Keystone macrocyclic chelating agents and complexes thereof

Abstract

The invention relates to chelating agents and complexes of metal ions with the chelating agents useful in various applications, including therapeutic and diagnostic applications.

Description

Attorney Docket No. 061809-5015-WOKEYSTONE MACROCYCLIC CHELATING AGENTS AND COMPLEXES THEREOF FIELD OF THE INVENTION

[0001] The invention relates to new macrocyclic chelating agents including at least three bridging chelating moieties and metal ion complexes of these agents of use in therapeutic and / or diagnostic applications. In one embodiment, the invention relates to highly stable complexes of the chelating agents of the invention with radionuclides, e.g., Ac(III).BACKGROUND

[0002] Current radioimmunotherapy practice makes use of two classes of chelating agents: acyclic species based on diethylenetriamine pentaacetic acid (DTP A) or macrocyclic derivatives similar to 1, 4, 7, 20-tetraazacy clododeccane N, N', N", N"' -tetraacetic acid (DOTA). The former display more rapid association kinetics, while the DOTA-like compounds tend to produce a more stable complex, with the caveat that complexation typically requires harsher conditions such as high temperatures. A list of radiometals currently under clinical investigation (according to clinicaltrials.gov) includes actinium-225, thorium-227, bismuth-213, copper-64, gallium-67, gallium-68, holmium-166, indium-111, lutetium-177, rubidium-82, samarium-153, zirconium-89, strontium-89, technetium-99m, lead-212, and yttrium-90.

[0003] Lanthanide and actinide radiometal cations, in the absence of chelation, are largely deposited in bone, a significant concern given the potential for bone marrow suppression. Toxicity concerns that have arisen recently following the use of MRI contrast agents such as Gd+3DTP A, clearly underscore the insufficient control of the metal cation biodistribution by this chelating group. Similarly, radiometal loss can lead to a loss of signal specificity by targeted radiodiagnostics. Therefore, there is a recognized, compelling need for improved chelating agents for use in radioimmunotherapy. Such chelating agents and complexes and methods of their use are provided by the present invention.

[0004] Targeted radiopharmaceutical therapy (TRT) is an important and growing treatment modality for cancer. In TRT, a radioactive isotope (radioisotope) is localized to tumor lesions via a cancer-seeking component, and the radioactive decay of the radioisotope generates high energy particles that can kill the tumor cells. Many of the most useful radioisotopes for TRT are metal ions, which can be attached to the cancer-seeking component via a bifunctional chelator (BFC). One particularly promising metallic radioisotope is225Ac(III), which hasDB1 / 159329947.1 1Attorney Docket No. 061809-5015-WObeen shown to have an extremely potent tumoricidal effect in e.g. late-stage prostate cancer patients. However, currently available bifunctional chelators are not ideal for chelation of225Ac(III) due to its large size, i.e. the large size of225Ac(III) requires a greater number of donor atoms to saturate the coordination sphere than for smaller metal ions. Important progress toward improved chelation of225Ac(III) was achieved in 2009 with the report of bpl8c6 (a.k.a. macropa). There, it was found that the decadentate, macrocyclic chelator demonstrated the unusual property of binding more strongly to La(III) than to Lu(III). Roca-Sabio et al., J. Am. Chem. Soc. 2009, 131, 9, 3331-3341. This was an unusual result because the smaller size (and thus higher charge density) of Lu(III) more typically results stronger chelate complexes than for La(III). A short time later, it was realized that the increasing stability with larger metal ions of macropa could be extended to Ac(III), which is c.a. 0.1 angstroms larger than La(III). Thiele et al., Angewandte Chemie 2017, 56, 46, 14712-14717. However, macropa is limited by relatively low thermodynamic stability due to containing many donor atoms that do not form strong coordination bonds (e.g., ether oxygens atoms, amines) with hard Lewis acid ions (e.g., lanthanides and actinides). Thus, the problem of providing a class of highly stable Ac(III) complexes with a chelating agent, particularly a functionalizable chelating agent, remains.SUMMARY OF INVENTION

[0005] Addressing the absence in the art of metal-free chelating agents able to form highly stable complexes with large metal ions, e.g., Ac(III), La(III), in various embodiments, the present invention provides a new class of chelating agents and metal complexes of these chelating agents which are particularly useful in therapeutic and / or diagnostic applications.

[0006] In exemplary embodiments, there is provided a new family of decadentate macrocyclic bifunctional chelators containing five anionic bidentate chelating units, which have been designed to specifically accommodate and stably complex very large metal ions, e.g., La(III) and Ac(III). In various embodiments, the structure of these macrocycles allows them to adopt the lowest energy coordination geometry for 10-coordination (bicapped square antiprism). For example, the La(III) complex of an exemplary macrocyclic chelating agent is shown to be totally inert to treatment with excess DTPA. The225Ac(III) complexes of the new 10-coordinate macrocycles, like the La(III) complex, are exceptionally stable, and so the invention described herein addresses the very real need for more effective chelation of critically important medical isotopes, e.g., actinium-225.134La(III) is also of interest for PETDB1 / 159329947.1 2Attorney Docket No. 061809-5015-WOimaging (via Ce-134). Like other 1,2-HOPO systems, the chelators presented here are also effective sensitizers of Eu(III) photoluminescence. Other metal ions of use for diagnostic and therapeutic applications are chelated by the chelating agents of the invention, providing highly stable metal ion complexes of these chelating agents.

[0007] The invention provides a new class of chiral chelating agents in which the chirality of the bridging scaffold moi eties joining pendant chelating moi eties in the chelating agents effectively preorganize the complex for metal binding. In an exemplary embodiment, the chirality of amino acid derived bridging scaffold moieties preorganize the chelating agent for metal binding, with one pendant chelating moiety oriented above the plane of the macrocycle and one pendant chelating moiety below the plane of the macrocycle. The structure of exemplary macrocyclic chelating agents permits an efficacious low energy geometry, e.g., the predicted DFT bicapped square antiprism geometry of both the Ac(III) complex and the La(III) complex (Fig. 3).

[0008] Exemplary compounds (chelating agents, chelates) of this invention comprise a mixture of bridging scaffold moieties, bridging chelating moieties, pendant chelating moieties, and pendant scaffold moieities linked together to have the structure:wherein L1, L2, L3, L4and L5are independently selected bridging scaffold moieties; Abl, Ab2and Ab3are independently selected bridging chelating moieties; Apland Ap2are independently selected pendant chelating moieties; and Lpland Lp2are independently selected pendant scaffold moieties. In some embodiments, the pendant scaffold moieties are independently selected from H, substituted or unsubstituted alkyl, a solubilizing group (e.g., a polyether, such as poly(ethylene glycol)), a reactive functional group, a targeting moiety, a linker to a reactive functional group, a linker to a targeting moiety, or a linking moiety as defined herein.

[0009] The bridging chelating moieties and pendant chelating moieties of the present invention are independently selected from:DB1 / 159329947.1 3Attorney Docket No. 061809-5015-WOwherein A and G are independently selected from carbon, nitrogen and oxygen. J is selected from carbon and nitrogen. Each R1and R2is independently selected from H, an enzymatically labile group, a hydrolytically labile group, a metabolically labile group, a photolytically labile group and a single negative charge. In an exemplary embodiment, at least one of R1and R2is other than H. Each R6, R7, R8, R9, and R10is independently selected from a bond to L1, L2, L3, L4, L5, Lpl, or Lp2, alkanediyl attached to L1, L2, L3, L4, or L5, H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, halogen, CN, -CF3, -C(O)R17, -SO2NR17R18, -NR17R18, -OR17, -S(O)2R17, -COOR17, -S(O)2OR17, -OC(O)R17, -C(O)NR17R18, -NR17C(O)R18, -NR17SO2R18, and -NO2, wherein at least two of R6, R7, R8, R9, and R10are optionally joined to form a ring system selected from substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl and substituted or unsubstituted heteroaryl. In an exemplary embodiment, at least one of R6, R7, R8, R9, and R10is other than H. R17and R18are independently selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl and substituted or unsubstituted heterocycloalkyl; and R17and R18, together with the atoms to which they are attached, are optionally joined to form a 5-, 6- or 7-membered ring. In an exemplary embodiment, at least one of R17and R18is other than H. When A is oxygen, R9isDB1 / 159329947.1 4Attorney Docket No. 061809-5015-WOnot present; and when G is oxygen, R7is not present. In some embodiements, at least one of R6, R7, R8, R9, and R10comprise a CH3 group.

[0010] In various embodiments, Ablis attached to L2and L3through two members selected from R6, R7, R8, R9and R10; Ab2is attached to L1and L3through two members selected from R6, R7, R8, R9and R10; and / or Ab3is attached to L1and L2through two members selected from R6, R7, R8, R9and R10. In various embodiments, Aplis attached to L4and Lplthrough a member selected from R6, R7, R8, R9and R10; and / or Ap2is attached to L5and Lp2through a member selected from R6, R7, R8, R9and R10.

[0011] Advantages of exemplary compounds of the invention include chelating agents chelating the metal (e.g., isotope) rapidly, so that forming the metal chelate is compatible with the practicalities of clinical laboratory preparation (e.g., rapid chelation of a decaying isotope). Exemplary compounds also bind the cation stably so that a minimal amount of metal ion is released from the chelator in vivo (e.g., prior to radioactive decay, or during elimination of the chelate). These apparently contradictory properties of the compounds of the invention are realized through pre-organized chelating groups retaining a degree of flexibility allowing them to chelate the metal.

[0012] Exemplary compounds of the present invention also comprise a linker to a reactive functional group, a linker to a targeting moiety, or a linking moiety comprising a reactive functional group and / or targeting moiety, therefore, exemplary chelating agents and their complexes provided herein can be directed to a site of interest for therapeutic and / or diagnostic purposes. A moiety comprising the linker-functional group, -targeting moiety cassette can be a component of a bridging scaffold moiety, a bridging chelating moiety, a pendent chelating moiety, one or more of Lpland Lp2pendant scaffold moi eties, or any combination thereof.

[0013] Exemplary chelating agents of the present invention and metal ion chelates thereof are particularly useful for targeted radioisotope applications of Ac(III), for example, and sensitized luminescence applications (e.g., a lanthanide, such as Eu(III) sensitized luminescence immunoassays). As shown in the Examples, chelating agents of the present invention stably coordinate metal cations and display facile complexation kinetics. In an exemplary embodiment, the compound of the invention is a complex formed between225Ac(III) or La(III).DB1 / 159329947.1 5Attorney Docket No. 061809-5015-WO

[0014] Additional embodiments, objects and advantages of the invention will be apparent from the detailed description that follows.BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 is the lowest energy idealized (by repulsion energy, n=6) 10-coordinate geometry for M(unidentate)10systems.

[0016] Figure 2 is lowest energy idealized (by DFT, B3LYP / / 6-31G(d,p) and MWB78) 10-coordinate geometry for M(bidentate)5systems, specifically [Ac(1,2-HOPO)5]2−here.

[0017] Figure 3 is a comparison of DFT-minimized structures of the parent (i.e. no sidearm) form of chelator 10 with Ac(III) (left) and La(III) (right). Both structures adopt the expected low energy bicapped square antiprism geometry.

[0018] Figure 4 is (A) isomer 1 of chelator 10 and associated La(III) structure, and (B) isomer 2 of chelator 10 and associated La(III) structure. The two structures are only different with regard to the orientation of the keystone bridging bidentate 1,2-HOPO unit (Abl).

[0019] Figure 5. HPLC chromatograms of macrocyclic bifunctional chelator 10 diluted in Tris buffer pH 7.5 (top, A), lanthanum chelate 11 diluted in 50 mM DTP A, pH 7.4 (middle, B), and chelate 11 diluted in 50 mM DTP A, pH 7.4 after 24 hrs (bottom, C).

[0020] Figure 6. Structures of lanthanum(III) complex 11 (La-10, left) and La-Lumi804 (La-13, right).

[0021] Figure 7. HPLC chromatograms of macrocyclic bifunctional chelator Lumi80413 diluted in in 50 mM DTP A, pH 7.4 (top, A), 13 treated with LaCl3diluted in 0.1M Tris buffer, pH 7.5, (top middle, B), 13 treated with LaCl3and diluted in 50 mM DTPA, pH 7.4 (bottom middle, C), and 13 treated with LaCl3and diluted in 50 mM DTPA after 72 hours (bottom, D).

[0022] Figure 8. Reverse Phase HPLC chromatogram resulting from incubation of chelator 30 with actinium-225 at 37 °C for 30 minutes. Results indicate <0.3% of the free actinium-225 radiometal at 3-5 minutes and 99.96% activity in a peak corresponding to actinium-225 bound to chelator 30, chelate 54, at 10 - 14 minutes by fraction collection and gamma counter detection (Counts Per Minute).

[0023] Figure 9. Reverse Phase HPLC chromatogram resulting from incubation of chelator 10 with actinium-225 at 37 °C for 30 minutes. Results indicate <0.3 % of the free actinium- DB1 / 159329947.1 6Attorney Docket No. 061809-5015-WO225 radiometal at 3-5 minutes, and 99.84% activity in a peak corresponding to actinium-225 bound to chelator 10, chelate 53, at 10 - 14 minutes by fraction collection and gamma counter detection (Counts Per Minute).

[0024] Figure 10. Reverse Phase HPLC chromatogram resulting from incubation of chelator 36 with actinium-225 at 37 °C for 30 minutes. Results indicate <0.3 % of the free actinium-225 radiometal at 3-5 minutes, and 99.96% activity in a peak corresponding to actinium-255 bound to chelator 36, chelate 55, at 10 - 14 minutes by fraction collection and gamma counter detection (Counts Per Minute).

[0025] Figure 11. Reverse Phase HPLC chromatogram resulting from incubation of chelator Lumi804 with actinium-225 at 37 °C for 30 minutes. Results indicate 19.17% of the free actinium-225 radiometal at 3-5 minutes, and 80.29% activity in a peak corresponding to actinium-225 bound to chelator Lumi804, chelate Lumi804-Ac, at 10 - 14 minutes by fraction collection and gamma counter detection (Counts Per Minute).DESCRIPTION OF EMBODIMENTSDefinitions

[0026] Where substituent groups are specified by their conventional chemical formulae, written from left to right, they optionally equally encompass the chemically identical substituents, which would result from writing the structure from right to left, e.g., -CH2O- is intended to also recite -OCH2-.

[0027] The term “alkyl”, by itself or as part of another substituent, means a straight or branched chain hydrocarbon, which may be fully saturated, mono- or polyunsaturated and includes mono-, di- and multivalent radicals. Examples of saturated hydrocarbon radicals include, but are not limited to, groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, cyclohexyl, (cyclohexyl)methyl, cyclopropylmethyl, homologs and isomers of, for example, n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like. An unsaturated alkyl group is one having one or more double bonds or triple bonds (i.e., alkenyl and alkynyl moieties). Examples of unsaturated alkyl groups include, but are not limited to, vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl, 3-(l,4-pentadienyl), ethynyl, 1- and 3-propynyl, 3-butynyl, and the higher homologs and isomers. The term “alkyl” can refer to “alkylene”, which by itself or as part of another substituent means a divalent radical derived from an alkane, as exemplified, but not limited, by -CH2CH2CH2CH2-. Typically, anDB1 / 159329947.1 7Attorney Docket No. 061809-5015-WOalkyl (or alkylene) group will have from 1 to 30 carbon atoms. A “lower alkyl” or “lower alkylene” is a shorter chain alkyl or alkylene group, generally having eight or fewer carbon atoms. In some embodiments, alkyl refers to an alkyl or combination of alkyls selected from Cl, C2, C3, C4, C5, C6, C7, C8, C9, C10, Cll, C12, C13, C14, C15, C16, C17, C18, C19, C20, C21, C22, C23, C24, C25, C26, C27, C28, C29 and C30 alkyl. In some embodiments, alkyl refers to C1-C25 alkyl. In some embodiments, alkyl refers to C1-C20 alkyl. In some embodiments, alkyl refers to C1-C15 alkyl. In some embodiments, alkyl refers to C1-C10 alkyl. In some embodiments, alkyl refers to Ci-Ce alkyl.

[0028] The term “heteroalkyl,” by itself or in combination with another term, means an alkyl in which one or more carbons are replaced with one or more heteroatoms selected from the group consisting of O, N, Si and S, (preferably O, N and S), wherein the nitrogen and sulfur atoms may optionally be oxidized and the nitrogen heteroatom may optionally be quaternized. The heteroatoms O, N, Si and S may be placed at any interior position of the heteroalkyl group or at the position at which the alkyl group is attached to the remainder of the molecule. In some embodiments, depending on whether a heteroatom terminates a chain or is in an interior position, the heteroatom may be bonded to one or more H or substituents such as (Ci, C2, C3, C4, Cs or Ce) alkyl according to the valence of the heteroatom. Examples of heteroalkyl groups include, but are not limited to, -CH2-CH2-O-CH3, -CH2-CH2-NH-CH3, -CH2-CH2-N(CH3)-CH3, -CH2-S-CH2-CH3, -CH2-CH2-S(O)-CH3, -CH2-CH2-S(O)2-CH3, -CH=CH-O-CH3, -Si(CH3)3, -CH2-CH=N-OCH3, and -CH=CH-N(CH3)-CH3. No more than two heteroatoms may be consecutive, as in, for example, -CH2-NH-OCH3 and -CH2-O-Si(CH3)3, and in some instances, this may place a limit on the number of heteroatom substitutions. Similarly, the term “heteroalkylene, is sub-generic to “heteroalkyl” andby itself or as part of another substituent means a divalent radical derived from heteroalkyl, as exemplified, but not limited by, -CH2-CH2-S-CH2-CH2- and -CH2-S-CH2-CH2-NH-CH2-. The designated number of carbons in heteroforms of alkyl, alkenyl and alkynyl includes the heteroatom count. For example, a (Ci, C2, C3, C4, Cs or Ce) heteroalkyl will contain, respectively, 1, 2, 3, 4, 5 or 6 atoms selected from C, N, O, Si and S such that the heteroalkyl contains at least one C atom and at least one heteroatom, for example 1-5 C and 1 N or 1-4 C and 2 N. Further, a heteroalkyl may also contain one or more carbonyl groups. In some embodiments, a heteroalkyl is any C2-C30 alkyl, C2-C25 alkyl, C2-C20 alkyl, C2-C15 alkyl, C2-C10 alkyl or C2-C6 alkyl in any of which one or more carbons are replaced by one or moreDB1 / 159329947.1 8Attorney Docket No. 061809-5015-WOheteroatoms selected from O, N, Si and S (or from O, N and S). In some embodiments, each of 1, 2, 3, 4 or 5 carbons is replaced with a heteroatom.

[0029] The terms “alkoxy,” “alkylamino” and “alkylthio” (or thioalkoxy) are used in their conventional sense, and refer to those alkyl and heteroalkyl groups attached to the remainder of the molecule via an oxygen atom, a nitrogen atom (e.g., an amine group), or a sulfur atom, respectively.

[0030] The terms “cycloalkyl” and “heterocycloalkyl”, by themselves or in combination with other terms, refer to cyclic versions of “alkyl” and “heteroalkyl”, respectively. Additionally, for heterocycloalkyl, a heteroatom can occupy the position at which the heterocycle is attached to the remainder of the molecule. Examples of cycloalkyl include, but are not limited to, cyclopentyl, cyclohexyl, 1 -cyclohexenyl, 3 -cyclohexenyl, cycloheptyl, and the like. Examples of heterocycloalkyl include, but are not limited to, 1 -(1, 2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl, 1-piperazinyl, 2-piperazinyl, and the like.

[0031] The term “aryl” means a polyunsaturated, aromatic substituent that can be a single ring or optionally multiple rings (preferably 1, 2 or 3 rings) that are fused together or linked covalently. In some embodiments, aryl is a 3, 4, 5, 6, 7 or 8 membered ring, which is optionally fused to one or two other 3, 4, 5, 6, 7 or 8 membered rings. The term “heteroaryl” refers to aryl groups (or rings) that contain 1, 2, 3 or 4 heteroatoms selected from N, O, and S, wherein the nitrogen and sulfur atoms are optionally oxidized, and the nitrogen atom(s) are optionally quatemized. A heteroaryl group can be attached to the remainder of the molecule through a heteroatom. Non-limiting examples of aryl and heteroaryl groups include phenyl, 1 -naphthyl, 2-naphthyl, 4-biphenyl, 1 -pyrrolyl, 2-pyrrolyl, 3 -pyrrolyl, 3-pyrazolyl, 2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl, 2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl, 1 -isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl, 3-quinolyl, and 6-quinolyl.

[0032] In some embodiments, any of alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl and heteroaryl is optionally substituted. That is, in some embodiments, any of these groups isDB1 / 159329947.1 9Attorney Docket No. 061809-5015-WOsubstituted or unsubstituted. In some embodiments, substituents for each type of radical are selected from those provided below.

[0033] Substituents for the alkyl, heteroalkyl, cycloalkyl and heterocycloalkyl radicals (including those groups often referred to as alkylene, alkenyl, heteroalkylene, heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, and heterocycloalkenyl) are generically referred to as “alkyl group substituents”. In some embodiments, an alkyl group substituent is selected from -halogen, -OR’, =0, =NR’, =N-0R’, -NR’R”, -SR’, -SiR’R”R’”, -OC(O)R’, -C(O)R’, -CO2R’, -CONR’R”, -0C(0)NR’R”, - NR”C(0)R’, -NR’-C(0)NR”R’”, -NR”C(0)2R’, -NR-C(NR’R”R’”)=NR””, -NR-C(NR’R”)= NR’”, -S(O)R’, -S(O)2R’, -S(O)2NR’R”, -NRSO2R’, -CN and -NO2in a number ranging from zero to (2m’+l), where m’ is the total number of carbon atoms in such radical. In one embodiment, R’, R”, R’” and R”” are each independently selected from hydrogen, alkyl (e.g., Ci, C2, C3, C4, Cs and Ce alkyl). In one embodiment, R’, R”, R’” and R”” each independently refer to hydrogen, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, e.g., aryl substituted with 1-3 halogens, substituted or unsubstituted alkyl, alkoxy or thioalkoxy groups, or arylalkyl groups. In one embodiment, R’, R”, R’” and R”” are each independently selected from hydrogen, alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, alkoxy, thioalkoxy groups, and arylalkyl. When R’ and R” are attached to the same nitrogen atom, they can be combined with the nitrogen atom to form a 5-, 6-, or 7-membered ring. For example, -NR’R” can include 1-pyrrolidinyl and 4-morpholinyl. In some embodiments, an alkyl group substituent is selected from substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl and substituted or unsubstituted heteroaryl.

[0034] Similar to the substituents described for the alkyl radical, substituents for the aryl and heteroaryl groups are generically referred to as “aryl group substituents”. In some embodiments, an aryl group substituent is selected from -halogen, -OR’, =0, =NR’, =N-0R’, -NR’R”, -SR’, -SiR’R”R’”, -OC(O)R’, -C(O)R’, -CO2R’, -CONR’R”, -0C(0)NR’R”, -NR”C(0)R’, -NR’-C(0)NR”R’”, -NR”C(0)2R’, -NR-C(NR’R”R’”)=NR””, -NR-C(NR’R”)= NR’”, -S(O)R’, -S(O)2R’, -S(O)2NR’R”, -NRSO2R’, -CN and -NO2, -R’, -N3, -CH(Ph)2, fluoro(Ci-C4)alkoxy, and fluoro(Ci-C4)alkyl, in a number ranging from zero to the total number of open valences on the aromatic ring system. In some embodiments, R’, R”, R’” and R”” are independently selected from hydrogen and alkyl (e.g., Ci, C2, C3, C4, Cs and Ce DB1 / 159329947.1 10Attorney Docket No. 061809-5015-WOalkyl). In some embodiments, R’, R”, R’” and R”” are independently selected from hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl and substituted or unsubstituted heteroaryl. In some embodiments, R’, R”, R’” and R”” are independently selected from hydrogen, alkyl, heteroalkyl, aryl and heteroaryl. In some embodiments, an aryl group substituent is selected from substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl and substituted or unsubstituted heteroaryl.

[0035] Two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be replaced with a substituent of the formula -T-C(O)-(CRR’)q-U-, wherein T and U are independently -NR-, -O-, -CRR’- or a single bond, and q is an integer of from 0 to 3. Alternatively, two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be replaced with a substituent of the formula -A-(CH2)r-B-, wherein A and B are independently -CRR’-, -O-, -NR-, -S-, -S(O)-, -S(O)2-, -S(O)2NR’- or a single bond, and r is an integer of from 1 to 4. One of the single bonds of the new ring so formed may optionally be replaced with a double bond. Alternatively, two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be replaced with a substituent of theformula -(CRR’)s-X-(CR”R’”)d-, where s and d are independently integers of from 0 to 3, and X is -O-, -NR’-, -S-, -S(O)-, -S(O)2-, or -S(O)2NR’-. The substituents R, R’, R” and R’” are preferably independently selected from hydrogen or substituted or unsubstituted (Ci-Ce) alkyl.

[0036] The term “acyl” refers to a species that includes the moiety -C(O)R, where R has the meaning defined herein. Exemplary species for R include H, halogen, substituted or unsubstituted alkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, and substituted or unsubstituted heterocycloalkyl. In some embodiments, R is selected from H and (C1-C6)alkyl.

[0037] The terms “halo” or “halogen,” by themselves or as part of another substituent, mean, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom. Additionally, terms such as “haloalkyl,” are meant to include monohaloalkyl and polyhaloalkyl. For example, the term “halo(Ci-C4)alkyl” is mean to include, but not be limited to, trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, 3 -bromopropyl, and the like. In some embodiments, halogen refers to an atom selected from F, Cl and Br.DB1 / 159329947.1 11Attorney Docket No. 061809-5015-WO

[0038] The term “heteroatom” includes oxygen (O), nitrogen (N), sulfur (S) and silicon (Si). In some embodiments, a heteroatom is selected from N and S. In some embodiments, the heteroatom is O.

[0039] Unless otherwise specified, the symbol “R” is a general abbreviation that represents a substituent group that is selected from acyl, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl and substituted or unsubstituted heteroaryl. When a compound includes more than one R, R’, R”, R’” and R”” group, they are each independently selected.

[0040] For groups with solvent exchangeable protons, the ionized form is equally contemplated. For example, -COOH also refers to -COO⁻ and -OH also refers to -O'.

[0041] The use of five bidentate chelators to achieve 10-coordination necessarily lowers the symmetry of the idealized system to, at most, C2 point symmetry (i.e. two pairs of symmetry-related bidentate chelators and one unique chelator whose midpoint lies on the C2 symmetry axis). The first pair of symmetry related bidentate chelators connect the two capping donor atoms to two of the eight square antiprismatic donor atoms. Here, that set of bidentate chelators will be called the ‘bridging’ chelators. The lone chelator split by the C2 symmetry axis will be called the ‘keystone’ chelator. The remaining pair of symmetry related bidentate chelators will be called ‘pendant’ chelators.

[0042] Any of the compounds disclosed herein can be made into a pharmaceutically acceptable salt. The term “pharmaceutically acceptable salts” includes salts of compounds that are prepared with relatively nontoxic acids or bases, depending on the particular substituents found on the compounds described herein. When compounds of the present invention contain relatively acidic functionalities, base addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired base, either neat or in a suitable inert solvent. Examples of pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amino, or magnesium salt, or a similar salt. When compounds of the present invention contain relatively basic functionalities, acid addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired acid, either neat or in a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include those derived from inorganic acids like hydrochloric, hydrobromic, nitric, carbonic,DB1 / 159329947.1 12Attorney Docket No. 061809-5015-WOmonohydrogencarbonic, phosphoric, monohydrogenphosphoric, dihydrogenphosphoric, sulfuric, monohydrogensulfuric, hydriodic, or phosphorous acids and the like, as well as the salts derived from relatively nontoxic organic acids like acetic, propionic, isobutyric, maleic, malonic, benzoic, succinic, suberic, fumaric, lactic, mandelic, phthalic, benzenesulfonic, p-tolylsulfonic, citric, tartaric, methanesulfonic, and the like. Also included are salts of amino acids such as arginate and the like, and salts of organic acids like glucuronic or galactunoric acids and the like (see, for example, Berge etal., Journal of Pharmaceutical Science, 66: 1-19 (1977)). Certain specific compounds of the present invention contain both basic and acidic functionalities that allow the compounds to be converted into either base or acid addition salts. The neutral forms of the compounds are preferably regenerated by contacting the salt with a base or acid and isolating the parent compound in the conventional manner. The parent form of the compound differs from the various salt forms in certain physical properties, such as solubility in polar solvents, but otherwise the salts are equivalent to the parent form of the compound for the purposes of the present invention.

[0043] In addition to salt forms, the present invention provides any of the compounds disclosed herein in a prodrug form. Prodrugs of the compounds described herein are those compounds that readily undergo chemical changes under physiological conditions to provide the compounds of the present invention.

[0044] Certain compounds of the present invention can exist in unsolvated forms as well as solvated forms, including hydrated forms. In general, the solvated forms are equivalent to unsolvated forms and are encompassed within the scope of the present invention. Certain compounds of the present invention may exist in multiple crystalline or amorphous forms. In general, all physical forms are equivalent for the uses contemplated by the present invention and are intended to be within the scope of the present invention.

[0045] The compounds of the present invention may also contain unnatural proportions of atomic isotopes at one or more of the atoms that constitute such compounds. For example, the compounds may be labeled with deuterium (2H) or radiolabeled with radioactive isotopes, such as for example tritium (3H), iodine-125 (125I) or carbon-14 (14C). All isotopic variations of the compounds of the present invention, whether radioactive or not, are intended to be encompassed within the scope of the present invention.

[0046] Certain compounds of the invention are complexed with a non-decaying metal ion which is recognized as serving as a proxy for a radioactive metal ion. For example, certainDB1 / 159329947.1 13Attorney Docket No. 061809-5015-WOcompounds of the invention are complexed with La (III), which is recognized in the art to be a suitable, nonradioative surrogate for the Ac(III) ion. The La(III) complex is of use to investigate the kinetics and thermodynamics of the chelating agent and complex as the teachings of these investigations can be extrapolated to complexes with the chelating agent and Ac(III). Thiele et al., Cancer Biotherapy and Radiopharmaceutical 33(8):

[0047] The symbol, displayed perpendicular to a bond, indicates the point at which the displayed moiety is attached to the remainder of the molecule.

[0048] In some embodiments, the definition of terms used herein is according to IUPAC. Compositions

[0049] The invention provides numerous compounds (chelating agents, chelates) and metal ion complexes thereof. Generally, a chelating agent comprises a plurality of chelating moieties (e.g., pendant chelating moieties and / or bridging chelating moieties) that are linked together by way of a scaffold moiety (e.g., pendant scaffold moieties and / or bridging scaffold moieties).

[0050] Compounds (chelating agents, chelates) of the present invention and metal ion complexes thereof are particularly useful for targeted radioisotope applications (e.g., with Ac(III) or Th(IV) and sensitized luminescence applications (such as Eu sensitized luminescence immunoassays). As supported in the Examples, compounds (chelating agents, chelates) of the present invention stably coordinate Ac(III)-like metal ions, display facile complexation kinetics, and provide Ac(III)-like metal ion complexes possessing exceptionally high aqueous stability. An “Ac(III)-like” metal ion is generally a stable, art-recognized proxy for radioactive Ac(III); the stable nature of the Ac(III)-like metal ion enabling investigation of the properties of the the chelating agent and its complex without the necessity of the safety procedures associated with handling Ac(III).

[0051] There are several factors to be considered in the design of an alpha chelating agent for anticancer therapy. Some of the key issues apart from the kinetics will be the high affinity for the target metal (such as Ac, Th or Eu) which at the same time needs to have a low exchange rate for other biologically significant metal ions. Accordingly, in the instant chelating agent design, the electronic properties of the target metal and chelating agent are considered and matched. The chelate should also be able to assume the appropriate coordination cavity size and geometry for the desired metal. A coordination number of 8 or greater is generallyDB1 / 159329947.1 14Attorney Docket No. 061809-5015-WOpreferred by actinide ions as they have a tendency to form stable complexes with chelating agents of high denticity.

[0052] A chelating agent can comprise numerous chelating moieties. Particularly useful chelating agents contain a number of chelating moieties sufficient to provide, for example, 6, 8 or 10 heteroatoms such as oxygen that coordinate with a metal ion to form a complex. The heteroatoms such as oxygen provide electron density for forming coordinate bonds with a positively charged ion, and such heteroatoms can thus be considered “donors”. In some embodiments, the plurality of chelating moieties of a chelating agent comprises a plurality of oxygen donors and a metal ion (such as a radionuclide) is chelated to the chelating agent via at least one of the oxygen donors. In some embodiments, a chelating agent comprises a plurality of oxygen donors and a metal ion (such as a radionuclide) is chelated to the chelating agent via a plurality or all of the oxygen donors.Chelating Agents / Chelates

[0053] In one aspect, the invention provides a compound (chelating agents, chelates) having the structure:wherein L1, L2, L3, L4and L5are independently selected bridging scaffold moieties; Abl, Ab2and Ab3are independently selected bridging chelating moieties; Apland Ap2are independently selected pendant chelating moieties; and Lpland Lp2are independently selected pendant scaffold moieties. In some embodiments, the pendant scaffold moieties are independently selected from H, substituted or unsubstituted alkyl, a solubilizing group (e.g., a polyether, such as poly(ethylene glycol)), a reactive functional group, a targeting moiety, a sidearm, a linker to a reactive functional group, and a linker to a targeting moiety.

[0054] In some embodiments, L4is selected from substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted cycloalkenyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted arylalkyl, substituted or unsubstituted biaryl,DB1 / 159329947.1 15Attorney Docket No. 061809-5015-WOsubstituted or unsubstituted heteroaryl, and a substituted or unsubstituted polycyclic ring system. In some embodiments, L4is selected from substituted or unsubstituted C1-C8 alkyl, and substituted or unsubstituted C1-C8 heteroalkyl in which 1, 2, or 3 carbon atoms are replaced with a heteroatom, wherein each heteroatom is independently selected from N and O. In some embodiments, L4is selected from substituted or unsubstituted C1-C6 alkyl, and substituted or unsubstituted C1-C6 heteroalkyl in which 1 or 2 carbon atoms are replaced with a heteroatom, wherein each heteroatom is independently selected from N and O. In some embodiments, L4is selected from substituted or unsubstituted C2-C5 alkyl, and substituted or unsubstituted C2-C5 heteroalkyl in which 1 carbon atom is replaced with a nitrogen atom.

[0055] In some embodiments, L5is selected from substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted cycloalkenyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted arylalkyl, substituted or unsubstituted biaryl, substituted or unsubstituted heteroaryl, and a substituted or unsubstituted polycyclic ring system. In some embodiments, L5is selected from substituted or unsubstituted C1-C8 alkyl, and substituted or unsubstituted C1-C8 heteroalkyl in which 1, 2, or 3 carbon atoms are replaced with a heteroatom, wherein each heteroatom is independently selected from N and O. In some embodiments, L5is selected from substituted or unsubstituted C1-C6 alkyl, and substituted or unsubstituted C1-C6 heteroalkyl in which 1 or 2 carbon atoms are replaced with a heteroatom, wherein each heteroatom is independently selected from N and O. In some embodiments, L5is selected from substituted or unsubstituted C2-C5 alkyl, and substituted or unsubstituted C2-C5 heteroalkyl in which 1 carbon atom is replaced with a nitrogen atom.

[0056] L1, L2, L3, bridging scaffold moieties, bridging chelating moieties, pendant chelating moieties, and pendant scaffold moieties are as defined herein.

[0057] All combinations of L1, L2, L3L4, L5, Abl, Ab2, Ab3, Apl, Ap2, Lpland Lp2are encompassed by this disclosure and specifically provided by the invention.

[0058] In some embodiments, the compound (chelating agent, and / or complex) comprises a sidearm, a linker to a reactive functional group, or a linker to a targeting moiety. In some embodiments, at least one of L1, L2, L3L4, L5, Abl, Ab2, Ab3, Apl, Ap2, Lpland Lp2is substituted with a sidearm, a reactive functional group, a targeting moiety, a linker to a reactive functional group, or a linker to a targeting moiety. In some embodiments, at leastDB1 / 159329947.1 16Attorney Docket No. 061809-5015-WOone of L1, L2, L3L4, L5, Abl, Ab2, Ab3, Apl, and Ap2is substituted with a sidearm, a reactive functional group, a targeting moiety, a linker to a reactive functional group, or a linker to a targeting moiety. In some embodiments, at least one of L1, L2, L3L4, and L5is substituted with a sidearm, a reactive functional group, a targeting moiety, a linker to a reactive functional group, or a linker to a targeting moiety. In some embodiments, at least one of L1, Lpl, and Lp2is, or is substituted with, a sidearm, a linker comprising a reactive linker moiety, or a targeting linker moiety.

[0059] The sidearm, the linker to a reactive functional group, and the linker to a targeting moiety are as defined herein. In some embodiments, the reactive functional group is a protected reactive functional group.

[0060] In some embodiments, the compound (chelating agent) comprises one or more modifying moieties. The modifying moieties can be the same or different.

[0061] In some embodiments, L4, L5, Apl, Ap2, Lpl, and Lp2are absent.Bridging and Pendant Scaffold Moieties

[0062] In some embodiments, one or more of L1, L2and L3has a structure according to formula (A):|_x6RL1Rb2(A)whereineach Llais independently selected from substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted cycloalkenyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted arylalkyl, substituted or unsubstituted biaryl, substituted or unsubstituted heteroaryl, and a substituted or unsubstituted polycyclic ring system;each L1band L1care independently selected from a bond, −C(O)−, substituted or unsubstituted alkyl, and substituted or unsubstituted heteroalkyl; andDB1 / 159329947.1 17Attorney Docket No. 061809-5015-WOeach RL1and RL2are independently selected from H, substituted or unsubstituted alkyl, and substituted or unsubstituted heteroalkylwhereinwhen L1has the structure according to formula (A), Lx6is selected from H, substituted or unsubstituted alkyl, a solubilizing group (e.g., a polyether, such as poly(ethylene glycol)), a reactive functional group, a targeting moiety, a sidearm, a linker to a reactive functional group, and a linker to a targeting moiety;when L2has the structure according to formula (A), Lx6is a bond to L4; and when L3has the structure according to formula (A), Lx6is a bond to L5.

[0063] Any combination of Lla, Llb, Llc, Lx6, RL1, and RL2are encompassed by this disclosure and specifically provided by the invention. When more than one of L1, L2and L3has the structure according to formula (A), each Lla, Llb, Llc, Lx6, RL1, and RL2may be the same or different.

[0064] In some embodiments, one or more of L1, L2and L3is substituted with a sidearm, a linker to a reactive functional group, or a linker to a targeting moiety. The sidearm or the linker can be a component of any one or more of Lla, Llb, and / or Llc. In an exemplary embodiment, the sidearm or the linker is a component of Lla. In an exemplary embodiment, the sidearm or the linker is Lx6.

[0065] In some embodiments, when L1has the structure according to formula (A), Lx6is selected from H, a sidearm, a reactive functional group, a targeting moiety, a linker to a reactive functional group, and a linker to a targeting moiety. In some embodiments, Lx6comprises a modifying moiety, e.g., a solubilizing group.

[0066] In some embodiments, when one or more of L1, L2and L3has the structure according to formula (A), L4, L5, and each Llaare independently selected from substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted cycloalkenyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted arylalkyl, substituted or unsubstituted biaryl, substituted or unsubstituted heteroaryl, and a substituted or unsubstituted polycyclic ring system. In some embodiments, L4and L5are each independently selected from substituted or unsubstituted C1-C8 alkyl, and substituted or unsubstituted C1-C8 heteroalkyl in which 1, 2, orDB1 / 159329947.1 18Attorney Docket No. 061809-5015-WO3 carbon atoms are replaced with a heteroatom, wherein each heteroatom is independently selected from N and O. In some embodiments, L4and L5are each independently selected from substituted or unsubstituted C1-C6 alkyl, and substituted or unsubstituted C1-C6 heteroalkyl in which 1 or 2 carbon atoms are replaced with a heteroatom, wherein each heteroatom is independently selected from N and O. In some embodiments, L4and L5are each independently selected from substituted or unsubstituted C2-C5 alkyl, and substituted or unsubstituted C2-C5 heteroalkyl in which 1 carbon atom is replaced with a nitrogen atom.

[0067] In some embodiments, one or more of L1, L2and L3has a structure according to Formula (B):|_x6I O L1aOV Nz XN—■V H HXX (B)wherein Lla, and Lx6are as defined herein. When more than one of L1, L2and L3has the structure according to formula (B), each Llamay be the same or different. In some embodiments, when L1has the structure according to formula (B), Lx6is selected from H, a sidearm, a linker to a reactive linker moiety, or a linker to a targeting moiety; optionally wherein said sidearm, said linker, and / or L1comprises a modifying moiety, e.g., a solubilizing moiety. When L2has the structure according to formula (B), Lx6is a bond to L4. When L3has the structure according to formula (B), Lx6is a bond to L5.

[0068] Exemplary moieties for Lla, Llb, Llc, Lx6include substituted or unsubstituted alkyl, and substituted or unsubstituted heteroalkyl. Exemplary substituted or unsubstituted alkyl moieties for Lla, Llb, Llcare C2-C5 substituted or unsubstituted alkyl moieties. Exemplary substituted or unsubstituted heteroalkyl moieties for Lla, Llb, Llcare C2-C5 substituted or unsubstituted heteroalkyl moieties in which 1 or 2 C atoms are replaced with a heteroatom selected from N and O. In some embodiments, Lx6is H.

[0069] In some embodiments, when one or more of L1, L2and L3has the structure according to Formula (B), L4, and L5are independently selected from substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, and -(CH2)aNRpC(O)-, wherein a is an integer selected from 2, 3, 4, 5, and 6 and Rpis independently selected from H, substituted or unsubstituted alkyl, and substituted or unsubstituted heteroalkyl. In an exemplary embodiment, Rpis H.DB1 / 159329947.1 19Attorney Docket No. 061809-5015-WO

[0070] In some embodiments, Llahas the structure:wherein Rel, Re2, Re3, and Re4are independently selected from H, a bond, cyano, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted cycloalkenyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl; and two members selected from Rel, Re2, Re3, and Re4, together with the atom to which they are attached, are optionally joined, to form a substituted or unsubstituted ring (or ring system) selected from substituted or unsubstituted cycloalkyl, substituted or unsubstituted cycloalkenyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl. In some embodiments, a member selected from Rel, Re2, Re3, and Re4is Lx6. In some embodiments, a member selected from Rel, Re2, Re3, and Re4comprises a sidearm. In some embodiments, Relor Re2and Re3or Re4are hydrogen. In some embodiments, Re2or Re3is hydrogen. In some embodiments, a member selected from Rel, Re2, Re3, and Re4is a bond to L4. In some embodiments, a member selected from Rel, Re2, Re3, and Re4is a bond to L5.

[0071] In an exemplary embodiment, Llahas the structure:wherein Re2and Re3are as defined herein.

[0072] In an exemplary embodiement Llahas the structure,k / VW' k / WV'DB1 / 159329947.1 20Attorney Docket No. 061809-5015-WOwherein n is an integer selected from 0, 1, 2, 3, 4, 5, 6, and 7; and B is independently selected from -CH2-, -NH-, -O-, alkyl, and heteroalkyl. In an exemplary embodiment, n is 0.

[0073] In an exemplary embodiment, at least one of Rel, Re2, Re3, and Re4is a sidearm, a linker to a reactive functional group, or a linker to a targeting moiety. In some embodiments, at least one of Rel, Re2, Re3, and Re4is Lx6.

[0074] In some embodiments, Llais selected from:DB1 / 159329947.1 21Attorney Docket No. 061809-5015-WOwherein m and n are each an integer independently selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, and 9. Each R is as defined herein. R1and Rlaare used interchangeably with reference to the structures above. In preferred embodiments, R1and / or Rlais / are independently selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, and a modifying moiety; X is O, S, or CH₂. Any position in these moieties can be substituted with a sidearm, a linker to a reactive functional group, or a linker to a targeting moiety. In an exemplary embodiment, one or more position on one of the moieties shown above is substituted with Lx6.

[0075] In an exemplary embodiment, Llais a member selected from 5 membered ring moieties and 6 membered ring moieties. In various emodiments, this moiety is substituted with one or more linker to a functional group or linker to a targeting moiety. In an exemplary embodiment, the linker is Lx6.

[0076] In another preferred embodiment, Llais a member selected from 5 membered ring moieties and 6 membered ring moieties, wherein the 5 membered ring moiety or the 6 membered ring moiety is part of a fused ring system. In various emodiments, this moiety isDB1 / 159329947.1 22Attorney Docket No. 061809-5015-WOsubstituted with one or more linker to a functional group or linker to a targeting moiety. In an exemplary embodiment, the linker is Lx6.

[0077] In some embodiments, Llais not unsubstituted Ci, C2, or linear C3 alkyl. In some embodiments, Llais not an unsubstituted, linear alkyl. In some embodiments, Llais not unsubstituted alkyl.

[0078] In an exemplary embodiment, Llais selected from:wherein R is as defined herein.

[0079] In an exemplary embodiment, Llais selected from:wherein n = 0, 1, 23, 4, 5, 6, or 7.

[0080] In an exemplary embodiment, Llais selected from:wherein n = 0, 1, 2 or 3; and R is as defined herein.

[0081] In some embodiments, Llais selected from:DB1 / 159329947.1 23Attorney Docket No. 061809-5015-WOwherein m and n are integers independently selected from 0, 1, 2, 3, 4, 5, and 6; and R is a reactive functional group, a targeting moiety, a reactive linking moiety, or a targeting linking moiety.

[0082] In some embodiments, Llais selected from:wherein m is an integer selected from 1, 2, 3, and 4; n is an integer selected from 0, 1, 2, and 3; and R is Lx6, a reactive functional group, a targeting moiety, a reactive linking moiety, or a targeting linking moiety.

[0083] In another preferred embodiment, Llahas the structure:wherein R and Rlaare defined herein.

[0084] In some embodiments, any implicit hydrogen atom in the Llamoieties shown above is optionally replaced by substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, or a modifying moiety. In various emodiments, an implicit hydrogen atom is replaced with one or more linker to a functional group or linker to a targeting moiety. In an exemplary embodiment, the linker is Lx6.DB1 / 159329947.1 24Attorney Docket No. 061809-5015-WO

[0085] In some embodiments, Llband Llcare independently selected from a bond, -C(O)-, substituted or unsubstituted alkyl, and substituted or unsubstituted heteroalkyl.

[0086] In some embodiments, Llband Llcare independently selected from a bond, -C(O)-, -(CH2)aC(O)-, and -O(CH2)aC(O)-; wherein a is an integer selected from 1, 2, 3, 4, 5, and 6.

[0087] In various embodiments, one or more of Llband Llcis substituted with one or more linker to a functional group or linker to a targeting moiety. In an exemplary embodiment, the linker is Lx6.

[0088] In a preferred embodiment, Llband Llcare each -C(O)-.

[0089] In some embodiments, one or more of L1, L2and L3has the structure:wherein n is an integer independently selected from 1, 2, 3, and 4.

[0090] In some embodiments, L4, and L5are independently selected from substituted or unsubstituted alkyl and substituted or unsubstituted heteroalkyl.

[0091] In some embodiments, L4, and L5are independently selected from Lx6.

[0092] In some embodiments L4and L5are bonds.

[0093] In some embodiments, L4, and L5are independently selected from substituted or unsubstituted Ci-Cs alkyl.

[0094] In some embodiments, L4and L5are independently selected from substituted or unsubstituted C2, C3, C4 and Cs alkyl; and L2bis selected from substituted or unsubstituted C2, C3, C4, and C5alkyl.

[0095] In some embodiments, one or both L4and L5are independently selected from (CH2)PNHC(O)-X in which X represents Aplor Ap2, and p is an integer selected from 1, 2, 3, 4, 5, 6, 7, or 8. In an exemplary embodiment p is 3, 4 or 5. In an exemplary embodiment, p is 4.

[0096] In some embodiments, one or more of L4, and L5is substituted with a sidearm, a linker to a reactive functional group, or a linker to a targeting moiety. In some embodiments, DB1 / 159329947.1 25Attorney Docket No. 061809-5015-WOL4is substituted with a sidearm, a linker to a reactive functional group, or a linker to a targeting moiety. In some embodiments, L5is substituted with a sidearm, a linker to a reactive functional group, or a linker to a targeting moiety.

[0097] In some embodiments, RL3and RL4are independently selected from hydrogen, substituted or unsubstituted alkyl, and substituted or unsubstituted heteroalkyl. In some embodiments, RL3and RL4are each H.Bridging and Pendant Chelating Moietieswherein A and G are independently selected from carbon, nitrogen and oxygen; J is selected from carbon and nitrogen. Each R1and R2is independently selected from H, an enzymatically labile group, a hydrolytically labile group, a metabolically labile group, a photolytically labile group and a single negative charge. Each R6, R7, R8, R9, and R10is independently selected from H, a bond to one or more of L1- L5, alkanediyl attached to one or more of L1- L5, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, halogen, CN, -CF3, -C(O)R17, -SO2NR17R18, -NR17R18, -OR17, -S(O)2R17, -COOR17, -S(O)2OR17, -OC(O)R17, -C(O)NR17R18, -NR17C(O)R18, -NR17SO2R18, and -NO2, wherein at least two of R6, R7, R8, R9, and R10are optionally joined to form a ring system selected from substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl and substituted or unsubstituted heteroaryl. R17and R18areDB1 / 159329947.1 26Attorney Docket No. 061809-5015-WOindependently selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl and substituted or unsubstituted heterocycloalkyl; and R17and R18, together with the atoms to which they are attached, are optionally joined to form a 5-, 6- or 7-membered ring.

[0099] In an exemplary embodiment, when A is oxygen, R9is not present; and when G is oxygen, R7is not present.

[0100] In some embodiments, when Ablhas a structure according to formula (I), Ablis attached to L2and L3through R6and R10; when Ablhas a structure according to formula (Ila), (lib), or (III), Ablis attached to L2and L3through R6and R9; when Ab2has a structure according to formula (I), Ab2is attached to L1and L3through R6and R10; when Ab2has a structure according to formula (Ila), (lib), or (III), Ab2is attached to L1and L3through R6and R9; when Ab3has a structure according to formula (I), Ab3is attached to L1and L2through R6and R10; when Ablhas a structure according to formula (Ila), (Illb), or (III), Ablis attached to L1and L2through R6and R9. When Aplhas a structure according to formula (I), Aplis attached to L4through R6or R10; when Aplhas a structure according to formula (Ila), (lib), or (III), Aplis attached to L4through R6or R9; when Aplhas a structure according to formula (IVa) or (IVb), Aplis attached to L4though R6, R7, R8, R9, or R10; when Ap2has a structure according to formula (I), Ap2is attached to L5through R6or R10; when Ap2has a structure according to formula (Ila), (lib), or (III), Ap2is attached to L5through R6or R9; and when Ap2has a structure according to formula (IVa) or (IVb), Ap2is attached to L5through R6, R7, R8, R9, or R10.

[0101] In some embodiements, at least one of R6, R7, R8, R9, and R10comprise a CH3 group. In an exemplary embodiment, Apland Ap2have a structure according to formula (Ila), and one or two of R6, R7, R8, and R9comprise a CH3 group.

[0102] In some embodiements, formula (lib) may take a tautomeric form of the thiohydroxypyridine coordinating unit. Preferred IUPAC names are 1 -hydroxy -2(1 H)-pyridinethione (thione form, right) and 2-pyridinethiol 1 -oxide (thiol form, left). Both forms are encompassed by this disclosure and specifically provided by the invention.DB1 / 159329947.1 1Attorney Docket No. 061809-5015-WO

[0103] In some embodiments, Abl, Ab2, Ab3, Apland Ap2are each independently selected from:

[0104] In a preferred embodiment, each Abl, Ab2, Ab3, Apland Ap2has a structure according to formula (2b).

[0105] In another preferred embodiment, each Abl, Ab2, Ab3, Apland Ap2has a structure according to formula (2a).

[0106] In another preferred embodiment, each Abl, Ab2, Ab3, Apland Ap2has a structure according to formula (1).

[0107] In various embodiments, there is provided a compound in which Abl, Ab2, Ab3, Apland Ap2are members independently selected from:wherein each R7, and R8are independently selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, halogen, CN, -CF3, −C(O)R17, -SO2NR17R18, -NR17R18, -OR17, -S(O)2R17, -COOR17, -S(O)2OR17, -OC(O)R17, -C(O)NR17R18,DB1 / 159329947.1 28Attorney Docket No. 061809-5015-WO-NR17C(O)R18, -NR17SO2R18, and -NO2, wherein R7and R8are optionally joined to form a ring system selected from substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl and substituted or unsubstituted heteroaryl. R17and R18are independently selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl and substituted or unsubstituted heterocycloalkyl; and R17and R18, together with the atoms to which they are attached, are optionally joined to form a 5-, 6- or 7-membered ring.

[0108] In some embodiments, each R7, and R8is independently selected from a bond to L1, L2or L3, and alkanediyl attached to L1, L2or L3.

[0109] In some embodiments, Abl, Ab2, Ab3, Apland Ap2are each independently selected from a chelating moiety comprising:DB1 / 159329947.1 29Attorney Docket No. 061809-5015-WOin which x and y are independently 0 or 1, with the proviso that x and y are not both 0 or both 1; Q is selected from O, S, and SH; and n is an integer selected from 1, 2, 3, 4, 5, and 6. This representation is intended to illustrate two different orientations of the chelating moieties when attached to another component of the chelating agent. In the figures above, the moiety terminated by the wavy line indicates a point of attachment between the chelating moiety and another component of the chelating agent, e.g., the moiety is a bond, a methylene, ethylene, propylene, butylene or higher order divalent moiety, or a heteroalkyl analogue of one of these moieties, attaching the chelating moiety to another component of the chelating agent. The divalent moiety can be substituted or unsubstituted.

[0110] In some embodiments, Abl, Ab2, Ab3, Apland Ap2are each independently selected from a chelating moiety comprising:This representation, and those in the paragraphs and figures below, illustrate the orientations of the chelating groups in two different embodiments of the chelating agents when attached to another component of the chelating agent. In the figures above, the moiety terminated by the wavy line indicates a point of attachment between the chelating moiety and another component of the chelating agent, e.g., the moiety is a bond, an alkylene or heteroalkylen (e.g. a methylene, ethylene, propylene, butylene or higher order divalent moiety, or a heteroalkyl analogue of one of these moieties), attaching the chelating moiety to another component of the chelating agent. The divalent moiety can be substituted or unsubstituted.

[0111] In some embodiments, a member selected from Abl, Ab2, Ab3, Apl, Ap2and a combination thereof is not a moiety comprising:DB1 / 159329947.1 30Attorney Docket No. 061809-5015-WO

[0112] In some embodiments, Abl, Ab2, Ab3, Apland Ap2are each independently selected from a moiety comprising:

[0113] In some embodiments, Apland Ap2are each independently selected from a moiety comprising:

[0114] In some embodiments, Apland Ap2are each independently selected from a moiety comprising:RandR

[0115] In some embodiments, Apland Ap2each are a moiety comprising:R9

[0116] In some embodiments, Apland Ap2each are a moiety comprising:DB1 / 159329947.1 31Attorney Docket No. 061809-5015-WOX^OH

[0117] In some embodiments, Apland Ap2each are a moiety comprising:-NX^OH

[0118] In some embodiments, one or both of Apland Ap2comprise a modifying moiety. Exemplary modifying moieties are as defined herein. In some embodiments, R9of Apl, Ap2, or Apland Ap2comprises a modifying moiety. In some embodiments, R9of Apl, Ap2, or Apland Ap2is -C(O)NR17R18, wherein R17is H and R18is a modifying moiety. In some embodiments, R6of Apl, Ap2, or Apland Ap2comprises a modifying moiety. In some embodiments, R6of Apl, Ap2, or Apland Ap2is -C(O)NR17R18, wherein R17is H and R18is a modifying moiety. In an exemplary embodiment, one or both of R6and R9is COOH, or COO’.Linker to Functional Group / Targeting Moiety

[0119] A “linker”, “linking member”, or “linking moiety”, as used herein, is a moiety that joins or potentially joins, covalently or noncovalently, a first moiety to a second moiety. In particular, a linker attaches or is configured to attach a chelating agent described herein to another molecule, such as a targeting moiety. In some embodiments, a linker attaches or is configured to attach a chelating agent described herein to a solid support. A linker comprising a reactive functional group that can be reacted with a reactive functional group on a structure of interest, e.g., a targeting moiety, a modifying moiety, to attach the structure of interest to the chelating agent through the linker is referred to as a “functionalized linker”. In exemplary embodiments, a linker is a functionalized linker. In exemplary embodiments, a chelating agent comprises one or more functionalized linkers. In some embodiments, a linker comprises a targeting moiety. In some embodiments, a linker to a targeting moiety comprises a bond to the targeting moiety. In some embodiments, the linker is a linker to a reactive functional moiety, or a linker to a targeting moiety. In some embodiments, the reactive functional group is a protected reactive functional group. In some embodiments, the linker isDB1 / 159329947.1 32Attorney Docket No. 061809-5015-WOa linker to a reactive functional group, or a linker to a targeting moiety. In an exemplary embodiment, the linker is a component of the sidearm as defined herein.

[0120] A linker can be any useful structure for that joins a chelating agent to a reactive functional group or a targeting moiety, such as an antibody. Examples of a linker include 0-order linkers (i.e., a bond), substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl and substituted or unsubstituted heteroaryl. Further exemplary linkers include substituted or unsubstituted (Ci, C2, C3, C4, Cs, Ce, C7, Cs, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19 C20, C21, C22, C23, C24, or C25) alkyl, substituted or unsubstituted (Ci, C2, C3, C4, Cs, Ce, C7, Cs, C9, C10, C11, C12, C13, C14, C15, Cie, C17, Cis, C19 C20, C21, C22, C23, C24, or C25) heteroalkyl, -C(O)NR’-, -C(O)O-, -C(O)S-, and -C(O)CR R, wherein R and R are members independently selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted Ci, C2, C3, C4, Cs, Ce, C7, Cs, C9 or C10 heteroalkyl with one or more heteroatoms (e.g., N, O, S, P), substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl and substituted or unsubstituted heterocycloalkyl. In some embodiments, a linker includes at least one heteroatom. Exemplary linkers also include one or more of -C(O)NH-, -C(O), -NH-, -S-, -O-. In an exemplary embodiment, the linker comprises substituted or unsubstituted (Ci, C2, C3, C4, Cs, Ce, C7, Cs, C9, C10, C11, C12, C13, C14, Cis, Cie, C17, Cis, C19 C20, C21, C22, C23, C24, or C25) heteroalkyl in which 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 C atoms are replaced with heteroatoms selected from N and O. In an exemplary embodiment, a linker is a heteroalkyl moiety substituted with a reactive functional group. Reactive Functional Groups

[0121] In one embodiment, a linker comprises a reactive functional group (or a “reactive functional moiety”, used synonymously), which can be reacted to covalently attach the linker to a targeting moiety. Reactive functional groups and classes of reactions useful in practicing the present invention are generally those that are well known in the art of bioconjugate chemistry. Currently favored classes of reactions available with reactive functional groups of the invention are those which proceed under relatively mild conditions. These include, but are not limited to nucleophilic substitutions (e.g., reactions of amines and alcohols with acyl halides and activated esters), electrophilic substitutions (e.g., enamine reactions) and additions to carbon-carbon and carbon-heteroatom multiple bonds (e.g., Michael reactions and Diels-Alder reactions). These and other useful reactions are discussed, for example, inDB1 / 159329947.1 33Attorney Docket No. 061809-5015-WOMarch, Advanced Organic Chemistry (3rd Ed., John Wiley & Sons, New York, 1985);Hermanson, Bioconjugate Techniques (Academic Press, San Diego, 1996); and Feeney et al., Modification of Proteins, Advances in Chemistry Series, Vol. 198 (American Chemical Society, Washington, D. C., 1982).

[0122] In some embodiments, a reactive functional group refers to a group selected from olefins, acetylenes, alcohols, phenols, ethers, oxides, halides, aldehydes, ketones, carboxylic acids, esters, amides, cyanates, isocyanates, thiocyanates, isothiocyanates, amines, hydrazines, hydrazones, hydrazides, diazo, diazonium, nitro, nitriles, mercaptans, sulfides, disulfides, sulfoxides, sulfones, sulfonic acids, sulfinic acids, acetals, ketals, anhydrides, sulfates, sulfenic acids isonitriles, amidines, imides, imidates, nitrones, hydroxylamines, oximes, hydroxamic acids thiohydroxamic acids, allenes, ortho esters, sulfites, enamines, ynamines, ureas, pseudoureas, semicarbazides, carbodiimides, carbamates, imines, azides, azo compounds, azoxy compounds, and nitroso compounds. Reactive functional groups also include those used to prepare bioconjugates, e.g., N-hydroxysuccinimide esters, maleimides and the like. Methods to prepare each of these functional groups are well known in the art and their application or modification for a particular purpose is within the ability of one of skill in the art see, for example, Sandler and Karo, eds., Organic Functional Group Preparations, (Academic Press, San Diego, 1989)).

[0123] A reactive functional group can be chosen according to a selected reaction partner. As an example, an activated ester, such as an NHS ester will be useful to label a protein via lysine residues. Sulfhydryl reactive groups, such as maleimides can be used to label proteins via amino acid residues carrying an SH-group (e.g., cystein). Antibodies may be labeled by first oxidizing their carbohydrate moi eties (e.g., with periodate) and reacting resulting aldehyde groups with a hydrazine containing chelating agent.

[0124] The reactive functional groups can be chosen such that they do not participate in, or interfere with, the reactions necessary to assemble the reactive chelating agent. Alternatively, a reactive functional group can be protected from participating in the reaction by means of a protecting group. Those of skill in the art understand how to protect a particular functional group so that it does not interfere with a chosen set of reaction conditions. For examples of useful protecting groups, see, for example, Greene et al., PROTECTIVE GROUPS IN ORGANIC SYNTHESIS, John Wiley & Sons, New York, 1991.DB1 / 159329947.1 34Attorney Docket No. 061809-5015-WOAmines and Amino-Reactive Groups

[0125] In one embodiment, a reactive functional group is selected from an amine, (such as a primary or secondary amine), hydrazine, hydrazide and sulfonylhydrazide. Amines can, for example, be acylated, alkylated or oxidized. Useful non-limiting examples of amino-reactive groups include N-hydroxysuccinimide (NHS) esters, sulfur-NHS esters, imidoesters, isocyanates, isothiocyanates, acylhalides, arylazides, p-nitrophenyl esters, aldehydes, sulfonyl chlorides, thiazolides and carboxyl groups.

[0126] NHS esters and sulfo-NHS esters react preferentially with primary (including aromatic) amino groups of a reaction partner. The imidazole groups of histidines are known to compete with primary amines for reaction, but the reaction products are unstable and readily hydrolyzed. The reaction involves the nucleophilic attack of an amine on the acid carboxyl of an NHS ester to form an amide, releasing the N-hydroxysuccinimide.

[0127] Imidoesters are the most specific acylating reagents for reaction with amine groups of a molecule such as a protein. At a pH between 7 and 10, imidoesters react only with primary amines. Primary amines attack imidates nucleophilically to produce an intermediate that breaks down to amidine at high pH or to a new imidate at low pH. The new imidate can react with another primary amine, thus crosslinking two amino groups, a case of a putatively monofunctional imidate reacting bifunctionally. The principal product of reaction with primary amines is an amidine that is a stronger base than the original amine. The positive charge of the original amino group is therefore retained. As a result, imidoesters do not affect the overall charge of the conjugate.

[0128] Isocyanates (and isothiocyanates) react with the primary amines of the conjugate components to form stable bonds. Their reactions with sulfhydryl, imidazole, and tyrosyl groups give relatively unstable products.

[0129] Acylazides are also used as amino-specific reagents in which nucleophilic amines of the reaction partner attack acidic carboxyl groups under slightly alkaline conditions, e.g. pH 8.5.

[0130] Arylhalides such as l,5-difluoro-2,4-dinitrobenzene react preferentially with the amino groups and tyrosine phenolic groups of the conjugate components, but also with its sulfhydryl and imidazole groups.DB1 / 159329947.1 35Attorney Docket No. 061809-5015-WO

[0131] p-Nitrophenyl esters of carboxylic acids are also useful amino-reactive groups. Although the reagent specificity is not very high, a- and s-amino groups appear to react most rapidly.

[0132] Aldehydes react with primary amines of the conjugate components (e.g., 8-amino group of lysine residues). Although unstable, Schiff bases are formed upon reaction of the protein amino groups with the aldehyde. Schiff bases, however, are stable, when conjugated to another double bond. The resonant interaction of both double bonds prevents hydrolysis of the Schiff linkage. Furthermore, amines at high local concentrations can attack the ethylenic double bond to form a stable Michael addition product. Alternatively, a stable bond may be formed by reductive amination.

[0133] Aromatic sulfonyl chlorides react with a variety of sites of the conjugate components, but reaction with the amino groups is the most important, resulting in a stable sulfonamide linkage.

[0134] Free carboxyl groups react with carbodiimides, soluble in both water and organic solvents, forming pseudoureas that can then couple to available amines yielding an amide linkage. Yamada et al., Biochemistry, 1981, 20: 4836-4842, e.g., teach how to modify a protein with carbodiimides.Sulfhydryl and Sulfhydryl-Reactive Groups

[0135] In another embodiment, a reactive functional group is selected from a sulfhydryl group (which can be converted to disulfides) and sulfhydryl-reactive group. Useful nonlimiting examples of sulfhydryl-reactive groups include maleimides, alkyl halides, acyl halides (including bromoacetamide or chloroacetamide), pyridyl disulfides, and thiophthalimides.

[0136] Maleimides react preferentially with the sulfhydryl group of the conjugate components to form stable thioether bonds. They also react at a much slower rate with primary amino groups and the imidazole groups of histidines. However, at pH 7 the maleimide group can be considered a sulfhydryl-specific group, since at this pH the reaction rate of simple thiols is 1000-fold greater than that of the corresponding amine.

[0137] Alkyl halides react with sulfhydryl groups, sulfides, imidazoles, and amino groups. At neutral to slightly alkaline pH, however, alkyl halides react primarily with sulfhydryl groups to form stable thioether bonds. At higher pH, reaction with amino groups is favored. DB1 / 159329947.1 36Attorney Docket No. 061809-5015-WO

[0138] Pyridyl disulfides react with free sulfhydryl groups via disulfide exchange to give mixed disulfides. As a result, pyridyl disulfides are relatively specific sulfhydryl-reactive groups.

[0139] Thiophthalimides react with free sulfhydryl groups to also form disulfides.Other Reactive Functional Groups

[0140] Other exemplary reactive functional groups include:(i) carboxyl groups and various derivatives thereof including, but not limited to, N- hydroxybenztriazole esters, acid halides, acyl imidazoles, thioesters, p-nitrophenyl esters, alkyl, alkenyl, alkynyl and aromatic esters;(ii) hydroxyl groups, which can be converted to esters, ethers, aldehydes, etc.;(iii) haloalkyl groups, wherein the halide can be displaced with a nucleophilic group such as, for example, an amine, a carboxylate anion, thiol anion, carbanion, or an alkoxide ion, thereby resulting in the covalent attachment of a new group at the site of the halogen atom;(iv) dienophile groups, which are capable of participating in Diels-Alder reactions such as, for example, maleimido groups;(v) aldehyde or ketone groups, such that subsequent derivatization is possible via formation of carbonyl derivatives such as, for example, imines, hydrazones, semicarbazones or oximes, or via such mechanisms as Grignard addition or alkyllithium addition;(vi) alkenes, which can undergo, for example, cycloadditions, acylation, Michael addition, etc;(vii) epoxides, which can react with, for example, amines and hydroxyl groups;(ix) phosphoramidites and other standard functional groups useful in nucleic acid synthesis and(x) any other functional group useful to form a covalent bond between the functionalized chelating agent and a molecular entity or a surface.DB1 / 159329947.1 37Attorney Docket No. 061809-5015-WOFunctional Groups with Non-specific Reactivities

[0141] In addition to the use of site-specific reactive moieties, the present invention contemplates the use of non-specific reactive groups to link a chelating agent to a targeting moiety. Non-specific groups include photoactivatable groups, for example.

[0142] Photoactivatable groups are ideally inert in the dark and are converted to reactive species in the presence of light. In one embodiment, photoactivatable groups are selected from precursors of nitrenes generated upon heating or photolysis of azides. Electrondeficient nitrenes are extremely reactive and can react with a variety of chemical bonds including N-H, O-H, C-H, and C=C. Although three types of azides (aryl, alkyl, and acyl derivatives) may be employed, arylazides are presently preferrred. The reactivity of arylazides upon photolysis is better with N-H and O-H than C-H bonds. Electron-deficient arylnitrenes rapidly ring-expand to form dehydroazepines, which tend to react with nucleophiles, rather than form C-H insertion products. The reactivity of arylazides can be increased by the presence of electron-withdrawing substituents such as nitro or hydroxyl groups in the ring. Such substituents push the absorption maximum of arylazides to longer wavelength. Unsubstituted arylazides have an absorption maximum in the range of 260-280 nm, while hydroxy and nitroarylazides absorb significant light beyond 305 nm. Therefore, hydroxy and nitroarylazides are most preferable since they allow to employ less harmful photolysis conditions for the affinity component than unsubstituted arylazides.

[0143] In another preferred embodiment, photoactivatable groups are selected from fluorinated arylazides. The photolysis products of fluorinated arylazides are arylnitrenes, all of which undergo the characteristic reactions of this group, including C-H bond insertion, with high efficiency (Keana et al., J. Org. Chem. 55: 3640-3647, 1990).

[0144] In another embodiment, photoactivatable groups are selected from benzophenone residues. Benzophenone reagents generally give higher crosslinking yields than arylazide reagents.

[0145] In another embodiment, photoactivatable groups are selected from diazo compounds, which form an electron-deficient carbene upon photolysis. These carbenes undergo a variety of reactions including insertion into C-H bonds, addition to double bonds (including aromatic systems), hydrogen attraction and coordination to nucleophilic centers to give carbon ions.DB1 / 159329947.1 38Attorney Docket No. 061809-5015-WO

[0146] In still another embodiment, photoactivatable groups are selected from diazopyruvates. For example, the p-nitrophenyl ester of p-nitrophenyl diazopyruvate reacts with aliphatic amines to give diazopyruvic acid amides that undergo ultraviolet photolysis to form aldehydes. The photolyzed diazopyruvate-modified affinity component will react like formaldehyde or glutaraldehyde forming intraprotein crosslinks.Sidearms

[0147] In some embodiments, the chelating agent described herein comprises a “sidearm”. A “sidearm”, as used herein, is a structure comprising a branching linking moiety (BL) and one or more of an extension linking moiety (EL), a reactive linking moiety (RL), and a targeting linking moiety (TL). In some embodiments, at least one of L1, L2, and L3comprise a sidearm.

[0148] A “branching linking moiety”, or “BL”, as used herein, is a linker joining, covalently or noncovalently, the remainder of the chelating agent to the remainder of the sidearm, the remainder of the chelating agent to the extension linking moiety, the remainder of the chelating agent to the reactive linking moiety, or the remainder of the chelating agent to the targeting linking moiety; or BL is a linker binding to, covalently or noncovalently, the remainder of the chelating agent only and is configured to join, covalently or noncovalently, the remainder of the chelating agent to the remainder of the sidearm, the remainder of the chelating agent to the extension linking moiety, the remainder of the chelating agent to the reactive linking moiety, or the remainder of the chelating agent to the targeting linking moiety.

[0149] An “extension linking moiety”, or “EL”, as used herein, is a linker joining, covalently or noncovalently, the branching linking moiety to the reactive linking moiety or the branching linking moiety to the targeting linking moiety; or EL is a linker binding to, covalently or noncovalently, the branching linking moiety only and is configured to join, covalently or noncovalently, the branching linking moiety to the reactive linking moiety or the branching linking moiety to the targeting linking moiety. In some embodiments, EL is linear. In some embodiments, EL is branched. In some embodiments, when there is more than one EL included in the sidearm, each EL may be the same or different.

[0150] A “reactive linking moiety”, or “RL”, as used herein, is a linker including at least one reactive functional group, which functional group, optionally, has been reacted to join, covalently or noncovalently, the branching linking moiety to the targeting linking moiety or DB1 / 159329947.1 39Attorney Docket No. 061809-5015-WOthe extension linking moiety to the targeting linking moiety; or RL is a linker including at least one reactive functional group, which functional group, optionally, has been reacted to bind to, covalently or noncovalently, the branching linking moiety or the extension linking moiety, and is configured to join, covalently or noncovalently, the branching linking moiety to the targeting linking moiety or the extension linking moiety to the targeting linking moiety. In some embodiments, RL is terminated with a second reactive functional group or a protected functional group.

[0151] A “targeting linking moiety”, or “TL”, as used herein, is a linker having a targeting moiety, the linker joining, covalently or noncovalently, the targeting moiety to the branching linking moiety, the extension linking moiety, or the reactive linking moiety.

[0152] In some embodiments, the chelating agent comprises a sidearm having the structure:wherein BL is a branching linking moiety covalently bonded to the chelating agent; EL is an extension linking moiety; RL is a reactive linking moiety; TL is a targeting linking moiety; m, n, and o are integers independently selected from 0, 1, 2, 3, 4, and 5. In some embodiments, n is an integer selected from 0, 1, and 2. In some embodiments, o is an integer selected from 0 or 1. In some embodiments, m is an integer selected from 0, 1, and 2; n is 1; and o is 0. In some embodiments, m is an integer selected from 0, 1, and 2; n is 0; and o is 1.

[0153] In some embodiments, the chelating agent comprises a sidearm. In some embodiments, the chelating agent comprises a sidearm comprising a reactive linking moiety RL. In exemplary embodiments, the chelating agent comprises a sidearm comprising a targeting linking moiety TL. In exemplary embodiments, a linker comprises a reactive functional group. In exemplary embodiments, a linker comprises a targeting moiety. In some embodiments, the chelating agent comprises a reactive linking moiety and a targeting linking moiety.

[0154] In some embodiments, the chelating agent comprises a sidearm comprising BL. In some embodiments, the chelating agent comprises a sidearm comprising BL and EL. In some embodiments, the chelating agent comprises a sidearm comprising BL and RL. In some embodiments, the chelating agent comprises a sidearm comprising BL and TL. In some embodiments, the chelating agent comprises a sidearm comprising BL, EL, and RL. In someDB1 / 159329947.1 40Attorney Docket No. 061809-5015-WOembodiments, the chelating agent comprises a sidearm comprising BL, RL, and TL. In some embodiments, the chelating agent comprises a sidearm comprising BL, EL, and TL. In some embodiments, the chelating agent comprises a sidearm comprising BL, EL, RL, and TL.

[0155] In some embodiments, one or more of EL, RL, TL are not present. In some embodiments, only BL is present (i.e., EL, RL, TL are not present). In some embodiments, two of the linking moieties are present in any permutation, e.g., BL and EL, BL and RL, BL and TL, EL and RL, EL and TL, etc. Those of ordinary skill in the art will be able to readily determine from the disclosure herein an appropriate combination of linking moieties and methods of preparing the relevant chelating agents.

[0156] Any linker described herein may be a linker comprising a reactive functional group configured to react with a reactive functional group on a targeting, or other, moiety to join the linker to that moiety. Any linker described herein may be a linker comprising a bond to a targeting moiety. As described, hereing, the term “targeting moiety” refers to a moiety targeting or directing the molecule to which it is attached (e.g., a chelating agent or a chelating agent complexed to a metal ion (such as a radionuclide)) to a particular location or molecule, most generally present in a subject to whom the molecule-targeting agent composition is administered. Thus, for example, a targeting moiety may be used to target a molecule to a specific target cell, protein or enzyme, to a particular cellular location, to a particular cell type or to a diseased tissue. As will be appreciated by those in the art, the localization of proteins within a cell by active or passive internalization after binding by a targeting agent, is a simple method for increasing effective concentration. For example, shuttling an imaging agent and / or therapeutic into the nucleus confines them to a smaller space thereby increasing concentration. Finally, the physiological target may simply be localized to a specific compartment, and the agents must be localized appropriately.

[0157] The targeting moiety can be a small molecule (e.g., MW < 500D), which includes both non-peptides and peptides. Examples of a targeting moiety also include peptides, polypeptides (including proteins, and in particular antibodies, which includes antibody fragments), nucleic acids, oligonucleotides, carbohydrates, lipids, hormones (including proteinaceous and steroid hormones (for instance, estradiol)), growth factors, lectins, receptors, receptor ligands, cofactors and the like. Targets of a targeting moiety can include a complementary nucleic acid, a receptor, an antibody, an antigen or a lectin, for example.DB1 / 159329947.1 41Attorney Docket No. 061809-5015-WO

[0158] In exemplary embodiments, a targeting moiety binds to a target with high binding affinity. In other words, a targeting moiety with high binding affinity to a target has a high specificity for or specifically binds to the target. In some embodiments, the targeting agentchelating agent, -metal ion complex composition has a dissociation constant Kd of about 10'7M or less. In exemplary embodiments, the composition has a dissociation constant Kd of about 10'8M or less, about 10'9M or less, about IO'10M or less, about 10'11M or less, about IO’12M or less, about 10'13M or less, about 10'14M or less or about 10'15M or less. A composition of the invention has a high binding affinity for a target if the targeting moiety has a high binding affinity for the target.

[0159] In exemplary embodiments, a targeting moiety is an antibody. An “antibody” refers to a protein comprising one or more polypeptides substantially encoded by all or part of the recognized immunoglobulin genes. The recognized immunoglobulin genes, for example in humans, include the kappa (K), lambda ( ) and heavy chain genetic loci, which together compose the myriad variable region genes, and the constant region genes mu (p), delta (5), gamma (y), epsilon (a) and alpha (a), which encode the IgM, IgD, IgG, IgE, and IgA isotypes respectively. Antibody herein is meant to include full length antibodies and antibody fragments, and may refer to a natural antibody from any organism, an engineered antibody or an antibody generated recombinantly for experimental, therapeutic or other purposes as further defined below. Antibody fragments include Fab, Fab’, F(ab’)2, Fv, scFv or other antigen-binding subsequences of antibodies and can include those produced by the modification of whole antibodies or those synthesized de novo using recombinant DNA technologies. The term “antibody” refers to both monoclonal and polyclonal antibodies. Antibodies can be antagonists, agonists, neutralizing, inhibitory or stimulatory.

[0160] While a targeting moiety (including TL) may be appended to a chelating agent, or its metal ion complex, in order to localize the composition to a specific region in a subject to whom it is administered, certain chelating agents (complexes) have a natural affinity for cells, tissue, organs or some other part of the animal. For example, a chelating agent designed using the principals and methods set forth herein can have a natural or intrinsic affinity for bone, e.g., affinity for calcium or phosphate. Thus, in some embodiments, a chelating agent (complex) of the invention does not comprise a separate targeting moiety, targeting linking moiety, or a linker to a targeting moiety, yet still exhibits inherent target specificity. A chelating agent (complex) of the invention lacking a targeting moiety can be also used in any method that does not require specific targeting.DB1 / 159329947.1 42Attorney Docket No. 061809-5015-WO

[0161] In some embodiments, a chelating agent comprises a linker to a solid support. That is, any linker described herein may be a linker comprising a reactive functional group or reactive linking moiety that could react with a reactive functional group on a solid support to join the chelating agent to the solid support. Any linker described herein may be a linker comprising a bond to a solid support. A “solid support” is any material that can be modified to contain discrete individual sites suitable for the attachment or association of a chelating agent. Suitable substrates include biodegradable beads, non-biodegradable beads, silica beads, magnetic beads, latex beads, glass beads, quartz beads, metal beads, gold beads, mica beads, plastic beads, ceramic beads, or combinations thereof. Of particular use are biocompatible polymers, including biodegradable polymers that are slowly removed from the system by enzymatic degradation. Example biodegradable materials include starch, crosslinked starch, polyethylene glycol), polyvinylpyrrolidine, polylactides (PLA), polyglycolides (PGA), poly(lactide-co-glycolides) (PLGA), polyanhydrides, polyorthoesters, poly(DTH iminocarbonate), poly (bisphenol A iminocarbonate), polycyanoacrylate, polyphosphazene, mixtures thereof and combinations thereof. Other suitable substances for forming the particles exist and can be used. In some embodiments, a solid support is a bead comprising a cross-linked starch, for example, cross-linked potato starch. Beads made from starch are completely biodegradable in the body, typically by serum amylase, a naturally occurring enzyme found in the body. In these embodiments, the chelating agent optionally further comprises a targeting moiety, a linker to a targeting moiety, or TL. In cases where a chelating agent that is attached to a solid support does not comprise a targeting moiety, the chelating agent can be localized directly by the practitioner, for example, by direct surgical implantation.

[0162] In some embodiments, a linker has the structure -BL-RL, wherein BL is selected from a bond, acyl, -NH-, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl and substituted or unsubstituted heteroaryl; and RL is selected from a reactive functional group, or a protected functional group.

[0163] In some embodiments, BL is selected from a bond, substituted or unsubstituted alkyl and substituted or unsubstituted heteroalkyl. In some embodiments, BL is heteroalkyl. In some embodiments, BL is (Ci, C2, C3, C4, Cs, Ce, C7, Cs, C9, C10, C11, C12, C13, C14, C15, Ci6, C17, Cis, C19 or C20) alkyl in which 1, 2, 3, 4, or 5 atoms are replaced with a heteroatom, suchDB1 / 159329947.1 43Attorney Docket No. 061809-5015-WOas nitrogen or oxygen (i.e., heteroalkyl). In some embodiments, BL is NH2 and EL, RL, and TL are not present.

[0164] In some embodiements, BL has the structure,HNwherein n is an integer selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.

[0165] In some embodiments, linker has the structure:HNmwherein EL and RL are defined herein, n is an integer selected from 0, 1, 2, 3, 4, 5, 6, 7, and 8 and m is an integer selected from 0, 1, 2, and 3.

[0166] In some embodiments, linker has the structure:HNwherein EL and RL are defined herein, and m is an integer selected from 0, 1, 2, and 3.

[0167] In some embodiments, linker has the structure:HNmDB1 / 159329947.1 44Attorney Docket No. 061809-5015-WOwherein EL and TL is defined herein, n is an integer selected from 0, 1, 2, 3, 4, 5, 6, 7, and 8 and m is an integer selected from 0, 1, 2, and 3.

[0168] In some embodiments, linker has the structure:Owherein EL and TL is defined herein, and m is an integer selected from 0, 1, 2, and 3.

[0169] In some embodiments, EL is selected from:wherein n is an integer selected from 1, 2, 3, 4, 5, and 6.

[0170] In some embodiments, RL is selected from -NH2, -C(O)OH, alkyl ester (e.g., methyl ester), N-hydroxysuccinimide (NHS) ester, sulfo-NHS ester, isothiocyanate, tetrafluorophenyl ester, tetrachlorophenylester, squareamide, and maleimide. In some embodiments, RL is selected from -NH2 and -C(O)OH.

[0171] In some embodiments, -BL-RL is selected from:DB1 / 159329947.1 45Attorney Docket No. 061809-5015-WO. In some embodiments, -BL-EL-RL is selected from:DB1 / 159329947.1 46Attorney Docket No. 061809-5015-WO

[0173] In a preferred embodiment according to paragraph

[0139] , any implied hydrogens can be selected from substituted or unsubstituted alkyl, and substituted or unsubstituted heteroalkyl of 1, 2, 3, 4, 5, 6, 7, 8, 9 members selected from C or a heteroatom.

[0174] In some embodiments, Lx6is a sidearm.

[0175] In some embodiments, EL is seleceted from substituted or unsubstituted alkyl, and substituted or unsubstituted heteroalkyl. In some embodiments, EL is a substituted or unsubstituted alkoxy alkyl.

[0176] In some embodiments, EL is a substituted or unsubstituted monoether. In some embodiments, EL is a substituted or unsubstituted polyether. In some embodiments, the polyether has from 2 to 10 (z.e., 2, 3, 4, 5, 6, 7, 8, 9, or 10) ether groups. In some embodiments, EL comprises a modifying moiety.

[0177] In some embodiments, the sidearm has a structure selected from:DB1 / 159329947.1 47Attorney Docket No. 061809-5015-WO

[0178] In a preferred embodiment, the sidearm has a structure selected from:

[0179] In another preferred embodiment, the sidearm has a structure selected from:

[0180] In another preferred embodiment, the sidearm has a structure selected from:DB1 / 159329947.1 48Attorney Docket No. 061809-5015-WO

[0181] In exemplary embodiments, the sidearm has the structure:5— BL— F-EH— TL' 'mwhere BL, EL, TL, and m are as defined herein. In an exemplary embodiment, m is an integer selected from 0, 1, 2, 3, 4, and 5.

[0182] In exemplary embodiments, a linker is a linker to a targeting moiety. In some embodiments, the targeting moiety is selected from a polypeptide, a nucleic acid, a lipid, a polysaccharide, a small molecule, a cofactor and a hormone. In exemplary embodiments, the targeting moiety is an antibody or antibody fragment.

[0183] In exemplary embodiments, the sidearm comprises TL. In some embodiments, the targeting moiety of TL is selected from a polypeptide, a nucleic acid, a lipid, a polysaccharide, a small molecule, a cofactor and a hormone. In exemplary embodiments, the targeting moiety of TL is an antibody or antibody fragment.

[0184] In a linker with multiple reactive functional groups, a particular functional group can be chosen such that it does not participate in, or interfere with, the reaction controlling the attachment of the functionalized spacer component to another chelating agent component. Alternatively, the reactive functional group can be protected from participating in the reaction by the presence of a protecting group. Those of skill in the art understand how to protect a particular functional group from interfering with a chosen set of reaction conditions. For examples of useful protecting groups, See Greene et al., PROTECTIVE GROUPS IN ORGANIC SYNTHESIS, John Wiley & Sons, New York, 1991.Modifying Moiety

[0185] In some embodiments, the compound (chelating agent, chelate) comprises one or more modifying moi eties. In some embodiments, one or more of L1, L2, L3, L4, L5, Abl, Ab2, Apl, Ap2, LP1, and LP2comprise(s) a modifying moiety. In some embodiments, one or more of Lla, Llb, Llc, RL1, and RL2, RL3, RL4, Abl, Ab2, Apl, and Ap2comprise(s) a modifying moiety. In some embodiments, the sidearm comprises a modifying moiety. In someDB1 / 159329947.1 49Attorney Docket No. 061809-5015-WOembodiments, the sidearm comprises a modifying moiety. In some embodiments, EL comprises a modifying moiety. Each of the modifying moieties can be the same or different.

[0186] The modifying moiety modifies various properties of the chelating agent and / or a complex formed between the chelating agent and a metal ion, such as solubility, charge, pharmokinetics, physical or spectra properties, or affinity for specific material. In some embodiments, the modifying moiety does not interact with the metal when the chelating agent is complexed to a metal. In some embodiments, the modifying moiety is a solubilizing group, a hormone-derived moiety, a prodrug moiety (for example, with a cleavable moiety), an oligonucleotide, ssDNA, dsDNA, RNA, or a peptide. The solubilizing group improves solubility of the chelating agent and / or a complex formed between the chelating agent and a metal ion in aqueous media. In some embodiments, the hormone (of the homone-derived moiety) is a steroid. In some embodiments, the steroid is estradiol. In some embodiments, the modifying moiety is an estradiol -derived moiety. Peptides of a hydrophilic and hydrophobic nature by virtue of their amino acid composition may be used to tune solubility of the chelating agent and / or a complex formed between the chelating agent and a metal ion. In an exemplary embodiment, the modifying moiety has no specific targeting role in the chelating agent or complex thereof, and it is distinct in function and / or structure from the targeting moiety.

[0187] In some embodiments, the modifying moiety is substituted or unsubstituted heteroalkyl. In some embodiments, the modifying moiety comprises a substituted or unsubstituted alkoxyalkyl. In some embodiments, the modifying moiety comprises a substituted or unsubstituted monoether. In some embodiments, the modifying moiety comprises a substituted or unsubstituted polyether. In some embodiments, the modifying moiety comprises an estradiol-derived moiety. In some embodiments, the modifying moiety comprises a polyether substituted with an estradiol-derived moiety.

[0188] In some embodiments, the modifying moiety comprises a glycol moiety, forexample, a member selected from:'^^O^°^NH2; andDB1 / 159329947.1 50Attorney Docket No. 061809-5015-WOCH3OH, wherein f is an integer selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, and 25.

[0189] In some embodiments, the modifying moiety comprises a peptide. In some embodiments, the modifying moiety is an amino acid or peptide. An exemplary modifying moiety comprises:H H N N N N H H

[0190] In some embodiments, the modifying moiety comprises an oligonucleotide.

[0191] In some embodiments, the modifying moiety is selected from:HerHO-Exemplary Chelating Agents

[0192] In some embodiements the invention provides for a mixture of multiple structures, for example, a mixture of isomers.

[0193] In some embodiements, the invention provides a chelating agent having the structure:DB1 / 159329947.1 51Attorney Docket No. 061809-5015-WOwherein Abl, Lpl, Lp2, and Llaare defined herein; and Lx6is a linker comprising a targeting linker moiety or a reactive linker moiety as defined herein.

[0194] In some embodiements, the invention provides for a chelating agent having the structure:wherein Abl, Lpl, and Lp2, are defined herein; and Lx6is a linker comprising a targeting linker moiety or a reactive linker moiety as defined herein.

[0195] In some embodiements, the invention provides for a chelating agent having a structure selected from:DB1 / 159329947.1 52Attorney Docket No. 061809-5015-WOwherein Abl, Lpl, and Lp2, are defined herein; and Lx6is a linker comprising a targeting linker moiety or a reactive linker moiety as defined herein.

[0196] In some embodiments, the invention provides a chelating agent or mixture of chelating agents having a structure selected from:

[0197] In a preferred embodiment, the invention provides a chelating agent or mixture of chelating agents having a structure selected from:DB1 / 159329947.1 53Attorney Docket No. 061809-5015-WO

[0198] In some embodiments, the invention provides a chelating agent having the structure: / \.. < HQ 0HN HN N"'d NH NHs >■■■- z;| d o joA p o, Ao> A AzN OH H0 N;V. -NH HN~. / O A „J 0NH-', or an isomer thereof.

[0199] In some preferred embodiments, the invention provides a chelating agent, or mixture of chelating agents having a structure selected from:DB1 / 159329947.1 54Attorney Docket No. 061809-5015-WO

[0200] In some embodiments, the invention provides a chelating agent having the structure:, or an isomer thereof.

[0201] In some embodiments, the invention provides a chelating agent having the structure:DB1 / 159329947.1 55Attorney Docket No. 061809-5015-WO

[0202] In some embodiments, the invention provides a chelating agent, or mixture of chelating agents having a structure selected from:

[0203] In some embodiments, the invention provides a chelating agent having the structure: DB1 / 159329947.1 56Attorney Docket No. 061809-5015-WOor an isomer thereof.

[0204] In some embodiments, the invention provides a chelating agent, or mixture of chelating agents having a structure selected from: / ;. \ P O,.?■ O Q.< O.„.". / N-d AH N HN., N G OH? j HO d OH; j HO b.. / HQ Q O OH }., MN HN HN" HN M,. NH NH O *• o 1 o -xo? O' p Q C--A O 0 A-O N -OH HO N f N-OH HO-fci )and

[0205] In some embodiments, the invention provides a chelating agent having the structure:DB1 / 159329947.1 57Attorney Docket No. 061809-5015-WOZ""'^ O Q.<. z-< V- --j<'>•••• N, NH Mhkxhj — d OH ( J Hd O. / HQ O V- HN H'N N““< NH NH\ w >■■■■■< / \ O ••.?O / o-\ p q Ao(' N- OH HO H }\-NH HN-dO \ / b7 \ X'' H> N" •.< r, or an isomer thereof.

[0206] In some embodiments, the invention provides a chelating agent having the structure:or an isomer thereof.

[0207] In some embodiments, the invention provides a chelating agent having the structure:DB1 / 159329947.1 58Attorney Docket No. 061809-5015-WO

[0208] In one aspect, the invention provides a complex of a compound (chelating agent, chelate) disclosed herein with a metal ion.

[0209] In an exemplary embodiment, the complex is a complex of a +2 metal cation, e.g., Ba2+, Sr2+, Ca+2or Mg+2. In an exemplary embodiment, the +2 metal cation protects a reactive moiety on a reagent contacting the complex from unproductive reaction with the complex during the course of modification of the complex with the reagent. An exemplary reactive moiety is an imide moiety, e.g, N-hydoxysuccinimide (NHS). In a preferred embodiment, the complex of the +2 metal cation is converted to an NHS ester by contacting the complex with a reagent comprising a NHS moiety. In a preferred embodiment, the +2 metal cation is displaced following reaction with the reagent, e.g., following NHS ester formation, by a cation of valence higher than +2, e.g., +3 or +4. In a preferred embodiment, the cation of higher valence is selected from an ion of lanthanides, transition metals and actinides.

[0210] Any of the combinations of compounds (chelating agent, chelate) disclosed herein and a metal ion disclosed herein are encompassed by this disclosure and specifically provided by the invention.

[0211] In some embodiments, the complex is luminescent.

[0212] In some embodiments, the complex includes a metal ion that is a known radioisotope.DB1 / 159329947.1 59Attorney Docket No. 061809-5015-WO

[0213] Exemplary complexes are shown in the Examples.

[0214] In another aspect, the invention provides a complex of a compound (chelating agent, chelate) disclosed herein with an element, or ion thereof, from periods 4, 5, 6 and 7 and / or from groups 13, 14, 15, 16. In another aspect, the invention provides a complex of a compound (chelating agent, chelate) disclosed herein with an element, or ion thereof, from periods 3, 4, 7, 8, 9, 10, 11, 13, 14, and 15. In some embodiments, the invention provides a complex of a compound (chelating agent, chelate) disclosed herein with an element, or ion thereof, from periods 3, 4, and 13.Metals

[0215] In some embodiments, the metal complexed by a chelating agent of the invention is an actinide. In some embodiments, the actinide is actinium (Ac). In some embodiments, the actinide is thorium (Th). In some embodiments, the metal is a lanthanide. In some embodiments, the lanthanide is terbium (Tb). In some embodiments, the lanthanide is europium (Eu). In some embodiments, the lanthanide is dysprosium (Dy). In some embodiments, the lanthanide is lutetium (Lu). In some embodiments, the lanthanide is samarium (Sm). In some embodiments, the lanthanide is lanthanum (La). In some embodiments, the lanthanide is gadolinium (Gd). In some embodiments the metal is yttrium (Y). In some embodiments, the metal is zirconium (Zr). In some embodiments, the metal ion is yttrium(III). In some embodiments, the metal ion is europium (III). In some embodiments, the metal ion is terbium(III). In some embodiments, the metal ion is zirconium (IV). In some embodiments, the metal ion is thorium(IV). In some embodiments, the metal ion is selected from Th4+, Zr4+, Eu3+, Dy3+, Tb3+, Lu3+, Ac3+, La3+and Y3+. In some embodiments, the metal (ion) is a radionuclide. In some embodiments, the metal ion is selected from225Ac(III), and227Th(IV). In some embodiments, the metal ion is89Zr(IV).

[0216] In some embodiments, the metal complexed by a chelating agent of the invention is177LU. In some embodiments, the metal is166Ho. In some embodiments, the metal is153Sm. In some embodiments, the metal is90Y. In some embodiments, the metal is86Y. In some embodiments, the metal is166Dy. In some embodiments, the metal is165Dy. In some embodiments, the metal is169Er. In some embodiments, the metal is175Yb. In some embodiments, the metal is225Ac. In some embodiments, the metal is149Tb. In some embodiments, the metal is153Gd. In some embodiments, the metal is230U.DB1 / 159329947.1 60Attorney Docket No. 061809-5015-WO

[0217] In some embodiments, the metal complexed by a chelating agent of the invention is111In. In some embodiments, the metal is67Ga. In some embodiments, the metal is67Cu. In some embodiments, the metal is64Cu. In some embodiments, the metal is186Re. In some embodiments, the metal is188Re. In some embodiments, the metal is111Ag. In some embodiments, the metal is109Pd. In some embodiments, the metal is212Pb. In some embodiments, the metal is203Pb. In some embodiments, the metal is212Bi. In some embodiments, the metal is213Bi. In some embodiments, the metal is195mPt. In some embodiments, the metal is201Tl. In some embodiments, the metal is55Co. In some embodiments, the metal is99mTc.

[0218] In some embodiments, the metal complexed by a chelating agent of the invention is selected from yttrium (Y), a lanthanoid, an actinoid, zirconium (Zr), iron (Fe), and indium (In). In some embodiments, the metal is selected from zirconium (Zr), iron (Fe), indium (In), europium (Eu), holmium (Ho), lutetium (Lu), yttrium (Y), terbium (Tb), ytterbium (Yb), gadolinium (Gd), samarium (Sm), dysprosium (Dy), erbium (Er), lanthanum (La), cerium (Ce), calcium (Ca), magnesium (Mg), strontium (Sr), Actinium (Ac) and barium (Ba), and thorium (Th). In some embodiments, the metal is selected from Eu, Tb, Sm, Dy, La, Th, Zr, and Ac. In some embodiments, the metal is Ac.

[0219] In some embodiments, the metal ion complexed by a chelating agent of the invention is selected from Zr(IV), Fe(III), Ga(III), In(III), Eu(III), Ho(III), Lu(III), Y(III), Tb(III), Yb(III), Gd(III), Sm(III), Dy(III), Er(III), and Th(IV). In some embodiments, the metal ion is selected from227Th(IV),89Zr(IV), and177Lu(III).

[0220] In some embodiments, the metal is a radionuclide.Radionuclides

[0221] The chelating moieties disclosed herein can be used to bind metal ions, in particular, a radionuclide. The term “radionuclide” or “radioisotope” refers to a radioactive isotope or element with an unstable nucleus that tends to undergo radioactive decay. Numerous decay modes are known in the art and include alpha decay, proton emission, neutron emission, double proton emission, spontaneous fission, cluster decay, β−decay, positron emission (β+decay), electron capture, bound state beta decay, double beta decay, double electron capture, electron capture with positron emission, double positron emission, isomeric transition and internal conversion.DB1 / 159329947.1 61Attorney Docket No. 061809-5015-WO

[0222] Exemplary radionuclides include alpha-emitters, which emit alpha particles during decay. In some embodiments, a radionuclide is an emitter of a gamma ray or a particle selected from an alpha particle, an electron and a positron.

[0223] In some embodiments, the radionuclide is an actinide. In some embodiments, the radionuclide is a lanthanide. In some embodiments, the radionuclide is a 3+ion. In some embodiments, the radionuclide is a 4+ion. In some embodiements the radionuclide is a 2+ion.

[0224] Of particular use in the complexes provided herein are radionuclides selected from isotopes of U, Pu, Fe, Cu, La, Ce, Sm, Gd, Tb, Dy, Ho, Er, Yb, Lu, Y, Th, Zr, In, Ga, Bi, Ra, At and Ac. In some embodiments, a radionuclide is selected form radium-223, thorium-227, lanthanum- 134, cerium-134, bismuth-213, lead-212, lutetium-177, and actinium-225. Other useful radioisotopes include bismuth-212, iodine-123, copper-64, iridium-192, osmium-194, rhodium-105, samarium-153, and yttrium-88, yttrium-90, and yttrium-91. In exemplary embodiments, the radionuclide is thorium, particularly selected from thorium-227 and thorium-232. In some embodiments, thorium-226 is excluded. In some embodiments, U is excluded. In some embodiments, uranium-230 is excluded. That is, in some embodiments, a radionuclide is not U, or a radionuclide is not uranium-230 or a radionuclide is not thorium-226.

[0225] In a preferred embodiment, the radionuclide is selected from Th(IV)-227, Zr(IV)-89, Lu(III)-177, Y(III)-90, Y(III)-86, Ac(III)-225, and In(III)-l 11.

[0226] In another preferred embodiment, the radionuclide is Ac(III)-225.

[0227] In some embodiments, the radionuclide is selected from Tb(III)-149, Sc(III)-47, Dy(III)-166, Er(III)-169, Gd(III)-l 53, Ho(III)-166, Sm(III)-153, Yb(III)-175, Ac(III)-225, Bi(III)-212 and Bi(III)-213.

[0228] In a preferred embodiment, the complex is luminescent and comprises a metal ion which is selected from Tb(III), Eu(III), Sm(III), Dy(III), and Yb(III).

[0229] 232Th exists in nature as an a-emitter with a half life of 1.4 x 1010yr. In aqueous solution, Th(IV) is the only oxidation state. Thorium(IV) ion is bigger than Pu(IV) and usually forms complexes with 9 or higher coordination number. For example, the crystal structure of both Th(IV) complexes of simple bidentate 1,2-HOPO and Me-3,2-HOPO have been determined as nine coordinated species.DB1 / 159329947.1 62Attorney Docket No. 061809-5015-WO

[0230] Similar to other actinide ions, thorium(IV) prefers forming complexes with oxygen, especially negative oxygen donor chelating agents. Thorium(IV) also prefers octadentate or higher multidentate chelating agents:ChelatingAcac NTA HEDTA* EDTA** DTPA TTHA AgentType Bi-dentate Tetra- Hexa- Hexa- Octa- Deca- Log Ki 7.85 16.9 18.5 25.3 30.34 31.9*with one alcoholic oxygen and three carboxyl groups; **with four carboxyl groups.

[0231] Other radionuclides with diagnostic and therapeutic value that can be used with the compounds disclosed herein can be found, for example, in U. S. Patent Nos. 5,482,698 and 5,601,800; and Boswell and Brechbiel, Nuclear Medicine and Biology, 2007 October, 34(7): 757-778 and the manuscript thereof made available in PMC 2008 October 1.Uses

[0232] The chelating agents and complexes disclosed herein can be used in a wide variety of therapeutic and diagnostic settings.

[0233] In one aspect, the invention provides a method of treating a disease in an animal comprising administering a complex disclosed herein to the animal, whereby the disease is ameliorated or eliminated.

[0234] In one aspect, the invention provides a method of diagnosing a disease in an animal comprising (a) administering a complex disclosed herein to the animal and (b) detecting the presence or absence of a signal emitted by the complex. In some embodiments, the detecting step comprises obtaining an image based on the signal.

[0235] In some embodiments, the disease is cancer.

[0236] In some embodiments, the complex comprises a linker to a targeting moiety and the method further comprises localizing the complex to a targeting site in the animal by binding the targeting moiety to the targeting site.

[0237] In some embodiments, the chelating agent or complex thereof is used to diagnose or treat a disease by localizing to disease areas leveraging the targeting linker moiety affinities for diseased cells and delivering a radioisotope that provides either imaging or treatment suitable decay products.DB1 / 159329947.1 63Attorney Docket No. 061809-5015-WO

[0238] In some embodiments, the complex having a targeting moiety (e.g., a sidearm comprising TL, or a linker to a targeting moiety) is used to diagnose and treating a disease by localizing to disease areas leveraging the targeting linker moiety affinities for diseased cells and delivering a radioisotope that provides imaging and treatment suitable decay products.

[0239] In some embodiments, the targeting moiety comprises a prostate specific membrane antigen (PSMA) targeting moiety. Targeting moieties binding to PSMA are known in the art. Non-limiting reference to useful targeting agents, methods of exploring structural diversity in PSMA and other targeting moieties, and methods of forming and using conjugates of these targeting moieties are set forth in, for example, Anderson et al., Bioorg Med Chem. 2007; 15(21): 6678-6686, which discloses various peptidomimetic targeting moieties. See, also, Benesova et al., J Nucl Med. 2015; 56:914-920; Anderson et al., Theranostics 2017; 7(7): 1928-1939, which discusses various phosphoramidate targeting moieties. Such targeting moieties are also the focus of, for example, Ganguly et al., Nucl Med Biol 2015; 42(10): 780-787, which discloses a phopsphoramidate peptide-mimetic; Schally et al., Eur J Endocrin. 1999; 141: 11-14; and Bouvet et al., EJNMMI Res. 2016; 6: 40, disclosing small molecule PSMA inhibitors which are radiolabeled. Wu et al., BiorgMed Chem 2007; 15(231): 7434-7443 provides PSMA inhibitor analogs of the natural substrate retaining elements of this substrate, i.e., folyl-y-Glu. This structure includes a serine residue at the Pl position, which provides a convenient locus for attachment of a phosphoramidate moiety, which can bind zinc used by PSMA. Other disclosures on PSMA inhibitors include Umbrecht et al., EJNMMI Res 2017: 7-9; and Goumi et al., Molecules 2017; 22: 525.

[0240] In some embodiements the invention provides a chelating agent having a targeting moiety selective for different types of cancer. Exemplary cancers include, but are not limited to, Acute Lymphoblastic Leukemia (ALL), Acute Myeloid Leukemia (AML), Adrenocortical Carcinoma, Kaposi Sarcoma (Soft Tissue Sarcoma), AIDS-Related Lymphoma (Lymphoma), Primary CNS Lymphoma (Lymphoma), Anal Cancer, Atypical Teratoid / Rhabdoid Tumor, Childhood, Central Nervous System (Brain Cancer), Basal Cell Carcinoma of the Skin, Bile Duct Cancer, Bladder Cancer, Bone Cancer (includes Ewing Sarcoma and Osteosarcoma and Malignant Fibrous Histiocytoma), Brain Tumors, Breast Cancer, Bronchial Tumors (Lung Cancer), Burkitt Lymphoma, Atypical Teratoid / Rhabdoid Tumor, Childhood (Brain Cancer), Medulloblastoma and Other CNS Embryonal Tumors, Childhood (Brain Cancer), Germ Cell Tumor, Childhood (Brain Cancer), Primary CNS Lymphoma, Cervical Cancer, Cholangiocarcinoma, Chordoma, Childhood (Bone Cancer), Chronic Lymphocytic Leukemia DB1 / 159329947.1 64Attorney Docket No. 061809-5015-WO(CLL), Chronic Myelogenous Leukemia (CML), Colorectal Cancer, Craniopharyngioma, Childhood (Brain Cancer), Cutaneous T-Cell Lymphoma, Lymphoma (Mycosis Fungoids and Sezary Syndrome), Ductal Carcinoma In Situ (DCIS), Diffuse Intrinsic Pontine Glioma (DIPG) (Brain Cancer), Embryonal Tumors, Medulloblastoma and Other Central Nervous System, Childhood (Brain Cancer), Endometrial Cancer (Uterine Cancer), Ependymoma, Childhood (Brain Cancer), Esophageal Cancer, Esthesioneuroblastoma (Head and Neck Cancer), Ewing Sarcoma (Bone Cancer), Extracranial Germ Cell Tumor, Extragonadal Germ Cell Tumor, Eye Cancer, Intraocular Melanoma, Retinoblastoma, Fallopian Tube Cancer, Gallbladder Cancer, Gastric (Stomach) Cancer, Gastrointestinal Neuroendocrine Tumors, Gastrointestinal Stromal Tumors (GIST) (Soft Tissue Sarcoma), Germ Cell Tumors, Childhood Central Nervous System Germ Cell Tumors (Brain Cancer), Childhood Extracranial Germ Cell Tumors, Extragonadal Germ Cell Tumors, Ovarian Germ Cell Tumors, Testicular Cancer, Gestational Trophoblastic Disease, Hairy Cell Leukemia, Head and Neck Cancer, Heart Tumors, Childhood, Hepatocellular (Liver) Cancer, Histiocytosis, Langerhans Cell, Hodgkin Lymphoma, Hypopharyngeal Cancer (Head and Neck Cancer), Intraocular Melanoma, Islet Cell Tumors, Pancreatic Neuroendocrine Tumors, Kaposi Sarcoma (Soft Tissue Sarcoma), Kidney (Renal Cell) Cancer, Langerhans Cell Histiocytosis, Laryngeal Cancer (Head and Neck Cancer), Leukemia, Lip and Oral Cavity Cancer (Head and Neck Cancer), Liver Cancer, Lung Cancer (Non-Small Cell, Small Cell, leuropulmonary Blastoma, Pulmonary Inflammatory Myofibroblastic Tumor, and Tracheobronchial Tumor), Lymphoma, Male Breast Cancer, Melanoma, Melanoma, Intraocular (Eye), Merkel Cell Carcinoma (Skin Cancer), Mesothelioma, Malignant, Metastatic Cancer, Metastatic Squamous Neck Cancer with Occult Primary,(Head and Neck Cancer), Midline Tract Carcinoma With NUT Gene Changes, Mouth Cancer (Head and Neck Cancer), Multiple Endocrine Neoplasia Syndromes, Multiple Myeloma / Plasma Cell Neoplasms, Mycosis Fungoides (Lymphoma), Myelodysplastic Syndromes, Myelodysplastic / Myeloproliferative Neoplasms, Myelogenous Leukemia, Chronic (CML), Myeloid Leukemia, Acute (AML), Myeloproliferative Neoplasms, Nasal Cavity and Paranasal Sinus Cancer (Head and Neck Cancer), Nasopharyngeal Cancer (Head and Neck Cancer), Neuroblastoma, Neuroendocrine Tumors (Gastrointestinal), Non-Hodgkin Lymphoma, Non-Small Cell Lung Cancer, Oral Cancer, Lip and Oral Cavity Cancer and Oropharyngeal Cancer (Head and Neck Cancer), Osteosarcoma and Undifferentiated Pleomorphic Sarcoma of Bone Treatment, Ovarian Cancer, Pancreatic Cancer, Pancreatic Neuroendocrine Tumors (Islet Cell Tumors), Papillomatosis (Childhood Laryngeal), Paraganglioma, Paranasal Sinus and Nasal Cavity DB1 / 159329947.1 65Attorney Docket No. 061809-5015-WOCancer (Head and Neck Cancer), Parathyroid Cancer, Penile Cancer, Pharyngeal Cancer (Head and Neck Cancer), Pheochromocytoma, Pituitary Tumor, Plasma Cell Neoplasm / Multiple Myeloma, Pleuropulmonary Blastoma (Lung Cancer), Pregnancy and Breast Cancer, Pregnancy and Hodgkin Lymphoma, Pregnancy and Non-Hodgkin Lymphoma, Primary Central Nervous System (CNS) Lymphoma, Primary Peritoneal Cancer, Prostate Cancer, Pulmonary Inflammatory Myofibroblastic Tumor (Lung Cancer), Rare Cancers of Childhood, Rectal Cancer, Recurrent Cancer, Renal Cell (Kidney) Cancer, Retinoblastoma, Rhabdomyosarcoma, Childhood (Soft Tissue Sarcoma), Salivary Gland Cancer(Head and Neck Cancer), Sarcoma, Childhood Rhabdomyosarcoma (Soft Tissue Sarcoma), Childhood Vascular Tumors (Soft Tissue Sarcoma), Ewing Sarcoma (Bone Cancer), Kaposi Sarcoma (Soft Tissue Sarcoma), Osteosarcoma (Bone Cancer), Soft Tissue Sarcoma, Uterine Sarcoma, Sezary Syndrome (Lymphoma), Skin Cancer, Small Cell Lung Cancer, Small Intestine Cancer, Soft Tissue Sarcoma, Squamous Cell Carcinoma of the Skin - see Skin Cancer, Squamous Neck Cancer with Occult Primary, Metastatic (Head and Neck Cancer), Stomach (Gastric) Cancer, T-Cell Lymphoma, Cutaneous -see Lymphoma (Mycosis Fungoides and Sezary Syndrome), Testicular Cancer, Throat Cancer (Head and Neck Cancer), Nasopharyngeal Cancer, Oropharyngeal Cancer, Hypopharyngeal Cancer, Thymoma and Thymic Carcinoma, Thyroid Cancer, Tracheobronchial Tumors (Lung Cancer), Transitional Cell Cancer of the Renal Pelvis and Ureter (Kidney (Renal Cell) Cancer), Ureter and Renal Pelvis, Transitional CellCancer (Kidney (Renal Cell) Cancer), Urethral Cancer, Uterine Cancer, Endometrial, Uterine Sarcoma, Vaginal Cancer, Vascular Tumors (Soft Tissue Sarcoma), Vulvar Cancer, Wilms Tumor and Other Childhood Kidney Tumors.

[0241] In some embodiments, the targeting linker moiety has an affinity known targets for cancer diagnosis or treatment including, but not limited to, 2B4, 4-1BB, 4-1BBL, A33, adenosine A2a receptor, Akt, Androgen receptor, Aurora A, Aurora B, B7-H3, B7-H4, Bcl-2, Bcr-Abl, BRAF, BTK, BTLA, BTN2A1, CAIX, CCR4, CD155, CD160, CD19, CD20, CD200, CD200R, CD25, CD27, CD28, CD30, CD33, CD36, CD38, CD40, CD40L, CD47, CD48, CD52, CD70, CD80, CD86, CD96, CDK4, CEA, CEACAM1, ChKl, ChK2, c-KIT, c-Met / HGFR, COX2, CSF-1R, CTLA-4, DDR2, DNAM-1, DR5, EGFR, EpCAM, EPHA3, ERK1, ERK2 / p38 MAPK, FAP, FGFR1, FGFR2, FGFR3, FGFR4, Flt-3, Gal-9, GITR, GITRL, HDAC1, HDAC2, HER2, HER3, HER4 / ERBB4, HGF, HHLA2, HVEM, ICOS, ICOS Ligand, IDO, IGF1R, KRAS, LAG-3, LIGHT, MDM2, MEK1, MEK2, mTOR, MucinDB1 / 159329947.1 66Attorney Docket No. 061809-5015-WO1, NRAS, NTRK1, NTRK2, NTRK3, 0X40, OX40L, p53, PARP1, PD1, PDGFR-a, PDGFR-P, PD-L1, PD-L2, PI3Ka, PI3KP, PI3Ky, PI3K5, PSMA, PTEN, RAF-1, RANKL, RET, SIRPa, SLAMF7, Syk, TDO, TIGIT, TIM-3, TMIGD2, TRAIL, TRAILR1, VEGF, VEGFR-1, VEGFR-2, VEGFR-3, and VISTA.

[0242] In some preferred embodiements, the chelating agent or complex thereof of the present invention is used with targeting agents against specific validated markers and those markers showing promise in clinical trials including, for example, SSGJ-707, adagrasib, elecestrant, elranatamab, magrolimab, orelabrutinib, patritumab deruxtecan (HER3-DXd), pirtobrutinib (LOXO-305), paziotinib, tislelizuma, ublituximab, ivosidenib, vorasidenib, bemcentinib, berzosertib, zenocutuzumab, and darovasertib.

[0243] In an exemplary embodiment, the invention provides a chelating agent (complex) with a linker to a targeting moiety (i.e., PSMA targeting moiety) having the structure:

[0244] Exemplary compounds disclosed herein are particularly well-suited for the preparation of stable, pre-labeled antibodies for use in the diagnosis and / or treatment of neoplasms, e.g., cancer, and other diseases. For example, antibodies with affinity for specific tumors or tumor-associated antigens are labeled with a diagnostic metal chelate, e.g., a radionuclide chelate, generally through covalent conjugation through reactive functional groups of complementary reactivity on the antibody and the chelating agent / chelate. The antibodies used as targeting moieties in the invention can be polyclonal or monoclonal, and can be selective for melanoma, colon cancer, breast cancer, prostate cancer, etc. Such antibodies are known in the art and are readily available. The method of preparation will depend upon the type of radionuclide and antibody used, and the radionuclide-labeled antibodies can be prepared according to methods known in the art. The labeled antibodies can be further stabilized through lyophilization. A stable, lyophilized, radiolabeled antibody DB1 / 159329947.1 67Attorney Docket No. 061809-5015-WOcan be reconstituted with suitable diluent at the time of intended use, thus greatly simplifying the on site preparation process.EXAMPLES

[0245] The chelating agents and metal chelates of the invention are synthesized by an appropriate combination of generally well-known synthetic methods. Techniques useful in synthesizing the chelating agents and metal chelates of the invention are both readily apparent and accessible to those of skill in the relevant art. The discussion below is offered to illustrate certain of the diverse methods available for use in assembling the chelating agents and metal chelates of the invention, but it is not intended to limit the scope of reactions or reaction sequences that are useful in preparing the chelating agents and metal chelates of the present invention.Example 1. Structure and design of 10-coordinate macrocycle.

[0246] For 10-coordinate systems, the bicapped square antiprism is the theoretically expected and experimentally found low energy structure. In the bicapped square antiprism geometry there are two distinct sets of donor atoms: eight equivalent atoms forming the square antiprism (related by D4d point symmetry) and two equivalent capping donor atoms at opposing poles of the 4-fold high symmetry axis (Fig. 1).

[0247] The use of five bidentate chelators to achieve 10-coordination necessarily lowers the symmetry of the idealized system to, at most, C2 point symmetry (i.e., two pairs of symmetry-related bidentate chelators and one unique chelator whose midpoint lies on the C2 symmetry axis). The first pair of symmetry related bidentate chelators connect the two capping donor atoms to two of the eight square antiprismatic donor atoms. Here, that set of bidentate chelators will be called the ‘bridging’ chelators (bridging chelating moieties). The lone chelator split by the C2 symmetry axis will be called the ‘keystone’ chelator (keystone bridging chelating moiety). The remaining pair of symmetry related bidentate chelators will be called ‘pendant’ chelators (pendant chelating moieties). The energy minimized DFT structure of 1,2-HOPOs around a central Ac(III) center was calculated, using the B3LYP functional method and the 6-3 lG(d,p) basis set for the light atoms and MWB78 effective core potential for the Ac(III) center (Fig. 2).

[0248] Making use of bivalent 1,2-HOPO units, it became apparent that the two bridging chelating moieties (Ab2and Ab3) and the keystone bridging chelating moiety (Abl) could beDB1 / 159329947.1 68Attorney Docket No. 061809-5015-WOlinked together to form a macrocycle. From there, the pendant chelating moieties were attached to the macrocycle via amino acid derived bridging scaffold moieties connecting the keystone bridging chelating moiety and each of the two bridging chelating moieties.Importantly, the chirality of the amino acid derived diamines preorganizes the complex for metal binding, with one pendant chelating moiety oriented above the plane of the macrocycle and one pendant chelating moiety below the plane of the macrocycle. The structure of the macrocyclic chelating agent permits the low energy bicapped square antiprism geometry from both the Ac(III) complex and the La(III) complex (Fig. 3).

[0249] To make a bifunctional chelator from the proposed 10-coordinate macrocycle, a diamine linking the two bridging chelating moieties can be modified to include an amine containing sidearm linker. Here, the same amino acid derived bridging scaffold moiety used for the pendant chelating moieties is also used for this purpose. However, it should be noted that the chiral center of this amino acid derivative generates two isomers in the synthesis of BFC 10 (Fig. 4a and 4b, also see Scheme 1, Compound 8 cyclization step with Compound 7). Structurally, the two isomers are very similar and are expected to have very similar properties. For simplicity, in the following synthetic schemes only isomer 1 and its derivatives are shown, but the schemes are intended in all cases to apply to both possible isomers.

[0250] The DFT minimized coordinates of both isomers for chelator 10 with both Ac(III) and La(III) are tabulated below. As before, the B3LYP functional method was used, treating the light atoms with the 6-3 lG(d,p) basis set, Ac(III) with MWB78 and La(III) with MWB28.Table 1. Atomic coordinates Ac-10 (isomer 1) and La-10 (isomer 1)Ac-10 (isomer 1 La-10 (isomer 1Atom Labelx-coord y-coord z-coord x-coord y-coord z-coord Ac or La -0.374 -0.041 -0.031 -0.330 -0.045 0.019 C -0.976 -3.559 -0.055 -1.089 -3.414 -0.052 C -1.450 -4.869 0.225 -1.665 -4.692 0.182 H -1.994 -4.994 1.154 -2.249 -4.797 1.089 C -1.233 -5.915 -0.653 -1.496 -5.730 -0.715 H -1.611 -6.907 -0.416 -1.953 -6.697 -0.517 C -0.546 -5.697 -1.852 -0.758 -5.534 -1.887 H -0.375 -6.470 -2.589 -0.629 -6.296 -2.644 C 5.611 0.464 -0.532 5.588 0.487 -0.543 H 6.516 0.728 -1.061 6.486 0.776 -1.072 C 5.608 -0.061 0.753 5.602 -0.086 0.719 H 6.527 -0.232 1.303 6.527 -0.275 1.253C 4.415 -0.391 1.387 4.417 -0.444 1.355DB1 / 159329947.1 69Attorney Docket No. 061809-5015-WOAc-10 (isomer 1 La-10 (isomer 1 Atom Labelx-coord y-coord z-coord x-coord y-coord z-coord C 3.171 -0.191 0.710 3.162 -0.221 0.702 C -1.440 0.628 -3.369 -1.401 0.563 -3.262 C -1.491 1.179 -4.687 -1.482 1.085 -4.591 C -2.709 1.270 -5.354 -2.700 1.089 -5.263 H -2.701 1.683 -6.357 -2.712 1.477 -6.276 C -3.888 0.880 -4.732 -3.857 0.652 -4.633 H -4.860 0.981 -5.194 -4.834 0.694 -5.095 C -1.054 5.567 1.837 -1.057 5.427 1.919 H -0.977 6.338 2.594 -0.977 6.192 2.679 C -1.703 5.752 0.612 -1.761 5.599 0.723 H -2.137 6.720 0.368 -2.233 6.555 0.504 C -1.797 4.707 -0.290 -1.866 4.555 -0.180 H -2.302 4.808 -1.245 -2.415 4.645 -1.111 C -1.230 3.434 -0.010 -1.255 3.296 0.073 N -0.245 -3.412 -1.252 -0.301 -3.295 -1.215 N 3.229 0.363 -0.590 3.204 0.373 -0.582 N -2.665 0.226 -2.793 -2.603 0.105 -2.677 N -0.536 3.326 1.215 -0.495 3.207 1.258 0 -1.140 -2.538 0.661 -1.204 -2.398 0.680 0 0.251 -2.191 -1.454 0.304 -2.117 -1.356 0 2.052 0.556 -1.180 2.020 0.571 -1.152 0 2.022 -0.456 1.168 2.019 -0.498 1.163 0 -0.397 0.469 -2.676 -0.354 0.472 -2.565 0 -2.577 -0.293 -1.571 -2.485 -0.393 -1.451 0 0.056 2.151 1.417 0.157 2.057 1.414 0 -1.280 2.417 -0.747 -1.316 2.278 -0.659 C -0.062 -4.431 -2.151 -0.171 -4.301 -2.138 C -0.479 4.339 2.137 -0.433 4.214 2.186 C -3.864 0.377 -3.433 -3.807 0.185 -3.321 C 4.411 0.675 -1.206 4.379 0.716 -1.195 C -2.807 -1.584 5.181 -2.696 -1.455 5.235 H -2.820 -2.032 6.169 -2.706 -1.889 6.230 C -3.979 -1.223 4.527 -3.863 -1.052 4.601 H -4.963 -1.375 4.950 -4.845 -1.162 5.043 C -3.926 -0.665 3.252 -3.813 -0.513 3.317 0 -0.433 -0.572 2.633 -0.338 -0.584 2.619 0 -2.595 0.107 1.460 -2.488 0.203 1.496 C -1.568 -1.406 4.575 -1.466 -1.345 4.595 C -0.350 -1.906 5.304 -0.259 -1.896 5.301 C 4.515 -0.932 2.789 4.541 -1.032 2.733 C -5.247 -0.361 2.566 -5.137 -0.201 2.646 C -5.203 0.111 -2.770 -5.132 -0.102 -2.644 C -0.275 1.738 -5.373 -0.300 1.697 -5.286 H 0.701 1.563 -3.618 0.657 1.674 -3.511 C 4.508 1.222 -2.619 4.464 1.311 -2.588 H 2.523 1.052 -2.787 2.479 1.127 -2.748 N 3.356 1.340 -3.312 3.305 1.446 -3.267N 0.828 1.866 -4.584 0.771 1.951 -4.485 DB1 / 159329947.1 70Attorney Docket No. 061809-5015-WOAc-10 (isomer 1 La-10 (isomer 1 Atom Labelx-coord y-coord z-coord x-coord y-coord z-coord H -4.224 -0.548 -1.144 -4.129 -0.640 -0.986 H -4.198 0.397 1.034 -4.106 0.513 1.078 H 0.692 -1.604 3.607 0.740 -1.726 3.559 H 2.502 -0.968 2.883 2.530 -1.064 2.860 N -5.169 -0.387 -1.513 -5.079 -0.527 -1.361 N -5.163 0.234 1.356 -5.066 0.376 1.426 N 0.796 -1.925 4.570 0.852 -2.028 4.527 N 3.340 -1.183 3.422 3.377 -1.305 3.375 0 -6.241 0.380 -3.385 -6.182 0.101 -3.266 0 -6.305 -0.664 3.128 -6.191 -0.491 3.222 0 -0.417 -2.294 6.477 -0.305 -2.231 6.492 0 5.618 -1.114 3.323 5.652 -1.232 3.244 0 5.620 1.509 -3.079 5.570 1.619 -3.048 0 -0.307 2.085 -6.560 -0.333 1.982 -6.490 C -6.351 -0.478 -0.677 -6.253 -0.558 -0.511 H -6.436 -1.494 -0.271 -6.313 -1.527 -0.001 H -7.219 -0.283 -1.312 -7.129 -0.448 -1.155 C -6.311 0.519 0.508 -6.225 0.557 0.564 H -7.203 0.346 1.120 -7.110 0.429 1.198 C 2.037 -2.517 5.031 2.083 -2.644 4.979 H 2.197 -3.473 4.513 2.223 -3.599 4.452 H 1.915 -2.732 6.096 1.961 -2.862 6.043 C 3.288 -1.641 4.808 3.346 -1.786 4.753 H 4.154 -2.303 4.928 4.204 -2.463 4.847 C 3.340 1.787 -4.704 3.278 1.913 -4.651 H 4.154 2.518 -4.783 4.080 2.657 -4.725 C 2.034 2.553 -4.999 1.956 2.658 -4.924 H 1.945 2.743 -6.071 1.843 2.844 -5.995 H 2.090 3.526 -4.489 2.003 3.631 -4.415 C 0.130 4.166 3.521 0.219 4.045 3.550 H 0.354 2.224 3.083 0.475 2.109 3.092 C 0.591 -4.229 -3.510 0.512 -4.107 -3.482 H 0.632 -2.270 -3.095 0.702 -2.168 -3.008 C 1.027 2.486 5.115 1.160 2.366 5.119 H 1.487 3.356 5.595 1.630 3.233 5.595 H 0.233 2.129 5.790 0.370 2.014 5.802 C 2.031 1.357 4.850 2.154 1.231 4.842 H 2.935 1.779 4.391 3.060 1.647 4.385 H 1.586 0.697 4.103 1.700 0.584 4.089 C 2.339 -1.306 -4.785 2.345 -1.196 -4.738 H 1.821 -0.679 -4.056 1.839 -0.579 -3.993 H 3.250 -1.665 -4.290 3.271 -1.548 -4.266 C 1.424 -2.501 -5.088 1.434 -2.396 -5.025 H 1.963 -3.340 -5.539 1.971 -3.234 -5.483 H 0.641 -2.199 -5.801 0.639 -2.102 -5.729 0 0.246 5.147 4.264 0.331 5.021 4.301 0 0.823 -5.207 -4.231 0.667 -5.075 -4.238N 0.453 2.902 3.850 0.576 2.785 3.859 DB1 / 159329947.1 71Attorney Docket No. 061809-5015-WOAc-10 (isomer 1 La-10 (isomer 1Atom Labelx-coord y-coord z-coord x-coord y-coord z-coord N 0.821 -2.947 -3.847 0.851 -2.839 -3.773 C 3.704 0.674 -5.722 3.652 0.817 -5.684 H 4.653 0.242 -5.381 4.616 0.405 -5.360 H 3.925 1.183 -6.671 3.847 1.339 -6.632 C 2.694 -0.452 -6.005 2.664 -0.330 -5.960 H 1.777 -0.030 -6.435 1.733 0.074 -6.377 H 3.128 -1.093 -6.787 3.102 -0.958 -6.750 C 2.395 0.529 6.085 2.512 0.384 6.067 H 1.495 0.034 6.470 1.606 -0.095 6.458 H 2.739 1.199 6.887 2.879 1.040 6.871 C 3.505 -0.512 5.851 3.597 -0.678 5.810 H 4.433 -0.004 5.562 4.533 -0.186 5.518 H 3.715 -1.010 6.809 3.804 -1.192 6.759 N -2.711 -0.450 2.665 -2.604 -0.349 2.700 C -1.491 -0.809 3.278 -1.389 -0.764 3.290 C -6.263 1.983 0.033 -6.209 1.968 -0.049 H -7.050 2.140 -0.715 -6.978 2.027 -0.830 H -5.311 2.141 -0.490 -5.247 2.114 -0.557 C -6.394 3.009 1.165 -6.415 3.084 0.983 H -7.423 3.003 1.554 -7.447 3.047 1.362 H -5.749 2.705 1.999 -5.764 2.897 1.846 C -6.005 4.429 0.733 -6.115 4.482 0.426 H -6.596 4.728 -0.147 -6.699 4.653 -0.492 H -4.954 4.427 0.412 -5.057 4.528 0.134 C -6.183 5.481 1.836 -6.406 5.615 1.420 H -7.245 5.553 2.109 -7.474 5.612 1.678 H -5.657 5.155 2.742 -5.866 5.428 2.357 N -5.705 6.834 1.512 -6.054 6.970 0.969 H -4.699 6.788 1.354 -5.048 7.004 0.809H -6.104 7.118 0.618 -6.475 7.130 0.054 Table 2. Atomic coordinates Ac-10 (isomer 2) and La-10 (isomer 2)Ac-10 (isomer 2) La-10 (isomer 2) Atom Labelx-coord y-coord z-coord x-coord y-coord z-coord Ac or La -0.372 0.039 0.012 -0.325 0.047 -0.035 C -1.351 -3.406 -0.038 -1.376 -3.258 -0.119 C -1.974 -4.658 0.219 -2.046 -4.492 0.108 H -2.509 -4.745 1.158 -2.626 -4.562 1.022 C -1.895 -5.701 -0.687 -1.957 -5.536 -0.796 H -2.372 -6.652 -0.461 -2.476 -6.471 -0.598 C -1.206 -5.535 -1.893 -1.210 -5.388 -1.969 H -1.138 -6.304 -2.652 -1.140 -6.153 -2.731 C 5.626 -0.024 -0.562 5.623 0.016 -0.525 H 6.546 0.155 -1.102 6.551 0.219 -1.044 C 5.590 -0.548 0.723 5.568 -0.556 0.737 H 6.496 -0.803 1.263 6.465 -0.831 1.281C 4.379 -0.765 1.373 4.347 -0.798 1.359DB1 / 159329947.1 72Attorney Docket No. 061809-5015-WOc 3.151 -0.451 0.710 3.127 -0.458 0.693 c -1.387 0.835 -3.319 -1.279 0.788 -3.329 c -1.414 1.436 -4.616 -1.306 1.366 -4.637 c -2.631 1.648 -5.255 -2.514 1.504 -5.312 H -2.606 2.097 -6.243 -2.486 1.933 -6.308 C -3.829 1.318 -4.632 -3.709 1.133 -4.709 H -4.797 1.496 -5.080 -4.674 1.265 -5.178 C -0.363 5.708 1.805 -0.586 5.556 1.847 H -0.179 6.479 2.541 -0.445 6.317 2.603 C -1.001 5.947 0.583 -1.279 5.777 0.653 H -1.324 6.953 0.327 -1.687 6.762 0.436 C -1.238 4.904 -0.293 -1.467 4.741 -0.243 H -1.746 5.046 -1.240 -2.019 4.866 -1.168 C -0.834 3.576 0.012 -0.954 3.442 0.014 N -0.619 -3.319 -1.243 -0.576 -3.197 -1.280 N 3.245 0.097 -0.591 3.240 0.130 -0.590 N -2.631 0.511 -2.737 -2.520 0.408 -2.771 N -0.149 3.406 1.233 -0.208 3.295 1.200 0 -1.383 -2.391 0.703 -1.417 -2.240 0.615 0 0.024 -2.167 -1.420 0.128 -2.075 -1.408 0 2.084 0.398 -1.167 2.087 0.439 -1.174 0 1.988 -0.610 1.181 1.957 -0.625 1.139 0 -0.352 0.566 -2.648 -0.251 0.583 -2.629 0 -2.563 -0.045 -1.529 -2.452 -0.140 -1.562 0 0.280 2.165 1.461 0.340 2.092 1.366 0 -1.021 2.558 -0.703 -1.092 2.428 -0.717 C -0.576 -4.328 -2.169 -0.528 -4.202 -2.211 C 0.052 4.423 2.129 -0.062 4.300 2.122 C -3.825 0.759 -3.357 -3.708 0.599 -3.422 C 4.443 0.300 -1.220 4.449 0.360 -1.190 C -2.875 -1.205 5.278 -2.862 -1.036 5.183 H -2.904 -1.616 6.281 -2.912 -1.427 6.194 C -4.027 -0.785 4.626 -3.990 -0.567 4.523 H -5.014 -0.860 5.063 -4.980 -0.585 4.959 C -3.957 -0.284 3.328 -3.893 -0.095 3.215 0 -0.476 -0.471 2.660 -0.430 -0.470 2.552 0 -2.604 0.352 1.499 -2.505 0.457 1.385 C -1.638 -1.147 4.644 -1.626 -1.059 4.545 C -0.455 -1.734 5.364 -0.480 -1.702 5.271 C 4.446 -1.307 2.776 4.400 -1.390 2.740 C -5.271 0.020 2.632 -5.191 0.231 2.505 C -5.171 0.495 -2.705 -5.057 0.326 -2.786 C -0.165 1.906 -5.309 -0.068 1.886 -5.308 H 0.819 1.577 -3.582 0.874 1.704 -3.534 C 4.575 0.844 -2.632 4.606 0.951 -2.578 H 2.580 0.877 -2.773 2.615 0.967 -2.758 N 3.430 1.087 -3.307 3.474 1.207 -3.266 N 0.958 1.898 -4.540 1.020 2.000 -4.499 H -4.185 -0.291 -1.144 -4.092 -0.406 -1.185H -4.231 0.653 1.034 -4.127 0.746 0.877 DB1 / 159329947.1 73Attorney Docket No. 061809-5015-WOH 0.575 -1.589 3.638 0.532 -1.692 3.527 H 2.440 -1.143 2.896 2.395 -1.222 2.847 N -5.136 -0.101 -1.492 -5.037 -0.244 -1.561 N -5.190 0.518 1.377 -5.091 0.658 1.225 N 0.667 -1.891 4.609 0.611 -1.974 4.504 N 3.260 -1.434 3.426 3.208 -1.542 3.372 0 -6.204 0.827 -3.295 -6.086 0.641 -3.394 0 -6.331 -0.221 3.219 -6.262 0.055 3.096 0 -0.531 -2.078 6.550 -0.557 -1.992 6.472 0 5.532 -1.596 3.295 5.481 -1.696 3.263 0 5.703 1.019 -3.106 5.742 1.148 -3.026 0 -0.186 2.296 -6.483 -0.067 2.213 -6.502 C -6.314 -0.348 -0.673 -6.224 -0.385 -0.730 H -7.184 -0.143 -1.306 -7.085 -0.225 -1.387 C -6.348 0.649 0.513 -6.240 0.730 0.345 H -7.236 0.481 1.127 -7.136 0.649 0.966 H -6.390 1.667 0.107 -6.254 1.700 -0.165 C 1.844 -2.609 5.061 1.765 -2.715 4.976 H 1.892 -3.580 4.548 1.803 -3.685 4.461 H 1.712 -2.801 6.129 1.610 -2.905 6.041 C 3.180 -1.876 4.816 3.116 -2.004 4.754 H 3.970 -2.628 4.929 3.894 -2.770 4.861 C 3.443 1.550 -4.694 3.508 1.691 -4.645 H 4.331 2.188 -4.778 4.388 2.343 -4.704 C 2.226 2.458 -4.962 2.279 2.582 -4.917 H 2.146 2.672 -6.031 2.197 2.795 -5.985 H 2.394 3.412 -4.441 2.428 3.536 -4.391 C 0.653 4.206 3.510 0.572 4.086 3.487 H 0.609 2.242 3.118 0.672 2.134 3.040 C 0.078 -4.171 -3.534 0.173 -4.053 -3.553 H 0.369 -2.243 -3.072 0.510 -2.137 -3.066 C 1.360 2.459 5.128 1.366 2.348 5.071 H 1.911 3.284 5.589 1.916 3.171 5.539 H 0.547 2.186 5.819 0.544 2.081 5.754 C 2.245 1.234 4.858 2.248 1.119 4.812 H 3.179 1.561 4.383 3.194 1.441 4.359 H 1.726 0.616 4.121 1.740 0.512 4.060 C 2.120 -1.423 -4.786 2.255 -1.305 -4.771 H 1.680 -0.757 -4.041 1.805 -0.651 -4.021 H 2.997 -1.880 -4.311 3.139 -1.753 -4.299 C 1.084 -2.515 -5.088 1.233 -2.408 -5.080 H 1.527 -3.397 -5.563 1.689 -3.287 -5.547 H 0.323 -2.125 -5.782 0.474 -2.028 -5.781 0 0.914 5.180 4.225 0.759 5.053 4.235 0 0.175 -5.150 -4.283 0.256 -5.026 -4.312 N 0.807 2.918 3.868 0.831 2.804 3.803 N 0.462 -2.919 -3.842 0.604 -2.811 -3.837 C 3.669 0.415 -5.728 3.768 0.573 -5.689 H 4.572 -0.119 -5.407 4.681 0.057 -5.366H 3.927 0.908 -6.676 4.021 1.081 -6.631 DB1 / 159329947.1 74Attorney Docket No. 061809-5015-WOc 2.541 -0.595 -6.003 2.666 -0.461 -5.981 H 1.666 -0.074 -6.411 1.783 0.042 -6.395 H 2.892 -1.268 -6.800 3.039 -1.121 -6.778 C 2.542 0.380 6.093 2.511 0.252 6.046 H 1.602 -0.016 6.497 1.559 -0.127 6.438 H 2.968 1.016 6.884 2.942 0.873 6.846 C 3.533 -0.773 5.848 3.479 -0.920 5.803 H 4.504 -0.366 5.541 4.463 -0.533 5.513 H 3.705 -1.285 6.805 3.627 -1.446 6.757 N -2.738 -0.165 2.719 -2.670 -0.044 2.605 C -1.539 -0.600 3.327 -1.497 -0.535 3.220 C -6.326 -1.813 -0.200 -6.275 -1.796 -0.119 H -7.135 -1.943 0.531 -7.065 -1.827 0.642 H -5.393 -2.005 0.343 -5.332 -1.978 0.412 C -6.469 -2.832 -1.338 -6.497 -2.903 -1.158 H -7.488 -2.785 -1.751 -7.514 -2.820 -1.569 H -5.794 -2.551 -2.155 -5.812 -2.745 -2.000 C -6.146 -4.266 -0.900 -6.277 -4.312 -0.594 H -6.767 -4.544 -0.034 -6.896 -4.456 0.306 H -5.103 -4.306 -0.556 -5.232 -4.406 -0.269 C -6.340 -5.309 -2.010 -6.589 -5.431 -1.597 H -7.397 -5.337 -2.308 -7.646 -5.377 -1.891 H -5.779 -5.002 -2.902 -6.009 -5.273 -2.515 N -5.925 -6.680 -1.678 -6.319 -6.801 -1.134 H -4.922 -6.675 -1.494 -5.323 -6.884 -0.937H -6.357 -6.949 -0.794 -6.780 -6.940 -0.235DB1 / 159329947.1 75Attorney Docket No. 061809-5015-WOScheme 1. Synthesis of a macrocyclic bifunctional chelator.Example 2. Synthesis of a macrocyclic bifunctional chelator (Scheme 1).

[0251] l-(Benzyloxy)-6-oxo-l,6-dihydropyridine-2,5-dicarbonyl dichloride 3, tert-butyl (S)-(5,6-bis(l-(benzyloxy)-6-oxo-5-(2-thioxothiazolidine-3-carbonyl)-l,6-dihydropyridine-2-carboxamido)hexyl)carbamate 8 and benzyl tert-butyl (6-aminohexane-l,5-diyl)(S)-dicarbamate were prepared as previously described (Tatum D, Xu J, Magda D, Butlin N. Macrocyclic ligands with pendant chelating moieties and complexes thereof.WO / 2019 / 173639, 2019: USA.). Reagents were purchased from Sigma-Aldrich Chemicals unless noted otherwise. 2,2,2-Trifluoro-l-(2-thioxothiazolidin-3-yl)ethan-l-one was prepared according to literature protocol (Nagao, Y., et al., J. Organometal. Chem. 2000, 611,DB1 / 159329947.1 76Attorney Docket No. 061809-5015-WO172 - 177.). N-[2-(trimethylsilyl)ethoxycarbonyloxy]succinimide was purchased from Chemlmpex, Inc.

[0252] Benzyl tert-butyl (2-(trimethylsilyl)ethyl) hexane-l.2.0-triylfSl-tri carbamate 1. Benzyl tert-butyl (6-aminohexane-l,5-diyl)(S)-dicarbamate (956 mg, 2.61 mmol) was dried overnight in a 100 mL flask equipped with a stir bar. Dichloromethane (16 mL) was added to form a solution, tri ethylamine (729 pL, 5.23 mmol) and N-[2-(trimethylsilyl)ethoxycarbonyloxy]succinimide (814 mg, 3.14 mmol) were added under an inert atmosphere. After 4 hr, the solution was washed with water (50 mL), solvents were removed under reduced pressure, and the residue was dried overnight in vacuo. The residue was purified using silica gel chromatography, using 1-2% methanol in di chloromethane to elute compound 1 that was dried in vacuo (1.282 g, 96.4%). FTMS+pESI: Calc, for C25H43N3O6NaSi (M+Na)+, 532.2813; found, 532.2821.

[0253] tert-butyl (2-(trimethylsilyl)ethyl) (2-aminohexane-L6-diyl)(S)-dicarbamate 2.Benzyl tert-butyl (2-(trimethylsilyl)ethyl) hexane-l, 2, 6-triyl(S)-tricarbamate 1 (1.059 g, 2.08 mmol) was dried overnight in a 100 mL flask equipped with a stir bar. Methanol (13 mL) was added to form a solution, whereupon 10% palladium on carbon was added. The flask was transferred to a Parr bomb, and the atmosphere was exchanged to 200 psi hydrogen. After 24 hr, the suspension was filtered through Celite filter aid, solvent was removed under reduced pressure, and the residue was dried overnight in vacuo to provide compound 2 (806 mg, 103%). FTMS+pESI: Calc, for CnlfcsNsCUSi (M+H)+, 376.2626; found, 376.2628.

[0254] tert-Butyl (2-(trimethylsilyl)ethyl) ((S)-2-(l-(benzyloxy)-2-oxo-6-(((S)-2,2,17,17-tetramethyl-6,15-dioxo-5,16-dioxa-7,14-diaza-2-silaoctadecan-9-yl)carbamoyl)-L2-dihydropyridine-3-carboxamido)hexane-L6-diyl)dicarbamate 4. l-(Benzyloxy)-6-oxo-l,6-dihydropyridine-2,5-dicarboxylic acid (248 mg, 858 pmol) was dried in vacuo in a 50 mL flask. The compound was suspended in dichloromethane (anhydrous, 5 mL), oxalyl chloride (500 pL, 5.83 mmol) was added, and dimethylformamide (anhydrous, ca. 5 pL) was added using a pipettor. A second aliquot of dimethylformamide was added after 15 minutes and a third after 30 minutes. Solvents were removed in vacuo after an additional 45 minutes and the crude yellow residue 3 was dried overnight. tert-Butyl (2-(trimethylsilyl)ethyl) (2-aminohexane-l,6-diyl)(S)-dicarbamate 2 (806 mg, 2.15 mmol) was dissolved in ca. 5 mL di chloromethane and mixed with K2CO3 (711 mg, 5.15 mmol) dissolved in 10 mL water. The resulting mixture was cooled on an ice bath with stirring. To this mixture, the yellowDB1 / 159329947.1 77Attorney Docket No. 061809-5015-WOresidue of diacid chloride 3 prepared above dissolved in 6 mL di chloromethane was added dropwise over 30 minutes. The mixture was allowed to warm to ambient temperature after 2 hours and was transferred to a separatory funnel to recover the organic phase. Solvents were removed under reduced pressure, and the residue was purified using silica gel chromatography, using 3.5% methanol in di chloromethane to elute compound 4 that was dried in vacuo (756 mg, 87.7%). FTMS+pESI: Calc, for C48H82N7Oi2Si2 (M+H)+, 1004.5555; found, 1004.5545.

[0255] l-(Benzyloxy)-6-(2-thioxothiazolidine-3-carbonyl)pyridin-2(lH)-one 5. 1-(Benzyloxy)-6-oxo-l,6-dihydropyridine-2-carboxylic acid (1.00 g, 4.08 mmol) and 4-(dimethylamino)pyridine (100 mg, 818 pmol) were dried together in a 100 mL flask. The solids were suspended in di chloromethane (anhydrous, 33 mL), N, N-diispropylethylamine (2.13 mL, 12.2 mmol) was added, and then 2,2,2-trifluoro-l-(2-thioxothiazolidin-3-yl)ethan-1-one (1.23 g, 773 pL, 5.71 mmol) was added using a syringe. The solution, which gradually turns from orange to yellow color, was allowed to stir for 3 hr. Solvents were removed and the residue was dried overnight. The residue was purified using silica gel chromatography, using 0-2% isopropyl alcohol in dichloromethane to elute compound 5 as a yellow solid after removing solvent. The residue was dissolved in dichloromethane (5 mL), layered with methyl t-butyl ether, and allowed to stand. The resulting crystals of compound 5 were filtered and dried in vacuo (1.004 g, 71.0%).

[0256] bis(2-(trimethylsilyl)ethyl) ((2S,2'S)-((l-(benzyloxy)-6-oxo-L6-dihydropyridine-2,5-dicarbonyl)bis azanediyl))bis 6- l- benzyloxy)-6-oxo-L6-dihvdropyridine-2-carboxamido)hexane-2,l-diyl))dicarbamate 6. tert-Butyl (2-(trimethylsilyl)ethyl) ((S)-2-(l-(benzyloxy)-2-oxo-6-(((S)-2, 2, 17,17-tetramethyl-6,15-di oxo-5, 16-dioxa-7, 14-diaza-2-silaoctadecan-9-yl)carbamoyl)- 1,2-dihydropyridine-3 -carboxamido)hexane- 1,6-diyl)dicarbamate 4 (629 mg, 626 pmol) and p-toluenesulfonic acid hydrate (324 mg, 1.69 mmol) were dissolved in ethanol (10 mL) in a 50 mL flask. The solution was heated at 70 °C using an oil bath for 90 minutes and allowed to cool, whereupon solvent was removed under reduced pressure. The residue was dissolved in dichloromethane (10 mL) and washed with IM NaOH (20 mL). Solvent was removed and the residue dried in vacuo. The residue was dissolved in dichloromethane (anhydrous, 6 mL) and compound 5 (477 mg, 1.38 mmol) was added. After 5 hours, solvents were removed under reduced pressure, and the residue was purified twice using silica gel chromatography, using 2-3.5% methanol in di chloromethane toDB1 / 159329947.1 78Attorney Docket No. 061809-5015-WOelute compound 6 that was dried in vacuo (366 mg, 46.5%). FTMS+pESI: Calc, for C64H84N9Oi4Si2 (M+H)+, 1258.5671; found, 1258.5674.

[0257] N2, N5-Bis((S)-l-amino-6-(l-(benzyloxy)-6-oxo-l,6-dihydropyridine-2-carboxamido)hexan-2-yl)-l-(benzyloxy)-6-oxo-l,6-dihydropyridine-2,5-dicarboxamide 7. A mixture of 5% triisopropylsilane (AK Scientific), 50% trifluoroacetic acid and 45% di chloromethane (15 mL) was added to compound 6 (366 mg, 291 pmol) in a 100 mL flask and the solution was stirred for 3 hr. Solvents were removed in vacuo to provide crude compound 7 that was used in the next step without purification. FTMS+pESI: Calc, for C52H60N9O10 (M+H)+, 970.4458; found, 970.4441.

[0258] tert-butyl (4-((lZ,4S,15S,24S,28Z)-9,19,30-tris(benzyloxy)-15,24-bis(4-(l-(benzyloxy)-6-oxo- 1,6-dihydropyridine-2-carboxamido)butyl)-2,7, 10,12,17,20,22,27,29-nonaoxo-3,6,9,13,16,19,23,26,30-nonaazatetracyclo[26.2.2.28’11.218’211hexatriaconta-l(31),8(34),ll(33),18(36),21(35),28(32)-hexaen-4-yl)butyl)carbamate (shown in Scheme 1) and tert-butyl (4-((lZ,5S,15S,24S,28Z)-9,19,30-tris(benzyloxy)-15,24-bis(4-(l-(benzyloxy)-6-oxo- 1,6-dihydropyridine-2-carboxamido)butyl)-2,7, 10,12,17,20,22,27,29-nonaoxo-3,6,9,13,16,19,23,26,30-nonaazatetracyclo[26.2.2.28’11.218’211hexatriaconta-l(31),8(34),ll(33),18(36),21(35),28(32)-hexaen-5-yl)butyl)carbamate (not shown in Scheme 1) 9. Compound 7 (291 pmol) was dissolved in anhydrous di chloromethane and anhydrous acetonitrile (1:1 mixture, 41 mL) and triethylamine (94 uL, 674 pmol) and transferred to a 50 mL Hamilton syringe, tert-butyl (S)-(5,6-bis(l-(benzyloxy)-6-oxo-5-(2-thioxothiazolidine-3-carbonyl)-l,6-dihydropyridine-2-carboxamido)hexyl)carbamate 8 (284 mg, 291 pmol) was dissolved in anhydrous dichloromethane and anhydrous acetonitrile (1:1 mixture, 41 mL) and transferred to another syringe. The two reactants were added to a stirring pot of dichloromethane (500 mL) at a rate of 0.25 - 0.50 mL / hr over 7 days using two syringe pumps. After 3 additional days, solvents were removed and the residue was dried overnight. The residue was purified using silica gel chromatography, using 0.1% triethylamine, 3.5 -5% methanol in dichloromethane to elute compound 9 as two isomers that were separated into 2 pure fractions, isomer A (first to elute), isomer B (last to elute), and one mixed fraction. Solvent was removed in vacuo and the resulting off white solids were dried in vacuo (isomer A: 142 mg, 28.6%; mixed isomers: 146 mg, 29.3%; isomer B: 80 mg, 16%). FTMS+pESI (isomer A): Calc, for C91H99N14O22 (M+H)+, 1707.7155; found, 1707.7143. FTMS+pESI (isomer B): Calc, for C91H99N14O22 (M+H)+, 1707.7155; found, 1707.7150.DB1 / 159329947.1 79Attorney Docket No. 061809-5015-WO

[0259] N, N,-(((4S,8Z,llZ,15S,24S)-4-(4-aminobutyl)-9,19,30-trihydroxy-2,7,10,12,17,20,22,27,29-nonaoxo-3,6,9,13,16,19,23,26,30-nonaazatetracvclor26.2.2.28’11.218’211hexatriaconta-l(31),8(34\l 1(33\18(36\21(35\28(32)-hexaene- 15,24-diyl)bis(butane-4, 1 -diyl))bis( 1 -hydroxy-6-oxo- 1,6-dihydropyridine-2-carboxamide) (Shown in Scheme 1) orN, N'-(((5S,8Z, HZ,15S,24S)-5-(4-aminobutyl)-9,19,30-trihydroxy-2,7,10,12,17,20,22,27,29-nonaoxo-3,6,9,13,16,19,23,26,30-nonaazatetracyclor26.2.2.28’11.218’211hexatriaconta-l(31),8(34\l 1(33\18(36\21(35\28(32)-hexaene- 15,24-diyl)bis(butane-4, 1 -diyl))bis( 1 -hydroxy-6-oxo- 1,6-dihydropyridine-2-carboxamide) (not shown in Scheme 1) 10. Compound 9 (isomer A, 122 mg, 71.4 pmol) was dissolved in a 1: 1 mixture of 12 M HC1 and acetic acid (2.0 mL). The resulting solution was stirred under a nitrogen atmosphere for 1 month. Solvents were removed under reduced pressure and the product was dissolved in methanol. Diethyl ether was added to form a precipitate, that was dried in vacuo overnight to provide compound 10 as the hydrochloride salt (50 mg, 60%). FTMS+pESI: Calc, for CsiHssFeNuOis (M+Fe+H)+, 1210.3403; found, 1210.3384. Calc, for C5iH55Fe2Ni4Oi8 (M+2Fe)+, 1263.2518; found, 1263.2501.Scheme 2. Synthesis of a macrocyclic bifunctional lanthanum chelate.Example 3. Synthesis of a macrocyclic bifunctional lanthanum chelate (Scheme 2).DB1 / 159329947.1 80Attorney Docket No. 061809-5015-WO

[0260] Lanthanum(III) complex ofN, N'-(((4S,8Z,llZ,15S,24S)-4-(4-aminobutyl)-9,19,30-trihydroxy-2,7,10,12,17,20,22,27,29-nonaoxo-3,6,9,13,16,19,23,26,30-nonaazatetracvclol26.2.2.28’11.218’211hexatriaconta-l(31),8(34\l 1(33\18(36\21(35\28(32)-hexaene- 15,24-diyl)bis(butane-4, 1 -diyl))bis( 1 -hydroxy-6-oxo- 1,6-dihydropyri dine-2-carboxamide) 11. The hydrochloride salt ofN, N'-(((4S,8Z, HZ,15S,24S)-4-(4-aminobutyl)-9,19,30-trihydroxy-2,7,10,12,17,20,22,27,29-nonaoxo-3,6,9,13,16,19,23,26,30-nonaazatetracyclo[26.2.2.28’11.218’21]hexatriaconta-l(31),8(34),l 1(33), 18(36), 21(35), 28(32)-hexaene- 15,24-diyl)bis(butane-4, 1 -diy l))bi s( 1 -hydroxy-6-oxo- 1,6-dihydropyridine-2-carboxamide) 10 (3.44 mg, 2.89 pmol) was dissolved in dimethylformamide (344 pL). LaCh in dimethylformamide (40 mM, 115 pL, 2.88 pmol) was added to this solution to form a precipitate that redissolved upon addition of buffer, pH 7.5 (0.1 M HEPES saline, 229 pL). After adding water (2.29 mL) the product was purified by semi-preparative HPLC using a gradient of 20 - 30% acetonitrile (Solvent B) in 0.1% trifluoroacetic acid (Solvent A).Product containing fractions were lyophilized and a small amount of product was dissolved in dimethylformamide for analysis. HPLC: 94.9%. UV-vis (diluted 1:100 in Tris buffered saline, 0.05% TWEEN, pH 7.5): Xmax = 385 nm. The concentration of the stock solution was calculated to be 77.9 pM assuming s385= 18,750 M^cm’1. FTMS-pESI: Calc, for C51H56N14O18La (M-H)-, 1291.2966; found, 1291.2976.Scheme 3. Synthesis of a macrocyclic bifunctional isothiocyanate chelator.Example 4. Synthesis of a macrocyclic bifunctional isothiocyanate chelator (Scheme 3).DB1 / 159329947.1 81Attorney Docket No. 061809-5015-WO

[0261] N,N'-(((4S,8Z,11Z,15S,24S)-9,19,30-trihydroxy-4-(4-(3-(4-isothiocyanatophenyl)thioureido)butyl)-2,7,10,12,17,20,22,27,29-nonaoxo-3,6,9,13,16,19,23,26,30-nonaazatetracvclo[26.2.2.28’11.218’211hexatriaconta-l(31),8(34),ll(33),18(36),21(35),28(32)-hexaene-15,24-diyl)bis(butane-4,l-diyl))bis(1-hydroxy-6-oxo-1,6-dihydropyridine-2-carboxamide) 12. The hydrochloride salt ofN, N'-(((4S,8Z,1 lZ,15S,24S)-4-(4-aminobutyl)-9,19,30-trihydroxy-2,7,10,12,17,20,22,27,29-nonaoxo-3,6,9,13,16,19,23,26,30-nonaazatetracyclo[26.2.2.28’11.218’21]hexatriaconta-1(31), 8(34), 11(33), 18(36), 21(35), 28(32)-hexaene-15,24-diyl)bis(butane-4,l-diyl))bis(l-hydroxy-6-oxo-l,6-dihydropyridine-2-carboxamide) 10 (4.75 mg, 3.98 pmol) was dissolved in dimethylformamide (100 pL) and triethylamine (3.0 pL, 22 pmol). 1,4-Phenylene diisothiocyanate (PDITC, 8.40 mg, 43.7 pmol) was dissolved in dimethylformamide (100 pL). The solution of 10 was added to the solution of PDITC and vortexed for 75 minutes, whereupon the reaction mixture was divided into two 2 mL microfuge tubes and a precipitate was formed by the addition of diethyl ether (ca. 1.5 mL per tube). Solvent was decanted after centrifugation at 11,500 rpm for 1 minute, and the pellets were washed with diethyl ether and allowed to air dry. The pellets were dissolved in dimethylformamide (20 pL per tube) and methanol (300 pL per tube). Diethyl ether (1.5 mL per tube) was added to form a precipitate. Solvent was decanted after centrifugation at 11,500 rpm for 1 minute, the pellets were washed with diethyl ether, allowed to air dry, and then dried in vacuo to provide 3.60 mg compound 12 (67%). FTMS-pESL Calc, for C59H63N16O18S2 (M-H); 1347.3953; found, 1347.3951.Example 5. HPLC assay measurements were conducted on solutions of macrocylic compounds in DMF, diluted 10-fold with 0, IM TrisHCl buffer, pH 7,5 or with 50 mM DTP A, pH 7,4.

[0262] Reverse phase HPLC was performed using an Agilent 1200 system with a Zorbax Eclipse XDB-C18 column (4.6 x 150 mm, 5 micron). The method employed a gradient of acetonitrile (10 - 80% over 25 minutes) in 0.1 M triethylammonium bicarbonate, pH 7.5 as mobile phase. Chromatograms of the chelating agent 10, lanthanum complex 11 formulated in DTP A, and lanthanum complex 11 formulated in DTP A for 24 hours display retention times of 6.7 minutes, 8.2 minutes, and 8.2 minutes, respectively (Figure 5, A-C). These data indicate that complex 11 remained stable for 24 hours upon dilution in 50mM DTP A, pH 7.4. Analysis of Lumi804 chelating agent 13 and Lumi804 chelating agent complexed with lanthanum (Figure 7, A, B) indicated retention times of 4.6 minutes and 7.2 minutes, DB1 / 159329947.1 82Attorney Docket No. 061809-5015-WOrespectively. Lumi804 chelating agent complexed with lanthanum 13-La diluted in 50 mM DTPA displayed a complex chromatogram with peaks at 4.6 minutes, 6.6 minutes, and 7.3 minutes (Figure 7, C). The 4.6-minute peak increased in size after 3 days (Figure 7, D). These data lead us to suggest that chelating agent 10 forms a more stable complex with La(III) cation than Lumi804 chelating agent 13.Example 6. Preparation of an antibody chelator conjugate 14.

[0263] To a solution of trastuzumab (Biosynth, Inc., 14.1 mg / mL, 94.0 pM, 120 pL) in 100 mM sodium bicarbonate buffer, pH 9, in an O-ring type microcentrifuge tube was added a solution of 4-isothiocyanatophenylthiourea derivative 12 (13.54 pL of 5 mM stock solution in dimethylformamide for 6 molar equivalents). The resulting solution was mixed at 800 rpm for two hours. The contents of the microtube were applied to a 1.0 mL Penefsky size exclusion column containing Sephadex G50 Fine equilibrated in 50 mM Tris, pH 7.6, 150 mM NaCl. The tube was centrifuged at ca. 700 rpm for 3 minutes and the eluent collected. The concentration of protein was measured by UV-vis spectrometry of a fortyfold dilution using extinction coefficients for trastuzumab at 280 nm of 1.4 mL / mg, and 21,000 and 17,000 M'1cm’1for the chelator at 280 nm and 365 nm, respectively. The resulting solution was found to be 13.2 mg / mL conjugate 14 in ca. 100 pL buffer. The degree of labeling was calculated to be 2.3 chelators per protein, using a molecular weight of 150,000 g / mol for trastuzumab.DB1 / 159329947.1 83Attorney Docket No. 061809-5015-WOScheme 4. Synthesis of a macrocyclic bifunctional chelator.Example 7. Synthesis of a macrocyclic bifunctional chelator (Scheme 4).

[0264] Methyl l-hydroxy-6-((2-methoxyethyl)carbamoyl)-2-oxo-l,2-dihydropyridine-3-carboxylate 16. Methyl l-(benzyloxy)-2-oxo-6-(2-thioxothiazolidine-3 -carbonyl)- 1,2-dihydropyridine-3 -carboxylate 15 (Tatum D, Xu J, Magda D, Butlin N. Macrocyclic ligands with pendant chelating moieties and complexes thereof, WO / 2019 / 173639, 2019: USA.) is dissolved in dichloromethane. 2 -Methoxy ethan-1 -amine is added and allowed to mix overnight. The product is purified using methanol and dichloromethane on silica gel, and solvent is removed under reduced pressure to provide the purified compound 16.

[0265] l-(Benzyloxy)-6-((2 -methoxy ethyl)carbamoyl)-2-oxo-l,2-dihydropyridine-3-carboxylic acid 17. Methyl 1 -hydroxy-6-((2-methoxyethyl)carbamoyl)-2-oxo- 1,2-dihydropyridine-3 -carboxylate 16 is dissolved in a mixture of methanol and tetrahydrofuran. Sodium hydroxide (IM in water) is added, and the mixture is stirred for several hours.Volatile solvents are removed under reduced pressure, water is added to form a solution, and hydrochloric acid (IM in water) is added to form a precipitate. The precipitate is filtered and dried in vacuo to provide compound 17.DB1 / 159329947.1 84Attorney Docket No. 061809-5015-WO

[0266] l-(Benzyloxy)-N-(2-methoxyethyl)-6-oxo-5-(2-thioxothiazolidine-3-carbonyl)-l,6-dihydroDyridine-2-carboxamide 18. l-(Benzyloxy)-6-((2 -methoxy ethyl)carbamoyl)-2-oxo-l,2-dihydropyridine-3 -carboxylic acid 17 and 4-dimethylaminopyridine are suspended in anhydrous dichloromethane, and diisopropylethylamine is added to form a solution. 2,2,2-Trifluoro-l-(2-thioxothiazolidin-3-yl)ethan-l-one is added and the reaction mixture is allowed to stir for several hours. The yellow product is purified using isopropyl alcohol and dichloromethane on silica gel, and solvent is removed under reduced pressure to provide the purified compound 18.

[0267] 2-(Trimethylsilyl)ethyl ((S)-2-(l-(benzyloxy)-6-(((S)-15-(l-(benzyloxy)-6-((2-methoxyethyl)carbamoyl)-2-oxo-l,2-dihvdroDyridin-3-yl)-2,2-dimethyl-6,15-dioxo-5-oxa-7,14-diaza-2-silapentadecan-9-yl)carbamoyl)-2-oxo-l,2-dihydropyridine-3-carboxamido)-6-(l-hydroxy-6-((2 -methoxy ethyl)carbamoyl)-2-oxo-l,2-dihydropyridine-3-carboxamido)hexyl)carbamate 19. Compound 19 is prepared as described above for compound 6 using compound 18 in place of compound 5.

[0268] N5-((S)-6-amino-5-(5-(((S)-l-amino-6-(l-hydroxy-6-((2-methoxyethyl)carbamoyl)-2-oxo- 1,2-dihydropyridine-3 -carboxamido)hexan-2-yl)carbamoyl)- 1 -(benzyloxy)-6-oxo- 1,6-dihvdroDyridine-2-carboxamido)hexyl)- 1 -(benzyloxy)-N2-(2-m ethoxy ethyl)-6-oxo- 1,6-dihydropyridine-2,5-dicarboxamide 20. Compound 20 is prepared as described above for compound 7.

[0269] tert-Butyl (4-((lZ,4S,15S,24S,28Z)-9,19,30-tris(benzyloxy)-15,24-bis(4-(l-(benzyloxy)-6-((2-methoxyethyl)carbamoyl)-2-oxo-l,2-dihydropyridine-3-carboxamido)butyl)-2,7,10,12,17,20,22,27,29-nonaoxo-3,6,9,13,16,19,23,26,30-nonaazatetracy clo[26, 2, 2, 28’11,218’211hexatriaconta-l (31),8(34),11(33), 18(36),21 (35),28(32)-hexaen-4-yl)butyl)carbamate (isomer shown in Scheme 4) and tert-butyl (4-((lZ,5S,15S,24S,28Z)-9,19,30-tris(benzyloxy)-15,24-bis(4-(l-(benzyloxy)-6-((2-methoxyethyl)carbamoyl)-2-oxo-l,2-dihydropyridine-3-carboxamido)butyl)-2,7,10,12,17,20,22,27,29-nonaoxo-3,6,9,13,16,19,23,26,30-nonaazatetracvclo[26.2.2.28’11.218’211hexatriaconta-l(31),8(34),l 1(33),18(36),21(35),28(32)-hexaen-5-yl)butyl)carbamate (isomer not shown in Scheme 4) 21. Compound 21 is prepared as described above for compound 9.

[0270] N5, N5'-(((4S,8Z,llZ,15S,24S)-4-(4-Aminobutyl)-9,19,30-trihvdroxy-2,7,10,12,17,20,22,27,29-nonaoxo-3,6,9,13, 16,19,23,26,30-DB1 / 159329947.1 85Attorney Docket No. 061809-5015-WOnonaazatetracyclor26.2.2.28’11.218’211hexatriaconta-l(31),8(34\ll(33\18(36),21(35),28(32)-hexaene-15,24-diyl)bis(butane-4,l-diyl))bis(l-hydroxy-N2-(2 -methoxy ethyl)-6-oxo-l, 6-dihydroDyridine-2,5-dicarboxamide) (isomer shown in Scheme 4) and N\N5'-(((5S,8Z,llZ,15S,24S)-5-(4-aminobutyl)-9,19,30-trihydroxy-2,7,10,12,17,20,22,27,29-nonaoxo-3,6,9,13,16,19,23,26,30-nonaazatetracyclol26.2.2.28’11.218’211hexatriaconta-l(31),8(34),ll(33),18(36),21(35),28(32)-hexaene-15,24-diyl)bis(butane-4,l-diyl))bis(l-hvdroxy-N2-(2-methoxyethyl)-6-oxo-l,6-dihvdropyridine-2,5-dicarboxamide) (isomer not shown in Scheme 4) (HC1 salt) 22. The hydrochloride salt of compound 22 is prepared as described above for compound 10.Scheme 5. Synthesis of a macrocyclic bifunctional chelator.Example 8. Synthesis of a macrocyclic bifunctional chelator (Scheme 5).DB1 / 159329947.1 86Attorney Docket No. 061809-5015-WO

[0271] 2-((tert-Butoxycarbonyl)amino)propane-1,3-diyl dimethanesulfonate 23. tert-Butyl (l,3-dihydroxypropan-2-yl)carbamate (Aldrich Chem.) (5.00 g, 26.1 mmol) was dissolved in dry dichloromethane (115 mL) and triethylamine (14.6 mL) and cooled on an ice bath.Methanesulfonyl chloride (6.07 mL, 78.4 mmol) was added, and the resulting suspension was stirred for one half hour prior to warming to ambient temperature for one hour. The suspension was cooled again on ice then washed successively with 5% K2CO3 (sat.) (20 mL), water (100 mL) and brine (50 mL). Solvent was removed under reduced pressure to provide the crude compound 23 (9.3g, 105%) that was used in the next step without further purification.

[0272] tert-Butyl (1,3-diazidopropan-2-yl)carbamate 24. Compound 23 (9.3 g, 26.8 mmol) and sodium azide (5.22 g, 80.3 mmol) were dissolved in dimethylformamide (125 mL). The solution was heated at 70 °C for 19 hr then allowed to cool to ambient temperature. Solvent was removed under reduced pressure, and the residue was dissolved in ethyl acetate (200 mL) and water (200 mL). The organic layer was washed with brine (100 mL) and solvent was removed under reduced pressure. The residue was purified using dichloromethane on silica gel. Solvent was removed under reduced pressure to provide the purified compound 24 (4.063 g, 62.8%). FTMS + pESI: Calc, for C8H16N7O2(M+H)+, 242.1360; found, 242.1360.

[0273] tert-Butyl (1,3-diaminopropan-2-yl)carbamate 25. Compound 24 (577 mg, 2.81 mmol) was dissolved in methanol (20 mL), 10% palladium on carbon (68 mg) was added, and the atmosphere was exchanged for hydrogen gas at 200 psi for 25 hr using a Parr apparatus. The resulting suspension was filtered using filter aid (Celite®), and solvent was removed under reduced pressure to provide compound 25 (533 mg, 100%). FTMS + pESI: Calc, for C8H20N3O2 (M+H)+, 190.1550; found, 190.1551.

[0274] Dimethyl 6,6'-(((2-((tert-butoxycarbonyl)amino)propane-1,3-diyl)bis(azanediyl))bis(carbonyl))bis(1-(benzyloxy)-2-oxo-1,2-dihydropyridine-3-carboxylate) 26. Compound 15 (2.278 g, 5.63 mmol) was dissolved in di chloromethane (60 mL) and added to compound 25 (533 mg, 2.82 mmol). After stirring for 20 hr, solvent was removed under reduced pressure. The residue was purified using 2% isopropyl alcohol in di chloromethane on silica gel (to recover unreacted compound 15), then 3.5% methanol in dichloromethane to elute product. Solvents were removed under reduced pressure to provide the purified compound 26 (2.029 g, 94.7%). FTMS + pESI: Calc, for C38H42N5O12 (M+H)+, 760.2824; found, 760.2834.DB1 / 159329947.1 87Attorney Docket No. 061809-5015-WO

[0275] 6,6'-(((2-((tert-Butoxycarbonyl)amino)propane-1,3-diyl)bis(azanediyl))bis(carbonyl))bis(1-(benzyloxy)-2-oxo-1,2-dihydropyridine-3-carboxylic acid) 27. Compound 26 (1.947 g, 2.56 mmol) was dissolved in methanol (40 mL). Sodium hydroxide solution (IM, ca. 8 mL) was added and the solution was allowed to stir and form a precipitate for 25 hr. Methanol was removed under reduced pressure and hydrochloric acid (IM, ca. 10 mL) was added to form a precipitate. The precipitate was filtered, washed with water (15 mL), and dried in vacuo to provide compound 27 (1.803 g, 96.3%). FTMS - pESL Calc, for C36H36N5O12 (M-H)-, 730.2366; found, 730.2368.

[0276] tert-Butyl (1,3-bis(1-(benzyloxy)-6-oxo-5-(2-thioxothiazolidine-3-carbonyl)-1,6-dihydropyridine-2-carboxamido)propan-2-yl)carbamate 28. Compound 27 (900 mg, 1.230 mmol) and 4-dimethylaminopyridine (60 mg, 0.49 mmol) were dried together overnight. The solids were suspended in anhydrous dichloromethane (20 mL), and diisopropylethylamine (1.285 mL, 7.38 mmol) was added to form a solution. 2,2,2-Trifluoro-l-(2-thioxothiazolidin-3-yl)ethan-l-one (499 pL, 3.69 mmol) was added and the reaction mixture was allowed to stir for three hours and form a precipitate. The yellow product was filtered, washed with di chloromethane (15 mL) and dried in vacuo to provide the purified compound 28 (1.108 g, 96.4%). FTMS + pESI: Calc, for C42H44N7O10S4 (M+H)+, 934.2027; found, 934.2020.

[0277] tert-Butyl ((5S,8Z,1 lZ,14S)-9,20,30-tris(benzyloxy)-5,14-bis(4-(l-(benzyloxy)-6-oxo- 1,6-dihydropyridine-2-carboxamido)butyl)-2,7, 10,12,17,19,22,28,31 -nonaoxo-3, 6,9, 13, 16,20,23, 27,30-nonaazatetracyclo[27.2,2, 28,11.218’211heptatriaconta- 1 (32), 8(35), 11 (34), 18(37),21 (36),29(33)-hexaen-25-yl)carbamate 29. Compound 28 (571 mg, 612 pmol) was dissolved in anhydrous dimethylformamide (44 mL) and transferred to a 50 mL Hamilton syringe. N2, N5-Bis((S)-l-amino-6-(l-(benzyloxy)-6-oxo-l,6-dihydropyridine-2-carboxamido)hexan-2-yl)-l-(benzyloxy)-6-oxo-l,6-dihydropyridine-2,5-dicarboxamide 7 (636 pmol) was dissolved in anhydrous dimethylformamide (22 mL), triethylamine (266 pL, 1.91 mmol) was added and the solution was transferred to another syringe. The two reactants were added to a stirring pot of dichloromethane (500 mL) at a rate of 0.25 - 0.50 mL / hr over 7 days using two syringe pumps. After 2 additional days, solvents were removed under reduced pressure and the residue was dried overnight. The crude product was purified using silica gel chromatography, using 0.1% tri ethylamine, 5 - 7.5% methanol in di chloromethane to elute compound 29. Solvent was removed in vacuo and the resulting off-white solid was dried in vacuo (344 mg, 36.2%). FTMS+pESI: Calc, for C88H93N14O20 (M+H)+, 1665.6685; found, 1665.6690.DB1 / 159329947.1 88Attorney Docket No. 061809-5015-WO

[0278] N,N'-(((1Z,5S,14S,29Z)-25-amino-9,20,30-trihydroxy-2,7,10,12,17,19,22,28,31-nonaoxo-3,6,9,13,16,20,23,27,30-nonaazatetracyclo[27.2.2.28,11.218,21]heptatriaconta-1(32),8(35),11(34),18(37),21(36),29(33)-hexaene-5,14-diyl)bis(butane-4,1-diyl))bis(1-hydroxy-6-oxo-1,6-dihydropyridine-2-carboxamide) 30. Compound 29 (287 mg, 172 pmol) was dissolved in a mixture of 12 M HC1 (5 mL) and acetic acid (4 mL). The resulting solution was allowed to stand under a nitrogen atmosphere for 5 weeks. Solvents were removed under reduced pressure and the product was suspended in methanol (6 mL). The suspension was filtered, washed with methanol (2 mL) and dried under reduced pressure. Additional product was recovered from the filtrate by reducing its volume (to 3 mL), transferring to 6 microtubes, and treating with diethyl ether (1.5 mL / tube) to form a precipitate that was dried in vacuo. The dried precipitates were combined to provide compound 30 as the hydrochloride salt (188.5 mg, 95%). FTMS+pESI: Calc. for C48H55N14O18(M+H)+, 1115.3813; found, 1115.3820.DB1 / 159329947.1 89Attorney Docket No. 061809-5015-WOScheme 6. Synthesis of a macrocyclic bifunctional chelator.Example 9. Synthesis of a macrocyclic bifunctional chelator (Scheme 6).

[0279] Methyl l-(benzyloxy)-6-((2-((2-(l-(benzyloxy)-5-(methoxycarbonyl)-6-oxo-l,6-dihydropyridine-2-carboxamido)ethyl)(2-((tert-butoxycarbonyl)amino)ethyl)amino)ethyl)carbamoyl)-2-oxo-l,2-dihydropyridine-3-carb oxy late 32. A solution of compound 15 (2.208 g, 5.46 mmol) in di chloromethane (anhydrous, 50 mL) was added to tert-butyl (2-(bis(2-aminoethyl)amino)ethyl)carbamate 31 (673 mg, 2.73 mmol) and stirred for 2 days. Solvent was removed under reduced pressure, and the residue was purified using 2% isopropyl alcohol in dichloromethane on silica gel (to recover unreacted compound 15), then 3.5% methanol in dichloromethane to elute product.DB1 / 159329947.1 90Attorney Docket No. 061809-5015-WOSolvents were removed under reduced pressure to provide the purified compound 32 (1.230 g, 55.2%). FTMS + pESI: Calc, for C41H49N6O12 (M+H)+, 817.3403; found, 817.3386.

[0280] 1 -(Benzyloxy)-6-((2-((2-( 1 -(benzyloxy)-5-carboxy-6-oxo- 1,6-dihydropyridine-2-carboxamido)ethyl)(2-((tert-butoxycarbonyl)amino)ethyl)amino)ethyl)carbamoyl)-2-oxo-l,2-dihvdropyridine-3 -carboxylic acid 33. Compound 32 (1.230 g, 1.506 mmol) was dissolved in methanol (30 mL). Sodium hydroxide solution (IM, ca 5 mL) was added and the solution was stirred for one hour, forming a precipitate. Water (20 mL) was added to form a solution that was stirred for an additional 23 hours. Methanol was removed under reduced pressure and hydrochloric acid (IM, ca. 6 mL) was added to form a sticky precipitate. The precipitate was filtered, and the residue from filter funnel and flask were dissolved in methanol. Solvent was removed under reduced pressure and the residue was dried in vacuo to provide compound 33 (831 mg, 66.9%). FTMS - pESI: Calc, for C39H45N6O12 (M+H)+, 789.3090; found, 789.3073.

[0281] tert-Butyl (2-(bis(2-(l-(benzyloxy)-6-oxo-5-(2-thioxothiazolidine-3-carbonyl)-l,6-dihydropyridine-2-carboxamido)ethyl)amino)ethyl)carbamate 34. Compound 33 (831 mg, 1.007 mmol) and 4-dimethylaminopyridine (49 mg, 0.40 mmol) were dried together overnight. The solids were suspended in anhydrous di chloromethane (18 mL), and diisopropylethylamine (1.052 mL, 6.04 mmol) was added to form a solution. 2,2,2-Trifluoro-l-(2-thioxothiazolidin-3-yl)ethan-l-one (409 pL, 3.02 mmol) was added and the reaction mixture was allowed to stir for five hours. Solvent was reduced to ca. 5 mL under reduced pressure, and methyl-tert-butyl ether (20 mL) was added to form an oily solid. Solvent was decanted and the residue dried in vacuo. The residue was purified using 2 - 5% isopropyl alcohol in di chloromethane on silica gel. Solvents were removed under reduced pressure to provide the purified compound 34 (946 mg, 94.8%). FTMS + pESI: Calc, for C45H51N8O10S4 (M+H)+, 991.2605; found, 991.2597.

[0282] tert-Butyl (2-((lZ,5S,14S,18Z,21Z,31Z)-9,20,32-tris(benzyloxy)-5,14-bis(4-(l-(benzyloxy)-6-oxo-l,6-dihydropyridine-2-carboxamido)butyl)-2,7,l 0,12, 17, 19,22,30,33-nonaoxo-3, 6,9, 13, 16,20,23, 26,29,32-decaazatetracyclo[29.2,2, 28,11.218’211nonatriaconta-1 (34), 8(37), 11 (36), 18(39),21 (38),31 (35)-hexaen-26-yl)ethyl)carbamate 35. Compound 34 (481 mg, 485 pmol) was dissolved in anhydrous dimethylformamide (24 mL) and transferred to a 25 mL Hamilton syringe. N2, N5-Bis((S)-l-amino-6-(l-(benzyloxy)-6-oxo-l,6-dihydropyridine-2-carboxamido)hexan-2-yl)-l-(benzyloxy)-6-oxo-l,6-dihydropyridine-2,5-DB1 / 159329947.1 91Attorney Docket No. 061809-5015-WOdicarboxamide 7 (500 pmol) was dissolved in anhydrous dimethylformamide (24 mL), triethylamine (209 pL, 1.50 mmol) was added, and the solution was transferred to another syringe. The two reactants were added to a stirring pot of dichloromethane (500 mL) at a rate of 0.25 mL / hr over 7 days using two syringe pumps. Solvents were removed under reduced pressure and the residue was dried overnight. The residue was purified using silica gel chromatography, using 0.1% triethylamine, 5 - 7.5% methanol in dichloromethane to elute compound 35. Solvent was removed in vacuo and the resulting off-white solid was dried in vacuo (253 mg, 29.4%). FTMS+pESI: Calc, for C91H100N15O20 (M+H)+, 1722.7264; found, 1722.7280.

[0283] N, N'-(((1Z,5S, 14S,3 lZ)-26-(2-Aminoethyl)-9,20,32-trihvdroxy-2,7,10,12,17,19,22,30,33-nonaoxo-3,6,9,13, 16,20,23,26,29,32-decaazatetracyclol29, 2.2.28,11.218’211nonatriaconta-l(34),8(37),ll(36),18(39),21(38),31(35)-hexaene-5,14-diyl)bis(butane-4,l-diyl))bis(l-hydroxy-6-oxo-l,6-dihydropyridine-2-carboxamide) 36. Compound 35 (253 mg, 147 pmol) was dissolved in a mixture of 12 M HC1 (5 mL) and acetic acid (4 mL). The resulting solution was allowed to stand under a nitrogen atmosphere for one month. Solvents were removed under reduced pressure and the product was suspended in methanol (4 mL). The suspension was filtered, washed with methanol (2 mL) and dried under reduced pressure. Additional product was recovered from the filtrate by reducing its volume (to 3 mL), transferring to 6 microtubes, and treating with diethyl ether (1.5 mL / tube) to form a precipitate that was dried in vacuo. The dried precipitates were combined to provide compound 36 as the dihydrochloride salt (160.4 mg, 87.6%).FTMS+pESI: Calc, for C51H62N15O18 (M+H)+, 1172.4392; found, 1172.4393.DB1 / 159329947.1 92Attorney Docket No. 061809-5015-WOScheme 7. Synthesis of a macrocyclic bifunctional lanthanum chelate.Example 10. Synthesis of a macrocyclic bifunctional lanthanum chelate (Scheme 7).

[0284] Lanthanum(III) complex ofN, N'-(((lZ,5S,14S,31Z)-26-(2-Aminoethyl)-9,20,32-trihydroxy-2,7,10,12,17,19,22,30,33-nonaoxo-3,6,9,13,16,20,23,26,29,32-decaazatetracyclo[29.2.2.28’n.218’21]nonatriaconta-l(34), 8(37), 11(36), 18(39), 21(38), 31(35)-hexaene-5,14-diyl)bis(butane-4,l-diyl))bis(l-hydroxy-6-oxo-l,6-dihydropyridine-2-carboxamide) 37. The hydrochloride salt of compound 36 (3.02 mg, 2.43 pmol) wasdissolved in dimethylformamide (302 pL). LaCh in dimethylformamide (40 mM, 72.7 pL, 2.91 pmol) was added to this solution to form a precipitate that redissolved upon addition of water (188 pL). After adding water (2.29 mL) the product was purified by semi-preparative HPLC using a gradient of 20 - 30% acetonitrile (Solvent B) in 0.1% trifluoroacetic acid (Solvent A). Product-containing fractions were lyophilized and a small amount of product 37 was submitted for mass spectral analysis. FTMS-pESI: Calc, for CsiHsvNisOisLa (M-H)', 1306.3075; found, 1306.3110.Scheme 8. Synthesis of a macrocyclic chelator conjugate.Example 11. Synthesis of a macrocyclic chelator conjugate (Scheme 8).DB1 / 159329947.1 93Attorney Docket No. 061809-5015-WO

[0285] di-tert-Butyl (((S)-l-(tert-butoxy)-l-oxo-6-(4-((2-((lZ,5S,14S,31Z)-9,20,32-trihydroxy-5,14-bis(4-(l-hydroxy-6-oxo-l,6-dihydropyridine-2-carboxamido)butyl)-2,7,10,12,17,19,22,30,33-nonaoxo-3,6,9,13,16,20,23,26,29,32-decaazatetracyclo[29, 2,2, 28’11.218’211nonatriaconta-l(34),8(37), 11(36), 18(39),21(38),31(35)-hexaen-26-yl)ethyl)carbamoyl)benzamido)hexan-2-yl)carbamoyl)-L-glutamate 39. The hydrochloride salt of compound 36 (3.2 mg, 2.6 pmol) was dissolved in dimethylformamide (200 pL) and tri ethylamine (4 pL, 29 pmol). CaCh in dimethylformamide (50 mM, 100 pL, 5 pmol) was added to this solution to form a suspension that was treated with di-tert-butyl (((S)-l-(tert-butoxy)-l-oxo-6-(4-(2-thioxothiazolidine-3-carbonyl)benzamido)hexan-2-yl)carbamoyl)-L-glutamate 38 (5.5 mg, 7.5 pmol, prepared as described in WO / 2022 / 087317) and placed on shaker for 24 hr. Diethyl ether (1.5 mL) was added to form a precipitate, that was centrifuged, the supernatant removed, the pellet washed with 1.5 mL diethyl ether and dried. Crude compound 39 was used in the next step without further purification. FTMS-pESI: Calc, for CssHiosCaNisCh? (M-H)', 1825.7028; found, 1825.7125.

[0286] (((S)-l-Carboxy-5-(4-((2-((lZ,5S,14S,31Z)-9,20,32-trihydroxy-5,14-bis(4-(l-hydroxy-6-oxo-l,6-dihydropyridine-2-carboxamido)butyl)-2,7,l 0,12, 17, 19,22,30,33-nonaoxo-3, 6,9, 13, 16,20,23, 26,29,32-decaazatetracyclo[29, 2,2, 28,11.218’211nonatriaconta-1 (34), 8(37), 11 (36), 18(39),21 (38),31 (35)-hexaen-26-yl)ethyl)carbamoyl)benzamido)pentyl)carbamoyl)-L-glutamic acid 40. Compound 39 was dissolved in triisopropylsilane (10 pL), trifluoroacetic acid (100 pL), and dichloromethane (90 pL) and placed on a shaker at 800 rpm for 24 hr. Solvents were removed under reduced pressure and the residue dissolved in dimethylformamide (200 pL) and water (467 pL). The crude product was purified by semi-preparative HPLC using a gradient of 20 - 30% acetonitrile (Solvent B) in 0.1% trifluoroacetic acid (Solvent A) at 40 °C. Product containing fractions were lyophilized and a small amount of product was submitted for mass spectral analysis. FTMS-pESI: Calc, for C71H83N18O27 (M-H); 1619.5681; found, 1619.5728.DB1 / 159329947.1 94Attorney Docket No. 061809-5015-WOScheme 9. Synthesis of a macrocyclic bifunctional chelator.Example 12. Synthesis of a macrocyclic bifunctional chelator (Scheme 9).

[0287] Benzyl tert-butyl (5-hydroxypentane-L4-diyl)(S)-dicarbamate 42. A solution of (S)-2-(((benzyloxy)carbonyl)amino)-5-((tert-butoxycarbonyl)amino)pentanoic acid 41 (Chemlmpex, 4.975 g, 13.6 mmol) in tetrahydrofuran (30 mL) was treated with carbonyl diimidazole (2.64 g, 16.3 mmol). After one hour, a solution of sodium borohydride (1.03 g, 27.2 mmol) in water (5 mL), cooled on ice, was added and the solution was stirred for 80 minutes. Volatile solvent was removed under reduced pressure and the residue was dissolved in ethyl acetate (75 mL) and aqueous hydrochloric acid (IM, 25 mL). The organic phase wasDB1 / 159329947.1 95Attorney Docket No. 061809-5015-WOseparated, washed with aqueous hydrochloric acid (IM, 25 mL), saturated sodium bicarbonate solution (2 x 25 mL), and saturated sodium chloride solution (25 mL). The organic phase was allowed to stand over sodium sulfate (anhydrous) overnight then concentrated under reduced pressure. The crude product was purified using silica gel chromatography using ethyl acetate as eluent, solvent was removed under reduced pressure, and the residue dried overnight in vacuo to provide compound 42 (4.252 g, (88.7%).FTMS+pESI: Calc, for C18H29N2O5 (M+H)+, 353.2071; found, 353.2074.

[0288] (S)-2-(((Benzyloxy)carbonyl)amino)-5-((tert-butoxycarbonyl)amino)pentyl methanesulfonate 43. A solution of compound 42 (4.252 g, 12.1 mmol) in triethylamine (3.36 mL, 24.2 mmol) and di chloromethane (60 mL) was cooled on an ice bath and methanesulfonyl chloride (1.21 mL, 15.7 mmol) was added over twenty minutes. After stirring an additional fifteen minutes the ice bath was removed and the solution was allowed to warm to ambient temperature for two hours. The solution was cooled again and a 5% solution of aqueous potassium bisulfate (9 mL) was added. The organic phase was separated and washed with water (20 mL) and saturated sodium chloride solution (25 mL). Solvent was removed under reduced pressure and the residue was dried in vacuo to provide crude compound 43 (5.382 g, 103%) that was used in the next step without further purification. FTMS+pESI: Calc, for C19H31N2O7S (M+H)+, 431.1846; found, 431.1846.

[0289] Benzyl tert-butyl (5-azidopentane-L4-diyl)(S)-dicarbamate 44. Compound 43 (5.209 g, 12.1 mmol) and sodium azide (1.180 g, 18.1 mmol) were dried together in vacuo.Dimethylformamide (Biotech grade, 30 mL) was added and the mixture was heated at 70 °C for 21 hours. Solvent was removed under reduced pressure and the residue was dissolved in ethyl acetate (100 mL) and water (50 mL). The organic layer was separated and washed with water (50 mL) and saturated sodium chloride solution (25 mL). The solution was dried over sodium sulfate (anhydrous) briefly and solvent was removed under reduced pressure. The crude product was purified by silica gel chromatography using 2% methanol in dichloromethane (0.1% triethylamine) as eluent. Solvent was removed under reduced pressure and the residue was dried in vacuo to provide compound 44 (4.568 g, 100%).FTMS+pESI: Calc, for C18H28N5O4(M+H)+, 378.2136; found, 378.2135.

[0290] Benzyl tert-butyl (5-aminopentane-L4-diyl)(S)-dicarbamate 45. Compound 44 (4.56 g, 12.1 mmol) was dried overnight in a 100 mL flask equipped with a stir bar. Methanol (45 mL) was added to form a solution, whereupon 5% palladium on calcium carbonate (LindlarDB1 / 159329947.1 96Attorney Docket No. 061809-5015-WOcatalyst, 450 mg) was added. The flask was transferred to a Parr bomb, and the atmosphere was exchanged to 200 psi hydrogen. After 23 hr, the suspension was filtered through Celite filter aid, solvent was removed under reduced pressure, and the residue was dried overnight in vacuo to provide compound 45 (3.818 g, 89.8%). FTMS+pESI: Calc, for C18H30N3O4 (M+H)+, 352.2231; found, 352.2232.

[0291] Benzyl tert-butyl (2-(trimethylsilyl)ethyl) pentane- L2,5-triyl(S)-tri carbamate 46. Compound 45 (3.82 g, 10.9 mmol) was dissolved in triethylamine (3.03 mL, 21.7 mmol) and anhydrous dichloromethane (50 mL). N-[2-(Trimethylsilyl)ethoxycarbonyloxy]succinimide (Chemlmpex, 3.38 g, 13.0 mmol) was added and the solution stirred for 3.5 hours. The solution was washed with water (50 mL) and solvent removed in vacuo. The crude product was purified by silica gel chromatography using 2% methanol in dichloromethane (0.1% triethylamine) as eluent. Solvent was removed under reduced pressure and the residue was dried in vacuo to provide compound 46 (4.946 g, 91.5%). FTMS+pESI: Calc, for C24H42N3O6Si (M+H)+, 496.2837; found, 496.2840.

[0292] tert-Butyl (2-(trimethylsilyl)ethyl) (2-aminopentane-L5-diyl)(S)-dicarbamate 47. Compound 46 (4.00 g, 8.07 mmol) was dissolved in methanol (50 mL) and 10% palladium on carbon (400 mg) was added. The flask was transferred to a Parr bomb, and the atmosphere was exchanged to 200 psi hydrogen. After 24 hr, the suspension was filtered through Celite filter aid, solvent was removed under reduced pressure, and the residue was dried overnight in vacuo to provide compound 47 (3.088 g, 106%). Crude compound 47 was used in the next step without further purification. FTMS+pESI: Calc, for C16H36N3O4Si (M+H)+, 362.2470; found, 362.2472.

[0293] tert-Butyl (2-(trimethylsilyl)ethyl) ((S)-2-(l-(benzyloxy)-2-oxo-6-(((S)-2,2,16,16-tetramethyl-6,14-dioxo-5,15-dioxa-7,13-diaza-2-silaheptadecan-9-yl)carbamoyl)-L2-dihydropyridine-3-carboxamido)pentane-L5-diyl)dicarbamate 48. l-(Benzyloxy)-6-oxo-l,6-dihydropyridine-2,5-dicarboxylic acid (1.060 g, 3.66 mmol) was dried in vacuo in a 100 mL flask. The compound was suspended in dichloromethane (anhydrous, 26 mL), oxalyl chloride (1.38 mL, 16.1 mmol) was added, and dimethylformamide (anhydrous, ca. 20 pL) was added using a pipettor. A second aliquot of dimethylformamide was added after 25 minutes and a third after another 50 minutes. Solvents were removed in vacuo after an additional 20 minutes and the crude yellow residue 3 was dried overnight. Compound 47 (2.914 g, 8.06 mmol) was dissolved in ca. 26 mL dichloromethane and mixed with K2CO3DB1 / 159329947.1 97Attorney Docket No. 061809-5015-WO(3.033 g, 22.0 mmol) dissolved in 52 mL water. The resulting mixture was cooled on an ice bath with stirring. To this mixture, the yellow residue of diacid chloride 3 prepared above dissolved in 26 mL dichloromethane was added in portions over 45 minutes using a syringe. The mixture was allowed to warm to ambient temperature after 2 hours and was transferred to a separatory funnel to recover the organic phase. Solvents were removed under reduced pressure, and the residue was purified using silica gel chromatography, using 0.1% triethylamine and 3.5% methanol in dichloromethane to elute compound 48 that was dried in vacuo (3.076 g, 86.1%). FTMS+pESI: Calc, for C46H78N7O12Si2(M+H)+, 976.5242; found, 976.5227.

[0294] bis 2- Trimethylsilyl)ethyl) ((2S,2'S)-((l-(benzyloxy)-6-oxo-L6-dihvdropyridine-2,5-dicarbonyl)bis(azanediyl))bis(5-(l-(benzyloxy)-6-oxo-L6-dihydropyridine-2-carboxamido)pentane-2,l-diyl))dicarbamate 49. Compound 48 (3.076 g, 3.15 mmol) and p-toluenesulfonic acid hydrate (1.618 g, 8.51 mmol) were dissolved in ethanol (54 mL) in a 250 mL flask. The solution was heated at 70 °C using an oil bath for 3 hours and allowed to cool, whereupon solvent was removed under reduced pressure. The residue was dissolved in dichloromethane (40 mL) and washed with 2M NaOH (20 mL). Saturated sodium chloride (30 mL) was added to promote separation of the organic layer. The organic layer was washed with water (10 mL), solvent was removed under reduced pressure, and the residue dried in vacuo. Compound 5 (2.40 g, 6.93 mmol) was dissolved in dichloromethane (anhydrous, 34 mL) and added to the diamine intermediate prepared above. After 24 hours, solvents were removed under reduced pressure, and the residue was purified twice using silica gel chromatography, using 0.1% triethylamine and 5% methanol in dichloromethane to provide compound 49 that was dried in vacuo (1.731 g, 44.7%). FTMS+pESI: Calc, for C62H80N9O14Si2(M+H)+, 1230.5358; found, 1230.5342.

[0295] N2, N5-Bis((S)-l-amino-5-(l-(benzyloxy)-6-oxo-L6-dihvdroDyridine-2-carboxamido)pentan-2-yl)-l-(benzyloxy)-6-oxo-L6-dihydropyridine-2,5-dicarboxamide 50.A mixture of 5% triisopropyl silane (AK Scientific), 50% trifluoroacetic acid and 45% dichloromethane (20 mL) was added to compound 49 (570 mg, 463 pmol) in a 50 mL flask and the solution was stirred for 4 hr. Solvents were removed in vacuo, and the residue was dried in vacuo. The residue was dissolved in dichloromethane (10 mL) and triethylamine (7 mL) and transferred to a separatory funnel using dichloromethane (10 mL). The organic layer was washed with water (10 mL), separated, and additional tri ethylamine (1 mL) was added to form a clear solution. Solvents were removed under reduced pressure and the DB1 / 159329947.1 98Attorney Docket No. 061809-5015-WOresidue was dried in vacuo to provide crude compound 50 (616 mg, 141%) that was used in the next step without further purification.

[0296] tert-Butyl ((5S,8Z,1 lZ,14S)-9,20,30-tris(benzyloxy)-5,14-bis(3-(l-(benzyloxy)-6-oxo- 1,6-dihydropyridine-2-carboxamido)propyl)-2,7, 10,12,17,19,22,28,31 -nonaoxo-3, 6,9, 13, 16,20,23, 27,30-nonaazatetracyclor27, 2,2, 28,11.218’21]heptatriaconta- 1 (32), 8(35), 11 (34), 18(37),21 (36),29(33)-hexaen-25-yl)carbamate 51. Compound 50 (463 pmol) was dissolved in dimethylformamide (Biotech grade, 16 mL) and triethylamine (194 pL, 1.39 mmol) and transferred to a 25 mL gastight syringe. tert-Butyl (l,3-bis(l-(benzyloxy)-6-oxo-5-(2-thioxothiazolidine-3-carbonyl)-l,6-dihydropyridine-2-carboxamido)propan-2-yl)carbamate 28 (432 mg, 463 pmol) was dissolved in dimethylformamide (32 mL) and transferred to a 50 mL gastight syringe. The two reactants were added to a stirring pot of dichloromethane (500 mL) at a rate of 0.25 - 0.50 mL / hr over 5 days using two syringe pumps. After 2 additional days, solvents were removed under reduced pressure and the residue was dried overnight. The residue was purified using silica gel chromatography, using 0.1% triethylamine, 5 - 7.5% methanol in di chloromethane to elute product. Solvent was removed in vacuo and the resulting solid was dried in vacuo to provide compound 51 as a mixture of isomers. FTMS+pESL Calc, for C86H89N14O20(M+H)+, 1637.6372; found, 1637.6378.

[0297] N, N'-(((lZ,5S,14S,29Z)-25-Amino-9,20,30-trihydroxy-2,7,10,12,17,19,22,28,31-nonaoxo-3,6,9,13,16,20,23,27,30-nonaazatetracyclo[27.2.2.28,11,218’211heptatriaconta-l(32),8(35),ll(34),18(37),21(36),29(33)-hexaene-5,14-diyl)bis(propane-3,l-diyl))bis(l-hydroxy-6-oxo-l,6-dihydropyridine-2-carboxamide) 52. Compound 52 is prepared using the same protocol as compound 36 above.DB1 / 159329947.1 99Attorney Docket No. 061809-5015-WOin HCI ACCI3Ammonium Acetate Buffer53in HCI ACCI3Ammonium Acetate Bufferin HCI ACCI3Ammonium Acetate BufferHjN'''"''- / 36 55in HCI ACCI3Ammonium Acetate BufferLumi804 Lumi804-AcScheme 10. Synthesis of macrocyclic chelates of actinium(III).DB1 / 159329947.1 100Attorney Docket No. 061809-5015-WOExample 13. Synthesis of macrocyclic chelates of actinium-225 (Scheme 10).

[0298] Experimental: Chelator stock solutions were prepared in dimethylformamide at a concentration of 1 pg / pL. Each chelator stock (10 pL) was transferred to a reaction vial and an additional 10 pL dimethylformamide was added. Ammonium acetate buffer (0.1M, pH 5.5 - 6.0, 80 pL) was added to each reaction vial, and the resulting solutions were allowed to stand for ca. 15 minutes. A stock solution (4 pL, 13.5 pCi) of actinium-225 nitrate (Brookhaven National Lab) in hydrochloric acid (0.2M) was added to each vial, and the reaction vial solutions were mixed by shaking at 37 °C for 30 minutes. Samples for RP-HPLC analysis (20 pL) were removed and DMF (25%, 5 pL) and DTPA solution (50 mM, pH 7, 5 pL) were added prior to analysis using an injection volume of 5 pL. RP-HPLC was performed using a Kinetex. Polar C-18 100 A, 150 mm 4.6, 2.6 pm column at a flow rate of 0.5 mL / minute at ambient temperature. A 25-minute gradient method was employed using ammonium acetate buffer (0.03M, pH 7) as Solvent A and 2% - 90% acetonitrile as Solvent B. Radioactive signal was detected by fraction collection using a Hidex gamma counter at secular equilibrium. Using these conditions, free actinium-225 has a retention time of 4 - 6 minutes.

[0299] The RP-HPLC profile resulting from incubation of chelator 30 with actinium-225 at 37 °C for 30 minutes, shown in Figure 8, shows <0.3% of the free radiometal at 3-5 minutes and 99.96% activity in a peak corresponding to actinium-225 bound to chelator 30, chelate 54, at 10 - 14 minutes by fraction collection and gamma counter detection.

[0300] The RP-HPLC profile resulting from incubation of chelator 10 with actinium-225 at 37 °C for 30 minutes, shown in Figure 9, shows <0.3 % of the free radiometal at 3-5 minutes and 99.84% activity in a peak corresponding to actinium-225 bound to chelator 10, chelate 53, at 10 - 14 minutes by fraction collection and gamma counter detection.

[0301] The RP-HPLC profile resulting from incubation of chelator 36 with actinium-225 at 37 °C for 30 minutes, shown in Figure 10, shows <0.3 % of the free radiometal at 3-5 minutes and 99.96% activity in a peak corresponding to actinium-255 bound to chelator 36, chelate 55, at 10 - 14 minutes by fraction collection and gamma counter detection.

[0302] The RP-HPLC profile resulting from incubation of chelator Lumi804 with actinium-225 at 37 °C for 30 minutes, shown in Figure 11, shows 19.17% of the free radiometal at 3-5 minutes and 80.29% activity in a peak corresponding to actinium-225 bound to chelatorDB1 / 159329947.1 101Attorney Docket No. 061809-5015-WOLumi804, chelate Lumi804-Ac, at 10 - 14 minutes by fraction collection and gamma counter detection.Scheme 11. Synthesis of a macrocyclic bifunctional chelator.Example 13. Synthesis of a macrocyclic bifunctional chelator (Scheme 11).

[0303] di-tert-Butyl (((1 -(benzyl oxy)-6-oxo-L6-dihydropyridine-2, 5-dicarbonyl)bis(azanediyl))bis(ethane-2,l-diyl))dicarbamate 56. l-(Benzyloxy)-6-oxo-l,6-dihydropyridine-2,5-dicarboxylic acid (289 mg, 1.00 mmol) was dried in vacuo in a 50 mLflask. The compound was suspended in dichloromethane (anhydrous, 6 mL), oxalyl chloride(377 pL, 4.40 mmol) was added, and dimethylformamide (anhydrous, ca. 10 pL) was addedusing a pipettor. A second aliquot of dimethylformamide was added after 35 minutes and athird after another 55 minutes. Solvents were removed in vacuo after an additional 20minutes and the crude yellow residue 3 was dried overnight. tert-Butyl (2-aminoethyl)carbamate (396 pL, 2.50 mmol) was dissolved in ca. 6 mL dichloromethane and mixed with K2CO3 (828 mg, 6.00 mmol) dissolved in 12 mL water. The resulting mixturewas cooled on an ice bath with stirring. To this mixture, the yellow residue of diacid chloride3 prepared above dissolved in 8 mL dichloromethane was added in portions over 40 minutes using a syringe. The mixture was allowed to warm to ambient temperature after one hour and was transferred to a separatory funnel to recover the organic phase. Solvents were removedunder reduced pressure, and the residue was purified using silica gel chromatography, using0.1% triethylamine and 5% methanol in dichloromethane to elute compound 56 that wasDB1 / 159329947.1 102Attorney Docket No. 061809-5015-WOdried in vacuo (574 mg, 100%). FTMS+pESI: Calc, for C28H40N5O8 (M+H)+, 574.2871; found, 574.2868.

[0304] N2, N5-bis(2 -Aminoethyl)- l-(benzyloxy)-6-oxo-l,6-dihydropyridine-2,5-dicarboxamide 57. A mixture of 5% triisopropylsilane (AK Scientific), 50% trifluoroacetic acid and 45% dichloromethane (20 mL) was added to compound 56 (266 mg, 464 umol) and the solution was stirred for 3.5 hr. Solvents were removed in vacuo, and the residue was dried in vacuo. The residue was dissolved in dichloromethane (10 mL) and triethylamine (4 mL) was added. Solvents were removed under reduced pressure and the residue was dried in vacuo to provide crude compound 57 that is used in the next step without further purification. FTMS+pESI: Calc, for C18H24N5O4 (M+H)+, 374.1823; found, 374.1826.

[0305] tert-Butyl ((lZ,29Z)-9,20,30-tris(benzyloxy)-2,7,10,12,17,19,22,28,31-nonaoxo-3,6,9,13,16,20,23,27,30-nonaazatetracyclo[27.2.2.28’11.218’211heptatriaconta- 1(32),8(35),1 l(34),18(37),21(36),29(33)-hexaen-25-yl)carbamate 58. Compound 57 is dissolved in dimethylformamide (Biotech grade) and triethylamine and transferred to a gastight syringe. tert-Butyl (l,3-bis(l-(benzyloxy)-6-oxo-5-(2-thioxothiazolidine-3-carbonyl)-l,6-dihydropyridine-2-carboxamido)propan-2-yl)carbamate 28 is dissolved in dimethylformamide and transferred to a gastight syringe. The two reactants are added to a stirring pot of dichloromethane over 5 days using two syringe pumps. Solvents are removed under reduced pressure and the residue is dried overnight. The residue is purified using silica gel chromatography. Solvent is removed in vacuo and the resulting solid is dried in vacuo to provide compound 58 (284 mg, (37.5%).

[0306] (lZ,29Z)-25-Amino-9,20,30-trihydroxy-3,6,9,13, 16,20,23,27,30-nonaazatetracyclo[27.2.2.28,11.218’211heptatriaconta-l(32),8(35),ll(34),18(37),21(36),29(33)-hexaene-2,7,10,12, 17, 19,22,28, 31 -nonaone 59. Compound 59 is prepared using the same protocol as compound 36 above.DB1 / 159329947.1 103Attorney Docket No. 061809-5015-WOScheme 12. Synthesis of a macrocyclic bifunctional chelator. The letters m or n represent an integer independently selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25.Example 14. Synthesis of a macrocyclic bifunctional chelator (Scheme 12).

[0307] Compound 30 is dissolved in dimethylformamide containing an equimolar amount of calcium chloride. An equimolar or greater amount of reagent 60 (BroadPharm, Inc.) and triethylamine are added, and the reaction mixture is mixed for one hour. Ethyl ether is added to form a precipitate, which is collected and washed with dichloromethane to provide the mal eimide derivative 61.3063Scheme 13. Synthesis of a macrocyclic bifunctional chelator. The letters m or n represent an integer independently selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25.Example 15. Synthesis of a macrocyclic bifunctional chelator (Scheme 13).

[0308] Compound 30 is dissolved in dimethylformamide containing an equimolar amount of calcium chloride. An equimolar or greater amount of reagent 62 (BroadPharm, Inc.) and triethylamine are added, and the reaction mixture is mixed for one hour. Ethyl ether is added to form a precipitate, which is collected and washed with dichloromethane to provide the active ester derivative 63.DB1 / 159329947.1 104Attorney Docket No. 061809-5015-WOScheme 14. Synthesis of a macrocyclic bifunctional chelator.Example 16. Synthesis of a macrocyclic bifunctional chelator (Scheme 14).

[0309] Methyl 1 -(benzyloxy)-6-((l -( 1 -(benzyloxy)-5-(methoxycarbonyl)-6-oxo- 1,6-dihy dropyridin-2-yl)-l 7, 17-dimethyl-l, 7, 15-tri oxo- 11,16-dioxa-2,8,14-tri azaoctadecan-6-yl)carbamoyl)-2-oxo-l,2-dihydropyridine-3 -carboxylate 65. Compound 65 is prepared from compound 15 and tert-butyl (2-(2-(2,5-diaminopentanamido)ethoxy)ethyl)carbamate 64 (prepared as described in WO2022 / 087317) as described above for compound 26.

[0310] l-(Benzyloxy)-6-((l-(l-(benzyloxy)-5-carboxy-6-oxo-l,6-dihydropyridin-2-yl)-17, 17-dimethyl- 1,7, 15-trioxo- 11,16-dioxa-2,8, 14-triazaoctadecan-6-yl)carbamoyl)-2-oxo-l,2-dihydropyridine-3 -carboxylic acid 66. Compound 66 is prepared from compound 65 as described above for compound 27.

[0311] tert-Butyl (2-(2-(2,5-bis(l-(benzyloxy)-6-oxo-5-(2-thioxothiazolidine-3-carbonyl)-l,6-dihydropyridine-2-carboxamido)pentanamido)ethoxy)ethyl)carbamate 67. Compound 67 is prepared from compound 66 as described above for compound 28.DB1 / 159329947.1 105Attorney Docket No. 061809-5015-WO

[0312] tert-Butyl (2-(2-((5S,8Z,llZ,14S,18Z,21Zl-9,20,31-tris(benzyloxyl-5,14-bis(4-(l-(benzyloxyl-6-oxo- 1,6-di hydropyri dine-2-carboxami do (butyl )-2, 7, 10,12,17,19,22,29,32-nonaoxo-3,6,9,13,16,20,23,28,31-nonaazatetracvclo[28.2.2.28’11.218’211octatriaconta- 1(331,8(361,11(351, 18(381,2 l(371,30(341-hexaene-24-carboxamidolethoxylethyllcarbamate 68. Compound 68 is prepared from compounds 67 and 7 as described above for compound 29.

[0313] N, N'-(((lZ,5S,14S,30Zl-24-((2-(2-aminoethoxylethyllcarbamoyll-9,20,31-trihydroxy-2,7, 10,12,17,19,22,29, 32-nonaoxo-3,6,9, 13,16,20,23,28,31 -nonaazatetracy clo[28, 2, 2, 28’11,218’211octatriaconta-l (331,8(361,11(351,18(381,21(371,30(341-hexaene-5,14-diyllbis(butane-4,l-diylllbis(l-hvdroxy-6-oxo-l,6-dihvdropyridine-2-carboxamidel 69. Compound 69 is prepared from compound 68 as described above for compound 30.Scheme 15. Synthesis of a macrocyclic bifunctional chelator.DB1 / 159329947.1 106Attorney Docket No. 061809-5015-WOExample 17. Synthesis of a macrocyclic bifunctional chelator (Scheme 15).

[0314] 2-(tert-Butyl) 5-methyl l-(benzyloxy)-6-oxo-l, 6-dihy dropyridine-2,5-di carboxylate 71. l-(Benzyloxy)-5-(methoxycarbonyl)-6-oxo-l,6-dihydropyridine-2-carboxylic acid 70 (WO2019 / 173639) is dissolved in dichloromethane, oxalyl chloride is added, and then a small amount of dimethylformamide. The resulting solution is stirred, and solvents are removed under reduced pressure. The residue is dissolved in dichloromethane, tert-butyl alcohol and triethylamine are added, and the solution is stirred. Solvents are removed under reduced pressure to provide compound 71.

[0315] l-(Benzyloxy)-6-(tert-butoxycarbonyl)-2-oxo-l,2-dihvdroDyridine-3 -carboxylic acid 72. Compound 72 is prepared from compound 71 as described above for compound 17.

[0316] tert-Butyl 1 -(benzyloxy)-6-oxo-5-(2-thioxothiazolidine-3 -carbonyl)- 1,6-dihydropyridine-2-carboxylate 73. Compound 73 is prepared from compound 72 as described above for compound 18.

[0317] tert-Butyl 1 -(benzyloxy)-5-((2-methoxyethyl)carbamoyl)-6-oxo- 1,6-dihydropyridine-2-carboxylate 74. Compound 74 is prepared from compound 73 as described above for compound 16.

[0318] l-(Benzyloxy)-5-((2 -methoxy ethyl)carbamoyl)-6-oxo-l, 6-dihy droDyridine-2-carboxylic acid 75. Compound 75 is prepared from compound 74 as described above for compound 17.

[0319] l-(Benzyloxy)-N-(2-methoxyethyl)-2-oxo-6-(2-thioxothiazolidine-3 -carbonyl)- 1,2-dihydropyridine-3 -carboxamide 76. Compound 76 is prepared from compound 75 as described above for compound 18.

[0320] bis(2-(Trimethylsilyl)ethyl) ((2S,2'S)-((l-(benzyloxy)-6-oxo-l,6-dihydropyridine- 2.5-dicarbonyl)bis(azanediyl))bis(6-(l-(benzyloxy)-5-((2 -methoxy ethyl)carbamoyl)-6-oxo- 1.6-dihy dropyridine-2-carboxamido)hexane-2,l-diyl))dicarbamate 77. Compound 77 is prepared from compound 4 and compound 76 as described above for compound 19.DB1 / 159329947.1 107Attorney Docket No. 061809-5015-WO

[0321] N2, N2'-((5S,5'S)-((l-(benzyloxy)-6-oxo-l,6-dihydropyridine-2,5-dicarbonyl)bis(azanediyl))bis(6-aminohexane-5,l-diyl))bis(l-(benzyloxy)-N5-(2-methoxyethyl)-6-oxo-l,6-dihvdroDyridine-2,5-dicarboxamide) 78. Compound 78 is prepared from compound 77 as described above for compound 20.

[0322] tert-Butyl (4-((lZ,4R,15S,24S,28Z)-9,19,30-tris(benzyloxy)-15,24-bis(4-(l-(benzyloxy)-5-((2-methoxyethyl)carbamoyl)-6-oxo-l,6-dihvdropyridine-2-carboxamido)butyl)-2,7,10,12,17,20,22,27,29-nonaoxo-3,6,9,13,16,19,23,26,30-nonaazatetracyclo[26.2.2.28’11.218’211hexatriaconta-l(31),8(34\l 1(33\18(36\21(35\28(32)-hexaen-4-yl)butyl)carbamate 79. Compound 79 is prepared from compound 8 and compound 78 as described above for compound 21.

[0323] N2, N2,-(((4R,8Z,llZ,15S,24S)-4-(4-aminobutyl)-9,19,30-trihydroxy-2,7,10,12,17,20,22,27,29-nonaoxo-3,6,9,13,16,19,23,26,30-nonaazatetracvclo[26.2.2.28’11.218’211hexatriaconta-l(31),8(34\ll(33\18(36),21(35),28(32)-hexaene-15,24-diyl)bis(butane-4,l-diyl))bis(l-hydroxy-N5-(2 -methoxy ethyl)-6-oxo-l, 6-dihydropyridine-2,5-dicarboxamide) 80. Compound 80 is prepared from compound 79 as described above for compound 22.DB1 / 159329947.1 108Attorney Docket No. 061809-5015-WOScheme 16. Synthesis of a macrocyclic bifunctional chelator.Example 18. Synthesis of a macrocyclic bifunctional chelator (Scheme 16).

[0324] Methyl (S)-5-(((benzyloxy)carbonyl)amino)-6-hydroxyhexanoate 82. Compound 82 is prepared from (S)-2-(((benzyloxy)carbonyl)amino)-6-methoxy-6-oxohexanoic acid 81 as described above for compound 42.

[0325] Methyl (S)-5-(((benzyloxy)carbonyl)amino)-6-((methylsulfonyl)oxy)hexanoate 83.Compound 83 is prepared from compound 82 as described above for compound 43.

[0326] Methyl (S)-6-azido-5-(((benzyloxy)carbonyl)amino)hexanoate 84. Compound 84 is prepared from compound 83 as described above for compound 44.

[0327] Methyl (S)-6-amino-5-(((benzyloxy)carbonyl)amino)hexanoate 85. Compound 85 is prepared from compound 84 as described above for compound 45.DB1 / 159329947.1 109Attorney Docket No. 061809-5015-WO

[0328] Methyl (S)-5-(((benzyloxy)carbonyl)amino)-6-(((2-(trimethylsilyl)ethoxy)carbonyl)amino)hexanoate 86. Compound 86 is prepared from compound 85 as described above for compound 46.

[0329] Methyl (S)-5-amino-6-(((2-(trimethylsilyl)ethoxy)carbonyl)amino)hexanoate 87. Compound 87 is prepared from compound 86 as described above for compound 47.

[0330] Dimethyl 5,5'-((l-(benzyloxy)-6-oxo-l,6-dihydropyridine-2,5-dicarbonyl)bis(azanediyl))(5S,5'S)-bis(6-(((2-(trimethylsilyl)ethoxy)carbonyl)amino)hexanoate) 88. Compound 88 is prepared from compound 3 and compound 87 as described above for compound 48.

[0331] Dimethyl 5,5'-((l-(benzyloxy)-6-oxo-l,6-dihydropyridine-2,5-dicarbonyDbi s(azanediyl))(5 S, 5 ' S)-bi s(6-aminohexanoate) 89. Compound 89 is prepared from compound 88 as described above for compound 50.

[0332] Dimethyl 4,4,-((lZ,5S,14S,29Z)-9,20,30-tris(benzyloxy)-25-((tert-butoxy carbonyl)amino)-2,7, 10, 12, 17, 19,22,28, 3 l-nonaoxo-3, 6,9, 13, 16,20,23, 27,30-nonaazatetracyclo[27.2,2, 28,11.218’211heptatriaconta-l(32),8(35), 11(34), 18(37),21(36),29(33)-hexaene-5,14-diyl)dibutyrate 90. Compound 90 is prepared from compound 28 and compound 89 as described above for compound 51.

[0333] 4,4'-((lZ,5S,14S,29Z)-25-Amino-9,20,30-trihydroxy-2,7,10,12,17,19,22,28,31-nonaoxo-3, 6,9, 13, 16,20,23, 27,30-nonaazatetracyclo[27.2.2.28,11.218,21]heptatriaconta-1(32),8(35),11(34),18(37),21(36),29(33)-hexaene-5,14-diyl)dibutyric acid 91. Compound 91 is prepared from compound 90 as described above for compound 52.DB1 / 159329947.1 110Attorney Docket No. 061809-5015-WOScheme 17. Synthesis of a macrocyclic bifunctional chelator.Example 19. Synthesis of a macrocyclic bifunctional chelator (Scheme 17).

[0334] 4,4'-((lZ,5S,14S,29Z)-9,20,30-Tris(benzyloxy)-25-((tert-butoxycarbonyl)amino)-2,7,10,12,17,19,22,28,31-nonaoxo-3,6,9,13,16,20,23,27,30-nonaazatetracyclo[27.2,2, 28,11.218’211heptatriaconta-l(32),8(35), 11(34), 18(37),21(36),29(33)-hexaene-5,14-diyl)dibutyric acid 92. Compound 92 is prepared from compound 90 as described above for compound 17.

[0335] tert-Butyl ((5S,8Z,1 lZ,14S)-9,20,30-tris(benzyloxy)-5,14-bis(4-(((3-(benzyloxy)- 1, 5-dimethyl-4-oxo- 1,4-dihydropyridin-2-yl)methyl)amino)-4-oxobutyl)- 2,7,10,12,17,19,22,28,31-nonaoxo-3,6,9,13,16,20,23,27,30-nonaazatetracyclo[27.2,2, 28,11.218’211heptatriaconta-l(32),8(35), 11(34), 18(37),21(36),29(33)-hexaen-25-yl)carbamate 93. Compound 92 is suspended in dichloromethane, 1-hydroxysuccinimide is added. N, N'-Dicyclohexylcarbodiimide is added and the solution is allowed to stir for one hour, whereupon diethyl ether is added to form a precipitate. TheDB1 / 159329947.1 111Attorney Docket No. 061809-5015-WOprecipitate is washed with diethylether and dried under reduced pressure. The residue is dissolved in dichloromethane and 2-(aminomethyl)-3-(benzyloxy)-l,5-dimethylpyridin-4(lH)-one (Liu, Z. D., et al., J. Med. Chem. 2002, 45, 631-639) is added, and the mixture is stirred for several hours. Solvents are removed under reduced pressure and the residue is purified by silica gel chromatography to provide compound 93.

[0336] 4,4,-((lZ,5S,14S,29Z)-25-Amino-9,20,30-trihydroxy-2,7,10,12,17,19,22,28,31-nonaoxo-3,6,9,13,16,20,23,27,30-nonaazatetracyclo[27.2.2.28,11,218’211heptatriaconta-l(32),8(35),ll(34),18(37),21(36),29(33)-hexaene-5,14-diyl)bis(N-((3-hydroxy-l,5-dimethyl-4-oxo- l,4-dihydropyridin-2-yl)methyl)butanamide) 94. Compound 94 is prepared from compound 93 as described above for compound 10.DB1 / 159329947.1 112Attorney Docket No. 061809-5015-WOScheme 18. Synthesis of a macrocyclic bifunctional chelator.Example 20. Synthesis of a macrocyclic bifunctional chelator (Scheme 18).

[0337] bis(2-(trimethylsilyl)ethyl) ((2S,2'S)-((l-(benzyloxy)-6-oxo-l,6-dihydropyridine-2,5- dicarbonyl)bis(azanediyl))bis(5-(2-(N-(benzyloxy)acetamido)acetamido)pentane-2,l- diyl))dicarbamate 96. Compound 96 is prepared from compound 48 and compound 95 (WO2023 / 150540 A8) as described above for compound 49.DB1 / 159329947.1 113Attorney Docket No. 061809-5015-WO

[0338] N2, N5-bis((S)-l-amino-5-(2-(N-(benzyloxy)acetamido)acetamido)pentan-2-yl)-l-(benzyloxy)-6-oxo-l,6-dihydropyridine-2,5-dicarboxamide 97. Compound 97 is prepared from compound 96 as described above for compound 50.

[0339] tert-Butyl ((lZ,5S,14S,29Z)-9,20,30-tris(benzyloxy)-5,14-bis(3-(2-(N- (benzyloxy)acetamido)acetamido)propyl)-2,7, 10,12,17,19,22,28,31 -nonaoxo-3,6,9,13,16,20,23,27,30-nonaazatetracvclo[27.2.2.28’11.218’211heptatriaconta- 1 (32), 8(35), 1 l(34),18(37),21(36),29(33)-hexaen-25-yl)carbamate 98. Compound 98 is prepared from compound 28 and compound 97 as described above for compound 51.

[0340] N, N'-(((lZ,5S,14S,29Z)-25-Amino-9,20,30-trihvdroxy-2,7,10,12,17,19,22,28,31-nonaoxo-3, 6,9, 13, 16,20,23, 27,30-nonaazatetracyclo[27.2,2, 28,11.218,211heptatriaconta-l(32),8(35),ll(34),18(37),21(36),29(33)-hexaene-5,14-diyl)bis(propane-3,l-diyl))bis(2-(N-hydroxy acetamido (acetamide) 99. Compound 99 is prepared from compound 98 as described above for compound 52.DB1 / 159329947.1 114Attorney Docket No. 061809-5015-WOScheme 19. Synthesis of a macrocyclic bifunctional chelator.Example 21. Synthesis of a macrocyclic bifunctional chelator (Scheme 19).

[0341] Bis(2-(trimethylsilyl)ethyl) ((2S,2'S)-((l-(benzyloxy)-6-oxo-E6-dihydropyridine- 2,5-dicarbonyl)bis(azanediyl))bis(5-(l-(benzyloxy)-3-methyl-2-oxopiperidine-3-carboxamido)pentane-2,l-diyl))dicarbamate 101. Compound 101 is prepared from compound 48 and compound 100 (F. Mangin, et al., ChemPlusChem 2024, 89, e202400062. https: / / doi.org / 10.1002 / cplu.202400062) as described above for compound 49.DB1 / 159329947.1 115Attorney Docket No. 061809-5015-WO

[0342] N2, N5-bis((2S)-l-amino-5-(l-(benzyloxy)-3-methyl-2-oxopiperidine-3-carboxamido)pentan-2-yl)-l-(benzyloxy)-6-oxo-l,6-dihydropyridine-2,5-dicarboxamide 102.Compound 102 is prepared from compound 101 as described above for compound 50.

[0343] tert-Butyl ((5S,8Z,1 lZ,14S)-9,20,30-tris(benzyloxy)-5,14-bis(3-(l-(benzyloxy)-3-methyl-2-oxopiperidine-3 -carboxamido)propyl)-2,7, 10,12,17,19,22,28,31 -nonaoxo-3,6,9,13,16,20,23,27,30-nonaazatetracvclo[27.2.2.28’11.218’211heptatriaconta- 1(32),8(35),1 l(34),18(37),21(36),29(33)-hexaen-25-yl)carbamate 103. Compound 103 is prepared from compound 28 and compound 102 as described above for compound 51.

[0344] N, N'-(((lZ,5S,14S,29Z)-25-amino-9,20,30-trihvdroxy-2,7,10,12,17,19,22,28,31-nonaoxo-3,6,9,13,16,20,23,27,30-nonaazatetracyclo[27.2.2.28,11,218’211heptatriaconta-l(32),8(35),ll(34),18(37),21(36),29(33)-hexaene-5,14-diyl)bis(propane-3,l-diyl))bis(l-hvdroxy-3-methyl-2-oxopiperidine-3-carboxamide) 104. Compound 104 is prepared from compound 103 as described above for compound 52.DB1 / 159329947.1 116Attorney Docket No. 061809-5015-WOScheme 20. Synthesis of a macrocyclic bifunctional chelator.Example 22. Synthesis of a macrocyclic bifunctional chelator (Scheme 20).

[0345] Bis(2-(trimethylsilyl)ethyl) ((2S,2'S)-((l-(benzyloxy)-6-oxo-l,6-dihydropyridine-2,5-dicarbonyl)bis(azanediyl))bis(5-(2-((l-(benzyloxy)-6-((2 -methoxy ethyl)carbamoyl)-2-oxo- 1,2-dihydropyri din-3 -yl)oxy)acetamido)pentane-2,l-diyl))dicarbamate 106. Compound 106 is prepared from compound 48 and compound 105 as described above for compound 49.

[0346] Bis(2-(trimethylsilyl)ethyl) ((2S,2'S)-((l-(benzyloxy)-6-oxo-l,6-dihydropyridine-2,5-dicarbonyl)bis(azanediyl))bis(5-(2-((l-(benzyloxy)-6-((2 -methoxy ethyl)carbamoyl)-2-oxo- 1,2-dihvdropyri din-3 -yl)oxy)acetamido)pentane-2,l-diyl))dicarbamate 107. Compound 107 is prepared from compound 106 as described above for compound 50.DB1 / 159329947.1 117Attorney Docket No. 061809-5015-WO

[0347] tert-Butyl ((5S,8Z,llZ,14S)-9,20,30-tris(benzyloxy)-5,14-bis(3-(2-((l-(benzyloxy)-6-((2-methoxyethyl)carbamoyl)-2-oxo-l,2-dihydropyri din-3 -yl)oxy)acetamido)propyl)-2,7,10,12,17,19,22,28,31-nonaoxo-3,6,9,13, 16,20,23,27,30-nonaazatetracyclo[27.2.2.28,11.218,21]heptatriaconta- 1(32),8(35),1 l(34),18(37),21(36),29(33)-hexaen-25-yl)carbamate 108. Compound 108 is prepared from compound 28 and compound 107 as described above for compound 51.

[0348] 5,5'-((((((lZ,5S,14S,29Z)-25-Amino-9,20,30-trihydroxy-2,7,10,12,17,19,22,28,31-nonaoxo-3, 6,9, 13, 16,20,23, 27,30-nonaazatetracyclo[27.2.2.28,11.218,21]heptatriaconta-1(32),8(35),1 l(34),18(37),21(36),29(33)-hexaene-5,14-diyl)bis(propane-3,l-diyl))bis(azanediyl))bis(2-oxoethane-2,l-diyl))bis(oxy))bis(l-hvdroxy-N-(2-methoxy ethyl)-6-oxo- l,6-dihydropyridine-2-carboxamide) 109. Compound 109 is prepared from compound 108 as described above for compound 52.

[0349] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.DB1 / 159329947.1 118

Claims

Attorney Docket No. 061809-5015-WOWE CLAIM:

1. A compound having a structure selected from:wherein L1, L2, L3, L4and L5are independently selected bridging scaffold moieties; Abl, Ab2and Ab3are independently selected bridging chelating moieties; Apland Ap2are independently selected pendant chelating moieties; Lpland Lp2are pendant scaffold moieties independently selected from H, substituted or unsubstituted alkyl, a solubilizing group (e.g., a polyether, such as poly(ethylene glycol)), a reactive functional group, a targeting moiety, a sidearm, a linker to a reactive functional group, or a linker to a targeting moiety.

2. The compound according to claim 1, wherein a member selected from L1, L2, L3and a combination thereof has a structure according to formula (A):|_x6RL1Rb2(A)whereinL4, L5, and each Llaare independently selected from substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted cycloalkenyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted arylalkyl, substituted or unsubstituted biaryl, substituted or unsubstituted heteroaryl, and a substituted or unsubstituted polycyclic ring system;each L1band L1care independently selected from a bond, −C(O)−, substituted or unsubstituted alkyl, and substituted or unsubstituted heteroalkyl; and each RL1and RL2are independently selected from H, substituted or unsubstituted alkyl, and substituted or unsubstituted heteroalkyl; and whereinDB1 / 159329947.1 119Attorney Docket No. 061809-5015-WOwhen L1has the structure according to formula (A), Lx6is selected from H, a sidearm, a linker to a reactive functional group, and a linker to a targeting moiety;when L2has the structure according to formula (A), Lx6is a bond to L4; and when L3has the structure according to formula (A), Lx6is a bond to L5, and when more than one of L1, L2and L3has the structure according to formula (A), each Lla, Llb, Llc, Lx6, RL1, and RL2may be the same or different.

3. The compound according to claim 2, wherein Llband Llcare independently selected from a bond, -C(O)-, -(CH2)aC(0)-, and -0(CH2)aC(0)-; wherein a is an integer selected from 1, 2, 3, 4, 5, and 6.

4. The compound according to claim 2, wherein Llband Llcare each -C(O)-.

5. The compound according to any one of claims 2-4, wherein RL1and RL2are each H.

6. The compound according to any preceding claim, wherein a member selected from L1, L2, L3and a combination thereof has a structure according to formula (B):|_x6Iwhereinwhen L1has the structure according to formula (B), Lx6is selected from H, a sidearm, a linker comprising a reactive linker moiety, or a targeting linker moiety, optionally wherein L1comprises a solubilizing moiety; andwhen L2has the structure according to formula (B), Lx6is a bond to L4;when L3has the structure according to formula (B), Lx6is a bond to L5;L4and L5are independently selected from substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, -(CH2)aNHC(0)- and -(CH2)aNRpC(0)-, wherein a is an integer selected from 2, 3, 4, 5, and 6 and Rpis independently selected from H, substituted or unsubstituted alkyl, and substituted or unsubstituted heteroalkyl.DB1 / 159329947.1 120Attorney Docket No. 061809-5015-WO7. The compound according to any one of claims 2-6, wherein Llahas the structure:whereineach Rel, Re2, Re3, and Re4are independently selected from H, cyano, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted cycloalkenyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl; andtwo members selected from Rel, Re2, Re3, and Re4, together with the atom to which they are attached, are optionally joined, to form a substituted or unsubstituted ring (or ring system) selected from substituted or unsubstituted cycloalkyl, substituted or unsubstituted cycloalkenyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl,optionally wherein a member selected from Rel, Re2, Re3, and Re4is Lx6; and optionally wherein a member selected from Rel, Re2, Re3, and Re4is a bond to L4or L5.

8. The compound according to any one of claims 2-7, wherein Llahas the structure:JVW s / WVwhereinn is an integer selected from 0, 1, 2, 3, 4, 5, 6, and 7; andB is independently selected from -CH2-, -NH-, -O-, alkyl, and heteroalkyl.DB1 / 159329947.1 121Attorney Docket No. 061809-5015-WO 9. The compound according to any one of claims 2-8, wherein Llais selected from:o oDB1 / 159329947.1 122Attorney Docket No. 061809-5015-WOwhereinm and n are each an integer independently selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, and 9; each R and Rlais independently selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, a reactive functional group, a modifying moiety, and targeting moiety; andX is O, S, or CH2.

10. The compound according to any one of claims 2-9, wherein Llahas the structure:DB1 / 159329947.1 123Attorney Docket No. 061809-5015-WO11. The compound according to any preceding claim, wherein Abl, Ab2, and Ab3arewhereinA and G are independently selected from carbon, nitrogen and oxygen;J is selected from carbon and nitrogen;each R1and R2is independently selected from H, an enzymatically labile group, a hydrolytically labile group, a metabolically labile group, a photolytically labile group and a single negative charge;each R6, R7, R8, R9, and R10is independently selected from a bond to a member selected from L1, L2, L3, L4, L5and a combination thereof, alkanediyl attached to a member selected from L1, L2, L3, L4, L5and a combination thereof, H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, halogen, CN, -CF3, -C(O)R17, -SO2NR17R18, -NR17R18, -OR17, -S(O)2R17, -COOR17, -S(O)2OR17, -OC(O)R17, -C(O)NR17R18, -NR17C(O)R18, -NR17SO2R18, and -NO2,whereinat least two of R6, R7, R8, R9, and R10are optionally joined to form a ring system selected from substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl and substituted or unsubstituted heteroaryl;R17and R18are independently selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl and substituted or unsubstituted heterocycloalkyl; andR17and R18, together with the atoms to which they are attached, are optionally joined to form a 5-, 6- or 7-membered ring.DB1 / 159329947.1 124Attorney Docket No. 061809-5015-WO12. The compound according to any preceding claim, wherein Apland Ap2are eachwhereinA and G are independently selected from carbon, nitrogen and oxygen;J is selected from carbon and nitrogen;each R1and R2is independently selected from H, an enzymatically labile group, a hydrolytically labile group, a metabolically labile group, a photolytically labile group and a single negative charge;each R6, R7, R8, R9, and R10is independently selected from a bond to a member selected from L1, L2, L3, L4, L5and a combination thereof, alkanediyl attached to a member selected from L1, L2, L3, L4, L5and a combination thereof, H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, halogen, CN, -CF3, -C(O)R17, -SO2NR17R18, -NR17R18, -OR17, -S(O)2R17, -COOR17, -S(O)2OR17, -OC(O)R17, -C(O)NR17R18, -NR17C(O)R18, -NR17SO2R18, and -NO2,whereinat least two of R6, R7, R8, R9, and R10are optionally joined to form a ring system selected from substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl and substituted or unsubstituted heteroaryl;DB1 / 159329947.1 125Attorney Docket No. 061809-5015-WOR17and R18are independently selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl and substituted or unsubstituted heterocycloalkyl; andR17and R18, together with the atoms to which they are attached, are optionally joined to form a 5-, 6- or 7-membered ring; andApland Ap2are attached to L4and L5, respectively, through a member selected from R6, R7, R8, R9and R10.

13. The compound according to any preceding claim, wherein Abl, Ab2, Ab3, Apland Ap2are each independently selected from:

14. The compound according to any preceding claim, wherein Abl, Ab2and Ab3are members independently selected from:whereineach R7, and R8is independently selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, halogen, CN, -CF3, −C(O)R17, -SO2NR17R18, -NR17R18, -OR17, -S(O)2R17, -COOR17, -S(O)2OR17, -OC(O)R17, -C(O)NR17R18, -NR17C(O)R18, -NR17SO2R18, and -NO2, whereinDB1 / 159329947.1 126Attorney Docket No. 061809-5015-WOR7, and R8, are optionally joined to form a ring system selected from substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl and substituted or unsubstituted heteroaryl; R17and R18are independently selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl and substituted or unsubstituted heterocycloalkyl; andR17and R18, together with the atoms to which they are attached, are optionally joined to form a 5-, 6- or 7-membered ring.

15. The compound according to any preceding claim, wherein Abl, Ab2and Ab3are each independently selected from:

16. The compound according to any preceding claim, wherein Abl, Ab2and Ab3are each independently selected from:

17. The compound according to any preceding claim, wherein Apland Ap2are each independently selected from:DB1 / 159329947.1 127Attorney Docket No. 061809-5015-WO18. The compound according to any preceding claim, wherein Apland Ap2are each independently selected from:RandR19. The compound according to any preceding claim, having the structure:whereinLpland Lp2are independently selected from H, -C(O)O-, a linker, a modifying moiety, an oligonucleotide, a peptide, an oligosacharide, polyethyleneglycol, and -C(O)NR17R18, wherein R17is H and R18is a modifying moiety, DB1 / 159329947.1 128Attorney Docket No. 061809-5015-WOoptionally wherein one or both of Lpland Lp2is COOH, or COO⁻;XOHJy°" OHAblis selected fromand —I—;ALlais VW' k / VW^ wherein n is an integer selected from 0, 1, 2, 3, 4, 5, 6, 7; and B is independently selected from -CH2-, -NH-, -O-, alkyl, and heteroalkyl; and Lx6is a linker comprising a sidearm, a reactive functional group, a targeting moiety.

20. The compound according to any preceding claim, wherein the compound comprises a sidearm, a linker to a reactive functional group, or a linker to a targeting moiety; optionally wherein the linker or the sidearmcomprises a solubilizing group.

21. The compound according to any preceding claim, wherein at least one of L1, L2, L3, L4, L5, Apland Ap2is substituted with a sidearm, a linker to a reactive functional group, or a linker to a targeting moiety.

22. The compound according to any preceding claim, wherein Llais substituted with a sidearm, a linker to a reactive functional group, or a linker to a targeting moiety.

23. The compound according to any preceding claim, wherein the sidearm has the structure:whereinBL is selected from substituted or unsubstituted alkyl and substituted or unsubstituted heteroalkyl in which 0, 1, 2 or 3 atoms are replaced with a heteroatom, such as nitrogen or oxygen,each EL is independently selected from a bond, acyl, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl and substituted or unsubstituted heteroaryl;m is an integer selected from 0, 1, 2, and 3;DB1 / 159329947.1 129Attorney Docket No. 061809-5015-WOn is an integer selected from 0 and 1;o is an integer selected from 0 and 1;whereinwhen n is 1, RL is selected from a reactive linker moiety, a protected reactive linker moiety and TL is not present; andwhen n is 0, RL is not present and TL is a targeting linker moiety.

24. The compound according to any preceding claim, wherein the sidearm has a structure selected from:Owhereinn is selected from 0, 1, 2, 3, 4, 5, 6, 7, and 8;m is selected from 0, 1, and 2;EL is selected from a bond, substituted or unsubstituted alkyl, and substituted or unsubstituted heteroalkyl; andRL is selected from a reactive linker moiety or a protected reactive linker moiety.

25. The compound according to any preceding claim, wherein the sidearm has a structure selected from:whereinn is selected from 0, 1, 2, 3, 4, 5, 6, 7, and 8;m is selected from 0, 1, and 2;DB1 / 159329947.1 130Attorney Docket No. 061809-5015-WOEL is selected from a bond, substituted or unsubstituted alkyl, and substituted or unsubstituted heteroalkyl; andTL is selected from a targeting linker moiety.

26. The compound according to any preceding claim, wherein the linker to the reactive functional group, or the sidearm has the structure:DB1 / 159329947.1 131Attorney Docket No. 061809-5015-WO27. The compound according to any preceeding claim, having a structure selected from:whereinLpland Lp2are independently selected from H, -C(O)O-, a linker, a modifying moiety, an oligonucleotide, a peptide, an oligosacharide, polyethyleneglycol, and -C(O)NR17R18, wherein R17is H and R18is a modifying moiety, optionally wherein one or both of Lpland Lp2is COOH, or COO⁻;OHJy°" OHAblis selected from —I— and; andLx6is a linker comprising a sidearm, a reactive linker moiety, or a targeting linker moiety.DB1 / 159329947.1 132Attorney Docket No. 061809-5015-WO28. The compound according to any preceding claim, having a structure selected from:structural isomers thereof.

29. The compound according to any preceeding claim, having a structure selected from:structural isomers thereof.DB1 / 159329947.1 133Attorney Docket No. 061809-5015-WO 30. The compound according to any preceeding claim, having a structure selected from:structural isomers therof.

31. The compound according to any preceding claim, having the structure:HNDB1 / 159329947.1 134Attorney Docket No. 061809-5015-WO32. A complex comprising a compound according to any preceding claim and a metal ion complexed therewith.

33. The complex according to claim 32, wherein the ion is an ion of a metal selected from a lanthanide and an actinide.

34. The complex according to claim 32, wherein the ion is an ion of a metal selected from zirconium (Zr), iron (Fe), indium (In), europium (Eu), holmium (Ho), lutetium (Lu), yttrium (Y), terbium (Tb), ytterbium (Yb), gadolinium (Gd), samarium (Sm), dysprosium (Dy), erbium (Er), thorium (Th), lanthanum (La), cerium (Ce), calcium (Ca), magnesium (Mg), strontium (Sr), Actinium (Ac) and barium (Ba).

35. The complex according to claim 32, wherein the complex is luminescent.

36. The complex according to claim 32, wherein the ion is an ion of a metal selected from Eu, Tb, Sm, Dy, La, Th, Zr, and Ac.

37. The complex according to claim 32, wherein the ion is an ion of Ac.

38. The complex according to claim 32, wherein the ion is a radionuclide.

39. The complex according to claim 32, wherein the metal ion is selected from: Zr(IV), Fe(III), Sc(III), In(III), Eu(III), Ho(III), Lu(III), Y(III), Tb(III), Yb(III), Gd(III), Sm(III), Dy(III), Er(III), Ac(III), Th(IV), La(III), Ce(III), Ce(IV), Ca(II), Mg(II), Sr(II), and Ba(II).

40. The complex according to claim 32, wherein the metal ion is selected from225Ac(III),134La(III),134Ce(III), and134Ce(IV).

41. A mixture of one or more isomers of complexes according to any preceding claim.DB1 / 159329947.1 135

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