BIFUNCTIONAL METAL CHELATING CONJUGATES

BACKGROUND

[0001] The present invention is generally directed to metal chelating conjugates for use as metallopharmaceutical diagnostic or therapeutic agents.

[0002] Metallopharmaceutical diagnostic and therapeutic agents are finding ever-increasing application in biological and medical research, and in diagnostic and therapeutic procedures. Generally, these agents contain a radioisotope or paramagnetic metal, which upon introduction to a subject, become localized in a specific organ, tissue or skeletal structure of choice. When the purpose of the procedure is diagnostic, images depicting in vivo distribution of the radioisotope or paramagnetic metal can be made by various means. The distribution and corresponding relative intensity of the detected radioisotope or paramagnetic metal not only indicates the space occupied by the targeted tissue, but may also indicate a presence of receptors, antigens, aberrations, pathological conditions, and/or the like. When the purpose of the procedure is therapeutic, the agent typically contains a radioisotope, and the radioactive agent delivers a dose of radiation to the local site.

[0003] Depending upon the target organ or tissue of interest and the desired diagnostic or therapeutic procedure, a range of metallopharmaceutical agents may be used. One common form is a conjugate including a radioactive or paramagnetic metal, a carrier agent for targeting the conjugate to a specific organ or tissue site, and a linkage for chemically linking the metal to the carrier. In such conjugates, the metal is typically associated with the conjugate in the form of a coordination complex, more typically as a chelate of a macrocycle. See, e.g., Liu, U.S. Patent No. 6,916,460.

[0004] In U.S. Patent No. 5,435,990, Cheng et al. disclose functionalized macrocyclic polyaminocarboxylate chelants that coordinate rare earth metal ions for use in therapeutic and/or diagnostic oncology procedures. Cheng et al. link their macrocyclic chelant to a carrier agent, in their case an antibody or antibody fragment, via a thiourea linkage. However, thioureas tend to exchange oxygen for sulfur under the reaction conditions for their formation, thereby obscuring the absolute molecular form of the product conjugate. Thiourea linkages may also create a risk of non-specific binding to tissues other than the intended target, which would undesirably result in the delivery of a dose of radiation to the incorrect site.

[0005] In U.S. Patent No. 6, 143,274, Tweedle et al. disclose a method for imaging mammalian tissue utilizing a non-ionic complex of a paramagnetic ion of a lanthanide element and a macrocyclic chelating agent. A non-ionic complex, however, is less stable than an anionic complex (i.e., the anionic complex tends to exhibit stronger electrostatic interaction between the cationic metal and anionic ligand).

SUMMARY

[0006] Among the several aspects of the present invention is the provision of conjugates for use in diagnostic and therapeutic procedures. Advantageously, conjugates of the invention may tend to accumulate in the specific organs, tissues or skeletal structures of diagnostic or therapeutic interest with a reduced risk of non-specific binding to non-target tissues, thereby allowing for the conjugates to be targeted to specific disease states, if desired.

[0007] In one aspect, the present invention is directed to a conjugate including a metal . coordinating moiety and one or more carriers for targeting the conjugate to a biological tissue or organ. In addition, the conjugate includes a linker that includes a urea linkage and that chemically links the metal coordinating moiety to the carrier(s). In some embodiments, a metal (e.g., a radioactive or paramagnetic metal) may be complexed by the metal coordinating moiety of the conjugate.

[0008] Another aspect of the invention is directed to a diagnostic or therapeutic method. In this method, a conjugate of the type disclosed herein is administered to a subject (e.g., patient).

[0009] Yet another aspect of the invention is directed to a kit for the preparation of a metal lopharmaceutical. The kit includes a conjugate of the type disclosed herein.

[0010] Still another aspect of the invention is directed to a kit including a protected metal coordinating moiety having an active urea, a deprotecting acid, a buffer, and a solution of a radioactive metal.

[0011] Other aspects of the invention will be in part apparent and in part pointed out hereinafter.

DETAILED DESCRIPTION OF VARIOUS EMBODIMENTS

[0012] The present invention provides conjugates that can rapidly form coordination complexes with metals for use in diagnostic or therapeutic metalloradiopharmaceuticals, or magnetic resonance imaging contrast agents. These conjugates may serve as bϊfunctional chelators (BFCs) for attaching metal ions to bio-directing carriers, sometimes referred to as biomolecules, that bind in vivo to a tissue type, organ or other biologically expressed composition or receptor. Target specific metallopharmaceuticals of the present invention are useful in the diagnosis of disease by magnetic resonance imaging or scintigraphy, or in the treatment of disease by systemic radiotherapy.

[0013] Generally, conjugates of the present invention include one or more bio-directing carriers and a metal coordinating moiety covalently joined, directly or indirectly, by a urea moiety. The urea moiety may be directly bonded to the bio-directing carrier(s), or indirectly bonded to the bio-directing carrier(s) through a series of atoms. Similarly and independently, the urea moiety may also be directly bonded to the metal coordinating moiety, or indirectly bonded to the metal coordinating moiety through a series of atoms. Schematically, a conjugate including a bio-directing carrier, the urea moiety, and the metal coordinating moiety of the present invention corresponds to Formula:

metal coordinating carrier

[0014] Formula A

[0015] wherein

[0016] S ₁ and S ₂ are spacers, each independently being a bond or a series of atoms, and

[0017] Zi and Z ₂ are independently hydrogen, aryl, Ci. ₇ alkyl, Ci _-7 hydroxyalkyl or Ci _-7 alkoxyalkyl.

[0018] In combination, the sequence -Si-N(Zi)C(O)N(Z ₂ )S ₂ - may be characterized as a linker, covalently linking the bio-directing carrier to the metal coordinating moiety. Viewed in this manner, the linker includes the urea moiety and spacers, Si and S ₂ , each of the spacers independently being a bond or a series of atoms linking the urea moiety to the metal coordinating moiety or to one or more bio-directing carriers, respectively. Alternatively, however, either or both of the spacers could be considered to be separate and independent components of the conjugate, or members of the metal coordinating moiety and the bio-directing carrier, respectively. For example, Si may be considered a part of the metal coordinating moiety and/or S ₂ may be considered a part of a bio-directing carrier without departing from the spirit of the present invention.

[0019] Although Formula A depicts only a single bio-directing carrier, it is contemplated that a conjugate may include multiple carriers. For instance, in some embodiments, multiple carriers may be connected to the urea linker via S ₂ . As another example, multiple carriers may be connected to the metal coordinating moiety via a plurality of separate and distinct linkers. In other words, a plurality of linkers may be connected to the metal coordinating moiety, and at least one carrier may be connected with each linker.

[0020] Prior to use in a diagnostic and/or therapeutic procedure, a conjugate corresponding to Formula A is generally complexed with a metal to form a metallopharmaceutical diagnostic or therapeutic agent of the present invention.

[0021] Bio-directing Carriers

[0022] As previously noted, conjugates of the present invention include one or more bio- directing carriers, also known as biomolecules, that direct the conjugate to the targeted tissue, organ, receptor or other biologically expressed composition. Ideally, each carrier is selective or specific for the targeted organ or tissue site.

[0023] Typical bio-directing carriers include hormones, amino acids, peptides, peptidomϊmetics, proteins, nucleosides, nucleotides, nucleic acids, enzymes, carbohydrates, glycomimetics, lipids, albumins, mono- and polyclonal antibodies, receptors, inclusion compounds such

as cyclodextriπs, and receptor binding molecules. Specific examples of carriers include steroid hormones for the treatment of breast and prostate lesions; somatostatin, bombesin, CCK, and neurotensin receptor binding molecules for the treatment of neuroendocrine tumors; CCK receptor binding molecules for the treatment of lung cancer; ST receptor and carcinoembryonic antigen (CEA) binding molecules for the treatment of colorectal cancer; dihyroxyindolecarboxylic acid and other melanin producing biosynthetic intermediates for the treatment of melanoma; integrin receptor and atherosclerotic plaque binding molecules for the treatment of vascular diseases; and amyloid plaque binding molecules for the treatment of brain lesions. Exemplary bio-directing carriers also include synthetic polymers such as polyaminoacids, polyols, polyamines, polyacids, oligonucleotides, aborols, dendrimers, and aptamers.

[0024] In some embodiments, the bio-directing carrier is selected from among imidazole, triazole, antibodies (e.g., NeutroSpect®, Zevalin®, and Herceptin®), proteins (e.g., TCII, HSA, annexin, and Hb), peptides (e.g., octreotide, bombesin, neurotensin, and angiotensin), nitrogen- containing simple or complex carbohydrates (e.g., glucosamine and glucose), nitrogen-containing vitamins (e.g., vitamin A, Bl, B2, B12, C, D2, D3, E, H, and K), nitrogen-containing hormones (e.g., estradiol, progesterone, and testosterone), nitrogen-containing active pharmaceuticals (e.g., celecoxib or other nitrogen-containing NSAIDS, AMD3100, CXCR4 and CCR5 antagonists) and nitrogen-containing steroids. In one example of these embodiments, the bio-directing carrier is selected from among imidazole, triazole, a peptide, a nitrogen-substituted simple or complex carbohydrate, a nitrogen- substituted vitamin, and a nitrogen-substituted small molecule. In another example, the bio-directing carrier may be imidazole, triazole, the N-termϊnus of a peptide, a nitrogen-substituted simple or complex carbohydrate, or a nitrogen-substituted vitamin. In still another example, the bio-directing carrier (or a terminal group thereof) may be imidazole or triazole.

[0025] As mentioned above, some embodiments of the invention may include conjugates having multiple bio-directing carriers. For instance, to increase specificity for a particular target tissue, organ receptor or other biologically expressed composition, multiple bio-directing carriers may be utilized. In such instances, the bio-directing carriers may be the same or different. For example, a single conjugate may possess multiple antibodies or antibody fragments, which are directed against a desired antigen or hapten. Typically, the antibodies used in the conjugate are monoclonal antibodies or antibody fragments that are directed against a desired antigen or hapten. Thus, for example, the conjugate may include two or more monoclonal antibodies having specificity for a desired epitope thereby increasing concentration of the conjugate at the desired site. Similarly, and independently, a conjugate may include two or more different bio-directing carriers each of which is targeted to a different site on the same target tissue or organ. By utilizing multiple bio-directing carriers in this manner, the conjugate advantageously concentrates at several areas of the target tissue or organ, potentially increasing the effectiveness of therapeutic treatment. Further, the conjugate may have a ratio of bio-directing carriers designed to

concentrate the conjugate at a target tissue or organ that optimally achieves the desired therapeutic and/or diagnostic results while minimizing non-target deposition.

[0026] Linker

[0027] As previously noted, one or more bio-directing carriers may be covalently bonded to the metal coordinating moiety via a linker including a urea group. In some embodiments, the linker corresponds to Formula B:

[0028] Formula B

[0029] wherein

[0030] S] and S ₂ are independently a covalent bond or a chain of atoms covalently linking the urea moiety to the metal coordinating moiety or to one or more bio-directing carriers, respectively; and

[0031] Zi and Z ₂ are independently selected from the group consisting of hydrogen, aryl, Ci _-7 alkyl, Ci _-7 hydroxyalkyl and Ci _-7 alkoxyalkyl. For example, Zi and Z ₂ may be selected from the group consisting of hydrogen, C ₁ . ₇ alkyl, alkoxyalkyl, and phenyl. By way of further example, Zi and Z ₂ may be selected from a more restrictive group (e.g., hydrogen, Ci _-4 alkyl and C ₁ . ₄ alkoxyalkyl). In some embodiments, Zj and Z ₂ may both be hydrogen.

[0032] Whether considered to be part of the linker, separate and independent component(s) of the conjugate, or part of the metal coordinating moiety and bio-directing carrier, respectively, the spacers, Si and S ₂ , are preferably designed to favorably impact biodistribution and potency as well as to provide separation between the metal coordinating moiety and the bio-directing carrier. For example, carbohydrates, polyalkylene glycols, peptides or other polyamino acids, and/or cyclodextrins may be employed as spacers to influence biodistribution of the conjugate, enhance or decrease the rate of blood clearance, and/or direct the route of elimination of the conjugate. In general, preferred spacers are those that result in moderate to fast blood clearance and enhanced renal excretion. Ideally, the spacers are not metabolized via the liver, but instead are cleared by the kidneys thereby diminishing the effects of the conjugates on liver tissue. It should be noted, however, that some embodiments of the invention may include one or more spacers that are metabolized via the liver.

[0033] When other than a covalent bond, Si and S ₂ include a chain of atoms. This chain may be linear, branched, cyclic or a combination thereof. In some embodiments, the chain includes no more than about 40 atoms, or even no more than about 20 atoms. In some embodiments, the chain includes from about 2 to about 15 atoms. The atoms included in this chain are typically selected from the group consisting of carbon, oxygen, nitrogen, sulfur, selenium, silicon and phosphorous. In some embodiments, the atoms may be selected from the erouo cnnskHno- nf _^ rhn _n] oxygen, nitrogen,

phosphorous and sulfur. While in other embodiments, the atoms may be selected from the group consisting of carbon, nitrogen, oxygen and phosphorous. In some embodiments, at least some of the chain atoms may be optionally substituted, with exemplary substituents including, but not limited to, one or more hydroxyl, -OR, and R substituents

[0034] In some embodiments, for example, Si and S ₂ are independently a bond (e.g., a single covalent bond), aryl or Ci. ₂ o alkylene optionally substituted with one or more carbaldehyde, keto, carboxyl (-CO ₂ H), cyano (-CN), halo, nitro (-NO ₂ ), amido (-C(O)R ₁ R ₂ ), sulfato (-OSO ₃ H), sulfite (- SO ₃ H) , phosphate (-OPO ₃ H ₂ ) , phosphite (-PO ₃ H ₂ ), hydroxyl (-OH), oxy, mercapto (-SH), thio (-SR ₁ ), sulfoxo (S(O)Ri) wherein R, Ri and R ₂ are independently C] _-2O alkyl optionally substituted with one or more sulfoxo, carboxyl, cyano, nitro, amido, hydroxyl, amino, sulfite, phosphite, sulfato, and phosphate. For these embodiments, each of Si and S ₂ may independently be a single bond, aryl optionally substituted with one or more of oxy, keto, halo, and amido, or Ci _-8 alkylene optionally substituted with one or more oxy and keto. In one example of these embodiments, Si and S ₂ are independently a single bond or C _M alkylene optionally substituted with oxy. While in another example of these embodiments, Si and S ₂ are each a single bond.

[0035] In some embodiments, S ₂ may be: (i) a C _2-20 alkyl chain or ring optionally substituted with one or more oxygen atoms as ether linkages or pendant with one or more hydroxyl groups as alcohols; (ii) a peptide chain or ring consisting of one or more amino acid residues such as alanine, isoleucine, leucine, valine, phenylalanine, tryptophan, tyrosine, asparagine, methionine, cysteine, serine, glutamine, threonine, aspartic acid, glutamic acid, arginine, histidine, lysine, glycine or proline, conjugated in a natural or unnatural way; or (Hi) one or more aromatic rings in chains or condensed in polycycles, optionally substituted with one or more sulfoxo, carboxyl, cyano, nitro, amido, hydroxyl, amino, sulfite, phosphite, sulfato, phosphate, Ci.io alkyl chain or ring optionally substituted with one or more sulfoxo, carboxyl, cyano, nitro, amido, hydroxyl, amino, sulfite, phosphite, sulfato, and phosphate.

[0036] Metals

[0037] Any metal capable of being detected in a diagnostic procedure in vivo or in vitro or useful in the therapeutic treatment of disease can be employed as a metal in conjugates of the present invention. Particularly, any radioactive metal ion or paramagnetic metal ion capable of producing a diagnostic result or therapeutic response in a human or animal body or in an in vitro diagnostic assay may be used. The selection of an appropriate metal based on the intended purpose is known by those skilled in the art. In some embodiments, the metal may be selected from the group consisting of Lu, Lu- 177, Y, Y-90, In, In-1 1 1, Tc, Tc=O, Tc-99m, Tc-99m=O, Re, Re-186, Re-188, Re=O, Re-186=O, Re-188=O, Ga ₅ Ga-67, Ga-68, Cu, Cu-62, Cu-64, Cu-67, Gd, Gd-153, Dy, Dy-165, Dy-166, Ho, Ho- 166, Eu, Eu-169, Sm, Sm-153, Pd, Pd-103, Pm, Pm-149, Tm, Tm-170, Bi, Bi-212, As and As-21 1. For example, the metal may be selected from the group consisting of Y-90, In-111, Tc-99m, Re- 186, Re-

188, Cu-64, Ga-67, Ga-68 and Lu-177. By way of another example, the metal may be selected from a more restrictive group (e.g., the group consisting of Y-90, In-1 1 1, Tc-99m, Re-186, Cu-64, Ga-67, and Lu-177; or the group consisting of Y-90, In-11 1, and Tc-99m).

[0038] Metal Coordinating Moiety

[0039] The metal coordinating moiety may be any moiety used to complex (also referred to as "coordinate") one or more metals under physiological conditions. Preferably, the metal coordinating moiety forms a thermodynamically and kinetically stable complex with the metal to keep the complex intact under physiological conditions; otherwise, systemic release of the coordinated metal may result.

[0040] In general, the metal coordinating moiety may be acyclic or cyclic. For example, metal coordinating moieties include polycarboxylic acids such as EDTA, DTPA, DCTA, DOTA, TETA, or analogs or homologs thereof. To provide greater stability under physiological conditions, however, macrocyclic moieties (e.g., triaza and tetraza macrocycles) are generally preferred. In some embodiments, the macrocyclic metal coordinating moiety is cyclen or tacn.

[0041] In some embodiments, the metal coordinating moiety includes a substituted heterocyclic ring where the heteroatom is nitrogen. Typically, the heterocyclic ring includes from about 9 to about 15 atoms, at least 3 of these ring atoms being nitrogen. In one example of these embodiments, the heterocyclic ring includes 3-5 ring nitrogen atoms where at least one of the ring nitrogen atoms is substituted. For these embodiments, the ring carbon atoms may be optionally substituted. One such macrocycle corresponds to Formula (1):

[0043] wherein

[0044] n is 0, 1 or 2;

[0045] m is 0-16, wherein when m is greater than 0, each A is independently selected from the group consisting of optionally substituted Ci _-2O alkyl and aryl.

[0046] When the metal coordinating moiety corresponds to Formula (1) and m is greater than zero, it is generally preferred that each A be a substituent that positively impacts stability and biodistribution. When present, each A may independently be substituted with one or more aryl, Ci. ₂ o alkyl, carbaldehyde, keto, carboxyl, cyano, halo, nitro, amido, sulfato, sulfito, phosphato, phosphito, hydroxyl, oxy, mercapto or thio substituents. When A is aryl or alkyl, each of these, in turn, may be optionally substituted with an aryl or Ci _-2 O alkyl moiety optionally substituted with one or more aryl,

carbaldehyde, keto, carboxyl, cyano, halo, nitro, amido, sulfato, sulfito, phosphato, phosphito, hydroxy!, oxy, mercapto and thio.

[0047] For the metal coordinating moieties of Formula (1), the A substituent, if present, is bonded to any of the ring carbon atoms. Further, each ring carbon atom may be substituted so that the number of possible A substituents varies with the number of ring carbon atoms. In one embodiment of metal coordinating moieties of Formula (1) having at least one A substituent, each A is independently aryl or Ci _-8 alkyl optionally substituted with one or more aryl, keto, carboxyl, cyano, nitro, Ci _-2O alkyl, amido, sulfato, sulfito, phosphato, phosphito, oxy and thio. For example, each A may be aryl or Ci_ ₆ alkyl optionally substituted with one or more aryl, keto, amido and oxy. By way of further example, each A may be methyl.

[0048] In general, as the value of n increases, the size of the macrocycle increases. In this manner, the size of the macrocycle may be controlled to match the desired size and coordination number of the metal to be coordinated.

[0049] In some embodiments where the metal coordinating moiety includes a substituted heterocyclic ring, the metal coordinating moiety corresponds to Formula (Ia)

[0050]

[0051] wherein

[0052] n is 0, 1 or 2;

[0053] m is 0-16, wherein when m is greater than 0, each A is Ci. ₂ o alkyl or aryl optionally substituted by one or more aryl, Ci _-2 O alkyl, carbaldehyde, keto, carboxyl, cyano, halo, nitro, amido, sulfato, sulfito, phosphato, phosphito, hydroxyl, oxy, mercapto or thio;

[0054] q is 0-3, wherein when q is greater than 0, each D is independently selected from the group consisting of fluoro, chloro, bromo, iodo, carboxyl, cyano, nitro, amido, hydroxyl, amino, sulfato, sulfito, phosphato, phosphito, aryl, and C _1-20 alkyl optionally substituted with one or more of C _L20 alkyl, carboxyl, cyano, nitro, amido, hydroxyl, amino, sulfato, sulfito, phosphato, and phosphito;

[0055] X ₁ , X ₂ , X ₃ , X ₄ are independently optionally substituted methylene where the substituents are selected from the group consisting of aryl, Ci _-2O alkyl, carbaldehyde, keto, carboxyl, cyano, halo, nitro, amido, sulfato, sulfito, phosphato, phosphito, hydroxyl, oxy, mercapto and thio;

[0056] Q ₂ -Q ₄ are independently selected from the group consisting of:

[0057] q ₂ is 0-4, wherein when q ₂ is greater than 0, each E is independently selected from the group consisting of fluoro, chloro, bromo, iodo, carboxyl, cyano, nitro, amido, hydroxyl, amino, sulfito, phosphito, and Ci_ ₂ o alkyl optionally substituted with one or more or Ci_ ₂ o alkyl, carboxy, cyano, nitro, amido, hydroxyl, sulfito, phospito, sulfato, and phosphato; and

[0058] Ti is hydroxyl or mercapto.

[0059] For metal coordinating moieties of Formula (Ia), the D substituent, if present, is independently bonded to any of the substitutable phenyl ring carbon atoms. In some embodiments, each D may be fluoro, chloro, bromo, iodo, carboxyl, cyano, nitro, amido, hydroxyl, amino, sulfito, phosphito, sulfato, phosphato, aryl, or C] _-8 alkyl optionally substituted with one or more of C] _-2O alkyl, carboxyl, cyano, nitro, amido, hydroxyl, amino, sulfito, phosphito, sulfato, and phosphate. For example, in some embodiments, each D may be bromo, iodo, carboxyl, or hydroxyl. In some embodiments, when Ti is hydroxyl, D may be a constituent other than hydroxyl at the position that is alpha to the point of attachment of Xi and beta to the point of attachment of T].

[0060] For metal coordinating moieties of Formula (Ia), the E substituent, if present, is independently bonded to any of the substitutable phenyl ring carbon atoms. In some embodiments, each E may independently be fluoro, chloro, bromo, iodo, carboxyl, cyano, nitro, amido, hydroxyl, amino, sulfito, phosphito, sulfato, phosphato, aryl; or C _1-8 alkyl optionally substituted with one or more of C] _-2O alkyl, carboxyl, cyano, nitro, amido, hydroxy!, amino, sulfito, phosphito, sulfato, and phosphato. For example, in some embodiments, each E may independently be bromo, iodo, carboxyl, or hydroxyl.

[0061] Typically, for metal coordinating moieties of Formula (Ia), X ₁ -X ₄ are independently methylene optionally substituted by d. ₆ alkyl, halo, or hydroxyl.

[0062] In some embodiments of the metal coordinating moieties of Formula (Ia), q ₂ is 0. Accordingly, Q ₂ , Q ₃ , and Q ₄ may independently be selected from the group consisting of:

[0063] In addition to the metal coordinating moieties including a heterocyclic ring, the metal coordinating moieties may alternatively include a heterosubstituted alkyl chain. Typically, the heterosubstituted alkyl chain includes from about 4 to about 10 atoms in the heterosubstituted alkyl chain, at least 2 of the atoms being nitrogen. In one example of metal coordinating moieties including a heterosubstituted alkyl chain, the chain includes 2-4 nitrogen atoms wherein at least one of the chain nitrogen atoms is substituted. For these embodiments, the chain carbon atoms may optionally be substituted. Typically, the nitrogen atoms including the heterosubstituted alkyl chain are separated from each other by two carbon atoms and thus the metal coordinating moiety may be depicted by the following Formula (2)

[0065] wherein

[0066] n is 0, 1 or 2; and

[0067] m is 0-8 wherein when m is greater than 0, each A is independently selected from the group consisting of optionally substituted C ₁ . ₂₀ alkyl and aryl.

[0068] When the metal coordinating moiety corresponds to Formula (2) and m is greater than 0, it is generally preferred that each A be a substituent that positively impacts stability and biodistribution. When present, each A may independently be substituted with one or more aryl, Ci _-2O alkyl, carbaldehyde, keto, carboxyl, cyano, halo, nitro, amido, sulfato, sulfϊto, phosphate, phosphito, hydroxyl, oxy, mercapto, or thio substituents. In addition, when A is aryl or alkyl, each of these, in turn, may be optionally substituted with an aryl or Ci _-2 O alkyl moiety optionally substituted with one or more aryl, carbaldehyde, keto, carboxyl, cyano, halo, nitro, amido, sulfato, sulfito, phosphato, phosphito, hydroxyl, oxy, mercapto and thio.

[0069] For metal coordinating moieties of Formula (2), the A substituent, if present, may be bonded to any of the ring carbon atoms. Each ring carbon atom may be substituted so that the number of possible A substituents varies with the number of ring carbon atoms. In one embodiment of metal

coordinating moieties of Formula (2) having at least one A substituent, each A is independently aryl or Ci _-S alkyl optionally substituted with one or more aryl, keto, carboxyl, cyano, nitro, Cuo alkyl, amido, sulfato, sulfito, phosphato, phosphite, oxy and thio. For example, each A may be aryl or C|_6 alkyl optionally substituted with one or more aryl, keto, amido and oxy. By way of further example, each A may be methyl.

[0070] In general, as the value of n increases, the length of the heterosubstituted alkyl chain increases. In this manner, the length of the heterosubstituted alkyl chain may be controlled to match the size and coordination capacity of the metal to be coordinated.

[0071] In some embodiments where the metal coordinating moiety includes a heterosubstituted alkyl chain, the metal coordinating moiety complies with the following Formula (2a)

[0073] wherein [0074] n is 0, 1 or 2;

[0075] m is 0-8 wherein when m is greater than 0, each A is C _\ . ₂ o alkyl or aryl optionally substituted by one or more aryl, C _]-2 o alkyl, carbaldehyde, keto, carboxyl, cyano, halo, nitro, amido, sulfato, sulfito, phosphato, phosphito, hydroxyl, oxy, mercapto or thio;

[0076] q is 0-3 wherein when q is greater than 0, each D is independently selected from the group consisting of fluoro, chloro, bromo, iodo, carboxyl, cyano, nitro, amido, hydroxyl, amino, sulfato, sulfito, phosphato, phosphito, aryl, and Cj _-2O alkyl optionally substituted with one or more of C ₁ . ₂₀ alkyl, carboxyl, cyano, nitro, amido, hydroxyl, amino, sulfato, sulfito, phosphato, and phosphito;

[0077] X], X ₂ , X3, X ₄ , and X ₅ are independently optionally substituted methylene where the substituents are selected from the group consisting of aryl, Ci _-2O alkyl, carbaldehyde, keto, carboxyl, cyano, halo, nitro, amido, sulfato, sulfito, phosphato, phosphito, hydroxyl, oxy, mercapto and thio;

[0078] Q ₂ -Qs are independently selected from the group consisting of:

[0079] q ₂ is 0-4 wherein when q ₂ is greater than 0, each E is independently selected from the group consisting of fluoro, chioro, bromo, iodo, carboxyl, cyano, nitro, amido, hydroxyl, amino, sulfito, phosphito, and C|. ₂ oalkyl optionally substituted with one or more or Ci _-2O alkyl, carboxy, cyano, nitro, amido, hydroxyl, sulfito, phospito, sulfato, and phosphato; and

[0080] T] is hydroxyl or mercapto.

[0081] For metal coordinating moieties of Formula (2a), the D substituent, if present, may be independently bonded to any of the substitutable phenyl ring carbon atoms. In some embodiments, each D may independently be fluoro, chioro, bromo, iodo, carboxyl, cyano, nitro, amido, hydroxyl, amino, sulfito, phosphito, sulfato, phosphato, aryl, or Ci _-8 alkyl optionally substituted with one or more of C _!-2 o alkyl, carboxyl, cyano, nitro, amido, hydroxyl, amino, sulfito, phosphito, sulfato, and phosphate. For instance, each D of some embodiments may independently be bromo, iodo, carboxyl, or hydroxyl. In some embodiments, when Ti is hydroxyl, D may be a constituent other than hydroxyl at the position that is alpha to the point of attachment of X ₁ and beta to the point of attachment of T].

[0082] For metal coordinating moieties of Formula (2a), the E substituent, if present, may be independently bonded to any of the substitutable phenyl ring carbon atoms. In some embodiments, each E may independently be fluoro, chioro, bromo, iodo, carboxy], cyano, nitro, amido, hydroxyl, amino, sulfito, phosphito, sulfato, phosphato, aryl, or Cj _-8 alkyl optionally substituted with one or more of Ci. ₂ o alkyl, carboxyl, cyano, nitro, amido, hydroxyl, amino, sulfito, phosphito, sulfato, and phosphato. For instance, each E may independently be bromo, iodo, carboxyl, or hydroxyl in some embodiments.

[0083] Typically, for metal coordinating moieties of Formula (2a), Xi-Xi are independently methylene optionally substituted by Ci _-6 alkyl, halo, or hydroxyl.

[0084] In some embodiments of metal coordinating moieties of Formula (2a), q ₂ is 0. Accordingly, Q ₂ , Q3, Q ₄ and Q ₅ are independently selected from the group consisting of:

[0085] For any of the above embodiments, the metal coordinating moiety may be complexed with a metal, M, thereby forming a metal complex.

[0086] In some embodiments where the metal coordinating moiety is a heterocyclic ring and complexed with a metal, M, the complex has the following Formula (3):

[0088] wherein

[0089] n is 0, 1 or 2;

[0090] m is 0-16 wherein when m is greater than 0, each A is C _1-2O alkyl or aryl optionally substituted by one or more aryl, C] _-20 alkyl, carbaldehyde, keto, carboxyl, cyano, halo, nitro, amido, sulfate, sulfϊto, phosphate, phosphite, hydroxyl, oxy, mercapto or thio;

[0091] q is 0-3 wherein when q is greater than 0, each D is independently selected from the group consisting of fluoro, chloro, bromo, iodo, carboxyl, cyano, nitro, amido, hydroxyl, amino, sulfate, sulfϊto, phosphate, phosphite, aryl, and Ci _-20 alkyl optionally substituted with one or more of Cj _-2O alkyl, carboxyl, cyano, nitro, amido, hydroxyl, amino, sulfate, sulfite, phosphate, and phosphito;

[0092] Xi, X ₂ , X3, X4 are independently optionally substituted methylene where the substituents are selected from the group consisting of aryl, C _]-2 o alkyl, carbaldehyde, keto, carboxyl, cyano, halo, nitro, amido, sulfato, sulfite, phosphate, phosphito, hydroxyl, oxy, mercapto and thio;

[0093] Q ₂ -Q ₄ are independently selected from the group consisting of:

[0094] q ₂ is 0-4 wherein when q ₂ is greater than 0, each E is independently selected from the group consisting of fluoro, chloro, bromo, iodo, carboxyl, cyano, nitro, amido, hydroxyl, amino, sulfito, phosphito, and C ₁ . ₂₀ alkyl optionally substituted with one or more or C]. ₂ o alkyl, carboxy, cyano, nitro, amido, hydroxyl, sulfito, phospito, sulfato, and phosphate;

[0095] Ti is hydroxyl or mercapto; and

[0096] M is selected from the group consisting of Lu, Lu-177, Y, Y-90, In, In-1 1 1 , Tc, Tc=O, Tc-99m, Tc-99m=O, Re, Re-186, Re-188, Re=O, Re-186=O, Re-188=O, Ga, Ga-67, Ga-68, Cu, Cu-62, Cu-64, Cu-67, Gd, Gd-153, Dy, Dy-165, Dy-166, Ho ₃ Ho-166, Eu, Eu-169, Sm ₅ Sm-153, Pd, Pd-] 03, Pm, Pm-149, Tm, Tm-170, Bi, Bi-212, As and As-21 1.

[0097] In some embodiments where the metal coordinating moiety is a heterosubstituted alkyl chain and is complexed with a metal, M, the complex has the following Formula (4):

[0099] wherein

[00100] n is 0, 1 or 2;

[00101] m is 0-8 wherein when m is greater than 0, each A is Ci _-2O alkyl or aryl optionally substituted by one or more aryl, Ci.20 alkyl, carbaldehyde, keto, carboxyl, cyano, halo, nitro, amido, sulfato, sulfite, phosphate, phosphito, hydroxyl, oxy, mercapto or thio;

[00102] q is 0-3 wherein when q is greater than 0, each D is independently selected from the group consisting of fluoro, chloro, bromo, iodo, carboxyl, cyano, nitro, amido, hydroxyl, amino, sulfate,

sulfito, phosphato, phosphito, aryl, and optionally substituted with one or more of Ci _-2O alkyl, carboxyl, cyano, nitro, amido., hydroxyl, amino, sulfato, sulfito, phosphato, and phosphito;

[00103] Xi, X ₂ , X ₃ , X ₄ and X ₅ are independently optionally substituted methylene where the substituents are selected from the group consisting of aryl, C ₁ . ₂₀ alkyl, carbaldehyde, keto, carboxyl, cyano, halo, nitro, amido, sulfato, sulfito, phosphato, phosphito, hydroxyl, oxy, mercapto and thio;

[00104] Q ₂ -Q ₅ are independently selected from the group consisting of:

[00105] q ₂ is 0-4, wherein when q ₂ is greater than 0, each E is independently selected from the group consisting of fluoro, chloro, bromo, iodo, carboxyl, cyano, nitro, amido, hydroxyl, amino, sulfito, phosphito, and C] _-2 O alkyl optionally substituted with one or more or Ci _-2O alkyl, carboxy, cyano, nitro, amido, hydroxyl, sulfito, phospito, sulfato, and phosphato;

[00106] T] is hydroxyl or mercapto; and

[00107] M is selected from the group consisting of Lu ₅ Lu-177, Y, Y-90, In, In-111, Tc, Tc=O, Tc-99m, Tc-99m=O, Re, Re-186, Re-188, Re=O, Re-186=O, Re-188=O, Ga, Ga-67, Ga-68, Cu, Cu-62, Cu-64, Cu-67, Gd, Gd-153, Dy, Dy-165, Dy-166, Ho, Ho-166, Eu, Eu-169, Sm, Sm-153, Pd, Pd-103, Pm, Pm-149, Tm, Tm-170, Bi, Bi-212, As and As-21 1.

[00108] Whether the complex corresponds to Formula (3) or Formula (4) typically depends on the particular metal selected for coordination. For example, for yttrium and lanthanides, the complex corresponding to Formula (3) is preferred. Formula (3) is also preferred for iron, copper, and manganese, while Formula (4) is the preferred complex for the remaining transition metals. The preferred complex for any particular metal is related to the potential for transmetallation with endogenous ion. Thus, Formula (3) provides greater stability with high exchange metals, including, but not limited to, yttrium, lanthanides, and gallium. Transmetallation with endogenous ions does not present as great a concern for regular transition metals. While complexes of Formula (3) have been mentioned above as being preferred for use with some metals, while complexes of Formula (4) have been mentioned above as being preferred for use with other metals, it is contemplated that complexes of Formulas (3) and (4) may be utilized with metals other than those listed for the respective complexes.

[00109] Macrocyclic metal coordinating moieties with three-dimensional cavities often form metal complexes with high stability. These complexes often exhibit selectivity for certain metal ions based on metal size and coordination chemistry, and capability to adopt a preorganized conformation in the uncomplexed form, which facilitates metal complexation. The selection of appropriate macrocyclic metal coordinating moieties and metals is known by those skilled in the art.

[00110] The value of n, and hence the size or length of the metal coordinating moiety, depends upon the particular metal to be coordinated. For yttrium and lanthanides, for example, n is generally 1. For transition metals, n is typically 0 or 1. For manganese and technetium, n is 0, 1 , or 2 depending on the value of X ₂ -X ₄ . It is, however, contemplated that other values of n may be appropriate for one or more of the metals discussed above.

[00111] General Synthesis

[00112] For illustrative purposes, the following reaction shows the activation of a metal chelator using carbonyl ditriazine (CDT):

[00113] To prevent the reaction of free hydroxyl groups prior to preparation of the conjugate, the hydroxyl groups of the metal coordinating moiety are protected. Any conventional means of protecting the hydroxyl groups is permissible. A variety of protecting groups for the hydroxyl groups and the synthesis thereof may be found in "Protective Groups in Organic Synthesis, 3rd Edition" by T. W. Greene and P.G.M. Wuts, John Wiley and Sons, 1999. Exemplary protecting groups include tert- butyl, methoxymethyl, 1 -ethoxymethyl, benzyloxymethyl, (beta-trimethylsilylethoxy)methyl, tetrahydropyranyl, 2,2,2-trichloroethyoxycarbonyl, t-butyl(diphenyl)silyl, trialkylsilyl, trichloromethoxycarbonyl and 2,2,2-trichloroethoxymethyl.

[00114] To create a reactive urea group from an amine, a mild activating agent is preferred. Exemplary activating agents include carbonyl ditriazine or carbonyl di imidazole (CDI), or mixtures thereof. Other activating agents include phosgene, bis(trichloromethyl)carbonate, and trichloromethyl chloroformate. The reactive intermediates can be isolated as solids, which are stable while under anhydrous conditions. Thus, such an active urea could be allowed to react with a synthetic or natural product (e.g., a biomolecule) to give a protected intermediate. The product may be isolated by precipitation from the reaction mixture usinε. for examnle. _an( j ether. Purification of

the product can be carried out, for example, by using normal or C18 reverse phase chromatography, as needed. This intermediate can be subsequently deprotected by application of an acid, such as trifϊic acid in trifluoroethanol, thereby unmasking the phenol hydroxyl and carboxylates.

[00115] For this embodiment, the bio-directing carrier and metal may be any of those previously recited. The radioisotope or paramagnetic metal ion is typically dissolved in a solution. The solution may be an aqueous acid or any other solution known in the art to dissolve a radioisotope or paramagnetic metal ion. The solution should allow for the stable storage of the metal in the kit and not interfere with the properties of the metal. Solubilization aids useful in the preparation of radiopharmaceuticals and in the diagnostic kits include, but are not limited to, ethanol, glycerin, polyethylene glycol, propylene glycol, polyoxyethylene sorbitan monooleate, sorbitan monoloeate, polysorbates, poly(oxyethylene)-poly(oxypropylene)-poly(oxyethylene) block copolymers (Pluronics) and lecithin. Preferred solubilizing aids are polyethylene glycol and Pluronics.

[00116] Metallopharmaceutical Compositions

[00117] MetaHopharmaceuticai compositions of the present invention include a conjugate, complexed to a metal, dispersed in a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier, also known in the art as an excipient, vehicle, auxiliary, adjuvant, or diluent, is typically a substance which is pharmaceutically inert, confers a suitable consistency or form to the composition, and does not diminish the therapeutic or diagnostic efficacy of the conjugate. The carrier is generally considered to be "pharmaceutically or pharmacologically acceptable" if it does not produce an unacceptably adverse, allergic or other untoward reaction when administered to a mammal, especially a human.

[00118] The selection of a pharmaceutically acceptable carrier tends, at least in part, to be a function of the desired route of administration. In general, metallopharmaceutical compositions of the invention can be formulated for any route of administration so long as the target tissue is available via that route. For example, suitable routes of administration include, but are not limited to, oral, parenteral (e.g., intravenous, intraarterial, subcutaneous, rectal, subcutaneous, intramuscular, intraorbital, intracapsular, intraspinal, intraperitoneal, or intrasternal), topical (nasal, transdermal, intraocular), intravesical, intrathecal, enteral, pulmonary, intralymphatic, intracavital, vaginal, transurethral, intradermal, aural, intramammary, buccal, orthotopic, intratracheal, intralesional, percutaneous, endoscopical, transmucosal, sublingual and intestinal administration.

[00119] Examples of pharmaceutically acceptable carriers for use in compositions of the present invention are well known to those of ordinary skill in the art and may be selected based upon a number of factors: the particular conjugate used, and its concentration, stability and intended bioavailability; the disease, disorder or condition being treated or diagnosed with the composition; the subject, its age, size and general condition; and the route of administration. Suitable nonaqueous,

pharmaceutical ly-acceptable polar solvents include, but are not limited to, alcohols (e.g., α-glycerol formal, β-glycerol formal, 1,3-butyleneglycol, aliphatic or aromatic alcohols having 2-30 carbon atoms such as methanol, ethanol, propanol, isopropanol, butanol, t-butanol, hexanoi, octanol, amylene hydrate, benzyl alcohol, glycerin (glycerol), glycol, hexylene glycol, tetrahydrofurfuryl alcohol, lauryl alcohol, cetyl alcohol, or stearyl alcohol, fatty acid esters of fatty alcohols such as polyalkylene glycols (e.g., polypropylene glycol, polyethylene glycol), sorbitan, sucrose and cholesterol); amides (e.g., dimethylacetamide (DMA), benzyl benzoate DMA, dimethylformamide, N-(β-hydroxyethyl)-lactamide, N, N-dimethylacetamide_amides, 2-pyrrolidinone, l-methyl-2-pyrrolidinone, or polyvinylpyrrolidone); esters (e.g., l-methyl-2-pyrrolidinone, 2-pyrrolidinone, acetate esters such as monoacetin, diacetin, and triacetin, aliphatic or aromatic esters such as ethyl caprylate or octanoate, alkyl oleate, benzyl benzoate, benzyl acetate, dimethylsulfoxide (DMSO) ₃ esters of glycerin such as mono, di, or tri-glyceryl citrates or tartrates, ethyl benzoate, ethyl acetate, ethyl carbonate, ethyl lactate, ethyl oleate, fatty acid esters of sorbitan, fatty acid derived PEG esters, glyceryl monostearate, glyceride esters such as mono, di, or triglycerides, fatty acid esters such as isopropyl myristrate, fatty acid derived PEG esters such as PEG- hydroxyoleate and PEG-hydroxystearate, N-methyl pyrrolidinone, pluronic 60, polyoxyethylene sorbitol oleic polyesters such as poly(ethoxylated) ₃₀ . ₆ o sorbitol poly(oleate) _2- 4, poly(oxyethylene) ₁₅ _ ₂ o monooleate, poly(oxyethylene)is.2o mono 12-hydroxystearate, and poly(oxyethylene)i _5-2 o mono ricinoleate, polyoxyethylene sorbitan esters such as polyoxyethylene-sorbitan monooleate, polyoxyethylene-sorbitan monopalmitate, polyoxyethylene-sorbitan monolaurate, polyoxyethylene-sorbitan monostearate, and Polysorbate® 20, 40, 60 or 80 from ICI Americas, Wilmington, DE, polyvinylpyrrolidone, alkyleneoxy modified fatty acid esters such as polyoxyl 40 hydrogenated castor oil and polyoxyethyiated castor oils (e.g., Cremophor® EL solution or Cremophor® RH 40 solution), saccharide fatty acid esters (i.e., the condensation product of a monosaccharide (e.g., pentoses such as ribose, ribulose, arabinose, xylose, lyxose and xylulose, hexoses such as glucose, fructose, galactose, mannose and sorbose, trioses, tetroses, heptoses, and octoses), disaccharide (e.g., sucrose, maltose, lactose and trehalose) or oligosaccharide or mixture thereof with a C ₄ -C ₂₂ fatty acid(s)(e.g., saturated fatty acids such as caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid and stearic acid, and unsaturated fatty acids such as palmitoleic acid, oleic acid, elaidic acid, erucic acid and linoleic acid)), or steroidal esters); alkyl, aryl, or cyclic ethers having 2-30 carbon atoms (e.g., diethyl ether, tetrahydrofuran, dimethyl isosorbide, diethylene glycol monoethyl ether); glycofurol (tetrahydrofurfuryl alcohol polyethylene glycol ether); ketones having 3-30 carbon atoms (e.g., acetone, methyl ethyl ketone, methyl isobutyl ketone); aliphatic, cycloaliphatic or aromatic hydrocarbons having 4-30 carbon atoms (e.g., benzene, cyclohexane, dichloromethane, dioxolanes, hexane, n-decane, n-dodecane, n-hexane, sulfolane, tetramethylenesulfon, tetramethylenesulfoxide, toluene, dimethylsulfoxide (DMSO), or tetramethylenesulfoxide); oils of mineral, vegetable, animal, essential or synthetic origin (e.g., mineral oils such as aliphatic or wax-based hydrocarbons, aromatic hydrocarbons, mixed aliphatic and aromatic based hydrocarbons, and refined

paraffin oil, vegetable oils such as linseed, tung, safflower, soybean, castor, cottonseed, groundnut, rapeseed, coconut, palm, olive, corn, corn germ, sesame, persic and peanut oil and glycerides such as mono-, di- or triglycerides, animal oils such as fish, marine, sperm, cod-liver, haliver, squalene, squalane, and shark liver oil, oleic oils, and polyoxyethylated castor oil); alkyl or aryl halides having 1- 30 carbon atoms and optionally more than one halogen substituent; methylene chloride; monoethanolamine; petroleum benzin; trolamine; omega-3 polyunsaturated fatty acids (e.g., alpha- linolenic acid, eicosapentaenoic acid, docosapentaenoic acid, or docosahexaenoic acid); polyglycol ester of 12-hydroxystearic acid and polyethylene glycol (Solutol® HS-15, from BASF, Ludwigshafen, Germany); polyoxyethylene glycerol; sodium laurate; sodium oleate; or sorbitan monooleate.

[00120] Other pharmaceutically acceptable solvents for use in the invention are well known to those of ordinary skill in the art, and are identified in The Chemotherapy Source Book (Williams & Wilkens Publishing), The Handbook of Pharmaceutical Excipients, (American Pharmaceutical Association, Washington, D.C., and The Pharmaceutical Society of Great Britain, London, England, 1968), Modern Pharmaceutics, (G. Banker et al., eds., 3d ed.)(Marcel Dekker, Inc., New York, New York, 1995), The Pharmacological Basis of Therapeutics, (Goodman & Gilman, McGraw Hill Publishing), Pharmaceutical Dosage Forms, (H. Lieberman et al., eds.)(Marcel Dekker, Inc., New York, New York, 1980), Remington's Pharmaceutical Sciences (A. Gennaro, ed., 19th ed.)(Mack Publishing, Easton, PA, 1995), The United States Pharmacopeia 24, The National Formulary 19, (National Publishing, Philadelphia, PA, 2000), A.J. Spiegel et al., and Use of Nonaqueous Solvents in Parenteral Products, JOURNAL OF PHARMACEUTICAL SCIENCES, Vol. 52, No. 10, pp. 917-927 (1963).

[00121] Dosage

[00122] Dosage and regimens for the administration of the pharmaceutical compositions of the invention can be readily determined by those with ordinary skill in diagnosing or treating disease. It is understood that the dosage of the conjugates will be dependent upon the age, sex, health, and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment, and the nature of the effect desired. For any mode of administration, the actual amount of conjugate delivered, as well as the dosing schedule necessary to achieve the advantageous effects described herein, will also depend, in part, on such factors as the bioavailability of the conjugate, the disorder being treated or diagnosed, the desired therapeutic or diagnostic dose, and other factors that will be apparent to those of skill in the art. The dose administered to an animal, particularly a human, in the context of the present invention should be sufficient to affect the desired therapeutic or diagnostic response in the animal over a reasonable period of time.

[00123] Radiolabeled scintigraphic imaging agents provided by the present invention are provided having a suitable amount of radioactivity. In forming diagnostic radioactive complexes, it is generally preferred to form radioactive complexes in solutions containing radioactivity at concentrations

of from about 0.01 millicurie (mCi) to 100 mCi per mL. Generally, the unit dose to be administered has a radioactivity of about 0.01 mCi to about 100 mCi, preferably about 1 mCi to about 30 mCi. The solution to be injected at unit dosage is from about 0.01 mL to about 10 mL. The amount of radiolabeled conjugate appropriate for administration is dependent upon the distribution profile of the chosen conjugate in the sense that a rapidly cleared conjugate may need to be administered in higher doses than one that clears less rapidly. In vivo distribution and localization can be tracked by standard scintigraphic techniques at an appropriate time subsequent to administration; typically between thirty minutes and 180 minutes depending upon the rate of accumulation at the target site with respect to the rate of clearance at the non-target tissue.

[00124] Typically, an In-1 1 1 diagnostic dose is 3-6 mCi while a typical Tc-99m does is 10- 30 mCi. Generally, radiotherapeutic doses of radiopharmaceuticals vary to a greater extent, depending on the tumor and number of injections of cycles. For example, cumulative doses of Y-90 range from about 100-600 mCi (20 -150 mCi/dose), while cumulative doses of Lu-177 range from about 200-800 mCi (50-200 mCi/dose).

[00125] Kits

[00126] For convenience, metallopharmaceutical compositions of the present invention may be provided to the user in the form of a kit containing some or all of the necessary components. The use of a kit is particularly convenient since some of the components, e.g., a radioisotope, have a limited shelf life, particularly when combined. Thus, the kit may include one or more of the following components (i) a conjugate, (ii) a metal coordinated to or for coordination by the conjugate, (iii) a carrier solution, and (iv) instructions for their combination and use. Depending on the metal, a reducing agent may be necessary to prepare the metal for reaction with the conjugate. Exemplary reducing agents include Ce (III), Fe (II), Cu (I), Ti (III), Sb (III), and Sn (II). Of these, Sn (II) is particularly preferred. Often the components of the kit are in unit dosage form (e.g., each component in a separate vial).

[00127] For reasons of stability, it may be preferred that the conjugate be provided in a dry, lyophilized state. The user may then reconstitute the conjugate by adding the carrier or other solution.

[00128] Because of the short half-life of suitable radionuclides, it will frequently be most convenient to provide the kit to the user without a radionuclide. The radionuclide is then ordered separately when needed for a procedure. Alternatively, if the radionuclide is included in the kit, the kit will most likely be shipped to the user just before it is needed.

[00129] In addition to the metal coordinating moiety, biomolecule, active urea, metal and deprotecting acid, the kit of the present invention typically includes a buffer. Exemplary buffers include citrate, phosphate and borate.

[00130] The kit optionally contains other components frequently intended to improve the ease of synthesis of the radiopharmaceutical by the practicing end user, the ease of manufacturing the kit,

the shelf-life of the kit, or the stability and shelf-life of the radiopharmaceutical. Such components of the present invention include lyophilization aids, e.g., mannitol, lactose, sorbitol, dextran, Ficoll, and polyvinylpyyrolidine (PVP); stabilization aids, e.g., ascorbic acid, cysteine, monothioglycerol, sodium bisulfite, sodium metabisulfite, gentisic acid, and inositol; and bacteriostats, e.g., benzyl alcohol, benzalkonium chloride, chlorbutanol, and methyl, propyl, or butyl paraben.

[00131] Typically, when the conjugate is formulated as a kit, the kit includes multiple vials consisting of a protected metal coordinating moiety having an active urea group, a deprotecting acid, a buffer, and a solution of a radioactive metal such as, but not limited to, In-111, Y-90 or Lu- 177. In practice, the user will take the vial containing the metal coordinating moiety and add a solution of a bio- directing carrier of interest bearing a reactive amino (NH ₂ ) group. Once conjugation is complete, the deprotecting acid is added to affect deprotection, followed by addition of the radioactive metal. The mixture is then buffered to complete complexation of the radioactive metal by the metal chelator.

[00132] Definitions

[00133] The compounds described herein may have asymmetric centers. Compounds of the present invention containing an asymmetrically substituted atom may be isolated in optically active or racemic form. Cis and trans geometric isomers of the compounds of the present invention are described and may be isolated as a mixture of isomers or as separated isomeric forms. AU chiral, diastereomeric, racemic forms and all geometric isomeric forms of a structure are intended, unless the specific stereochemistry or isomeric form is specifically indicated. All processes used to prepare compounds of the present invention and intermediates made therein are considered to be part of the present invention.

[00134] The present invention includes all isotopes of atoms occurring in the present compounds. Isotopes include those atoms having the same atomic number but different mass numbers.

[00135] Unless otherwise indicated, the alkyl groups described herein are preferably lower alkyl containing from one to eight carbon atoms in the principal chain and up to 20 carbon atoms. They may be straight or branched chain or cyclic and include methyl, ethyl, propyl, isopropyl, butyl, hexyl and the like.

[00136] The term "amido" as used herein includes substituted amido moieties where the substituents include, but are not limited to, one or more of aryl and Ci _-2O alkyl, each of which may be optionally substituted by one or more aryl, carbaldehyde, keto, carboxyl, cyano, halo, nitro, Ci _-2O alkyl, sulfato, sulfito, phosphato, phosphite, hydroxyl, oxy, mercapto, and thio substituents.

[00137] The term "amino" as used herein includes substituted amino moieties where the substituents include, but are not limited to, one or more of aryl and Ci _-2O alkyl, each of which may be optionally substituted by one or more aryl, carbaldehyde, keto, carboxyl, cyano, halo, nitro, C] _-20 alkyl, sulfato, sulfito, phosphato, phosphito, hydroxyl, oxy, mercapto, and thio substituents.

[00138] The terms "aryl" or "ar" as used herein alone or as part of another group denote optionally substituted homocyclic aromatic groups, preferably monocyclic or bicyclic groups containing from 6 to 12 carbons in the ring portion, such as phenyl, biphenyl, naphthyl, substituted phenyl, substituted biphenyl or substituted naphthyl. Phenyl and substituted phenyl are the more preferred aryl.

[00139] The term "complex" refers to a metal coordinating moiety of the invention, e.g. Formula (1), complexed or coordinated with a metal. The metal is typically a radioactive isotope or paramagnetic metal ion.

[00140] The term "conjugate" refers to a metal coordinating moiety of the invention, e.g. Formula (1), bonded to a bio-directing carrier (biomolecule) whether or not the metal coordinating moiety is complexed with a metal. For the present invention, the metal coordinating moiety is bonded to the bio-directing carrier directly or indirectly by a urea moiety.

[00141] The terms "halogen" or "halo" as used herein alone or as part of another group refer to chlorine, bromine, fluorine, and iodine.

[00142] The term "heteroatom" shall mean atoms other than carbon and hydrogen.

[00143] The terms "heterocyclo" or "heterocyclic" as used herein alone or as part of another group denote optionally substituted, fully saturated or unsaturated, monocyclic or bicyclic, aromatic or nonaromatic groups having at least one heteroatom in at least one ring. The heterocyclo group preferably has 1 to 5 nitrogen atoms in the ring, and may be bonded to the remainder of the molecule through a carbon atom. Exemplary heterocyclics include macrocyclics, cyclen, tacn, DOTA, DOTMA, DOTP, and TETA.

[00144] The "heterosubstituted alkyl" moieties described herein are alkyl groups, in which a carbon atom is covalently bonded to at least one heteroatom and optionally with hydrogen, the heteroatom being, for example, a nitrogen atom.

[00145] The term "metallopharmaceutical" as used herein refers to a pharmaceutically acceptable compound including a metal, wherein the compound is useful for imaging or treatment.

[00146] Examples

[00147] Example 1: Synthesis of l,4,7,10-Tetraazacyclododecane-l,4,7-triacetic acid, 10-r(2-butoxy-5-(N — ftCNCbl-5'-rfl3-amino)-4,7.10-trioxatridencanecarbamatel))- carbonylamino ^' tphenvDmethvll, tri-butyl ester (8)

[00148] Synthesis of 2-t-butoxy-5-nitrobenzyl bromide (1)

TBTA

1 Koshlands

Reagent I

[00149] f-Butyl trichloroacetimidate (TBTA)

[00150] Potassium f-butoxide (IM in f-butanol), 69mL 0.069mole, was dissolved in diethyl ether, 69mL to form a solution. This solution was added dropwise, over 30 minutes, to a 0 ⁰ C solution of trichloroacetonitrile, 100g 0.69mole, in diethyl ether, 69mL. The mixture was allowed to warm to room temperature over one hour and stirred for an additional hour with heating at reflux. The mixture was cooled to room temperature and evaporated under reduced pressure to an oil. The oil was dissolved in hexanes, 14OmL, and filtered to remove potassium salts. The filtrate was then evaporated under reduced pressure and the residue vacuum distilled collecting the fraction distilling at 2.4mm Hg and 40°C. Yield 105g, 69% based on trichloroacetonitrile. ¹ H nmr (300MHz CDCl ₃ ): 1.58, (s, 9H), 8.21 (br, s, IH). ¹³ C (75.45 MHz, CDCl ₃ ) 27.23, 83.86, 92.78, 160.33.

[00151] 2-t-Butoxy-5-nitrobenzyl bromide CO

[00152] A suspension of 2-hydroxy-5-nitrobenzylbromide, 19.4g 0.0836mole, cyclohexane, 334mL, and dichloromethane, 167mL, was stirred under nitrogen. To this suspension was added a solution off-butyl trichloroacetimidate, 73.08g 0.334mole, in cyclohexane, 669mL, dropwise over 3.5 hours. The mixture was stirred for one hour after completion of the addition and boron trifluoride etherate, 20OuL, was added. The mixture was allowed to stir overnight. A large amount of precipitate, trichloroacetamide, formed. The reaction mixture was treated with sodium bicarbonate, 4.0Og 0.0418 mole, stirred for one hour and filtered. The solids were washed with diethyl ether and the combined filtrates concentrated to an oil under reduced pressure. The oil was treated with hexanes, 10OmL, and

the solution stirred until crystals formed. After cooling to -20 ⁰ C and stirring for an additional hour, the resulting solid was collected by filtration, washed with cold, fresh hexane, suctioned dry and vacuum dried. Yield 13.2g, 55% based on 2-hydroxy-5-nitrobenzyl bromide. CaIc C 45.85, H 4.90, N 4.86, Br 27.73. Found C 45.39, H 5.07, N 4.94, Br 27.66. ¹ H nmr (300MHz CDCl ₃ ) 1.58 (s, 9H), 4.48 (s, 2H), 7.10 (d, JH = 9Hz, IH), 8.1 1 (dd, J=9Hz, J=2.7Hz, IH), 8.22 (d, J=2.7Hz, IH). ¹³ C (75.45 MHz, CDCl ₃ ) 28.92, 81.59, 1 16.86, 125.07, 126.34, 129.98, 140.69, 159.97.

[00153] Synthesis of 1,4, 7,10-Tetraazacyclododecane-1,4, 7-triacetic acid, 10-[(2-t-butoxy- S-nitrophenyl)methylJ-, tri-t-butyl ester (3)_

[00154] .Synthesis of 1.4.7.10-Tetraazacyclododecane-1.4,7-triacetic acid, tri-f-butyl ester Hydrobromide (D

[00155] Cyclen, 32.Og 0.186mole, and sodium acetate trihydrate, 75.8g 0.557mole, were stirred with dimethylacetamide, 60OmL, for one hour. To this mixture was added a solution of ?-butyl bromoacetate, 109g 0.557mole, in dimethylacetamide, 15OmL, dropwise, over four hours. The rate of the addition was adjusted so as to keep the temperature of the reaction mixture less than 25°C. The mixture was allowed to stir over two nights. After cooling to -10 ⁰ C and stirring for two hours, the resulting solid was collected by filtration, washed with cold, fresh dimethylacetamide, 5OmL, and suctioned dry. The solid was dissolved in chloroform, 0.5L, and the solution washed with water, 3x200mL. The organic phase was collected, dried with magnesium sulfate, filtered and concentrated,

under reduced pressure, to 30OmL. Hexanes, 30OmL, was added and the solution stirred for one hour at room temperature. After a few minutes crystallization began. The resulting slurry was cooled to -20 ⁰ C, stirred for two hours and filtered. The solid was washed with cold, fresh chloroform-hexanes, 5OmL 1 :1, suctioned dry and vacuum dried overnight at room temperature. Yield 69g, 62% based on cyclen. ¹ H nmr (300MHz, CDCl ₃ ) 1.44 (s, 9H), 1.45 (s, 18H), 2.87-2.90 (br, m, 12H), 3.07-3.08 (br, m, 4H), 3.27 (s, 2H), 3.56 (s, 4H), 9.97 (br,s , 2H). ¹³ C nmr (75.45MHz, CDCl ₃ ) 28.15, 28.18, 47.44, 48.68, 49.1 1, 51.15, 51.25, 58.1 1, 81.54, 81.70, 169.32, 170.21.

[00156] Synthesis of l,4.7.10-Tetraazacvclododecane-1.4,7-triacetic acid. 10-IT2-f-butoxy-5- nitrophenvOmethyli-. tri-f-butyl ester, sodium bromide complex (3)

[00157] 1 ,4,7, 10-Tetraazacyclododecane-1 ,4,7-triacetic acid, tri-?-butyl ester hydrobromide, 8.46g 0.0142mole, was stirred with aqueous sodium hydroxide, 0.1N 20OmL, and diethyl ether, 20OmL. When the entire solid had dissolved, the organic phase was collected and the aqueous phase washed with diethyl ether, 2x200mL. The combined organic extracts were dried with magnesium sulfate, filtered and evaporated, under reduced pressure, to an oil. The oil was dissolved in acetonitrile, 135mL. To this solution was added sodium bicarbonate, 1.19g 0.0142mole ₅ followed by 2-/-butoxy-5- nitrobenzyl bromide, 4.5Og 0.0156mole. The mixture was warmed to 35°C, and stirred overnight under argon. When the reaction was complete by nmr, 12-14 hours total, the mixture was filtered and the filtrate concentrated under reduced pressure to give an oil. The oil was suspended in diethyl ether, 5OmL, and a white precipitate formed after stirring. The solid was collected by filtration, suctioned dry and dried in a vacuum overnight. Yield 11.7g, 98% based on starting 1 ,4,7, 10-Tetraazacyclododecane- 1 ,4,7-tri acetic acid, tri-/-butyl ester hydrobromide. Anal. CaIc. C 52.73, H 7.77, N 8.31, Br 9.48. Found C 52.31, H 7.68, N 8.26, Br 9.67. ¹ H nmr (300MHz, CDCl ₃ ) 1.45 (s, 27H), 1.51 (s, 9H), 1.78 (br, s, 2H), 2.20 (m, 4H), 2.33 (br, 4H), 2.73 (br, 4H), 2.93 (complex, br, 6H), 3.10 (m, 2H), 3.29 (s, IH), 3.37 (s, IH), 3.57 (s, 2H), 7.15 (d, ³ J _H-H =9Hz, IH), 8.07 (d of d, ³ J _H . _H =9Hz, ⁴ J _H . _H =2.7Hz, IH), 8.88 (d, ⁴ J _H - _H =2.7HZ). ¹³ C nmr (75.45MHz, CDCl ₃ ) 28.15, 28.20, 29.48, 50.00 (br), 55.97, 56.28, 81.83, 82.68, 83.29, 1 18.27, 124.18, 127.44, 131.13, 141.95, 161.31, 172.67, 173.62.

[00158] Synthesis of 1,4, 7,10-Tetraazacyclododecane-1,4, 7-triacetic acid, 10-[(2-t-butoxy- S-(triazolyl- and imidazolylcarbonylamino)phenyl)methyl]~, tri-t-butyl ester (5)_ and (61

[00159] Synthesis of 1.4.7.10-Tetraazacvclododecane-l .4,7-triacetic acid, 10-rf2-t-butoxy-5- aminophenVPmethylK tri-t-butyl ester, sodium bromide complex, pentahvdrate. (4)

[00160] Raney nickel-water slurry, ca. 0.4g, methanol, 2OmL, and hydrazine hydrate, 1.15mL, were placed in an argon-flushed 25OmL round bottom flask. The mixture was heated to reflux and a solution consisting of 1 ,4,7, 10-tetraazacyclododecane-l, 4,7-triacetic acid, 10-[(2-£-butoxy-5- nitrophenyl)methyl]-, tri-?-butyl ester, sodium bromide complex, 4.0Og 0.0047mole, methanol, 2OmL, was added dropwise. The addition took 30 minutes. The mixture was heated for an additional 10 minutes. An aliquot was removed, evaporated and dissolved in CDCl ₃ . ¹ H nmr showed the reaction to be greater than 95mole% complete. The reaction mixture was cooled to room temperature, filtered on celite. The filtrate was evaporated and dissolved in chloroform, 14mL, filtered to remove some fine solids and treated with diethyl ether, 8OmL. After stirring for a few minutes, crystallization began. The mixture was cooled to -10 ⁰ C, stirred for one hour and the solid collected by filtration, washed with fresh ether, suctioned dry and vacuum dried. Yield 3.57g 85%, based on starting 1 ,4,7, 10- tetraazacyclododecane-l, 4,7-triacetic acid, 10-[(2-f-butoxy-5-nitrophenyl)methyl]-, tri-f-butyl ester, sodium bromide complex. Anal. CaIc. C 50.22, H 8.54, N 7.91 , Br 9.03. Found C 50.49, H 7.68, N 7.80, Br 8.86. ¹ H nmr (300MHz, CDCl ₃ ) 1-28 (s, 9H), 1.45 (s, 9H), 1.47 (s, 18H), 2.22 (m, 4H), 2.36 (br, 6H), 2.80 (br, 6H), 2.97 (s, br, 4H), 3.40 (s, 2H), 3.44 (s, 2H), 6.45 (d of d, ³ J _H-H =9HZ, ⁴ J _H . _H =2.7HZ, IH), 6.75 (d, ³ J _H . _H =9HZ, IH), 6.92 (d, ⁴ J _H . _H =2.7HZ). 13C nmr (75.45MHz, CDCl ₃ ) 28.25, 28.41, 29.50, 50.00 (br), 54.00, 56.15, 56.49, 79.43, 82.55, 82.88, 115.03, 1 17.73, 124.07, 131.90, 142.69, 146.74, 172.64, 173.38.

[00161] Synthesis of 1.4.7. lO-Tetraazacyclododecane-l, 4,7-triacetic acid, 10-[Y2-f-butoxy-5- (imidazolylcarbonylamino)phenyl)methyl]-, tri-f-butyl ester,sodium bromide complex (5)

[00162] 1 ,4,7, 10-Tetraazacyclododecane-1 , 4,7-triacetic acid, 10-[(2-A-butoxy-5- aminophenyl)methyl]-, tri-/-butyl ester, sodium bromide complex, pentahydrate, l .OOg 0.001 lmole, was dissolved in dichloromethane, 4mL, and carbonyldiimidazole, 0.26g O.lόmole, was added. ¹ H nmr showed the reaction to be complete by disappearance of the aniline chemical shifts. The mixture was evaporated and the resulting oil stirred with diethyl ether, 25mL. The resulting solid was collected by filtration, washed with fresh ether and vacuum dried. Yield 0.77g, 77% based on starting 1,4,7, 10- tetraazacyclododecane-l, 4,7-triacetic acid, 10-[(2-f-butoxy-5-aminophenyl)methyl]-, tri-/-butyl ester, sodium bromide complex, pentahydrate. ¹ H nmr (300MHz, CDCl ₃ ) 1.33 (s, 9H), 1.44 (s, 27H), 2.24 (br, m, 6H), 2.58 (br, m, 10H), 3.00 (br, s, 2H), 3.05 (br, s, 4H), 3.72 (s, 2H), 7.02 (d, ³ J _H-H = 8.7Hz, I H), 7.06 (s, IH), 7.88 (d of d, ³ J _H-H = 8.7Hz, V _H = 2.1Hz, IH), 8.01 (d, ⁴ J _H . _H =2.1Hz, IH), 8.52 (s, IH), 8.57 (s, IH), 10.59 (br, s, IH). ¹³ C nmr (75.45MHz, CDCl ₃ ) 28.24, 28.34, 29.64, 50.9(br), 56.13, 56.70, 79.92, 82.84, 83.00, 117.92, 122.30, 122.42, 126.10, 128.32, 130.02, 132.57, 137.26, 147.82, 151.59, 172.41, 173.1 1.

[00163] Synthesis of 1.4,7.10-Tetraazacyclododecane-K4,7-triacetic acid, 10-IY2-f-butoxy-5- (triazolylcarbonylamincOphenvDmethyl ^' j-, tri-f-butyl ester (6)

[00164] Carbonyldi-l ,2,4-triazole, 0.14g 0.0009mole, was dissolved in dichloromethane, 1OmL. To this was added, dropwise, a dichloromethane, 5mL, solution of 1,4,7,10- tetraazacyclododecane-l,4,7-triacetic acid, 10-[(2-f-butoxy-5-aminophenyl)methyl]-, tri-r-butyl ester, sodium bromide complex, pentahydrate, 0.5Og O.OOOβmole. The mixture was allowed to stir for two hours and diethyl ether, 5OmL, was added to precipitate the product. Yield 0.3 g 60% based on 1 ,4,7,10- tetraazacyclododecane-l ,4,7-triacetic acid, 10-[(2~f-butoxy-5-aminophenyl)methyl]~, tri-f-butyl ester, sodium bromide complex, pentahydrate. ¹ H nmr (300MHz, CDCl ₃ ) 1.38 (s, 9H), 1.44 (s, 27H), 2.19 (br, m- 4H), 2.38 (br, m, 4H), 2.80 (br, m, 8H), 3.00 (s, 6H), 3.57 (s, 2H), 7.12 (d, ³ J _H-H = 8.1 Hz, IH), 7.72 (d of d, ³ J _H -H = 8.7Hz, ⁴ J _H-H = 2.7Hz, IH), 7.84 ( ⁴ J _H-H = 2.7Hz, IH), 7.91 (s, IH), 8.87 (s, IH). ¹³ C nmr (75.45MHz, CDCI ₃ ) 28.23, 28.44, 29.54, 49.9(br), 56.01, 56.44, 80.39, 82.56, 83.07, 119.75, 122.96, 123.01, 131.03, 132.23, 143.57, 144.71, 152.30, 152.70, 172.66, 173.72.

[00165] 1,4, 7,10-Tetraazacyclododecane-1,4, 7-triacetic acid, 10-[(2~t-butoxy-5-(N- {CNCbl-5'-f(13-amino)-4,7,10-trioxa-tridecanecarbamateϊ)}-c arbonvlamino)phenvl)methylI (9)

CNCbl-5'-[(13-amino)-4,7,10-trioxa-tridecaπecarbamate]), J7

1,4,7,10-Tetraa2acyclododecane-1,4,7-triacetic acid, 10-[(2-t-butoxy-5-(N-{CNCbl-5'-[(13-amino)-4,7,10-Uioxa- tridecanecarbamateDJ-carbonylaminoiphenyOmethyQ-, tri-t-butyl ester, 8

1,4,7,10-Tetraazacyclododecaπe-1,4,7-trlacetlc acid, 10-[(2-t- butoxy-S-fN-CCNCbl-S'-KI 3-amino)-4,7,10-trioxa- tridecanecarbamate])}-carbonylamino)phenyl)methyl], 9

[00166] Synthesis of 1.4,7.10-Tetraazacyclododecane-1.4.7-triacetic acid, 10-[Y2-t-butoxy-5- (N-(CNCbt-5'-r(l 3-aminoV4,7, 10-trioxa-tridecanecarbamateDI-carbonylamino^phenvπmethyll- , tri-t- bυtyl ester (8)

[00167] CNCb]-5'-[(13-amino)-4,7,10-trioxa-tridecanecarbamate] was dissolved in dry dimethylsulfoxide, I . OmL, under argon. To this was added l ,4,7,10-tetraazacyclododecane-l,4,7- triacetic acid, 10-[(2-?-butoxy-5-(triazolylcarbonylamino)phenyl)methyl]-, tri-/-butyl ester, 0.1 Og 0.000 lmole. The mixture was allowed to until HPLC showed the reaction was complete. The mixture was precipitated using dichloromethane, 5mL, and collected by filtration. The crude solid was washed with fresh dichloromethane, 25mL, diethyl ether, 25mL, and suctioned dry. The solid was dissolved in methanol and purified by C-18 column chromatography. Pure fractions were combined, concentrated under reduced pressure and the product isolated by precipitation with acetone. Yield 0.12g 41% based on starting l,4,7,10-tetraazacyclododecane-l,4,7~triacetic acid, 10-[(2-t-butoxy-5-(N-{CNCb!-5'-[(13- amino)-4,7,10-trioxa-tridecanecarbamate])}-carbonylamino)phe nyl)methyl]-, tri-^-butyl ester. The product was characterized by HPLC, single eluting peak at 15min, and mass spectrometry (M+3H) ³⁺ =774.1 theory = 774.1.

[00168] Synthesis of 1.4.7.10-Tetraazacyclododecane-1.4.7-triacetic acid, 10-f(2-hvdroxy-5- rN-{CNCbl-5'-[C13-amino ^' )-4,7,10-trioxa-tridecanecarbamate1) ^' )-carbonylamino ^' )phenyl')methyl1- (9)

[00169] l,4,7,10-Tetraazacyclododecane-l,4,7-triacetic acid, 10-[(2-t-butoxy-5-(N-{CNCbl- 5'-[(13-amino)-4,7,10-trioxa-tridecanecarbamate])}-carbonyla mino)phenyl)methyl]-, tri-Nbutyl ester, 0.003g 1.3μmoles, was dissolved in trifluoroethanol, 1.OmL. To this was added triflic acid, 2.3μL, and the mixture stirred for lOminutes. HPLC showed a complete disappearance of the starting material,

replaced by a single peak eluting at lO.δminutes. The mixture was evaporated and redissolved in water and evaporated several times. Yield 2.4mg 89% based on starting 1, 4,7,10-tetraazacycIododecane- 1,4,7- triacetic acid, 10-[(2-t-butoxy-5-(N-{CNCbi-5'-[(13-amino)-4 _: ,7,10-trioxa-tridecanecarbamate])}- carbonylamino)phenyl)methyl]-, tri-?-butyl ester. Mass spectrometry shows (M+2H) ²⁺ = 1048.4 Theory = 1048.6.

[00170] Synthesis of 1 A7.10-Tetraazacyclododecane-1.4.7-triacetic acid. 10-|Y2-hydroxy-5- OJ-(N ^E -lvsf3ybombesin(l-14 ^' )l-carbonylamino)phenvDmethyπ (11)

11

[00171] Synthesis of 1,4.7.10-Tetraazacvciododecane-1.4.7-triacetic acid. 10-f (2-hydroxy-5- rN-IN^lysfSVbombesinCl-MH-carbonylaminofahenvDmethvn, tri-/-butyl ester (10)

[00172] l,4,7,10-Tetraazacyclododecane-l,4,7-triacetic acid, 10-[(2-f-butoxy-5- (imidazolylcarbonylamino)phenyl)methyl]-, tri-r-butyl ester, 3.0mg 3.8millimoles, was dissolved in anhydrous DMSO. To this was added lys(3)-bombesin(l -14), 5mg 3.1millimoles. The mixture was stirred for four hours. HPLC of an aliquot revealed the reaction was not complete. An additional aliquot

of 1 ,4,7, 10-tetraazacyclododecane-l,4,7-triacetic acid, 10-[(2-?-butoxy-5-

(imidazolylcarbonylamino)phenyl)methyl]-, tri-f-butyl ester, 1.5mg 1.9millimoles, was added and the mixture was allowed to stir overnight. The crude product was isolated by precipitation with diethyl ether and purified by reverse phase chromatography. Yield 3.0mg 41 % based on starting lys(3)-bombesin(l- 14). LCMS shows (M+2H) ²⁺ = 1 155.6 (Theory = 1 155.1).

[00173] Synthesis of l,4,7.10-Tetraazacvclododecane-1.4,7-triacetic acid, 10-fY2-hydroxy-5- (N-(N ^ε -lys(3')-bombesin('l-14')>-carbonylamino)phenvnmethvπ (11)

[00174] A sample of 1,4,7, 10-tetraazacyclododecane-l ,4,7-triacetic acid, 10-[(2-hydroxy-5- (N-{N ^ε -lys(3)-bombesin(l-14)}-carbonylamino)phenyl)methyl], tri-f-butyl ester, 3.0mg 0.0013millimole, was suspended in deionized water, 0.0 I mL. To this was added trifluoroacetic acid, 0.5mL ₅ and the mixture was allowed to stir overnight. The solvent was evaporated and the residue treated with fresh water and evaporated several times. The residue was purified by reverse phase HPLC, 5μ C 18. Yield 0.00Ig 37% based on starting 1,4,7, 10-tetraazacyclododecane-l ,4,7-triacetic acid, 10-[(2-hydroxy-5-(N- {N ^e -iys(3)-bombesin(l-14)}-carbonylamino)phenyl)methyl], tri-f-butyl ester. LCMS shows (M+2H) ²⁺ = 1043.3 (Theory = 1043.0).