Radioactive halogen prosthetic moieties and radiolabeled biomolecules

Abstract

This application relates to radioactive halogen prosthetic moieties and precursors thereof, as well as radiolabeled biomolecules containing such radioactive halogen prosthetic moieties. The biomolecules have affinity for specific cell types and can specifically bind certain cells, such as cancer cells. Related biomolecules include antibodies, monoclonal antibodies, antibody fragments, peptides, other proteins, nanoparticles, aptamers, and pharmacological moieties used to target prostate-specific membrane antigen (PSMA).

Description

[Technical Field]

[0001] FIELD OF THE INVENTION The present invention relates to radioactive halogen prosthetic moieties useful for radiolabeling biomolecules, as well as methods for preparing such radioactive halogen prosthetic moieties and radiolabeled biomolecules. The present disclosure also provides precursors to such radioactive halogen prosthetic moieties. Radioactive halogen prosthetic moieties can effectively retain radioactivity from biomolecules that are internalized within cells, making such moieties and corresponding radiolabeled biomolecules useful for the diagnosis and treatment of diseases, particularly cancer. [Background technology]

[0002] background Radioactive iodine labeling is one of the simplest methods for radiolabeling biomolecules. Several radioisotopes of iodine are available for cancer imaging and targeted radiotherapy. Radioisotopes of iodine are supplied as alkaline solutions, in which iodine is in its -1 oxidation state (I -iodide). Standard methods for radioiodine labeling of biomolecules require the oxidation of iodine to the +1 oxidation state for electrophilic substitution at tyrosine amino acids present in biomolecules such as antibodies, other proteins, and peptides. Challenges with radioiodine-labeled monoclonal antibodies (mAbs) and peptides include their in vivo instability to intracellular proteolysis after internalization, deiodination, and loss of radioactivity from tumor cells as a result of both processes. It is widely recognized that after internalization (which can occur as a result of receptor and binding to certain antigens), radioiodine-labeled antibodies and peptides are proteolytically degraded inside the cell to radioiodotyrosine, which is efficiently transported out of the cell by membrane amino acid transporters. The released radioiodotyrosine is deiodinated by deiodinase enzymes found in tissues, and the free radioiodine redistributes and accumulates in organs with expression of the sodium iodide symporter, particularly the thyroid, stomach, and salivary glands. Thus, the amount of radiolabel retained in the tumor is reduced, while the uptake of radioactivity in normal tissues is increased.

[0003] One of the disadvantages of antibodies is their long half-life in the bloodstream, which leads to high background levels after systemic administration and, as a result, low tumor-to-background ratios. Furthermore, conventional antibodies diffuse somewhat slowly into solid tumors, which prevents the antibody from reaching and binding to receptors / antigens uniformly throughout the tumor mass.

[0004] Although some prosthetic agents have been identified in the art, they are unstable and difficult to manufacture in commercial quantities.In addition, the uptake of antibody into tumor cells, especially brain metastases, is low due to the size of antibody, which is particularly problematic for brain tumors due to the delivery restriction imposed by the blood-brain barrier.Therefore, there is a need for additional prosthetic agents that can be used for radiolabeling biomolecules for targeted radiotherapy and imaging applications. Summary of the Invention [Means for solving the problem]

[0005] Summary of the Invention The present invention relates to prosthetic agents, precursors thereof, and compositions for radiolabeling biological molecules (also called macromolecules) with radioactive halogen atoms, particularly radioactive iodine. Advantageously, such methods, compounds, and compositions minimize the loss of radioactive halogen due to dehalogenation in vivo after in vivo administration, maintain the biological activity of the biological molecule, maximize retention in diseased cells, such as cancer cells, and / or minimize retention of radioactivity in normal tissues.

[0006] Biomolecules have affinity for specific cell types. That is, biomolecules can specifically bind certain cells, such as cancer cells. Certain compositions of the present invention include radiolabeled biomolecules. Such biomolecules include antibodies, monoclonal antibodies, antibody fragments, peptides, other proteins, nanoparticles, and aptamers. Examples of biomolecules for purposes of the present invention include diabodies, scFv fragments, DARPins, fibronectin type III-based scaffolds, affibodies, VHH molecules (also known as single-domain antibody fragments (sdAbs) and nanobodies), nucleic acid or protein aptamers, and nanoparticles. In addition, larger molecules such as proteins greater than 50 kDa, including antibodies, monoclonal antibodies, chimeric antibodies, humanized antibodies, and F(ab')2 fragments, can be used in the methods disclosed herein. Furthermore, nanoparticles less than 50 nm in size can be used in the methods disclosed herein. In some embodiments, the principles disclosed herein are particularly relevant to, but not limited to, VHH molecules and other types of small protein constructs, as more fully described herein.

[0007] As such, the present disclosure provides such radiolabeling moieties (herein referred to as "radioactive halogen prosthetic moieties"), and precursors for providing such prosthetic moieties (herein referred to as "radioactive halogen precursors"). The present disclosure further provides radiolabeled macromolecules (e.g., biomolecules) comprising such radioactive halogen prosthetic moieties and one or more macromolecules. In some such embodiments, these radiolabeled macromolecules are targeted radiotherapeutic agents. The prosthetic moieties and radiolabeled macromolecules of the present invention are useful, for example, for diagnosing disease and for targeted radiotherapy.

[0008] The present disclosure includes, without limitation, the following embodiments.

[0009] Embodiment 1: A compound of the formula: [ka] wherein MC is a multidentate chelating moiety; R is H, an ester, or a carboxylic acid; A is -R-R-Y or Y; B is H, an alkoxy group, or a non-radioactive halogen; R is a direct bond, an alkyl group, or an oxygen-containing moiety (e.g., -O-, -O-CH-, -O-CHCH-, etc.); R is a direct bond or an aromatic moiety; Y is a radioactive halogen or a moiety that can be converted to a radioactive halogen (also referred to herein as a "precursor" to a radioactive halogen); MMCM is a polymeric conjugation moiety; and L is a direct bond or a linker. by a radioactive halogen prosthetic moiety or its precursor.

[0010] Embodiment 2: The radiohalogen prosthetic moiety or precursor of the preceding embodiment, wherein MC is a cyclic multidentate chelating moiety.

[0011] Embodiment 3: The radiohalogen prosthetic moiety or precursor of embodiment 1, wherein MC is an acyclic multidentate chelating moiety.

[0012] Embodiment 4: The radiohalogen prosthetic moiety or precursor of embodiment 1, wherein MC is a modified DOTA moiety.

[0013] Embodiment 5: The radiohalogen prosthetic moiety or precursor of the preceding embodiment, wherein MC is DOTA-tris(t-Bu ester) or DOTA-tris(COOH).

[0014] Embodiment 6: The radiohalogen prosthetic moiety or precursor of embodiment 1, wherein MC is a modified NOTA moiety.

[0015] Embodiment 7: The radiohalogen prosthetic moiety or precursor of the preceding embodiment, wherein MC is NOTA-bis(t-bu ester) or NOTA-bis(COOH).

[0016] Embodiment 8: The radioactive halogen prosthetic moiety or precursor of any preceding embodiment, wherein MC comprises a metal.

[0017] Embodiment 9: The metal is a mixture of non-radioactive metals (e.g., including lutetium, yttrium, indium, or gallium) and radioactive metals (e.g., 177 Lu, 64 Cu, 67 Cu, 111 In, 90 Y, 225 Ac, 213 Bi, 212 Pb, 212 Bi, 67 Ga, 68 Ga, 89 Zr and 227 The radioactive halogen prosthetic moiety or precursor of the preceding embodiment is selected from the group consisting of:

[0018] Embodiment 10: The radioactive halogen prosthetic moiety or precursor of any of embodiments 1-7, wherein MC does not comprise a metal.

[0019] Embodiment 11: The radiohalogen prosthetic moiety or precursor of any preceding embodiment, wherein M C is attached through a nitrogen atom present thereon, or M C is attached through a backbone carbon.

[0020] Embodiment 12: A compound of the formula: [ka] The radioactive halogen prosthetic moiety or precursor of embodiment 1, represented by:

[0021] Embodiment 13: A compound of the formula: [ka] The radioactive halogen prosthetic moiety or precursor of embodiment 1, represented by:

[0022] Embodiment 14: A compound of the formula: [ka] The radioactive halogen prosthetic moiety or precursor of embodiment 1, represented by:

[0023] Embodiment 15: A compound of the formula: [ka] The radioactive halogen prosthetic moiety or precursor of embodiment 1, represented by:

[0024] Embodiment 16: A compound of the formula: [ka] The radioactive halogen prosthetic moiety or precursor of embodiment 1, represented by:

[0025] Embodiment 17: A compound of the formula: [ka] The radioactive halogen prosthetic moiety or precursor of embodiment 1, represented by:

[0026] Embodiment 18: A compound of the formula: [ka] The radioactive halogen prosthetic moiety or precursor of embodiment 1, represented by:

[0027] Embodiment 19: A compound of the formula: [ka] The radioactive halogen prosthetic moiety or precursor of embodiment 1, represented by:

[0028] Embodiment 20: A compound of the formula: [ka] The radioactive halogen prosthetic moiety or precursor of embodiment 1, represented by:

[0029] Embodiment 21: A compound of the formula: [ka] The radioactive halogen prosthetic moiety or precursor of embodiment 1, represented by:

[0030] Embodiment 22: Y is 18 F, 75 Br, 76 Br, 77 Br, 123 I, 124 I, 125 I, 131 I and 211 The radioactive halogen prosthetic moiety or precursor of any preceding embodiment is selected from the group consisting of At.

[0031] Embodiment 23: The radioactive halogen prosthetic moiety or precursor of any of Embodiments 1-22, wherein Y is selected from the group consisting of a trialkyltin moiety, a trialkylsilicon moiety, a trialkylgermanium moiety, a HgX moiety (X = halogen, such as CH3COO, CF3COO, etc.), a Tl(OCOCF3)2 moiety, boric acid (B(OH)2), Bpin (pivaloylboronate), an aryl iodonium salt, and an iodonium ylide (including, for example, a diazonium salt or a triazene), among others.

[0032] Embodiment 24: The radiohalogen prosthetic moiety or precursor of any of embodiments 12-23, wherein the metal is not complexed within the multidentate chelating moiety.

[0033] Embodiment 25: The radiohalogen prosthetic moiety or precursor of any of Embodiments 12-23, further comprising a metal complexed within the multidentate chelating moiety.

[0034] Embodiment 26: The metal is a mixture of non-radioactive metals (e.g., including lutetium, yttrium, indium, or gallium) and radioactive metals (e.g., 177 Lu, 64 Cu, 67 Cu, 111 In, 90 Y, 225 Ac, 213 Bi, 212 Pb, 212 Bi, 67 Ga, 68 Ga, 89 Zr and 227 24. The radioactive halogen prosthetic moiety or precursor of any of embodiments 12-23, selected from the group consisting of:

[0035] Embodiment 27: CO2 as shown t The CO2 shown includes the case where all of the Bu groups are deprotected. t The radioactive halogen prosthetic moiety or precursor of any one of the preceding embodiments, wherein one or more of the Bu groups are deprotected (i.e., replaced with a COOH group).

[0036] Embodiment 28: A radiolabeled biomolecule or intermediate comprising the radioactive halogen prosthetic moiety or precursor of any preceding embodiment attached to the biomolecule.

[0037] Embodiment 29: The radiolabeled biomolecule or intermediate of the preceding embodiment, wherein the biomolecule is selected from the group consisting of an antibody, an antibody fragment, a VHH molecule, an aptamer or a variant thereof.

[0038] Embodiment 30: The radiolabeled biomolecule or intermediate of any preceding embodiment, wherein the biomolecule is a VHH.

[0039] Embodiment 31: The radiolabeled biomolecule or intermediate of the preceding embodiment, wherein said VHH targets HER2.

[0040] Embodiment 32: The radiolabeled biomolecule or intermediate of embodiment 28, wherein the biomolecule comprises a carbamate- or urea-containing pharmacological moiety.

[0041] Embodiment 33: The radiolabeled biomolecule or intermediate of the preceding embodiments, wherein the carbamate- or urea-containing pharmacological moiety is a moiety used to target PSMA in prostate cancer.

[0042] It should be noted that in the specific structures shown in Examples 12-21, the type of chelating moiety shown is not intended to be limiting, and that, for example, cases where the radioactive halogen prosthetic moiety or precursor is shown to include an N-hydroxysuccinimide (NHS) ester, or an analog containing a tetrafluorophenol (TFP) ester, an isothiocyanate group, or a maleimide group (or other chelating moiety) instead of an NHS ester are also encompassed herein. Similarly, cases where the radioactive halogen prosthetic moiety or precursor is shown to include a TFP ester, or an analog containing an NHS ester, an isothiocyanate group, or a maleimide group (or other chelating moiety) instead of a TFP ester are also encompassed herein.

[0043] It is further noted that in certain embodiments, aromatic / aryl rings are incorporated within the disclosed moieties. The term "aryl," as used herein, means a stable monocyclic, bicyclic, or tricyclic carbon ring, each ring being up to eight members, with at least one ring being aromatic, as defined by the Huckel 4n+2 rule. In the embodiments provided herein, the aromatic / aryl rings are generally designated as homoaromatic / homoaryl; however, the present disclosure is intended to encompass heteroaromatic / heteroaryl rings as well. Thus, in some embodiments, one or more (e.g., 1 to 4) of the carbon atoms designated in the aromatic / aryl ring of the formulas provided herein may be replaced with a heteroatom selected from O, S, and N. In some preferred embodiments, such rings may contain one heteroatom that is N. Exemplary aryl groups include, but are not limited to, phenyl, naphthyl, tetrahydronaphthyl, and biphenyl, and exemplary aromatic groups include, but are not limited to, benzene, indole, furan, pyridine, and pyrazine, as well as substituted derivatives thereof.

[0044] These and other features, aspects, and advantages of the present disclosure will become apparent upon reading the following detailed description. The present invention includes any combination of two, three, four, or more of the above-described embodiments, and any combination of two, three, four, or more features or elements set forth in this disclosure, regardless of whether such features or elements are explicitly combined in specific embodiments described herein. The present disclosure is intended to be read as a whole, and therefore, in any of its various aspects and embodiments, any separable features or elements of the disclosed invention should be considered as intended to be combinable unless the context clearly dictates otherwise. Other aspects and advantages of the present invention will become apparent from the following. DETAILED DESCRIPTION OF THE INVENTION

[0045] Detailed Description The present disclosure generally provides certain radioactive halogen prosthetic moieties, precursors thereof, and radiolabeled macromolecules containing the radioactive halogen prosthetic moieties. Certain associated chemical and biological components of these moieties, precursors, and radiolabeled macromolecules are disclosed in U.S. Patent No. 9,839,704 to Zalutsky et al., which is incorporated herein by reference in its entirety.

[0046] For example, as seen in International Patent Application Publication No. WO2018 / 178936, which is incorporated herein by reference in its entirety, certain exemplary radiohalogen precursors and radiohalogen prosthetic moieties have the following formula: MC-Cm-L4-Cm-T (Formula X) wherein the substituents are defined as referenced therein (generally, "MC" represents a multidentate metal chelating moiety, "Cm" represents a conjugate moiety such as a thiourea, amide, or thioether, "L4" represents a bond, a substituted or unsubstituted alkyl chain, a substituted or unsubstituted alkenyl chain, a substituted or unsubstituted alkynyl chain optionally having an NH, CO, or S at one or both termini, or a polyethylene glycol (PEG) chain, and "T" represents a radiohalogen prosthetic moiety or precursor thereof). The present application provides certain modifications and highlighted features of various prosthetic moieties and radiolabeled macromolecules containing such moieties, such as those disclosed in the references, and their precursors, as more fully outlined herein below.

[0047] The present disclosure specifically provides radiohalogen precursor moieties and radiohalogen prosthetic moieties that are somewhat similar in structure to that of Formula X, but do not include the Cm-L4-Cm linker functional group. In accordance with the present disclosure, radiohalogen precursor moieties and radiohalogen prosthetic moieties are provided in a simplified structure (shown below) referred to herein as Formula A. MC-T (formula A)

[0048] MC is a polydentate metal chelating moiety. MC can be any polydentate chelating moiety and can be cyclic or acyclic. The composition of MC can vary. MC can be uncomplexed (lacking the metal) or complexed with a stable (non-radioactive) or radioactive form of the metal. In some embodiments, the metal is a metal of any charge, with +2, +3, or +4 charges being preferred. In certain embodiments, the metal is a trivalent metal (M), such as lutetium, yttrium, indium, actinium, or gallium. +3 ) and in certain embodiments, specific radiometals that can be complexed with MC include, but are not limited to: 177 Lu, 64 Cu, 67 Cu 111 In, 90 Y, 225 Ac, 213 Bi, 212 Pb, 212 Bi, 67 Ga, 68 Ga, 89 Zr and 227 The radioactive metals include those selected from the group consisting of Th, Th, and Th. This list is not exhaustive, and although these exemplified radioactive and non-radioactive metals are trivalent, it should be noted that certain MCs that can be used in accordance with the present invention can bind metals of other valencies, and such metals and MCs containing such metals are also encompassed herein.

[0049] In some embodiments, MC is a macrocyclic ligand consisting of a ring containing 8 or more atoms and having at least three negatively charged substituents, such as carboxyl or phosphonate groups. Exemplary macrocyclic ligands suitable as MC groups include 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA) and 1,4,7-triazacyclononane-1,4,7-tri(methylenephosphonic acid) (NOTP). In other embodiments, MC is MeO-DOTA, as disclosed in Gali et al., Anticancer Research (2001), 21(4A), 2785-2792, which is incorporated herein by reference.

[0050] The present disclosure particularly recognizes the advantages of using functionalized MC groups, such as modified DOTA or modified NOTA. One exemplary modified DOTA is DOTA-tris(t-Bu ester), i.e., DOTA containing a tert-butyl acetate group associated with each nitrogen atom other than the nitrogen atom used for connecting to "T." Such a compound suitable for the reaction to yield MC-T is also referred to as tri-tert-butyl 1,4,7,10-tetraazacyclododecane-1,4,7-tricarboxylate. Removal of the tert-butyl protecting group results in the deprotected form, which may be referred to as 1,4,7,10-tetraazacyclododecane-1,4,7-tricarboxylic acid. Additional modified DOTAs that may find use in certain embodiments of the present disclosure are acyclic analogs, such as acyclic analogs of DOTA or acyclic analogs of modified DOTA. One exemplary tert-butyl acetate-functionalized acyclic DOTA that can react to form MC-T is tert-butyl (2-((2-((2-aminoethyl)(tert-butoxycarbonyl)amino)ethyl)(tert-butoxycarbonyl)amino)ethyl)glycinate. Removal of the tert-butyl protecting group results in a deprotected form, which can be referred to as (2-((2-((2-aminoethyl)(carboxy)amino)ethyl)(carboxy)amino)ethyl)glycine. Similarly, in some embodiments, a modified analog of NOTA is used that contains a tert-butyl acetate group associated with each nitrogen atom other than the nitrogen atom used for connecting to "T." Such a modified NOTA can be di-tert-butyl 1,4,7-triazonane-1,4-dicarboxylate, which can react through the nitrogen atom not attached to the tert-butyl group to provide MC-T.

[0051] Without intending to be limited by theory, it is believed that such acetate substituents (or acetate substituents upon deprotection of modified DOTA / NOTA MC groups) provide some degree of stabilization of the DOTA / NOTA (or analog) chelating moiety. In all figures herein that provide tert-butyl acetate groups in association with MC groups, they are shown by t-butyl, but it is understood that the deprotected form (containing an acetate group in place of the tert-butyl acetate group) is also intended to be encompassed herein.

[0052] Certain such modified "MC" groups are shown below in Formulas B-1, B-2, and B-3, respectively, with one possible point of attachment to "T" shown on each shown "MC" moiety, to provide related radiohalogen precursor moieties and radiohalogen prosthetic moieties, and radiolabeled biomolecules containing them. [ka] [ka]

[0053] According to the present disclosure, MC is connected to the remainder of the prosthetic / precursor moiety (including "T") through any relevant atom, for example (but not limited to) through a carbon or nitrogen associated with MC, to provide the corresponding radiohalogen precursor and prosthetic moiety. For the modified DOTA and NOTA groups referred to herein, MC may advantageously be connected to T through a nitrogen present on the modified DOTA / NOTA (as shown in Formulas B-1, B-2, and B-3 above) or through other positions on MC (including, for example, through one of the MC backbone carbons).

[0054] T is a radiohalogenated template or a radiohalogen precursor template. T can be, for example, a compound of formula C shown below (a compound containing MMCM, as described in more detail below), and its connection to MC is indicated by a wavy line. Typically, in the disclosed structures, T is directly attached to MC, i.e., by a direct bond between the carbon atom shown adjacent to R and a moiety on MC (e.g., MC is DOTA / NOTA or modified DOTA / NOTA, and the carbon atom shown below can be directly attached to a nitrogen atom of the backbone DOTA / NOTA structure). [ka]

[0055] In formula C, the following definitions are applicable: R1 = H, ester or carboxylic acid. A is R2-R3-Y or Y. B is H, an alkoxy (eg, methoxy, ethoxy, propoxy, or butoxy) group, or a halogen (eg, a non-radioactive halogen such as selected from the group consisting of Cl, F, I, and Br). R2 = direct bond, alkyl group, or oxygen-containing moiety. Certain examples of "oxygen-containing moieties" as provided herein include, but are not limited to, -O-, -O-CH2-, -O-CH2CH2-, and the like. R3 = direct bond, alkyl group or aromatic moiety (e.g., phenyl ring). Y = a) radioactive halogen (e.g., 18 F, 75 Br, 76 Br, 77 Br, 123 I, 124 I, 125 I, 131 I and 211At (wherein the designated moiety is a radioactive halogen prosthetic moiety); or a) a precursor of a radioactive halogen (wherein the designated moiety is a precursor suitable for radiohalogenation, also referred to herein as a "radioactive halogen precursor moiety"). Precursors suitable for radiohalogenation can vary, including, for example, alkylmetal moieties containing three C alkyl group ligands on the metal (e.g., trialkyltin moieties, e.g., BuSn or MeSn, trialkylsilicon moieties, and trialkylgermanium moieties). Other suitable precursors for radiohalogenation include, but are not limited to, HgX moieties (X = halogen, e.g., CHCOO, CFCOO, etc.), Tl(OCOCF) moieties, boronic acids (B(OH)), Bpin (pivaloylboronate), aryl iodonium salts, iodonium ylides, diazonium salts, triazenes, etc., which can be further processed to yield the radioactive halogen prosthetic moiety. MMCM = polymeric conjugation moiety that couples the compound / radical of formula C to a polymer. L1 = direct bond or linker.

[0056] In certain embodiments, R2 is -O-CH2- and R3 is a phenyl ring, such that R2-R3 are benzyloxy groups attached to the central phenyl ring of formula C. The positions of the substituents on the phenyl ring relative to each other can vary. The L1-MMCM on the aromatic ring relative to the other substituents (connections to A, B, and MC) can vary. In certain embodiments, the L1-MMCM group is meta to the connection to MC. In some embodiments, the central phenyl ring of the compound does not contain a substituent ortho to L1-MMCM (such that the carbon to which L1-MMCM is attached is ortho to two unsubstituted carbon atoms). In some preferred embodiments, A is ortho to the carbon attached to MC. When A is ortho to the carbon attached to MC, in some such embodiments, B is ortho to the A substituent and is H. In other preferred embodiments, A is meta to the carbon attached to MC. When A is meta to the carbon attached to MC, in some such embodiments, B is ortho to the carbon attached to MC (and meta to the A substituent), and in certain embodiments, B is alkoxy (e.g., methoxy).

[0057] MMCM can vary and can be any component suitable for attachment to a biomolecule. In some embodiments, the MMCM is an activated ester. An activated ester is defined herein as an ester that can conjugate with an amine group present on a polymer / biomolecule (e.g., a peptide or protein) under mild conditions, i.e., conditions that do not result in loss of the biological function of the polymer / biomolecule. Exemplary such MMCM groups include, but are not limited to, N-hydroxysuccinimide (NHS) esters or tetrafluorophenol (TFP) esters, isothiocyanate groups, or maleimide groups. Such MMCMs generally result in random (non-site-specific) labeling of amine groups on proteins or peptides. In other embodiments, the MMCM results in site-specific conjugation, performed using an enzyme such as sortase, which results in conjugation to only one site on the protein (either the N-terminus or C-terminus of the protein). In the case of sortase, the MMCM is, for example, the tripeptide GlyGlyGly. Enzymes that can be used to achieve suitable conjugation include, but are not limited to, transglutaminases, lipoic acid ligases, farnesyltransferases, and many others, such as those disclosed in Massa et al., Exp. Opin. Drug Del. 2016, 13(8), pp. 1149-1163; Zhang et al., Chem. Soc. Rev. 2018, 47, pp. 9106-9136; Falck et al., Antibodies 2018, 7(4), pp. 1-19; and van Berkel et al., Drug Disc. Today: Technologies 2018, 30, pp. 3-10, all of which are incorporated by reference in their entireties.

[0058] MMCM can be directly attached to the aromatic ring (L1 = direct bond) or can be attached to the aromatic ring via a linker (L1), which can be, for example, a substituted or unsubstituted alkyl chain, a substituted or unsubstituted alkenyl chain, a substituted or unsubstituted alkynyl chain, or a spacer such as a short polyethylene glycol (PEG) chain (1-10 ethylene glycol units).

[0059] In accordance with the present disclosure, certain structural features associated with such radiohalogen precursors and prosthetic moieties have been identified as providing various advantageous functions, as outlined more thoroughly herein below. Provided herein are radiohalogen precursors and prosthetic moieties, and radiolabeled biomolecules, comprising various combinations of these identified structural features.

[0060] In one embodiment, R1 is advantageously an ester group, such as CO2, as shown in formula C-1 below: t Bu. In such embodiments, this R group between the aromatic ring on "T" and the "MC" group may provide a free COOH at this site, especially after deprotection, to provide further stabilization of the metal complex. [ka]

[0061] In one embodiment, the central aromatic ring of T is functionalized with an alkoxy group, particularly a methoxy (OCH3) group, as the substituent B. This alkoxy group can be present at various positions on the phenyl ring, but the alkoxy group is advantageously located on the carbon ortho to the carbon to which the CHR1-MC group is attached, so that the alkoxy is relatively close to the MC moiety. Surprisingly, it has been found that this group can provide additional stabilization of the metal complex (e.g., modified DOTA or NOTA group), especially upon deprotection of the metal complex ester substituent. Advantageously, such compounds containing an alkoxy group on the phenyl ring have four substituents on the aromatic ring. See, for example, Formula C-2 below. [ka]

[0062] In some embodiments, the halogenation site or precursor moiety is located on an aromatic ring that is bonded (directly or indirectly) to the central aryl ring (for example, including, but not limited to, being connected to the central aryl ring via an O-alkyl-moiety), as shown in Formula C-3. The location of the halogen or trialkylmetal moiety on the phenyl ring can vary, but in certain embodiments, the halogen or trialkylmetal moiety is para to its connection to the remainder of the molecule. In some such embodiments, B is H, as shown in Formula C-3. In another embodiment, the halogen site or precursor moiety is on an aromatic ring that is connected to the central aryl ring, and the aromatic ring is directly bonded to the central aryl ring, as shown in Formula C-4. In some such embodiments, B is H, as shown in Formula C-4.

[0063] It should be noted that in embodiments where the halogenation site or precursor moiety is on an aromatic ring bonded to a central aryl ring, the aromatic ring may contain no other substituents, or in some embodiments, may contain one or more additional substituents at various positions relative to the halogenation site or precursor moiety. For example, in some embodiments, the aromatic ring further comprises guanidinomethyl. Thus, for example, formulas C-3 and C-4 below are shown with an aromatic ring substituted only with I, but the disclosure is not limited thereto, and other substituents may be present on the ring in various embodiments. In one particular embodiment, a moiety of formula C-3 is provided in which the aryl ring is substituted with both I and a guanidinomethyl group. [ka]

[0064] In some embodiments, the halogenation site / precursor moiety is directly on the central aryl ring (e.g., connected via a direct bond), as clearly shown in formulas C-5 and C-6. In some such embodiments, B is H, as shown in formula C-5. In formula C-5, the iodination site is proximal to the chelating ester, and these types of structures may advantageously allow for weak charge stabilization to the chelating moiety. In some such embodiments, B is advantageously OCH3, as shown in formula C-6. [ka] [ka]

[0065] In one specific embodiment, a DOTA-SIB radiohalogen prosthetic moiety according to formula D-1 below is provided. [ka]

[0066] As shown, this radiohalogen prosthetic moiety of Formula D-1 contains a single carbon between the functionalized phenyl ring of T and the nitrogen on the modified DOTA chelating moiety (MC). In Formula D-1, CO2 is present on the carbon between the functionalized phenyl ring of T and the nitrogen on the modified DOTA chelating moiety. t The Bu group provides additional charge stabilization of the chelating complex (especially resulting in a free COOH group on the modified DOTA after deprotection). Additionally, the methoxy substituent on the central aryl ring provides additional charge stabilization for the modified DOTA chelating moiety. In the radiohalogen prosthetic moiety of formula D-1, the halogenation site is directly on the central aryl (benzene) ring.

[0067] Formula D-2 provides a similar NOTA-SIB hybrid radiohalogen prosthetic moiety. [ka]

[0068] In another specific embodiment, a DOTA-SIB radiohalogen prosthetic moiety according to formula D-3 below is provided. [ka]

[0069] As shown, this radiohalogen prosthetic moiety of formula D-3 contains a single carbon between the functionalized phenyl ring of T and the nitrogen on the modified DOTA chelating moiety (MC). In formula D-3, CO2 is present on the carbon between the functionalized phenyl ring of T and the nitrogen on the modified DOTA chelating moiety. t The Bu group provides additional charge stabilization of the chelating complex (especially resulting in a free COOH group on the modified DOTA after deprotection). The O-alkyl subunit immediately adjacent to the central aryl ring provides additional charge stabilization to the chelating moiety MC. The halogen in this formula D-3 structure is located on the aromatic ring substituted on the O-alkyl subunit, rather than on the central aromatic ring.

[0070] Again, as referenced above with respect to general formula C-3, in further embodiments, compounds of formula D-3 may further include one or more additional substituents on the aromatic ring substituted on the O-alkyl subunit. For example, in one embodiment, the aromatic ring substituted on the O-alkyl subunit of formula D-3 includes, in addition to the indicated I, a guanidinomethyl group (which may be ortho or meta to the I substituent on the ring). A specific example is shown below as formula D-3' (wherein X and Y may be located at any of the five positions on the aromatic ring). When X is an iodine radioisotope or another radioactive halogen (or precursor thereof), Y is guanidinomethyl, and vice versa. The N-hydroxysuccinimidyl ester may be replaced with other groups, such as a TFP ester group, as referenced throughout. [ka]

[0071] In a further specific embodiment, a DOTA-SIB radiohalogen prosthetic moiety according to formula D-4 below is provided. [ka]

[0072] As shown, this radiohalogen prosthetic moiety of formula D-4 contains a single carbon between the functionalized phenyl ring of T and the nitrogen on the modified DOTA chelating moiety (MC). In formula D-4, CO2 is present on the carbon between the functionalized phenyl ring of T and the nitrogen on the modified DOTA chelating moiety. t The Bu group provides additional charge stabilization of the chelating complex (especially providing a free COOH group on the functionalized DOTA after deprotection). The halogen in formula D-4 is proximal to the modified DOTA, allowing weak charge stabilization to the metal complex.

[0073] In a further specific embodiment, a DOTA-SIB radiohalogen prosthetic moiety according to formula D-5 below is provided. [ka]

[0074] As shown, this radiohalogen prosthetic moiety of formula D-5 is equivalent in structure to that of formula D-4, but comprises an acyclic modified DOTA (rather than the cyclic form of formula D-4).

[0075] In some specific embodiments, L1-MC is a tetrafluorophenol (TFP) ester, as shown, for example, in Formulas D-6 through D-8 below. Again, Formula D-7 can be modified to include one or more additional substituents on the -O-alkyl-phenyl group (e.g., guanidinomethyl groups) in addition to the exemplified I substituents, for example, as described above with respect to Formula D-3. [ka] [ka]

[0076] As shown in formulas D1-D8, ester-containing R1 substituents, such as CO2 t While the Bu group is generally preferred, other embodiments provide radiohalogen prosthetic moieties or precursors thereof in which R is H, as shown in Formula D-9 below. In some embodiments, such moieties can be prepared in fewer steps than other equivalent compounds (e.g., those containing an ester group at the R position). [ka]

[0077] In addition to the above general and specific formulas D1-D6 (shown as radioactive halogen prosthetic moieties including radioactive iodine), the present disclosure also includes analogs of D1-D6 with other halogens, for example, analogs of D1-D6 in which radioactive iodine (I) is substituted with another radioactive halogen (such as, but not limited to, 18 F), and analogs that serve as precursors to the moieties of formulas D1-D6 (i.e., radiohalogen precursor moieties), such as those where I is replaced with an alkylmetal moiety (including, but not limited to, Bu3Sn, e.g., t It is understood that this includes those replaced with precursor moieties such as methyl methyl silyl ether (BuSn). As referenced above, precursors can include, for example, trialkyltin moieties, trialkylsilicon moieties, trialkylgermanium moieties, alkylmetal moieties such as HgX moieties (X = halogen, CHCOO, CFCOO, etc.) or Tl(OCOCF) moieties, boronic acids (B(OH)), Bpin (pivaloylboronates), aryl iodonium salts and iodonium ylides (including, for example, diazonium salts or triazenes), among others, which can be further processed to provide radioactive halogen prosthetic moieties.

[0078] The present disclosure further provides methods for providing radioactive halogen prosthetic moieties and radioactive (e.g., radioiodine) labeled prosthetic moieties that can be conjugated (via MMCM) to biomolecules, for example, in a site-specific manner (contained within the formulae referenced herein above). The present disclosure provides methods for conjugating the resulting radioactive (e.g., iodine radionuclides, bromine radionuclides, 18 F or 211 Further provided are radiolabeled (At) biomolecules. Related biomolecules include those generally disclosed in International Patent Application Publication No. WO2018 / 178936, the entire contents of which are incorporated herein by reference. Biomolecules can vary widely. In some embodiments, the biomolecule is selected from the group consisting of an antibody, an antibody fragment, a VHH molecule, an aptamer, or a variant thereof. A specific example is a VHH, such as a VHH that targets HER2. In some embodiments, the biomolecule comprises a carbamate- or urea-containing pharmacological moiety (e.g., used to target prostate-specific membrane antigen (PSMA)). Such radiolabeled biomolecules can be provided according to the present disclosure, for example, by replacing the protein / peptide with an appropriate carbamate or urea and using the same labeling template as described for proteins, for example. See, for example, Yang et al., J. Med. Chem. 2016, 59, pp. 206-218; Chen et al., J. Med. Chem. 2008, 51, pp. 7933-7943; and Eiber et al., J. Nucl., Med. 2017, pp. 67S-76S, all of which are incorporated by reference in their entireties.

[0079] The present disclosure further provides a pharmaceutical composition comprising a radiolabeled biomolecule as disclosed herein (e.g., a labeled biomolecule as described / illustrated above) in association with one or more pharmaceutically acceptable adjuvants, diluents and / or carriers. In a further aspect of the present disclosure, a method of treating cancer is provided, comprising administering to an individual in need thereof an effective amount of a radiolabeled biomolecule as disclosed herein and / or an effective amount of a pharmaceutical composition as disclosed herein.

[0080] The following examples are offered by way of illustration and not by way of limitation. [Example]

[0081] Example 1 Preparation of tri-tert-butyl 2,2',2''-(10-(2-(tert-butoxy)-1-(5-(((2,5-dioxopyrrolidin-1-yl)oxy)carbonyl)-2-methoxy-3-(tributylstannyl)phenyl)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetate

[0082] a. Benzyl 4-(benzyloxy)-3-methylbenzoate [ka]

[0083] To a round-bottom flask was added 4-hydroxy-3-methylbenzoic acid (2.0 g, 1 equiv., 13 mmol), potassium carbonate (7.3 g, 4.0 equiv., 53 mmol), benzyl bromide (22 g, 16 mL, 10 equiv., 0.13 mol), and N,N-dimethylformamide (75 mL). The reaction mixture was heated at 80° C. for 16 hours, at which point the mixture was cooled to room temperature and partitioned with water and ethyl acetate. The organics were separated, dried over MgSO4, and concentrated to dryness. The crude oil was purified using a 50 g SNAP ULTRA® silica gel column (hexane:EA, 9:1) to give benzyl 4-(benzyloxy)-3-methylbenzoate (4.0 g, 92%) as a pure oil. LRMS (M+H) (333), (M+Na) (355).

[0084] b. 4-(benzyloxy)-3-methylbenzoic acid [ka]

[0085] To a 100 mL round-bottom flask was added benzyl 4-(benzyloxy)-3-methylbenzoate (4.0 g, 1 equiv., 12 mmol), 1,4-dioxane (25 mL), water (25 mL), and lithium hydroxide (1.2 g, 4.00 equiv., 48 mmol). The cloudy mixture was allowed to react at 25 °C for 16 h, at which point the mixture was concentrated to one-half volume in vacuo using a rotary evaporator. The resulting crude material was diluted with water and extracted with ether. The ether layer was discarded, the aqueous layer was cooled to 0-5 °C, and the mixture was acidified using concentrated HCl. The resulting cloudy white mixture was extracted with ethyl acetate, ethyl acetate solution, dried over MgSO4, and concentrated to dryness to give 4-(benzyloxy)-3-methylbenzoic acid (2.7 g, 93%) as a nearly pure white solid. The product was used without further purification. LRMS (MH) (241).

[0086] c. 2-(Trimethylsilyl)ethyl 4-(benzyloxy)-3-methylbenzoate [ka]

[0087] 4-(Benzyloxy)-3-methylbenzoic acid (2.6 g, 1 equiv., 11 mmol), DMAP (0.13 g, 0.1 equiv., 1.1 mmol), EDC (3.1 g, 1.5 equiv., 16 mmol), and dichloromethane (75 mL) were added to a 100 ml round-bottom flask, and the resulting solution was stirred at room temperature for approximately 5 minutes. To this stirred solution, 2-(trimethylsilyl)ethan-1-ol (1.9 g, 1.5 equiv., 16 mmol) was added, and the reaction was stirred at 25 °C for 16 hours. The reaction mixture was partitioned between saturated water and CHCl. ​​The organics were separated and dried over anhydrous NaSO. The volatiles were concentrated and chromatographed using a 50 g BIOTAGE SNAP ULTRA column (hexane: EtOAc (1:1)) to give 2-(trimethylsilyl)ethyl 4-(benzyloxy)-3-methylbenzoate (3.0 g, 82%) as a clear oil. LRMS (M+Na) (365 + ). [ka]

[0088] d. 2-(Trimethylsilyl)ethyl 4-hydroxy-3-methylbenzoate

[0089] A solution of 2-(trimethylsilyl)ethyl 4-(benzyloxy)-3-methylbenzoate (2.0 g, 1 eq., 5.8 mmol) in 50 mL of ethanol was added to a round-bottom flask. The homogeneous solution was then degassed under house vacuum for 5 minutes. The solution was then mixed with palladium on carbon (0.20 g, 0.032 eq., 0.19 mmol), and degassing was repeated. After 5 minutes, the mixture was purged with hydrogen gas using a balloon. The reaction was allowed to proceed for 2 hours, occasionally refilling the balloon. TLC indicated the reaction was complete, and the mixture was degassed under vacuum. The reaction was then purged with argon, and the palladium catalyst was filtered off using Celite. The Celite was washed with MeOH, and the filtrate was concentrated to dryness to give 2-(trimethylsilyl)ethyl 4-hydroxy-3-methylbenzoate (1.3 g, 88%) as an oily solid. LRMS(M+H)(253+), (MH)(251).

[0090] e. 2-(Trimethylsilyl)ethyl 4-acetoxy-3-methylbenzoate [ka]

[0091] To a round-bottom flask was added TEA (1.2 g, 1.6 mL, 2 equiv., 12 mmol), 2-(trimethylsilyl)ethyl 4-hydroxy-3-methylbenzoate (1.5 g, 1 equiv., 5.9 mmol), pyridine (1.4 g, 1.4 mL, 3 equiv., 18 mmol), and acetonitrile (50 mL). The reaction mixture was stirred at room temperature and charged with acetic anhydride (2.4 g, 2.2 mL, 4 equiv., 24 mmol). The reaction was stirred at room temperature for 16 hours and then partitioned between water and ethyl acetate. The organics were separated, dried over MgSO4, and concentrated to dryness. The crude oil was purified using a 25 g SNAP ULTRA® silica gel column (hexane:EA, 9:1) to give 2-(trimethylsilyl)ethyl 4-acetoxy-3-methylbenzoate (1.5 g, 86%) as a pure oil. LRMS(M+Na)(317).

[0092] f. 2-(Trimethylsilyl)ethyl 4-acetoxy-3-(bromomethyl)benzoate [ka]

[0093] To an oven-dried round-bottom flask were added 2-(trimethylsilyl)ethyl 4-acetoxy-3-methylbenzoate (1.5 g, 1 equiv., 5.1 mmol), 1,2-dichloroethane (50 mL), and NBS (1.8 g, 2.0 equiv., 10 mmol). The resulting solution was stirred at room temperature for 5 minutes. This mixture was heated to 90°C and mixed with AIBN (0.17 g, 0.2 equiv., 1.0 mmol), and the reaction was allowed to react for 2.5 hours. After 2.5 hours, the reaction was concentrated to dryness to give an oily solid. The crude product was chromatographed using a Biotage 10 g SNAP ULTRA column (hexane:EtOAc 10:1) to give 2-(trimethylsilyl)ethyl 4-acetoxy-3-(bromomethyl)benzoate (1.8 g, 95%) as a low-melting solid with approximately 70% purity. The material was inherently unstable and was used as is in the next reaction.

[0094] g. 2-(Trimethylsilyl)ethyl 4-acetoxy-3-(2-(tert-butoxy)-2-oxoethyl)benzoate [ka]

[0095] To a round-bottom flask was added 2-(trimethylsilyl)ethyl 4-acetoxy-3-(bromomethyl)benzoate (1.8 g, 1 equiv., 4.8 mmol), heptane (100 mL), and tri-tert-butylborate (3.3 g, 3.0 equiv., 14 mmol), and the solution was purged with argon for 5 minutes. The flask was then charged with chloro(1,5-cyclooctadiene)rhodium(I) dimer (0.24 g, 0.1 equiv., 0.48 mmol) and purged with additional argon. The solution was then purged with carbon monoxide for 2 minutes and sealed using a rubber septum. The reaction flask was fitted with a balloon filled with carbon monoxide and heated at 75 °C for 24 hours. The reaction mixture was filtered through Celite, concentrated to dryness, and the crude black oil was purified using a 25 g SNAP Ultra Biotage column (hexane:EtOAc (5:1)) to give 2-(trimethylsilyl)ethyl 4-acetoxy-3-(2-(tert-butoxy)-2-oxoethyl)benzoate (562 mg, 30%) as an oil. LRMS (M+H) (395), (M+Na) (417).

[0096] h. 2-(Trimethylsilyl)ethyl 3-(2-(tert-butoxy)-2-oxoethyl)-4-hydroxybenzoate [ka]

[0097] A round-bottom flask was charged with 2-(trimethylsilyl)ethyl 4-acetoxy-3-(2-(tert-butoxy)-2-oxoethyl)benzoate (500 mg, 1 equiv., 1.27 mmol) and methanol (50 mL). The solution was cooled to 0-5°C and purged with ammonia gas for 3 minutes. The flask was fitted with an argon-filled balloon, and the reaction was stirred for 4.0 hours. After 4 hours, tlc analysis indicated the reaction was complete, and the reaction mixture was concentrated to dryness to give 2-(trimethylsilyl)ethyl 3-(2-(tert-butoxy)-2-oxoethyl)-4-hydroxybenzoate (466 mg, 104%), which was carried on to the next step without purification. LRMS (M+Na) (375), (MH) (351).

[0098] i. 2-(Trimethylsilyl)ethyl 3-bromo-5-(2-(tert-butoxy)-2-oxoethyl)-4-hydroxybenzoate [ka]

[0099] To a round-bottom flask was added 2-(trimethylsilyl)ethyl 3-(2-(tert-butoxy)-2-oxoethyl)-4-hydroxybenzoate (400 mg, 1 equiv., 1.13 mmol) and N,N-dimethylformamide (20 mL), and the reaction mixture was cooled to 0-5 °C. The reaction solution was then mixed with NBS (606 mg, 3.0 equiv., 3.40 mmol), and the reaction was stirred at room temperature for 3.0 h. TLC analysis indicated the reaction was complete, and then the reaction was partitioned between EtOAc and water. The EtOAc layer was separated, dried over MgSO4, and concentrated to dryness to give 2-(trimethylsilyl)ethyl 3-bromo-5-(2-(tert-butoxy)-2-oxoethyl)-4-hydroxybenzoate (533 mg, 109%) as a crude solid, along with some residual DMF. LRMS(M+Na)(453, 455), (MH)(429, 431).

[0100] j. 2-(Trimethylsilyl)ethyl 3-bromo-5-(2-(tert-butoxy)-2-oxoethyl)-4-methoxybenzoate [ka]

[0101] A round-bottom flask was charged with 2-(trimethylsilyl)ethyl 3-bromo-5-(2-(tert-butoxy)-2-oxoethyl)-4-hydroxybenzoate (533 mg, 1 equiv., 1.24 mmol), potassium carbonate (854 mg, 5.0 equiv., 6.18 mmol), dimethyl sulfate (468 mg, 354 μL, 3.0 equiv., 3.71 mmol), and N,N-dimethylformamide (20 mL). The reaction mixture was heated at 50° C. for 2 h, at which point the mixture was cooled to room temperature and partitioned between water and ethyl acetate. The organics were separated, dried over MgSO4, and concentrated to dryness. The crude oil was purified using a 10 g SNAP ULTRA® silica gel column (hexane:EA, 9:1) to give 2-(trimethylsilyl)ethyl 3-bromo-5-(2-(tert-butoxy)-2-oxoethyl)-4-methoxybenzoate (301 mg, 54.7%) as an oil. LRMS (M+Na) (467, 469).

[0102] k. Tri-tert-butyl 2,2',2''-(10-(1-(3-bromo-2-methoxy-5-((2-(trimethylsilyl)ethoxy)carbonyl)phenyl)-2-(tert-butoxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetate [ka]

[0103] A round-bottom flask was charged with 2-(trimethylsilyl)ethyl 3-bromo-5-(2-(tert-butoxy)-2-oxoethyl)-4-methoxybenzoate (300 mg, 1 equivalent, 674 μmol), NBS (180 mg, 1.5 equivalents, 1.01 mmol), AIBN (11.1 mg, 0.1 equivalents, 67.4 μmol), and 1,2-dichloroethane (20 mL), and the reaction mixture was heated to reflux for 1.0 h. The reaction mixture was then concentrated to dryness, and the resulting oily solid was mixed with ether, and the insoluble material was filtered off. The filtrate was concentrated to give a crude oil that was a mixture of 2-(trimethylsilyl)ethyl 3-bromo-5-(1-bromo-2-(tert-butoxy)-2-oxoethyl)-4-methoxybenzoate and the starting material. This material was mixed with tri-tert-butyl 2,2',2''-(1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetate (347 mg, 1 equiv., 674 μmol), acetonitrile (20 mL), and potassium carbonate (372 mg, 4.0 equiv., 2.69 mmol). The mixture was then heated at 50°C for 16 h. The reaction mixture was filtered to remove excess potassium carbonate, and the crude product obtained by evaporation of acetonitrile from the filtrate was dissolved in methylene chloride and filtered through a bed of silica gel using a 9:1 solution of dichloromethane and MeOH. The solution was concentrated to give the desired product, tri-tert-butyl 2,2′,2″-(10-(1-(3-bromo-2-methoxy-5-((2-(trimethylsilyl)ethoxy)carbonyl)phenyl)-2-(tert-butoxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetate (445 mg, 69.0%) as a crude solid. LRMS (M+H), (957, 959), (M+Na), (979, 981).

[0104] l. Tri-tert-butyl 2,2',2''-(10-(2-(tert-butoxy)-1-(2-methoxy-3-(tributylstannyl)-5-((2-(trimethylsilyl)ethoxy)carbonyl)phenyl)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetate [ka]

[0105] A round-bottom flask was charged with tri-tert-butyl 2,2',2''-(10-(1-(3-bromo-2-methoxy-5-((2-(trimethylsilyl)ethoxy)carbonyl)phenyl)-2-(tert-butoxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetate (444 mg, 1 equivalent, 463 μmol), 1,4-dioxane (20 mL), and 1,1,1,2,2,2-hexabutyldistannane (1.34 g, 5.0 equivalents, 2.32 mmol), and the solution was stirred at 100°C for 15 minutes. The reaction mixture was then mixed with bis(triphenylphosphine)palladium(II) chloride (65.1 mg, 0.2 equiv., 92.7 μmol), and the reaction was allowed to proceed for a total of approximately 6.0 h. The reaction mixture was poured onto ice and partitioned between EtOAc and water. The black mixture was filtered through Celite, and the filter cake was washed with additional EtOAc. The organics were separated, dried over MgSO4, and then concentrated. The crude oil was filtered through a pad of silica gel, eluting with (hexane: EtOAc (5:1)) to give tri-tert-butyl 2,2',2''-(10-(2-(tert-butoxy)-1-(2-methoxy-3-(tributylstannyl)-5-((2-(trimethylsilyl)ethoxy)carbonyl)phenyl)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl) triacetate (243 mg, 44.9%) as a crude oil contaminated with hexabutylditin. LRMS (M+Na) (1191, 1189, 1192, 1187, 1188).

[0106] m. Tri-tert-butyl 2,2',2''-(10-(2-(tert-butoxy)-1-(5-(((2,5-dioxopyrrolidin-1-yl)oxy)carbonyl)-2-methoxy-3-(tributylstannyl)phenyl)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetate [ka]

[0107] To an oven-dried round-bottom flask was added tri-tert-butyl 2,2',2''-(10-(2-(tert-butoxy)-1-(2-methoxy-3-(tributylstannyl)-5-((2-(trimethylsilyl)ethoxy)carbonyl)phenyl)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetate (200 mg, 1 equiv., 171 μmol), anhydrous tetrahydrofuran (10 mL), and TBAF (179 mg, 0.68 mL, 4.0 equiv., 685 μmol). The reaction was stirred at room temperature for 72 hours and monitored by TLC. After 72 hours, the reaction mixture was concentrated to dryness. The crude mixture was redissolved in anhydrous dichloromethane (20 mL) and mixed with 1-hydroxypyrrolidine-2,5-dione (98.5 mg, 5.0 equiv., 856 μmol), DMAP (20.9 mg, 1 equiv., 171 μmol), and EDC (328 mg, 10 equiv., 1.71 mmol). The resulting homogeneous solution was stirred at room temperature for 64 hours and concentrated to dryness. The resulting oil was partitioned between ethyl acetate and water. The water was separated and discarded, and the organics were washed with additional saturated NaCl. The organics were dried over anhydrous NaSO and concentrated to dryness. The crude foamy solid was chromatographed using flash chromatography (9:1 CHCl:MeOH). The resulting fractions were then rechromatographed using preparative TLC. The product-containing band was isolated, mixed with a 9:1 CHCl:MeOH mixture, and filtered to remove the silica gel. The solvent was removed in vacuo to give tri-tert-butyl 2,2',2''-(10-(2-(tert-butoxy)-1-(5-(((2,5-dioxopyrrolidin-1-yl)oxy)carbonyl)-2-methoxy-3-(tributylstannyl)phenyl)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetate (18 mg, 8.1%) as a semi-solid. LRMS (M+Na), (1188, 1186, 1189, 1187, 1184).

[0108] All publications, patents, and patent applications mentioned in this specification are indicative of the level of those skilled in the art to which this invention pertains. All publications, patents, and patent applications are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. Although the foregoing invention has been described in some detail by way of illustration and example, for purposes of clarity of understanding, it will be apparent that certain changes and modifications can be made within the scope of the embodiments. The present invention provides, for example, the following items. (Item 1) The following formula: [ka] (In the formula, MC is a multidentate chelating moiety; R 1 is H, an ester, or a carboxylic acid; A is -R 2 -R 3 -Y or Y; B is H, an alkoxy group, or a non-radioactive halogen; R 2 is a direct bond, an alkyl group, or an oxygen-containing moiety; R 3 is a direct bond or an aromatic moiety; Y is a radioactive halogen or a precursor of a radioactive halogen; MMCM is a polymeric conjugation moiety; L 1 is a direct bond or a linker) by a radioactive halogen prosthetic moiety or its precursor. (Item 2) 2. The radiohalogen prosthetic moiety or precursor thereof according to item 1, wherein MC is a cyclic multidentate chelating moiety. (Item 3) 2. The radioactive halogen prosthetic moiety or precursor thereof according to item 1, wherein MC is an acyclic multidentate chelating moiety. (Item 4) 2. The radioactive halogen prosthetic moiety or precursor thereof according to item 1, wherein MC is a modified DOTA moiety. (Item 5) 2. The radioactive halogen prosthetic moiety or precursor thereof according to item 1, wherein MC is DOTA-tris(t-Bu ester) or DOTA-tris(COOH). (Item 6) 2. The radioactive halogen prosthetic moiety or precursor thereof according to item 1, wherein MC is a modified NOTA moiety. (Item 7) 2. The radioactive halogen prosthetic moiety or precursor thereof according to item 1, wherein MC is NOTA-bis(t-bu ester) or NOTA-bis(COOH). (Item 8) 8. The radioactive halogen prosthetic moiety or precursor thereof according to any of items 1 to 7, wherein MC comprises a metal. (Item 9) The metals include non-radioactive lutetium, yttrium, indium and gallium, and radioactive 177 Lu, 64 Cu, 67 Cu, 111 In, 90 Y、 225 Ac, 213 Bi, 212 Pb, 212 Bi, 67 Ga, 68 Ga, 89 Zr and 227 9. The radioactive halogen prosthetic moiety or precursor thereof according to item 8, selected from the group consisting of Th. (Item 10) 8. The radioactive halogen prosthetic moiety or precursor thereof according to any of items 1 to 7, wherein MC does not contain a metal. (Item 11) 11. The radioactive halogen prosthetic moiety or precursor thereof according to any of items 1 to 10, wherein M C is attached via a nitrogen atom present thereon. (Item 12) The following formula:

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Claims

1. The following formula: 【Chemistry 42】 Radioactive halogen precursors.

2. A radioactive halogen precursor as described in claim 1, which contains a metal that forms a complex.

3. The metals include non-radioactive lutetium, yttrium, indium and gallium, and radioactive １７７ Lu, ６４ Cu, ６７ Cu, １１１ In, ９０ Y. ２２５ Ac, ２１３ Bi, ２１２ Pb, ２１２ Bi, ６７ Ga, ６８ Ga, ８９ Zr and ２２７ 3. The radioactive halogen precursor of claim 2, wherein the radioactive halogen precursor is selected from the group consisting of Th.

4. The radioactive halogen precursor of claim 1, which does not contain any complexed metals.

5. The following formula: 【Chemistry 43】 A radioactive halogen prosthetic moiety represented by the formula: wherein Y is a radioactive halogen.

6. Y is, １８ F. ７５ Br, ７６ Br, ７７ Br, １２３ I, １２４ I, １２５ I, １３１ I and ２１１ 6. The radioactive halogen prosthetic moiety of claim 5 selected from the group consisting of At.

7. The CO ２ ｔ 7. A radiohalogen prosthetic moiety according to any one of claims 5 to 6, wherein the Bu group has been deprotected so that the moiety contains a COOH (acetic acid) group in that position.

8. A radiolabeled biomolecule comprising a radioactive halogen prosthetic moiety according to any one of claims 5 to 7 attached to the biomolecule.

9. 9. The radiolabeled biomolecule of claim 8, wherein the biomolecule is selected from the group consisting of an antibody, an antibody fragment, a VHH molecule, an aptamer, or a variant thereof.

10. The radiolabeled biomolecule of claim 9 , wherein the biomolecule is a VHH.

11. The radiolabeled biomolecule of claim 10, wherein the VHH targets HER2.

12. 9. The radiolabeled biomolecule of claim 8, wherein the biomolecule comprises a carbamate or urea-containing pharmacological moiety.

13. 13. The radiolabeled biomolecule of claim 12, wherein the carbamate or urea-containing pharmacological moiety is a moiety used to target PSMA in prostate cancer.

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