniere. Y.. et al.. Inorg. Chem., 39, 3499-3505 (2000). The dicarboxylate of 1 ,10-phenanthroline, 1 ,10-phenanthroline-2,9-dicarboxylic acid (PDA), often has been employed to incorporate 1 ,10-phenanthroline moieties in multidentate chelating agents. For example, PDA can be used in the synthesis of phenanthrolino-18-crown-6 diester ligands. See Wang, T., J., et al., J. Heterocyclic Chem., 31(1), 1-10 (1994).

PDA PDA also forms a number of coordination complexes itself. Spectroscopic studies of PDA complexes with Eu and Fe species have been reported. See Dyson, R. M. et aL Polyhedron, 18, 3243-3251 (1999); Kόnig, E., and Ritter, G., J. Inorg. Nucl. Chem., 43, 2273-2280 (1981); Sammes, P. G., et al., J. Chem. Soc. Chem. Commun., 1282-1283, (1992); Templeton, E. F. G., and Pollak. A., J. Lumin., 43, 195-205, (1989). The crystal structure of PDA and Mg²⁺ also has been reported. See Park, K.-M., et al., Acta Cryst, E57, m154-m156, (2001). Additionally, the synthesis, characterization, crystal structure, and solution studies of a PDA complex with Co²⁺ has been reported. See Moghimi et al., Inorg. Chem., 42, 1616-1624 (2003). The rigidity of the phenanthroline ring structure is one parameter in determining which ions phenanthroline-based chelating agents will preferably bind. Without wishing to be bound to any one particular theory, it is suggested that the distance between the donor groups (i.e., the N atoms of the heteroaromatic, e.g., pyridine, ring system) is more rigorously predetermined than in the case of more flexible polyalkylamines. PDA has the shape and dimensions to preferentially form a complex with metal ions of about the same size as Ca²⁺, La³⁺, and Gd³⁺. Because of the rigidity of PDA, many unwanted metal ions, such as Mg²⁺, which might interfere with Ca²⁺, and Zn²⁺, which might interfere with Gd³⁺, are too small to contact all four donor atoms available in PDA and are therefore complexed only very weakly by PDA. Demonstrating this, Figures 1A and 1 B show the spacefilling molecular models of PDA complexes with Ca²⁺ and Mg²⁺, respectively.

ILL Novel Metal Complexing Liqands

The presently disclosed subject matter provides, in some embodiments, a family of metal chelating ligands having a fused heteroaromatic ring system. In some embodiments, variation in the structure of the non-ring donor groups (i.e., the chelating groups pendant to the ring system) allows for the formation of ligands with different size metal binding sites. Changes in the fused heteroaromatic ring system, i.e., the backbone, also can affect the bonding angles of the ligand. Further, variation in the non-ring donor groups and in the electronic nature of the substituent groups along the ligand backbone can affect the electron donating ability of the N atoms of the pyridine rings, which, in turn, can affect complex stability. Thus, the presently disclosed subject matter provides a ligand structure that leads to variants which allow for an increase in complex stability and selectivity for metal ions on the basis of the size and coordination chemistry of the metal ion.

In some embodiments, the carboxylate groups of 1 ,10-phenanthroline-2,9- dicarboxylate (PDA) can be replaced with donor groups, such as amides (see, for example, Compound a) and aminomethyl groups (see, for example, Compound b).

compound a

compound b

Further, the ring system can be extended to accommodate phenolate groups (see, for example, Compound c). Also, one or more of the ring structures comprising the ring system can be reduced to change the angle of the donor groups (see, for example, Compound d). Accordingly, the ring system of the presently disclosed metal complexing ligands can comprise a variety of ring structures to increase the angle of the donor groups to complex large metal ions (see, for example, Compound e).

compound c compound d

compound e

Additionally, in some embodiments, the ring system can be functionalized to modify the solubility of the ligand or to alter the electronic environment of the metal ion binding group(s). Alternatively, a ring substituent can be used as a reactive site to further modify the ligand or to attach the ligand to a carrier, substrate or targeting moiety. For example, Compound f can be reacted with ethylenediamine to give Compound g, a ligand comprising an additional fused aromatic ring.

compound f compound g

Alternatively, substitution with a water-solubilizing group such as a sulfonic acid, a salt of a sulfonic acid, an amine, the salt of an amine, carboxy, carboxyalkyl, carboxyalkoxy, and the like provides a ligand with greater aqueous solubility. An example of a ligand with a water-solubilizing group is compound h, below.

compound h Typically, the substitution of alkyl, alkoxy, perfluoroalkyl, cyano, dialkylamino, aryl, heteroaryl, or alkyl ester groups can be used to confer more solubility to the ligands in non-polar solvents and/or make the ligands more lipophilic. In particular, ring substituents that are 1 -(acyloxy)alkyl esters of carboxylic acid (such as an acetyloxymethyl ester) are readily hydrolyzable esters that can confer solubility or cell membrane permeability to the ligands. The esters are readily cleaved by intracellular esterases. See U.S. Patent No. 6,013,802 to Hoyland, B. M.. et al.

Further, the presently disclosed metal complexing ligands can be pentadentate rather than tetradentate (see, for example, Compound i, dipyridinoacridinediacetate, DPADA). Because DPADA is pentadentate, i.e., it has five donor atoms attached to the metal ions rather than four, as in the tetradentate PDA. Thus, DPADA should form more stable complexes compared to PDA.

compound i d ipyrid inoacrid ined iacetate (DPADA)

The presently disclosed subject matter also provides ligands that preferably complex with larger metal ions. This characteristic is due to the radius of the arc formed by the donor atoms, such that metal ions with a radius of about 1.0 A fit best. The radius can be made smaller by inserting one or more extra bridging atoms, as provided in Compounds j, k, and I.

compound j compound k

2-[(8-hydroxyquinolin-2-yl)methyl]quinolin-8-olate compound I In some embodiments, the addition of an additional bridging atom, even when it is not part of the aromatic backbone, as illustrated in compound j, allows for six-membered chelate rings as opposed to five membered chelate rings. As provided herein, a chelate ring comprises the ring formed by the metal ion in its complex, plus the atoms, e.g., C, N, O, S, and the like, that complete the ring. Examples of chelate ring sizes are given by the two ligands shown below, in which the atoms forming the chelate rings are numbered to indicate the size of the ring. In general, ligands that form all five-membered chelate rings favor complexing with large metal ions, while alternating five- and six-membered rings produces a better fit for small metal ions.

und j

III.A. Metal Chelating Ligands of Formula (I) In some embodiments, the presently disclosed subject matter provides metal chelating ligands of Formula (I):

(Rn)m

D₁ -Ar-D₂ (I) wherein Ar is an aromatic moiety containing at least two pyridine rings selected from the group consisting of:

D₁ and D₂ are not one Of-CO₂H, a carboxylate salt, an alkyl ester of -CO₂H, an acyloxymethyl ester of -CO₂H, and C(=O)NH₂.

In some embodiments, Ar is 1 ,10-phenanthroline, m = 6, and the compound of Formula (I) is a compound of Formula (II):

wherein D₁ and D₂ are independently selected from the group consisting of -CO₂H, a carboxylate salt, an alkyl ester of -CO₂H, an acyloxymethyl ester of

-CO₂H, -CH₂CO₂H, an acyloxymethyl ester Of -CH₂CO₂H, a salt of -CH₂CO₂ ^", an alkyl ester of -CH₂CO₂H, -C(=O)NH_2l -CH₂C(=O)NH₂, phosphate, methyl phosphate, phosphonate, methyl phosphonate, amino, alkylamino, dialkylamino, an amine salt, an amino-substituted alkyl, the salt of an amino-substituted alkyl, hydroxyl, alkylhydroxyl, alkoxyl, mercapto, and alkylmercapto; and

Riι R2, R3, R4, R5, and R₆ are independently selected from the group consisting of H, alkyl, substituted alkyl, branched alkyl, perfluoroalkyl, halo, nitro, cyano, amino, thio, alkylamino, dialkylamino, alkylthio, alkoxyl, sulfate, carboxylate, carbamoyl, alkylcarbamoyl, dialkylcarbamoyl, acyloxyl, acylamino, aroylamino, phosphate, substituted sulfonate, sulfonic acid, aryl, substituted aryl, aralkyloxyl, aryloxyl, arylthio, alkoxycarbonyl, and cycloalkyl; wherein any two R groups on adjacent carbons optionally can form an additional fused ring or ring system.

In some embodiments, the compound is a compound of Formula (II) wherein D₁ and D₂ contain additional atoms between Ar and the chelating moiety of Di and D₂ and the compound is a compound of Formula (Ha):

wherein Di and D₂ are independently selected from the group consisting of

-CH₂CO₂H, an acyloxymethyl ester of -CH₂CO₂H, a salt Of-CH₂CO₂ ^", an alkyl ester Of-CH₂CO₂H, -CH₂C(=O)NH₂, methyl phosphate, methyl phosphonate, an amino- substituted alkyl, the salt of amino-substituted alkyl, alkylhydroxyl, and alkylmercapto; and

Ri, R₂, R₃, R₄, R₅, and R₆ are independently selected from the group consisting of H, alkyl, substituted alkyl, branched alkyl, perfluoroalkyl, halo, nitro, cyano, amino, thio, alkylamino, dialkylamino, alkylthio, alkoxyl, sulfate, carboxylate, carbamoyl, alkylcarbamoyl, dialkylcarbamoyl, acyloxyl, acylamino, aroylamino, phosphate, substituted sulfonate, sulfonic acid, aryl, substituted aryl, aralkyloxyl, aryloxyl, arylthio, alkoxycarbonyl, and cycloalkyl; wherein any two R groups on adjacent carbons optionally can form an additional fused ring or ring system. In some embodiments, Ar is

m = 10, and the compound of Formula (I) is a compound of Formula (III):

wherein Di and D₂ are independently selected from the group consisting of -CO₂H, a carboxylate salt, an alkyl ester of -CO₂H, an acyloxymethyl ester of -CO₂H, -CH₂CO₂H, an acyloxymethyl ester Of -CH₂CO₂H, a salt Of -CH₂CO₂ ^", an alkyl ester of -CH₂CO₂H, -C(=O)NH₂, -CH₂C(=O)NH₂, phosphate, methyl phosphate, phosphonate, methyl phosphonate, amino, alkylamino, dialkylamino, an amine salt, an amino-substituted alkyl, the salt of an amino-substituted alkyl, hydroxyl, alkylhydroxyl, alkoxyl, mercapto and alkylmercapto; and

Ri, R2, R3, R4, R5, Re, R7, Rs, R9, and R₁₀ are each independently selected from the group consisting of H, alkyl, substituted alkyl, branched alkyl, perfluoroalkyl, halo, nitro, cyano, amino, thio, alkylamino, dialkylamino, alkylthio, alkoxyl, sulfate, carboxylate, carbamoyl, alkylcarbamoyl, dialkylcarbamoyl, acyloxyl, acylamino, aroylamino, phosphate, substituted sulfonate, sulfonic acid, aryl, substituted aryl, aralkyloxyl, aryloxyl, arylthio, alkoxycarbonyl, and cycloalkyl; wherein any two R groups on adjacent carbons optionally can form an additional fused ring or ring system. In some embodiments, the compound of Formula (I) is a compound of Formula (III) and D₁ and D₂ are each OH. In some embodiments, Ar is:

m = 8, and the compound of Formula (I) is a compound of Formula (IV):

wherein D₁ and D₂ are independently selected from the group consisting of -CO₂H, a carboxylate salt, an alkyl ester of -CO₂H, an acyloxymethyl ester of -CO₂H, -CH₂CO₂H, a salt of -CH₂CO₂ ^", an alkyl ester of -CH₂CO₂H, an acyloxymethyl ester of -CH₂CO₂H, -C(=O)NH₂, -CH₂C(=O)NH₂, phosphate, methyl phosphate, phosphonate, methyl phosphonate, amino, alkylamino, dialkylamino, an amine salt, an amino-substituted alkyl, the salt of an amino-substituted alkyl, hydroxyl, alkylhydroxyl, alkoxyl, mercapto and alkylmercapto; and

Ri _> R2, R3, R4, R5, Re, R7, and R₈ are independently selected from the group consisting of H, alkyl, substituted alkyl, branched alkyl, perfluoroalkyl, halo, nitro, cyano, amino, thio, alkylamino, dialkylamino, alkylthio, alkoxyl, sulfate, carboxylate, carbamoyl, alkylcarbamoyl, dialkylcarbamoyl, acyloxyl, acylamino, aroylamino, phosphate, substituted sulfonate, sulfonic acid, aryl, substituted aryl, aralkyloxyl, aryloxyl, arylthio, alkoxycarbonyl, and cycloalkyl; wherein any two R groups on adjacent carbons optionally can form an additional fused ring or ring system or wherein either or both of R₃ and R₄ together, and R₅ and RQ together can be (=0). In some embodiments, the compound of Formula (I) is a ligand having a metal binding site that is larger than that of PDA and the compound of Formula (I) is a compound of Formula (V):

wherein D₁ and D₂ are independently selected from the group consisting of

-CO₂H, a carboxylate salt, an alkyl ester of -CO₂H, an acyloxymethyl ester of -CO₂H, -CH₂CO₂H, a salt of -CH₂CO₂ ^", an alkyl ester of -CH₂CO₂H, an acyloxymethyl ester of -CH₂CO₂H, -C(=O)NH₂, -CH₂C(=O)NH₂, phosphate, methyl phosphate, phosphonate, methyl phosphonate, amino, alkylamino, dialkylamino, an amine salt, an amino-substituted alkyl, the salt of an amino-substituted alkyl, hydroxyl, alkylhydroxyl, alkoxyl, mercapto, and alkylmercapto; and wherein R₁, R₂, R₃, R₄, R₅, and R₆ are independently selected from the group consisting of H, alkyl, substituted alkyl, branched alkyl, perfluoroalkyl, halo, nitro, cyano, amino, thio, alkylamino, dialkylamino, alkylthio, alkoxyl, sulfate, carboxylate, carbamoyl, alkylcarbamoyl, dialkylcarbamoyl, acyloxyl, acylamino, aroylamino, phosphate, substituted sulfonate, sulfonic acid, aryl, substituted aryl, aralkyloxyl, aryloxyl, arylthio, alkoxycarbonyl, and cycloalkyl; wherein any two R groups on adjacent carbons optionally can form an additional fused ring or ring system or wherein R₃ and R₄ together can together be (=0).

In some embodiments, Ar is:

m = 9, and the compound of Formula (1) is a compound of Formula (Vl):

wherein Di and D₂ are independently selected from the group consisting of -CO₂H, a carboxylate salt, an alkyl ester of -CO₂H, an acyloxymethyl ester of -CO₂H, -CH₂CO₂H₁ a salt of -CH₂CO₂ ^', an alkyl ester of -CH₂CO₂H, an acyloxymethyl ester of -CH₂CO₂H, -C(=0)NH₂₎ -CH₂C(=O)NH₂, phosphate, methyl phosphate, phosphonate, methyl phosphonate, amino, alkylamino, dialkylamino, an amine salt, an amino-substituted alkyl, the salt of an amino-substituted alkyl, hydroxy!, alkylhydroxyl, alkoxyl, mercapto, and alkylmercapto; and wherein Ri, R₂, R₃, R₄ and R₅ are independently selected from the group consisting of H, alkyl, substituted alkyl, branched alkyl, perfluoroalkyl, halo, nitro, cyano, amino, thio, alkylamino, dialkylamino, alkylthio, alkoxyl, sulfate, carboxylate, carbamoyl, alkylcarbamoyl, dialkylcarbamoyl, acyloxyl, acylamino, aroylamino, phosphate, substituted sulfonate, sulfonic acid, aryl, substituted aryl, aralkyloxyl, aryloxyl, arylthio, alkoxycarbonyl, and cycloalkyl; wherein any two R groups on adjacent carbons optionally can form an additional fused ring or ring system.

In some embodiments, Ar is:

m is 8, and the compound of Formula (I) is a compound of Formula (VII):

wherein D₁ and D₂ are independently selected from the group consisting of

-CO₂H, a carboxylate salt, an alkyl ester of -CO₂H, an acyloxymethyl ester of -CO₂H, -CH₂CO₂H, a salt of -CH₂CO₂ ^", an alkyl ester of -CH₂CO₂H, an acyloxymethyl ester of -CH₂CO₂H, -C(=O)NH₂, -CH₂C(=O)NH₂, phosphate, methyl phosphate, phosphonate, methyl phosphonate, amino, alkylamino, dialkylamino, an amine salt, an amino-substituted alkyl, the salt of an amino-substituted alkyl, hydroxy!, alkylhydroxyl, alkoxyl, mercapto, and alkylmercapto; and wherein R₁, R₂, R₃, R₄, R₅, R₆, R₇ and R₈ are each independently selected from the group consisting of H, alkyl, substituted alkyl, branched alkyl, perfluoroalkyl, halo, nitro, cyano, amino, thio, alkylamino, dialkylamino, alkylthio, alkoxyl, sulfate, carboxylate, carbamoyl, alkylcarbamoyl, dialkylcarbamoyl, acyloxyl, acylamino, aroylamino, phosphate, substituted sulfonate, sulfonic acid, aryl, substituted aryl, aralkyloxyl, aryloxyl, arylthio, alkoxycarbonyl, and cycloalkyl; wherein any two R groups on adjacent carbons optionally can form an additional fused ring or ring system.

In some embodiments, Ar is:

m is 12, and the compound of Formula (I) is a compound of Formula (VIII):

wherein D₁ and D₂ are independently selected from the group consisting of -CO₂H, a carboxylate salt, an alkyl ester of -CO₂H, an acyloxymethyl ester of -CO₂H, -CH₂CO₂H, a salt of -CH₂CO₂ ^", an alkyl ester of -CH₂CO₂H, an acyloxymethyl ester Of-CH₂CO₂H, -C(=O)NH₂, -CH₂C(=O)NH₂, phosphate, methyl phosphate, phosphonate, methyl phosphonate, amino, alkylamino, dialkylamino, an amine salt, an amino-substituted alkyl, the salt of an amino-substituted alkyl, hydroxyl, alkylhydroxyl, alkoxyl, mercapto, and alkylmercapto; and wherein R₁, R₂, R₃, R₄, R₅, Re, R7, Rs, R9, R10, R11, and R₁₂ are each independently selected from the group consisting of H, alkyl, substituted alkyl, branched alkyl, perfluoroalkyl, halo, nitro, cyano, amino, thio, alkylamino, dialkylamino, alkylthio, alkoxyl, sulfate, carboxylate, carbamoyl, alkylcarbamoyl, dialkylcarbamoyl, acyloxyl, acylamino, aroylamino, phosphate, substituted sulfonate, sulfonic acid, aryl, substituted aryl, aralkyloxyl, aryloxyl, arylthio, alkoxycarbonyl, and cycloalkyl; wherein any two R groups on adjacent carbons optionally can form an additional fused ring or ring system or wherein R₆ and R₇ together can together be (=0). The ligands of the presently disclosed subject matter can be prepared according to or analogously to methods disclosed herein and previously disclosed. Additional modifications can be performed according to organic chemical group formations as known in the art of synthetic organic chemistry, and as would be apparent to one of ordinary skill in the art upon a review of the present disclosure. For example, PDA can be synthesized according to published procedures. See Moqhimi, A., et al.. Inorg. Chem., 42, 1616-1624 (2003); Angeloff A., et al.. Euro. J. Inorg. Chem., 1 , 1985-1996 (2000) and Chandler, et al., J. Heterocyclic Chem, 18, 599 (1981). To summarize, PDA can be synthesized from 2,9- dimethyl-1 ,10-phenanthroline in two steps. The first step involves oxidation using

SeO₂ to form 2, 9-bisformyl-9,10-phenanthroline. A second oxidation to form the di-carboxylate can be accomplished with HNO₃.

Additional functional transformations of 2, 9-dimethyl-1 ,10-phenanthroline are described in published European Patent Application 0288256 to Toner, incorporated herein by reference in its entirety. For example, 2,9-diformyl-1 ,10- phenanthroline can be transformed to 2,9-bis(hydroxymethyl)-1 ,10-phenanthroline by reduction with sodium borohydride. Alternatively, the bis-hydroxymethyl compound can be formed from PDA via the actived pentafluorophenyl ester (synthesized by reaction of PDA with pentafluorophenyl trifluoroacetate), followed by subsequent sodium borohydride reduction. See Esposito, V., etal., Eur. J. Org.

Chem., 4228-4233 (2002). The bis-hydroxymethyl compound can be used in the synthesis of 2, 9-bis(bromomethyl)-1 ,10-phenanthroline using HBr in acetic acid or can be acetylated using acetic anhydride. The bromide atoms of 2,9- bis(bromomethyl)-1 ,10-phenanthroline can be displaced by a variety of nucleophiles, such as, for example, amines. Amine displacement of the bromide atoms leads to the formation of ligands wherein D₁ and D₂ are amino-substituted alkyl groups.

The bis-hydroxymethyl compound can be reacted with chlorophosphites (including protected chlorophosphites such as, for example, 2-(trimethylsilyl)ethyl dichlorophosphite) to form the phosphate-containing ligands of the presently disclosed subject matter. Phosphonates can be synthesized by treating 2,9- bis(bromomethyl)-1 ,10-phenanthroline with trialkyl phosphite esters (i.e., a P(OR)₃ compound) under Michaelis-Arbuzov reaction conditions.

Additionally, PDA can be used to form a variety of esters and amide ligands either using carbodiimide chemistry or by reacting the dicarboxylate with thionyl chloride to form an acyl chloride, which can be reacted with various alcohols, phenols, and amines. To perform some transformations, it can be desirable to protect the amines of the heteroaryl group, as will be appreciated by one of skill in the art.

The utility of the ligands of the presently disclosed subject matter can be optionally enhanced by attaching the ligand to a carrier, substrate or targeting moiety. Such carriers, substrates and targeting moieties can include a variety of polymers and biomolecules. Attachment of the ligand to a polymeric material or biomolecule can be used to impart ion-sensing properties to that substance and/or to solubilize, insolubilize or otherwise modify the properties of the ligand, the substance, or both. Typical examples of such substances include, but are not limited to, antibodies, amino acids, proteins, peptides, polypeptides, enzymes, enzyme substrates, lipids, phospholipids, hormones, lymphokines, metabolites, antigens, haptens, lectins, avidin, streptavidin, toxins, poisons, environmental pollutants, carbohydrates, oligosaccharides, polysaccharides, glycoproteins, glycolipids, nucleotides, oligonucleotides, nucleic acids and derivatized nucleic acids (including deoxyribo- and ribonucleic acids), DNA and RNA fragments and derivatized fragments (including single and multi-stranded fragments), natural and synthetic drugs, receptors, virus particles, bacterial particles, virus components, biological cells, cellular components (including cellular membranes and organelles), natural and synthetic lipid vesicles, polymers, polymer particles, polymer membranes, conducting and non-conducting metals and non-metals, and glass and plastic surfaces and particles. Where the substance is glass, it is optionally incorporated into an optical fiber or is coated on an optical fiber.

Generally, the desired ligand-polymer or ligand-biomolecule conjugate is most easily prepared when the ligand is substituted by an amino or carboxylate or other chemically reactive R group such as a thiol, amine, anhydride, ester or alkyl halide. Synthetic procedures for preparing such conjugates are known to one of skill in the art. See, Brinklev, Bioconj. Chem., 3, 2 (1992).

In some embodiments, the ligand of Formula (I) is further substituted with a carrier, substrate or targeting moiety as described above through a linkage to an

R_n group, wherein R_n is as defined hereinabove. 111. B. Metal Ions

In some embodiments, the metal ion for complexation with the metal complexing ligands described hereinabove is selected from a metal atom having at least two, three, four, five, six, and seven coordination sites or more. In some embodiments, the metal atom is selected from transition metals, e.g., a metal selected from one of Groups 3-12 of the Periodic Table, or from the lanthanide or actinide series. The metal ion also can be an alkali metal ion or an alkaline metal ion. A non-limiting list of metal ions, including exemplary and non-limiting oxidation states, which are suitable for the presently disclosed subject matter, include, but are not limited to: Ca²⁺, Mg ²⁺, Co³⁺, Cr³⁺, Hg²⁺, Pd²⁺, Pt²⁺, Pd⁴⁺, Pt ⁴⁺,

Rh³⁺, Ir³⁺, Ru³⁺, Co²⁺, Ni²⁺, Cu²⁺, Zn²⁺, Cd₂₊, Pb²⁺, Mn²⁺, Fe³⁺, Fe²⁺, Tc⁺⁴, Au³⁺, Au⁺, Ag⁺, Cu⁺, MoO₂ ²⁺, Ti³⁺, Ti⁴⁺, Bi³⁺, CH₃Hg⁺, Al³⁺, Ga³⁺, Ce³⁺, UO₂ ²⁺, Y³⁺, Eu³⁺, Gd³⁺, Cs⁺, Sr²⁺, Pu⁴⁺, and La³⁺.

As used herein, the term "paramagnetic metal ion" refers to a metal ion that is magnetized parallel or antiparallel to a magnetic field to an extent proportional to the field. Generally, paramagnetic metal ions are metal ions that have unpaired electrons. Examples of suitable paramagnetic metal ions, include, but are not limited to, gadolinium III (Gd⁺³ or Gd(III)), iron Mil (Fe⁺³ or Fe(III)), manganese Il (Mn⁺² or Mn(II)), yttrium III (Yt⁺³ or Yt(III)), dysprosium (Dy⁺³ or Dy(III)), and chromium (Cr⁺³ or Cr(III)). In some embodiments, the paramagnetic ion is the lanthanide atom Gd(III), due to its high magnetic moment, a symmetric electronic ground state, and its current approval for diagnostic use in humans.

A metal ion of the presently disclosed subject matter is selected in part based on the use for which the ligand and/or the resulting coordination complex is/are intended. In some embodiments of the presently disclosed subject matter, the coordination complex prepared from a compound of Formula (I) is used as a radiopharmaceutical compound. In such embodiments, the metal ion used in the coordination complex can be a radionuclide. In some embodiments, the radionuclide is selected from the group including, but not limited to, ^99mTc, ¹¹¹In, ⁶⁷Ga, ²⁰¹TI, and ⁶⁴Cu. In some embodiments, the radiopharmaceutical is used as a diagnostic agent, such as an imaging and/or contrast enhancement agent. In some embodiments, the radiopharmaceutical is used to treat a condition, such as, for example, cancer, in a subject in need thereof.

In some embodiments of the presently disclosed subject matter, the coordination complex prepared from a compound of Formula (I) is used as a MRI imaging agent as described herein below. In such embodiments, the metal ion used in the coordination complex can be a paramagnetic ion, which can optionally be Gd³⁺.

In some embodiments, the metal complexing ligand of Formula (I) is used as a fluorescent sensor to monitor and/or quantify metal ions in biological systems. In such embodiments, the compound of Formula (I) preferentially binds one of

Ca²⁺, Fe³⁺, Mg²⁺, Zn²⁺, and Mn²⁺.

In some embodiments of the presently disclosed subject matter, the metal complexing ligand of Formula (I) is used to remove radioactive ions from industrial waste samples, environmental samples, or subjects suffering from exposure to radioactive ions. In such embodiments, the metal ion can optionally be selected from the group including, but not limited to, Pu⁴⁺, Cs⁺, Sr²⁺, and UO₂ ²⁺.

In some embodiments, the metal complexing ligand of Formula (I) is used in chelation therapy to treat cases of poisoning with non-radioactive metals, such as, but not limited to, mercury, lead and cadmium. In some embodiments of the presently disclosed subject matter, the metal complexing ligand of Formula (I) is used to recover economically valuable metals during mining operations or from metal-bearing waste. Such metals ions can include ions of copper, platinum, zinc, silver, nickel and the like.

JV₁ Nuclear Medicine Including Diagnostic Imaging and/or Contrast

Enhancement Agents, and Therapeutic Agents IVA Complexes of Radioisotopes for Positron Emission Tomography and

Radioscintigraphy

Nuclear medicine is a branch of medicine related to the diagnostic and therapeutic use of radioactive compounds. A common use of nuclear medicine is in imaging the distribution of a radiopharmaceutical in a specific organ system with, for example, a scintillation camera for diagnostic purposes. Generally, scintillation cameras detect gamma rays generated by the positrons emitted by the decaying radioactive isotopes of the radiopharmaceuticals. Diagnostic techniques related to nuclear medicine include single photon emission computed tomography (SPECT), positron emission tomography (PET) and other radioscintigraphic techniques.

In one example of the use of PET, glucose molecules are tagged with a radioisotope and are injected into the bloodstream. The gamma radiation emitted by the decay of the radioisotope reveals areas of active glucose uptake and offers a gauge of cell metabolism and function. In therapeutic applications, radiopharmaceuticals, e.g., those containing, for example, radioactive iodine or gallium, localize to certain organs and deliver cytotoxic radiation doses to tumors.

Thus, radiopharmaceuticals contain radionuclides, i.e., isotopes of artificial or natural origin that exhibit radioactivity. It is usually desirable that the radioisotope have a relatively short half-life (of about a few hours to about a day) so that the isotope will last long enough to be incorporated into the radiopharmaceutical, undergo quality assurance testing, and be used, but not long enough to create waste issues or to produce undesirably excessive doses of radiation to a patient. Often, radiopharmaceuticals also contain a complexing ligand or ligands (e.g., gallium (⁶⁷Ga) citrate, technetium (^99mTc) succimer, indium

(¹¹¹In) pentate (¹¹¹In-DTPA)). The nature of the ligand can be used to dictate the sites/organs to which the radiopharmaceutical is directed. Technetium (^99mTc) medronate, which contains a phosphate-containing ligand, is absorbed preferentially at sites of new bone formation. Technetium (^99mTc) bicisate was designed as a neutral, lipophilic coordination complex to cross the blood-brain barrier and to be used in the evaluation of stroke and other brain lesions.

Some radiopharmaceuticals also contain additional targeting moieties. Indium (¹¹¹In) satumomabpendetide is an antibody conjugate of an antibody that is specific for tumors. In some cases the radionuclide is incorporated directly into an organic molecule via covalent bonds (e.g., (¹⁸F)-2-fluoro-2-deoxy-D-glucose) or is a more simple salt (e.g., sodium iodide (¹³¹I), Indium (¹¹¹In) chloride, thallium (²⁰¹TI) chloride). Radionuclides can be produced using nuclear reactors, cyclotrons, or radioisotope generators.

Accordingly, in some embodiments, the presently disclosed subject matter provides a ligand for use in diagnostic applications, such as but not limited to an imaging agent or a ligand for complexing radioisotopes for use in positron emission tomography and radioscintigraphy. Further, in some embodiments, the presently disclosed subject matter provides a ligand for use in therapeutic applications, such as but not limited to a ligand for complexing radioisotopes for treating a target tissue, such as a tumor. Thus, the presently disclosed subject matter provides radiopharmaceuticals comprising a ligand and a radionuclide. The nature of the ligand can be used to dictate the sites/organs to which the radiopharmaceutical is directed. For example, a phosphate-containing ligand can be absorbed preferentially at sites of new bone formation. By way of additional example it is also provided that the metal complexes formed by the presently disclosed metal complexing ligands can cross the blood-brain barrier, such but not limited to providing a neutral, lipophilic coordination complex.

Radiopharmaceuticals in accordance with the presently disclosed subject matter can also comprise additional targeting moieties. Representative, non- limiting examples include but are not limited to an antibody that is specific for a target tissue, such as a tumor.

In some embodiments of the presently disclosed subject matter, the ligand is a compound of Formula (I):

(Rn)_n.

Di and D₂ are independently selected from the group consisting Of-CO₂H, a carboxylate salt, an alkyl ester of -CO₂H, an acyloxymethyl ester of -CO₂H, CH₂CO₂H, an acyloxymethyl ester Of-CH₂CO₂H, a salt Of-CH₂CO₂ ^", an alkyl ester of -CH₂CO₂H, -C(=0)NH₂, -CH₂C(=O)NH₂, phosphate, methyl phosphate, phosphonate, methyl phosphonate, amino, alkylamino, dialkylamino, an amine salt, an amino-substituted alkyl, the salt of an amino-substituted alkyl, hydroxy], alkylhydroxyl, alkoxyl, mercapto, and alkylmercapto; m is an integer corresponding to the number of sites on a given Ar that can bear an additional substituent (or the number of H atoms on the Ar portion of the DrAr-D₂ molecule); each n is an integer from 1 to m; and each R is independently selected from the group consisting of H, alkyl, substituted alkyl, branched alkyl, perfluoroalkyl, halo, nitro, cyano, amino, thio, alkylamino, dialkylamino, alkylthio, alkoxyl, sulfate, carboxylate, carbamoyl, alkylcarbamoyl, dialkylcarbamoyl, acyloxyl, acylamino, aroylamino, phosphate, substituted sulfonate, sulfonic acid, aryl, substituted aryl, aralkyloxyl, aryloxyl, arylthio, alkoxycarbonyl, and cycloalkyl; wherein any two adjacent R groups optionally can form an additional fused ring or ring system or wherein any two R groups substituted on the same carbon can together be (=0).

IV.B. Magnetic Resonance Imaging (MRl) Contrast Agents

Magnetic resonance imaging (MRI) is a diagnostic and research procedure that uses high magnetic fields and radio frequency signals to produce visible images. The most abundant molecular species in biological tissues is water. The quantum mechanical "spin" of the water proton nuclei gives rise to the signal in all imaging experiments. Briefly, contrast agents shift the MRI signal of the proton (H) in H₂O, which produces a contrast between high-water content tissues, e.g., plasma, and low-water content tissues, e.g., fat.

In MRI, the sample to be imaged is placed in a strong static magnetic field (e. g., a magnetic field between 1 to 12 Tesla) and the spins are excited with a pulse of radio frequency (RF) radiation to produce a net magnetization in the sample. Various magnetic field gradients and other RF pulses then act on the spins to code spatial information into the recorded signals. MRI is able to generate structural information in three dimensions in relatively short time spans.

In the design of MRI agents, consideration must be given to a variety of properties that might affect the physiological outcome apart from the ability to provide contrast enhancement. Two fundamental properties that are considered are biocompatibility and proton relaxation enhancement. Biocompatibility is influenced by several factors including toxicity, stability (thermodynamic and kinetic), pharmacokinetics and biodistribution. Proton relaxation enhancement (or relaxivity) is chiefly governed by the choice of metal and rotational correlation times. One feature to be considered during the design stage is the selection of the metal atom, which determines the measured relaxivity of the complex. Paramagnetic metal ions, as a result of their unpaired electrons, act as potent relaxation enhancement agents. Such metal ions decrease the T₁ and T₂ relaxation times of nearby spins. Some paramagnetic ions decrease the T-i without causing substantial linebroadening (e.g., gadolinium(lll), (Gd³⁺)), while others induce drastic linebroadening (e.g., superparamagnetic iron oxide). The mechanism Of T₁ relaxation is generally a through space dipole-dipole interaction between the unpaired electrons of the paramagnet (i.e., the metal atom with an unpaired electron) and bulk water molecules (i.e., water molecules that are not "bound" to the metal atom) that are in fast exchange with water molecules in the metal's inner coordination sphere (i.e., water molecules that are bound to the metal atom).

The lanthanide atom Gd³⁺ is the most frequently chosen metal atom for MRI contrast agents because it has a very high magnetic moment and a symmetric electronic ground state. Transition metals such as high spin Mn(II) and Fe(III) also are candidates due to their high magnetic moments. Gd³⁺ has seven unpaired electrons, which gives it the greatest power of any metal ion to shift the MRI signal of the proton in H₂O. Gd³⁺ itself is toxic, however. A suitable ligand or chelator must therefore be used to complex the Gd³⁺, thereby preventing it from exerting its toxic effect. Further, several factors influence the stability of chelate complexes, including enthalpy and entropy effects (e.g., number, charge and basicity of coordinating groups, ligand field and conformational effects). Additionally, various molecular design features of the ligand can be directly correlated with physiological results. For example, the presence of a single methyl group on a given ligand structure can have a pronounced effect on clearance rate. While the addition of a halo group, e.g., bromine, can force a given complex from a purely extracellular role to an effective agent that collects, for example, in hepatocytes.

The ligand diethylenetriaminepentaacetate (DTPA), the structure of which is shown below, forms a stable complex, i.e., chelates, with lanthanide ions, e.g., the rare-earth element gadolinium (Gd³⁺), and thus acts to detoxify lanthanide ions.

DTPA

The stability constant (K) (also referred to as the "formation constant) for Gd(DTPA)^2' is very high (logK=22.4) (the higher the logK, the more stable the complex). This thermodynamic parameter indicates the fraction of Gd³⁺ ions that are in the unbound state will be quite small. The water-soluble Gd(DTPA)^2" chelate is stable, nontoxic, and one of the most widely used contrast enhancement agents in experimental and clinical imaging research. See, e.g., Caravan et al., Chemical Reviews, 99, 2293-2352 (1999); Runge et al.. Magn, Reson. Imag., 3, 85 (1991); Russell et al.. AJR, 152, 813 (1989); Meyer et al.. Invest. Radiol., 25, S53 (1990)).

In addition to DTPA, other chelators, including 1 ,4,7,10- tetraazacyclododecane'-N, N¹N", N'"-tetracetic acid (DOTA), and derivatives thereof have been used. See U.S. Pat. Nos. 5,155,215; 5,087,440; 5,219,553; 5,188,816; 4,885,363; 5,358,704; 5,262,532; and MeyeretaL, Invest. Radiol., 25, S53 (1990). The Gd-DOTA complex has been thoroughly studied in laboratory tests involving animals and humans. The complex is conformationally rigid, has an extremely high formation constant (logK = 28.5), and at physiological pH possess very slow dissociation kinetics. Recently, the Gd(DOTA) complex was approved as an MRI contrast agent for use in adults and infants in France and has been administered to over 4500 patients.

A drawback with the DTPA complex of Gd³⁺ is that a relatively large amount of the Gd³⁺/DTPA complex, e.g., about 7 g, typically is injected to produce a good contrast. The ability of a complex of Gd³⁺ to shift the MRI signal of water is a function of the number of water molecules directly bound to the Gd³⁺. Complexes of Gd³⁺ known in the art for use as MRI agents, including the Gd³⁺/DTPA complex, have only one water molecule directly bound to Gd³⁺. The DTPA ligand has eight points of attachment to Gd³⁺. The Gd³⁺ ion has a coordination number (CN.) of nine (the total number of atoms directly attached to Gd³⁺). Therefore, if a ligand occupies eight sites on the Gd³⁺, only one binding site for a water molecule remains. In contrast, the ligand EDTA (shown below) occupies only six binding sites on the Gd³⁺, which means that a Gd³⁺/EDTA complex is capable of binding only three water molecules. Thus, in principle, EDTA would be attractive for use as an MRI contrast agent. Because it has only six points of attachment, however, the EDTA ligand binds Gd³⁺ weakly. As a result, if a Gd³⁺/EDTA complex were to be injected into a subject for use as an MRI contrast agent, the Gd³⁺ could be released into the subject. Thus, the Gd³⁴VEDTA complex is not suitable for use as an MRI contrast agent.

EDTA The amount of complex needed in MRI decreases dramatically with more water molecules bound to the Gd³⁺ in its complex. Thus, there is a need in the art for a ligand that has as few donor atoms as possible, e.g., few points of attachment to the Gd³⁺, but still binds the Gd³⁺ strongly enough not to release the Gd³⁺ into the subject, and that allows as many water molecules as possible to be bound to the Gd³⁺.

As described hereinabove, the CN. of Gd³⁺ usually is nine, so the total number of donor atoms that Gd³⁺ can accommodate is nine. PDA is tetradentate, i.e. it has only four donor atoms, which leaves five available sites for binding water molecules on the Gd³⁺. See Figure 2A. This characteristic makes a Gd³⁺ complex with one PDA and five waters more effective at making a contrast in the MRI than any currently existing Gd-based MRI agent known in the art. Other ligands of the presently disclosed subject matter, such as compounds of Formula (V), including dipyridinoacridinediacetate (DPADA), are pentatdentate. These ligands can form complexes with Gd³⁺ that leave four available sites for binding water molecules on the Gd³⁺. See Figure 2B. Accordingly, the presently disclosed ligands can accommodate the attachment of, in some embodiments, four water molecules, and, in some embodiments, five water molecules to Gd³⁺, while having sufficient stability of the Gd³⁺ ligand to prevent Gd³⁺ toxicity.

In some embodiments of the presently disclosed subject matter, the ligand comprises an MRI imaging agent, wherein the MRI imaging agent is a Gd-based MRI imaging agent and the ligand of the imaging agent is a compound of Formula (I) wherein Ar is:

and Di and D₂ are each carboxylate and the compound of Formula (I) is a compound of Formula (Mb) or Formula (Via) below:

wherein each R substituent (i.e., R₁, R₂, R3, R₄, R5, Re, R7, Re, and Rg) is independently selected from the group consisting of H, alkyl, substituted alkyl, branched alkyl, perfluoroalkyl, halo, nitro, cyano, amino, thio, alkylamino, dialkylamino, alkylthio, alkoxyl, sulfate, carboxylate, carbamoyl, alkylcarbamoyl, dialkylcarbamoyl, acyloxyl, acylamino, aroylamino, phosphate, substituted sulfonate, sulfonic acid, aryl, substituted aryl, aralkyloxyl, aryloxyl, arylthio, alkoxycarbonyl, and cycloalkyl; and wherein any two R groups on adjacent carbons optionally can be an additional fused ring or ring system.

Thus, the presently disclosed subject matter provides for substitution on the fused aromatic ring structure to regulate the solubility and/or hydrophobic/hydrophilic balance of the complex as desired, for example, so that the contrast agent is adequately distributed in the subject. An aspect of complexes of Formula (Mb) and Formula (Via) is that they have five (e.g., PDA) or four (e.g., DPADA) water molecules bound to the Gd³⁺. The molecular models of the Gd³⁺ complexes of PDA and DPADA are shown in Figures 2a and 2b, respectively, including the complexed water molecules.

An important aspect related to the fitness of Gd³⁺ MRI agents for use in the human body is that the Gd³⁺ atom not bind to proteins in vivo. One particular problem associated with Gd³⁺ MRI agents is the binding of protein phosphonate groups to chelation sites on the Gd³⁺ that are occupied only by water molecules. To prevent any undesirable binding to proteins, metal complexing ligands for MRI agents can be designed using phosphate or phosphonate metal chelating groups. Such groups can bind Gd³⁺ more strongly than the carboxylate groups of PDA, and, thus, their use can greatly limit the possibility of protein phosphonate groups binding to the MRI agent in vivo.

In some embodiments of the presently disclosed subject matter, the ligand comprises an MRI imaging agent, wherein the MRI imaging agent is a Gd-based MRI imaging agent and the ligand of the imaging agent is a compound of Formula (I) wherein Di and D₂ are each selected from the group consisting of phosphate, methyl phosphate, phosphonate, and methyl phosphonate. In some embodiments, Ar is:

and the compound of Formula (I) is a compound of Formula (II). In some embodiments, the ligand for use as a gadolinium-based MRI agent is a compound selected from:

wherein each R substituent (i.e., R-i, R₂, R₃, R₄, R₅, and R₆) is independently selected from the group consisting of H, alkyl, substituted alkyl, branched alkyl, perfluoroalkyl, halo, nitro, cyano, amino, thio, alkylamino, dialkylamino, alkylthio, alkoxyl, sulfate, carboxylate, carbamoyl, alkylcarbamoyl, dialkylcarbamoyl, acyloxyl, acylamino, aroylamino, phosphate, substituted sulfonate, sulfonic acid, aryl, substituted aryl, aralkyloxyl, aryloxyl, arylthio, alkoxycarbonyl, and cycloalkyl; and wherein any two R groups on adjacent carbons optionally can be an additional fused ring or ring system.

V₁ Fluorescent Ligands for Monitoring Metal Ions in Biological Systems

In some embodiments, the presently disclosed ligands can be used to monitor metal ions, such as Ca²⁺ and Zn²⁺, by fluorescence as the metal ions move about in a biological system, such as a cell. Fluorescence is the phenomenon in which a molecule or ion absorbs radiation, e.g., light, at one wavelength and emits radiation, e.g., light, at a longer wavelength. The presence and/or location of the Ca²⁺ and/or Zn²⁺ ion can be detected by the fluorescence. The intensity of the fluorescence is used as a measure of calcium concentration, which is particularly important in biological systems, such as living cells. Ca²⁺ and Zn²⁺ are important metal ions in biological systems. Ca²⁺ is the universal switch that turns on enzymes, releases neurotransmitters to cross synapses, responds to neurotransmitters to propagate the nervous impulse further beyond the synapse, controls cell division, and apoptosis (cell suicide when cells find themselves to be faulty or unneeded). See Messerschmidt, A.. Cyqler. M.. and Bode, W. (Eds.) Handbook of Metalloproteins; Wiley: Hoboken, N. J., Vol. 3, pp. 443-756 (2004); and Dudev. T. and Lim., C. Chem. Rev., 103, 773 (2000), each of which is incorporated herein by reference in its entirety. Likewise, Zn²⁺ plays an important structural and catalytic role in biological systems. For example, zinc influences DNA synthesis, microtubule polymerization, gene expression, immune system function, nervous system function, apoptosis, and the formation of β-amyloid, a protein associated with Alzheimer's disease. See Muruvama, S., et aL, J. Am. Chem. Soc, 124, 10650-10651 (2002). Accordingly, in some embodiments, the presently disclosed metal complexing ligands can be used as a fluorescent ligand for Zn²⁺, to, for example, detect Zn²⁺ deposited in the brain of a subject afflicted with or suspected of being afflicted with Alzheimer's disease.

Many indicators for Ca²⁺ ions to date have utilized a tetracarboxylate chelating group based upon the structure of 1 ,2-bis-2-aminophenoxyethane-N, N, N', N'-tetraacetic acid (BAPTA), usually in conjunction with a covalently attached fluorophore. Upon binding Ca²⁺ in the BAPTA chelate of the indicator, the fluorescence properties of the attached fluorophore are generally affected in some measurable way (i.e. emission is enhanced or decreased, the wavelength of excitation or emission is altered, and the like). Once the dissociation constant for a specific indicator-Ca²⁺ complex is known, a measurement of the fluorescence properties of a sample containing the indicator allows a determination of in situ Ca²⁺. See The Handbook-A Guide to Fluorescent Probes and Labeling Technologies, available at http://probes.invitrogen.com/handbook/. Specific examples of BAPTA-based fluorescent indicators include quin-2, Fura-2 and indo- 1. See Tsien, R. Y.. Biochemistry, 19, 2396, (1980); Grvnkiewicz, G., et a!.. J.

Biol., Chem., 260, 3440-3450, (1985); U.S. Patent No. 4,603,209 to Tsien et al.

Further, recent examples OfZn²⁺ specific probes include molecules formed from the covalent attachment of an N,N,N',N'-tetrakis(2-pyridylmethyl)ethylene- diamine chelator with a benzofuran fluorophore (see Muruvama. S.. et al.. J. Am. Chem. Soc, 124, 10650-10651 (2002)), quinoline derivatives of 1,2-bis(2- benzylaminoethoxy)ethane (see Kawakami. J., et al.. Anal. ScL, 19, 1353-1354 (2003)), and 2-(9-anthrylmethylamino)ethyl-appended 1 ,4,7,10-tetraaza- cyclododecane chelator (seeAoki, S., et a!.. Inorg. Chem., 42, 1023-1030 (2003)).

For general guidance on the use of fluorescent ligands for tracking metal ions in biological systems, see Kimura, E. and Aoki, S.. BioMetals, 14, 191 (2001); Harano. T., et a!.. J. Amer. Chem. Soc, 124, 6555 (2002); Burdette. S. C. and

Lippard, S. J.. Coord. Chem. Rev., 216, 333 (2001); Burdette, S. C. and Lippard,

S. J., Proc. Natl. Acad. ScL, 100, 3605 (2003); Shultz, M. P., et a!.. J. Amer.

Chem. Soc, 125, 10591 (2003): Burdette, S. C. et a!.. J. Amer. Chem. Soc, 123,

7831 (2001); and Snitsarev, V., et al., Biophys. J., 80, 1538 (2001), each of which is incorporated herein by reference in its entirety.

An example of the use of Fura-2 to track Ca²⁺ in a cell is shown in Figure 3, in which Ca²⁺ in a neuron from a subject suffering from Alzheimer's disease is shown. The actual death of neurons in Alzheimer's disease is caused by excessive Ca²⁺ concentrations within the neuron, which occurs when the plaques associated with Alzheimer's disease cause rupture of the membrane surrounding the neuron.

An aspect of the presently disclosed ligands is that their large aromatic system leads to what is called the chelation-enhanced fluorescence (CHEF) effect.

A system, such as the aromatic 1 ,10-phenanthroline system of PDA, where nitrogen atoms are part of the system, will not fluoresce, or will fluoresce only weakly with no metal ion bound to it. The nitrogen atoms have unshared pairs of electrons that quench the fluorescence of the aromatic system. Thus, such ligands will not exhibit measurable fluorescence until a metal ion attaches to the ligand and forms chemical bonds with the nitrogen atoms. When a metal ion, such as Ca or Zn , forms chemical bonds to the nitrogen atoms, the ability to fluoresce is greatly enhanced. The strength of the fluorescence is proportional to the concentration of the Ca²⁺ OrZn²⁺ present. As such, the presently disclosed ligands can be used as analytical tools for measuring metal ion concentration inside living cells in real time and mapping the location of the metal ion in the living cell as it fulfills its biological role in switching on processes, such as enzyme action or cell division.

In some embodiments, the presently disclosed ligands can be used as fluorescent detectors for metal ions, such as Ca²⁺ and Zn²⁺, in a biological system, such as a living cell. The specific indicator used in an assay or experiment is selected based on the desired affinity for the target ion as determined by the expected concentration range in the sample, the desired spectral properties and the desired ion selectivity. Initially, the suitability of a ligand as an indicator of ion concentration is commonly tested by mixing a constant amount of ligand with a measured amount of the target ion under the expected experimental conditions.

The interference of a non-target ion can be tested by a comparable titration of the ligand with that ion. Although Ca²⁺ or Zn²⁺ are the preferred target ions, any ion that yields a detectable change in absorption wavelengths, emission wavelengths, fluorescence lifetimes or other measurable optical property over the concentration range of interest can potentially be measured using one of the ligands of the presently disclosed subject matter.

Preferred ligands are those that are sufficiently water-soluble so that they are useful for assays conducted in aqueous or partially aqueous solution. Modifications that are designed to enhance permeability of the indicator through the membranes of living cells, such as acyloxymethyl esters and acetates can require the ligand to be predissolved in an organic solvent, such as dimethylsulfoxide (DMSO), before addition to a cell suspension, where the ligand then readily enters the cells. Intracellular enzymes cleave the esters to the more polar acids and phenols that are then well retained inside the cells. Once a ligand has been prepared that is suitable for the desired target ion, the optimal concentration range of ligand response is determined by preparing aliquots of a dilute ligand solution and adding increasing amounts of the target ion.

For example, if a 25 μM metal ion concentration gives a ten-fold increase in fluorescence emission intensity, the target ion is then tested at concentrations above and below this level to give an estimate of the concentrations of target metal ion over which the indicator is most sensitive (e.g. 1 , 5, 10 and 50 μM target ion).

Based on the results of this evaluation, further testing is run to determine the dissociation constant (K_d) of the indicator for the selected target ion(s).

To determine the K_d for the ion-ligand complex, aliquots of a dilute indicator solution are prepared and the target metal ion is added to each so as to give a regular gradient of increasing concentration, from zero to the saturating concentration. The data are then plotted as the change in fluorescence intensity versus ion concentration using a double log plot to give the K_d as the reciprocal log of the x-intercept. The most useful range of analyte concentration is about one log unit above and below the dissociation constant of the ion-ligand complex. The dissociation constant can be affected by the presence of other ions, particularly ions that have similar ionic radii and charge. The dissociation constant also can be affected by other conditions such as ionic strength, pH, temperature, viscosity, presence of organic solvents, and incorporation of the ligand in a membrane or polymeric matrix, or conjugation or binding of the ligand to a protein or other biological molecule. Any or all of these effects need to be taken into account when calibrating a ligand.

The ligand is combined with a sample in a way that will facilitate detection of the target ion concentration in the sample. The sample is generally a fluid or liquid suspension that is known or suspected to contain the target ion. Representative samples include intracellular fluids such as in blood cells, cultured cells, muscle tissue, neurons, and the like; extracellular fluids in areas immediately outside of cells; in vesicles; in vascular tissue of plants and animals; in biological fluids such as blood, saliva, and urine; in biological fermentation media; in environmental samples such as water soil, waste water and sea water; and in chemical reactors. The sample is optionally clarified before use, such as by filtration or centrifugation. Test samples that contain greater concentrations of metals can be diluted preceding the assay. The metal ions also can be concentrated or separated wholly, or in part, such as by chromatography, electrophoresis, or selective extraction. For example, samples that might contain otherwise undetectable concentrations of the target ion can be concentrated before the assay, such as by evaporation, ion exchange chromatography, or selective extraction into organic solvents.

The observation of a detectable change in the fluorescence properties of the ligand is optionally used to simply identify the presence of the target ion in a test sample. Alternatively, the detectable fluorescence response is quantified and used to measure the concentration of the target ion in the test sample. Quantification is typically performed by comparison of the fluorescence response to a standard, or calibration curve. The standard curve is generated according to methods known in the art using varying and known amounts of the target ion in standard solutions, or by comparison with a reference dye or dyed particle that has been standardized versus the target ion-ligand complex.

The optical response of the ligand to the ion can be detected by various approaches that include measuring absorbance or fluorescence changes with an instrument, visually, or by use of a fluorescence-sensing device. Several examples of fluorescence sensing devices are known, such as fluorometers, fluorescence microscopes, fluorescence microtiter plate readers, laser scanners, and flow cytometers was well as by cameras and other imaging equipment. These measurements can be made remotely by incorporation of the ligand as part of a fiber optic probe. The ligand can be covalently attached to the fiber optic probe material, typically glass or functionalized glass (e.g., aminopropyl glass) or the ligand can be attached to the fiber optic probe via an intermediate polymer, such as polyacrylamide. The ligand solution alternatively can be incorporated non- covalently within a fiber optic probe, as long as the target ion can come into contact with the indicator solution. Vl. Industrial and Environmental Metal Ion Recovery VIA Radioactive Metal Ion Extractants

The increased use of nuclear energy has resulted in an ever-increasing amount of radioactive waste. The safe storage of such waste, which contains dilute amounts of long-lived radioactive components, such as Pu⁴⁺, ⁹⁰Sr, and

¹³⁷Cs, along with other organic and inorganic species, is costly, and the potential for environmental and public health effects is a constant concern. Reducing the volume of nuclear waste can be greatly aided by the selective removal of the radioactive metal ions. See Gramer, C. J. and Raymond, K. N., Inorg. Chem., 43, 6397-6402, (2004); Gorden. A. E. V.. et aL Chem. Rev., 103, 4207-4282, (2003).

Nuclear waste exists in numerous forms and locations worldwide. The largest inventory of highly radioactive materials is produced from the reprocessing of spent nuclear fuel. The fission process produces a number of undesirable, highly radioactive elements that accumulate in the nuclear fuel. For the reuse or recycling of the unused fissionable material left in the fuel, usually, either uranium-

235 or plutonium-239, a separation process is employed to partition the fissionable material from the undesirable fission products. This separation process typically is accomplished by the leaching or dissolution of a portion or all of the spent nuclear fuel material, followed by chemical separation. Early chemical separation processes were based on precipitation, where, for example, BiPO₄ was used to coprecipitate plutonium for weapons-grade plutonium production. More recently, and by far the most common, solvent extraction processes utilize tri-n-butyl phosphate to chelate uranium and/or plutonium in aqueous solutions, and the complexes are extracted into organic solvents, such as kerosene. The remaining acidic liquid waste, still containing highly radioactive fission products and trace transuranic elements, particularly Cs⁺, has been accumulated and stored in various forms around the world for the past half century.

A number of other ligands have been investigated for use in extracting the remaining radioactive metal ions from nuclear fuel waste. An example of such an extractant is the ligand L1 , shown in Scheme 1 below, which selectively removes cesium (Cs) from radioactive waste solutions. The use of crown ether compounds and cation exchangers for extracting cesium and strontium from nuclear waste is the subject of U.S. Patent No 4,749,518 to Davis, M. W., et al. Metal dicarbollides also have been used to extract cesium and strontium from nuclear waste. See U.S. Patent No. 5,603,074 to Miller et al.; U. S. Patent No. 5666,641 to Abnev et aL; U.S. Patent Nos. 5,666,642 and 5,698,169 to Hawthorn, M.F.. et al.

L1 L1 complex of Cs⁺

Scheme 1. Complexation of Cs⁺ by the ligand L1.

In some embodiments, ligands of the presently disclosed subject matter can be used to extract metal ions of interest from nuclear fuel waste, or radioactively contaminated water or soil samples. The extraction process can be a liquid/liquid extraction or a liquid/solid extraction. In some embodiments, the metal ion is a contaminant of the industrial or environmental sample. In some embodiments, the contaminant interferes with the purity of another component of the sample. In some embodiments, the contaminant poses an environmental threat or a potential environmental threat.

For example, ligands soluble in organic solvents but not in aqueous solutions can be used in liquid/liquid extractions of aqueous nuclear fuel waste. Methods of liquid/liquid extraction of cesium from nuclear waste using crown ether ligands have been described previously. See U.S. Patent No.4,749,518 to Davis,

M. W., et al.. U.S. Patent No. 5,888,398 to Dietz, M. L. et al. The inclusion of an organophilic counter ion in such systems to balance the charge of the metal ion in the organic phase has been used to enhance liquid/liquid extractions. Another method of enhancing extraction has been to add high molecular weight organic acids to the organic phase. A third approach to enhancing liquid/liquid extractions of this type has been to use an aliphatic organic phase (e.g. a ketone or alcohol) containing a substantial amount of dissolved water. See Horowitz, et al.. Solvent Ext. Ion Exch., 8, 199 (1990); and Dietz, M. L. et al., Solvent Extr. Ion Exch., 14, 1-12, (1996). For the most efficient ion extraction, the concentration of the ligand should be as high as possible based on the limits of the ligand's solubility or dispersibility in the organic solvent. After equilibration of the two liquid phases to allow maximal extraction of the metal ion into the organic phase, the two phases are separated. The ligand-metal complex in the organic phase can be optionally recycled by stripping the metal ion. Such a recycling process can, in some embodiments, involve a second liquid/liquid extraction.

In liquid/solid extractions, metal chelators can be attached to a variety of supports, such as a polymer and placed in contact with a solution containing metal ions. Alternatively, a suitable hydrophilic ligand could be added to an aqueous waste solution containing metal ions and the metal complexes formed subsequently removed from solution via precipitation.

The presently disclosed subject matter provides for the use of ligands for the selective complexation of metal ions of particular sizes, from the relative small Mg²⁺, to the relative large Cs⁺. For example, the following approach depicted in Scheme 2 can be adopted for selectively modifying the ligand architecture to accommodate metal ions of different sizes.

Scheme 2. Modification of Ligand Architecture to Accommodate Metal Ions of Different Sizes

In some embodiments, the ligands are designed to selectively complex metal ions found in radioactive waste, such as plutonium, strontium, and cesium. Plutonium, a relatively large metal ion, is complexed by ligands. The cesium ion is larger than the plutonium ion, and with its single positive charge is complexed only weakly by most ligands. Thus, to complex relatively large metal ions, such as Cs⁺, a strategy of increasing the angle of the non-ring donor groups to accommodate metal ions having a relatively large ionic radius is adopted.

In some embodiments, the presently disclosed subject matter provides compounds of Formula (I) that can optionally be used for chelating Cs⁺ or other target metal ions, wherein the Ar of Formula (I) is

and the compound of Formula (I) is a compound of Formula (V):

wherein Di and D₂ are independently selected from the group consisting of -CO₂H, a carboxylate salt, an alkyl ester of -CO₂H, an acyloxymethyl ester of -CO₂H, -CH₂CO₂H, a salt of -CH₂CO₂ ^', an alkyl ester of -CH₂CO₂H, an acyloxymethyl ester of -CH₂CO₂H, -C(=O)NH₂, -CH₂C(=O)NH₂, phosphate, methyl phosphate, phosphonate, methyl phosphonate, amino, alkylamino, dialkylamino, an amine salt, an amino-substituted alkyl, the salt of an amino-substituted alkyl, hydroxyl, alkylhydroxyl, alkoxyl, mercapto and alkylmercapto; and wherein Ri, R₂, R3, R₄, R5, and R₆ are independently selected from the group consisting of H, alkyl, substituted alkyl, branched alkyl, perfluoroalkyl, halo, nitro, cyano, amino, thio, alkylamino, dialkylamino, alkylthio, alkoxyl, sulfate, carboxylate, carbamoyl, alkylcarbamoyl, dialkylcarbamoyl, acyloxyl, acylamino, aroylamino, phosphate, substituted sulfonate, sulfonic acid, aryl, substituted aryl, aralkyloxyl, aryloxyl, arylthio, alkoxycarbonyl, and cycloalkyl, optionally, any two adjacent R groups can form an additional fused ring or ring system or wherein R₃ and R₄ together can together be (=0).

In some embodiments, D₁ and D₂ are C(=O)NH₂.

VLB. Non-radioactive Metal Extraction

Most metallurgical processes are associated with the generation of wastes and residues containing various non-radioactive metals, such as arsenic, cadmium, chromium, nickel, lead, copper, mercury, silver, gold, and zinc, among others. In many cases, such wastes must be treated or disposed of in safe designated sites to meet laws governing environmental standards. In some cases, the wastes or residues are suitable as raw material for the recovery of economically valuable metals. Hydrometallurgy is a growing field involving the art or process of assaying or reducing ores by means of liquid reagents.

The ligands of the presently disclosed subject matter have many hydrometallurgical uses. In addition to the chelation and extraction of radioactive metal ions, the ligands of the presently disclosed subject matter also are useful in recovering or removing metal ions from the leach streams or process streams created through mining processes. In some embodiments, the ligands are useful in chelating metal ions of economic interest, such as ions from zinc, copper, silver, titanium, nickel, cobalt, indium, platinum, gold and the like. In some embodiments, the ligands are useful in removing contaminating metal ions from process streams to produce waste containing less metal. These metal ions can include metal ions that might be undesirable environmentally. Such metal ions include, but are not limited to, lead, arsenic, and cadmium, among others. In some embodiments, the presence of the contaminating metal interferes with the purity of another, potentially useful, component of the process stream.

In some embodiments, the ligands of Formula (I) can be used to remove or recover metal ions through liquid/liquid extraction processes. In some embodiments, the ligands of Formula (I) can be conjugated to a solid support (which can be fixed or freely moving within the liquid, e.g. beads) and be used to remove or recover metal ions through liquid/solid extraction. For example, the ligand can be conjugated to a polymer support, such as polystyrene. Metal ions in process or waste streams passed over the conjugated surface are complexed by the ligand and removed from solution. Subsequent treatment of the surface can serve to release the metal for final recovery and/or regeneration of the surface with the free ligand. As with the radioactive metal chelators described hereinabove, the fine-tuning of the size of the ligand binding pocket or of the nature of the donor atoms (oxygen, nitrogen, or sulfur) can render the ligands specific for a particular metal ion of interest, if necessary and/or desired.

VII. Chelation Therapy to Remove a Metal Ion or Metal Ions

Metal chelators also have been adapted to isolate ingested or inhaled metal ions, including radioactive metal ions and/or nonradioactive potentially toxic metal ions, such as Hg²⁺, Pb²⁺, As³⁺, Al³⁺, Fe³⁺, and Cd²⁺. Unlike organic poisons, which the body is able to metabolize, toxic metals are either excreted or immobilized. While small amounts of some metals are essential to biological functioning, immobilization of metals in the body through either acute or chronic exposure to the metal can lead to a variety of health issues. Lead intoxication, such as from exposure to lead-based paint, causes problems in the development of the brain and nervous systems of young children and fetuses. In adults, lead poisoning, often from occupational exposure through activities such as welding, smelting, or home remodeling and renovation, can lead to a host of symptoms, including increased blood pressure, fertility problems, nerve disorders, muscle and joint pain, and memory and concentration problems. Mercury, exposure to which can come from contaminated air and water from industrial activities or from dental amalgams, particularly causes problems with the kidneys and the brain. Mercury poisoning also can come from methylmercury, especially from consumption of contaminated fish. Cadmium poisoning, often from occupational exposure to fumes from ore processing or smelting, battery manufacture, electroplating, or paints, or in some cases non-occupational exposure from food or water contamination, can lead to pulmonary edema, lung and prostate cancer, bone osteoporosis, and anemia. Further, immobilization of radioactive metals in the body leads to radiation damage and to radiation-induced cancer. Because of the similarities in the coordination chemistry of Pu⁴⁺ and Fe³⁺, Pu replaces Fe in mammalian iron transport and storage proteins, making Pu especially well retained in the body following contamination. See Xu, J. et al., J. Med. Chem., 38, 2606- 2614 (1995). Because Pu coordination chemistry is so similar to that of iron, ligands for the decorporation of plutonium based on siderophore motifs have been examined, as have various hydrophilic DTPA- and EDTA-based ligands. See

Gorden, A. E. V., et al., Chem. Rev., 103, 4207-4282 (2003). Few metal chelation therapeutics exist. One such treatment for lead poisoning is succimer, or meso-2,3-dimercaptosuccinic acid (DMSA). Succimer is orally available and sold in the U.S. under the brand name CHEMET® (Ovation Pharmaceuticals, Inc., Deerfield, Illinois, United States of America). One disadvantage of DMSA therapy is that the molecule is too lipophobic to remove lead from intracellular sites. See Kalia K., and Flora, S.J., J. Occup. Health, 47(1),

1-21 (2005). 2,3-Dimercaprol (BAL) has been used in lead and arsenic poisoning.

Intravenous EDTA therapy also has been used for the treatment of lead, mercury, aluminum and cadmium poisoning.

Chelation therapy agents require a high selectivity toward the metal ion of interest. A non-selective ligand such as DTPA can deplete essential biological metal ions from patients, causing serious health problems. The use of a chelating agent with reasonable water solubility also is required so that the agent and its metal complex can stay in circulation and the complex can be cleared via the kidneys. In some embodiments, the presently disclosed ligands are used to chelate a radioactive metal ion in a subject in need of chelation therapy to remove a radioactive metal ion or ions. In some embodiments, the ligands are used to chelate a non-radioactive metal ion in a subject in need of chelation therapy to remove the metal ion. Such a subject can have accidentally been poisoned with radioactive or non-radioactive metal ions through the accidental inhalation or ingestion of contaminated air, water, or food or exposed to the metal ion through occupational exposure. Alternatively, the subject can be the victim of planned radioactive contamination through nuclear fallout brought on by an act of war or terrorism (such as the use of a "dirty" bomb).

In some embodiments, the ligand is present in the form of a pharmaceutically acceptable salt. Examples of pharmaceutically acceptable salts include gluconate, lactate, acetate, tartarate, citrate, phosphate, borate, nitrate, sulfate, maleate and hydrochloride salts. The compound can be formulated in a pharmaceutically acceptable carrier to enhance the absorption of the compound.

Pharmaceutical formulations can be prepared for oral, intravenous, or aerosol administration.

Pharmaceutical formulations comprise a ligand described herein or a pharmaceutically acceptable salt thereof in any pharmaceutically acceptable carrier. Optionally, the carrier is pharmaceutically acceptable in humans. If a solution is desired, water is the carrier of choice with respect to water-soluble compounds or salts. With respect to the water-insoluble compounds or salts, an organic vehicle, such as glycerol, propylene glycol, polyethylene glycol, or mixtures thereof, can be suitable. In the latter instance, the organic vehicle can contain a substantial amount of water. The solution in either instance can then be sterilized in a suitable manner known to those of skill in the art, and typically by filtration through a 0.22-micron filter. The solution can be lyophilized for ease of storage.

In addition to ligands or their salts, the pharmaceutical formulations can contain other additives, such as pH-adjusting additives. In particular, useful pH- adjusting agents include acids, such as hydrochloric acid, bases or buffers such as sodium lactate, sodium acetate, sodium phosphate, sodium citrate, sodium borate, or sodium gluconate. Further, the formulations can contain antimicrobial preservatives. Useful antimicrobial preservatives include methylparaben, propylparaben, and benzyl alcohol. The antimicrobial preservative is typically employed when the formulation is placed in a vial designed for multi-dose use. When the compound or salt is substantially water-insoluble, a sufficient amount of emulsifying agent, which is physiologically acceptable, can be employed in sufficient quantity to emulsify the compound or salt in an aqueous carrier.

Particularly useful emulsifying agents include phosphatidyl cholines and lecithin.

As used herein, the term "water-soluble" is meant to define any composition that is soluble in water in an amount of about 50 mg/mL, or greater. Also, as used herein, the term "water-insoluble" is meant to define any composition that has a solubility in water of less than about 20 mg/mL.

In accordance with the present methods, useful ligands of the presently disclosed subject matter can be administered orally as a solid or as a liquid, or can be administered intramuscularly or intravenously as a solution, suspension, or emulsion.

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Claims

CLAIMS What is claimed is:

1. A metal complexing ligand comprising: (a) a fused-ring heteroaromatic backbone, the heteroaromatic backbone comprising one or more donor atoms for complexing a metal ion;

(b) one or more substituents on the fused-ring heteroaromatic backbone, the one or more substituents providing one or more donor groups for complexing a metal ion; and

(c) wherein the fused-ring heteroaromatic backbone and the one or more substituents are chosen to provide for the selective binding to a metal ion having a predetermined ionic radius, subject to the proviso that the ligand is not 1 ,10- phenanthroline-2,9-dicarboxylate (PDA).

2. The metal complexing ligand of Claim 1 , wherein the fused-ring heteroaromatic backbone and the one or more substituents are capable of forming: (a) a five-membered chelate ring with a metal ion;

(b) a six-membered chelate ring with a metal ion; and

(c) combinations thereof.

3. The metal complexing ligand of Claim 1 , wherein the metal complexing ligand is selected from the group consisting of a tetradentate ligand and a pentadentate ligand.

4. The metal complexing ligand of Claim 1 , wherein the metal complexing ligand has a characteristic selected from the group consisting of a solubility in water, a solubility in a non-polar solvent, a solubility in a lipid, a cell membrane permeability, and combinations thereof.

5. A coordination complex comprising:

(a) a metal complexing ligand comprising:

(i) a fused-ring heteroaromatic backbone, the heteroaromatic backbone comprising one or more donor atoms for complexing a metal ion; (ii) one or more substituents on the fused-ring heteroaromatic backbone, the one or more substituents providing one or more donor groups for complexing a metal ion; and

(iii) wherein the fused-ring heteroaromatic backbone and the one or more substituents are chosen to provide for the selective binding to a metal ion having a predetermined ionic radius, subject to the proviso that the ligand is not 1 ,10-phenanthroline-2,9-dicarboxylate

(PDA); and

(b) a metal ion.

6. A conjugate comprising: (a) one of:

(i) a metal complexing ligand comprising a fused-ring heteroaromatic backbone, the heteroaromatic backbone comprising one or more donor atoms for complexing a metal ion; one or more substituents on the fused-ring heteroaromatic backbone, the one or more substituents providing one or more donor groups for complexing a metal ion; and wherein the fused-ring heteroaromatic backbone and the one or more substituents are chosen to provide for the selective binding to a metal ion having a predetermined ionic radius; and (ii) a coordination complex comprising a metal complexing ligand comprising a fused-ring heteroaromatic backbone, the heteroaromatic backbone comprising one or more donor atoms for complexing a metal ion; one or more substituents on the fused-ring heteroaromatic backbone, the one or more substituents providing one or more donor groups for complexing a metal ion; and wherein the fused-ring heteroaromatic backbone and the one or more substituents are chosen to provide for the selective binding to a metal ion having a predetermined ionic radius; and

(b) one of a carrier, a substrate, and a targeting moiety.

7. A metal complexing ligand of Formula (I):

(Rn)_n,

D₁ -Ar -D, (I)

wherein Ar is an aromatic moiety containing at least two pyridine rings selected from the group consisting of:

Di and D₂ are independently selected from the group consisting Of-CO₂H, a carboxylate salt, an alkyl ester of -CO₂H, an acyloxymethyl ester of -CO₂H, -CH₂CO₂H, an acyloxymethyl ester Of-CH₂CO₂H, a salt of -CH₂CO₂ ^", an alkyl ester of -CH₂CO₂H, -C(=O)NH₂, -CH₂C(=O)NH₂, phosphate, methyl phosphate, phosphonate, methyl phosphonate, amino, alkylamino, dialkylamino, an amine salt, an amino-substituted alkyl, the salt of an amino-substituted alkyl, hydroxyl, alkylhydroxyl, alkoxyl, mercapto, and alkylmercapto; m is an integer corresponding to the number of sites on a given Ar that can bear an additional substituent (or the number of H atoms on the Ar portion of the D_rAr-D₂ molecule); each n is an integer between 1 and m; and each R is independently selected from the group consisting of H, alkyl, substituted alkyl, branched alkyl, perfluoroalkyl, halo, nitro, cyano, amino, thio, alkylamino, dialkylamino, alkylthio, alkoxyl, sulfate, carboxylate, carbamoyl, alkylcarbamoyl, dialkylcarbamoyl, acyloxyl, acylamino, aroylamino, phosphate, substituted sulfonate, sulfonic acid, aryl, substituted aryl, aralkyloxyl, aryloxyl, arylthio, alkoxycarbonyl, and cycloalkyl; wherein any two R groups on adjacent carbons can form an additional fused ring or ring system or wherein any two R groups substituted on the same carbon can together be (=0); subject to the proviso that when Ar is:

Di and D₂ are not one Of-CO₂H, a carboxylate salt, an alkyl ester Of-CO₂H, an acyloxymethyl ester Of -CO₂H, and C(=O)NH₂.

8. A coordination complex comprising, (a) a metal complexing ligand of Formula (I); and

(b) a metal ion.

9. The coordination complex of Claim 8, wherein the metal ion is selected from the group consisting of an alkali metal ion, an alkaline metal ion, a transition metal ion, a lanthanide metal ion, and an actinide metal ion.

10. A conjugate comprising:

(a) one of

(i) a metal complexing ligand of Formula (I); and (ii) a coordination complex comprising a metal complexing ligand of Formula (I) and a metal ion; and

(b) one of a carrier, a substrate, and a targeting moiety.

11. The conjugate of Claim 6 or Claim 10, wherein the carrier, substrate or targeting moiety is selected from the group consisting of antibodies, amino acids, proteins, peptides, polypeptides, enzymes, enzyme substrates, lipids, phospholipids, hormones, lymphokines, metabolites, antigens, haptens, lectins, avidin, streptavidin, toxins, poisons, environmental pollutants, carbohydrates, oligosaccharides, polysaccharides, glycoproteins, glycolipids, nucleotides, oligonucleotides, nucleic acids and derivatized nucleic acids (including deoxyribo- and ribonucleic acids), DNA and RNA fragments and derivatized fragments (including single and multi-stranded fragments), natural and synthetic drugs, receptors, virus particles, bacterial particles, virus components, biological cells, cellular components (including cellular membranes and organelles), natural and synthetic lipid vesicles, polymers, polymer particles, polymer membranes, conducting and non-conducting metals and non-metals, glass, and plastic surfaces and particles.

12. A contrast enhancement agent useful for providing a visible image of a biological sample comprising:

(a) a metal complexing ligand comprising:

(i) a fused-ring heteroaromatic backbone, the heteroaromatic backbone comprising one or more donor atoms for complexing a metal ion; (ii) one or more substituents on the fused-ring heteroaromatic backbone, the one or more substituents providing one or more donor groups for complexing a metal ion; and (iii) wherein the fused-ring heteroaromatic backbone and the one or more substituents are chosen to provide for the selective binding to a metal ion having a predetermined ionic radius; and

(b) at least one metal ion, wherein the at least one metal ion is selected from the group consisting of a paramagnetic metal ion and a radionuclide.

13. A contrast enhancement agent useful for providing a visible image of a biological sample comprising:

(a) a metal complexing ligand of Formula (I); and (b) at least one metal ion, wherein the at least one metal ion is selected from the group consisting of a paramagnetic metal ion and a radionuclide.

14. The contrast enhancement agent of Claim 12 or Claim 13, wherein the paramagnetic metal ion is selected from the group consisting of a transition element, a lanthanide element, and an actinide element.

15. The contrast enhancement agent of Claim 14, wherein the paramagnetic metal ion is selected from the group consisting of Gd(III), Mn(II), Cu(II), Cr(III), Fe(II), Fe(III), Co(II), Er(II), Ni(II), Eu(III) and Dy(III).

16. The contrast enhancement agent of Claim 12 or Claim 13, wherein the radionuclide is selected from the group including, but not limited to, ^99mTc, ¹¹¹In, ⁶⁷Ga, ²⁰¹TI, ²¹²Bi and ⁶⁴Cu.

17. The contrast enhancement agent of Claim 12 or Claim 13, wherein the agent is in lyophilized form.

18. A kit for obtaining a visible image of a biological sample comprising at least a two-component system of a lyophilized contrast enhancement agent and an aqueous diluent, comprising:

(a) a first component comprising a lyophilized contrast enhancement agent, wherein the agent comprises a metal complexing ligand comprising:

(iii) wherein the fused-ring heteroaromatic backbone and the one or more substituents are chosen to provide for the selective binding to a metal ion having a predetermined ionic radius; and at least one metal ion, wherein the at least one metal ion is selected from the group consisting of a paramagnetic metal ion and a radionuclide; and

(b) a second component comprising a pharmaceutically acceptable diluent.

19. A kit for obtaining a visible image of a biological sample comprising at least a two-component system of a lyophilized contrast enhancement agent and an aqueous diluent, comprising: (a) a first component comprising a lyophilized contrast enhancement agent, wherein the agent comprises a ligand of Formula (I) and at least one metal ion, wherein the at least one metal ion is selected from the group consisting of a paramagnetic metal ion and a radionuclide; and (b) a second component comprising a pharmaceutically acceptable diluent.

20. The kit of Claim 18 or Claim 19, wherein the pharmaceutically acceptable diluent comprises a phosphate buffered saline.

21. A composition comprising an imaging agent comprising: (a) a metal complexing ligand comprising:

(i) a fused-ring heteroaromatic backbone, the heteroaromatic backbone comprising one or more donor atoms for complexing a metal ion;

(ii) one or more substituents on the fused-ring heteroaromatic backbone, the one or more substituents providing one or more donor groups for complexing a metal ion; and (iii) wherein the fused-ring heteroaromatic backbone and the one or more substituents are chosen to provide for the selective binding to a metal ion having a predetermined ionic radius;

(b) at least one metal ion, wherein the at least one metal ion is selected from the group consisting of a paramagnetic metal ion and a radionuclide; and

(c) a pharmaceutically acceptable carrier.

22. A composition comprising an imaging agent comprising a metal complexing ligand of Formula (I); at least one metal ion, wherein the at least one metal ion is selected from the group consisting of a paramagnetic metal ion and a radionuclide; and a pharmaceutically acceptable carrier.

23. A method of imaging one of a cell, a tissue, and a patient, the method comprising administering to one of a cell, tissue, and patient and rendering a magnetic resonance image of the one of a cell, tissue, and patient an imaging agent comprising:

(a) a metal complexing ligand comprising:

(ii) one or more substituents on the fused-ring heteroaromatic backbone, the one or more substituents providing one or more donor groups for complexing a metal ion; and (iii) wherein the fused-ring heteroaromatic backbone and the one or more substituents are chosen to provide for the selective binding to a metal ion having a predetermined ionic radius; and

24. A method of imaging one of a cell, a tissue, and a patient, the method comprising:

(a) administering to one of a cell, tissue, and patient an imaging agent comprising a metal complexing ligand of Formula (I) and at least one metal ion, wherein the at least one metal ion is selected from the group consisting of a paramagnetic metal ion and a radionuclide; and

(b) rendering a magnetic resonance image of the one of a cell, tissue, and patient.

25. A method for detecting a target substance associated with a disease state, the method comprising administering to one of a cell, a tissue, and a patient to produce an image of the one of a cell, a tissue, and a patient an imaging agent comprising at least one metal ion, wherein the at least one metal ion is selected from the group consisting of a paramagnetic metal ion and a radionuclide, and a metal complexing ligand comprising:

(a) a fused-ring heteroaromatic backbone, the heteroaromatic backbone comprising one or more donor atoms for complexing a metal ion;

(c) wherein the fused-ring heteroaromatic backbone and the one or more substituents are chosen to provide for the selective binding to a metal ion having a predetermined ionic radius.

26. A method for detecting a target substance associated with a disease state, the method comprising administering an imaging agent comprising at least one metal ion, wherein the at least one metal ion is selected from the group consisting of a paramagnetic metal ion and a radionuclide, and a metal complexing ligand of Formula (I) to one of a cell, a tissue, and a patient to produce an image of the one of a cell, a tissue, and a patient.

27. A radiopharmaceutical, comprising: (a) a metal complexing ligand comprising:

(iii) wherein the fused-ring heteroaromatic backbone and the one or more substituents are chosen to provide for the selective binding to a metal ion having a predetermined ionic radius; and (b) at least one radionuclide.

28. A radiopharmaceutical, comprising: (a) a metal complexing ligand of Formula (I); and

(b) at least one radionuclide.

29. The radiopharmaceutical of Claim 27 or Claim 28, wherein the radionuclide is selected from the group including, but not limited to, ^99mTc, ¹¹¹In, ⁶⁷Ga, ²¹²Bi and ⁶⁴Cu.

30. A method of treating a disease in a subject in need of treatment thereof, the method comprising administering to the subject a radiopharmaceutical, the radiopharmaceutical comprising: (a) a metal complexing ligand comprising: (i) a fused-ring heteroaromatic backbone, the heteroaromatic backbone comprising one or more donor atoms for complexing a metal ion; (ii) one or more substituents on the fused-ring heteroaromatic backbone, the one or more substituents providing one or more donor groups for complexing a metal ion; and

31. A method of treating a disease in a subject in need of treatment thereof, the method comprising administering to the subject a radiopharmaceutical, the radiopharmaceutical comprising:

(a) a metal complexing ligand of Formula (I); and

(b) at least one radionuclide.

32. A method for detecting a metal ion in a sample, comprising:

(a) providing a sample;

(b) adding to the sample, in an amount sufficient to generate a detectable fluorescence response to the metal ion, a metal complexing ligand comprising: (i) a fused-ring heteroaromatic backbone, the heteroaromatic backbone comprising one or more donor atoms for complexing a metal ion; (ii) one or more substituents on the fused-ring heteroaromatic backbone, the one or more substituents providing one or more donor groups for complexing a metal ion; and (iii) wherein the fused-ring heteroaromatic backbone and the one or more substituents are chosen to provide for the selective binding to a metal ion having a predetermined ionic radius; (c) illuminating the sample to generate a fluorescence response; and (d) observing the fluorescence response.

33. A method for detecting a metal ion in a sample, comprising: (a) providing a sample;

(b) adding to the sample, in an amount sufficient to generate a detectable fluorescence response to the metal ion, a metal complexing ligand according to Formula (I);

(c) illuminating the sample to generate a fluorescence response; and

(d) observing the fluorescence response.

34. The method of Claim 32 or Claim 33, further comprising quantifying the fluorescence response.

35. The method of Claim 32 or Claim 33, wherein metal ion is selected from the group consisting of Ca²⁺, Fe³⁺, Mg²⁺, Zn²⁺, and Mn²⁺.

36. The method of Claim 32 or Claim 33, wherein the sample comprises a living cell, a biological fluid, a tissue, an extract of a cell or tissue, and combinations thereof.

37. A method for removing a metal ion from an industrial or environmental sample, the method comprising: (a) providing an industrial or environmental sample; (b) contacting the industrial or environmental samples with a metal complexing ligand to form a ligand metal complex, wherein the metal complexing ligand comprises

(iii) wherein the fused-ring heteroaromatic backbone and the one or more substituents are chosen to provide for the selective binding to a metal ion having a predetermined ionic radius; and (c) separating the ligand metal complex from the sample.

38. A method for removing a metal ion from an industrial or environmental sample, the method comprising:

(a) providing an industrial or environmental sample; (b) contacting the industrial or environmental sample with a metal complexing ligand of Formula (I) to form a ligand metal complex; and

(c) separating the ligand metal complex from the sample.

39. The method of Claim 37 or Claim 38, wherein the metal ion is radioactive.

40. The method of Claim 39, wherein the radioactive metal ion is selected from the group consisting of ¹³⁷Cs, Pu⁴⁺, and ⁹⁰Sr.

41. The method of Claim 39, wherein the industrial or environmental sample is selected from the group consisting of spent nuclear fuel waste, water samples, and soil samples.

42. The method of Claim 37 or Claim 38, wherein the metal ion is an ion of a metal that has economic value.

43. The method of Claim 42, wherein the metal ion is an ion of a metal selected from the group consisting of zinc, copper, silver, titanium, nickel, cobalt, indium, platinum, and gold.

44. A method for removing a metal ion from a subject in need of such treatment, the method comprising contacting the subject with

(a) a metal complexing ligand comprising: (i) a fused-ring heteroaromatic backbone, the heteroaromatic backbone comprising one or more donor atoms for complexing a metal ion; (ii) one or more substituents on the fused-ring heteroaromatic backbone, the one or more substituents providing one or more donor groups for complexing a metal ion; and

(iii) wherein the fused-ring heteroaromatic backbone and the one or more substituents are chosen to provide for the selective binding to a metal ion having a predetermined ionic radius; and

(b) a pharmaceutically acceptable carrier.

45. A method for removing metal ions from a subject in need of such treatment, the method comprising contacting the subject with (a) a metal complexing ligand of Formula (I); and

(b) a pharmaceutically acceptable carrier.

46. The method of Claim 44 or Claim 45, wherein the metal ion is a radioactive metal ion.

47. The method of Claim 44 or Claim 45, wherein the metal ion is a toxic metal ion.

48. The method of Claim 47, wherein the metal ion is the ion of a metal selected from the group consisting of Pb, Hg, Cd, As, Al, and Fe.

49. A method for preparing a metal complexing ligand, wherein the metal complexing ligand comprises a fused-ring heteroaromatic backbone, the heteroaromatic backbone comprising one or more donor atoms for complexing a metal ion; one or more substituents on the fused-ring heteroaromatic backbone, the one or more substituents providing one or more donor groups for complexing a metal ion; and wherein the fused-ring heteroaromatic backbone and the one or more substituents are chosen to provide for the selective binding to a metal ion having a predetermined ionic radius; the method comprising varying one of or both of the fused-ring heteroaromatic backbone and the one or more substituents to regulate a characteristic selected from the group consisting of:

(a) a bonding angle of the ligand;

(b) a size of a metal binding site of the ligand;

(c) an electron-donating ability of the one or more donor atoms of the heteroaromatic backbone;

(d) a stability of a complex of the ligand and a predetermined metal ion; and

(e) combinations thereof.

50. A method for designing a metal complexing ligand, wherein the metal complexing ligand comprises a fused-ring heteroaromatic backbone, the heteroaromatic backbone comprising one or more donor atoms for complexing a metal ion; one or more substituents on the fused-ring heteroaromatic backbone, the one or more substituents providing one or more donor groups for complexing a metal ion; and wherein the fused-ring heteroaromatic backbone and the one or more substituents are chosen to provide for the selective binding to a metal ion having a predetermined ionic radius; the method comprising varying one of or both of the fused-ring heteroaromatic backbone and the one or more substituents to regulate a characteristic selected from the group consisting of: (a) a bonding angle of the ligand;

(b) a size of a metal binding site of the ligand;

(d) a stability of a complex of the ligand and a predetermined metal ion; and

(e) combinations thereof.

donor atom four donor atoms donor atoms

Figure 1A Figure 1B