WO2008124467A1

WO2008124467A1 - Bifunctional hydroxamic acid ligands and method of synthesis

Info

Publication number: WO2008124467A1
Application number: PCT/US2008/059213
Authority: WO
Inventors: Paul Jurek
Original assignee: Macrocyclics
Priority date: 2007-04-06
Filing date: 2008-04-03
Publication date: 2008-10-16

Abstract

A process for synthesizing a bifunctional hydroxamic acid ligand. The process includes providing a hydroxamic acid ligand, and reacting the hydroxamic acid ligand with a reactive linking moiety. The reactive linking moiety includes at least one isothiocyanato group.

Description

BIFUNCTIONAL HYDROXAMIC ACID LIGANDS AND METHOD OP

SYNTHESIS

CROSS -REFlRENCE TO RELATED APPLICATION

This Application claims the benefit of U.S. Provisional Application Serial No. 60/910,479 filed on April 6, 2007, entitled "BIFUNCTIONAL HYDROXAMIC ACID LIGANDS AND METHOD OF SYNTHESIS," commonly assigned with the present invention and incorporated herein by reference. TECHNICAL FIELD OF THE INVENTION

The present invention is directed to bifunctional hydroxamic acid ligands and processes for synthesizing the bifunctional hydroxamic acid ligands and for prepared radiological contrast agent using the bifunctional hydroxamic acid ligands. BACKGROUND OF THE INVENTION

Positron Emission Tomography (PET) imaging has become an important diagnostic tool for accurate and sensitive detection of small tumors as well as a means of assessing metabolic function. As an example, ¹⁸F-labeled deoxyglucose

(FDG) is one of the most utilized PET imaging agent approved by the U.S. Federal Drug Administration for human use. Nevertheless, the production and availably of useful positron emitting isotopes for PET has been a limiting factor in the growth of this imaging modality. For instance, the very short half life (ti_/2) of the F-18 isotope (110 minutes) requires high-yield and rapid processes to synthesis FDG, and close proximity to a cyclotron facility. Consequently, there is a long-felt need to refine techniques for the isolation and purification of longer lived isotopes.

The potential to produce PET imaging agents using Zr- 89, which has a half -life of 78.4 hours, could substantially expand the scope of PET. A synthesis of a Zr- 89 Immuno PET agent, where a modified desferrioxamine ligand is conjugated to a monoclonal antibody for targeted imaging, has been described by van Dongen et al . , The Journal of Nuclear Medicine, 44, 2003, 1271-1281 ("van Dongen"), and is incorporated by reference herein in its entirety. SUMMARY OF THE INVENTION

One embodiment of the present disclosure is a process for synthesizing a bifunctional hydroxamic acid ligand. The process includes providing a hydroxamic acid ligand, and reacting the hydroxamic acid ligand with a reactive linking moiety. The reactive linking moiety includes at least one isothiocyanato group. Another embodiment of the present disclosure is a process for preparing a radiological contrast agent . The process comprises forming a bifunctional hydroxamic acid ligand- targeting vector conjugate. Forming the conjugate includes providing a bifunctional hydroxamic acid ligand and providing a targeting vector. The bifunctional hydroxamic acid ligand includes at least two hydroxamic acid groups and at least one isothiocyanato group. The targeting vector includes one or more terminal amine functional groups. Forming the conjugate also includes mixing the bifunctional hydroxamic acid ligand and the targeting vector such that the bifunctional hydroxamic acid ligand is conjugated to the one or more terminal amine functional groups by a thiourea group.

Another embodiment of the present disclosure is a bifunctional hydroxamic acid ligand. The bifunctional hydroxamic acid ligand comprises first functional groups that include hydroxamic acid groups configured to complex to a metal ion and second functional groups that include an isothiocyanato group.

Another embodiment of the present disclosure is a radiological contrast agent. The contrast agent comprises a bifunctional hydroxamic acid ligand having the above- described first and second functional groups, a target vector covalently bonded to the isothiocyanato group and a metal ion complexed to the hydroxamic acid groups.

The foregoing has outlined preferred and alternative features of the present invention so that those skilled in the art may better understand the detailed description of the invention that follows. Additional features of the invention will be described hereinafter that form the subject of the claims of the invention. Those skilled in the art should appreciate that they can readily use the disclosed conception and specific embodiment as a basis for designing or modifying other structures for carrying out the same purposes of the present invention. Those skilled in the art should also realize that such equivalent constructions do not depart from the scope of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

For a more complete understanding of the present invention, reference is now made to the following descriptions taken in conjunction with the accompanying drawings, in which:

FIG. 1 presents a flow diagram showing selected steps in an example synthesis of a bifunctional hydroxamic acid ligand of the present disclosure;

FIG. 2 presents a flow diagram showing selected steps in an example preparation of a radiological contrast agent of the present disclosure; and

FIG. 3 illustrates selected steps in an example synthesis of a bifunctional hydroxamic acid ligand, and an example synthesis of a radiological contrast agent.

DETAILED DESCRIPTION

The present invention benefits from recognizing certain problems with van Dongen's process to synthesize a Zr-89 Immuno PET agent. van Dongen performed a complicated 6 -step synthesis to prepare a Zr-89 Immuno PET agent in which a modified desferrioxamine ligand is conjugated to a monoclonal antibody for targeted imaging. In the first step, the terminal amine of desferrioxamine was functionalized to introduce a carboxylic acid moiety, a key functional group for monoclonal antibody (Mab) attachment.

Due to the reactive nature of the hydroxamic acid groups, before antibody attachment, Fe⁺³ was introduced (step 2) to coordinate to the oxygen atoms of the hydroxyl amines . The complexing iron serves as a protecting group, to thereby prevent undesirable side reactions in the following step. Step 3 introduced an activated tetrafluorophenyl ester (TFP) functionality which is highly reactive with the amine groups (e.g., -NH₂ residues of lysine) present in the MAb. Once the conjugation of the desferrioxamine ligand, via the activated TFP ester, to the MAb is completed (step 4) , it is then necessary to remove the iron (step 5) . This is followed by the final step 6 to introduce the Zr-89 isotope to produce the Zr-89 Immuno PET agent.

Although useful, van Dongen's process is lengthy and requires a high degree of expertise. In addition, the use of iron as a protecting group is problematic in that it must then be removed following covalent attachment of the desferrioxamine ligand to the Mab. The iron removal step can be detrimental to targeting properties of the targeting vector. Examples of such detriments to targeting properties of the targeting vector include denaturing of the protein and reduction of immunoreactivity .

The present disclosure presents novel bifunctional hydroxamic acid ligands and radiological contrast agents that include the bifunctional hydroxamic acid ligand, and, novel processes for the synthesis of such bifunctional hydroxamic acid ligands and preparation of the contrast agent .

The term bifunctional hydroxamic acid ligand, as used herein, refers to an organic molecule having at least two different types of functional groups: a first type for complexation to a metal ion and a second type for conjugation to a targeting vector. The first type of functional group includes hydroxamic acid groups (-C(O)- N(OH)-), while the second type of functional group includes an isothiocyanato group (S=C=N-) . In cases where there is more than one isothiocyanato group, one of the isothiocyanato groups can be covalently bonded to the hydroxamic acid ligand. For instance, one of the isothiocyanato groups can be covalently bonded to a nitrogen atom of one of the hydroxamic acid groups.

The synthesis of the bifunctional hydroxamic acid ligands includes the use of a reactive linking moiety to facilitate the direct conjugation, via covalent bonding, to the terminal amine functionality (e.g., a lysine residue) of a targeting vector (e.g., peptides, proteins or Mabs) . The reactive linking moiety can be introduced early in the synthesis of a radiological contrast agent (e.g., in the first of a three reaction step embodiment of a process to prepare an Immuno PET contrast agent) . Additionally, several synthetic transformations are eliminated compared to van Dongen ' s process, thereby shortening the time to prepare the bifunctional hydroxamic acid ligands.

The processes for preparing radiological contrast agents, as disclosed herein, provide a more efficient synthetic transformation under milder reactions conditions than van Dongen's process. Unlike van Dongen ' s process, a reactive linking moiety that includes an isothiocyanato group can be used for direct conjugation to a targeting vector (e.g., the terminal amine groups of the Mab) . For instance, an activated bifunctional isothiocyante can be synthesized in a single step, and then directly conjugated to the targeting vector without the need for hydroxamic acid group protection and deprotection steps. Therefore, unlike van Dongen's process, the introduction of iron as a protecting group is not required. As such, the present process can be carried out in a substantially iron-free environment thereby avoid the above-described potential detriments to the targeting properties of the targeting vector . FIG. 1 presents a flow diagram of an example process 100 for synthesizing the bifunctional hydroxamic acid ligand. The process comprises a step 110 of providing a hydroxamic acid ligand. The term hydroxamic acid ligand, as used herein, refers to chemical having general molecular formula of:

(1) R¹- (CO-NH-OH)_n,

where CO is a carbonyl group, NH-OH is a hydroxylamine, R is an linear or macrocylic organic group having a molecular weight of about 1000 gm/mol or less, and n is between 1 and 4.

The hydroxamic acid ligand provided in step 110 can be obtained from commercial sources, or, can be synthesized using processes well known to those skilled in the art. The hydroxamic acid ligand can be an acyclic or macrocyclic molecule. One preferred hydroxamic acid ligand is desferrioxamine :

Desferrioxamine is a preferred hydroxamic acid ligand because this molecule is readily available commercially, is known to chelate Fe, Zr-89, Ga-68 and similar metals with high affinity, and its use and side-effects in humans and other animals have been studied. Desferrioxamine advantageously forms highly stable complexes with zirconium that are compatible with in vivo use.

Other example hydroxamic acid ligands include piperazine derivatives that have one or more hydroxamic acid groups covalently attached as pendant arms to the piperazine ring (e.g., H2L¹ or H2L²) :

Santos et al. (J. CHEM. SOC. DALTON TRANS. 1993, 927-932) has described the synthesis of a series of such hydroxamic acid ligand-containing piperazine derivatives, which is incorporated in its entirety by reference herein. Still other example hydroxamic acid ligands include macrocyclic tetraaza and triaza structures that have one or more hydroxamic acid groups covalently attached as pendant arms to the ring (e.g., TETMAHA, DOCYDMAHA, DOTRMAHA), or, linear ethylenediameine derivative (CDTMAHA) :

^TETMAHA DOCYDMAHA

DOI'RMΛHA CDTMΛHΛ

The synthesis of such macrocyclic hydroxamic acid ligands are described in Santos et al. J. CHEM. SOC. DALTON TRANS. 1995, 2565-2573, and Santos, BioMetals 12, 209-218, 1999, which are incorporated in their entirety by reference herein . The process 100 for synthesizing the bifunctional hydroxamic acid ligand also includes a step 120 of reacting the hydroxamic acid ligand provided in step 110, with a reactive linking moiety to produce the bifunctional hydroxamic acid ligand. The reactive linking moiety includes at least one isothiocyanato group (SCN) . In such cases, the bifunctional hydroxamic acid ligand produced is a bifunctional hydroxamic acid isothiocyante . In some preferred embodiments, the reactive linking moiety has the general molecular formula: (3) R³-SCN_m where R³ is an organic group having a molecular weight of about 1000 gm/mol or less, and m equals 1 or 2.

A reactive linking moiety having a SCN group is advantageous because the SCN group can be reacted with the amine group of the targeting vector under conditions that do not affect the targeting properties of the targeting vector. In some cases the reactive linking moiety includes two SCN groups (m=2) . A reactive linking moiety with two SCN groups is desirable because the first SCN group can be covalently bonded to an amine of the hydroxamic acid ligand, while the second SCN group can be covalently bonded to an amine of the targeting vector. An example reactive linking moiety having two SCN groups is 1,4- phenylenediisothiocyanate .

Other examples of suitable reactive linking moieties include included bis-NHS-esters and NHS-malimidio derivatives such as illustrated below (where n equals 1 to 2) :

In some embodiments, reacting the hydroxamic acid ligand with a reactive linking moiety in step 120 further includes a step 130 of forming a thiourea group ( -NHCSNH-) that covalently links the hydroxamic acid ligand to the reactive linking moiety. In some cases, forming the thiorea group include a step 135 of mixing a solution of hydroxamic acid ligand with a solution of an isothiocyanato-containing reactive linking moiety under alkaline conditions (e.g., a pH of greater than about 7) . In some embodiments, the mixing step 135 includes providing a mole ratio of hydroxamic acid ligand: isothiocyanato- containing reactive linking moiety equals about 1:2. For example, desferrioxamine can be dissolved in water, alcohol (e.g., isopropanol) , or mixture of water and alcohol to provide an about 0.2 to 0.5 mM solution, and 1,4- phenylenediisothiocyanate can dissolved in an organic solvent (e.g., chloroform) to provide an about 1 to 2 mM solution. The two solutions can be mixed together with small volume of base (e.g., 50 to 100 μL of triethylamine) added to make the mixture alkaline.

Forming the thiourea group in step 130 can also include a step 140 of incubating the mixture formed in step 135 under alkaline conditions for a period sufficient to permit a complete reaction between the hydroxamic acid ligand and reactive linking moiety. For instance, in some embodiments an about 20 to 40 min reaction period at room temperature (20-22⁰C), to facilitate the formation of an thiourea group between desferrioxamine and 1,4- phenylenediisothiocyanate) . In other embodiments, however, the covalent link between the hydroxamic acid ligand and the reactive linking moiety does not involve a thiourea group. For instance the hydroxamic acid ligand and the reactive linking moiety can be covalently bonded to each other by an NHS ester that forms a covalent amide linkage instead of a thiourea linkage .

In some embodiments, the process 100 further includes a step 145 of purifying the bifunctional hydroxamic acid ligand produced in step 140. For instance, purifying the bifunctional hydroxamic acid ligand in step 145 can include precipitating the bifunctional hydroxamic acid ligand produced in step 140. For instance, after the incubation period in step 140, the reacted mixture can be acidified with a volume of dilute acid (e.g., about 0.1 mM HCl), and vacuum distilled at about 30 to 35°C to remove the organic solvent, resulting in a precipitate corresponding to the bifunctional hydroxamic acid ligand. Purifying the bifunctional hydroxamic acid ligand in step 145 can further include isolating the precipitate from the remaining volume of water and alcohol using conventional procedures such as crystallization, filtration or centrifugation . Purifying the bifunctional hydroxamic acid ligand in step 145 can also include preparative flash chromatography to separate the bifunctional hydroxamic acid ligand product from the reactants (e.g., any un-reacted desferrioxamine and 1,4- phenylenediisothiocyanate) .

FIG. 2 presents a flow diagram of an example process 200 for preparing a radiological contrast agent of the present disclosure. The process 200 comprises a step 205 of forming a bifunctional hydroxamic acid ligand-targeting vector conjugate. Forming the conjugate (step 205) includes a step 210 of providing a bifunctional hydroxamic acid ligand. The bifunctional hydroxamic acid ligand includes two or more hydroxamic acid groups and at least one isothiocyanato group. The bifunctional hydroxamic acid ligand can be synthesized in accordance with the process 100 described in the context of FIG. 1. Forming the conjugate (step 205) also includes a step 220 of providing a targeting vector. One of ordinary skill in the art would be familiar with the processes to synthesize or isolate targeting vectors such as peptides, proteins or other biomolecules . In some preferred embodiments, the target vector is a Mab . One skilled in the art would be familiar with the procedures to produce and isolate a Mab that is targeted to bind with high affinity to particular cell types in an animal (e.g., tumor cells) or to a portion of fluid or tissue withdrawn from the animal .

The targeting vector includes one or more terminal amine functional groups (e.g., one or more lysine residues) . Having a terminal amine functional group is advantageous because this facilitates the covalent coupling to the bifunctional hydroxamic acid ligand (e.g., a bifunctional hydroxamic acid isothiocyante, such as a desferrioxamine isothiocyante) . Forming the conjugate (step 205) also includes a step 230 of mixing solutions of the bifunctional hydroxamic acid ligand and a solution of the targeting vector together under conditions to facilitate conjugation between the bifunctional hydroxamic acid ligand and the targeting vector. In some embodiments, the bifunctional hydroxamic acid ligand is dissolved in an organic solvent (e.g., methyl cyanide, dimethyl suloxide) and a protein targeting vector is dissolved in an alkaline aqueous solution (e.g., pH about 9 to 10) . The two solutions are combined to provide a mole ratio of about 1:1 (e.g., about 50 to 100 nmol of both the bifunctional hydroxamic acid ligand and targeting vector) . In step 240, the mixture is incubated under alkaline conditions (e.g., pH of greater than about 7, and more preferably, about 9 to 10) and at room temperature for a period (e.g., about 10 to 60 min) sufficient to permit the conjugation reaction to reach substantial completion. As noted above, the mixture can be substantially iron- free because the hydroxamic acid groups of the bifunctional hydroxamic acid ligand do not need to be protected, via iron chelation, during the conjugation. In step 245, an acid buffer is added to the mixture. For instance, in some embodiments, 2 , 5-dihydroxybenzoic acid and sulfuric acid or similar acid are added to adjust the mixture to a pH about 4 to 5.

The end-product is a bifunctional hydroxamic acid ligand-targeting vector conjugate. In some embodiments, where the bifunctional hydroxamic acid ligand includes an isothiocyante group available for the conjugation reaction, the bifunctional hydroxamic acid ligand is conjugated to the targeting vector by a thiourea group. That is, a nitrogen atom of an amine group of the targeting vector (e.g., a lysine residue in a protein targeting vector) and the isothiocyanato group of the bifunctional hydroxamic acid ligand both participate in forming the thiourea group.

In some embodiments, the bifunctional hydroxamic acid ligand-targeting vector conjugate includes two thiourea groups. For instance, when the reactive linking moiety includes two isothiocyante groups, there can be one thiourea group covalently linking the hydroxamic acid ligand to the reactive linking moiety, and a second thiourea group covalently linking the reactive linking moiety to the targeting vector. As an example, when the hydroxamic acid ligand includes or is desferrioxamine and the reactive linking moiety includes or is 1,4- phenylenediisothiocyanate, a first thiourea group covalently links the desferrioxamine to the 1,4- phenylenediisothiocyanate, and a second thiourea group links the 1 , 4-phenylenediisothiocyanate to an amine of the targeting vector.

The process 200 also includes a step 250 of forming a metal -bifunctional hydroxamic acid ligand-targeting vector complex. In some embodiments, the metal ion is preferably a transition metal having a radionuclide half-life of at least about 60 minutes. More preferably the metal is a PET isotope such as Zr-89 or Ga-68. In other embodiments, however, the metal ion can be a non radioactive transition metal such as iron, or a lanthanide.

In some embodiments, forming the complex (step 250) includes mixing, in step 255, the solution of the bifunctional hydroxamic acid ligand-targeting vector conjugate prepared in step 205 with a metal ion. In some preferred embodiments, metal ions are provided in an aqueous solution as a low molecular weight (e.g., about 1000 gm/mol or less) monocarboxylic acid-metal ion complex at neutral pH (e.g., a pH about 7 to 7.5) and room temperature. In some preferred embodiments, to facilitate substantially complete complexation to the bifunctional hydroxamic acid ligand-targeting vector, the mole-ratio of metal ion to bifunctional hydroxamic acid ligand-targeting vector conjugate in the mixture equals about 1000:1 or greater. For instance, in some cases, about 6 x 10^~5 mol of a metal-oxalic acid complex is mixed with 33 nmol of a desferrioxamine-protein conjugate in an aqueous solution buffered with Na₂CO₃ and 4- (2-hydroxyethyl) -1- piperazineethanesulfonic acid (HEPES) at a pH of about 7.3 at room temperature for a period sufficient to allow complex formation.

Forming the complex (step 250) can further include a step 260 of purifying the complex. For instance in some cases the metal -hydroxamic acid ligand-targeting vector complex is separated from the monocarboxylic acid-metal ion complex using size-exclusion chromatography.

FIG. 3 illustrates selected steps in an example synthesis of a bifunctional hydroxamic acid ligand using desferrioxamine-B as the hydroxamic acid ligand and 1,4- phenylenediisothiocyanate as the reactive linking moiety. FIG. 3 also illustrates selected steps in an example synthesis of a MAb-N-thiourea-Idesferal-⁸⁹Zr contrast agent. In step 1, desferrioxamine-B, and 1,4- phenylenediisothiocyanate are reacted to yield a bifunctional hydroxamic acid isothiocyante intermediate (I) in a single step. E.g., in one embodiment, desferrioxamine mesylate (0.2g, 0.37 mmol) was dissolved in an isopropanol/water solution (32 mL/4 mL) . A chloroform solution (19 mL) of 1 , 4 -phenylenediisothiocyanate (0.29g, 1.5 mmol) was then added in one portion followed by the immediate addition of triethylamine (80 μL) . This reaction mixture was then stirred for 30 minutes at room temperature. A 0. IM HCl solution (22.5 ml) was then added to give a 2 -phase system. The organic layer was separated and concentrated under vacuum at 30-35⁰C to remove chloroform. A white precipitate formed in the remaining isopropanol solution which was filtered. The isopropanol filtrate was then purified by preparative flash chromatography on a C- 18 column eluting with H₂0/CH₃CN to remove unreacted 1, 4-phenylendiisothiocyanate. The product- containing eluent was then freeze-dried to give an about 41% product yield. In step 2, the bifunctional hydroxamic acid isothiocyante intermediate (I) can be conjugated directly to the targeting vector via e.g., a -NH₂ residues of lysine present in a monoclonal antibody (Mab) to form a desferrioxamine-protein conjugate (II) . Notably, conjugation can be accomplished without having to perform additions steps to protect the hydroxamic acid using iron chelation, and then to de-protect the hydroxamic acid by removing the chelated iron. E.g., in some embodiments, to 1 mL (33 nmol) of mAb solution (5 mg/rtiL) , pH 9.5-9.8

(adjusted with 0.1 mol/L Na₂CO₃) and 20 μL (63 nmol) of the intermediate (I) (2.5 mg/mL in MeCN or DMSO) are combined to obtain final intermediate : mAb ratio of 1:1 (based on 54% reaction efficiency) . After 30 min, a 2 , 5-dihydroxybenzoic acid (gentisic acid) solution was added (50 μL , 100 mg/mL in 0.32 mol/L Na2CO3) to the reaction mixture, and the pH was adjusted to 4.3-4.5 with 30 μL of 0.25 mol/L H₂SO₄.

In step 3, a metal ion (e.g., Zr-89, Ga-68, Fe) can be complexed to the desferrioxamine-protein conjugate (II) to form a metal -desferrioxamine protein conjugate (III) . E.g., in some embodiments, to 600 μL of ⁸⁹Zr oxalic acid solution (1 mol/L oxalic acid), 130 μL of 0.9% NaCl, 270 μL of 2 mol/L Na₂CO₃, and 3 mL of 0.5 mol/L HEPES (pH 7.2-7.4) are added, followed by 2 mL (33 nmol) of the desferrioxamine-protein conjugate (II) (2.5 mg/mL in 0.9% NaCl/gentisic acid (5 mg/mL), pH 5), final pH 7.2-7.4. The reaction volume can be varied, provided that the amounts of oxalic acid, Na₂CO₃, and HEPES buffer are adjusted accordingly. After 30 min, the reaction mixture (6 mL) is divided over 3 PD-10 columns (eluent: 0.9% NaCl/gentisic acid (5 mg/mL), pH 5) . The first 2.5 mL (2-mL sample volume and first 0.5 mL) are discarded, and the metal- desferrioxamine protein conjugate (III) was collected in next 3 mL .

Although the present invention has been described in detail, those skilled in the art should understand that they can make various changes, substitutions and alterations herein without departing from the scope of the invention in its broadest form.

Claims

WHAT IS CLAIMED IS:

1. A process for synthesizing a bifunctional hydroxamic acid ligand, comprising: providing a hydroxamic acid ligand; and reacting the hydroxamic acid ligand with a reactive linking moiety, wherein the reactive linking moiety includes at least one isothiocyanato group.

2. The process of claim 1, wherein the hydroxamic acid ligand is desferrioxamine, and wherein the reactive linking moiety includes two isothiocyanato groups.

3. The process of claim 1, wherein the reactive linking moiety is 1 , 4 -phenylenediisothiocyanate .

4. The process of claim 1, wherein reacting the hydroxamic acid ligand with a reactive linking moiety is performed under alkaline conditions.

5. The process of claim 1, wherein reacting the hydroxamic acid ligand with a reactive linking moiety includes forming a thiourea group that covalently bonds the hydroxamic acid ligand to the reactive linking moiety.

6. A process for preparing a radiological contrast agent, comprising: forming a bifunctional hydroxamic acid ligand-targeting vector conjugate including: providing a bifunctional hydroxamic acid ligand, wherein the bifunctional hydroxamic acid ligand includes at least two hydroxamic acid groups and at least one isothiocyanato group; providing a targeting vector, wherein the targeting vector includes one or more terminal amine functional group ; and mixing the bifunctional hydroxamic acid ligand and the targeting vector such that the bifunctional hydroxamic acid ligand is conjugated to the one or more terminal amine functional group by a thiourea group.

7. The process of Claim 6, wherein providing the bifunctional hydroxamic acid ligand includes: providing a hydroxamic acid ligand; and reacting the hydroxamic acid ligand with a reactive linking moiety, wherein the reactive linking moiety includes at least one isothiocyanato group.

8. The process of Claim 6, wherein an amine residue of the targeting vector and the one isothiocyanato group participate in the thiourea group.

9. The process of Claim 6, wherein the bifunctional hydroxamic acid ligand includes desferrioxamine covalently linked to 1 , 4-phenylenediisothiocyanate by a second thiourea group, and the bifunctional hydroxamic acid ligand is conjugated to the targeting vector by the thiourea group formed between the one or more terminal amine functional group and the 1 , 4 -phenylenediisothiocyanate .

10. The process of Claim 6, wherein the mixture of the bifunctional hydroxamic acid ligand and the targeting vector is incubated at room temperature under alkaline conditions .

11. The process of Claim 6, wherein the targeting vector is a monoclonal antibody.

12. The process of Claim 6, further including forming a metal -hydroxamic acid ligand-targeting vector complex, including mixing the bifunctional hydroxamic acid ligand- targeting vector conjugate with metal ions.

13. The process of Claim 12, wherein the metal ions are complexed to a monocarboxlic acid.

14. The process of Claim 12, wherein the mixture of the bifunctional hydroxamic acid ligand-targeting vector conjugate and the metal ions are incubated at neutral pH and room temperature.

15. The process of Claim 12, wherein the metal ions are a transition metal ions having a radionuclide half-life of at least about 60 minutes.

16. The process of Claim 12, wherein the metal ions are one of Zr-89 or Ga-68.

17. A bifunctional hydroxamic acid ligand, comprising : first functional groups including hydroxamic acid groups configured to complex to a metal ion; and second functional groups including an isothiocyanato group .

18. The bifunctional hydroxamic acid ligand of claim 17, wherein the first functional groups including the hydroxamic acid groups of desferrioxamine and the second functional groups include a 1 , 4-phenylenediisothiocyanate group, wherein one of the isothiocyanato groups is covalent bonded to a nitrogen atom of one of the hydroxamic acid groups.

19. A radiological contrast agent, comprising: a bifunctional hydroxamic acid ligand having: first functional groups including hydroxamic acid groups; and second functional groups including an isothiocyanato group; a target vector covalently bonded to the isothiocyanato group; and a metal ion complexed to the hydroxamic acid groups.

20. The radiological contrast agent of claim 19, wherein the bifunctional hydroxamic acid ligand includes desferrioxamine covalently bonded to a first isothiocyanato group of 1 , 4-phenylenediisothiocyanate, a second isothiocyanato group of 1,4- phenylenediisothiocyanate is the isothiocyanato group that is covalently bonded to a lysine residue of the target vector, and hydroxamic acid groups of the desferrioxamine are the hydroxamic acid groups complexed to the metal ion.