Halogenated metal chelators and uses thereof

Abstract

The present disclosure provides novel halogenated (e.g., fluorinated) chelators and metal complexes. The disclosed composition can be used in imaging and therapeutic methods, such as in PET for imaging targeted alpha therapies (TATs).

Description

HALOGENATED METAL CHELATORS AND USES THEREOFCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 63 / 643,542, filed May 7, 2024, the content of which is hereby incorporated by reference in its entirety.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with government support under grant number 162648 awarded by the National Institutes of Health. The government has certain rights in the invention.BACKGROUND

[0003] Nuclear medicine uses radioactivity to image and treat diseases, primarily via the use of radiopharmaceuticals. The application of a given radiopharmaceutical depends on the primary emissions of the incorporated radionuclide.

[0004] Targeted alpha therapy (TAT) presents a promising approach to cancer treatment, leveraging high linear energy transfer and potent therapeutic efficacy of alpha-emitting isotopes. For instance, actinium-225 (225Ac), a leading radioisotope in this field, shows significant potential for treating cancers like metastatic castration-resistant prostate cancer (mCRPC). TAT agents comprise a radiometal like225Ac that emits alpha particles, a targeting vector such as a prostate specific membrane antigen (PSMA) ligand that directs the TAT to the tumor, a chelator to hold the radiometal in place, and a linker to connect the chelator to the targeting agent. One challenge in the development of TATs is the development of chelators for alpha-emitting metals that offer a high chelating efficiency under mild conditions and provide stable chelation in vivo. Another challenge in the development of TATs is how to assess their biodistribution since the radiometals are used in low dose and most of them do not emit positrons or gamma photons that can be imaged and accurate biodistribution information is critical for developing effective TATs. Lastly, an important challenge in the clinical use of TATs is assessing the tumor uptake and organ dosimetry in humans as this information is critical for determining the optimal therapeutic dose. Currently, imaging TATs is not possible, because the radiometals usedin TAT do not have positron or gamma emissions. Further, there have been significant challenges in identifying suitable diagnostic partnerradionuclides for alpha-emitting therapeutic nuclides like212Pb (ti 2 = 10.6 h),212Bi (ti / 2 = 60.6 min),213Bi (ti / 2 = 45.6 min), and225Ac (ti 2 = 10 d). Alpha emitters like Actinium-225 (Ac-225) present unique challenges such as being difficult to chelate due to their large size and having few direct gamma emissions to image their biodistribution.

[0005] There remains a need for radiopharmaceuticals that are suitable for imaging and therapeutic applications.BRIEF SUMMARY OF THE INVENTION

[0006] In one aspect, the present disclosure provides a compound of formula (I), (III), or (IV), or a salt thereof,X is halogen;Y is H or L-RTL is a bond or a linker group,RTcomprises an antibody, antibody fragment, a Fibroblast Activation Protein alpha (FAP-alpha) binding moiety, a binding peptide, a binding polypeptide, a binding protein, an enzyme, a nucleobase-containing moiety, a lectin, or a small molecule targeting moiety; andR1is H or Ci-4alkyl.

[0007] In one aspect, the present disclosure provides a metal complex of formula (II) or (V), or a salt thereof,whereinX is halogen;Y is H or L-RTL is a bond or a linker group,RTcomprises an antibody, antibody fragment, a Fibroblast Activation Protein alpha (FAP-alpha) binding moiety, a binding peptide, a binding polypeptide, a binding protein, an enzyme, a nucleobase-containing moiety, a lectin, or a small molecule targeting moiety; andM is a metal.

[0008] In one aspect, the present disclosure provides a pharmaceutical composition comprising the metal complex of formula (II) disclosed herein, or a salt thereof, and a pharmaceutically acceptable excipient, carrier, or diluent.

[0009] In one aspect, the present disclosure provides a process for the preparation of a complex of formula (II), or a salt thereof,the process comprising the steps of:(a) converting a compound of formula (III) to a compound of formula (IV),(b) converting the compound of formula (IV) to the compound of formula (II) by:(b-i) converting the compound of formula (IV) to a compound of formula (V), then converting the compound of formula (V) to the compound of formula (II),or(b-ii) converting the compound of formula (IV) to a compound of formula (I), then converting the compound of formula (I) to the compound of formula (II),whereinX is halogen;Y is H or L-RTL is a bond or a linker group,RTcomprises an antibody, antibody fragment, a Fibroblast Activation Protein alpha(FAP-alpha) binding moiety, a binding peptide, a binding polypeptide, a binding protein, an enzyme, a nucleobase-containing moiety, a lectin, or a small molecule targeting moiety;R1is H or Ci-4alkyl; andM is a metal.

[0010] In one aspect, the present disclosure provides a method of treating a disease by radiation in a subject in need thereof, the method comprising administering to the subject an effective amount of a radioactive metal complex of formula (1I-A), or a salt thereof, thereby the disease in the subject is treated,whereinX is halogen,Y is H or L-RTis a bond or a linker group,RTcomprises an antibody, antibody fragment, a Fibroblast Activation Protein alpha (FAP-alpha) binding moiety, a binding peptide, a binding polypeptide, a binding protein, an enzyme, a nucleobase-containing moiety, a lectin, or a small molecule targeting moiety, andMi is an alpha emitting metal.

[0011] In one aspect, the present disclosure provides a method of diagnosing a disease in a subject in need thereof, the method comprising: administering to the subject an effective amount of a metal complex of formula (II-B), or a salt thereof,whereinX is halogen,Y is H or L-RTL is a bond or a linker group,RTcomprises an antibody, antibody fragment, a Fibroblast Activation Protein alpha (FAP-alpha) binding moiety, a binding peptide, a binding polypeptide, a binding protein, an enzyme, a nucleobase-containing moiety, a lectin, or a small molecule targeting moiety,M2 is a positron or gamma emitting metal; and determining the biodistribution of the metal complex in the subject.

[0012] In one aspect, the present disclosure provides a method of imaging a subject, the method comprising: administering to the subject an effective amount of a metal complex of formula (II-B), or a salt thereof,whereinX is halogen,Y is H or L-RTL is a bond or a linker group,RTcomprises an antibody, antibody fragment, a Fibroblast Activation Protein alpha (FAP-alpha) binding moiety, a binding peptide, a binding polypeptide, a binding protein, an enzyme, a nucleobase-containing moiety, a lectin, or a small molecule targeting moiety,M2 is a positron or gamma emitting metal; and detecting the metal complex in the subject, thereby generating an image of the subject.

[0013] In one aspect, the present disclosure provides a method of diagnosing a disease in a subject in need thereof, the method comprising: administering to the subject an effective amount of a metal complex of formula (ILC), or a salt thereof,whereinY is H or L-RTL is a bond or a linker group,RTcomprises an antibody, antibody fragment, a Fibroblast Activation Protein alpha (FAP-alpha) binding moiety, a binding peptide, a binding polypeptide, a binding protein, an enzyme, a nucleobase-containing moiety, a lectin, or a small molecule targeting moiety,M is a metal; and determining the biodistribution of the metal complex in the subject.

[0014] In one aspect, the present disclosure provides a method of imaging a subject, the method comprising: administering to the subject an effective amount of a metal complex of formula (ILC), or a salt thereof,(I C) whereinY is H or L-RTL is a bond or a linker group,RTcomprises an antibody, antibody fragment, a Fibroblast Activation Protein alpha (FAP-alpha) binding moiety, a binding peptide, a binding polypeptide, a binding protein, an enzyme, a nucleobase-containing moiety, a lectin, or a small molecule targeting moiety,M is a metal; and detecting the metal complex in the subject, thereby generating an image of the subject.BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Non-limiting embodiments of the present invention will be described by way of example with reference to the accompanying figures, which are schematic and are not intended to be drawn to scale. In the figures, each identical or nearly identical component illustrated is typically represented by a single numeral. For purposes of clarity, not every component is labeled in every figure, nor is every component of each embodiment of the invention shown where illustration is not necessary to allow those of ordinary skill in the art to understand the invention.

[0016] FIG. 1 shows novel halogenated (e g., fluorinated) chelators and metal complexes, and their uses thereof.

[0017] FIG. 2 shows the human serum stability studies with macropa and macropaF radioactive 203Pb complexes in serum.

[0018] FIG. 3 shows PSMA-617 cysteine derivatives and the dual -modality macropaF -PSMA targeting agents.

[0019] FIG. 4 shows the labeling efficiency (radiochemical conversion, RCC%) to225Ac upon increasing concentrations of the macropaF-PSMA ligand.

[0020] FIG. 5 shows serum stability of the [225Ac]macropaF-PSMA complex.

[0021] FIG. 6 shows representative radio-TLC analysis of the223Ac complex.

[0022] FIG. 7 shows HPLC chromatographs of [18F]macropaF-S-PSMA and [18F][La(macropaF-S-PSMA)]+. Two traces indicate radiodetector signal and UV signal. Thesechromatographs indicate coelution of the 18F-labeled agents with the19F (ie. non-radioactive / cold) reference standards.

[0023] FIGS. 8A shows binding curves of [225Ac][Ac(macropaF-S-PSMA)]+to cells expressing PSMA (RM1.PSMA) and control (RM1.WT).

[0024] Fig. 8B shows quantification of the specific and nonspecific binding of [225Ac][Ac(macropaF-S-PSMA)]+to PSMA and non-PSMA expressing cells.

[0025] FIG. 9 shows crystal structures and bond distances of macropa and macropaF La3+complexes. The similar coordination geometry supports similar coordination properties of both ligands.

[0026] FIG. 10 shows therH NMR spectra (400 MHz, 298 K, D2O, pD = 7, MeCN as internal reference) of (a) macropa-F, (b) [Bi(macropa-F)]+, and (c) [Pb(macropa-F)]. The diagnostic aliphatic resonances are labelled with asterisks for each compound and indicate the diastereotopic benzylic methylene linker protons.

[0027] FIG. 11 shows the X-ray crystal structure of [Bi(macropa-F)]1. Ellipsoids are drawn at the 50% probability level. Hydrogen atoms, counter ions, and solvent molecules are excluded for clarity.

[0028] FIG. 12 shows (a) Normalized radio-HPLC traces of the independently injected radiofluorinated complexes [18F][Pb(macropa-F)] and [18F][Bi(macropa-F)]+. (b) Analytical HPLC traces of the independently injected non-radioactive reference metal complexes [Pb(macropa-F)] and [Bi(macropa-F)]+indicate matching retention times with the18F-labeled complexes.

[0029] FIG. 13 shows a summary of the radiolabelling efficiency experiments and kinetic stability studies of macropa and macropa-F with203Pb and207Bi. Experiments were performed in triplicate, (a) and (b) Concentration-dependent radiolabelling studies with different concentrations of macropa and macropa-F and203Pb or207Bi. (c) Human serum stability studies of [203Pb][Pb(macropa)] and [203Pb][Pb(macropa-F)] complexes at different time points, (d) Human serum stability studies of [207Bi][Bi(macropa)+and [207Bi][Bi(macropa-F)]+] complexes at different time points.

[0030] FIG. 14 shows the225Ac and212Pb decay chains.

[0031] FIG. 15 shows a comparison of macropa and macropaF: A) Chemical structures and properties; B)225Ac binding curves; C) Serum stability of225Ac complexes.

[0032] FIG. 16 shows radiochemical synthesis of [18F][La(macropaF-S-PSMA): dinitro macropa (3a) was synthesized and used for radiofluorination. Next, a PSMA vector was conjugated using a thiol linker. Product was confirmed by coelution with reference standard on radioHPLC.

[0033] FIG. 17 shows225Ac complexation of macropaF-S-PSMA.

[0034] FIG. 18 shows an alternative conjugation strategy: a precursor for radiofluorination containing an alkyne group has been synthesized for conjugation via click chemistry.

[0035] FIG. 19 shows the proposed radiochemical synthesis of [18F][M(macropaF-FAPI)] agents.

[0036] FIG. 20 shows an alternative macropaF-FAPI ligands with different linkers.

[0037] FIG. 21 shows the biodistribution of [225Ac][Ac(macropaF-SPSMA)]+evaluated 5 h post-injection in nude mice bearing both RM1.PSMA and RM1.WT tumors. Data are presented as mean ± standard deviation of the percentage of injected dose per gram of organ (%ID / g, n = 3).DETAILED DESCRIPTION OF THE INVENTION

[0038] The following terms are used throughout as defined below.

[0039] As used herein and in the appended claims, singular articles such as “a” and “an” and “the” and similar referents in the context of describing the elements (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the embodiments and does not pose a limitation on the scope of the claims unless otherwise stated. No language in the specification should be construed as indicating any non-claimed element as essential.

[0040] As used herein, “about” will be understood by persons of ordinary skill in the art and will vary to some extent depending upon the context in which it is used. If there are uses of the term which are not clear to persons of ordinary skill in the art, given the context in which it is used,“about” will mean up to plus or minus 10% of the particular term — for example, “about 10 wt. %” would be understood to mean “9 wt. % to 11 wt. %.” It is to be understood that when “about” precedes a term, the term is to be construed as disclosing “about” the term as well as the term without modification by “about” — for example, “about 10 wt. %” discloses “9 wt. % to 11 wt. %” as well as disclosing “10 wt. % .”

[0041] Generally, reference to a certain element such as hydrogen or H is meant to include all isotopes of that element. For example, if an R group is defined to include hydrogen or H, it also includes deuterium and tritium. Compounds comprising radioisotopes such as tritium,14C,32P and35S are thus within the scope of the present technology. Procedures for inserting such labels into the compounds of the present technology will be readily apparent to those skilled in the art based on the disclosure herein.

[0042] As used herein, Cm-Cn, such as C1-C12, Ci-Cs, or Ci-Ce when used before a group refers to that group containing m to n carbon atoms.

[0043] Alkyl groups include straight chain and branched chain alkyl groups having from 1 to 12 carbon atoms, and typically from 1 to 10 carbons or, in some embodiments, from 1 to 8, 1 to 6, or 1 to 4 carbon atoms. Examples of straight chain alkyl groups include groups such as methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, n-heptyl, and n-octyl groups. Examples of branched alkyl groups include, but are not limited to, isopropyl, iso-butyl, sec-butyl, tert-butyl, neopentyl, isopentyl, and 2,2-dimethylpropyl groups. Alkyl groups may be substituted or unsubstituted. Representative substituted alkyl groups may be substituted one or more times with substituents such as those listed above, and include without limitation haloalkyl (e.g., trifluoromethyl), hydroxyalkyl, thioalkyl, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, alkoxyalkyl, carboxyalkyl, and the like.

[0044] Cycloalkyl groups include mono-, bi- or tricyclic alkyl groups having from 3 to 12 carbon atoms in the ring(s), or, in some embodiments, 3 to 10, 3 to 8, or 3 to 4, 5, or 6 carbon atoms. Exemplary monocyclic cycloalkyl groups include, but not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl groups. In some embodiments, the cycloalkyl group has 3 to 8 ring members, whereas in other embodiments the number of ring carbon atoms range from 3 to 5, 3 to 6, or 3 to 7. Bi- and tricyclic ring systems include both bridged cycloalkyl groups and fused rings, such as, but not limited to, bicyclo[2.1.1]hexane, adamantyl, decalinyl, and the like. Cycloalkyl groups may be substituted or unsubstituted. Substitutedcycloalkyl groups may be substituted one or more times with, non-hydrogen and non-carbon groups as defined above. However, substituted cycloalkyl groups also include rings that are substituted with straight or branched chain alkyl groups as defined above. Representative substituted cycloalkyl groups may be mono-substituted or substituted more than once, such as, but not limited to, 2,2-, 2,3-, 2,4-2, 5- or 2, 6-di substituted cyclohexyl groups, which may be substituted with substituents such as those listed above.

[0045] Alkenyl groups include straight and branched chain alkyl groups as defined above, except that at least one double bond exists between two carbon atoms. Alkenyl groups have from 2 to 12 carbon atoms, and typically from 2 to 10 carbons or, in some embodiments, from 2 to 8, 2 to 6, or 2 to 4 carbon atoms. In some embodiments, the alkenyl group has one, two, or three carboncarbon double bonds. Examples include, but are not limited to vinyl, allyl, — CH=CH(CH3), — CH=C(CH3)2, — C(CH3)=CH2, — C(CH3)=CH(CH3), — C(CH2CH3)=CH2, among others. Alkenyl groups may be substituted or unsubstituted. Representative substituted alkenyl groups may be mono-substituted or substituted more than once, such as, but not limited to, mono-, di- or tri -substituted with substituents such as those listed above.

[0046] Alkynyl groups include straight and branched chain alkyl groups as defined above, except that at least one triple bond exists between two carbon atoms. Alkynyl groups have from 2 to 12 carbon atoms, and typically from 2 to 10 carbons or, in some embodiments, from 2 to 8, 2 to 6, or 2 to 4 carbon atoms. In some embodiments, the alkynyl group has one, two, or three carboncarbon triple bonds. Examples include, but are not limited to — C=CH, — C=CCH3, — CH2CACCH3, — C=CCH2CH(CH2CH3)2, among others. Alkynyl groups may be substituted or unsubstituted. Representative substituted alkynyl groups may be mono-substituted or substituted more than once, such as, but not limited to, mono-, di- or tri -substituted with substituents such as those listed above.

[0047] Aryl groups are cyclic aromatic hydrocarbons that do not contain heteroatoms. Aryl groups herein include monocyclic, bicyclic and tricyclic ring systems. Thus, aryl groups include, but are not limited to, phenyl, azulenyl, heptalenyl, biphenyl, fluorenyl, phenanthrenyl, anthracenyl, indenyl, indanyl, pentalenyl, and naphthyl groups. In some embodiments, aryl groups contain 6-14 carbons, and in others from 6 to 12 or even 6-10 carbon atoms in the ring portions of the groups. In some embodiments, the aryl groups are phenyl or naphthyl. Aryl groups may be substituted or unsubstituted. The phrase “aryl groups” includes groups containing fused rings, suchas fused aromatic-aliphatic ring systems (e.g., indanyl, tetrahydronaphthyl, and the like). Representative substituted aryl groups may be mono-substituted or substituted more than once. For example, monosubstituted aryl groups include, but are not limited to, 2-, 3-, 4-, 5-, or 6- substituted phenyl or naphthyl groups, which may be substituted with substituents such as those listed above.

[0048] Heterocyclyl groups include aromatic (also referred to as heteroaryl) and non-aromatic ring compounds containing 3 or more ring members, of which one or more is a heteroatom such as, but not limited to, N, O, and S. In some embodiments, the heterocyclyl group contains 1, 2, 3 or 4 heteroatoms. In some embodiments, heterocyclyl groups include mono-, bi- and tricyclic rings having 3 to 16 ring members, whereas other such groups have 3 to 6, 3 to 10, 3 to 12, or 3 to 14 ring members. Heterocyclyl groups encompass aromatic, partially unsaturated and saturated ring systems, such as, for example, imidazolyl, imidazolinyl and imidazolidinyl groups. The phrase “heterocyclyl group” includes fused ring species including those comprising fused aromatic and non-aromatic groups, such as, for example, benzotri azolyl, 2,3-dihydrobenzo[l,4]dioxinyl, and benzo[l,3]dioxolyl. The phrase also includes bridged polycyclic ring systems containing a heteroatom such as, but not limited to, quinuclidyl. Heterocyclyl groups may be substituted or unsubstituted. Heterocyclyl groups include, but are not limited to, aziridinyl, azetidinyl, pyrrolidinyl, imidazolidinyl, pyrazolidinyl, thiazolidinyl, tetrahydrothiophenyl, tetrahydrofuranyl, dioxolyl, furanyl, thiophenyl, pyrrolyl, pyrrolinyl, imidazolyl, imidazolinyl, pyrazolyl, pyrazolinyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, thiazolyl, thiazolinyl, isothiazolyl, thiadiazolyl, oxadiazolyl, piperidyl, piperazinyl, morpholinyl, thiomorpholinyl, tetrahydropyranyl, tetrahydrothiopyranyl, oxathiane, dioxyl, dithianyl, pyranyl, pyridyl, pyrimidinyl, pyridazinyl, pyrazinyl, triazinyl, dihydropyridyl, dihydrodithiinyl, dihydrodithionyl, homopiperazinyl, quinuclidyl, indolyl, indolinyl, isoindolyl, azaindolyl (pyrrolopyridyl), indazolyl, indolizinyl, benzotriazolyl, benzimidazolyl, benzofuranyl, benzothiophenyl, benzthiazolyl, benzoxadiazolyl, benzoxazinyl, benzodithiinyl, benzoxathiinyl, benzothiazinyl, benzoxazolyl, benzothiazolyl, benzothiadiazolyl, benzo[l,3]dioxolyl, pyrazolopyridyl, imidazopyridyl (azabenzimidazolyl), tri azol opyridyl, isoxazolopyridyl, purinyl, xanthinyl, adeninyl, guaninyl, quinolinyl, isoquinolinyl, quinolizinyl, quinoxalinyl, quinazolinyl, cinnolinyl, phthalazinyl, naphthyridinyl, pteridinyl, thianaphthyl, dihydrobenzothiazinyl, dihydrobenzofuranyl, dihydroindolyl, dihydrobenzodioxinyl, tetrahydroindolyl, tetrahydroindazolyl, tetrahydrobenzimidazolyl,tetrahydrobenzotri azolyl , tetrahydropyrrol opyri dyl , tetrahy dropyrazol opyri dyl , tetrahydroimidazopyridyl, tetrahydrotriazolopyridyl, and tetrahydroquinolinyl groups. Representative substituted heterocyclyl groups may be mono-substituted or substituted more than once, such as, but not limited to, pyridyl or morpholinyl groups, which are 2-, 3-, 4-, 5-, or 6- substituted, or disubstituted with various substituents such as those listed above.

[0049] Heteroaryl groups are aromatic ring compounds containing 5 or more ring members, of which, one or more is a heteroatom such as, but not limited to, N, O, and S. Heteroaryl groups include, but are not limited to, groups such as pyrrolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, thiazolyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, thiophenyl, benzothiophenyl, furanyl, benzofuranyl, indolyl, azaindolyl (pyrrolopyridinyl), indazolyl, benzimidazolyl, imidazopyridinyl (azabenzimidazolyl), pyrazolopyridinyl, triazolopyridinyl, benzotri azolyl, benzoxazolyl, benzothiazolyl, benzothiadiazolyl, imidazopyridinyl, isoxazolopyridinyl, thianaphthyl, purinyl, xanthinyl, adeninyl, guaninyl, quinolinyl, isoquinolinyl, tetrahydroquinolinyl, quinoxalinyl, and quinazolinyl groups. Heteroaryl groups include fused ring compounds in which all rings are aromatic such as indolyl groups and include fused ring compounds in which only one of the rings is aromatic, such as 2,3-dihydro indolyl groups. Heteroaryl groups may be substituted or unsubstituted. Thus, the phrase “heteroaryl groups” includes fused ring compounds as well as includes heteroaryl groups that have other groups bonded to one of the ring members, such as alkyl groups. Representative substituted heteroaryl groups may be substituted one or more times with various substituents such as those listed above.

[0050] Groups described herein having two or more points of attachment (i.e., divalent, trivalent, or polyvalent) within the compound of the present technology are designated by use of the suffix, “ene.” For example, divalent alkyl groups are alkylene groups, divalent aryl groups are arylene groups, divalent heteroaryl groups are divalent heteroarylene groups, and so forth. Substituted groups having a single point of attachment to the compound of the present technology are not referred to using the “ene” designation. Thus, e.g., chloroethyl is not referred to herein as chloroethylene. Such groups may further be substituted or unsubstituted.

[0051] Alkoxy groups are hydroxyl groups ( — OH) in which the bond to the hydrogen atom is replaced by a bond to a carbon atom of a substituted or unsubstituted alkyl group as defined above. Examples of linear alkoxy groups include but are not limited to methoxy, ethoxy, propoxy, butoxy, pentoxy, hexoxy, and the like. Examples of branched alkoxy groups include but are not limited toisopropoxy, sec-butoxy, tert-butoxy, isopentoxy, isohexoxy, and the like. Examples of cycloalkoxy groups include but are not limited to cyclopropyloxy, cyclobutyloxy, cyclopentyloxy, cyclohexyloxy, and the like. Alkoxy groups may be substituted or unsubstituted. Representative substituted alkoxy groups may be substituted one or more times with substituents such as those listed above.

[0052] The term “carboxylic acid” as used herein refers to a compound with a — C(O)OH group. The term “carboxylate” as used herein refers to a — C(O)O group. A “protected carboxylate” refers to a — C(O)O-G where G is a carboxylate protecting group. Carboxylate protecting groups are well known to one of ordinary skill in the art. An extensive list of protecting groups for the carboxylate group functionality may be found in Protective Groups in Organic Synthesis, Greene, T. W.; Wuts, P. G. M., John Wiley & Sons, New York, N.Y., (3rd Edition, 1999) which can be added or removed using the procedures set forth therein and which is hereby incorporated by reference in its entirety and for any and all purposes as if fully set forth herein.

[0053] The term “thiol” refers to — SH groups, while sulfides include — SR80groups, sulfoxides include — S(O)R81groups, sulfones include — SO2R82groups, and sulfonyls include — SO2OR83. R80, R81, R82, and R83are each independently a substituted or unsubstituted alkyl, cycloalkyl, alkenyl, alkynyl, aryl aralkyl, heterocyclyl or heterocyclylalkyl group as defined herein. In some embodiments the sulfide is an alkylthio group, — S-alkyl.

[0054] The term “halogen” or “halo” as used herein refers to bromine, chlorine, fluorine, or iodine. In some embodiments, the halogen is fluorine. In other embodiments, the halogen is chlorine or bromine.

[0055] The term “nitro” as used herein refers to an — NO2 group.

[0056] The term “hydroxyl” as used herein can refer to — OH or its ionized form, — O .

[0057] As will be understood by one skilled in the art, for any and all purposes, particularly in terms of providing a written description, all ranges disclosed herein also encompass any and all possible subranges and combinations of subranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art all language such as “up to,” “at least,” “greater than,” “less than,” and the like include the number recited and refer to ranges which can be subsequentlybroken down into subranges as discussed above. Finally, as will be understood by one skilled in the art, a range includes each individual member. Thus, for example, a group having 1-3 atoms refers to groups having 1, 2, or 3 atoms. Similarly, a group having 1-5 atoms refers to groups having 1, 2, 3, 4, or 5 atoms, and so forth.

[0058] Pharmaceutically acceptable salts of compounds described herein are within the scope of the present technology and include acid or base addition salts which retain the desired pharmacological activity and is not biologically undesirable (e.g., the salt is not unduly toxic, allergenic, or irritating, and is bioavailable). When the compound of the present technology has a basic group, such as, for example, an amino group, pharmaceutically acceptable salts can be formed with inorganic acids (such as hydrochloric acid, hydroboric acid, nitric acid, sulfuric acid, and phosphoric acid), organic acids (e.g., alginate, formic acid, acetic acid, benzoic acid, gluconic acid, fumaric acid, oxalic acid, tartaric acid, lactic acid, maleic acid, citric acid, succinic acid, malic acid, methanesulfonic acid, benzenesulfonic acid, naphthalene sulfonic acid, and p-toluenesulfonic acid) or acidic amino acids (such as aspartic acid and glutamic acid). When the compound of the present technology has an acidic group, such as for example, a carboxylic acid group, it can form salts with metals, such as alkali and earth alkali metals (e.g., Na+, Li+, K+, Ca2+, Mg2+, Zn2+), ammonia or organic amines (e.g. dicyclohexylamine, trimethylamine, triethylamine, pyridine, picoline, ethanolamine, diethanolamine, triethanolamine) or basic amino acids (e.g., arginine, lysine and ornithine). Such salts can be prepared in situ during isolation and purification of the compounds or by separately reacting the purified compound in its free base or free acid form with a suitable acid or base, respectively, and isolating the salt thus formed.

[0059] Those of skill in the art will appreciate that compounds of the present technology may exhibit the phenomena of tautomerism, conformational isomerism, geometric isomerism and / or stereoisomerism. As the formula drawings within the specification and claims can represent only one of the possible tautomeric, conformational isomeric, stereochemical or geometric isomeric forms, it should be understood that the present technology encompasses any tautomeric, conformational isomeric, stereochemical and / or geometric isomeric forms of the compounds having one or more of the utilities described herein, as well as mixtures of these various different forms.

[0060] Because of the limits of representing compounds by structural formulas, it is to be understood that all chemical formulas of the compounds described herein represent all tautomeric forms of compounds and are within the scope of the present technology.

[0061] Stereoisomers of compounds (also known as optical isomers) include all chiral, diastereomeric, and racemic forms of a structure, unless the specific stereochemistry is expressly indicated. Thus, compounds used in the present technology include enriched or resolved optical isomers at any or all asymmetric atoms as are apparent from the depictions. Both racemic and diastereomeric mixtures, as well as the individual optical isomers can be isolated or synthesized so as to be substantially free of their enantiomeric or diastereomeric partners, and these stereoisomers are all within the scope of the present technology.

[0062] The compounds of the present technology may exist as solvates, especially hydrates. Hydrates may form during manufacture of the compounds or compositions comprising the compounds, or hydrates may form over time due to the hygroscopic nature of the compounds. Compounds of the present technology may exist as organic solvates as well, including DMF, ether, and alcohol solvates among others. The identification and preparation of any particular solvate is within the skill of the ordinary artisan of synthetic organic or medicinal chemistry.

[0063] Throughout this disclosure, various publications, patents and published patent specifications are referenced by an identifying citation. Also within this disclosure are Arabic numerals referring to referenced citations, the full bibliographic details of which are provided immediately preceding the claims. The disclosures of these publications, patents and published patent specifications are hereby incorporated by reference into the present disclosure to more fully describe the present technology.

[0064] Cancer remains one of the most pressing global health challenges, impacting millions of lives each year with its devastating consequences. Despite notable advancements in treatment modalities like surgery, conventional chemotherapy, and external radiation therapy, effectively managing cancer continues to present formidable hurdles. Traditional therapies often lack specificity, resulting in indiscriminate damage to healthy tissues and significant adverse effects. In recent years, the emergence of targeted therapies has offered a promising avenue to tackle these challenges. By selectively targeting cancer cells while sparing normal tissues, these therapies aim to maximize efficacy and minimize toxicity. Among the innovative approaches is targetedradionuclide therapy (TRT), which holds the potential for highly localized and potent cytotoxicity against cancer cells.

[0065] TRT differs significantly from external radiation therapy, which administers radiation to patients through a high-energy X-ray beam targeted at the primary tumor location. Instead, TRT functions similarly to targeted chemotherapy, in which a targeted drug is administered systemically. TRT utilizes molecules labeled with a radionuclide to deliver a toxic level of radiation to disease sites throughout the body.

[0066] Recent years have witnessed the approval of several targeted radionuclide therapy (TRT) drugs by regulatory agencies. These include Lutathera (177Lu DOTATATE)9, and Pluvicto (177LU vipivotide tetraxetan). In addition, there has been a significant surge in the number of TRT drugs in clinical trials. According to data from ClinicalTrials.gov, the number of TRT clinical trials has escalated exponentially from 35 in 2003 to over 1,000 by the present day. This remarkable increase underscores the growing recognition of TRT as a promising cancer treatment. It reflects its rising prominence in the field of oncology and suggests its potential for broader adoption in the future. Such advancements hold promise for improving outcomes and enhancing the quality of life for cancer patients. One instance is177Lu-DOTATATE, a TRT for neuroendocrine tumors approved in 2018, which demonstrated significantly improved progression-free survival compared to conventional treatments, such as chemotherapy or somatostatin analogs.

[0067] Within TRT, targeted alpha therapy (TAT) has garnered significant attention for its highly effective approach to cancer treatment. This is evidenced by the number of ongoing clinical trials involving a-emitting radionuclides12and the substantial interest within the commercial sector. TAT uses a-particles with high linear energy transfer (LET= 80-100 keV / pm) and a short path length (40-100 pm)12, aligning well with the cell-specific nature of molecular targeting. Unlike targeted beta therapy, such as Pluvicto, TAT employs a-emitters like actinium-225 (225Ac, ti / 2 = 9.9 days) or lead-212 (212Pb, ti / 2 = 10.6 h) resulting in more potent cytotoxic effects on cancer cells (FIG. 14).

[0068] However, developing TAT presents unique challenges. The relatively large size of a- emitters complicates effective complexation and conjugation to targeting vectors. Additionally, the limited y-emissions combined with the low doses used in TAT complicate precise imaging for biodistribution analysis, thus challenging therapy development and utilization. Despite their therapeutic promise, developing successful TATs requires accurate pharmacokinetic andbiodistribution data to maximize tumor targeting while minimizing off-target toxicity. However, direct imaging of these radionuclides is challenging due to their low doses and complex emission profiles.

[0069] Currently, direct imaging of225Ac or212Pb radiopharmaceuticals using Single-Photon Emission Computed Tomography (SPECT) is exceedingly challenging due to the low doses used in TAT (approximately lOOx lower than PET or SPECT imaging agents), the fact that at early timepoints the parent and the y-emitting daughters have not reached secular equilibrium, and the fact that the recoil energy from a-decay causes the daughters to dissociate from the parent radiopharmaceutical and diffuse away. While there have been a few reports conducting SPECT imaging after administration of225Ac or212Pb radiopharmaceuticals, it is unclear what specific chemical entities are being imaged given the multiple emissions. Moreover, the accuracy at early time-points and in low-dose organs remains poor. The only way to obtain direct pharmacokinetic information in animals for225Ac and212Pb is by ex vivo organ biodistribution using y-counting, which requires highly skilled personnel, and it is very labor-intensive.

[0070] The present disclosure addresses the challenges of the conventional TAT technologies. In particular embodiments, the present disclosure provides18F-labeled macropaF-based compounds, structurally analogous to TAT agents, which can be used to accurately demonstrate the biodistribution and dosimetry of225Ac- and212Pb-labeled radiopharmaceuticals. Fluorine-18 (18F) (ti / 2 = 1.83 h) is an ideal PET radionuclide due to its high resolution, widespread availability, and well-established clinical use, making it a strong candidate for developing PET-based surrogates of TAT agents. This strategy will enhance the development of TATs with improved tumor-to-background ratios and inform personalized treatment plans.

[0071] Fluorinated macropa chelator, macropaF, demonstrates high affinity and stability with radiometals, making it an excellent candidate for complexation with a-emitting radionuclides while, at the same time, being amenable to18F-labeling. Using [18F]macropaF surrogates will enable PET-based prediction of TAT pharmacokinetics and dosimetry. As non-limiting examples, prostate-specific membrane antigen (PSMA) and fibroblast activation protein inhibitors (FAPI) — clinically validated targets in PET and TAT — are used as model systems demonstrating the efficacy of this technology.

[0072] Accurate organ biodistribution and pharmacokinetic information about225Ac and212Pb radiopharmaceuticals within hours post-administration would be highly useful for designing TATswith higher tumor-to-background ratios, especially for agents with rapid clearance such as small molecules or peptides. In human studies, such information would help improve dosimetry and enable personalized treatment.

[0073] Advantageously, the present disclosure establishes a novel PET-based approach for predicting the biodistribution of225Ac- and212Pb-TAT agents, addressing a critical gap in TAT optimization. Specific benefits of the present disclosure include, for example, (1) enabling early- stage pharmacokinetic assessment, accelerating TAT development, (2) providing accurate images of tumor and organ biodistribution, improving dosimetry and reducing toxicity of new TAT, and (3) providing a clinically translatable strategy for dosimetry with rapidly circulating peptide- and small molecule-based TATs, such as PSMA and FAPI ligands.

[0074] Compositions

[0075] In one aspect, the present disclosure provides a compound of formula (I), (III), or (IV), or a salt thereof,X is halogen;Y is H or L-RTL is a bond or a linker group,RTcomprises an antibody, antibody fragment, a Fibroblast Activation Protein alpha (FAP-alpha) binding moiety, a binding peptide, a binding polypeptide, a binding protein, an enzyme, a nucleobase-containing moiety, a lectin, or a small molecule targeting moiety; and R1is H or Ci-4alkyl.

[0076] In some embodiments, Y is L-RT; and RTcomprises belimumab, Mogamulizumab, Blinatumomab, Ibritumomab tiuxetan, Obinutuzumab, Ofatumumab, Rituximab, Inotuzumab ozogamicin, Moxetumomab pasudotox, Brentuximab vedotin, Daratumumab, Ipilimumab, Cetuximab, Necitumumab, Panitumumab, Dinutuximab, Pertuzumab, Trastuzumab, Trastuzumab emtansine, Siltuximab, Cemiplimab, Nivolumab, Pembrolizumab, Olaratumab, Atezolizumab, Avelumab, Durvalumab, Capromab pendetide, Elotuzumab, Denosumab, Ziv-aflibercept, Bevacizumab, Ramucirumab, Tositumomab, Gemtuzumab ozogamicin, Alemtuzumab, Cixutumumab, Girentuximab, Nimotuzumab, Catumaxomab, Etaracizumab, an antigen-binding fragment of any thereof, a prostate specific membrane antigen (“PSMA”) binding peptide, a somatostatin receptor agonist, a bombesin receptor agonist, a seprase binding compound, or a binding fragment of any thereof.

[0077] In some embodiments of formular (I), Y is L-RT; and L comprises an amide group (- NH-C(O)-), a thiourea group (-NH-C(S)-NH-), a thioether group (— S— ), an imine group (-CH=N- ), an alkylene group (-(CH2)n-), wherein n is 1-20, a polyethylene glycol group (-(CFECFEC m-), wherein m is 1-20,or a combination thereof; or L iswherein * indicates attachment to RT.

[0078] In some embodiments of formular (I), Y is

[0079] In some embodiments, X is F or18F.

[0080] As used in the chemical formulas herein, reference to an element by name (such as halogen or metal) is understood to include all possible combinations of any isotopes of that element at any abundance level. Reference to an element by symbol without atomic weight (e.g., F) means an element with natural distribution of its isotopes. Reference to an isotope of an element by a symbol with atomic weight (e.g.,18F), or as an “isotope,” refers to the natural abundance or an enriched state of such isotope. It is understood that certain isotopes, such as225Ac are synthetic elements.

[0081] In some embodiments of formula (III) or (IV), R1is H or methyl.

[0082] In some embodiments, the compound iso, .0

[0083] In one aspect, the present disclosure provides a metal complex of formula (II) or (V), or a salt thereof,whereinX is halogen;Y is H or L-RTL is a bond or a linker group,RTcomprises an antibody, antibody fragment, a Fibroblast Activation Protein alpha(FAP-alpha) binding moiety, a binding peptide, a binding polypeptide, a binding protein, an enzyme, a nucleobase-containing moiety, a lectin, or a small molecule targeting moiety; and M is a metal.

[0084] The metal may be a radioactive isotope, or a nonradioactive metal. In some embodiments, the metal may be suitable for use in alpha therapy, such as Bi, Po, At, Rn, Fr, Ra, Ac, or Th.

[0085] In some embodiments, X is F or18F.

[0086] In some embodiments, M is actinium-225 (223Ac3+), radium-223 (233Ra2+), bismuth-213 (213Bi3+), lead-212 (212Pb2+and / or212Pb4+), terbium-149 (149Tb3+), fermium-255 (255Fm3+),thorium-227 (227Th4+), thorium-226 (226Th4+), astatine-21 1 (211At+), astatine-217 (217At+), uranium-230, La, or Ce.

[0087] In some embodiments, X is F; and M is actinium-225 (225Ac3+), radium-223 (233Ra2+), bismuth-213 (213Bi3+), lead-212 (212Pb2+and / or212Pb4+), terbium-149 (149Tb3+), fermium-255 (255Fm3+), thorium-227 (227Th4+), thorium-226 (226Th4+), astatine-211 (211At+), astatine-217 (217At+), or urani um-230.

[0088] In some embodiments, the metal may be a surrogate metal for PET imaging, such as132La,134La, and / or134Ce. In some embodiments, X is18F; and M is La or Ce.

[0089] In some embodiments of formula (II), Y is

[0090] In some embodiments, the metal complex is

[0091] In some embodiments, L is a linker and RTis a targeting moiety. Suitable linkers and targeting moieties include those disclosed in U.S. Patent No. 11,279,698, the content of which is incorporated herein by reference in its entirety. In some embodiments, RTincludes an antibody, as well as antigen-binding fragments of such antibody and any equivalent embodiments, as would be known to those of ordinary skill in the art.

[0092] In some embodiments, it may be that the binding peptide comprises a prostate specific membrane antigen (“PSMA”) binding peptide, a somatostatin receptor agonist, a bombesin receptor agonist, a seprase binding compound, or a binding fragment thereof. Exemplary PSMA binding peptides include, but are not limited to, those disclosed in U.S. Patent No. 11,279,698, the content of which is incorporated herein by reference in its entirety.

[0093] In some embodiments of the present disclosure, a modified antibody, modified antibody fragment, or modified binding peptide is provided that comprises a linkage arising from conjugation of a compound of Formula (I) or pharmaceutically acceptable salt thereof, with an antibody, antibody fragment, or binding peptide. In any embodiment disclosed herein, it may be that the antibody includes belimumab, Mogamulizumab, Blinatumomab, Ibritumomab tiuxetan, Obinutuzumab, Ofatumumab, Rituximab, Inotuzumab ozogamicin, Moxetumomab pasudotox, Brentuximab vedotin, Daratumumab, Ipilimumab, Cetuximab, Necitumumab, Panitumumab, Dinutuximab, Pertuzumab, Trastuzumab, Trastuzumab emtansine, Siltuximab, Cemiplimab, Nivolumab, Pembrolizumab, Olaratumab, Atezolizumab, Avelumab, Durvalumab, Capromab pendetide, Elotuzumab, Denosumab, Ziv-aflibercept, Bevacizumab, Ramucirumab, Tositumomab, Gemtuzumab ozogamicin, Alemtuzumab, Cixutumumab, Girentuximab, Nimotuzumab, Catumaxomab, or Etaracizumab. In any embodiment disclosed herein, it may be that the antibody fragment includes an antigen-binding fragment of belimumab, Mogamulizumab, Blinatumomab, Ibritumomab tiuxetan, Obinutuzumab, Ofatumumab, Rituximab, Inotuzumab ozogamicin, Moxetumomab pasudotox, Brentuximab vedotin, Daratumumab, Ipilimumab, Cetuximab, Necitumumab, Panitumumab, Dinutuximab, Pertuzumab, Trastuzumab, Trastuzumab emtansine, Siltuximab, Cemiplimab, Nivolumab, Pembrolizumab, Olaratumab, Atezolizumab, Avelumab, Durvalumab, Capromab pendetide, Elotuzumab, Denosumab, Ziv-aflibercept, Bevacizumab, Ramucirumab, Tositumomab, Gemtuzumab ozogamicin, Alemtuzumab, Cixutumumab, Girentuximab, Nimotuzumab, Catumaxomab, or Etaracizumab. In any embodiment disclosed herein, it may be that the binding peptide includes a prostate specific membrane antigen (“PSMA”) binding peptide, a somatostatin receptor agonist, a bombesin receptor agonist, a seprase binding compound, or a binding fragment thereof.

[0094] As an example of a modified antibody, modified antibody fragment, or modified binding peptide of the present technology, it may be that the linkage is a thiocyante linkage; wherein the thiocyanate linkage arises from conjugation of the compound with the antibody, antibody fragment, or binding peptide.

[0095] As used herein, the term “Fibroblast Activation Protein alpha (FAP-alpha) binding moiety” includes any suitable peptide or small molecule ligands for FAP.

[0096] As used herein, the term “small molecule targeting moiety” refers to any moieties with a molecular weight less than 1 kDa that selectively bind to a cell component (such as cellularmembrane or receptor). For example, the small molecule moiety can target tumor cells by selectively binding to tumor-specific antigens or receptors expressed on the plasma membrane.

[0097] A person of ordinary skill in the art will recognize that numerous chemical conjugation strategies provide ready access to compounds of the present technology, whereby exposed amino acid residues on a protein (e.g., an antibody) undergo well-known reactions with reactive moieties on a prosthetic molecule. For example, amide coupling is a well-known route, where — as an example — lysine residues on the antibody surface react with terminal activated carboxylic acid esters to generate stable amide bonds. Amide coupling is typically mediated by any of several coupling reagents (e.g., HATU, EDC, DCC, HOBT, PyBOP, etc ), which are detailed elsewhere. (See generally Eric Valeur & Mark Bradley, Amide Bond Formation: Beyond the Myth of Coupling Reagents, 38 CHEM. SOC. REV. 606 (2009).) These and other amide coupling strategies are described in a recent review by Tsuchikama. (Kyoji Tsuchikama & Zhiqiang An, Antibody-Drug Conjugates: Recent Advances in Conjugation and Linker Chemistries, 9 PROTEIN CELL 33, 36 (2018); see also, e.g., A. C. Lazar et al., Analysis of the Composition of Immunoconjugates Using Size-Exclusion Chromatography Coupled to Mass Spectrometry, 19 RAPID COMMUN. MASS SPECTROM. 1806 (2005).)

[0098] Additionally, a person of ordinary skill in the art will recognize that cysteine coupling reactions may be employed to conjugate prosthetic molecules with thiol -reactive termini to protein surfaces through exposed thiol side chains on cysteine residues on the protein (e.g., antibody) surface. (See generally Tsuchikama & An, supra, at 36-37; see also, e g., Pierre Adumeau et al., Thiol-Reactive Bifunctional Chelators for the Creation of Site-Selectively Modified Radioimmunoconjugates with Improved Stability, 29 BIOCONJUGATE CHEM. 1364 (2018).) Because cysteine residues readily form disulfide linkages with nearby cysteine residues under physiological conditions, rather than existing as free thiols, some cysteine coupling strategies may rely upon selective reduction of disulfides to generate a higher number of reactive free thiols. (See id.) Cysteine coupling techniques known in the art include, but are not limited to, cys alkylation reactions, cysteine rebridging reactions, and cys-aryl coupling using organometallic palladium reagents. (See, e.g., C. R. Behrens et al., Antibody-Drug Conjugates (ADCs) Derived from Interchain Cysteine Cross-Linking Demonstrates Improved Homogeneity and Other Pharmacological Properties Over Conventional Heterogeneous ADCs, 12 MOL. PHARM. 3986(2015); Vinogradova et al., Organometallic Palladium Reagents for Cysteine Bioconjugation, 526 NATURE 687 (2015); see also Tsuchikama, supra, at 37 (collecting examples).)

[0099] Protein conjugation strategies using non-natural amino acid side chains are also well- known in the art. For example, “click chemistries” provide access to conjugated proteins, by rapid and selective chemical transformations under a diverse range of reaction conditions. Click chemistries are known to yield peptide conjugates with limited by-product formation, despite the presence of unprotected functional groups, in aqueous conditions. One important non-limiting example of a click reaction in the formation of conjugated peptides is the copper(I)-catalyzed azide-alkyne 1,3-dipolar cycloaddition reaction (CuAAC). (See Liyuan Liang & Didier Astruc, The C fper(7)-Catalysed Alkyne-dz / t / c Cycloaddition (CuAAC) “Click” Reaction and Its Applications: An Overview, 255 COORD. CHEM. REV. 2933 (2011); see also, e.g., Herman S. Gill & Jan Marik, Preparation of V&F-labeled Peptides using the Cqp / ?er(7)-Catalyzed Azide- Alkyne 1, 3 -Dipolar Cycloaddition, 6 NATURE PROTOCOLS 1718 (2011).) The CuAAC click reaction may be carried out in the presence of ligands to enhance reaction rates. Such ligands may include, for example, polydentate nitrogen donors, including amines (e.g., tris(triazolyl)methyl amines) and pyridines. (See Liang & Astruc, supra, at 2934 (collecting examples); P. L. Goias et al., 39 MACROMOLECULES 6451 (2006).) Other widely-utilized click reactions include, but are not limited to, thiol-ene, oxime, Diels-Alder, Michael addition, and pyridyl sulfide reactions.

[0100] Copper-free (Cu-free) click methods are also known in the art for delivery of therapeutic and / or diagnostic agents, such as radionuclides (e.g.,81F), chemotherapeutic agents, dyes, contrast agents, fluorescent labels, chemiluminescent labels, or other labels, to protein surfaces. Cu-free click methods may permit stable covalent linkage between target molecules and prosthetic groups. Cu-free click chemistry may include reacting an antibody or antigen-binding fragment, which has been modified with a non-natural amino acid side chain that includes an activating moiety such as a cyclooctyne (e.g., dibenzocyclooctyne (DBCO)), a nitrone or an azide group, with a prosthetic group that presents a corresponding or complementary reactive moiety, such as an azide, nitrone or cyclooctyne (e.g., DBCO). (See, e.g., David. J. Donnelly et al., Synthesis and Biologic Evaluation of a Novel18F -Labeled Adnectin as a PET Radioligand for Imaging POLI Expression, 59 J. NUCL. MED. 529 (2018).) For example, where the targeting molecule comprises a cyclooctyne, the prosthetic group may include an azide, nitrone, or similar reactivemoiety. Where the targeting molecule includes an azide or nitrone, the prosthetic group may present a complementary cyclooctyne, alkyne, or similar reactive moiety. Cu-free click reactions may be carried out at room temperature, in aqueous solution, in the presence of phosphate-buffered saline (PBS). The prosthetic group may be radiolabeled (e.g., with18F) or may be conjugated to any alternative diagnostic and / or therapeutic agent (e g., a chelating agent). (See id. at 531.)

[0101] The present technology also provides compositions (e.g., pharmaceutical compositions) and medicaments comprising any of one of the embodiments of the compounds of Formula (II, any one of the modified antibodies, modified antibody fragments, or modified binding peptides of the present technology disclosed herein, and a pharmaceutically acceptable excipient, carrier, or diluent. The compositions may be used in the methods and treatments described herein.

[0102] In one aspect, the present disclosure provides a pharmaceutical composition comprising the metal complex of formula (II) disclosed herein, or a salt thereof, and a pharmaceutically acceptable excipient, carrier, or diluent.

[0103] Preparation Process

[0104] The present disclosure provides synthetic processes to make18F -labeled surrogates of a-emitting radiopharmaceuticals without substantially altering their structure and use PET to obtain accurate pharmacokinetic information and biodistribution with high temporal and spatial precision. Specifically, we hypothesize that an18F-labeled version of the macropa chelator can be made, which is useful, for example, to image TATs by PET (FIG. 1).

[0105] In one aspect, the present disclosure provides a process for the preparation of a complex of formula (II), or a salt thereof,the process comprising the steps of(a) converting a compound of formula (III) to a compound of formula (IV),(III) (IV)(b) converting the compound of formula (IV) to the compound of formula (II) by:(b-i) converting the compound of formula (IV) to a compound of formula (V), then converting the compound of formula (V) to the compound of formula (II),(b-ii) converting the compound of formula (IV) to a compound of formula (I), then converting the compound of formula (I) to the compound of formula (II),whereinX is halogen;Y is H or L-RTL is a bond or a linker group,RTcomprises an antibody, antibody fragment, a Fibroblast Activation Protein alpha (FAP-alpha) binding moiety, a binding peptide, a binding polypeptide, a binding protein, an enzyme, a nucleobase-containing moiety, a lectin, or a small molecule targeting moiety;R1is H or Ci-4alkyl; andM is a metal.

[0106] In some embodiments, step (a) may be carried out in the presence of a halogenating reagent and a base. In some embodiments, the halogenating reagent may comprise a metal halide, such as KF, or K18F. A person of ordinary skill in the art will recognize that numerous bases provide ready access to compounds of the present method, such as LiOH, KOH, or NaOH.

[0107] In some embodiments, step (b) may be carried out in the presence of a metal complex, such .

[0108] In some embodiments, X is F or18F.

[0109] In particular embodiments, the present disclosure shows that fluorinated macropa, macropaF, has essentially the same chelating properties as macropa, making it an excellent chelator for a-emitting metals such as225Ac and212Pb. The present disclosure also demonstrates that [18F]macropaF can be synthesized quickly and efficiently. Further, preliminary data show that [18F]macropaF can be efficiently conjugated to a PSMA vector and that225Ac complexes of macropaF are highly stable in buffer and in human serum.

[0110] Methods

[0111] The metal complexes as described here can be used as effective TAT agent for treatment of a disease, such as cancer. In one aspect, the present disclosure provides a method of treating a disease by radiation in a subject in need thereof, the method comprising administering to the subject an effective amount of a radioactive metal complex of formula (II-A), or a salt thereof, thereby the disease in the subject is treated,(II-A) whereinX is halogen, Y is H or L-RTL is a bond or a linker group,RTcomprises an antibody, antibody fragment, a Fibroblast Activation Protein alpha (FAP-alpha) binding moiety, a binding peptide, a binding polypeptide, a binding protein, an enzyme, a nucleobase-containing moiety, a lectin, or a small molecule targeting moiety, and Mi is an alpha emitting metal.

[0112] In some embodiments, suitable alpha emitting metals include, but are not limited to, Bi, Po, At, Rn, Fr, Ra, Pb, Ac, and Th.

[0113] In some embodiments, Mi is225Ac,213Bi,212Bi, or212Pb.

[0114] The metal complexes of the present disclosure can also be used for diagnosing a disease, for example, by a suitable imaging technology. In particular embodiments, the distribution of the synthetic18F-macropa conjugates can be detected to accurately reflect the pharmacokinetics and biodistribution of the same agents labeled with212Pb and223Ac. As non-limiting examples, [, 8F]macropaF-PSMA and [18F]macropaF-FAPI can be used as chelators for the metal complex, which is then used for imaging studies. These vectors are selected because there is a lot of interest in developing TAT for PSMA and FAPI and due to their fast pharmacokinetics compatible withour proposed PET / TAT approach. For example, [225Ac]DOTA-PSMA-617 was previously shown to reach maximum uptake in tumors and other organs within 4 hours post-injection. Since the present disclosure uses18F (1.83 h half-life), it is better suited for fast-clearing agents like small molecule and peptide-based vectors than for slowly circulating agents such as antibodies.

[0115] Prostate-specific membrane antigen (PSMA) is a well-established target for prostate cancer imaging and therapy. There are currently numerous ongoing studies evaluating225Ac- and212Pb-labeled PSMA agents for cancer treatment (e.g., NCT04597411 (225Ac-PSMA-617, phase I), NCT05983198 (225Ac-PSMA-R2, phase I / II), NCT06402331 (225Ac-PSMA-I&, phase II), NCT06217822 (223Ac-PSMA-Trillium, phase I),212Pb-PSMA). These agents are usually paired with68Ga- or18F-labeled PSMA ligands such as68Ga-PSMA-l 1,68Ga-PSMA-I&T,18F-DCFPyL, and18F-PSMA-1007 to detect PSMA overexpression and identify patients who may benefit from the TRT. However, these PET agents do not provide precise information about the pharmacokinetics or biodistribution of the TRT since they contain different structures, chelators and metals .

[0116] Fibroblast activation protein inhibitors (FAPIs) represent another innovative class of radiopharmaceuticals, targeting cancer-associated fibroblasts in the tumor stroma. Several FAPI PET probes, such as18F-A1F-F API-74,68Ga-F API-04 and64Cu-F API-04 have demonstrated high tumor-to-background ratio for imaging a wide range of stromal-rich tumors. There are several studies proposing the use of225Ac-FAPI and212Pb-FAPI for cancer treatment. Similar to PMSA, agents such as FAPI-46 have fast pharmacokinetics and are suitable for validating our approach.

[0117] The present diagnostic or imaging methods can be compared to, or also enable, other approaches. Current approaches that use “imageable radiometals” such as64Cu,68Ga and89Zr have been used or proposed as theranostic pairs for a emitters, but these metals have very different size, charge and chemical hardness requiring different chelators, which may result in different pharmacokinetics and biodistribution. Recent research focuses on pairing a-emitters with elementally matched imageable isotopes such as203Pb or226Ac or isotopes of similar size such as132La,133La or134Ce. While highly promising, access, cost and specific activity of these radiometals are limited compared to18F. Furthermore, many of these radiometals decay by y- emission (some with multiple or low percent emissions) requiring less quantitative and sensitive SPECT imaging.

[0118] As an alternative,18F was proposed as imaging surrogate due to its high availability and excellent imaging properties (i.e., 100% positron emission and short positron range). These approaches include the use of18F-labeled metal fluorides such as [18F]A1F3 or [18F]ScFa, which also require different chelators. Alternatively, others have attached18F to a prosthetic group such as R / Si / B-18F, which facilitates labeling but substantially changes the molecular weight and the physicochemical properties of the radiopharmaceutical altering its pharmacokinetics and biodistribution. The present disclosure differs from these theranostic approaches in that minimal modifications are made to the radiopharmaceutical structure. By adding a single fluorine atom to the macropa chelator, the changes to the molecule’s physicochemical properties including molecular weight, logD, p , etc. are minimal. Furthermore, macropaF containing non-radioactive fluorine (19F) of the present disclosure can be used in TATs thus providing the exact molecular entity for TAT and for imaging.

[0119] While18F-labeling of macropa can be challenging compared to “swapping the radiometal” approaches (i.e., requires heating, organic solvents, and HPLC purification), these challenges are routinely overcome in PET production facilities throughout the world. As demonstrated herein, the18F-labeling of the proposed agents was successfully carried out to prepare the present chelators and metal complexes .

[0120] In summary, there is a pressing need for imaging surrogates to evaluate the pharmacokinetics and biodistribution of TATs, as this information can drive the development of novel TATs with higher tumor-to-background ratios and improve personalized treatment. To address this need, to the present disclosure in specific embodiments developed and evaluated18F- labeled macropa-based TAT surrogates. The key advantages of the present approach are that it uses the widely available and highly efficient imaging isotope18F, that it employs the same molecular structure for imaging and therapy, ensuring consistent pharmacokinetics and biodistribution, and that it can be generalized to any TAT containing small molecule or peptide- based targeting vectors.

[0121] In one aspect, the present disclosure provides a method of diagnosing a disease in a subject in need thereof, the method comprising: administering to the subject an effective amount of a metal complex of formula (II-B), or a salt thereof,(II-B) whereinX is halogen,Y is H or L-RTL is a bond or a linker group,R1comprises an antibody, antibody fragment, a Fibroblast Activation Protein alpha (FAP-alpha) binding moiety, a binding peptide, a binding polypeptide, a binding protein, an enzyme, a nucleobase-containing moiety, a lectin, or a small molecule targeting moiety,M2 is a positron or gamma emitting metal; and determining the biodistribution of the metal complex in the subject.

[0122] In one aspect, the present disclosure provides a method of imaging a subject, the method comprising: administering to the subject an effective amount of a metal complex of formula (II-B), or a salt thereof,whereinX is halogen,Y is H or L-RTL is a bond or a linker group,RTcomprises an antibody, antibody fragment, a Fibroblast Activation Protein alpha (FAP-alpha) binding moiety, a binding peptide, a binding polypeptide, a binding protein, an enzyme, a nucleobase-containing moiety, a lectin, or a small molecule targeting moiety,M2 is a positron or gamma emitting metal; and detecting the metal complex in the subject, thereby generating an image of the subject.

[0123] In some embodiments, M2 is226Ac,203Pb,132La, or134Ce.

[0124] In one aspect, the present disclosure provides a method of diagnosing a disease in a subject in need thereof, the method comprising: administering to the subject an effective amount of a metal complex of formula (ILC), or a salt thereof,whereinY is H or L-RTL is a bond or a linker group,RTcomprises an antibody, antibody fragment, a Fibroblast Activation Protein alpha (FAP-alpha) binding moiety, a binding peptide, a binding polypeptide, a binding protein, an enzyme, a nucleobase-containing moiety, a lectin, or a small molecule targeting moiety,M is a metal; and determining the biodistribution of the metal complex in the subject.

[0125] In one aspect, the present disclosure provides a method of imaging a subject, the method comprising: administering to the subject an effective amount of a metal complex of formula (II-C), or a salt thereof,whereinY is H or L-RTL is a bond or a linker group,RTcomprises an antibody, antibody fragment, a Fibroblast Activation Protein alpha (FAP-alpha) binding moiety, a binding peptide, a binding polypeptide, a binding protein, an enzyme, a nucleobase-containing moiety, a lectin, or a small molecule targeting moiety,M is a metal; and detecting the metal complex in the subject, thereby generating an image of the subject.

[0126] In some embodiments, M of formula (II-C) is a non-radioactive metal.

[0127] In some embodiments, detecting the metal complex comprises performing a positron emission tomography (PET) or a single photon emission computed tomography (SPECT).

[0128] In some embodiments, the disease is cancer or the subject has cancer.

[0129] Since a cancer cell targeting agent can be selected to target any of a wide variety of cancers, the cancer considered herein for treatment is not limited. The cancer can be essentially any type of cancer. For example, antibodies or peptide vectors can be produced to target any of a wide variety of cancers. The targeting compositions described herein are typically administered by injection into the bloodstream, but other modes of administration, such as oral or topical administration, are also considered. In some embodiments, the targeting composition may be administered locally, at the site where the target cells are present, i.e., in a specific tissue, organ,or fluid (e g., blood, cerebrospinal fluid, etc.). Any cancer that can be targeted through the bloodstream is of particular consideration herein. Some examples of applicable body parts containing cancer cells include the breasts, lungs, stomach, intestines, prostate, ovaries, cervix, pancreas, kidney, liver, skin, lymphs, bones, bladder, uterus, colon, rectum, and brain. The cancer can also include the presence of one or more carcinomas, sarcomas, lymphomas, blastomas, or teratomas (germ cell tumors). The cancer may also be a form of leukemia.

[0130] In some embodiments, the subject is a human. In some embodiments, the subject is a human having cancer. As non-limiting examples, the cancer can be breast cancer (such as a triple negative breast cancer) or prostate cancer (such as a metastatic castration-resistant prostate cancer (mCRPC)). As demonstrated herein, the present technology can include administering225Ac- labeled agents (such as macropaF agents) for treating cancer and administering18F-labeled agents (such as18F labeled macropaF agents) for imaging cancer as well as using the combination of both for developing improved TATs. In addition, the present methods can also use imageable metals with macropaF (such as203Pb,226Ac, or134Ce) for diagnosis or imaging applications.

[0131] In some embodiments, Y of the metal complex of formula (II-A), (II-B), or (II-C) may be any of the Y disclosed herein.

[0132] In some embodiments, Y of the metal complex of formula (II-A), (II-B), or (II-C) is

[0133] In any of the above embodiments, the effective amount may be determined in relation to a subject. “Effective amount” refers to the amount of a compound or composition required to produce a desired effect. One non-limiting example of an effective amount includes amounts or dosages that yield acceptable toxicity and bioavailability levels for therapeutic (pharmaceutical) use including, but not limited to, the treatment of e.g., one or more of a glioma, a breast cancer, an adrenal cortical cancer, a cervical carcinoma, a vulvar carcinoma, an endometrial carcinoma, a primary ovarian carcinoma, a metastatic ovarian carcinoma, a non-small cell lung cancer, a small cell lung cancer, a bladder cancer, a colon cancer, a primary gastric adenocarcinoma, a primary colorectal adenocarcinoma, a renal cell carcinoma, and a prostate cancer. Another example of an effective amount includes amounts or dosages that are capable of reducing symptoms associated with e.g., one or more of a glioma, a breast cancer, an adrenal cortical cancer, a cervical carcinoma, a vulvar carcinoma, an endometrial carcinoma, a primary ovarian carcinoma, a metastatic ovarian carcinoma, a non-small cell lung cancer, a small cell lung cancer, a bladder cancer, a colon cancer, a primary gastric adenocarcinoma, a primary colorectal adenocarcinoma, a renal cell carcinoma, and a prostate cancer, such as, for example, reduction in proliferation and / or metastasis of prostate cancer, breast cancer, or bladder cancer. The effective amount may be from about 0.01 pg to about 1 mg of the compound per gram of the composition, and preferably from about 0.1 pg to about 500 pg of the compound per gram of the composition. As used herein, a “subject” or “patient” is a mammal, such as a cat, dog, rodent or primate. Typically the subject is a human, and, preferably, a human suffering from or suspected of suffering from one or more of a glioma, a breast cancer, an adrenal cortical cancer, a cervical carcinoma, a vulvar carcinoma, an endometrial carcinoma, a primary ovarian carcinoma, a metastatic ovarian carcinoma, a non-small cell lung cancer, a small cell lung cancer, a bladder cancer, a colon cancer (such as colon adenocarcinoma), a primary gastric adenocarcinoma, a primary colorectal adenocarcinoma, a renal cell carcinoma, and a prostate cancer. The term “subject” and “patient” can be used interchangeably.

[0134] In any of the embodiments of the present technology described herein, the pharmaceutical composition may be packaged in unit dosage form. The unit dosage form is effective in treating one or more of a glioma, a breast cancer, an adrenal cortical cancer, a cervical carcinoma, a vulvar carcinoma, an endometrial carcinoma, a primary ovarian carcinoma, a metastatic ovarian carcinoma, a non-small cell lung cancer, a small cell lung cancer, a bladder cancer, a colon cancer (such as colon adenocarcinoma), a primary gastric adenocarcinoma, aprimary colorectal adenocarcinoma, a renal cell carcinoma, and a prostate cancer. Generally, a unit dosage including a compound of the present technology will vary depending on patient considerations. Such considerations include, for example, age, protocol, condition, sex, extent of disease, contraindications, concomitant therapies and the like. An exemplary unit dosage based on these considerations may also be adjusted or modified by a physician skilled in the art. For example, a unit dosage for a patient comprising a compound of the present technology may vary from 1 x 104g / kg to 1 g / kg, preferably, 1 * 103g / kg to 1.0 g / kg. Dosage of a compound of the present technology may also vary from 0.01 mg / kg to 100 mg / kg or, preferably, from 0.1 mg / kg to 10 mg / kg. Suitable doses for TAT and PET may range between 3,700 Bq and 3,700,000 Bq per patient injection. Suitable unit dosage forms, include, but are not limited to powders, tablets, pills, capsules, lozenges, suppositories, patches, nasal sprays, injectibles, implantable sustained-release formulations, mucoadherent films, topical varnishes, lipid complexes, etc.

[0135] The pharmaceutical compositions may be prepared by mixing one or more of the compounds of the present disclosure, or any one of the modified antibodies, modified antibody fragments, or modified binding peptides of the present technology, or any embodiment of the tripartite compound of the present technology, pharmaceutically acceptable salts thereof, stereoisomers thereof, tautomers thereof, or solvates thereof, with pharmaceutically acceptable carriers, excipients, binders, diluents or the like to prevent and treat disorders associated with cancer and / or a mammalian tissue overexpressing PSMA. The compounds and compositions described herein may be used to prepare formulations and medicaments that treat e.g., prostate cancer, breast cancer, or bladder cancer. Such compositions may be in the form of, for example, granules, powders, tablets, capsules, syrup, suppositories, injections, emulsions, elixirs, suspensions or solutions. The instant compositions may be formulated for various routes of administration, for example, by oral, parenteral, topical, rectal, nasal, vaginal administration, or via implanted reservoir. Parenteral or systemic administration includes, but is not limited to, subcutaneous, intravenous, intraperitoneal, and intramuscular, injections. Typically, TATs and PET agents are administered intravenously (e.g., by injection). The following dosage forms are given by way of example and should not be construed as limiting the instant present technology.

[0136] For oral, buccal, and sublingual administration, powders, suspensions, granules, tablets, pills, capsules, gelcaps, and caplets are acceptable as solid dosage forms. These can be prepared, for example, by mixing one or more compounds of the instant present technology, orpharmaceutically acceptable salts or tautomers thereof, with at least one additive such as a starch or other additive. Suitable additives are sucrose, lactose, cellulose sugar, mannitol, maltitol, dextran, starch, agar, alginates, chitins, chitosans, pectins, tragacanth gum, gum arabic, gelatins, collagens, casein, albumin, synthetic or semi-synthetic polymers or glycerides. Optionally, oral dosage forms can contain other ingredients to aid in administration, such as an inactive diluent, or lubricants such as magnesium stearate, or preservatives such as paraben or sorbic acid, or antioxidants such as ascorbic acid, tocopherol or cysteine, a disintegrating agent, binders, thickeners, buffers, sweeteners, flavoring agents or perfuming agents. Tablets and pills may be further treated with suitable coating materials known in the art.

[0137] Liquid dosage forms for oral administration may be in the form of pharmaceutically acceptable emulsions, syrups, elixirs, suspensions, and solutions, which may contain an inactive diluent, such as water. Pharmaceutical formulations and medicaments may be prepared as liquid suspensions or solutions using a sterile liquid, such as, but not limited to, an oil, water, an alcohol, and combinations of these. Pharmaceutically suitable surfactants, suspending agents, emulsifying agents, may be added for oral or parenteral administration.

[0138] As noted above, suspensions may include oils. Such oils include, but are not limited to, peanut oil, sesame oil, cottonseed oil, com oil and olive oil. Suspension preparation may also contain esters of fatty acids such as ethyl oleate, isopropyl myristate, fatty acid glycerides and acetylated fatty acid glycerides. Suspension formulations may include alcohols, such as, but not limited to, ethanol, isopropyl alcohol, hexadecyl alcohol, glycerol and propylene glycol. Ethers, such as but not limited to, poly(ethyleneglycol), petroleum hydrocarbons such as mineral oil and petrolatum; and water may also be used in suspension formulations.

[0139] Injectable dosage forms generally include aqueous suspensions or oil suspensions which may be prepared using a suitable dispersant or wetting agent and a suspending agent. For example, the therapeutic or diagnostic agents of the present disclosure can be dissolved in sterile saline for injection. Injectable forms may be in solution phase or in the form of a suspension, which is prepared with a solvent or diluent. Acceptable solvents or vehicles include sterilized water, Ringer's solution, or an isotonic aqueous saline solution. Alternatively, sterile oils may be employed as solvents or suspending agents. Typically, the oil or fatty acid is non-volatile, including natural or synthetic oils, fatty acids, mono-, di- or tri-glycerides.

[0140] For injection (such as intravenous injection), the pharmaceutical formulation and / or medicament may be a powder suitable for reconstitution with an appropriate solution as described above. Examples of these include, but are not limited to, freeze dried, rotary dried or spray dried powders, amorphous powders, granules, precipitates, or particulates. For injection, the formulations may optionally contain stabilizers, pH modifiers, surfactants, bioavailability modifiers and combinations of these.

[0141] Compounds of the present technology may be administered to the lungs by inhalation through the nose or mouth. Suitable pharmaceutical formulations for inhalation include solutions, sprays, dry powders, or aerosols containing any appropriate solvents and optionally other compounds such as, but not limited to, stabilizers, antimicrobial agents, antioxidants, pH modifiers, surfactants, bioavailability modifiers and combinations of these. The carriers and stabilizers vary with the requirements of the particular compound, but typically include nonionic surfactants (Tweens, Pluronics, or polyethylene glycol), innocuous proteins like serum albumin, sorbitan esters, oleic acid, lecithin, amino acids such as glycine, buffers, salts, sugars or sugar alcohols. Aqueous and nonaqueous (e.g., in a fluorocarbon propellant) aerosols are typically used for delivery of compounds of the present technology by inhalation.

[0142] Besides those representative dosage forms described above, pharmaceutically acceptable excipients and carriers are generally known to those skilled in the art and are thus included in the instant present technology. Such excipients and carriers are described, for example, in “Remingtons Pharmaceutical Sciences” Mack Pub. Co., New Jersey (1991), which is incorporated herein by reference. The instant compositions may also include, for example, micelles or liposomes, or some other encapsulated form.

[0143] Specific dosages may be adjusted depending on conditions of disease, the age, body weight, general health conditions, sex, and diet of the subject, dose intervals, administration routes, excretion rate, and combinations of drugs. Any of the above dosage forms containing effective amounts are well within the bounds of routine experimentation and therefore, well within the scope of the instant present technology.

[0144] Various assays and model systems can be readily employed to determine the therapeutic effectiveness of the treatment according to the present technology.

[0145] For the indicated condition, test subjects will exhibit a 10%, 20%, 30%, 50% or greater reduction, up to a 75-90%, or 95% or greater, reduction, in one or more symptom(s) caused by, orassociated with, the disorder in the subject, compared to placebo-treated or other suitable control subjects.

[0146] As is well known in the art, the dosage of the active ingredient(s) generally depends on the disorder or condition being treated, the extent of the disorder or condition, the method of administration, size of the patient, and potential side effects. In different embodiments, depending on these and other factors, a suitable dosage of the targeting composition may be precisely, at least, above, up to, or less than, for example, 1 mg, 10 mg, 50 mg, 100 mg, 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, 1000 mg, 1200 mg, or 1500 mg, or a dosage within a range bounded by any of the foregoing exemplary dosages. In typical cases, the mass of radiopharmaceuticals can be less than 10 micrograms. Furthermore, the composition can be administered in the indicated amount by any suitable schedule, e.g., once, twice, or three times a day or on alternate days for a total treatment time of one, two, three, four, or five days, or one, two, three, or four weeks, or one, two, three, four, five, or six months, or within a time frame therebetween. Alternatively, or in addition, the composition can be administered until a desired change in the disorder or condition is realized, or when a preventative effect is believed to be provided.

[0147] The examples herein are provided to illustrate advantages of the present technology and to further assist a person of ordinary skill in the art with preparing or using the compounds of the present technology or salts, pharmaceutical compositions, derivatives, prodrugs, or tautomeric forms thereof. The examples herein are also presented in order to more fully illustrate the preferred aspects of the present technology. The examples should in no way be construed as limiting the scope of the present technology, as defined by the appended claims. The examples can include or incorporate any of the variations, aspects or embodiments of the present technology described above. The variations, aspects or embodiments described above may also further each include or incorporate the variations of any or all other variations, aspects or embodiments of the present technology.EXAMPLES

[0148] Disclosed herein are halogenated (e.g., fluorinated) chelators and metal complexes that can be applied in imaging and therapeutic methods, such as in PET for imaging targeted alpha therapies (TATs), in single photon emission computed tomography (SPECT), in monitoring tumor vs. other tissues, and / or in monitoring cancer treatment. Notably, the disclosed chelators aresuitable for medically relevant radiometals, including actinium and lanthanum, and other large metals such as lead, bismuth, and radium.

[0149] In one aspect of the present disclosure, new fluorine- 18 radiolabeled macropa derivatives are disclosed. Fluorine- 18 represents a high-resolution PET isotope. The disclosed macropa derivatives may serve as exceptional chelators for alpha-emitting radiometals and may be utilized as imaging (e.g., PET and SPECT) agents and for the metal complexation of radioactive metals relevant for targeted alpha therapy. In some embodiments, the disclosed compounds possess the fluorine- 18 feature for imaging and radioactive therapeutic metal complexation in one molecule. In addition, the disclosed compounds possess the possibility for targeting vector bioconjugation by using a variety of targeting moieties, such as thiol-functionalized targeting vectors.Example 1

[0150] As an example, Scheme 1 shows a synthetic procedure for the synthesis of macropa derivatives for fluorine- 18 radiolabeling.Scheme 1. Synthesis of macropa derivatives for fluorine-18 radiolabelingurea-H2O, TFAA, DCM ^ H2SO4HNO3NO2MeOH H2SO4°2

[0151] The resulting macropa-F derivative can be radiolabeled with lead-203, as shown in Scheme 2. The following experimental conditions are followed. Formation of the products were monitored using radio-TLC and radio-HPLC.18F-labeled compounds generated were validated through co-inj ection with their respective non-radioactive19F standard references.• 5 pL of stock solution of ligands with 10'2-10'7M• 44 of pL Buffer: NFFrOAc (1 M, pH 7.0)• 1 pL of [203Pb]PbCl2(0.312 MBq) in HC1 (IM HC1)• Radiolabeling conditions: room temperature, 5 min• analysis via radio-TLC (50 mM EDTA (pH 5.0))Scheme 2. Radiolabeling of Macropa and Macropa-F with Lead-203 (tl / 2=51.9 h, 279 keV(81%))

[0152] The radiolabeled [203Pb]Pb-macropa and [203Pb]Pb-macropa-F show high radiochemical conversion with ligand concentrations above 1 pM, and high kinetic stability of radioactive complexes in human serum up to five days (FIG. 2).

[0153] The precursor for fluorine- 18 radiolabeling can be radiolabeled with F-18, as shown in Scheme 3.Scheme 3. Radiolabeling of precursor with fluorine- 18

[0154] [18F]macropa-F was synthesized using a TRACERlab Fx2N synthesizer from GE Healthcare Technologies Inc. In particular, the mononitro functionalized precursor (macropa-Me2- NCh, 8 mg, 13 pmol) in DMSO (0.3 mL) was added to an azeotropically dried mixture of [18F]KF / K222 (K2CO3: 12 mg, K222: 15 mg, ~15 GBq) and was heated at 130 °C for 15 min. After cooling to room temperature, the mixture was treated with lithium hydroxide (0.2 M, 250 pL) and ethanol (250 pL), stirred for 10 min, and then neutralized with an aqueous solution of hydrochloric acid (0.1 M, 1 mL). The reaction mixture was diluted with deionized water with 0.1% v / v acetic acid. The crude solution was purified by semi-preparative HPLC. The RCC from the first radiolabelling step was 69 ± 12% (n = 4), determined by analytical HPLC. The isolated RCY was calculated to be 20 ± 1% (n = 3). The radiochemical purity of [18F]macropa-F was 86 ± 5%.

[0155] After HPLC purification, the fraction containing [18F]macropa-F (-110 MBq) solution was combined with solid PbCE (2 mg, 7.19 pmol) in ammonium acetate buffer (pH 4.5, 10 mM, 1 mL). The mixture was stirred for 5 min at room temperature. Subsequently, the solution wasloaded onto a Cl 8 cartridge. Then, the product on the cartridge was washed with a solution of NH4OAC buffer (pH = 4.5, 10 mM, 10 mL). Afterwards, the product was eluted with a solution of DMSO (0.6 mL) and NH4OAC buffer (pH 4.5, 10 mM, 2.4 mL). [18F][Pb(macropa-F)] was obtained with a RCY of 73 ± 6% (n = 3) and a radiochemical purity exceeding 95%. The proof- of-identity and the purity were evaluated by comparison of retention times with the non-radioactive reference [Pb(macropa-F)].Example 2

[0156] In this study, a new ligand, [18F]macropa-F is provided, which is an analogue of the 18-membered macrocyclic chelator macropa and can be used for metal complexation. The18F is directly incorporated into the chelator backbone thus minimizing the structural footprint and its effect on its pharmacology. In parallel to this, the non-radioactive fluorine-containing macropa-F was labelled with203Pb and207Bi, analogues of their alpha-emitting radioisotopes212Pb and 2i2 / 2i3gj Finally, the stabilities of the new radioactive complexes were analysed in human serum, validating this approach for the development of new a-emitting radiotheranostic agents.

[0157] The macropa-Mei-NOz precursor was targeted because of the high affinity of macropa-like chelators for a-emitters, as well as the known susceptibility of aromatic nitro compounds to radiofluorination reactions. The synthesis of macropa-Mei-NOi is shown in Scheme 4. The picolinic-acid methyl ester functionalized crown ether 1 was alkylated with the mesylated picolinic acid methyl ester 2 to obtain the analytically pure and macropa-Mei-NOi with a yield of 37% after column chromatography and high-performance liquid chromatography (HPLC). In addition, the fluorine-containing analogue macropa-F was synthesized.Scheme 4. a) Synthesis route of a macropa-derived precursor macropa-Mei-NOi for fluorine- 18 radiolabelling and b) synthesis of the corresponding non-radioactive reference 4 and macropa-F for radiometal complexation

[0158] This compound was targeted because it could be used as a non-radioactive surrogate of the18F-labelled version and for the complexation of the alpha emitters. Macropa-F was synthesized in two steps, using the fluorinated picolinic pendent arm 3, and it was obtained in high purity after reversed-phase chromatographic purification. With macropa-F in hand, we sought to investigate how the fluorine substituent in the picolinate pendent donor would affect metal ion coordination. Given the relevance of212Pb and213Bi for alpha therapy, the complexation properties of this ligand with Pb2+and Bi3+were investigated withXH NMR spectroscopy and high-resolution electrospray ionization mass spectrometry (HR-ESI-MS). The NMR spectra of macropa-F in the presence of Bi3+, Pb2+, and La3+, a surrogate for225Ac, in an aqueous solution at pH = 7 are shown in FIG. 10. Consistent with prior NMR studies on macropa complexes, the NMR spectra of macropa-F with these metal ions show significant changes in the chemical shifts upon coordination. For example, the benzylic methylene proton signals split into two doublets, consistent with complex formation.25Further, complex formation was verified by HR-ESI-MS measurements, which displays the expected m / z molecular ion peaks for the complexes. Notably, the molecular ion peak for the [M+H]+ion of [Pb(macropa-F)] at mlz = 757.2119 showed the characteristic isotopic pattern arising from the naturally abundant isotopes204Pb,206Pb,207Pb, and208Pb. A crystal structure of [Bi(macropa-F)]+was also obtained (FIG. 11). Compared to the previously reported crystal structure of [Bi(macropa)]+, that of [Bi(macropa-F)]+is very similar. The coordination geometry of the Bi(III) ion can best be described as a six-coordinate pentagonal pyramid. This asymmetric geometry is most likely a consequence of the stereochemically active 6s2lone pair, which occupies the axial position of a pentagonal bipyramid. Importantly, the interatomic distances between the Bi(III) ion and the ligand donor atoms within [Bi(macropa-F)]+are nearly identical to those found in [Bi(macropa)]+, indicating that the F atom does not negatively alter the coordination properties of this ligand.

[0159] To validate the ability of macropa-Mez-NOz to be radiofluorinated, it was treated with [18F]KF and [2.2.2]cryptand (K222) in DMSO at 130 °C, followed by the addition of an aqueous solution of lithium hydroxide in ethanol (Scheme 4a). Under these conditions, the methyl esters were cleaved, and [18F]macropa-F was isolated with a radiochemical yield (RCY) of 20% after semi-preparative HPLC purification. Notably, the esters, as protecting groups, are necessary for18F radiolabelling; the free acid form of this ligand cannot be radiolabelled under these conditions. The subsequent addition of non-radioactive PbCh and Bi(NCh)3 5HzO salts gave the radiofluorinated metal complexes [18F] [Pb(macropa-F)] and [18F][Bi(macropa-F)]+in high radiochemical purity (>95%) after Cis-cartridge purification with RCYs of 73% and 57%, respectively. The radio-HPLC chromatograms of the radioactive complexes and the UV-detected HPLC chromatograms of the non-radioactive complexes display peaks corresponding to the major products with equivalent retention times, confirming their chemical identities (FIG. 12). To verify the stability of the radiolabelled complexes, they were incubated in human serum at 37 °C for up to 4 h. Under these conditions, no degradation of the complexes occurs, indicating that radiofluorination of the macrocycle is a suitable approach for generating a stable PET agent in vivo.

[0160] Next, we investigated the ability of the modified chelators to bind to therapeutically relevant radiometals. Given the efficacy of macropa for the alpha therapeutic radionuclides213Bi and212Pb, as well as their short physical half-lives that could be compatible with that of18F, we considered them for this application. In place of213Bi and212Pb, however, we used the longer-lived surrogate radioisotopes203Pb (ti / 2 = 51.9 h) and207Bi (ti / 2 = 31.6 a), which both decay via electron capture, for investigating radiolabelling and long-term stabilities of the complexes. Macropa and macropa-F were radiolabelled with203Pb in ammonium acetate buffer (pH = 7, 1.0 M) and with207Bi in 2-(N-morpholino)ethanesulfonic acid (MES) buffer (pH = 5.53, 0.5 M) at room temperature. As shown in FIG. 13, quantitative radiolabelling with both ligands and radiometals is achieved at ligand concentrations as low as 1 M. Importantly, macropa-F performs comparably well to macropa, indicating that the F atom functionalization does not negatively impact its ability to bind to radiometal ions. To validate the chemical identities of the203Pb radiocomplexes formed, HPLC of the non-radioactive and radioactive complexes was carried out,showing nearly identical retention times for both species. Incubation of the radiometal complexes [203Pb] [Pb(macropa-F)] and [207Bi] [Bi(macropa-F)]+(final ligand concentration in buffer: 5 pM) in human serum (190 pL) at 37 °C revealed that these complexes remain >95% intact over the course of 120 h (FIG. 13), demonstrating them to be suitably stable for in vivo applications.

[0161] Lastly, the water-octanol distribution coefficients (logZ>7.4) of the macropa and macropa-F complexes were measured to determine their respective lipophilicities. The logLh.4 values of [203Pb] [Pb(macropa)] and [203Pb][Pb(macropa-F)J were determined to be -0.78 ± 0.04 and -0.37 ± 0.01, whereas those for [207Bi][Bi(macropa)]+and [207Bi][Bi(macropa-F)]+were measured to be -2.09 ± 0.12 and -1.83 ± 0.24. As these data show, the F atom only has a minimal effect on the complex lipophilicity.

[0162] In summary, we successfully developed radiofluorinated [18F] macropa-F metal complexes that could potentially be applied for PET imaging. In addition, macropa-F still enables radiolabelling with radioisotopes of Bi3+and Pb2+, which are therapeutically relevant. These results demonstrate the potential of developing new theranostic agents via the use of18F-functionalized radiometal chelators. Given the versatility and widespread availability of18F, this approach could provide access to theranostic radiopharmaceuticals for many different alpha emitters. Further work is needed to validate this strategy. In particular, the development of bioconjugateable versions of macropa-F are needed, along with the corresponding in vivo studies, in order to validate the dual use of18F for PET imaging in conjunction with alpha emitters for therapy.Example 3

[0163] Methods and Results

[0164] As another example, Scheme 5 shows a synthetic procedure for the synthesis of [18F][La(macropaF-NO2)]+(5) and conjugation with organic thiol as mimic of biological targeting vector. The dinitro substituted macropa precursor featuring two nitro substituents on the paraposition of each picolinate pendent arm and the carboxylic acid groups capped as methyl esters (macropa-Me2-(NO2)2 ester, 3) was synthesized in one step and 86% yield using conventional organic chemistry methods. Leveraging this innovative precursor, we performed18F- denitrofluorination of the precursor followed by ester hydrolysis to produce18F-labeled fluoronitro- macropa ([18F]macropaF-NO2, 4) with high radiochemical yields (50-60% for each step). Subsequently, the purified18F-labeled chelator was incubated with the non-radioactive actiniumsurrogate lanthanum(III) chloride (LaCE) resulting in the rapid formation of the [18F][La(macropaF-NO2)]+complex (5) in quantitative yield. After purification using a C18 cartridge, the complex was conjugated with thiol ethyl thioglycolate (HS-R, 6) (as a mimic of a biological targeting vector) at the carbon attached to the nitro group yielding the desired product (7). The18F-labeling was carried out using a GE Fx2N automated synthesizer and subsequently purified via semi-preparative HPLC.18F-labeled compounds generated at each step were validated through co-inj ection with their respective non-radioactive19F standard references.Scheme 5. Radiochemical synthesis of [18F][La(macropaF)]+(5) and conjugation with organic thiol as mimic of biological targeting vector.

[0165] In addition, Scheme 6 shows the synthesis of the non-radioactive reference compound and the corresponding thiol conjugation.

[0166] FIG. 9 shows the crystal structures and bond distances of the macropa and macropaF La3+complexes. The nearly identical bond distances indicated similar coordination properties.Scheme 6. Synthesis of the non-radioactive reference compound and thiol conjugation.

[0168] The crown ether 1 (150 mg, 572 pmol, 1 eq.) and the picolinic methyl ester 2 (332 mg,1.14 mmol, 2 eq.) were dissolved in anhydrous acetonitrile (30 mL). Sodium carbonate (364 mg, 3.43 mmol, 6 eq.) was added as a solid to the crown ether solution. The yellow suspension was heated up to 80 °C. The reaction mixture was stirred for 18 hours at 80 °C. After full conversion of the picolinic acid (monitored by TLC: EtOAc: hexanes: 1 : 1), the reaction mixture was filtrated to obtain a brown solution. Then, the organic solvent was completely removed under reduced pressure. The remaining brown oil was further purified by using the Biotage flash column chromatography (25 g SiCh cartridge, DCM:MeOH 99: 1 to 85: 15). Fractions indicating a single spot on the TLC (DCM:MeOH: 9: 1) were combined and the solvent was removed under reduced pressure. After removal of the solvent, the product 3 (320 mg, 492 pmol, 86%) was obtained as a brown solid.

[0169] 1H NMR (400 MHz, CDCh, 298 K): <5 (ppm)= 8.75 (d, J= 2.2 Hz, 2 H, Harom), 8.60 (d, J= 2.3 Hz, 2 H, Harom), 4.10 (s, 4 H, CH2), 4.06 (s, 6 H, CH3), 3.66 (t, J= 5.5 Hz, 8 H, CH2), 3.63 (s, J= 5.5 Hz, 8 H, CH2), 2.89 (t, J= 5.5 Hz, 8 H, CH2). ^CfH} NMR (126 MHz, CDCh, 298 K): d (ppm)= 166.7, 164.3, 155.7, 149.8, 118.8, 116.3, 70.9, 69.7, 61.2, 54.9, 53.6.

[0170] The ability of the novel macropa derivative to efficiently chelate lanthanum, bismuth, and lead was confirmed via NMR and supported by X-ray crystallography. Importantly, attempts to label the [Laimacropa-lNChhJ] complex directly with fluorine- 18 did not afford the desired product justifying the multi step synthesis approach.

[0171] Conclusion

[0172] In summary, this represents the first instance of a chelator designed for alpha-emitting metals that incorporates fluorine and a handle for bioconjugation directly into the chelator structure. The fluorine may be non-radioactive fluorine- 19 or a radioactive fluorine- 18.

[0173] Despite the multi step approach, the synthesis and purification of the fluorine- 18 version can be accomplished within 3-4 hours offering the opportunity to perform imaging and biodistribution studies with18F out to several hours post agent administration.

[0174] This also represents the first example of incorporating18F directly on the chelator through a C-F bond. Significantly, the disclosed compounds have advantages over alternative compounds that incorporate the fluorine to a different part of the molecule, such as through a Si-F bond or a M-F bond. While these alternative approaches have the advantage of easier labeling / synthesis, they suffer from drawbacks because the final products do not resemble the therapeutic products. Addition of a18F to the metal changes its charge, which changes its pharmacology. Addition of fluorine through a Si-F bond makes the molecules much more lipophilic, which changes their pharmacology.

[0175] This minimalistic approach offers the opportunity to use the same chelator structure and metal for therapeutic and imaging purposes. This novel chelator is ideally suited to study the kinetics and biodistribution of TATs that have short and medium circulation times such as small molecules and peptides.Example 4

[0176] The radiolabeled macropa derivatives disclosed herein can be conjugated with alternative thiol derivatives, such as a cysteine functionalized PSMA-617 derivative (HS-Cys- PSMA). The HS-Cys-PSMA was prepared through solid phase peptide synthesis.

[0177] Disclosed herein are macropa based agents, such as macropaF-S-PSMA, that can be independently labeled with fluorine-18 (18F) for PET imaging or225Ac for alpha-emitting therapy (FIG. 3). This radiopharmaceutical design uniquely enables imaging and therapeutic components to share an identical structure, thereby streamlining clinical applications and enhancing treatment precision.

[0178] Methods and Results

[0179] }8F radiolabelins (diagnostic) [18F]macropa-F-NC>2 was synthesized as described in Example 3. It was subsequently conjugated with HS-Cys-PSMA in 25 mM NH4HCO3 buffer (pH= 7.5) at 40 °C for 30 min, yielding [18F]macropa-F-SPSMA ([18F]8) in 48% RCY. The product was purified by preparative HPLC, achieving >99% radiochemical purity, with the entire process completed in approximately 2 hours. The purified [18F]8 was then complexed with LaCh to form the PSMA-targeting18F-labeled PET imaging probe [18F][La(macropa-F-SPSMA)]+([18F]9) with> 95% RCY and >95% radiochemical purity, without the need for further purification (Scheme7). Proof-of-identity and the radiochemical purity were evaluated by comparison of retention times with those of the corresponding non-radioactive references (FIG. 7).Scheme 7. Synthesis of PSMA-targeting18F-labeled PET imaging probe.

[0180] Corresponding19F-references:Scheme 8. Thiol conjugation of macropaF-NCh with HS-Cys-PSMA

[0181] The chelator 4 (3 mg, 5.04 pmol, 1 eq.) was dissolved in a freshly prepared ammonium bicarbonate solution (25 mM, pH=7.5, 1 mL). In a separate vial, HS-Cys-PSMA (4.97 mg, 6.55 pmol, 1.3 eq.) and the reducing agent tris(2-carboxyethyl)phosphine hydrochloride (2.90 mg, 10.1 pmol, 2 eq.) were dissolved in ammonium bicarbonate solution (25 mM, pH=7.5, 1 mL). In the next step, both colorless solutions were mixed and warmed up to 40 °C. The solution was stirred for 16 hours at 40 °C the progress of the reaction was monitored via analytical HPLC. Then, the crude mixture was lyophilized. The remaining colorless residue was redissolved in deionized water containing 0.1% TFA (0.5 mL) and was purified by using the Biotage equipped with a Cis-functionalized cartridge on the reversed-phase mode (10 g SiCh cartridge, FhO+O. / o TFA:MeOH 95:5 to 0:80). The fractions were analyzed via analytical HPLC (H2O+0.1% TFA:MeOH 95:5 to 0: 100) and fractions with the same retention time and high purity were combined. The combined fractions were concentrated and lyophilized. Finally, the conjugated chelator 8 was observed as a colorless powder (5.1 mg, 3.90 pmol, 77%) (Scheme 8).

[0182] HR-ESI-MS (MeCN):Scheme 9. Synthesis of lanthanum complex [La(macropaF-S-PSMA)]+, 9

[0183] In a vial, the chelator 8 (2.00 mg, 1.53 pmol, 1 eq.) was dissolved in deionized water (200 pL) and the pH was adjusted to pH=7 by using a solution of sodium hydroxide (0.1 N). In a separate vial, the lanthanum chloride heptahydrate (0.57 mg, 1.53 pmol, 1 eq) was dissolved in deionized water (200 pL) and the pH was adjusted to pH=7. Then, both solutions were combined and the colorless solution was kept for 10 minutes at room temperature to achieve complete complexation. Afterwards, the solution was lyophilized to obtain the metal complex as a colorless solid (Scheme 9).

[0184] HR-ESI-MS (MeCN):

[0185] 225Ac radiolabeling (therapeutic). The macropaF-S-PSMA ligand was radiolabeled with225Ac in NH4OAC buffer (pH 6) at 37 °C, using ligand concentrations from 100 pM to 1 nM, yielding [225Ac][Ac(macropaF-S-PSMA)]+(Scheme 10). Complexation efficiency was assessed using radio-TLC developed with 50 mM EDTA (pH 6) and read at secular equilibrium. Serum stability tests were conducted in human serum at 37 °C over a period of up to 7 days. MacropaF-S-PSMA exhibited very high complexation efficiency with225Ac, achieving 100% radiolabeling efficiency (RE) at ligand concentrations of 100 nM or greater (agent specific activity = 20 pCi / nmol). Curve fitting analysis indicated that 50% RE occurred at 38 ± 2 nM (FIG. 4). Serum stability studies showed 80 ± 2% of225Ac retention with the precursor after 7 days of incubation, suggesting good stability of [225Ac][Ac(macropaF-S-PSMA)]+under physiological conditions (FIG. 5 and FIG. 6).Scheme 10.

[0186] PSMA-Binding Properties

[0187] Saturation binding studies were performed using PSMA expressing RM1.PSMA cells and the radiolabeled compound [225Ac][Ac(macropaF-S-PSMA)]+to assess its PSMA-binding specificity and affinity. RM1.PSMA cells were plated in a 96-well culture plates at a density of 5,000 cells per 100 pl per well and incubated for 24 h in DMEM supplemented with 10%FBS under 5% CO2 at 37 °C. After removing the media, cells were incubated at 0 °C for 60 min with a serial dilution (150 pM to 600 nM) of [225Ac] [Ac(macropaF- S-PSMA)] ' (specific activity: 20 pCi / nmol). Non-specific binding was assessed in parallel by co-incubation with 500 pM of a PSMA-blocking peptide (PSMA-617 without DOTA, compound 11). After incubation, cells were washed 3 times with cold DMEM and the amount radiolabeled compound attached to the cells was quantified using a Phosphorimager screen which included reference standards. The equilibrium dissociation constant (KD) was calculated by the GraphPad Prism software using “one site-total and nonspecific binding” analysis, yielding a KD of 55.25 nM (95% CI: 27.84-110.4 nM). These results are consistent with previously reported values. To further confirm specificity, a double-blinded study was conducted using PSMA expressing RM1.PSMA cells and PSMA-negative RM1.WT cells. Both cell lines were incubated with [225Ac][Ac(macropaF-S-PSMA)]+(60 nM, 20 pCi / nmol) for 60 min at 0°C in the presence and absence of excess high-affinity PSMA inhibitor 2-PMPA (100 pM, Ki = 0.9 nM). In RM1.PSMA cells, radioligand binding was significantly reduced by the presence of excess 2-PMPA, indicating specific PSMA-mediated uptake. In contrast, RM1.WT cells showed negligible binding with or without the inhibitor, further confirming the PSMA specificity of the radioligand (FIGS. 8A-8B).

[0188] Conclusion

[0189] This work characterized the binding of PSMA targeting macropaF-S-PSMA to225Ac for the first time, demonstrating exceptional complexation efficiency and high stability under serum challenge. Additionally,18F labeling of macropaF-S-PSMA was achieved with good RCY and high radiochemical purity. The resulting18F-labeled ligand was readily complexed with lanthanum, making it a valuable imaging agent. This strategy may overcome key challenges in the evaluation of225Ac-containing radiopharmaceuticals by capitalizing on the PET imaging capabilities of18F while maintaining structural identity between the therapeutic and imaging agents. By developing structurally identical agents, this innovative design represents a significant advancement towards accurate tracking of therapeutic agent biodistribution, minimizing discrepancies, and ultimately enhancing the precision and efficacy of PSMA-targeted treatments.

[0190] Example 5

[0191] We have demonstrated that macropaF, a macropa derivative containing a fluorine group in the 3 position of the pyridine of one of the pendant arms has essentially the same chelation properties as macropa (FIG. 15, also see Example 2). We also showed that similar to macropa, macropaF metal complexes have very high stability in buffer and in serum. Finaly, the addition of the fluorine to macropa, does not substantially change the logD of the complexes, strongly suggesting that TATs containing macropaF will have very similar pharmacokinetics and biodistribution as those containing macropa.

[0192] Next, we successfully produced [18F]macropaF-S-PSMA, as shown in Example 4. This novel18F-labeled agent was complexed with La(III) to form [18F][La(macropaF-S-PSMA)] which was confirmed by coelution with reference standard on radioHPLC (FIGS. 7 and 16).

[0193] With the cold macropaF-S-PSMA on hand, we performed the complexation with22?Ac in NFUOAc (pH 6) buffer at 37 °C at various ligand concentrations ranging from 100 pM to 1 nM (FIG. 17). These experiments showed very high chelation efficiency (ECso ~30 nM). We also tested the stability of this novel22?Ac-labeled PSMA agent in buffer and in human serum and found it be highly stable (100% and 80% after 7 days, respectively). Compared to DOTA, macropa-based agents provide superior complexation and stability.

[0194] Novel [18F]macropaF-PSMA and [18F]macropaF-FAPI imaging agents can be developed that are structurally matched to the225Ac- and212Pb- therapeutic agents. The18F-labeled ligand will be complexed with the (nonradioactive) metalsnatLa (a surrogate for Ac) andnatPb to produce imaging agents. In parallel, the (nonradioactive)19F ligands will be complexed with the a-emitting radiometals225Ac and212Pb to create the therapeutic agents. Following synthesis, PET, SPECT and ex vivo y-counting biodistribution can be conducted to study the consistency between the different labels and detection methods.

[0195] Optimization of [l8F]macropaF-PSMA synthesis

[0196] 18F is typically introduced at the final step in radiotracer synthesis, but its conditions (i.e., anhydrous polar organic solvents and heat) are not compatible with the labeling of molecules like PSMA-617. To address this limitation, we have developed a method to conjugate [18F]macropaF to a PSMA targeting vector using a thiol-based linker (FIG. 16). This conjugation method allows for conjugation of a -NO2 precursor to a peptide containing a free thiol in aqueous media under mild conditions. We have also shown that this18F-ligand readily forms a complex with La(III) in aqueous buffer. The conditions for conjugation and metal complexation can be optimized and a simple purification method can be developed that uses a solid-phase extraction cartridge instead of HPLC. At the end of the synthesis, radiochemical yield, molar activity and radiochemical purity can be determined.

[0197] 18F-labeling method: reactions are first evaluated via manual labeling using small amounts of18F (< 30 mCi). Briefly, [18F]fluoride in water is loaded onto a QMA cartridge and eluted with a mixture of K222, K2CO3 in 1 : 1 water-acetonitrile. The [18F]fluoride is dried with heating under a stream of nitrogen. Following drying the precursor is added. Multiple temperatures, times, bases and solvents are screened. Upon identification of suitable conditions, the synthesis is scaled up and transferred to an automated synthesizer (GE Fx2N TracerLab)equipped with radioHPLC. Purification is done using semiprep HPLC or solid phase extraction cartridge. The product is reformulated using a solution compatible for injection. Product identity is confirmed by coelution with reference standard on analytical radioHPLC and radioTLC. Radiochemical conversion is determined integrating the peaks on radioHPLC from the crude. Radiochemical yield is determined based on the isolated18F product divided by the starting18F activity. Molar activity is determined from the UV peak using a calibration curve.

[0198] FIG. 18 shows an alternative strategy for thiol conjugation using PSMA through click chemistry using a macropaF precursor bearing an alkyne. This precursor can be radiofluorinated using similar conditions as the NO2 precursor and conjugated to a commercially available PSMA vector containing an azide (N3) group.

[0199] Challenges due to short half-life of18F:18F can be introduced before conjugation because many peptide base targeting vectors are not compatible with standard radiofluorination conditions (i.e., dry organic solvents and heating). Should the synthesis and purification take longer than expected (~2 h), the labeling procedure can start with higher amounts of18F (3 Ci or more), as routinely done using automated synthesizers.

[0200] Radiolabel macropaF-PSMA with22sAc and212Pb

[0201] The chelation of macropaF-S-PSMA with225Ac and212Pb can be optimized. Preliminary data show high chelation efficiency for225Ac (FIG. 5), and similar conditions can be applied to212Pb. For each radiometal, chelation efficiency and molar activity can be determined.

[0202] 225AC labeling method: the225Ac was can be obtained from229Th decay and can be free of227AC. Upon receipt, it is reconstituted in 0.1 M of HC1 as the stock solution. From this stock solution, working solutions are prepared prior to each experiment. Radiolabeling experiments are conducted in 0.5 M NH4OAC buffer (pH 6) at 37 °C for 5 min with specified ligand concentration.

[0203] 212Pb labeling method: a224Ra / 212Pb generator can be acquired with up to 16 mCi. The generator is eluted using 2 M HC1 into a reaction vessel containing 0.5 M NH4OAC buffer (pH 6). Radiolabeling reactions are conducted in the same buffer at 37 °C for 5-60 min.

[0204] 225AC Chelation efficiency assay: to solutions of ligand concentration ranging from 1 mM to 1 nM in 100-200 pL of NH4OAC (pH 6) buffer l-10pL of225Ac stock solution is added. Solutions are incubated for 1 h at 37 °C or room temperature. Radiolabeling efficiency wasassessed using radio-TLC developed with 50 mM EDTA (pH 6) and analyzed at secular equilibrium with an ARL-2000 TLC scanner or a Typhoon 9000 phosphor imager.

[0205] ^55655 the stability and binding affinity in vitro

[0206] Each radiolabeled compound (18F,225Ac,212Pb) is evaluated for stability in formulation buffer and in human serum (8 h for18F, 48 h212Pb and 10 days for225Ac) using radioHPLC or radioTLC. Preliminary data with [225Ac][Ac(macropaF-S-PSMA)] shows that this complex is fully stable in buffer for 7 days and >80% stable in human serum up to 7 days (FIG. 5).

[0207] Complex stability assay: the complex solutions are mixed with human serum in 1 :3 ratio and incubated at 37 °C. Stability is monitored by radio-TLC at 0, 1, 18, 48 and 168 hours (168 h monitored only for225Ac).

[0208] The study also evaluates binding affinity and specificity to mouse tumor cells (RM1) genetically engineered to express human PSMA (PSMA+ cells) as compared to wild-type RM1 (PSMA- cells). Binding affinity will be determined by displacing with variable amounts of cold agent whereas specificity will be determined by comparing binding in PSMA+ vs. PSMA- cells.

[0209] Competitive binding assay: Cells are plated in 96 well plates (5 x 103cells per well). 5 nCi of the complex solution in 100 pL of media is added to each well and incubated at 0 °C for 30 min. To determine binding affinity serial dilutions of the cold agent ranging from 1 nM to 10 mM are added to the media. After 30 min, the solution is aspirated and the cells washed with media 3 times. After the final wash the activity bound is measured using a Phosphorimager. Binding affinity is determined as the concentration that produces 50% maximum binding.

[0210] Potential low binding affinity: The chelator or the linker may not affect the binding affinity since they are in opposite sides of the molecule. If PSMA affinity is substantially reduced the linker can be altered as proposed in FIG. 18. The RM1.PSMA / WT system offers a cellular uptake assay in directly match cells. The study can additionally verify these findings in PSMA+ / - human cell lines available to us, including the PSMA+ LnCAP and PSMA- PC3 lines.

[0211] Radiochemical synthesis of ! 'dd aci'opal -l'API

[0212] Unlike PSMA, the FAPI vector is a small molecule compatible with late-stage radiofluorination. A number of FAPI ligands have been developed with FAPI-46 being among the most promising for TAT due to its high tumor retention. [18F]macropaF-FAPI can be synthesizedbased on FAPI-46 from a NCh-substituted precursor as shown in FIG. 19. The precursor and standard can be custom-made compounds. The reaction conditions such as solvent, temperature and base can be optimized. The final compound can be complexed with non-radioactive La(III) or Pb(II) and purified using a solid phase extraction cartridge. At the end of the synthesis, radiochemical yield, molar activity and radiochemical purity can be determined.

[0213] Radiolabeling macropaF-FAPI with225Ac and212Pb

[0214] Using conditions optimized as described above, macropaF-FAPI can be chelated with223Ac and212Pb to assess chelation efficiency. For each radiometal, chelation efficiency, purity and molar activity can be determined.

[0215] Stability and binding affinity in vitro

[0216] The study also includes the stability of each radiolabeled agent in formulation buffer and in human serum. For example, the binding affinity and specificity in U87MG cells (which have high expression of FAP) and HT1080 cells (which have low FAP expression) can be evaluated. These cells have been selected as previous studies have described high and low binding with FAPI agents, respectively. Plus, these cells can be used to grow tumors in mice. Binding affinity will be determined by displacing with variable amounts of cold agent, whereas specificity will be determined by comparing binding in high v . low FAP expressing cells.

[0217] In some cases, macropaF-FAPI agent can show low binding affinity or stability, and agents based on other FAPI ligands with different linkers (such as the ones shown in FIG. 20) can be explored.

[0218] PET imaging and ex vivo biodistribution of [,8FlmacropaF-PSMA

[0219] The study also evaluates the organ and tumor concentration of [18F][La(macropaF-S- PSMA)] and [18F][Pb(macropaF-S-PSMA)] using PET and ex vivo y-counting. PET can be performed at multiple time points up to 8h post injection. Ex vivo organ biodistribution can be performed at matching timepoints in different cohort of animals to compare the sensitivity and accuracy of each method.

[0220] Even though the targeting moiety of these agents is the same, the different metal charge (La3+and Pb2+) may affect their pharmacokinetics and therefore it is important to use the correct pairing.

[0221] Mouse model: nude mice with RM1.PSMA and RM1.WT tumors can be used. In some cases, only male mice are used since PSMA tumors predominantly affect males.

[0222] Tumor inoculation: Four-week-old male athymic nude mice will be subcutaneously inoculated with RM1.PSMA cells (expressing PSMA) on the right shoulder and RM1.WT cells (lacking PSMA expression) on the left shoulder (inoculation dose of 2 x 106cells in 50 pL). Tumor sizes will be measured every 3-4 days using a caliper once tumors establish to palpable sizes (3-4 mm in diameter). Tumor bearing mice can be imaged approximately one week later when tumor sizes reach 5 - 15 mm in diameter.

[0223] PET imaging18F: At day 7-10 post-xenograft, when tumors reach a suitable size, the18F-labeled agent are administered via tail-vein injection under isoflurane anesthesia and perform 10 min static scans at 0.2, 0.5, 1, 2, 4 and 8h mid-point post injection (n = 6 animals) using a Sedecal Argus PET / CT. Each animal will receive 100-200 pCi of18F radiotracer.

[0224] Ex vivo biodistribution18F: At day 7-10 post-xenograft, when tumors reach a suitable size, the18F-labeled agents are administered to mice and harvest the tumors (PSMA+ and PSMA- ) and main organs (i.e., heart, liver, spleen, kidney, small intestine, muscle, bone and blood) for ex vivo y-counting at 0.2, 0.5, 1, 2, 4 and 8 h post injection (n = 6 animals per time point). Tissue dissection and y-counting are performed immediately after euthanasia. Animals for euthanasia at 0.2, 0.5 and 1 h post injection receive 100 pCi per animal. Animals for euthanasia at 4 h and 8 h post injection receive 200 and 400 pCi per animal, respectively, to account for decay. Tissues are counted for 1 min on an automated y- counter using a 460-560 keV energy window. Based on our calibration curve, the limit of quantification (LoQ) of this method (LoQ = / / blank +10obiank) is 0.15 nCi per sample. Assuming that a 25 g mouse euthanized 8 h p.i. received 400 pCi (16 pCi / g) and that the smallest tissue sample weights 100 mg, this method is able to quantify organ concentrations as low 1.5 nCi / g at the time of measurement or an SUV of 0.00094. For a sample measured 9 h post injection, this corresponds to an SUV of 0.003 after decay correction. This means that18F bioD is able to accurately quantify any tissue that has an SUV > 0.003. The range of SUV for tumors is expected to be in the 1-10 SUV range and for most tissues in the 0.1-1 SUV range.

[0225] Biodistribution of225Ac- and212Pb macropaF-PSMA agents

[0226] The study evaluates the organ and tumor concentration of [225Ac][Ac(macropaF-S- PSMA)] and [212Pb][Pb(macropaF-S-PSMA)] by SPECT and ex vivo y-counting comparing the sensitivity and accuracy of each method.

[0227] Mouse model: same as described above.

[0228] SPECT imaging: 10 min static scans imaging are performed at 0.2, 1, 4 and 24 h for212Pb and the same time points plus 48 and 168 h for225Ac (n = 6 animals). Each animal receives 1 pCi of the radiometal agent.225Ac is imaged in Triumph Trifoil PET / SPECT / CT preclinical scanner. This scanner has low and mid energy y-camera capable to image at 10-250 keV range that can capture emissions from225Ac (221Fr peak at 218 keV) and212Pb (238 keV).

[0229] A dose of 1 uCi may not be detectable by SPECT: A dose of 1 pCi per animal is selected as this corresponds to 40 nCi / g (1,480 kBq / kg) for a 25 g mouse, which is in the upper range of the doses typically used for TAT. Doses higher than that are likely to cause excessive toxicity killing the animals. Should SPECT not produce accurate results, ex vivo biodistribution data using y-counting can be used.

[0230] It is possible that the tumors is gone by 168 h post injection: even though the goal is not to assess the therapeutic efficacy of these compounds, to the study uses conditions that mimic the conditions used for TAT. Should the PSMA+ tumors disappear by 168 h p.i., tumors can be analyzed separately.

[0231] Ex vivo biodistribution225Ac: The animals are euthanized and the tumors and main organs are harvested for ex vivo y-counting at 0.2, 0.5, 1, 2, 4, 8, 24, 48 and 168 h post injection (n = 6 animals per time point). Tissue dissection can be performed immediately after euthanasia and the tissues stored at 4 °C. Tissues are counted using y-counting >5 h later to allow for secular equilibrium of the daughters. Each animal receives 1 pCi of225Ac. Tissues can be counted for 5 min on an automated y- counter using an energy window 400-480 keV to capture the213Bi y- emission (peak at 440 keV). Based on the calibration curve, this method is linear over 4 orders of magnitude and the limit of quantification of this method is 0.6 nCi per sample. Assuming that a 25 g mouse receives 1 pCi (40 nCi / g) and that smallest tissue sample weights 100 mg, this method will be able to quantify organ concentrations as low 6 nCi / g at the time of measurement or an SUV of 0.15 SUV. For a sample measured 7 days post injection, this corresponds to an SUV at the timeof injection of 0.24. This means that223Ac bioD will be able to accurately quantify any tissue that has an SUV > 0.24.[002321 Ex vivo biodistribution212Pb: The animals are euthanized and the tumors and main organs are harvested for ex vivo y-counting at 0.2, 0.5, 1, 2, 4, 8 and 24 h post injection (n = 6 animals per time point), y-counting can be performed > 4 h after dissection to allow for secular equilibrium. Each animal receives 1 pCi of212Pb. Tissues can be counted for 5 min on an automated y- counter using 207-267 keV energy window (peak at 238 keV). Assuming similar sensitivity as225Ac, we will be able to quantify organ concentrations as low 6 nCi / g at the time of measurement or an SUV of 0.15 SUV. For a sample measured 24 h post injection, this corresponds to an SUV at the time of injection of 0.74. This means that212Pb bioD will be able to accurately quantify any tissue that has an SUV > 0.74.

[0233] 212Pb has an elementally matched SPECT imaging isotope203Pb which may be a better approach: the main advantages of18F over203Pb are that18F is: i) more readily available, ii) less expensive than203Pb, iii)18F PET provides better resolution and quantification than203Pb SPECT. a comparative experiment using [203Pb][Pb(macropaF-S-PSMA] can be performed.

[0234] Example 6

[0235] FIG. 21 shows the biodistribution of [225Ac][Ac(macropaF-SPSMA)]+evaluated 5 h post-injection in nude mice bearing both RM1.PSMA and RM1.WT tumors.

[0236] Procedure

[0237] The 6-8-week-old male nude mice were subcutaneously inoculated with 2.5 x 105RM1.PSMA cells in the right flank and 2.5 x 105RM1.WT cells in the left flank. Tumor growth was monitored daily using caliper measurements. Mice were enrolled in the biodistribution studies when tumor diameter reached approximately 7-9 mm, typically 10 days post-inoculation.

[0238] For biodistribution studies, the radioactive complex [225Ac][Ac(macropaF-S-PSMA)]+was diluted in PBS and injected via the tail vein at a dose of 40 kBq per mouse, corresponding to 50 pmol of the peptide. Injection was carried out under 2% isoflurane anesthesia. Mice were euthanized at 5 h post-injection, and organs of interest (blood, bone, brain, heart, kidney, large bowel, liver, lung, muscle, salivary glands, skin, small bowel, spleen, stomach, tumor.PSMA, tumor. wild-type) were harvested, weighed, and counted for radioactivity after reaching secularequilibrium (energy window peak at 440 keV, 5min / sample). Radioligand uptake was expressed as %ID / g for each organ.[002391 Results

[0240] The organ distribution profile demonstrated high concentration of the radioactive223Ac complex in kidneys and spleen, in addition to the target tumor. Notably, tracer accumulation in the PSMA-expressing tumor was approximately 11-fold higher than in PSMA-negative tumor ( 1.29 ± 0.46 vs. 0.12 ± 0.04 %ID / g, p value < 0.05), confirming target specificity. At 5 h post-injection, the tumor-to-blood ratio was 26.7 for the PSMA-positive tumor. Overall, the biodistribution pattern of [223Ac][Ac(macropaF-SPSMA)]+is consistent with previously reported data for [177LU][LU-PSM -617] and [225Ac][Ac-mcp-M-PSMA], supporting its potential for targeted alpha-therapy (FIG. 21).

Claims

CLAIMSWhat is claimed is:

1. A compound of formula (I), (III), or (IV), or a salt thereof,X is halogen;Y is H or L-RTL is a bond or a linker group,RTcomprises an antibody, antibody fragment, a Fibroblast Activation Protein alpha (FAP-alpha) binding moiety, a binding peptide, a binding polypeptide, a binding protein, an enzyme, a nucleobase-containing moiety, a lectin, or a small molecule targeting moiety; andR1is H or Ci-4alkyl.

2. The compound of formula (I) of claim 1, or a salt thereof, whereinY is L-RT; andRTcomprises belimumab, Mogamulizumab, Blinatumomab, Ibritumomab tiuxetan, Obinutuzumab, Ofatumumab, Rituximab, Inotuzumab ozogamicin, Moxetumomab pasudotox,Brentuximab vedotin, Daratumumab, Ipilimumab, Cetuximab, Necitumumab, Panitumumab, Dinutuximab, Pertuzumab, Trastuzumab, Trastuzumab emtansine, Siltuximab, Cemiplimab, Nivolumab, Pembrolizumab, Olaratumab, Atezolizumab, Avelumab, Durvalumab, Capromab pendetide, Elotuzumab, Denosumab, Ziv-aflibercept, Bevacizumab, Ramucirumab, Tositumomab, Gemtuzumab ozogamicin, Alemtuzumab, Cixutumumab, Girentuximab, Nimotuzumab, Catumaxomab, Etaracizumab, an antigen-binding fragment of any thereof, a prostate specific membrane antigen (“PSMA”) binding peptide, a somatostatin receptor agonist, a bombesin receptor agonist, a seprase binding compound, or a binding fragment of any thereof.

3. The compound of formular (I) of any one of claims 1-2, or a salt thereof, whereinY is L-RT; andL comprises an amide group (-NH-C(O)-), a thiourea group (-NH-C(S)-NH-), a thioether group (— S— ), an imine group (-CH=N-), an alkylene group (-(CH2)n-), wherein n is 1-20, a polyethylene glycol group (-(CH2CH2O)m-), wherein m is 1-20,combination thereof; orL iswherein * indicates attachment to RT.

4. The compound of formular (I) of any one of claims 1-3, or a salt thereof, wherein5. The compound of any one of claims 1-4, or a salt thereof, wherein X is F or18F.

6. The compound of formula (III) or (IV) of claim 1, or a salt thereof, wherein R1is H or methyl.

7. The compound of claim 1 or a salt thereof, wherein the compound iso, .

08. A metal complex of formula (II) or (V), or a salt thereof,whereinX is halogen;Y is H or L-RTL is a bond or a linker group,RTcomprises an antibody, antibody fragment, a Fibroblast Activation Protein alpha(FAP-alpha) binding moiety, a binding peptide, a binding polypeptide, a binding protein, an enzyme, a nucleobase-containing moiety, a lectin, or a small molecule targeting moiety; and M is a metal.

9. The metal complex of claim 8, or a salt thereof, wherein X is F or18F.

10. The metal complex of claim 8, or a salt thereof,, wherein M is actinium-225 (225Ac3+), radium-223 (233Ra2+), bismuth-213 (213Bi3+), lead-212 (212Pb2+and / or212Pb4+), terbium-149 (149Tb3+), fermium-255 (235Fm3+), thorium-227 (227Th4+), thorium-226 (226Th4+), astatine-211 (211At+), astatine-217 (217At+), uranium-230, La, or Ce.11 . The metal complex of claim 8, or a salt thereof, whereinX is F; andM is actinium-225 (22?Ac3), radium-223 (233Ra2+), bismuth-213 (213Bi3+), lead-212 (212Pb2+and / or212Pb4+), terbium-149 (149Tb3+), fermium-255 (255Fm3+), thorium-227 (227Th4+), thorium-226 (226Th4+), astatine-211 (211At+), astatine-217 (217At+), or uranium-230.

12. The metal complex of claim 8, or a salt thereof, whereinX is18F; andM is La or Ce.

13. The metal complex of formula (II) of any one of claims 8-12, or a salt thereof, wherein Y is14. The metal complex of claim 8, or a salt thereof, wherein the metal complex is15. A pharmaceutical composition comprising the metal complex of formula (II) according to any one of claims 8-14, or a salt thereof, and a pharmaceutically acceptable excipient, carrier, or diluent.

16. A process for the preparation of a complex of formula (II), or a salt thereof,the process comprising the steps of:(a) converting a compound of formula (III) to a compound of formula (IV),(b) converting the compound of formula (IV) to the compound of formula (II) by:(b-i) converting the compound of formula (IV) to a compound of formula (V), then converting the compound of formula (V) to the compound of formula (II),(V) (II) or(b-ii) converting the compound of formula (IV) to a compound of formula (I), then converting the compound of formula (I) to the compound of formula (II),whereinX is halogen;Y is H or L-RTis a bond or a linker group,RTcomprises an antibody, antibody fragment, a Fibroblast Activation Protein alpha (FAP-alpha) binding moiety, a binding peptide, a binding polypeptide, a binding protein, an enzyme, a nucleobase-containing moiety, a lectin, or a small molecule targeting moiety;R1is H or Ci-4alkyl; andM is a metal.

17. The process of claim 16, wherein X is F or18F.

18. A method of treating a disease by radiation in a subject in need thereof, the method comprising administering to the subject an effective amount of a radioactive metal complex of formula (ILA), or a salt thereof, thereby the disease in the subject is treated,(II-A) whereinX is halogen,Y is H or L-RTis a bond or a linker group,RTcomprises an antibody, antibody fragment, a Fibroblast Activation Protein alpha (FAP-alpha) binding moiety, a binding peptide, a binding polypeptide, a binding protein, an enzyme, a nucleobase-containing moiety, a lectin, or a small molecule targeting moiety, andMi is an alpha emitting metal.

19. The method of claim 18, wherein Mi is225Ac,213Bi,212Bi, or212Pb.

20. A method of diagnosing a disease in a subject in need thereof, the method comprising: administering to the subject an effective amount of a metal complex of formula (II-B), or a salt thereof,(II-B) whereinX is halogen,Y is H or L-RTL is a bond or a linker group,R1comprises an antibody, antibody fragment, a Fibroblast Activation Protein alpha (FAP-alpha) binding moiety, a binding peptide, a binding polypeptide, a binding protein, an enzyme, a nucleobase-containing moiety, a lectin, or a small molecule targeting moiety,M2 is a positron or gamma emitting metal; and determining the biodistribution of the metal complex in the subject.21 A method of imaging a subject, the method comprising: administering to the subject an effective amount of a metal complex of formula (II-B), or a salt thereof,whereinX is halogen,Y is H or L-RTL is a bond or a linker group,RTcomprises an antibody, antibody fragment, a Fibroblast Activation Protein alpha (FAP-alpha) binding moiety, a binding peptide, a binding polypeptide, a binding protein, an enzyme, a nucleobase-containing moiety, a lectin, or a small molecule targeting moiety,M2 is a positron or gamma emitting metal; and detecting the metal complex in the subject, thereby generating an image of the subject.

22. The method of any one of claims 20-21, wherein M2 is226Ac,203Pb,132La, or134Ce.

23. A method of diagnosing a disease in a subject in need thereof, the method comprising: administering to the subject an effective amount of a metal complex of formula (II-C), or a salt thereof,(II-C) whereinY is H or L-RTL is a bond or a linker group,RTcomprises an antibody, antibody fragment, a Fibroblast Activation Protein alpha (FAP-alpha) binding moiety, a binding peptide, a binding polypeptide, a binding protein, an enzyme, a nucleobase-containing moiety, a lectin, or a small molecule targeting moiety,M is a metal; anddetermining the biodistribution of the metal complex in the subject.

24. A method of imaging a subject, the method comprising: administering to the subject an effective amount of a metal complex of formula (II-C), or a salt thereof,whereinY is H or L-RTL is a bond or a linker group,RTcomprises an antibody, antibody fragment, a Fibroblast Activation Protein alpha (FAP-alpha) binding moiety, a binding peptide, a binding polypeptide, a binding protein, an enzyme, a nucleobase-containing moiety, a lectin, or a small molecule targeting moiety,M is a metal; and detecting the metal complex in the subject, thereby generating an image of the subject.

25. The method of any one of claims 23-24, wherein M is a non-radioactive metal.

26. The method of any one of claims 20-25, wherein detecting the metal complex comprises performing a positron emission tomography (PET) or a single photon emission computed tomography (SPECT).

27. The method of any one of 18-24, wherein the disease is cancer or the subject has cancer.

28. The method of any one of claims 18-27, wherein Y of the metal complex of formula (II-A), (II-B), or (II-C) is

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