AU7587091A

AU7587091A - A fonctionalized complexand

Info

Publication number: AU7587091A
Application number: AU75870/91A
Authority: AU
Inventors: Martin W. Brechbiel; Otto A. Gansow
Original assignee: US Government; US Department of Commerce
Current assignee: US Department of Commerce
Priority date: 1990-03-26
Filing date: 1991-03-14
Publication date: 1991-10-21
Also published as: WO1991014458A1; CA2079206A1; EP0522007A1; EP0522007A4

Description

A FϋNCTIONALIZED COMPLEXAND

BACKGROUND OF THE INVENTION Technical Field The present invention relates to macrocyclic chelates and methods of use thereof. Specifically, the invention relates to a substituted 1,4,7,10- tetraazacyclododecane triacetic acid wherein the three triacetic acids are linked at three of the nitrogens of the macrocycle, and a hydrogen is attached to the fourth nitrogen of the macrocycle. The chelate exists as a mixture of the four possible structures (or isomers) . The present invention also relates to the various uses of these particular chelating agents.

Background Information Macrocycles have been studied for their usefulness as chelating agents for numerous metal ions that have therapeutic, diagnostic, or other uses. A macrocycle of considerable value as a chelate is the 1,4,7,10-tetraazacyclododecane-N, N', N'¹, N"¹- tetraacetic acid (DOTA) . DOTA type compounds have been linked to biomolecules to form delivery systems for the chelated metal ion to specific sites in vivo.

U.S. Patent 4,678,667 to Meares et al., discloses such chelating agents. The chelating agents of this patent can include a bifunctional DOTA compound that is a Cu(II) chelate. The usefulness is therefore limited to the effects of the copper metal ion. The synthesis reportedly also lacks in efficiency resulting in low yields and not always reproducible results. U.S. Patent 4,622,420 to Meares et al. discloses acyclic bifunctional chelating agents of the ethylenediamine N, N, N¹, N*-tetraacetic acid (EDTA) types useful for binding metals, other than copper, such as Indium. These compounds are useful for imaging tumors.

U.S. Patent 4,652,519 to arshawsky et al. also discloses EDTA type bifunctional chelates and a process for their production. These compounds were offered as an alternative to the Meares compounds discussed above.

U.S. Patents 4,453,106 and 4,474,509 to Gansow et al. disclose the use of metal chelate conjugated monoclonal antibodies and specific metal chelate conjugated monoclonal antibodies, respectively. These disclosures provide compounds and methods for treating cellular disorders. Advantages of the use of chimeric and monoclonal antibodies have been discussed

(Morrison, S.L. , Hospital Practice (Office Edition) 24:64-65, 72-74, 77-80 (1989). Radiometal chelate conjugated monoclonal antibodies specific to a target cell are used to deliver alpha, beta, or Auger electon emitting metal ion. These disclosures are not related to DOTA compounds.

European Patent Application 0 088 695 to McKearn et al. discloses the oxidation of monoclonal antibody and linking of ligand by imine formation and subsequent cyanoborohydride reduction to form conjugates.

PCT application (WO 89/01476) to Parker et al. discloses macrocycle ligand/chelates and conjugates thereof. European Patent Application 0 292 689 to

Tweedle et al. discloses triacetate 1,4,7 10-tetraaza macrocycles but does not discuss the linkage of these compounds to haptens.

Other U.S. patents which disclose various chelating agents or conjugates include U.S.P. 4,442,305, U.S.P. 4,530,963, U.S.P. 4,732,974, and U.S.P. 4,585,559.

The value of having a ligand conjugate to chelate metal ions of therapeutic, diagnostic, or other uses lies in their commercial importance. This commercial importance is created by the fact that many metal ions have desirable characteristics, but the delivery systems lack specificity to target the metal ions, or do not adequately bind the metal ions. Examples of the usefulness of specific metal ions follow. The usefulness of radionuclide materials in cancer therapy is disclosed in the article, Kozak et al., "Radionuclide-Conjugated Monoclonal Antibodies: A Synthesis of Immunology, in Organic Synthesis and Nuclear Science," Trends in Biotechnology 4(10): 259- 264 (1985) . This article discusses the use of antibody conjugates to deliver either alpha or beta radiation.

Other uses for chelated metal ions are discussed in Magerstadt et al. "Gd(DOTA) : An Alternative to Gd(DTPA) as a T._/2 Relaxation Agent for NMR Imaging or Speσtroscopy," Magnetic Resonance in

Medicine 3:808-812 (1986). Specifically, this article discloses the usefulness of gadolinium as a relaxation agent for NMR imaging.

The efficacy of a linking group, within chelated metal complexes, has been discussed by Paik et al., J. Nucl. Med. 30:1693-1701 (1989). Other articles which may be of some interest include: McMurry et al., "Template and Stepwise Synthesis of a Macrobicyclic Catechoylamide Ferric Ion Sequestering Agent," J. Amer. Chem. Soc. 109: 3451-53 (1987), Raymond et al., "Macrocyclic Catechol

Containing Ligands," Pure & Appl. Chem. 60: 545-48 (1988), McMurry et al., "Macrobicyclic Ion (III) Sequestering Agents," J. Am. Chem. Soc. 109: 7196-98 (1987), McMurry et al., "Molecular Recognition and Metal Ion Template Synthesis," Science 244: 938-43 (1989), Kiggin et al., "Functionalized Oligocyclic Large Cavities- A Novel Siderophore," Angew. Chem. Int. Ed. Engl. 23: 714-15 (1984), Kiggin et al., "Large Oligocyclic Cavities for Strong Cation Complexation, Tetrahedron 42: 1859-72 (1986), Sun et al., "New

Multidentate Ligands. 28. Synthesis and Evaluation of New Macrocyclic Ligands Containing Bidentate Endocyclic Catechol Donor Groups," Inorg. Chem. 25: 4780-85 (1985), Moi et al., "The Peptide Way to Macrocyclic Bifunctional Chelating Agents: Synthesis of 2-(p-

Nitrobenzyl) -1,4,7,10- Tetraazacyclododecane -N, N* , N' * , N* * • -Tetraacetic Acid and Study of Its Yttrium (III) Complex, " J. Am. Chem. Soc. 110:6266-67 (1988), and Cox et al. , "Synthesis of a Kinetically Stable Yttrium-90 Labelled Macrocycle Antibody Conjugate," J. Chem. Soc.. Chem. Commun. , pp. 797-98 (1989) .

All patents and publications referred to herein are hereby incorporated by reference.

The industry is lacking a macrocyclic chelate that can be efficiently produced in high yields, that can be linked to proteins, and that has desirable chelating qualities for numerous metal ions wherein the formation kinetics thereof and reaction conditions required for metal complexation are favorable for use with monoclonal antibodies.

SUMMARY OF THE INVENTION The invention is the ligand and its metal chelates having a general Formula of I and la, respectively:

ⁱ Ic wherein three of the R groups are -CH-COOH with the fourth R being kept as -H; n is an integer from 1 to 5;

X equals -NO, or -NH_; and and M is a metal ion being a member selected from the group of elements consisting of Y, In, Bi, Pb, Cu, Ag,

Au, Pt, and the Lanthanides.

A preferred embodiment includes the situation where, in the ligand, X is either -N0_ or -NH,, and n is an integer from 1 to 2.

Another preferred embodiment includes the situation where, in the chelate, X is either -NO or -

NH_, and n is 1 to 2.

An additional embodiment includes the situation where, in the chelate described in the paragraph directly above, M is a member selected from the group consisting of Bi, Pb, Y, Cu, Gd, Eu and Tb. A further embodiment includes the situation where, in the chelate, n is 2, X is -NH_, and M is a member selected from the group consisting of Pb 203,

Pb²¹², Bi²¹², -90 and Cu 67

An additional embodiment includes the chelate described in the paragraph directly above wherein M is

212 a member selected from the group consisting of Pb and Bi²¹².

Another embodiment includes the situation where, in the chelate, n is 2, X is -NH₂, and X is

-NH_?, and M is a member selected from the group consisting of Eu and Tb.

The invention also includes ligand-hapten conjugates of the general Formula II and chelate-hapten conjugates of the general Formula Ila:

_χl IIcu. wherein three of the R groups are -CH₂COOH with the fourth R group being kept as -H; n is an integer from 1 to 5;

X¹ equals -NH-Q where Q is selected from the group consisting of hormones, steroids, enzymes and proteins, a subset of proteins being monoclonal antibodies, chimeric antibodies, and fragments thereof; and M is a metal ion being a member selected from the group consisting of Y, In, Bi, Pb, Cu, Ag, Au, Pt, and the Lanthanides.

A further embodiment includes the ligand- hapten conjugate wherein n is 2 and X' is-NH-Q.

Another embodiment includes the ligand hapten- conjugate wherein Q is a protein, said protein being a monoclonal antibody, chimeric antibody, or fragments thereof. A further embodiment of this invention is a ligand-hapten conjugate, as is drawn in Formula II (shown above) , wherein three groups are -CH-COOH, and the fourth is -H, n is an integer from 1 to 5, and X' is -NH-L-Q where Q is a hapten chosen from the group consisting of hormones, steroids, enzymes, proteins, monoclonal antibodies, chimeric antibodies, or fragments thereof, and L is a covalent linking group.

An additional embodiment of this invention is a chelate-hapten conjugate, as is drawn in Formula Ila (shown above) , wherein three groups are

-CH₂COOH, and the fourth is -H, n is an integer from 1 to 5, X' is -NH-L-Q where Q is a hapten selected from the group consisting of hormones, steroids, enzymes, pro'-ϋins, monoclonal antibodies, chimeric antibodies, or fragments thereof, M is a metal ion selected from the group consisting of Bi, Pb, Y, Cu, Ag, Au, Pt and the lanthanides, and L is a covalent linking group. In either of the two embodiements directly above, L may be an organic radical or a substituted aliphatic hyrdocarbon chain, optionally interrupted by one or more hetero atoms selected from -0- or -S-, or by one or more -NR*- groups (where R* is a hydrogen atom or a C, _ alkyl group), -CONR¹- groups, - NR'CO- groups, cycloaliphatic groups, aromatic groups, or heteroaromatic groups, or a mixture thereof. The invention also includes methods for using these compounds for treatment of cellular disorders and for diagnostic tests. Thus, the above compounds may be used as therapeutic and diagnostic agents.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 illustrates the chemical pathway used to produce the preferred embodiment of the invention. DETAILED DESCRIPTION OF THE INVENTION Compounds of this invention include the substituted 1,4,7,10 tetraazacyclododecane triacetic acid represented in the general Formula I shown above or specifically by compound 5 of Figure 1. The general formula is a 12 membered ring tetraaza macrocycle, with the nitrogens in the 1,4,7,10 positions. The acetic acid groups are attached to three of the nitrogens, and a hydrogen is attached to the remaining secondary nitrogen. This description encompasses all four possible structures or iso ers contained in the mixture. Furthermore, each of the nitrogens is bridged by an ethylene group. The substituted triacetic acid ligands represented by Formula I complexes metals (Formula la) . Metal complexes are formed by placing the ligand into solution with an appropriate metal salt having the metal to be chelated. Metal salts have to be selected so as to prevent the hydrolysis of the metal. Also, reaction conditions in an aqueous medium have to be chosen such that the metal is not hydrolyzed. For example, a lead nitrate, bismuth iodide complex, or yttrium acetate salts can be used to form a metal chelate with lead, bismuth, or yttrium, respectively. General examples of suitable salts include any soluble divalent metal complex or any trivalent metal complex that is not hydrolyzed at pH 4 to about 9. The most desirable metal ions for chelation with general Formula I are members from the group consisting of bismuth, lead, copper, yttrium, platinum, gold, silver, gallium, and any of the elements of the lanthanide series. The most desirable elements of the lanthanide series are (1) gadolinium, for use in NMR imaging and as a relaxation agent in NMR imaging, and (2) terbium and (3) europium, because of their use as chromophores in time resolved fluorescence speσtroscopy. These fluorescent compounds can be useful in an in vitro diagnostic assay.

The aniline substituent of compound 5 is desirable as a substituent that can be used to conjugate the compound to haptens. The aniline group can be linked to an oxidized carbohydrate on the protein as an imine, and the linkage can subsequently be reduced to an a ine by cyanoborohydride.

The haptens suitable for linking with the compounds of general Formula I or la can vary widely. The most desirable haptens are members selected from the group consisting of hormones, steroids, enzymes, proteins, monoclonal or chimeric antibodies, and fragments thereof. These haptens are desirable because of their site specificity to tumors and/or various organs of the body. The preferred hapten for use in treating cellular disorders or various disease conditions is a specific protein type, a monoclonal antibody or chimeric antibody, or fragments thereof.

In Formulas la and Ila, the compounds of this invention can have a value of n equal to an integer from 1 to 5. In a preferred embodiment, n equals 2. It is desirable for n to equal 2 versus 1 because the chelating ligand is separated form the protein more and possesses increased free rotation. The increased free rotation allows a metal to chelate with the macromolecule more easily. When n is 3 or greater, the synthesis of the compound becomes lengthy.

Figure 1 illustrates the preferred reaction pathway or process for forming the compound of this invention. The process first provides a cyclic triamide with a substituent on the carbon backbone framework of the molecule. The embodiment of Figure 1 has n=l as the initial substituent for linkage. The process then provides a tetraaza macromolecule having the substituent in the 2 position as shown. Alkylation with bromacetic acid forms the three nitrogen to carbon bonds of the three carboxymethylene substituents.

The desired diactive ester 1 is formed sequentially from iminodiacetic acid. The a ine is first blocked using the reagent BOC-ON or any other suitable blocking agent such as EMOC, in the presence of triethylamine which serves to deprotonate the starting material. The subsequent nitrogen blocked diacetic acid or other such nitrogen blocked compound is then coupled to N-hydroxysuccinimide, or any of the suitable compounds such as phenols, or N- hydroxydicarboximides which form a reactive ester. The choice of compounds which form active esters or blocking groups in within the scope of the art. The coupling is done by a carbodii ide. This step produces the nitrogen blocked active ester.

Ring formation under high dilution conditions between amino acid amides (compounds la) with the nitrogen blocked active ester (compound 1) is then performed. This step forms the triamide macrocycle (compound 2) . The amine nitrogen of compound 2 is deblocked with hydrochloric acid i dioxane. This forms the HC1 salt of the triamide macrocycle. The salt is then reduced with borane/tetrahydrofuran. The resulting borane adduct is cleaved by hydrochloric acid to form the substituted tetraaza macrocycle (compound 3) . This macrocycle can then be alkylated with three equivalents of haloacetic acid in the presence of base to form a nitrobenzyl tetraaza macrocycle triacetate (compound 4) . The nitro group of compound 4 can be reduced with hydrogen over a palladium on carbon catalyst to produce the aniline or the aminobenzyl structure depicted as compound 5.

The chelating agent formed by the tri- alkylation of the cyclen described in the process of Figure 1 is novel and separate from those previously described in that, when reacted with trivalent metal ions, an electrically neutral (zero charged) metal chelate is formed. The advantages of such zero charged chelates in medical applications is well understood and has been recently described, for example, by Tweedle, et. al. (E.P.A. 0 292 698), and includes reduced osmolality of the complex and increased lipophilicity, hence solubility in lipid tissues. This, in turn, confers an increased ability of the metal complexes to clear from the interior of cells. This last feature is of importance since often monoclonal antibodies are catabolized into normal liver cell interiors, and for diagnostic imaging in the abdomen, such clearance from normal liver cells is to be desired.

A particular advantage of the tri-alkylated ligands of Formula I lies in their ability to rapidly form metal chelates at aqueous solution pH values 4-7. The more complex DOTA macrocycles, in which the hydrogen that is attached to the secondary nitrogen of Formula I is replaced by a carboxymethylene group, are harder to label with metals since they react exceedingly slow with trivalent metals such as indium or yttrium at these pH values, thus, if a radiometal is used, providing an undesirable long term radiation does during metal chelate formation to any hapten to which the ligand is conjugated. The pH region 4-7 is especially desirable for formation of trivalent metal ion chelate conjugated monoclonal antibodies in that the antibody is stable therein, and the metal ions are less susceptible to hydrolysis as occurs at higher pH values, thus markedly simplifying the radiometal labeling of ligand conjugated antibodies or fragments as compared to such procedures for hapten conjugated DOTA ligands.

In its preferred embodiment, the coupling of compound 5 to antibodies is through imine formation with oxidized carbohydrate on protein followed by in situ reduction to the amine. An advantage of this methodology is that when coupling to proteins and, in particular, when coupling to antibodies, the carbohydrate of the antibody can be oxidized prior to the coupling reaction. The aniline reacts with the aldehyde that is formed on the protein. The imine that is formed can be reduced by cyanoborohydride to form a covalent amine linkage to the antibody that is site specific. This position is distant from the antigen binding sites of the monoclonal antibody and thus minimizes any deleterious effects of the protein modification.

An embodiment of the invention involves a ligand-hapten conjugate of general Formula II.

This conjugate complexes metal ions. For complexation, especially of radioactive metals, it is desirable to expose metal ions to the protein conjugate in a concentrated metal solution for a short a period of time as possible. Certain metals, such as divalent metal ions, react rapidly and directly with the conjugate. The kinetics of the formation reactions for these compounds are so rapid that it is desirable to have the ligand-hapten conjugate available in the pharmacy immediately prior to use. The conjugate can then be mixed in the radionuclide to form the complex and, subsequently, the metal chelate conjugate formed can be purified, for example, by size exclusion high pressure liquid chromatography. A desirable hapten for the ligand conjugate can be selected from the group consisting of hormones, steroids, enzymes, and proteins.

The most commercially useful embodiments of the invention are metal chelate-hapten conjugates having the general Formula Ila where: (1) n is an integer from 1 to 5, (2) X is -NH-Q- with Q being a hapten selected from the group consisting of hormones. steroids, proteins, monoclonal antibodies, chimeric antibodies, and fragments thereof, and (3) M is a metal ion being selected from the group of elements consisting of Bi, Pb, Cu, Ag, Au, Y, Pt, and the lanthanides. These chelate conjugates can deliver radioactive metal ions such as Pb 212, Bi212, Y90, and Cu 67 to treat specific cellular disorders. A further embodiment of this invention is a ligand-hapten conjugate, as is drawn in Formula II (shown above) , wherein three groups are -CH-COOH, and the fourth is - H, n is an integer from 1 to 5, and X' is -NH-L-Q where Q is a hapten chosen from the group consisting of hormones, steroids, enzymes, proteins, monoclonal antibodies, chimeric antibodies, or fragments there and L is a covalent linking group.

An additional embodiment of this invention is a chelate-hapten conjugate as is drawn in Formula Ila (shown above) wherein three groups are -CH₂COOH, and the fourth is -H, n is an integer from 1 to 5, X' is - NH-L-Q where Q is a hapten selected from the group consisting of hormones, steroids, enzymes, proteins, monoclonal antibodies, chimeric antibodies, or fragments thereof, M is a metal ion selected from the group consisting of Bi, Pb, Y, Cu, Ag, Au, Pt and the lanthanides, and L is a covalent linking group.

In either of the two embodiments directly above, L may be an organic radical or a substituted aliphatic hyrdocarbon chain, optionally interrupted by one or more hetero atoms selected from -0- or -S-, or by one or more -NR¹- groups (where R* is a hydrogen atom or a C, _ alkyl group), -CONR'- groups, - NR'CO- groups, cycloaliphatic groups, aromatic groups. or heteroaromatic groups, or a mixture thereof.

The invention includes a process for treating cellular disorders. The process uses the chelate conjugate with a hapten having a selective binding site at the cellular disorder. For example, Q can be a monoclonal antibody wherein the antibody is created and directed against an epitope found specifically on the tumor cells. Thus, when Y 90 is transported to the antigen site and decays, a beta irradiation is produced. If desired, Bi 212 can be introduced in those cases where the disorder to be treated, such as with leukemic cells, can be reached within the 1 hour half-life of the isotope. Most desirably, at least 95 percent of the radionuclide remains in the chelate. In an acidic medium, such as the stomach, at least about 70 percent is retained.

The invention also includes a process for diagnostic testing. This process uses a chelate conjugate having general Formula Ila wherein M is selected from the group consisting of Pb 203, In111,

67 68 Ga , Ga . The usefulness of metal ions for both in vitro and in vivo diagnostic procedures is disclosed in

U.S. Patent 4,454,106.

The most desirable embodiment of this diagnostic process uses Pb 203. Pb203 has a 52.1 hour half-life as a gamma emitter. Pb 203 has a unique property in that it decays in a high percentage by single photon emission. This gamma emission is preferred and dominant over all other emissions. This single photon emission makes Pb 203 useful for single photon emission computed tomography (SPECT) which is a diagnostic tool. Thus, when Pb 203 is linked by use of a chelate to a hapten, which specifically localizes in a tumor, then that particular localization can be dimensionally mapped for diagnostic purposes in vivo by

SPECT. Alternatively, the emission can be used in vitro in radioimmunoassay.

The present invention can be illustrated by the use of the following non-limiting examples.

EXAMPLE 1

Synthesis of p-Nitrobenzyl l,4.7_r10 tetraazacyclododecane triacetic acids

The cyclen (compound 3 of Figure 1) was alkylated at 45^βC with three sequential additions of 1 equivalent of bromoacetic acid in the presence of 5 M sodium hydroxide. The base was added via an autoburette, and the pH of the reaction solution was maintained at 8.5. The reaction was allowed to stir at 45^βC overnight after the final addition. The following day the solution was acidified to pH 2.0 with 3 N HCl. The solution was loaded onto a 2.6 x 30 cm AG50wX8 200/400 mesh H+ form ion exchange column and washed with water until the eluant was neutral. The crude product was eluted form the column with one liter of 2 M NH.OH. The solution was rotary evaporated to a solid. The solid was taken up in water (25 mL total with rinsing ) and loaded onto a 1.6 x 20 cm AGlwX8 200/400 mesh HOAc form ion exchange column. The product was eluted with a two liter gradient of 0.0 to 0.3 M HOAc. The relevant fractions were combined, concentrated to 25 mL, and then freeze dried to give a fluffy white solid product. EXAMPLE 2 Labeling of Antibody With Metal Chelate

The procedures and reagents described above for the preferred embodiment of making the compounds are used for this example.

The monoclonal antibody B72.3 binds specifically to a glycoprotein on LS-174T cells. This glycoprotein is also present in humans who have colon cancer. This antibody is labeled with the ligand of compound 5 of Figure 1 as follows:

One mL of B72.3 (10 mg/mL) was cooled in an ice bath and combined with 20 μL of 2.5 M sodium acetate (pH 6). Sodium periodate (NaI0₄, 3.5-4.7 mg, Aldrich) was added, giving a NaI0₄ concentration of 16.22 mM. After the solid was dissolved, the clear solution was left on ice in the refrigerator for 1 1/2 hrs. The solution was then passed through a 2 x 10 cm Sephadex G-50 column, eluted with acetate-buffered saline (pH 6) . Ten fractions (1 mL each) were collected, and the concentration of B72.3 in each fraction was determined by UV absorption at 220 nm. The fractions containing oxidized B72.3 (6.7-9.0 mg in 2.2 L) were collected and reacted with excess compound 5 at a molar ratio of 1:50-60. The mixture was mixed, allowed to stand at room temperature for one hr, and finally left in the refrigerator overnight. The next day, sodium cyanoborohydride (Aldrich, 20-25 μL of 1 M solution) was added to the chelate-conjugated B72.3 to reduce the Schiff base to the saturated amine. This mixture was allowed to stand at room temperature for 4- 5 hr. Before labeling with metal, the protein is dialyzed against a solution comprising 0.02 M N- morpholinoethanesulfonic acid and 0.01 M NaCl at pH 5.9. The protein in solution is labeled with Y⁹⁰ by reaction with an acetate solution of the isotope followed by passage through a TSK 3000 size exclusion column. This is a high pressure liquid chromatography technique. The compound is mixed with an excipient and is used in a model system in athymic mice which have been implanted with LS-174T cells to develop a tumor in the flank of the animal. The antibody localizes specifically to these tumor cells to deliver its radiation. EXAMPLE 3

In Vivo Use of the Labeled Protein Conjugate

The protein conjugate in solution in labeled with indium or gadolinium, and the chelate conjugate is injected or introduced into body fluids of a mammal. The antibody then localizes delivering the indium or gadolinium to the tumor site, and conventional gamma camera or magnetic resonance imaging techniques are employed to respectively visualize the malignancy.

Claims

CLAIMS :

1. A ligand comprising: a general Formula I:

wherein three of the R groups are -CH₂C00H with the fourth R group being -H; wherein n is an integer from 1 to 5; and

X is either -N0₂ or -NH₂-

2. The ligand of claim 1 wherein n is 1 to 2 and X is either -NO or -NH₂.

3. A chelate comprising a general Formula

wherein three of the R groups are -CH-COOH with the fourth R group being -H; wherein n is an integer from 1 to 5; and

X is either -N0₂ or -NH₂ ^': and M is a metal ion being a member selected from the group of elements consisting of Bi, Pb, Y, Cu, Au, Ag,

Pt, Ga, In, and the Lanthanides.

4. The chelate of claim 3 wherein n is 1 to 2 and X is either -N0₂ or -NH₂.

5. The chelate of claim 4 wherein M is a member selected from the group consisting of Bi, Pb, Y, Cu, Gd, Eu, and Tb.

6. The chelate of claim 3 wherein n is 2, X is -NH-, and M is a member selected from the group consi •sIti-ng o.f_= „P-b_.203, -P,b.212, „Bi.212, „Y90, and_, C_Λu67.

7. The chelate of claim 6, wherein M is a member sseelected from the group consisting of Pb 212 and

8. The chelate of claim 4, wherein n is 2, X is -NH_ and M is a member selected from the group consisting of Eu and Tb.

9. A ligand-hapten conjugate comprising: a general Formula II:

IX wherein three R groups are -CH-COOH and the fourth R group is -H; n is an integer from 1 to 5; and

X' consists of -NH-Q with Q being a hapten selected from the group of hormones, steroids, enzymes, proteins, monoclonal antibodies, chimeric antibodies, or fragments thereof.

10. The ligand-hapten conjugate of claim 9 wherein n is 2 and X' is -NH-Q.

11. The ligand-hapten conjugate of claim 9 wherein Q is a protein, said protein being a monoclonal antibody, chimeric antibody, or fragments thereof.

12. A chelate-hapten conjugate comprising: a general Formula Ila:

11 O

wherein three R groups are -CH₂COOH and the fourth is

H; n is an integer from 1 to 5; and

X¹ is -NH-Q with Q being a hapten selected from the group consisting of hormones, steroids, enzymes, proteins, monoclonal antibodies, chimeric antibodies, or fragments thereof; and M is a metal ion being a member selected from the group of elements consisting of Bi, Pb, Y, Cu, Ag, Au, Pt, and the Lanthanides.

13. The ligand-hapten conjugate of claim 9, where X¹ is -NH-L-Q wherein L is a covalent linking group.

14. The covalent linking group, L, of claim 13 wherein L is an organic radical or a substituted aliphatic hydrocarbon chain.

15. The substituted hydrocarbon chain of claim 14 where said chain may be interrupted by one or more hetero atoms selected from the group consisting of -O- or -S-, or by one or more -NR' groups, where R' is a hydrogen atom or a C, _c alkyl group, -CONR* groups, - NR'CO- groups, cycloaliphatic groups, aromatic groups or heteroaromatic groups, or a mixture thereof.

16. The chelate-hapten conjugate of claim 12 wherein X^» is -NH-L-Q wherein L is a covalent linking group.

17. The covalent linking group L of claim 15 wherein L is an organic radical or a substituted aliphatic hydrocarbon chain.

18. The substituted hydrocarbon chain of claim 17 wherein said chain may be interrupted by one or more hetero atoms selected from the group consisting of -0- or -S-, or by one or more -NR¹ groups, where R' is a hydrogen atom or a C, _c alkyl group, -CONR¹ groups, -NR'CO- groups, cycloaliphatic groups, aromatic groups or heteroaromatic groups, or a mixture thereof.

19. The method of using the chelate-hapten conjugate of claim 12 where said conjugate is administered to the patient as a diagnostic or therapeutic agent.

20. The method of using the chelate-hapten conjugate of claim 15 wherein said conjugate is administered to the patient as a diagnostic or therapeutic agent.