Chelating agents for radioactive metals and methods of making and using same

Abstract

A chelating agent having the general structure (I), wherein each R1 is independently OH, NH, or SH, X is O, S, or NR3, and R3 is H or CH2C(=O)R1. Methods of making and using the chelating agents, metal chelating compounds, and biological constructs for delivering targeted radiation therapy using the chelating agents are provided. JPEG2024517879000033.jpg47170

Description

[Technical field]

[0001] Some embodiments relate to improved chelating agents. Some embodiments relate to improved biological targeting constructs incorporating chelating agents. Some embodiments relate to chelating agents that are coupled to a targeting moiety and can be coupled to a radioisotope to provide targeted in vivo delivery of the radioisotope to a desired location within a mammalian subject. [Background technology]

[0002] Radionuclides have potential utility in cancer diagnosis and treatment, especially when they can be selectively delivered to a target location in the subject's body. Targeted delivery of radionuclides can be achieved by using engineered constructs that safely hold the radionuclides for in vivo delivery and selectively deliver the radionuclides to desired locations in the body, with fairly low levels of delivery to non-target areas of the body.

[0003] For such purposes, targeting constructs have been developed that utilize a targeting moiety that targets a desired area of ​​the body (e.g., a tumor-associated antigen) covalently linked to a chelator to protect the radionuclide. The targeting moiety can be linked to the chelator via a linker. Such targeting constructs are sometimes called radioimmunoconjugates. Radioimmunoconjugates are used to chelate the desired radionuclide for in vivo delivery, for example, to provide diagnostic imaging, targeted radionuclide therapy using the construct, or both (i.e., as a theranostic construct). In some cases, chelators can also be used for in vivo delivery of a suitable companion radionuclide for diagnosis.

[0004] Historically, approaches to the design of chelating ligands with high metal ion selectivity have been based on several fundamental principles, such as the selection of appropriate donor groups based on Pearson's Hard and Soft Acids and Bases rule, the effect of cavity size, consideration of macrocyclic / chelating effects, and optimal chelating ring size. However, the impact of the distribution of inverted donor groups on the common backbone has yet to be explored. Considering the inherent challenges in the synthesis of ligands with symmetric donor substitutions, and the requirement to often "break" symmetry in the pursuit of bifunctional derivatives suitable for pharmaceutical applications, an assessment of the precise impact of asymmetric / inverted substitutions on metal ion chelation is warranted. The development of new ligand systems with "inverted" symmetry offers several advantages in terms of synthesis; protecting group strategies can be avoided by rational synthetic design, and the incorporation of bifunctional handles within the core framework is synthetically more accessible.

[0005] H4noneunpa, having the structure (A), was previously published by the present inventors. 8 , also published as Oxyaapa by Hu et al. 13 .

[0006] [ka]

[0007] In the context of nuclear medicine, significant improvements in the treatment of oncogenic diseases have been achieved with the advent of several peptide-based radiopharmaceuticals targeting somatostatin receptor subtype 2 (SSTR2), a receptor that is commonly overexpressed in neuroendocrine tumors (NETs). 14 [ 111 In][In(DTPA-D-Phe-Octreotide)](OctreoScan™) and 68 Ga][Ga(DOTATATE)] are two established SSTR2 agonists used clinically for the diagnosis of NETs. 14 Most notably, 177Lu][Lu(DOTATATE)] (Lutathera®) reached clinical FDA approval as a peptide receptor radionuclide therapy (PRRT) for the treatment of gastroenteropancreatic NETs in 2018 15 However, β - As a result of the inherent nuclear decay characteristics of the emitting radionuclides [low linear energy transfer (approximately 0.2 keV / m), long distance (0.5-10 mm)], the therapeutic range in this situation is limited to the primary tumor site and large metastases. 4、16 .

[0008] Targeted alpha therapy (TAT) has emerged as a powerful tool for the treatment of both primary and metastatic tumors. 225 Ac]Ac 3+ is the corresponding half-life (t 1 / 2 =10 days) and the high therapeutic efficacy of its breakdown products (E α = 5-9 MeV, 50-100 m range) making it one of the most prominent candidates for transition to clinical practice. 17 To achieve this goal, 225 Ac]Ac 3+ High dentate chelating ligands for stable chelation of ions are needed, which can further sequester imaging isotopes for diagnosis and evaluation of patient suitability prior to radionuclide therapy. One potential imaging companion is the radioactive lanthanide [ 155 Tb]Tb 3+ This means that 225 Ac]Ac 3+ (e.g., CN=9; ionic radius=1.095 Å (Tb 3+ ), 1.220Å(Ac 3+ )) 18、19 , its low-energy gamma emission (E γ = 87 keV (32%), 105 keV (25%)) and equivalent half-lives (t 1 / 2 = 5.32 days), making it suitable for SPECT / CT diagnosis. 155 Tb]Tb 3+ is a radioisotope 149 Tb, 152 Tb, 155Tb, and 161 It is part of the so-called "Terbium theranostic quartet" that also includes Tb and has been shown to be effective in both PET and SPECT imaging modalities, as well as in all three therapeutic decay types (α, β - , Meitner-Auger electrons) 21~23 Therefore, different Tb 3+ Interchange between radioisotopes provides true theranostic radiopharmaceuticals with identical pharmacokinetic and biodistribution properties.

[0009] There is a general desire for improved chelating agents that are useful in the targeted in vivo delivery of radiometals using appropriate targeting constructs. The foregoing examples of the related art and limitations associated therewith are intended to be illustrative and not exhaustive. Other limitations of the related art will become apparent to those of ordinary skill in the art upon reading the specification and studying the drawings. Summary of the Invention

[0010] The following embodiments and aspects thereof are described and illustrated in conjunction with systems, tools, and methods that are intended to be exemplary and illustrative, not limiting in scope. In various embodiments, one or more of the above-mentioned problems are reduced or eliminated, while other embodiments are directed to other improvements.

[0011] Novel chelating agents are provided having the general structure (I), where each R1 is independently OH, NH, or SH, X is O, S, or NR3, and R3 is H or CH2C(=O)R1. In some embodiments, the chelating agent is a bifunctional molecule having the structure (II), where each R1 is independently OH, NH, or SH, or a functional group, each R2 is independently H or a functional group, X is O, S, or NR3, and one or more of the R1 or R2 groups are functional groups that allow the chelating agent to be attached to a biological targeting moiety, and R3 is H or CH2C(=O)R1. In some embodiments, the functional group is an ester, amide, imide, thioamide, thioester, or guanidinium group. In some embodiments, the radioisotope targeting construct has the structure (II), and the chelating agent is attached to the biological targeting moiety via one R1 group or one R2 group.

[0012] [ka]

[0013] In some embodiments, the chelator has structure (III) or is a bifunctional molecule having structure (III), where each R1 is independently OH, NH, or SH, or a functional group; each R2 is independently H or a functional group; each R4 is independently H or a functional group, or both R4 taken together form a cyclohexyl moiety; each R5 is independently H or a functional group, or both R5 taken together form a cyclohexyl moiety; X is O, S, or NR3, where one or more of the R1, R2, R4, or R5 groups are functional groups that allow the chelator to be attached to a biological targeting moiety, and R3 is H or CH2C(=O)R1. In some embodiments, the functional group is an ester, amide, imide, thioamide, thioester, or guanidinium group. In some embodiments, the radioisotope targeting construct has structure (III) and the chelator is attached to the biological targeting moiety via one R1 group, one R2 group, one R4 group, or one R5 group.

[0014] [ka]

[0015] The chelator may be conjugated to a biological targeting moiety to facilitate targeted in vivo delivery of the radioisotope. 227 Th, 225 Ac, 155 Tb, 177 Lu, 111 In, 132 La, 235 La, 90 Y, 68 Ga, 44 Sc, 203 Pb, 212 It could be Pb etc.

[0016] In some embodiments, the chelator has the following structure (6):

[0017] [ka]

[0018] A method of administering an in vivo radioisotope targeting construct comprising a chelating agent is provided. The in vivo radioisotope targeting construct can be administered to a mammalian subject. The targeting moiety of the in vivo radioisotope targeting construct can be used to enhance accumulation of the radioisotope at a selected location in the body (e.g., the location of a cancerous cell) compared to other locations in the body. Imaging procedures can be performed to evaluate the localization of the in vivo radioisotope targeting construct in the body. The in vivo radioisotope targeting construct can be used to cause cell death at a selected location in the body by exposing the cell to radiation from the radioisotope. The cell can be a cancer cell. The mammalian subject can be a human.

[0019] In addition to the exemplary aspects and embodiments described above, further aspects and embodiments will become apparent by reference to the drawings and by study of the following detailed description. [Brief description of the drawings]

[0020] Exemplary embodiments are shown in the referenced figures in the drawings. It is intended that the embodiments and drawings disclosed herein are to be considered illustrative and not restrictive. [Figure 1] 1 shows the structure of an exemplary in vivo targeting chelate construct. [Diagram 2] The chemical structure of H4noneunpaX(6) and the 1H (left) and 13C (right) assignments are shown. [Diagram 3] The 1H NMR spectrum of H4noneunpaX 4HCl (6) (400 MHz, DO, 298 K) is shown. [Figure 4] The 13C{1H} NMR spectrum (75 MHz, D2O, 298 K) of H4noneunpaX 4HCl (6) is shown. [Diagram 5]The 1H-1H COSY NMR spectrum (300 MHz, DO, 298 K) of H4noneunpaX (6) is shown. [Figure 6] The 1H-13C HSQC NMR spectrum (300 MHz, 75 MHz, DO, 298 K) of H4noneunpaX 4HCl (6) is shown. [Figure 7] The 1H-13C HMBC NMR spectrum (300 MHz, 75 MHz, DO, 298 K) of H4noneunpaX 4HCl (6) is shown. [Figure 8] Stacked 1H NMR spectra (400 MHz, DO, 298 K, pD 7.0) of H4noneunpaX and the corresponding complexes of La3+, Lu3+ and In3+ are shown. [In(noneunpaX)]- was characterized at pD = 4.5. [Figure 9] The 1H NMR spectrum of [La(noneunpaX)]- (400 MHz, DO, 298 K, pH 7) is shown, along with the chemical structure of [La(noneunpaX)]- and 1H assignments. [Figure 10] The 1H-1H COSY NMR spectrum of [La(noneunpaX)]- (400 MHz, DO, 298 K, pH 7.0) is shown. [Figure 11] The 1H-1H COSY NMR spectrum (400 MHz, DO, 298 K, pH 7.0) of [La(noneunpaX)]- (alkyl expansion). [Figure 12] The 1H-1H COSY NMR spectrum (400 MHz, DO, 298 K, pH 7.0) of [La(noneunpaX)]- is shown (aromatic expansion). [Figure 13] The 1H NMR spectrum of [Lu(noneunpaX)]- (400 MHz, DO, 298 K, pH 7.0) is shown, along with the chemical structure of [Lu(noneunpaX)]- and 1H assignments. [Figure 14] The 1H-1H COSY NMR spectrum of [Lu(noneunpaX)]- (400 MHz, DO, 298 K, pH 7.0) is shown. [Figure 15] The 1H-1H COSY NMR spectrum (400 MHz, DO, 298 K, pH 7.0) of [Lu(noneunpaX)]- is shown (alkyl expansion). [Figure 16] The 1H-1H COSY NMR spectrum (400 MHz, DO, 298 K, pH 7.0) of [Lu(noneunpaX)]- is shown (aromatic expansion). [Figure 17] The 1H NMR spectrum (400 MHz, DO, 298 K, pH 4.5) of [In(noneunpaX)]- is shown, along with the chemical structure of [In(noneunpaX)]- and 1H assignments. [Figure 18] The 1H-1H COSY NMR spectrum (400 MHz, DO, 298 K, pH 4.5) of [In(noneunpaX)]- is shown. [Figure 19] Calculated absorbance curves for eight species of H4noneunpaX determined by combined potentiometric-spectrophotometric titration ([L] = 9.57 × 10-4 M, l = 0.2 cm) and acidic in-batch spectrophotometric titration ([L] = 1.0 × 10-4 M, l = 1.0 cm) at T = 298 K and I = 0.16 M NaCl are shown. [Figure 20] FIG. 1 shows a speciation diagram of H4noneunpaX as a function of pH, with the dotted line indicating pH 7.4. [Figure 21] The speciation diagram of H4noneunpaX with La3+ ([La3+] = [H4noneunpaX] = 1 × 10-4 M) at 298 K, I = 0.16 M NaCl, and the dashed line indicates physiological conditions (pH 7.4). [Figure 22] Figure 1 shows the speciation diagram of H4noneunpaX with Sm3+ ([Sm3+] = [H4noneunpaX] = 1 × 10-4 M) at 298 K, I = 0.16 M NaCl, and the dashed line indicates physiological conditions (pH 7.4). [Figure 23]Figure 1 shows the speciation diagram of H4noneunpaX with Gd3+ ([Gd3+] = [H4noneunpaX] = 1 × 10-4 M) at 298 K, I = 0.16 M NaCl, and the dashed line indicates physiological conditions (pH 7.4). [Figure 24] The speciation diagram of H4noneunpaX with Dy3+ ([Dy3+] = [H4noneunpaX] = 1 × 10-4 M) at 298 K, I = 0.16 M NaCl, and the dashed line indicates physiological conditions (pH 7.4). [Diagram 25] The speciation diagram of H4noneunpaX with Lu3+ ([Lu3+] = [H4noneunpaX] = 1 × 10-4 M) at 298 K, I = 0.16 M NaCl, and the dashed line indicates physiological conditions (pH 7.4). [Figure 26] The speciation diagram of H4noneunpaX with In3+ ([In3+] = [H4noneunpaX] = 1 × 10-4 M) at 298 K, I = 0.16 M NaCl, and the dashed line indicates physiological conditions (pH 7.4). [Figure 27] The speciation diagram of H4noneunpaX with Sc3+ ([Sc3+] = [H4noneunpaX] = 1 × 10-4 M) at 298 K, I = 0.16 M NaCl is shown, and the dashed line indicates physiological conditions (pH 7.4). [Figure 28] [44Sc]Sc3+ (1.2MBq) in NaOAc (0.1M, pH 4.5) (panel (A)), [111In]In3+ (1.0MBq) in NH4OAc (0.5M, pH 5.8) (panel (B)), [155Tb]Tb3+ (40kBq) in NH4OAc (0.5M, pH 6.0) (panel (C)), [ Concentration-dependent radiolabeling studies of H4noneunpa, H4noneunpaX, and DOTA with [177Lu]Lu3+ (150 kBq) (panel (D)), [213Bi]Bi3+ (680 kBq) in MES (1.0 M, pH 5.5), and (F) [225Ac]Ac3+ (40 kBq) in NHOAc (1.0 M, pH 7.3) (panel (E)). [Figure 29] Human serum stability studies of H4noneunpa and H4noneunpaX using [111In]In3+ (54 GBq / μmol) (panel (A)), [155Tb]Tb3+ (1.0 GBq / μmol) (panel (B)), [177Lu]Lu3+ (2.0 GBq / μmol) (panel (C)), and [225Ac]Ac3+ (134 MBq / μmol) (panel (D)). [Diagram 30] DFT-optimized structures of [La(noneunpaX)]- in panel (A) (left-handed orientation) and panel (B) (front-facing orientation), and [Lu(noneunpaX)]- in panel (c) (left-handed orientation) and panel (D) (front-facing orientation) are shown. Selected hydrogens have been omitted for clarity. [Diagram 31] (A) [44Sc]Sc3+ (400 kBq) in NaOAc (0.1 M, pH 4.5), (B) [111In]In3+ (1.06 MBq) in NH4OAc (0.5 M, pH 5.5), (C) [177Lu]Lu3+ (350-500 kBq) in NH4OAc (0.5 M, pH 6.0), (D) NH4OAc (0.2 M, pH 7 Figure 1 shows concentration-dependent radiolabeling of bifunctional H4noneunpaX with (A) [133 / 135La]La3+ (400 kBq) in (0.0), (E) [155Tb]Tb3+ (140 kBq) in NH4OAc (0.5 M, pH 7.0), and (F) [225Ac]Ac3+ (40 kBq) in NH4OAc (0.5 M, pH 7.2). [Diagram 32] Molar activity study of H4noneunpaX-Bn-NH2 with [177Lu]LuCl3 (20MBq) in NHOAc (0.5M, pH 6.0) monitored over 10 min at room temperature. Highest molar activity (250GBq / μmol), ligand to metal ratio ([L]:[M]; [174:1]) (left panel). Human serum stability challenge of [177Lu][Lu(noneunpaX-Bn-NH2]- (5.2GBq / μmol) was performed at 37°C and monitored over 7 days by radioactive iTLC (n=3) (right panel). [Diagram 33]Radiolabeling studies of bifunctional H4noneunpaX and the corresponding peptide conjugates are shown: (A) Concentration-dependent radiolabeling with [225Ac]Ac3+ (40 kBq) in NHOAc buffer (0.5 M, pH 7) (RT, 10 min). (B) Human serum stability challenge of [225Ac]Ac3+ labeled compounds. (C) Concentration-dependent radiolabeling with [155Tb]Tb3+ (120 kBq) in NHOAc buffer (0.5 M, pH 6) (RT, 10 min). (D) Human serum stability challenge of [155Tb]Tb3+ labeled bioconjugates. [Diagram 34] Shown are radioactive HPLC traces of [155Tb]Tb3+ control and [155Tb][Tb3+] labeled H4noneunpaX-Bn-NH2 and H4noneunpaX-Bn-NCS. Method: A: HO (0.1% TFA), B: MeCN (0.1% TFA); 100% A to 10% B over 15 min, 1 mL / min. [Diagram 35] Radioactive HPLC trace for [155Tb][Tb(noneunpaX-Ahx-Tyr3-TATE)] is shown. Method: A: HO (0.1% TFA), B: MeCN (0.1% TFA); 100% A to 60% B over 15 min, 1 mL / min. [Diagram 36] Shown is a radioactive HPLC trace for [155Tb][Tb(noneunpaX-PEG2-Tyr3-TATE)]. Method: A: HO (0.1% TFA), B: MeCN (0.1% TFA); 100% A to 60% B over 15 min, 1 mL / min. [Figure 37] Coronal views of maximum intensity projections (MIPs) from quantitative dynamic SPECT / CT scans 0–1 h after administration of [155Tb][Tb(noneunpaX-Ahx-Tyr3-TATE)] (top) and [155Tb][Tb(noneunpaX-PEG2-Tyr3-TATE)] (bottom) in NRG mice bearing AR42J exocrine tumor xenografts (left shoulder). [Figure 38]Sagittal views of maximum intensity projections (MIPs) from SPECT / CT scans 1, 2, and 4 hours after administration of [155Tb][Tb(noneunpaX-Ahx-Tyr3-TATE)] (top) and [155Tb][Tb(noneunpaX-PEG2-Tyr3-TATE)] (bottom) in an NRG mouse bearing an AR42J pancreatic exocrine tumor xenograft (left shoulder). [Figure 39] Representative time-activity plots of (A) [155Tb][Tb(noneunpaX-Ahx-Tyr3-TATE)] and (B) [155Tb][Tb(noneunpaX-PEG-Tyr3-TATE)] in NRG mice bearing AR42J exocrine / pancreatic tumor xenografts. Standard uptake values ​​(SUVs) were extracted for ROIs in relevant organs from calibrated SPECT / CT images. [Diagram 40] Figure 1 shows decay-corrected biodistribution studies of [155Tb][Tb(noneunpaX-Ahx-Tyr3-TATE)] 2 and 4 hours post-injection, and [155Tb][Tb(noneunpaX-PEG2-Tyr3-TATE)] 4 hours post-injection in NRG mice bearing AR42J exocrine / pancreatic tumor xenografts. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0021] Throughout the following description, specific details are set forth to provide a more thorough understanding to those skilled in the art. However, well-known elements may not have been shown or described in detail to avoid unnecessarily obscuring the present disclosure. Thus, the description and drawings should be regarded in an illustrative sense, rather than a restrictive sense.

[0022] Here, we developed a novel chelating ligand, H4noneunpaX, with an inverted functional group configuration and compared the characteristics of this novel ligand with H4noneunpa. This ligand has a trivalent Ln 3+ It exhibits preferential complexation with ions to form a single isomeric species in solution, which exhibits high thermodynamic stability and kinetic inertness. 111In]In 3+ , [ 155 Tb]Tb 3+ , [ 177 Lu]Lu 3+ , and [ 225 Ac]Ac 3+ Radiolabeling studies of H4noneunpaX with β-lactamase showed excellent compatibility and achieved high molar activity under mild conditions (room temperature, 10 min). As a proof of principle, a bifunctional derivative of H4noneunpaX was prepared by a facile synthetic approach and modified with two Tyr ligands for targeting neuroendocrine tumors (NETs). 3 -Octreotate peptide analogue. 111 In]In 3+ , [ 177 Lu]Lu 3+ , [ 133 / 135 La]La 3+ , [ 155 Tb]Tb 3+ , and [ 225 Ac]Ac 3+ Further radiolabeling studies of these bioconjugates using 225 Ac]Ac 3+ / [ 155 Tb]Tb 3+ -Promising results for the development of theranostic pairs in cancer-bearing mice 155 Tb]Tb 3+ SPECT / CT and biodistribution studies of the radiolabeled tracer demonstrated excellent performance, with good tumor uptake, low residual background organ activity and no evidence of degradation in vivo.

[0023] Therefore, the objective of this study was to investigate the effect of donor group substitution on the common scaffold and to study the effect of asymmetric / inverted substitution on metal ion affinity. H4noneunpaX with structure (6) was prepared and evaluated in comparison with its symmetric counterparts H4noneunpa / H4oxyaapa. To further evaluate the utility of this approach, bifunctional analogs of H4noneunapX were prepared to first demonstrate the synthetic availability of bifunctional asymmetric / inverted chelators and to investigate their in vivo performance.

[0024] [ka]

[0025] As used herein, the term prevention includes preventing a disease, minimizing the severity of a disease, or preventing the worsening of a disease. As used herein, the term treat or treatment includes reversing a disease or reducing the severity of a disease.

[0026] As used herein, the term antibody includes all forms of antibodies, including polyclonal, monoclonal, chimeric, humanized, single chain, multimeric, etc. The term antigen-binding fragment of an antibody refers to any portion of an antibody capable of binding to an antigen, including, by way of example only, but not limited to, Fab fragments, F(ab')2 fragments, Fv fragments, scFv fragments, minibodies, diabodies, etc. Reference to a specific antibody includes reference to any antibody that has been determined by any regulatory authority to be biosimilar to that specific antibody.

[0027] As used herein, the term peptidomimetic means a small protein-like molecule designed to mimic a peptide, and includes, but is not limited to, modified peptides, peptide foldamers, structural mimetics, and mechanistic mimetics.

[0028] Chelator compositions for radioactive metals are disclosed. Methods of using and making the compositions are also disclosed. The compositions can be used as therapeutic and / or diagnostic agents. The inventors have now determined that chelators having the general structure (6) are capable of coordinating radioisotopes under mild conditions to produce complexes that are stable under in vivo conditions, making such chelators particularly suitable for applications in, for example, radiotherapeutic, diagnostic, and / or theranostic constructs. The chelators can be attached directly or via a linker to a biological targeting moiety to generate constructs suitable for use in such applications.

[0029] In some embodiments, H4noneunpaX can be directly linked to a biological targeting moiety by directly linking the biological targeting moiety or linker to one of the carboxyl groups of structure (6), optionally with a linker interposed between H4noneunpaX and the biological targeting moiety. Furthermore, in some embodiments, one or more of the oxygen atoms of the carboxyl group are replaced by different heteroatoms, e.g., N or S. Thus, in various embodiments, a bifunctional H4noneunpaX can be used to link the chelator to the biological targeting moiety. n The functional groups provided on the oneunpaX chelator can be carboxyl, ester, amide, imide, thioamide, thioester, guanidinium, and the like.

[0030] In some embodiments, the chelator has the following structure (I): wherein each R1 is independently OH, NH, or SH, X is O, S, or NR3, and R3 is H or CH2C(=O)R1.

[0031] [ka]

[0032] In some embodiments, the chelator is a bifunctional molecule having the structure (II), where each R1 is independently OH, NH, or SH, or a functional group, each R2 is independently H or a functional group, X is O, S, or NR3, and one or more of the R1 or R2 groups is a functional group that allows the chelator to be attached to a biological targeting moiety, and R3 is H or CH2C(=O)R1.

[0033] [ka]

[0034] In some such embodiments, the functional group is a carboxyl, ester, amide, imide, thioamide, thioester, guanidinium, or the like. In some embodiments, the radioisotope targeting construct has the structure (II), where one of the R1 groups or one of the R2 groups is a biological targeting moiety.

[0035] In some embodiments, the chelator has structure (III) or is a bifunctional molecule having structure (III), where each R1 is independently OH, NH, or SH, or a functional group; each R2 is independently H or a functional group; each R4 is independently H or a functional group, or both R4 together form a cyclohexyl moiety; each R5 is independently H or a functional group, or both R5 together form a cyclohexyl moiety; X is O, S, or NR3, where one or more of R1, R2, R4, or R5 groups is a functional group that allows the chelator to be bound to a biological targeting moiety, and R3 is H or CH2C(=O)R1. In some embodiments, when both R4 together form a cyclohexyl moiety, each R5 is independently H or a functional group. In some embodiments, when both R5 together form a cyclohexyl moiety, each R4 is independently H or a functional group. In some embodiments, the functional group is an ester, an amide, an imide, a thioamide, a thioester, or a guanidinium group. In some embodiments, the radioisotope targeting construct has structure (III), and the chelator is linked to the biological targeting moiety via one R1 group, one R2 group, one R4 group, or one R5 group. In some embodiments, the radioisotope targeting construct has structure (III), and one of the R1 groups, one of the R2 groups, one of the R4 groups, or one of the R5 groups is a biological targeting moiety.

[0036] [ka]

[0037] For example, in some embodiments, the chelator has one of the structures shown below.

[0038] [ka]

[0039] Other groups, such as Milenic et al., have found that DTPA derivatives containing a cyclohexyl moiety in the chelator backbone exhibit comparable specific activity when incorporated into chelator-antibody conjugates. 58 It is therefore reasonable to predict that compounds incorporating a cyclohexyl moiety into the backbone of the chelator will likely exhibit similar binding properties to NoneunpaX, having the structure (6).

[0040] In some embodiments, the chelating agent is NoneunpaX having the structure (6). In some embodiments, such as shown in FIG. 1, the in vivo targeting chelate construct 120 has a targeting moiety 122 attached to a chelator 126. In some embodiments, including the illustrated embodiment, a linker 124 is interposed between the targeting moiety 122 and the chelator 126. The targeting moiety 122, the linker 124 (if present), and the chelator 126 together comprise an in vivo targeting construct 130. Further, the chelator 126 is used to chelate a radionuclide 128 suitable for in vivo imaging and / or radiotherapy. The radionuclide 128, together with the in vivo targeting construct 130, provides the in vivo targeting chelate construct 120 suitable for targeted in vivo delivery of the radionuclide 128 payload assisted by the targeting moiety 122.

[0041] Any moiety suitable for directing the targeted delivery of the targeting chelate construct 120 in vivo may be used as the targeting moiety 122. In some embodiments, the targeting moiety 122 of the targeting construct 120 is a hapten, an antigen, an aptamer, an affibody molecule, an enzyme, a protein, a peptide, an antibody, an antigen-binding fragment of an antibody, a peptidomimetic, a receptor ligand, a steroid, a hormone, a growth factor, a cytokine, a molecule that recognizes a cell surface receptor (including molecules involved in cell growth, metabolism, or function), a lipid, a lipophilic group, a carbohydrate, or any other molecule or targeting moiety that can selectively direct the construct to a particular location in the body. The targeting moiety may be produced in any suitable manner, for example, as a biologic, semi-synthetically, or synthetically.

[0042] Examples of targeting moieties developed for delivering radioisotope targeting constructs to desired locations within a mammalian subject in vivo include antibodies that target specific markers associated with a particular type of cancer, peptidomimetics that target proteins highly expressed in cancer cells, and the like. Illustrative, non-limiting examples of suitable targeting moieties are listed in Table 1 (Lau, J.; Rousseau, E.; Kwon, D.; Lin, K.; Benard, F.; Chen, X., Insight into the Development of PET Radiopharmaceuticals for Oncology. Cancers 2020, 12, 1312, which is incorporated herein by reference in its entirety). Some targeting moieties selectively interact with biological targets, including antigens, proteins, carbohydrates, or other molecules (e.g., tumor-associated antigens) present on the surface of cells that are overexpressed in cancer cells compared to normal cells. Illustrative, non-limiting examples of suitable targets are listed in Table 1. Suitable targets and / or targeting moieties for radiopharmaceuticals, whether currently known or discovered or developed in the future, will be known to those skilled in the art. In some embodiments, the targeting moiety 122 is an antibody or an antigen-binding fragment of an antibody. In some embodiments, the targeting moiety 122 is a peptidomimetic. In some embodiments, the targeting moiety 122 is one of the targeting moieties listed in Table 1, and any chelating agent present in the referenced molecule is replaced by H4noneunpaX as the chelating agent. In some embodiments, the targeting moiety 122 selectively interacts with one of the targets listed in Table 1.

[0043] [Table 1-1]

[0044] [Table 1-2]

[0045] [Table 1-3]

[0046] [Table 1-4]

[0047] Any suitable linker may be used as the linker 124 to attach the chelator 126 to the targeting moiety 122. For example, and by way of example only, suitable linkers include the following: 1 to 10 carbon atoms (C1 to C 10 ) (containing 2, 3, 4, 5, 6, 7, 8 or 9 carbon atoms) which may be saturated or unsaturated and may be substituted with one or more heteroatoms or may have one or more substituents; the hydrocarbon linker may be linear, cyclic, and / or branched (e.g., 8-aminooctanoic acid, 6-aminohexanoic acid); Aromatic linkers containing an aromatic moiety such as a benzyl group (e.g. aminophenylacetic acid); an amino acid linker having 1 to 10 amino acid residues (including 2, 3, 4, 5, 6, 7, 8, or 9 amino acid residues), any one or more of which may be a natural amino acid residue, a D-amino acid residue, or other non-natural residue, examples of which include GlyGly, GluGluGlu, GlySerGlySer; a cyclized linker or cyclized ring structure, which may be a cyclized amino acid linker (e.g. aminocyclohexane carboxylic acid); · PEG-linker of any suitable length; cationic linkers, whether formed from amino acid residues or other residues (e.g. Pip, 4-(2-aminoethyl)-1-carboxymethyl-piperazine (Acp)); anionic linkers, whether formed from amino acid residues or other residues (e.g. AspAsp, GluGlu); Carbohydrate-containing linkers; ·Click chemistry linkers (triazoles); any other suitable linker; · or any combination or modification of the foregoing.

[0048] Examples of linkers developed in the art for other radiopharmaceutical targeting constructs are known to those skilled in the art. Hydrophilic or charged linkers (e.g., PEG linkers or cationic / anionic linkers) can be used to increase the overall water solubility of the targeting construct. Amino acid side chain substitutions and / or inclusion of carbohydrate moieties can be performed to improve or modify the solubility and / or pharmacokinetics of the targeting construct. Those skilled in the art can develop and optimize appropriate linkers for specific applications, if desired. Examples of linkers developed in the art for other radiopharmaceutical targeting constructs are described by Benesova et al., Barnaski et al., and Kuo et al., by way of example only and not limitation. (Benesova, M.; Schafer, M.; Bauder-Wust, U.; Afshar-Oromieh, A.; Kratochwil, C.; Mier, W.; Haberkorn, U.; Kopka, K.; Eder, M., Preclinical Evaluation of a Tailor-Made DOTA-Conjugated PSMA Inhibitor with Optimized Linker Moiety for Imaging and Endoradiotherapy of Prostate Cancer.J.Nucl.Med.2015,56,914-920, Baranski,A.-C.;Schafer,M.;Bauder-Wust,U.;Wacker,A.;Schmidt,J.;Liolios,C.;Mier,W.;Haberkorn,U.;Eisenhut,M.;Kopka,K.;Eder,M.,Improving the Imaging Contrast of 68Ga-PSMA-11 by Targeted Linker Design:Charged Spacer Moieties Enhance the Pharmacokinetic Properties.Bioconjugate Chem.2017,28,2485-2492, Kuo, H.-T.;Pan,J.;Zhang,Z.;Lau,J.;Merkens,H.;Zhang,C.;Colpo,N.;Lin,K.-S.;Benard,F.,Effects of linker modification on tumor-to-kidney contrast of 68 See, for example, Ga-labeled PSMA-targeted imaging probes. Mol Pharm. 2018, 15, 3502-3511; Benesova, M.; Bauder-Wust, U.; Schafer, M.; Klika, KD; Mier, W.; Haberkorn, U.; Kopka, K.; Eder, M., Linker Modification Strategies To Control the Prostate-Specific Membrane Antigen (PSMA)-Targeting and Pharmacokinetic Properties of DOTA-Conjugated PSMA Inhibitors. J. Med. Chem. 2016, 59, 1761-1775, each of which is incorporated herein by reference in its entirety. Those skilled in the art can develop and optimize linkers suitable for a particular application.

[0049] In some embodiments, a construct such as construct 120 is prepared by performing a suitable reaction to couple the targeting moiety 122 and the chelator 126, for example by a suitable chemical reaction, to obtain an in vivo targeted construct 130, optionally having a linker 124 interposed between the targeting moiety 122 and the chelator 126. A radionuclide 128 is then added and attached to the chelator 126 (e.g., at a later time in a hospital or clinic) to form the desired in vivo targeted metal chelate construct 120. In other embodiments, the radionuclide 128 may first be chelated with the chelator 126, which is then conjugated to the targeting moiety 122 in any suitable manner to obtain the in vivo targeted chelate construct 120.

[0050] In some embodiments, the radionuclide 128 is bound to the chelator 126 (included as part of the construct 130) under mild temperature conditions (e.g., less than about 65° C., 60° C., 55° C., 50° C., 45° C., 40° C., 35° C., or 30° C.). In some embodiments, the mild temperature conditions are between about 10° C. and 65° C., including any value or subrange therebetween (e.g., 15° C., 16° C., 17° C., 18° C., 19° C., 20° C., 21° C., 22° C., 23° C., 24° C., 25° C., 26° C., 27° C., 28° C., 29° C., 30° C., 35° C., 40° C., 45° C., 50° C., 55° C., or 60° C.). In some embodiments, the radionuclide 128 is conjugated to the chelator 126 or construct 130 at room temperature, i.e., in the range of about 15°C to about 25°C (including any temperature value therebetween, e.g., 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, or 24°C).

[0051] In some embodiments, the radionuclide 128 or construct 130 is combined with the chelator 126 to form a metal chelate compound under mild pH conditions, e.g., about 5.0 to about 7.4 (including any value or subrange therebetween, e.g., 5.2, 5.4, 5.6, 5.8, 6.0, 6.2, 6.4, 6.6, 6.8, 7.0, or 7.2). In some embodiments, the radionuclide 128 is conjugated to the chelator 126 at about neutral pH, i.e., about pH 7.0, e.g., about 6.8 to 7.2 (including any value therebetween, e.g., 6.9, 7.0, or 7.1). In some embodiments, the radionuclide 128 is conjugated to the chelator 126 at about physiological pH, i.e., about pH 7.4, e.g., about 7.2 to 7.6 (including any value therebetween, e.g., 7.3, 7.4, or 7.5). In some embodiments, the radionuclide 128 is combined with the chelator 126 or construct 130 in an aqueous solution. In some embodiments, the aqueous solution is free or substantially free of alcohol, such as ethanol.

[0052] In some embodiments, the radionuclide 128 is combined with the chelator 126 or construct 130 for an incubation period that allows the chelated metal complex to form. In some embodiments, the incubation period is from about 5 minutes to about 6 hours, including any period therebetween (e.g., 10, 15, 20, 25, 30, 45, 60, or 90 minutes, or 2, 3, 4, or 5 hours). In some embodiments, the incubation period is from about 5 minutes to about 30 minutes.

[0053] In some embodiments, the concentration of the chelator 126 or construct 130 present when conjugated to the radionuclide 128 is about 10 -4 ~10 -7 M (any value in between, e.g. 10 -5 or 10 -6M). The concentration of chelator 126 or construct 130 used may be adjusted depending on the rate of complex formation between the particular chelator 126 and radionuclide 128 used in any particular embodiment. Similarly, the temperature at which the radionuclide 128 is combined with the chelator 126 or construct 130 may be varied depending on the rate of complex formation.

[0054] In some embodiments, the in vivo radioisotope targeting chelate construct 120 is present in mammal serum, optionally in human serum. In some embodiments, the in vivo radioisotope targeting chelate construct 120 is stable in mammal serum, optionally in human serum. In some embodiments, the in vivo radioisotope targeting chelate construct 120 is present in mammal serum in a mammalian body, optionally in human serum in a human body. In some embodiments, the in vivo radioisotope targeting chelate construct 120 is present in mammal blood, optionally in human blood. In some embodiments, the in vivo radioisotope targeting chelate construct 120 is present in mammal blood in a mammalian body, optionally in human blood in a human body. In some embodiments, the in vivo radioisotope targeting chelate construct 120 is present in a mammalian body, optionally in a human body. In some embodiments, the in vivo radioisotope targeting chelate construct 120 is present in a mammalian cell, optionally a human cell.

[0055] In some embodiments, the radionuclide 128 is delivered to a selected location within the body of a mammalian subject by administering to the subject an in vivo radioisotope targeting chelate construct 120 incorporating the radionuclide 128 and a targeting moiety 122 that specifically targets the in vivo radioisotope targeting chelate construct 120 containing the bound radionuclide 128 to the selected location within the body of the subject. In some embodiments, the method includes the targeting moiety 122 enhancing accumulation of the in vivo radioisotope targeting chelate construct 120 at the selected location within the body compared to other locations within the body, allowing for selective delivery of a dose of radiation to the selected location. In some embodiments, the in vivo radioisotope targeting chelate construct 120 is used to cause cell death at the selected location by delivering a targeted dose of radiation. In some embodiments, the cells that die at the selected location are cancer cells. In some embodiments, the radiation is alpha radiation, while in other embodiments beta radiation or gamma radiation may be used.

[0056] In some embodiments, the in vivo radioisotope targeting chelate construct 120 is internalized by cells within the mammalian subject, for example by endocytosis or otherwise. Thus, in some embodiments, the in vivo radioisotope targeting chelate construct 120 is present within a mammalian cell. In some embodiments, the in vivo radioisotope targeting chelate construct 120 is present within a human cell.

[0057] In some embodiments, the in vivo radioisotope targeting chelate construct 120 is prepared prior to administration to the subject by combining the in vivo radioisotope targeting construct 130 having the targeting moiety 122, the chelator 126, and optionally the linker 124 with the radionuclide 128 to form the in vivo radioisotope targeting chelate construct 120. In some embodiments, the combining step is performed at a moderate temperature, for example, at a temperature ranging from about 10° C. to about 65° C. (including any value therebetween, for example, 15° C., 16° C., 17° C., 18° C., 19° C., 20° C., 21° C., 22° C., 23° C., 24° C., 25° C., 26° C., 27° C., 28° C., 29° C., 30° C., 35° C., 40° C., 45° C., 50° C., 55° C., or 60° C.). In some embodiments, the combining step is performed at a mild pH, e.g., at about neutral pH or about physiological pH. In some embodiments, the mild pH is a pH between about 5.0 and about 7.4 (including any value therebetween, e.g., 5.2, 5.4, 5.6, 5.8, 6.0, 6.2, 6.4, 6.6, 6.8, 7.0, or 7.4). In some embodiments, the mild pH is about 6.0. In some embodiments, the combining step is performed at a physiological pH, e.g., in the range of about 7.0 to 7.4 (including any value therebetween, e.g., 7.1, 7.2, or 7.3). In some embodiments, the radionuclide 128 is combined with the in vivo radioisotope targeting construct 130 in an aqueous solution. In some embodiments, the aqueous solution is free or substantially free of alcohol, such as ethanol. In some embodiments, the combining step is carried out for a period of about 5 to about 30 minutes (including any value therebetween, eg, 10, 15, 20, or 25 minutes).

[0058] In some embodiments, the in vivo targeting chelate construct 120 is used in diagnostic applications. For example, the in vivo targeting chelate construct 120 can be administered to a subject in any suitable manner, and any suitable imaging technique or procedure can be used to assess the localization of the targeting chelate construct 120 in the body by the targeting moiety 122 by visualizing the location of the bound radionuclide 128 (e.g., positron emission tomography (PET) imaging or single photon emission computed tomography (SPECT) imaging). Such imaging procedures can be performed, for example, to diagnose a subject as having a particular disorder or type of cancer, or to identify the area of ​​the subject's body that is affected by a particular disorder or type of cancer. In some embodiments, localization of the targeting chelating construct 120 to a target organ, region, or multiple sites within the body as assessed by such imaging techniques may indicate that the subject has a particular form of cancer, and / or may be used to assess the extent of cancer and / or the locations within the body where cancerous cells are or may be located, and / or may be used to assess the extent of metastasis of the cancer.

[0059] In some embodiments, constructs such as targeting chelate construct 120 are used in therapeutic applications, for example to perform targeted radionuclide therapy. For example, targeting chelate construct 120 may be administered to a subject in any suitable manner, and the targeting effect imparted by targeting moiety 122 may be used to deliver chelated radionuclide 128 to a desired location within the subject's body. In some embodiments, radiation from radionuclide 128 is used to kill cells at the desired location. In some embodiments, the cells killed at the desired location are cancer cells. In some embodiments, targeting construct 120 is used to perform targeted radionuclide therapy. In some embodiments, targeting construct 120 is used to perform targeted alpha therapy.

[0060] In some embodiments, a pharmaceutical composition is provided, comprising a construct, such as targeting construct 120, and a pharma- ceutically acceptable carrier. The pharmaceutical composition may include any suitable excipients, vehicles, buffers, diluents, binders, thickeners, lubricants, preservatives, etc., and may be provided in any desired state, e.g., as a liquid, suspension, emulsion, paste, etc. In some embodiments, the pharmaceutical composition may be administered in any suitable manner, e.g., orally, intravenously, intramuscularly, subcutaneously, intraperitoneally, intratumorally, by inhalation, etc.

[0061] In some embodiments, a method of prophylactically and / or therapeutically treating a subject having or suspected of having cancer is provided. In some embodiments, the method comprises administering to the subject an in vivo targeting chelate construct 120 or a pharmaceutical composition comprising such a targeting chelate construct 120. In some embodiments, the method comprises administering to the subject a therapeutically and / or prophylactically effective amount of the targeting chelate construct 120.

[0062] In some embodiments, the subject is a mammal. In some embodiments, the subject is a human. In alternative embodiments, the subject is a livestock or pet (e.g., horse, cow, sheep, goat, cat, dog, rabbit, etc.). In some embodiments, the subject is a monkey.

[0063] Although exemplary embodiments are described herein with respect to targeting and killing of cancer cells, such constructs may be used for the selective killing and / or elimination of other unwanted cell types, e.g., bacteria, fungi, cells involved in autoimmune disorders, virally infected cells, parasites, and the like.

[0064] In some embodiments, metals that can be used as metal 128 include actinides, lanthanides, rare earth metals, or main group metals. In some embodiments, the lanthanide is La, Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, or Lu. In some embodiments, the lanthanide is Gd, Lu, Pr, Nd, Ho, Er, or Yb. In some embodiments, the lanthanide is a radioactive lanthanide. In some embodiments, the actinide is Ac, Th, Pa, U, Np, Pu, Am, Cm, Bk, Cf, Es, Fm, Md, No, or Lr. In some embodiments, the actinide is Ac, Th, or U. In some embodiments, the actinide is a radioactive actinide. In some embodiments, the rare earth metal is Sc, Y, La, Ce, Pr, Nd, Pm, Sm, Gd, Tb, Dy, Ho, Er, Tm, Yb, or Lu. In some embodiments, the metal is a trivalent lanthanide ion.

[0065] In some embodiments, the metal is a radioisotope. In some embodiments, the radioisotope may be any desired radioisotope (e.g., 225 Ac, 227 Th, 226 Th, 211 At, 44 Sc, 90 Y, 89 Zr, 177 Lu, 111 In, 86 / 89 / 90 Y, 211 At, 211 Fr, 212 / 213 Bi, 153 Sm, 161 / 166 Ho, 165 / 166 Dy, 161 / 155 Tb, 140 La, 142 / 143 / 145 Pr, 159 Gd, 169 / 175 Yb, 167 / 170 Tm, 169 Er, 149 Pm, 150 EU, 68 Ga, 137 Cs, 141 Ce, etc.

[0066] In some embodiments, the metal is actinium (Ac), ruthenium (Lu), bismuth (Bi), gallium (Ga), indium (In), terbium (Tb), thorium (Th), or cesium (Cs). 3+ ), lutetium(III)(Lu 3+ ), Bismuth(III)(Bi 3+ ), Gallium(III)(Ga 3+ ), Indium(III)(In 3+ ), Terbium (Tb 3+ ), Thorium(III)(Th 3+ ), or cesium(I)(Cs 1+ In some embodiments, the metal is 225 Ac, 177 Lu, 213 Bi, 232 Th, 230 Th, 228 Th, 68 Ga, 161 Tb, 155 Tb, 152 Tb, 149 Tb, 111 In, or 137 In some embodiments, the metal is 227 Th, 225 Ac, 155 Tb, 177 Lu, 111 In, 132 La, 235 La, 90 Y, 68 Ga, 44 Sc, 203 Pb, or 212 It is Pb.

[0067] In some embodiments, H4noneunpaX binds to a metal ion to form a coordination complex. In some embodiments, the coordination complex is called a metal chelator. In some embodiments, H4noneunpaX as a metal chelator or chelating ligand is a counterion to one or more cations, such as Na + , K + , Ca 2+ etc. In some embodiments, the metal chelate or chelating ligand is fully protonated. In some embodiments, the metal chelate or chelating ligand is in its free acid form. In some embodiments, the metal chelate or chelating ligand is in a partially protonated state.

[0068] In some embodiments, the coordination complex is present in mammalian serum, optionally in human serum. In some embodiments, the coordination complex is stable in mammalian serum, optionally in human serum. In some embodiments, the coordination complex is present in mammalian serum in a mammalian body, and optionally in human serum in a human body. In some embodiments, the coordination complex is present in blood, optionally in human blood. In some embodiments, the coordination complex is stable in mammalian blood, optionally in human blood. In some embodiments, the coordination complex is present in mammalian blood in a mammalian body, and optionally in human blood in a human body. In some embodiments, the coordination complex is present in a mammalian body, and optionally in a human body. In some embodiments, the coordination complex is present in a cell of a mammalian subject, and optionally in a cell of a human subject. EXAMPLES

[0069] Further embodiments are described with reference to the following examples, which are illustrative and not limiting in nature. Example 1.0 - Synthesis and characterization of H4noneunpaX The nine-coordinate chelating ligand H4noneunpa has been reported previously.8、13 , was synthesized according to established procedures 8 The preparation of H4noneunpaX was carried out following the linear synthetic route outlined in Scheme 1.

[0070] [ka]

[0071] The synthesis of H4noneunpaX proceeded by N-alkylation of commercially available 2-(2-aminoethoxy)ethan-1-ol (1) with tert-butyl bromoacetate (TBBA) under mild conditions to give the di-tert-butyl ester (2) in high yield. Successive mesylation of compound (2) followed by displacement with sodium azide proceeded without difficulty to give the corresponding azide (3) in high yield. Diisopropylethylamine (dipea) was selected as a suitable base for the mesylation of compound (2) to precipitate the HCl equivalents generated in the reaction and thus minimize the formation of the corresponding chloride by-product. Staudinger reduction of azide (3) was used to generate the corresponding primary amine (4), which could be used directly without further purification. The amine (4) was subsequently N-alkylated with two equivalents of methyl 6-(bromomethyl)picolinate to give the protected ligand (5) in moderate yield. DIPEA was found to be a better base than K2CO3 in this reaction. Attempts to isolate compound (5) using K2CO3 as the base likely led to the product being released from solution. + The purification was hampered by difficulties due to chelating ions. This difficulty was alleviated by the use of an organic base, which produced the product in high yield and purity. Final deprotection of compound (5) was achieved by ester hydrolysis in 4 M HCl, followed by reversed-phase high performance liquid chromatography (HPLC). Coevaporation of the purified ligand with 3 M HCl then afforded H4noneunpaX (6) as the HCl salt.

[0072] The purified ligands and all synthetic intermediates were analyzed by NMR spectroscopy (1 H, 13 C{ 1 The product was fully characterized by NMR (H, COSY, HSQC) and mass spectrometry (LR / HR-MS). Elemental analysis (EA) and HPLC of the final ligand were performed to confirm the purity of the isolated product. The results for H4noneunpaX are shown in Figures 3-7.

[0073] Di-tert-butyl 2,2'-((2-(2-hydroxyethoxy)ethyl)azanediyl)diacetate (2): tert-Butyl bromoacetate (4.30 mL, 5.57 g, 28.6 mmol) was added slowly to a solution of 2-(2-aminoethyoxyl)ethan-1-ol (1) (1.53 g, 14.3 mmol) and diisopropylethylamine (5.00 mL, 3.69 g, 28.6 mmol) dissolved in dry MeCN (150 mL). The reaction mixture was heated to 50° C. and stirred overnight. Upon completion, the volatiles were removed in vacuo and the resulting residue was redissolved in EtOAc (150 mL). After standing at room temperature for 10 min, the white precipitate that had formed was removed by vacuum filtration, washed with cold EtOAc (50 mL) and discarded. The filtrate was washed with deionized water (3×150 mL) and the combined aqueous phases were back-extracted with EtOAc (2×150 mL). The combined organics were evaporated in vacuo to give the title compound as a pale yellow oil (4.69 g, 99%). 1 H NMR(300MHz,CDCl3,298K)3.66(2H,t,J=4.6Hz,1-CH2),3.60(2H,t,J=5.2Hz,3-CH2),3.52(2H,t,J=4.6Hz,2-CH2),3.47(4H,s,5-CH2),2.96(1H,br s,7-OH), 2.92(2H,t,J=5.2Hz,4-CH2), 1.42(18H,s,6-C(CH3)3). 13 C{ 1 H}NMR(75MHz, CDCl3,298K) 171.0(6-C), 81.2(7-C), 72.4(2-C), 70.2(3-C), 61.9(1-C), 56.8(5-C), 53.4(4-C), 28.3(8-C). ESI-MS(MeOH)334.26[M+H] + .

[0074] Di-tert-butyl 2,2'-((2-(2-azidoethoxy)ethyl)azanediyl)diacetate (3): Methanesulfonyl chloride (1.34 mL, 1.98 g, 17.3 mmol) was added slowly to a solution of di-tert-butyl 2,2'-((2-(2-hydroxyethoxy)ethyl)azanediyl)diacetate (2) (5.26 g, 15.7 mmol) and diisopropylethylamine (3.01 mL, 2.24 g, 17.3 mmol) in EtOAc (15 mL) at 0°C. After 10 min, the suspension was allowed to warm to room temperature and stirred for an additional 3 h. Upon completion, the suspension was cooled to 0°C and the white solid was isolated by vacuum filtration. The filtrate was diluted with EtOAc (50 mL) and washed with deionized water (3 x 50 mL). The organic phase was dried over Na2SO4 and evaporated in vacuo to give the corresponding mesylate as a pale yellow oil which was used without further purification.

[0075] NaN3 (3.08 mg, 47.2 mmol) was added to a solution of the crude mesylate in dry DMF (20 mL) and the suspension was heated at 80 °C overnight. Upon completion, the solution was cooled to room temperature and diluted with deionized water (50 mL). The aqueous phase was extracted with DCM (3 x 50 mL) and the combined organics were washed with 5% aqueous LiCl (50 mL). The volatiles were removed in vacuo and the resulting residue was purified by silica gel chromatography (Combiflash automated purification system; A: DCM, B: MeOH; 100% to 5% B). The title compound was obtained as a pale yellow oil (3.25 g, 58%). 1 H NMR(300MHz,CDCl3,298K)3.62-3.57(4H,m,2- and 3-CH2), 3.46(4H,s,5-CH2), 3.3 3(2H,t,J=5.0Hz,1-CH2), 2.92(2H,t,J=5.6Hz,4-CH2), 1.42(18H,s,6-(C(CH3)3). 13 C{ 1H}NMR(75MHz, CDCl3,298K) 170.9(6-C), 81.0(7-C), 70.9(3-C), 69.8(2-C), 56.9(5-C), 53.6(4-C), 50.8(1-C), 28.3(8-C). ESI-MS(MeOH)359.21[M+H] + .

[0076] Di-tert-butyl 2,2'-((2-(2-aminoethoxy)ethyl)azanediyl)diacetate (4): Di-tert-butyl 2,2'-((2-(2-aminoethoxy)ethyl)azanediyl)diacetate (3) (2.81 g, 7.85 mmol) was dissolved in dry THF (30 mL) and cooled to 0 °C. PPh3 (2.47 mg, 9.42 mmol) was slowly added to the reaction mixture under Ar and the resulting solution was allowed to warm to room temperature and stirred for 5 h. The resulting solution was then added dropwise to deionized water (350 mL) and the suspension was stirred overnight. The THF was removed in vacuo and the off-white suspension was allowed to settle at room temperature for 1 h. The white precipitate was removed by vacuum filtration and the aqueous phase was concentrated in vacuo to approximately 100 mL. The aqueous phase was extracted with CH2Cl2 (4 x 75 mL) and the combined organic phases were evaporated in vacuo to give the title compound as a pale yellow oil (2.38 g, 91%). 1 H NMR(300MHz,CDCl3,298K)6.72(3H,br s,7-NH2), 3.71(2H,t,J=4.5Hz,2-CH2), 3.58(2H,t,J=5.1Hz,3-CH2), 3.10(2H ,t,J=4.5Hz,1-CH2), 2.91(2H,t,J=5.1Hz,4-CH2), 1.42(18H,s,6-(C(CH3)3). 13 C{ 1 H}NMR(75MHz, CDCl3,298K) 171.0(6-C), 81.6(7-C), 68.8(3-C), 67.9(2-C), 56.2(5-C), 53.3(4-C), 40.5(1-C), 28.3(8-C). ESI-MS(MeOH)333.19[M+H] + .

[0077] ( tBu)2(Me)2noneunpaX (5). Methyl (6-bromomethyl)picolinate (515 mg, 2.824 mmol) was added to a solution of di-tert-butyl 2,2'-((2-(2-aminoethoxy)ethyl)azanediyl)diacetate (4) (372 mg, 1.12 mmol) in dry MeCN (15 mL) under argon. Diisopropylethylamine (585 μL, 434 mg, 3.36 mmol) was added and the resulting pale yellow solution was stirred at ambient temperature for 1 h. The reaction mixture was then heated to 50 °C and stirred overnight. After completion, the volatiles were removed in vacuo and the resulting residue was redissolved in CHCl (25 mL). The organic phase was washed with deionized water (3 × 25 mL) and brine (25 mL), dried over NaSO, and evaporated in vacuo. The crude residue was purified by flash column chromatography on neutral alumina (Combiflash automated purification system; A: CH2Cl2, B: MeOH; 100% A to 5% B). The product was obtained as a pale yellow oil (459 mg, 65%). 1 H NMR(300MHz,CDCl3,298K)7.90(2H,dd, 3 J=7.3Hz, 4 J=1.1Hz,10-CH), 7.80(2H,dd, 3 J=8.1Hz, 4 J=1.4Hz,8-CH), 7.75(2H,t, 3 J=7.5Hz,9-CH),3.94(4H,s,7-CH2),3.90(6H,s,11-CH3),3.49(2H,t, 3 J=5.6Hz,2-CH2), 3.44(2H,t, 3 J=5.8Hz,3-CH2), 3.40(4H,s,5-CH2), 2.84(2H,t, 3 J=5.8Hz,4-CH2), 2.74(2H,t, 3 J=5.6Hz,1-CH2), 1.35(18H,s,6-C(CH3)3). 13 C{ 1H}NMR(75MHz,CDCl3,298K)170.8(8-C), 165.9(15-C), 160.8(14-C), 147.3(10-C), 137.5(12-C), 126.1(11-C), 123. 6(13-C), 80.9(7-C), 70.5(3-C), 69.4(2-C), 60.9(9-C), 56.7(5-C), 54.1(1-C), 53.6(4-C), 52.9(16-C), 28.2(8-C). ESI-MS(MeOH)653.3[M+Na] + .

[0078] H4noneunpaX·4HCl·5H2O(6). ( t Bu)2(Me)2noneunpaX (5) (168 mg, 0.257 mmol) was dissolved in 4 M HCl (5 mL) and heated at 60 °C overnight. The volatiles were removed in vacuo and the resulting off-white solid was purified by RP-HPLC (A: HO (0.1% TFA), B: MeCN; 100% A to 20% B, 30 min, t R 21.5 min.) Coevaporation with 3M HCl afforded pure H4noneunpaX as a white HCl salt (146 mg, 87%). 1 H NMR(400MHz,D2O,298K)7.95(2H,d, 3 J=7.7Hz,1-CH), 7.89(2H,t, 3 J=7.7Hz,2-CH), 7.57(2H,d, 3 J=7.7Hz,3-CH), 4.76(4H,s,3-CH2), 4.21(4H,s,9-CH2), 3.94(2H,t, 3 J=4.4Hz,6-CH2), 3.84(2H,t, 3 J=4.4Hz,7-CH2), 3.75(2H,t, 3 J=4.4Hz,5-CH2), 3.63(2H,t, 3 J = 4.4Hz, 8-CH2). 13 C{ 1H}NMR(75MHz,CDCl3,298K)167.9(13-C), 166.7(1-C), 149.7(6-C), 146.3(2-C), 140.1(3-C), 128 .5(5-C), 125.4(4-C), 65.3(10-C), 64.6(9-C), 58.6(7-C), 56.2(11-C), 56.0(8-C), 55.5(12-C). ESI-MS(H2O)491.1[M+H] + HR-ESI-MS(H2O)calcd.for[C 22 H 26 N4O 9+ H] + :491.1700;found[M+H] + :491.1779. Elemental analysis:calcd.% for H4noneunpaX·4HCl·5H2O(C 22 H 26 N4O9 4HCl 5H2O = 726.178 gmol -1 ):C37.59,H5.36,N7.97;found:C37.65,H5.43,N7.97.

[0079] Example 2.0 - Metal Complexation and NMR Characterization The coordination properties of H4noneunpaX were examined using a series of non-radioactive trivalent metal ions (La 3+ , Lu 3+ , and In 3+ ) complexation, and NMR spectroscopy ( 1 The metal complexes of H4noneunpaX were prepared by mixing equimolar amounts of the ligand with the appropriate metal salt in DO and adjusting the pD to neutral with NaOD (0.1 M). The corresponding solutions were then filtered and 1 The metal complexes were analyzed directly by H NMR spectroscopy (Figure 8). Further confirmation of metal complex formation was achieved using high-resolution electrospray ionization mass spectrometry (HR-ESI-MS), where each metal complex was characterized by the [M+2H] + This was confirmed by the presence of a monocationic peak corresponding to the species (Table 2).

[0080] [Table 2]

[0081] Complete 1 H NMR characterization data ( 1 H,COSY) was obtained. Na[La(noneunpaX)]. The results are shown in Figures 9-12. La(NO3)3·6H2O (2.9 mg, 6.75 μmol, 1.0 equiv.) was added directly to a solution of H4noneunpaX·4HCl·5H2O (4.9 mg, 6.75 μmol, 1.0 equiv.) in DO (300 μL). The solution was thoroughly mixed at 1000 rpm using a vortex mixer and the pD was adjusted to approximately 7.0 using dilute NaOD solution to obtain the corresponding [La(noneunpaX)]. - The complex was obtained. The white precipitate was filtered and the filtrate was directly characterized without further purification. 1 H NMR(400MHz,D2O,298K,pD7.0)7.87(2H,t, 3 J = 7.7 Hz, i- and m-CH), 7.77 (2H, d, 3 J = 7.5 Hz, j- and n-CH), 7.42 (2H, d, 3 J = 7.7 Hz, h- and l-CH), 4.19 (2H, d, 2 J = 16.4 Hz, g- and k-CH), 3.93 (2H, d, 2 J = 16.4 Hz, g'- and k'-CH), 3.53 (2H, t, 3 J=5.0Hz,d-CH2), 3.48(2H,d, 3 J=4.9Hz,e-CH2), 3.37(2H,d, 2 J = 16.3 Hz, a- and b-CH), 3.31 (2H, d, 2 J = 16.3 Hz, a'- and b'-CH), 3.04 (2H, br t, f-CH2), 2.71 (2H, t, 3 J=5.0Hz,c-CH2). LR-ESI-MS(H2O)627.0[M+2H] + , 624.8[M] - HR-ESI-MS(H2O)calcd.for[C 22 H22 LaN4O9+2H] + :627.0536;found[M+2H] + :627.0600.

[0082] Na[Lu(noneunpaX)]. The results are shown in Figures 13-16. Lu(NO3)3·H2O (2.4 mg, 6.75 μmol, 1.0 equiv.) was added directly to a solution of H4noneunpaX·4HCl·5H2O (4.9 mg, 6.75 μmol, 1.0 equiv.) in DO (300 μL). The solution was thoroughly mixed at 1000 rpm using a vortex mixer and the pD was adjusted to approximately 6.5 using dilute NaOD solution to obtain the corresponding [Lu(noneunpaX)]. - The complex was obtained. The solution was filtered and analyzed without further purification. 1 H NMR(400MHz,D2O,298K,pD6.5)8.03(1H,t, 3 J=7.8Hz,i-CH), 7.96(1H,t, 3 J = 7.7Hz, m-CH), 7.90 (1H, d, 3 J = 7.7Hz, n-CH), 7.83 (1H, d, 3 J=7.8Hz,j-CH), 7.61(1H,d, 3 J = 7.8Hz, h-CH), 7.43 (1H, d, 3 J=7.7Hz,l-CH), 4.95(1H,d, 2 J = 15.8Hz, g-CH), 4.17 (1H, d, 2 J = 15.8 Hz, g'-CH), 4.01 (1H, d, 2 J=15.6Hz, k-CH), 3.97(1H,d, 2 J=17.4Hz,a-CH), 3.90(1H,d, 2 J=17.4Hz,a'-CH), 3.77(1H,ddd,c-CH), 3.66(1H,dd,d-CH), 3.45(1H,d, 2 J = 18.4 Hz, b-CH), 3.43-3.40 (1H, m, e-CH), 3.36-3.24 (2H, m, e'- and d'-CH), 3.27 (1H, d, 2 J = 15.6 Hz, k'-CH), 3.19 (1H, d,2 J=18.4Hz,b'-CH), 3.10(1H,ddd,f-CH), 2.80(1H,d, 2 J=12.9Hz,f'-CH), 2.65(1H,dd,c'-CH). LR-ESI-MS(H2O)663.1[M+2H] + , 661.1[M] - HR-ESI-MS(H2O)calcd.for[C 22 H 22 LuN4O9+2H] + :663.0873;found[M+2H] + :663.0946.

[0083] Na[In(noneunpaX)]. The results are shown in Figures 17 and 18. In(NO3)3·H2O (2.1 mg, 6.75 μmol, 1.0 equiv.) was added directly to a solution of H4noneunpaX·4HCl·5H2O (4.9 mg, 6.75 μmol, 1.0 equiv.) in DO (300 μL). The solution was thoroughly mixed at 1000 rpm using a vortex mixer and the pD was adjusted to approximately 4.5 using dilute NaOD solution to obtain the corresponding [In(noneunpaX)]. - The complex was obtained. The solution was filtered and analyzed without further purification. 1 H NMR(400MHz,D2O,298K,pD5.5)8.32(2H,t, 3 J = 5.2 Hz, i- and m-CH), 8.28 (2H, d, 3 J = 5.0 Hz, j- and n-CH), 7.88 (2H, d, 3 J = 5.0 Hz, h- and l-CH), 4.82 (2H, d, 2 J = 10.7 Hz, g and k-CH2), 4.28 (2H, d, 2 J=10.7Hz, g'- and k'-CH2), 3.74 (4H, s, a / a'- and b / b'-CH2), 3.56 (4H, br s, d / d'- and e / e'-CH2), 3.38 (2H, br s, f / f'-CH2), 2.80 (2H, br s, c / c'-CH2). LR-ESI-MS(H2O) 603.0[M+2H] + , 625.0[M+H+Na] +HR-ESI-MS(H2O)calcd.for[C 22 H 22 InN4O9+2H] + :603.0506;found[M+2H] + :603.0572.

[0084] [La(noneunpaX)] - of 1 The H NMR spectrum shows the formation of a single symmetric isomer in solution, with characteristic diastereotopic splitting of the methylene protons associated with the four pendant donor arms of the ligand, suggesting coordination of all four donor groups to the metal center. Clearly resolved resonances are observed for the protons in the ethylene bridge backbone, indicating the formation of a metal complex coordinated by the central ether oxygen. In contrast, Lu 3+ The NMR spectrum for the complex reveals a single asymmetric isomer, with chemically distinct resonances for each picolinate donor arm found in the aromatic region. Notably, one picolinate donor arm (H l , H m , H n ) is La 3+ It maintains the same chemical shifts as seen in the complex, but with the addition of a second picolinate arm (H h , H i , H j ) shows a significant downfield shift, and Lu 3+ This indicates closer coordination to the metal center and a change in ligand conformation at this position. This aspect is characterized by a large binding constant ( 2 J AB = 15.8 Hz, showing a characteristic diastereotopic splitting of adjacent methylene protons (H g and H g’ ) were further observed, with two different chemical environments (Δδ g / g’ =0.78 ppm). All remaining methylene protons associated with the pendant donor arms also become non-equivalent to metal ion complexation and appear as diastereotopic doublets, which are 1 H- 1It can be clearly distinguished in the H COSY NMR spectrum (Figures 14-16). [La(noneunpaX)] - In contrast, Lu 3+ The complex consists of interconnected protons (e.g., H c and H c’ ), indicating a strong complexation environment and close coordination to the metal center.

[0085] Metal complexes of H4noneunpaX share similar spectral characteristics to those reported for H4noneunpa, thereby confirming the presence of La, as indicated by the pendant donor arm resonances and diastereotopic splitting of the backbone ethylene bridge. 3+ and Lu 3+ A fully saturated metal coordination sphere was achieved with the ion. Interestingly, for H4noneunpa, the opposite trend in metal complex symmetry was achieved, with [La(noneunpa)] - The complexes appear as asymmetric isomers, whereas [Lu(noneunpa)] - The complex was completely symmetric. 8 .

[0086] [In(noneunpaX)] using NMR spectroscopy - Analysis of the complexes was hindered by their low solubility at neutral and basic pH and therefore, 1 The H NMR spectrum was recorded under acidic conditions (pH 4.0). a / a’ and H b / b’This indicates the formation of a different symmetric isomer in which both picolinate donors are coordinated to the metal center while the two acetate donor arms remain unbound, as indicated by the presence of a 4H singlet resonance at 3.50 ppm corresponding to . This assignment is also supported by the resonances observed for the backbone methylene protons, which appear as broad signals in the NMR spectrum. At higher pH values ​​(pH ca. 5.0-8.5), a second species was observed that may correspond to a fully coordinated complex, although a complete spectral analysis was not possible.

[0087] Example 3.0 - Solution Thermodynamic Stability Study and Sc 3+ , In 3+ , Lu 3+ , Dy 3+ , Gd 3+ , Sm 3+ , La 3+ Complexation equilibrium with Due to the competition between a given metal ion and protons for the same coordinating group during metal complex reactions, the protonation constants of the chelating ligands must be determined independently prior to the investigation of the thermodynamic stability of different metal complexes. Combined potentiometric and UV-vis spectrophotometric titrations were used to evaluate the protonation constants of H4noneunpaX from pH about 2 to 11.5, while acidic in-batch UV-vis spectrophotometric titrations were used to determine the protonation constants for the most acidic protons, which were below the threshold of the pH electrode (pH < 2). HyperSpec 24 and Hyperquad 25 All eight protonation constants were determined for H4noneunpaX by refining the experimental data using (Table 3).

[0088] [Table 3]

[0089] The protonation constants obtained for H4noneunpaX follow the typical trends observed for similar polyaminocarboxylate-based ligands. 6、12、13 , the first two dissociation events (species H8L4+ and H7L 3+ ) can be attributed to the protonated pyridyl nitrogen donor. The deprotonation of each successive pyridine donor, with pKas of approximately 0.72 and 1.01, respectively, is accompanied by a spectral change in the ligand absorption spectrum, and a large decrease in the ligand absorbance is observed for the first dissociation event (Figures 19 and 20). The deprotonation of two acetate carboxyl groups (logK6=2.14 and logK5=2.38) is followed by two picolinate donors (logK4=2.83 and logK3=3.79), with the last two deprotonations being assigned to the dipyridyl (logK2=7.08) and iminodiacetate (logK1=8.84) terminal amines, respectively. The protonation constants measured for the species HL are comparable to those of other ligands containing an iminodiacetate moiety (e.g., N-(2-hydroxyethyl)iminodiacetic acid (HEIDA), logK1=8.68). 26 In contrast, the second terminal amine in H4noneunpaX is equivalent to the equivalent donor group in H4noneunpa / Oxyaapa (logK2=7.63). 13 (logK2=7.08), which may be due to the stronger electron-withdrawing effect induced by placing two picolinate donors on the same nitrogen atom, in addition to steric constraints at this position that may disfavor protonation. Compared to H4noneunpa / Oxyaapa, the greater difference in basicity between the terminal amines of H4noneunpaX may favor metal ion coordination, whereby the less basic dipyridyl nitrogen favors complex formation at lower pH, while the more basic iminodiacetate nitrogen acts as a stronger donor group for metal ions.

[0090] Medically important metal ions (e.g., Sc 3+ , In 3+ , Lu 3+ , Dy 3+ , Gd 3+ , Sm 3+ , La 3+To assess the affinity of H4noneunpaX for metal ions, complexation equilibria were studied using both potentiometric-spectrophotometric complex titrations and, in all cases, acidic in-batch UV-vis spectrophotometric titrations at pH below 2. H4noneunpaX was observed to exhibit high affinity for all metal ions investigated, with metal complex formation beginning at a pH below about 1. Table 4 shows the stability constants for each metal ion.

[0091] [Table 4]

[0092] H4noneunpaX has a high stability constant (logK ML ) and pM values ​​(Table 4), trivalent Ln 3+ It shows high thermodynamic selectivity for complex formation with ions. Notably, these values ​​are in line with those previously determined for H4noneunpa / Oxyaapa. 13 In direct comparison, H4noneunpaX is Ln 3+ The series shows slightly higher stability constants, suggesting a small thermodynamic advantage for this inverted conformation, which may be the result of some degree of preorganization of the ligand binding cavity. 3+ The pM values ​​obtained for H4noneunpaX bearing ions exceed those of the current gold standard chelators DOTA and DTPA by several log units (e.g., pLu = 17.1 and 19.1, respectively). 11 Although stability constants and pM values ​​are useful parameters to compare the metal-trapping abilities of different chelating ligands, they do not always correlate well with in vivo stability, and another aspect to consider is the speciation behavior of a given metal complex as a function of pH.

[0093] Speciation plots for metal complexation of H4noneunpaX with trivalent lanthanide ions show that at acidic pH, protonated MHL species are formed, followed by [ML] species, which predominate over a wide pH range. -The interesting relationship is that only one conversion to the hydroxo species occurs, and even under very basic conditions (pH = 11.5), there is no hydroxo species (Figures 21-27). This is consistent with the previously reported Ln 3+ This is in contrast to many similar chelating ligands bearing ions (H4octapa, H4pypa, H4octox), all of which exhibit the formation of one or more hydroxo species (e.g., [M(OH)L], [M(OH)2L)]) under basic conditions (pH > 9.0), typically resulting from the deprotonation of coordinated water molecules in the complex. 7、9、12 Under physiological conditions (pH 7.4), a single species ([ML] - ) but all Ln 3+ It has been observed for the complexes that different isomers may exhibit different pharmacokinetic properties and biodistribution profiles, which is preferable for in vivo applications.

[0094] Potentiometric-spectrophotometric complex titrations of H4noneunpaX with smaller trivalent metal ions yielded Ln 3+ As typically observed due to the higher charge density of these metal centers, Ln 3+ Compared with Sc, a higher stability constant is observed. 3+ and In 3+ was determined (Table 4). 3+ In the case of , complex formation occurs below a pH of about 0.5 to give a neutral MHL species, which deprotonates with increasing pH to give the anionic [ML] - This transformation results in the Ln 3+ The complexes have a similar pKa range (pKa = 2.28-3.22) and can be attributed to the deprotonation of one of the carboxylate donors. 3+ The complexes exhibit different thermodynamic behavior in solution. The MHL species has a higher pK a It is deprotonated to an anionic form [ML] - Seed (pK a= 6.17(3)), which is attributed to the tertiary amine of the iminodiacetate moiety, which remains protonated at the beginning of the complex formation. This showed a single species at pH 4.0, [In(noneunpaX)] - In agreement with the NMR characterization of the complex, the two acetate donor groups remain unbound to the metal center. 3+ As in the case of the complexes, formation of hydroxo species is observed at higher pH, which is explained, without being bound by theory, by considering the mismatch between the size of these small metal ions and the ligand binding cavity.

[0095] All potentiometric titrations were performed using a Metrohm Titrando 809 and a Metrohm Dosino 800 with a Ross composite electrode. Direct titrations were recorded using a Varian Cary 60 UV-vis spectrophotometer (200-350 nm spectral range) equipped with an optical dip probe (0.2 cm path length), while acidic in-batch experiments were measured in standardized glass cuvettes (1 cm path length). A temperature-controlled (298 K) 20 mL glass cell with an inlet-outlet adapter for nitrogen gas purging (purified by 10% NaOH solution to eliminate CO2 before and during each titration) was used as the titration cell. The hydrogen ion concentration of the electrode was calibrated daily by direct titration of HCl with freshly prepared NaOH solution and the results were recorded according to the Gran procedure. 55 The standard potential (E°) and ionic product of water (pK ) were measured at 298 K using 0.16 M NaCl as the supporting electrolyte. w ) was determined. The solution under study was titrated with a carbonate-free NaOH solution (approximately 0.16 M) standardized against freshly recrystallized potassium hydrogen phthalate.

[0096] The protonation equilibrium of H4noneunpaX was determined by the addition of ligand ([L] = 9.57 × 10 -4H4noneunpaX was assessed by combined potentiometric-spectrophotometric titration of solutions containing 0.1 M NaOH. Electromotive force (EMF) values ​​and UV-vis spectra were recorded after each addition of NaOH, and the instrument was synchronized to have a consistent time interval (30 s) between each addition / equilibration and data acquisition. Further acidic in-batch UV-vis spectrophotometric studies were performed to obtain the protonation constant for the most acidic ionizable proton of H4noneunpaX, which was below the pH electrode threshold (pH < 2). Different amounts of HCl (0.1 M and 3.0 M) were used to titrate H4noneunpaX to a constant molar concentration ([L] = 1 × 10) while maintaining as constant an ionic strength as possible (0.16 M NaCl). -4 Separate solutions of the ligands were prepared in H + The equilibrium concentrations of ions were calculated and followed the Hammett acidity function (H0) for determining the acidity in highly concentrated acid solutions. All eight protonation constants for H4noneunpaX were calculated according to HypSpec2014 24 and Hyperquad 2013 25 It was determined by refining the experimental data using software.

[0097] H4noneunpaX and Sc 3+ , In 3+ , Lu 3+ , Dy 3+ , Gd 3+ , Sm 3+ , and La 3+ The complexation equilibrium with was evaluated using both potentiometric-spectrophotometric complex titrations, and acidic in-batch UV-vis spectrophotometric titrations at pH below 2. For potentiometric-spectrophotometric complex titrations (spectral range: 200–350 nm), a 1:1 molar ratio of metal ion to ligand ([M 3+ ] ≒ [H4noneunpaX] ≒ 8.40 × 10 -4 A solution containing 1:1 molar ratio of metal ion to ligand ([M]) was titrated against NaOH solution at T = 298 K and I = 0.2 cm. Acidic in-batch UV-vis spectrophotometry (spectral range: 200-350 nm) was performed with different amounts of HCl (0.1 M and 3.0 M) at 1:1 molar ratio of metal ion to ligand ([M]). 3+] ≒ [H4noneunpaX] ≒ 1 × 10 -4 Measurements were performed using a set of solutions containing 1000 mM NaOH, yielding samples with a pH range of 0-2. Measurements were recorded at T=298 K, I=1.0 cm, and the Hammett acidity function (H0) was used to determine pH in strongly acidic solutions rather than electrode potential. Metal solutions were prepared from atomic absorption spectroscopy (AAS) standards previously evaluated using Gran's method, in which an equimolar solution of a given metal ion and Na2H2EDTA is titrated against NaOH to determine the acid concentration.

[0098] All potential measurements were performed using Hyperquad 2013. 25 The spectroscopic measurements were performed according to the HypSpec2014 24 The calculation was performed using Sc 3+ , In 3+ , Lu 3+ , Dy 3+ , Gd 3+ , Sm 3+ , and La 3+ The proton dissociation constants corresponding to the hydrolysis of aqueous ions were obtained from Baes and Mesmer. Hyss software 56 was used to generate a speciation diagram with calculated protonation and stability constants.

[0099] The species produced in the investigated system are governed by the general equilibrium equation pM + qH + rL = M p H q L r (charges omitted), where a complex containing a metal ion M, a proton H, and a ligand L can be defined according to the general formula M p H q L r The stoichiometric index p may also be 0 in the case of protonation equilibrium, and negative values ​​of q refer to the removal of a proton from the coordinated water, equivalent to the addition of a hydroxide ion during the formation of the complex. p H q L rThe overall equilibrium constant for the formation of pM is denoted as logβ. The stepwise equilibrium constant, logK, corresponds to the difference in log units between the overall constants for successively protonated (or hydroxide) species. pM is expressed as (-log[M n+ ] free ), and [M n+ ]=1μM, [L x- ] = 10 μM, calculated from the stability constants obtained for each studied system at pH 7.4 and 25 °C. 57 .

[0100] Example 4.0 - Radiolabeling Studies [ 44 Sc]Sc 3+ , [ 111 In]In 3+ , [ 177 Lu]Lu 3+ , [ 155 Tb]Tb 3+ , [ 213 Bi]Bi 3+ , and [ 225 Ac]Ac 3+ Radiolabeling studies with were performed to investigate the variation in metal ion affinity with changes in ionic radius and coordination number over a wide size range. 225 Ac(t 1 / 2 = 9.92 days) is of great interest for applications in targeted alpha therapy (TAT) due to its long half-life and the potency of the particulate radiation emitted within its decay scheme. 225 Ac]Ac 3+ Appropriate companion radionuclides for CT are required to perform accurate staging of disease progression and assessment of patient suitability for treatment.

[0101] Relatively long-lived radionuclides [ 111 In]In 3+ (t 1 / 2 = 2.83 days) 27 and 155 Tb]Tb 3+ (t 1 / 2 = 5.51 days) 20are the high abundance low energy gamma rays (171 and 245 keV[ 111 In] 27 ;44, 87, and 105 keV[ 155 Tb] 20 ) emission, making it suitable for SPECT imaging, and [ 225 Ac]Ac 3+ This is well suited to the long half-life of 44 Sc]Sc 3+ is due to its long physical half-life (t 1 / 2 =3.97 hours) and a high positron branching ratio (E β+ = 632 keV, 94.3%), making it a promising candidate for PET imaging, and in addition, companion radioisotopes [ 47 Sc]Sc 3 (t 1 / 2 = 3.35 days) is β - Therapy (E β- = 162 keV, 100%) 28 .

[0102] Concentration-dependent radiolabeling studies showed that H4noneunpaX, 213 Bi]Bi 3+ It was shown to be a very versatile chelator, exhibiting high affinity for all metal ions tested, except for [Figure 28]. All reactions with H4noneunpa and H4noneunpaX were carried out at room temperature and monitored after 5 min and within 10 min after generator elution. 213 Bi]Bi 3+ The reactions with DOTA were carried out at elevated temperatures and monitored for 30–60 min.

[0103] Importantly, quantitative radiochemical conversion (RCC) was achieved within 10 min at room temperature, a notable advantage over the widely applied "gold standard" chelator DOTA. Optimal radiochemical yields were obtained under very mild conditions (pH 7.0, room temperature, 10 min), which are compatible with thermosensitive biological targeting vectors (monoclonal antibodies). H4noneunpaX showed coordination properties comparable to H4noneunpa; each chelator exhibited [ 111 In]In 3+ (54GBq / μmol), [ 155 Tb]Tb 3+ (1.0GBq / μmol), [ 177 Lu]Lu 3+ (2.0 GBq / μmol) and [ 225 Ac]Ac 3+ (134 MBq / μmol) was successfully radiolabeled at high molar activity.

[0104] [ 44 Sc]Sc 3+ Concentration-dependent radiolabeling studies of H4noneunpa and H4noneunpaX with β-lactams showed low radiometal ion compatibility; thereby, low RCYs were achieved at high ligand concentrations. These results indicate that β-lactams prefer coordination numbers between 6 and 8. 18 Sc 3+ This was expected based on the small ionic radius (0.870 Å, CN=8) and chemical hardness of the ion. However, these results further suggest that there is no significant difference in metal ion affinity between symmetric and asymmetric ligands based on this framework.

[0105] Each chelate compound 213 Bi]Bi 3+ The stark contrast in radiolabeling efficiency shown for Bi was somewhat surprising given the similarity in donor atoms and trends observed for other trivalent metal ions. However, without wishing to be bound by theory, this deviation may be due to the fact that Bi 3+ This can be rationalized by considering the coordination properties of the ions. 3+ Ac 3+Similar ionic radius (Bi 3+ and Ac 3+ 1.170 Å (CN=8) vs. 1.220 Å (CN=9), respectively. 18、19 However, it is of moderate chemical hardness and shows a stronger preference for moderately hard donor groups (e.g., nitrogen, pyridine) over hard ion donors (oxygen, phenolate). 3+ also has the stereochemically active 6s in some of its coordination complexes. 2 are known to exhibit lone pairs of electrons, which can have a significant effect on the preferred conformational geometry of chelating ligands and their effective denticities. 29、30 .

[0106] Example 5.0 - Human serum stability study H4noneunpaX and H4noneunpa and [ 111 In]In 3+ , [ 155 Tb]Tb 3+ , [ 177 Lu]Lu 3+ , and [ 225 Ac]Ac 3+ A series of serum stability challenge assays were performed to determine the kinetic inactivity of the resulting complexes with H4noneunpa in the presence of competing endogenous metal-binding proteins. The results are shown in Figure 29. In the case of H4noneunpaX, incubation of the radiolabeled complexes in human serum at 37°C showed no release of bound radiometal over a period of 5-7 days, indicating high kinetic inactivity and potential for in vivo applications. Radiolabeled complexes of H4noneunpa showed similar kinetic inactivity to H4noneunpaX and maintained >97% radiochemical purity over a period of 5-7 days, with the exception of [ 225 Ac][Ac(noneunpa)] - showed an initial decrease in radiochemical integrity of approximately 10% over the course of the study. All experiments were performed at 37° C. and monitored by radio-iTLC (n=3).

[0107] Example 6.0 - Computational Studies To gain insight into the solution structures of metal complexes of H4noneunpaX, density functional theory (DFT) calculations were performed using Gaussian16 (rev. B.01) in the polarizable continuum model (PCM). 31、32 The relatively small core quasi-relativistic effective core potentials (ECP28 / 60MWB) and metal ions (La 3+ , Lu 3+ , Bi 3+ Geometry optimization was performed using a hybrid Perdew-Burke-Ernzerhof (PBE(0)) exchange-correlation functional, using the relevant valence basis functions of , while the light atoms (C, H, N, O) were modeled up to the Def2TZVP level of theory. 33-35 Optimized structures were determined for all complexes using Cartesian coordinates and calculated thermodynamic energy values. Avogadro (version 1.2.0) was used to generate initial geometries and provide input coordinates for the calculations. 51 Solvation effects were modeled using an integral equation polarizable continuum model (IEF-PCM) for all metal complexes. 32 To verify that the resulting structure was a true energy minimum on the potential energy surface, a vibrational frequency analysis was performed on the final optimized geometry. NBO (version 3.1) in Gaussian16 36 A natural bond orbital (NBO) analysis was performed using [Bi(noneunpaX)] - 6s in 2 Contour plots of electron density including lone pairs were generated using the Multiwfn software. 52 Created using.

[0108] The distribution of donor arm substituents within the framework of H4noneunpaX gives rise to four possible conformational isomers upon metal ion coordination (with a coordination number of 9) resulting from the relative orientation of the five-membered chelate ring formed between the ethylene bridge of the ligand and the metal center (λ vs. δ). La 3+ , Lu3+ , and In 3+ The solution structure of H4noneunpa / Oxyaapa with 8、13 .

[0109] H4noneunpaX's La 3+ and Lu 3+ Geometry optimization of the complex produced a structure with a fully saturated metal coordination sphere, whereby all nine donor atoms from the ligand were bound to the metal center. [La(noneunpaX)] - In the case of [Lu(noneunpaX)], the geometry optimization favors a single conformer with the lowest energy symmetric δδ configuration, whereas in the case of [Lu(noneunpaX)] - The complexes favored different asymmetric isomers with a twisted λδ conformation (Figure 30). Interestingly, this was due to the fact that La 3+ In complexes, an asymmetric arrangement is formed, and Lu 3+ This is in contrast to the complex formation reported for H4noneunpa, which was found to form a completely symmetric conformation in the complex.

[0110] [La(noneunpaX)] - The structure of La shows a high degree of symmetry, with the two picolinic acid donors adopting the same relative configuration with both pyridine rings buried (parallel), while the two acetate donors also adopt the same relative configuration with antiparallel orientation relative to the central plane of symmetry. This characterization demonstrated the formation of a single symmetric isomer in solution. 3+ This is consistent with the experimental NMR results for the complex. Furthermore, the DFT calculated structure shows that the methylene protons (H a / H b and H a’ / H b’ ) are shown in nearly identical chemical environments and therefore have similar chemical shifts (δ ab = 3.37 ppm, δ a’b’ = 3.31 ppm 1 It gives rise to two pairs of diastereotopic doublets in the H NMR spectrum. In contrast, [Lu(noneunpaX)] -The solution-phase structure of shows a single asymmetric isomer, and a change in the backbone geometry to the λδ conformation induces a shift in the relative arrangement of the picolinic acid donors to an orthogonal configuration. This conformational shift also occurs in the Lu 3+ Complex 1 This is further supported by H NMR characterization, which shows clearly resolved diastereotopic doublets for the four methylene protons adjacent to the picolinate donor arms. g / H g’ (Δδ=0.78 ppm) and H k / H k’ The large difference in chemical shift for (Δδ=0.74 ppm) is due to the stiffness [Lu(noneunpaX)] - This can be clearly explained by the different local environments adopted in the complexes (Figure 30, panel (C)).

[0111] Example 7.0 - Synthesis and characterization of bifunctional H4noneunpaX-Bn-NCS A synthetic approach was developed towards the bifunctional H4noneunpaX-Bn-NCS having the structure (13).

[0112] [ka]

[0113] The synthetic approach involved a simple stepwise synthesis as outlined in Scheme 2, chosen to emphasize single modifications and functional group interconversions at each step to simplify purification of synthetic intermediates, minimize potential by-product formation, and enable scalability of the reactions.

[0114] [ka]

[0115] The synthesis of bifunctional H4noneunpaX-Bn-NCS (13) proceeded via a similar route to H4noneunpaX utilizing the same synthetic intermediates (compounds (1)-(4)) to provide primary amine (4), which was protected using 2-nitrobenzenesulfonyl chloride to give the corresponding nosyl-protected sulfonamide (7) in high yield. N-Alkylation of sulfonamide (7) with methyl(6-bromomethyl)picolinate under basic conditions was achieved by gentle heating of the reaction mixture overnight to produce the single alkylation product in high yield without difficulty. Compound (8) was treated with thiophenol to remove the nosyl protecting group to give the corresponding secondary amine (9). An excess of thiophenol (3 equivalents) was found to be necessary to achieve complete deprotection of compound (8), thus avoiding tedious purification of the desired product. Subsequent N-alkylation of secondary amines (9) using structurally modified derivatives of the protected picolinic acid electrophile (S1) was achieved by gentle heating overnight to give protected bifunctional ligands (10) bearing alkyne adducts suitable for click-based bioconjugation. The alkyne-derivatized picolinate electrophile (S1) was prepared separately starting from chelidamic acid as shown in Scheme 3. The alkyne (10) was subsequently reacted with a bifunctional azide linker (S2) using Husigen's 1,3-dipolar cycloaddition (click chemistry) using CuSO4·5H2O as an in situ source of Cu(I) catalyst and sodium ascorbate as the reducing agent. The synthesis of the bifunctional azide linker (S2) is also outlined in Scheme 4. Compound (11) was first treated with excess sodium sulfide to precipitate traces of Cu(I / II) chelated by the protecting ligand, followed by deprotection of the methyl, tert-butyl, and Boc protecting groups using 3M HCl. Purification of H4noneunpaX-Bn-NH2 (12) was achieved by RP-HPLC, and the product was isolated as the corresponding HCl salt by coevaporation with 3M HCl. Thiophosgene 37Aniline (12) was converted to isothiocyanate (13) using a standardized literature approach with the product isolated by RP-HPLC.

[0116] [ka]

[0117] Di-tert-butyl 2,2'-((2-(2-((2-nitrophenyl)sulfonamido)ethoxy)ethyl)azanediyl)diacetate (7). Di-tert-butyl 2,2'-((2-(2-aminoethoxy)ethyl)azanediyl)diacetate (4) (2.35 g, 7.16 mmol) was dissolved in dry CHCl (40 mL) and cooled to 0 °C. Triethylamine (2.00 mL, 1.45 g, 14.3 mmol, 2 equiv.) was added to the reaction mixture followed by the slow addition of 2-nitrobenzenesulfonyl chloride (1.59 g, 7.16 mmol). The resulting pale yellow solution was stirred at 0 °C for 1 h, then allowed to warm to room temperature and stirred for an additional 5 h. Upon completion, the reaction mixture was diluted with CHCl (40 mL) and extracted with deionized water (2 x 50 mL) and brine (50 mL). The organic phase was evaporated in vacuo and the resulting residue was purified by silica gel chromatography (Combiflash automated purification system; A: hexanes, B: EtOAc, 100% A to 40% B) to give the title compound as a pale yellow oil (3.51 g, 96%). 1 H NMR (400 MHz, CDCl3, 298 K) 8.14-8.12 (1H, m, 11-CH), 7.85-7.82 (1H, m, 9-CH), 7.75-7.70 (2H, m, 8- and 11-CH), 6.13 (1H, t, 3 J = 5.6 Hz, 7-NH), 3.54-3.49 (4H, m, 4- and 5-CH2), 3.45 (4H, s, 2-CH2), 3.28 (2H, q, 3 J=5.5Hz,6-CH2), 2.87(2H,t, 3 J=5.6Hz,3-CH2), 1.45(18H,s,1-C(CH3)3). 13 C{ 1H}NMR(100MHz,CDCl3,298K)170.7(3-C), 148.1(14-C), 134.0(11-C), 133.4(9-C), 132.6(12-C), 130.9( 13-C), 125.2(10-C), 81.1(2-C), 70.1(6-C), 68.9(7-C), 56.7(4-C), 53.2(5-C), 43.7(8-C), 28.2(1-C). ESI-MS(MeOH)518.7[M+H] + .R f = 0.50 (hexane / EtOAc; 2:1).

[0118] Di-tert-butyl-2,2'-((2-(2-((N-((6-(methoxycarbonyl)pyridin-2-yl)methyl)-2-nitrophenyl)sulfonamido)ethoxy)ethyl)azanediyl)diacetate (8): Methyl (6-bromomethyl)picolinate (884 mg, 3.84 mmol) was added to a solution of di-tert-butyl 2,2'-((2-(2-((2-nitrophenyl)sulfonamido)ethoxy)ethyl)azanediyl)diacetate (7) (1.98 g, 3.84 mmol) in dry MeCN (60 mL). The solution was stirred at room temperature for 15 min, after which K2CO3 (1.06 g, 7.68 mmol, 2.0 equiv) was added and the resulting suspension was heated at 50 °C overnight. After completion, inorganic salts were separated by centrifugation, washed with CHCl (3×10 mL) and the combined organic phases were evaporated in vacuum. The resulting residue was redissolved in CHCl (75 mL), washed with deionized water (3×75 mL) and brine (75 mL) and dried over NaSO. The organic phase was evaporated in vacuum and the resulting residue was purified by silica gel chromatography (Combiflash automated purification system; A: CHCl, B: MeOH; 100% A to 5% B). The title product was obtained as a pale yellow oil (2.26 g, 88%). 1 H NMR(400MHz,CDCl3,298K)8.13-8.1(1H,m,11-CH),7.98(1H,d, 3 J=7.1Hz,14-CH),7.81(1H,t, 3 J=7.1Hz,13-CH), 7.68(1H,d, 3J=7.1Hz,12-CH)、7.66-7.62(3H,m,8-,9-および10-CH)、4.83(2H,s,7-CH2)、3.95(3H,s,15-CH3)、3.54(2H,t, 3 J=5.3Hz,6-CH2)、3.47(2H,t, 3 J=5.3Hz,5-CH2)、3.37(4H,s,2-CH2)、3.35(2H,t, 3 J=6.0Hz,4-CH2)、2.71(2H, 3 J=6.0Hz,3-CH2)、1.41(18H,s,1-C(CH3)3)。 13 C{ 1 H}NMR(100MHz,CDCl3,298K)170.6(3-C)、165.4(21-C)、157.4(16-C)、148.1(20-C)、147.4(14-C)、137.9(18-C)、133.5(11-C)、133.4(9-C)、131.8(12-C)、131.0(13-C)、125.4(17-C)、124.3(10-C)、124.0(19-C)、80.9(2-C)、70.0(6-C)、68.6(7-C)、56.5(4-C)、53.9(15-C)、53.3(5-C)、52.8(22-C)、48.1(8-C)、28.1(1-C)。ESI-MS(MeOH)667.3[M+H] + 。R f =0.30(CH2Cl2 / MeOH;99:1)。

[0119] Di-tert-butyl 2,2'-((2-(2-(((6-(methoxycarbonyl)pyridin-2-yl)methyl)amino)ethoxy)ethyl)azanediyl)diacetate (9). Thiophenol (1.05 mL, 10.2 mmol, 3.0 equiv) was added to a suspension of di-tert-butyl 2,2'-((2-(2-((N-((6-(methoxycarbonyl)pyridin-2-yl)methyl)-2-nitrophenyl)sulfonamido)ethoxy)ethyl)azanediyl)diacetate (8) (2.26 g, 3.39 mmol) and K2CO3 (937 mg, 6.78 mmol, 2.0 equiv) in dry MeCN (60 mL). The reaction mixture was heated to 50 °C and stirred for 2 h. After completion, inorganic salts were separated by centrifugation, washed with CHCl (3×10 mL) and the combined organic phases were evaporated in vacuum. The resulting residue was redissolved in CHCl (50 mL) and washed with deionized water (2×50 mL) and brine (50 mL). The organic phase was evaporated in vacuum and the resulting oil was purified by silica gel chromatography (Combiflash automated purification system; A: CHCl, B: MeOH; 100% A to 10% B) to give the title product as a pale yellow oil (1.61 g, 98%). 1 H NMR(400MHz,CDCl3,298K)8.00(1H,d, 3 J=7.4Hz,11-CH), 7.81(1H,t, 3 J=7.4Hz,10-CH), 7.66(1H,d, 3 J=7.4Hz,9-CH), 4.05(2H,s,8-CH2), 3.99(3H,s,12-CH3), 3.61-3.56(4H,m,4- and 5-CH2), 3.49(4H,s,2-CH2), 2.94(2H,t, 3 J=5.8Hz,6-CH2), 2.83(2H,t, 3 J=5.2Hz,3-CH2), 2.26(1H,br s,7-NH), 1.44(18H,s,1-C(CH3)3). 13 C{ 1H}NMR(100MHz,CDCl3,298K)170.8(3-C), 165.8(15-C), 160.5(10-C), 147.4(14-C), 137.5(12-C), 125.6(11-C), 123. 5(13-C), 80.9(2-C), 70.1(6-C), 70.0(7-C), 56.6(4-C), 54.9(9-C), 53.4(5-C), 52.8(16-C), 48.8(8-C), 28.1(1-C). ESI-MS(MeOH)482.2[M+H] + .R f = 0.20 (CH2Cl2 / MeOH; 9:1).

[0120] ( t Bu)2(Me)2noneunpaX(OCHCCH) (10). Methyl 6-(bromomethyl)-4-(prop-2-yn-1-yloxy)picolinate (122 mg, 0.415 mmol) was added to a suspension of di-tert-butyl 2,2'-((2-(2-(((6-(methoxycarbonyl)pyridin-2-yl)methyl)amino)ethoxy)ethyl)azanediyl)diacetate (200 mg, 0.415 mmol) and K2CO3 (115 mg, 0.830 mmol) in dry MeCN (10 mL). The reaction mixture was heated to 50 °C and stirred overnight. After completion, the reaction mixture was cooled to room temperature and the inorganic salts were removed by centrifugation. The separated salts were washed with CHCl2 (3 × 10 mL) and the combined organic phase was evaporated in vacuum. The resulting residue was redissolved in CH2Cl2 (25 mL) and washed with deionized water (2 x 25 mL) and brine (25 mL). The volatiles were removed in vacuo and the crude product was purified by silica gel chromatography (Combiflash automated purification system; A: CH2Cl2, B: MeOH; 100% A to 5% B). The title product was obtained as a pale yellow oil (249 mg, 88%). 1 H NMR(400MHz,CDCl3,298K)7.98(1H,dd, 3 J=7.5Hz, 4 J=0.9Hz,10-CH), 7.88(1H,d, 3 J=7.5Hz,8-CH), 7.78(1H,t, 3J = 7.5 Hz, 9-CH), 7.59 (1H, d, 4 J = 2.5 Hz, 14-CH), 7.52 (1H, br d, 4 J = 2.5 Hz, 13-CH), 4.81 (2H, d, 4 J = 2.4 Hz, 16-CH2) 4.01 (2H, br s, 7-CH2), 3.98 (3H, s, 11-CH3), 3.97 (5H, br m, 12-CH2 and 15-CH3), 3.57 (2H, t, 3 J = 5.5 Hz, 4-CH2), 3.52 (2H, t, 3 J = 5.9 Hz, 5-CH2), 3.45 (4H, s, 2-CH2), 2.91 (2H, t, 3 J = 5.9 Hz, 6-CH2), 2.81 (2H, t, 3 J = 5.5 Hz, 3-CH2), 2.60 (1H, t, 4 J = 2.4 Hz, 17-CH), 1.42 (18H, s, 1-C(CH3)3). 13 C{ 1 H} NMR (100 MHz, CDCl3, 298 K) 170.7 (3-C), 165.8 (15-C), 165.7 (23-C), 165.1 (20-C), 162.6 (10-C), 160.6 (18-C), 148.9 (22-C) 147.3 (14-C), 137.4 (12-C), 126.1 (11-C), 123.6 (13-C), 111.8 (19-C), 111.0 (21-C), 80.8 (2-C), 77.1 (26-C), 76.9 (27-C), 70.4 (6-C), 69.3 (7-C), 60.8 (9-C), 60.6 (17-C), 56.6 (4-C), 55.9 (25-C), 54.0 (8-C), 53.5 (5-C), 53.0 (16-C), 52.9 (24-C), 28.1 (1-C). ESI-MS (MeOH) 685.3 [M + H] + .

[0121] ( t (Bu)2(Me)2noneunpaX(Bn-NHBoc) (11). A solution of Cu(OAc)2·H2O (66 mg, 0.329 mmol, 1.0 equiv) in deionized water (1 mL) was added to t BuOH (1 mL) containing tA mixture containing 1,2-Bu)2(Me)2noneunpaX(OCH2CCH) (225 mg, 0.329 mmol) and tert-butyl(4-(2-azidoethyl)phenyl)-carbamate (86 mg, 0.329 mmol) was added. Sodium ascorbate (65 mg, 0.329 mmol, 1.0 equiv.) was added to the reaction mixture, which gradually darkened over 10 min to give a red-brown solution. The solution was warmed to 40 °C and stirred for 48 h. Upon completion, the dark brown mixture was treated with a solution of Na2S·9H2O (790 mg, 3.29 mmol, 10 equiv.) dissolved in deionized water (4 mL). Black CuS precipitated immediately after addition and the suspension was stirred for an additional hour at room temperature. The precipitate was separated by centrifugation and the supernatant was collected and evaporated under reduced pressure to remove excess t BuOH was removed. The resulting aqueous phase was diluted with deionized water (10 mL) and extracted with CH2Cl2 (3 x 15 mL). The combined organic phase was washed with deionized water (10 mL) and brine (10 mL). Volatiles were removed in vacuo and the crude material was purified by silica gel chromatography (Combiflash automated purification system; A: CH2Cl2, B: MeOH; 100% A to 10% B) to give the title compound as a white foamy solid (211 mg, 76%). 1 H NMR(400MHz,CDCl3,298K)8.00(1H,d, 3 J=7.5Hz,10-CH), 7.90(1H,d, 3 J=7.5Hz,18-CH), 7.86(1H,t, 3 J=7.5Hz,9-CH), 7.60(1H,d, 4 J=2.2Hz,14-CH), 7.53(1H,d, 4 J=2.2Hz,13-CH), 7.45(1H,s,17-CH), 7.27(2H,d, 3 J=8.2Hz,21-CH), 6.98(2H,d, 3 J=8.2Hz,20-CH), 6.66(1H,br s,22-NH), 5.28(2H,s,25-CH2), 4.59(2H,t, 3J=7.1Hz,18-CH2), 4.02(2H,s,12-CH2), 3.99(3H,s,11-CH3), 3.98(3H,s,15-CH3), 3.96(2H,s,7-CH2), 3.58(2H,t, 3 J=5.6Hz, 4-CH2), 3.53(2H,t, 3 J=5.8Hz,5-CH2), 3.46(4H,s,4-CH2), 3.17(2H,t, 3 J=7.1Hz,19-CH2), 2.91(2H,t, 3 J=5.8Hz,6-CH2), 2.81(2H,t, 3 J=5.6Hz,3-CH2), 1.52(9H,s,23-C(CH3)3), 1.43(18H,s,1-C(CH3)3). ESI-MS(MeOH)947.5[M+H] + .

[0122] H4noneunpaX-Bn-NH2(12). t Bu)2(Me)2noneunpaX(Bn-NHBoc) (200 mg, 0.211 mmol) was dissolved in 4 M HCl (3 mL) and heated at 60 °C overnight. Upon completion, the volatiles were evaporated in vacuo and the resulting residue was purified by RP-HPLC (A: HO (0.1% TFA), B: MeCN; 95% A to 30% B over 40 min, R t = 20.3–22.3 min). The purified H4noneunpaX-triazole-Bn-NH2 was coevaporated with 3 M HCl (3 × 2 mL) to give the corresponding HCl salt as a white solid (120 mg, 81%). 1 H NMR (400 MHz, DO, 298 K) 8.03 (1H, s, 14-CH), 7.94-7.83 (2H, m, 8- and 9-CH), 7.62 (1H, dd, 3 J=6.3Hz, 4 J=2.0Hz,7-CH), 7.58(1H,d, 4 J=2.7Hz,12-CH), 7.24(1H,d, 4 J=2.7Hz,11-CH), 7.22(2H,d, 3 J=7.4Hz,17-CH), 7.15(2H,d, 3J=7.4Hz,18-CH), 5.29(2H,s,13-CH2), 4.74-4.65(6H,m,6-,10- and 15-CH2), 4.22(4H,s,1-CH2), 3. 97(2H,t,4-CH2), 3.89(2H,t,3-CH2), 3.76(2H,t,5-CH2), 3.66(2H,t,2-CH2), 3.23(2H,t,16-CH2). 13 C{ 1 H}NMR(100MHz,D2O,298K)168.1(17-C), 168.0(1-C), 166.4(13-C), 164.7(20-C), 150.4(8-C), 150.1(1 5-C), 147.3(12-C), 146.0(19-C), 140.9(22-C), 140.7(10-C), 138.5(29-C), 130.4(27-C), 128.5(26-C) , 128.4(9-C), 126.2(23-C), 125.5(11-C), 123.3(28-C), 115.8(16-C), 112.8(18-C), 65.4(5-C), 64.4(4) -C), 62.1(21-C), 58.3(7-C), 57.7(14-C), 56.4(3-C), 56.2(6-C), 55.6(2-C), 51.9(24-C), 35.2(25-C). LR-ESI-MS(H2O)707.6[M+H] + .

[0123] H4noneunpaX-Bn-NCS (13). Thiophosgene (120 μL, 1.56 mmol) in CHCl3 (660 μL) was added to a solution of H4noneunpaX-Bn-NH2·4HCl (88 mg, 0.103 mmol) in deionized water (1 mL). The biphasic mixture was vigorously stirred overnight at room temperature in the dark, after which the two immiscible layers were allowed to separate. The aqueous phase was collected and washed with CHCl3 (2 × 1 mL), then lyophilized to give the crude isothiocyanate product. The off-white residue was purified by semi-preparative RP-HPLC (A: HO (0.1% TFA), B: MeCN (0.1% TFA)): 0–6 min (95% A to 24% B); 6–30 min (24% B to 39% B); 30–35 min (39% B to 100% B); R t= 25.4 min. Appropriate fractions were pooled and lyophilized to give the purified product as an off-white foamy solid (67 mg, 87%). ESI-MS (HO / MeCN) 749.3 [M+H] + , 747.3[MH] - .

[0124] Dimethyl-4-hydroxypyridine-2,6-dicarboxylate (S3). Chelidamic acid monohydrate (3.00 g, 14.9 mmol) was suspended in dry MeOH (50 mL) and H2SO4 (0.5 mL) was added. The reaction mixture was heated at 50° C. overnight, then cooled to room temperature and the volatiles were removed in vacuo. The crude residue was dissolved in saturated NaHCO3 solution (150 mL) and extracted with EtOAc (3×150 mL). The combined organic phases were evaporated in vacuo to give the title compound as a white solid (3.02 g, 95%). 1 H NMR (300MHz, CDCl3, 298K) 7.49 (2H,s,2-CH), 3.97 (6H,s,1-CH3). 13 C{ 1 H}NMR (75MHz, CDCl3, 298K) 187.5(5-C), 163.5(2-C), 143.1(3-C), 118.6(4-C), 53.7(1-C).

[0125] Dimethyl 4-(prop-2-yn-1-yloxy)pyridine-2,6-dicarboxylate (S4). Propargyl bromide (80 wt%) (1.75 mL, 15.7 mmol, 1.1 equiv) was added to a suspension of dimethyl-4-hydroxypyridine-2,6-dicarboxylate (3.02 g, 14.3 mmol) and K2CO3 (3.95 g, 28.6 mmol, 2.0 equiv) in dry DMF (50 mL). The reaction mixture was heated at 50 °C overnight, then cooled to room temperature and the salts were removed by vacuum filtration. The filtrate was evaporated in vacuo and the resulting residue was purified by silica gel chromatography (Combiflash automated purification system; A: CH2Cl2, B: MeOH; 100% A to 5% B). The title compound was obtained as a pale yellow solid (3.20 g, 90%). 1H NMR(400MHz,CDCl3,298K)7.89(2H,s,2-CH), 4.87(2H,d, 4 J=2.4Hz,3-CH2), 4.01(6H,s,1-CH3), 2.62(1H,t, 4 J = 2.4 Hz, 4-CH). 13 C{ 1 H}NMR(75MHz, CDCl3,298K) 165.7(2-C), 165.1(5-C), 150.0(3-C), 115.0(4-C), 77.7(7-C), 76.3(8-C), 56.5(6-C), 53.4(1-C).

[0126] Methyl (6-hydroxymethyl)-4-(prop-2-yn-1-yloxy)picolinate (S5). NaBH4 (190 mg, 4.92 mmol) was added in portions to a solution of compound 2 (1.22 g, 4.90 mmol) in dry MeOH / CHCl2 (40 mL, 1:1) with vigorous stirring at 0°C. The reaction mixture was stirred at 0°C for 30 min, then warmed to room temperature and stirred for an additional 3.5 h. Upon completion, the reaction solution was cooled to 0°C and quenched with deionized water (25 mL). The volatiles were removed in vacuo and the resulting aqueous phase was extracted with CHCl2 (3×30 mL). The combined organic phase was dried over NaSO4, filtered and evaporated in vacuo to give an off-white solid. The crude material was purified by silica gel chromatography (CombiFlash automated purification system; A: CH2Cl2, B: MeOH; 100% A to 5% B) to afford the title compound as a white crystalline solid (721 mg, 67%). 1 H NMR(400MHz,CDCl3,298K)7.57(1H,d, 4 J=2.4Hz,2-CH), 7.16(1H,d, 4 J=2.4Hz,5-CH), 4.79(2H,s,6-CH2), 4.78(2H,d, 4 J=3-CH2), 4.22(1H,br s,7-OH), 3.93(3H,s,1-CH3), 2.61(1H,t, 4 J = 2.4 Hz, 4-CH). 13 C{ 1H}NMR(100MHz,CDCl3,298K)165.4(2-C), 165.2(5-C)(7-C), 162.9(-C), 148.6(3-C), 1 11.2(4-C), 109.9(6-C), 77.1(10-C), 76.8(11-C), 64.7(8-C), 56.0(9-C), 53.0(1-C).

[0127] Methyl 6-(bromomethyl)-4-(prop-2-yn-1-yloxy)picolinate (S1). Phosphorus tribromide (85 mL, 245 mg, 0.905 mmol, 1.1 equiv) was added dropwise to a cooled solution of methyl 6-(hydroxymethyl)-4-(prop-2-yn-1-yloxy)picolinate (182 mg, 0.823 mmol) in dry CHCl3 (5 mL). The reaction mixture was passively warmed to room temperature and stirred for 3 h. Upon completion, the yellow reaction mixture was quenched with saturated NH4Cl (20 mL) and extracted with CHCl2 (4 x 20 mL). The combined organic phases were evaporated in vacuo to give the title compound as a white solid (230 mg, 99%). 1 H NMR(300MHz,CDCl3,298K)7.63(1H,d, 4 J=2.4Hz,2-CH), 7.22(1H,d, 4 J=2.4Hz,5-CH), 4.79(2H,d, 4 J=2.4Hz,3-CH), 4.56(2H,s,6-CH2), 3.96(3H,s,1-CH3), 2.60(1H,t, 4 J = 2.4 Hz, 4-CH). 13 C{ 1 H}NMR(75MHz,CDCl3,298K)165.4(2-C), 165.3(5-C), 159.1(7-C), 149.4(3-C), 113 .7(6-C), 111.7(4-C), 77.5(10-C), 76.5(11-C), 56.2(9-C), 53.3(1-C), 33.3(8-C). ESI-MS(MeOH)284.0.

[0128] tert-Butyl (4-(2-hydroxyethyl)phenyl)carbamate (S6). Di-tert-butyl dicarbonate (3.50 g, 16.0 mmol, 1.1 equiv) was added to a solution of 2-(4-aminophenyl)ethanol (2.01 g, 14.6 mmol) and triethylamine (2.0 mL, 1.46 g, 14.6 mmol) in dry THF (50 mL). The reaction mixture was stirred at room temperature overnight and then heated to 50° C. for an additional 5 h. Upon completion, the volatiles were removed in vacuo to give the title product as an off-white solid (3.35 g, 97%) which was used without further purification. 1 H NMR(300MHz,CDCl3,298K)7.29(2H,d,J=8.4Hz,3-CH),7.13(2H,d,J=8.4Hz,4-CH),6.49(1H,br s,2-NH), 3.80(2H,t,J=6.6Hz,6-CH2), 2.80(2H,t,J=6.6Hz,5-CH2), 1.50(9H,s,1-C(CH3)3). 13 C{ 1 H}NMR(75MHz,CDCl3,298K)153.0(3-C), 136.9(4-C), 133.2(7-C), 129.7(6-C), 119.1(5-C), 80.6(2-C), 63.8(9-C), 38.6(8-C), 28.5(1-C). ESI-MS(MeOH)260.2[M+Na] + .

[0129] tert-Butyl (4-(2-azidoethyl)phenyl)carbamate (S2). Methanesulfonyl chloride (825 mL, 1.22 g, 10.6 mmol, 1.1 equiv) was added dropwise to a solution of tert-butyl (4-(2-hydroxyethyl)phenyl)carbamate (2.29 g, 9.68 mmol) and DIPEA (1.85 mL, 1.37 g, 10.6 mmol, 1.1 equiv) in dry EtOAc (20 mL) at 0° C. The solution was stirred on ice for 10 min, then warmed to room temperature and stirred for an additional 1 h. Upon completion, the reaction mixture was cooled to 0° C., filtered, and the precipitate was washed with cold EtOAc (70 mL). The filtrate was concentrated in vacuo to afford the corresponding mesylate as a pale yellow oil (S7), which was used without further purification.1 H NMR(300MHz,CDCl3,298K)7.31(2H,d,J=8.5Hz,3-CH),7.12(2H,d,J=8.5Hz,4-CH),6.67(1H,br s,2-NH), 4.34(2H,t,J=7.1Hz,6-CH2), 2.96(2H,t,J=7.1Hz,5-CH2), 2.83(3H,s,7-CH3), 1.49(9H,s,1-C(CH3)3). 13 C{ 1 H}NMR(75MHz,CDCl3,298K)152.9(3-C), 137.5(4-C), 130.8(7-C), 129.6(6- C), 118.9(5-C), 80.5(2-C), 70.5(9-C), 37.4(10-C), 35.0(8-C), 28.4(1-C). R f =0.75 (hexanes / EtOAc; 1:1). 4-((tert-butoxycarbonyl)amino)phenethyl methanesulfonate (S7) (3.03 g, 9.66 mmol) was dissolved in dry DMF (15 mL) and NaN3 (703 mg, 10.6 mmol, 1.1 equiv) was added. The reaction mixture was heated to 90° C. and stirred overnight. Upon completion, the volatiles were removed in vacuo and the resulting residue was partitioned between deionized water (50 mL) and EtOAc (50 mL). The aqueous phase was further extracted with EtOac (2×50 mL) and the combined organic phases were evaporated in vacuo to give the title compound as a pale yellow oil (2.18 g, 86%). 1 H NMR(300MHz,CDCl3,298K)7.30(2H,d, 3 J=8.4Hz,3-CH), 7.08(2H,d, 3 J=8.4Hz,2-CH), 6.91(1H,br s,2-NH), 3.42(2H,t, 3 J=7.2Hz,5-CH2), 2.79(2H,t, 3 J=7.2Hz,6-CH2), 1.48(9H,s,1-C(CH3)3). 13 C{ 1H}NMR(75MHz, CDCl3,298K) 153.0(3-C), 137.4(4-C), 132.5(7-C), 129.3(6-C), 118.9(5-C), 80.4(2-C), 52.6(9-C), 34.8(8-C), 28.4(1-C). ESI-MS(MeOH)285.16[M+Na] + .R f = 0.95 (hexane / EtOAc; 1:1).

[0130] Example 8.0 - Radiolabeling studies of bifunctional H4noneunpaX-Bn-NH2. To evaluate the effect of bifunctionalisation via one picolinic acid donor group on radiometal ion chelation, a concentration-dependent radiolabelling study of H4noneunpaX-Bn-NH2 was carried out (Figure 31). All reactions were carried out at ambient temperature and EDTA (50 mM, pH 7.0) was used as TLC eluent. 225 Ac]Ac 3+ Except in the case of , radiochemical yields (RCYs) were determined by iTLC using SA paper plates and EDTA (50 mM, pH 5.0) as the eluent.

[0131] [ 44 Sc]Sc 3+ Radiolabeling of H4noneunpaX-Bn-NH2 with 1000 keV (400 kBq) showed comparable results to the parent chelating ligand, achieving quantitative RCC within 10 min at room temperature, thus suggesting that functionalization using this approach did not result in significant changes to the coordination environment. Without wishing to be bound by theory, it is believed that the low ligand concentrations (10 -5 The moderate improvement in RCC for M) can be attributed to the bifunctional adduct imposing a degree of preorganization on the binding cavity, thereby favoring metal complexation under these conditions.

[0132] [ 177 Lu]Lu 3+ Radiolabeling studies of H4noneunpaX-Bn-NH2 with 1,2-dichlorophenyl ether also showed results comparable to the unmodified chelator;-3 ~10 -6 Quantitative RCY was achieved across the M range. 111 In]In 3+ , [ 177 Lu]Lu 3+ , [ 133 / 135 La]La 3+ , [ 155 Tb]Tb 3+ , and [ 225 Ac]Ac 3+ Further screening of the radiolabeling properties of H4noneunpaX-Bn-NH2 with 10 rH2O2 showed results comparable to the unmodified chelator, and quantitative RCC was performed with each respective radioactive metal ion at 10 -6 This was achieved at a ligand concentration of M. Notably, the amount of radioactivity used for assessment of RCC with each radionuclide corresponds to a similar molar equivalent of the radioactive metal ion, thereby allowing a more direct comparison of the concentration dependencies with the different radionuclides.

[0133] moreover,[ 177 Lu][Lu(noneunpaX-Bn-NH2)] - The maximum molar activity of 20 MBq of [ 177 Lu]Lu 3+ was used to determine the minimum amount of chelating compound required to achieve quantitative RCY (Figure 32, left panel). 177 Lu]Lu 3+ Quantitative radiolabeling of (20 MBq) was achieved using 80 pmol of H4noneunpa-Bn-NH2, corresponding to a molar activity of 250 GBq / μmol and a ligand-to-metal ratio of 174:1. 177 Lu][Lu(noneunpaX-Bn-NH2)] - Human serum stability studies of showed no transchelation of bound radioactivity over a period of 7 days, thereby confirming that modification of the pendant donor arm does not affect the overall stability of the metal complex (Figure 32 right panel).

[0134] Example 9.0 - Bioconjugate Studies As a proof of concept to evaluate the suitability of H4noneunpaX for in vivo applications, two structural analogs of H4noneunpaX-Bn-NCS were synthesized by covalently coupling the SSTR-2 targeting peptide Tyr 3 -Octoleotate (Tyr 3 The Tyr-TATE was prepared and evaluated in mice bearing AR42J exocrine / pancreatic tumor xenografts. 3 -TATE has been a promising candidate due to its ubiquitous use for targeting neuroendocrine tumors (NETs) and because of its compatibility with existing clinically applied radiotracers, i.e. 177 was chosen as a suitable model targeting vector to allow direct comparison with [Lu][Lu(DOTATATE)] 15 .

[0135] H4noneunpaX-Ahx / PEG2-Tyr 3 -Synthesis of TATE. Linear resin-bound Tyr 3 The synthesis of -octreotate peptide was carried out according to Noor et al. 38 A standardized Fmoc-based semi-automated peptide solid phase synthesis (SPPS) was performed on preloaded Wang resin using an approach similar to that reported by. The resin-bound linear peptide was prepared according to the sequence: [DPhe-Cys(Acm)-Tyr(tBu)-DTrp(tBu)-Lys(Boc)-Thr(tBu)-Cys(Acm)-Thr(tBu)-OH] (Scheme 5). After generation of the resin-bound octapeptide sequence, N α -Fmoc-Ahx-COH or N α -Fmoc-PEG-COH was incorporated as a covalent linker to give the Fmoc-protected linear peptides (14) and (15), respectively. Cyclization of the linear peptides was achieved by treatment with iodine in DMF, which successively removed the acetamidomethyl (Acm) protecting group and afforded the Cys 2 and Cys 7Global deprotection and cleavage from the resin was performed using a standardized TFA cleavage cocktail to afford the cyclized Fmoc-protected peptides (16) and (17) (Fmoc-Ahx-Tyr). 3 -TATE(16) and Fmoc-PEG2-Tyr 3 Fmoc-Ahx-Tyr-TATE (17) was obtained, which was isolated by RP-HPLC. ESI-MS (HO / MeCN): 3 -TATE 1384.4[M+H] + , 1382.7[MH] - ;Fmoc-PEG2-Tyr 3 -TATE 1416.8[M+H] + , 1414.7[MH] - .

[0136] To allow selective functionalization of the N-terminus of each peptide, Lys 5 The side chain primary amine group on the above was first protected using di-tert-butyl dicarbonate, followed by N-protection with 20% piperidine in DMF. α -Fmoc cleavage gave compounds (18) and (19). This approach uses Tyr as a chelator prior to the incorporation of the bifunctional chelator. 3 -octreotate peptide was chosen to maximize its purity. ESI-MS (HO / MeCN): H2N-Ahx-Boc(Lys 5 )-Tyr 3 -TATE(18)1262.9[M+H] + , 1260.8[MH] - ;H2N-PEG2-Boc(Lys 5 )-Tyr 3 -TATE(19)1294.8[M+H] + , 1292.7[MH] - .

[0137] Conjugation of H4noneunpaX-Bn-NCS to the free N-termini of (18) and (19) was achieved under mildly basic conditions in solution to afford N(Boc)-Lys 5Final cleavage of the protecting groups afforded the corresponding chelate-peptide bioconjugates (20) and (21). 3 -TATE(20) and H4noneunpaX-PEG2-Tyr 3 -TATE (21) was purified by RP-HPLC and mass spectrometry (ESI / MALDI) was performed to confirm isolation of the desired product.

[0138] [ka]

[0139] H4noneunpaX-Ahx-Tyr 3 Synthesis of -TATE More specifically, H4noneunpaX-Bn-NCS (3.7 mg, 4.95 μmol) was dissolved in H2N-Ahx-Boc(Lys) in dry DMF (1 mL). 6 )-Tyr 3 A solution of 1,2-dichloro-2,4-trimethylphenylacetate (6.3 mg, 4.95 μmol) was added to the solution. The solution was stirred at room temperature for 10 min, then DIPEA (10 μL, 57 μmol) was added. The resulting mixture was stirred at room temperature overnight, then treated with 20% TFA in CHCl (1 mL) and stirred for an additional 2 h. After completion, the volatiles were evaporated under a stream of N gas, and the resulting residue was diluted with H0 (0.1% TFA). The crude bioconjugate was purified by semi-preparative RP-HPLC (A: H0 (0.1% TFA), B: MeCN (0.1% TFA) (3 mL / min); Method: 0-6 min (95% A to 29% B); 6-25 min (ISO29% B); 25-30 min (29% B to 100% B); R t = 22.6 min. Appropriate fractions were pooled and lyophilized to give H4noneunpaX-Ahx-Tyr 3 -TATE (20) was obtained as a white solid. ESI-MS (HO / MeCN (1:1)) 1911.9 [M+H] + .

[0140] H4noneunpaX-PEG2-Tyr 3 -Synthesis of TATE. H4noneunpaX-Bn-NCS (2.3 mg, 3.07 μmol) was dissolved in dry DMF (500 μL) and diluted with H2N-PEG2-Boc(Lys 6 )-Tyr 3 A solution of 1,2-dichloro-2,4-trimethylphenylacetate (1.0 mg, 3.07 μmol) was added to the solution. The solution was stirred at room temperature for 10 min, then DIPEA (10 μL, 57 μmol) was added. The resulting mixture was stirred at room temperature overnight, then treated with 20% TFA in CHCl (1 mL) and stirred for an additional 2 h. After completion, the volatiles were evaporated under a stream of N gas, and the resulting residue was diluted with H0 (0.1% TFA). The crude bioconjugate was purified by semi-preparative RP-HPLC (A: H0 (0.1% TFA), B: MeCN (0.1% TFA) (3 mL / min); Method: 0–6 min (95% A to 29% B); 6–25 min (ISO29% B); 25–30 min (29% B to 100% B); R t = 19.1 min. Appropriate fractions were pooled and lyophilized to give H4noneunpaX-PEG2-Tyr 3 -TATE (21) was obtained as a white solid. ESI-MS (HO / MeCN (1:1)) 1943.3 [M+H] + , 972.7[M+2H] 2+ .

[0141] Radiolabeling studies of chelator-bioconjugates As shown in Figure 33, 155 Tb]Tb 3+ and 255 Ac]Ac 3+ Using H4noneunpaX-Ahx-Tyr 3 -TATE and H4noneunpaX-PEG2-Tyr 3 Concentration-dependent radiolabeling of chelate-TATE was evaluated. Each of the chelator-bioconjugates was radiolabeled at 10 ng / mL for 10 min at ambient temperature. -5Quantitative RCC was achieved at concentrations of 1000 M. The decrease in RCC at lower concentrations compared to the free bifunctional chelator is typical for peptide-based bioconjugates and is due to the steric influence of the targeting vector on the metal binding cavity in addition to the lower solubility of the construct in aqueous solution and therefore the availability of metal ion coordination. Evaluation of serum stability for each of the radiolabeled bioconjugates showed excellent stability over the course of the study, with no significant change in radiochemical purity from the initial time point.

[0142] For concentration-dependent radiolabeling studies, the following general protocol was applied for radiolabeling with different radioactive metal ions. Stock solutions of each respective chelating ligand were prepared at 1 × 10 -2 The antibodies were prepared in ultrapure deionized water at a concentration of 10 M. Prior to radiolabeling studies, the antibodies were diluted in 10 mL of ...% ethanol. -3 ~10 -6 A serial dilution series of each chelator was prepared over a concentration range of 10 M. Aliquots (10 μL) of each stock solution were added to NaOAc (0.1 M), NHOAc (0.5 or 1.0 M), or MES buffer (1.0 M) (90 μL) to obtain reaction solutions appropriate for the study. Aliquots (1–10 μL) of each respective radionuclide were added under the following conditions: [ 44 Sc]Sc 3+ (1.2MBq), [ 111 In]In 3+ (1.0MBq), [ 132 / 135 La]La 3+ (400 kBq), [ 155 Tb]Tb 3+ (40 kBq), [ 177 Lu]Lu 3+ (150 kBq), [ 213 Bi]Bi 3+ (680 kBq), [ 225 Ac]Ac 3+(40 kBq). The reaction was carried out at room temperature and monitored after 5 min and within 10 min of generator elution. 213 Bi]Bi 3+ Reactions with DOTA were carried out at elevated temperatures (85-90°C) and monitored for 30-60 min. Radiochemical yields (RCYs) were determined by iTLC using SA-paper plates and EDTA (50 mM, pH 5.0 or 7.0) as the eluent (n=4). 213 Bi]Bi 3+ In this case, the RCY uses a high-purity germanium (HPGe) detector, 213 This was further confirmed through gamma spectrometry by analysis of baseline and solvent front TLC peaks by monitoring the 440 keV gamma emission of Bi. In all studies, separate control reactions were performed in parallel by addition of radioactivity to a solution containing buffer (90 μL) and deionized water (10 μL).

[0143] Human serum stability studies were performed by adding each radiolabeled complex or bioconjugate (100 µL) to a vial containing human serum albumin (100 µL) and incubating the resulting solution at 37 °C for 5-7 days. Radiochemical purity (RCP) was determined by iTLC using SA-paper plates and EDTA (50 mM, pH 5.5 or 7.0) as the eluent, whereby transchelated radioactivity was measured at the solvent front (R f = 1.0), whereas the inactive metal complexes migrate with the baseline (R f = 0). All studies were performed in triplicate and the mean %RCP was used to evaluate each compound.

[0144] [ 177 A dose escalation study of [Lu][Lu(noneunpaX-Bn-NH2)] in NHOAc buffer (40 μL, 0.5 M, pH 6.0) was performed. 177 [Lu]LuCl3 (20 MBq) to a solution containing H4noneunpaX-Bn-NH2 (2–8 μL, 10 -4The reaction was carried out by sequential addition of 100 μM, 20–80 pmol of EDTA (50 mM, pH 5.5) at room temperature for 10 min between additions. After that, 1 μL of the solution was spotted onto an SA-paper TLC plate and the RCY was determined by developing with EDTA (50 mM, pH 5.5).

[0145] LogD 7.4 measurement Prior to investigation of the radiotracer performance in vivo, the lipophilicity of the radiolabeled chelator-bioconjugates was determined by measuring the partition coefficient between n-octanol and PBS (0.01 M, pH 7.4) (Table 5). All four radiolabeled tracers were moderately hydrophilic, with logD 7.4 The values ​​ranged from -1.933 to -2.585, which is the SSTR2 antagonist analogue of DOTATATE [ 161 Tb][Tb(DOTA-LM3)](logD 7.4 =-2.5±0.1) reported 39 As expected, the higher lipophilicity of the aliphatic hexyl covalent linker is comparable to logD 7.4 Reflected in the measurements, both Ahx-Tyr 3 -TATE tracer is PEG2-Tyr 3 It is noteworthy that [ 155 Tb]Tb 3+ and[ 225 Ac]Ac 3+ The exchange between the two bioconjugates was logD 7.4 The results showed that the theranostic pair had no significant effect on the α-aminobutyric acid (ABA) values, which may indicate comparable biodistribution profiles in vivo using this theranostic pair.

[0146] [ 155 Tb]Tb 3+ or[ 225 Ac]Ac 3+ Radiolabeled H4noneunpaX-Ahx-Tyr 3 -TATE or H4noneunpaX-PEG2-Tyr 3An aliquot of -TATE (10 μL) was added to a biphasic mixture of n-octanol (700 μL) and PBS (700 μL, pH 7.4). The mixture was vortexed for 2 min at room temperature and then separated by centrifugation (10 min, 3000 RPM). Aliquots of n-octanol (100 μL) and PBS (100 μL) were collected and the activity in each portion was determined by gamma spectroscopy. LogD 7.4 Measurements were performed in 5-6 replicates per radioactive tracer. 7.4 log 10 It is defined as [(n-octanol phase) / (buffer phase)].

[0147] [Table 5]

[0148] In vivo SPECT / CT and biodistribution studies Dynamic SPECT / CT scans of mice bearing AR42J tumor xenografts were acquired over a 1-hour period to determine whether both [ 155 The pharmacokinetic profiles of the [Tb]Tb-labeled radiotracers were evaluated (Figure 37). Additional static SPECT / CT images were recorded at 2 and 4 hours post-administration, and standard uptake values ​​(SUVs) for regions of interest (ROIs) were extracted from the quantitative image scans to generate time-activity curves for each radiotracer (Figures 38 and 39).

[0149] In both cases, 155 Tb]Tb 3+ The labeled radiotracers show rapid clearance from the blood circulation over the first hour after administration, with tumor uptake beginning within the first 5 minutes p.i. Both tracers follow the typical pharmacokinetic profile observed for hydrophilic octreotate-based bioconjugates, with fast clearance and accumulation in the kidney and bladder. 40、41 In comparison, both tracers showed very similar overall distributions over the course of the study, with 155Tb][Tb(noneunpaX-PEG2-Tyr 3 -TATE)] is 155 Tb][Tb(noneunpaX-Ahx-Tyr 3 Compared to 100-TATE), 100-TATE showed slightly faster accumulation in AR42J tumor xenografts with faster clearance over time. This observation is clearly in line with the expected trend and explains the difference in lipophilicity between these bioconjugates. Notably, good contrast in the tumor area was seen after 45 min pi, with maximum uptake peaking at 2 h pi.

[0150] Clearance of both radiotracers occurs primarily via the renal pathway, as observed by high uptake in the kidney and bladder, and low uptake in the liver. SPECT / CT images show further clearance via the bile duct, whereby increased activity is seen in the gallbladder beginning at 2 hours pi, with uptake in the small intestine (ileum, duodenum) at later time points. 177 Lu][Lu(DOTATATE)] 40 In contrast to the reabsorption of radioactive tracers in the gastrointestinal tract (enterohepatic circulation), which is common for drugs that exhibit clearance in the bile, 42 Due to this, this additional release pathway may explain the sustained tumor uptake over 1-2 h. Evaluation of the time-activity curves showed that the differences in hydrophilicity of each tracer were clearly reflected in the pharmacokinetic profiles; 155 Tb][Tb(noneunpaX-PEG2-Tyr 3 -TATE)] shows higher renal excretion, whereas [ 155 Tb][Tb(noneunpaX-Ahx-Tyr 3 -TATE)] indicates greater gallbladder uptake.

[0151] SSTR2 is expressed at low levels in normal healthy tissues, including the pancreas, lung, stomach, adrenal gland, and kidney, which are clearly visualized in the SPECT / CT images and biodistribution data determined for each tracer (Figure 40). However, 177 Lu][Lu(DOTATATE)] [pancreas (10.9%ID / g), lung (17.4%ID / g), and adrenal gland (8.95%ID / g)] 40 Compared to [ 155 Tb][Tb(noneunpaX-Ahx-Tyr 3 -TATE)] and [ 155 Tb][Tb(noneunpaX-PEG2-Tyr 3 For α-TATE), significantly lower accumulation was observed in the pancreas (2.39±0.18%ID / g and 2.43±0.68%ID / g), lungs (6.93±1.60%ID / g and 7.91±0.47%ID / g), and adrenal glands (4.84±1.11%ID / g and 6.28±1.12%ID / g) 4 hours after injection. 177 Lu][Lu(DOTATATE)](6.31%ID / g) 40 A higher uptake in the kidney (19.3 ± 2.78% ID / g and 20.5 ± 3.57% ID / g) was observed compared with [ 155 Tb][Tb(noneunpaX-Ahx-Tyr 3 Biodistribution studies at 2 hours pi for (-TATE) and time-activity curves for both tracers indicate better clearance than retention in the kidney.

[0152] The relatively low uptake in AR42J tumors at 4 hours post-administration (11.0±2.62%ID / g and 8.51±0.90%ID / g) is primarily a result of the large tumor size used in these studies. As can be visualized from the SPECT / CT images, the tumors appear heterogeneous, with areas of necrotic tissue and no uptake of the radiotracer; therefore, the %ID / g in the tumors is likely lower than achievable. However, the primary goal of these studies was to evaluate the in vivo suitability of H4noneunpaX as a novel bifunctional chelating ligand. Overall, the imaging and biodistribution studies show good initial results for this chelator, with no evidence of in vivo degradation or release of binding activity (seen as uptake in bone over time). Furthermore, both Tyr 3 -TATE analogs are 177 Compared with [Lu][Lu(DOTATATE)], it showed improved pharmacokinetics with lower non-target tissue accumulation and is of interest for further development and evaluation in vivo.

[0153] For the aforementioned study, 155 Tb][Tb(nonenunpaX-Ahx-Tyr 3 -TATE)] and [ 155 Tb][Tb(nonenunpaX-PEG2-Tyr 3 -TATE)] were prepared at high molar activities (23.6 MBq / nmol and 22.5 MBq / nmol, respectively) suitable for in vivo SPECT / CT imaging and biodistribution studies. 155 Tb]Tb 3+ An aliquot of (28 MBq, 40 μL) was diluted with H4noneunpaX-Ahx-Tyr in NH4OAc buffer (10 μL, 0.5 M, pH 5.5). 3 -TATE (6 μL, 2 × 10 -3 The pH was adjusted to neutral by the addition of NaOH (1 M, 1 μL) and the resulting solution was incubated at 37 °C for 15 min to ensure quantitative incorporation of radioactivity. The same radiolabeling protocol was followed for [ 155Tb][Tb(nonenunpaX-PEG2-Tyr 3 -TATE)]. Quality control measurements were performed by iTLC measurements and radio-HPLC; Method: A: HO (0.1% TFA), B: MeCN (0.1% TFA), 100% A to 60% B; 15 min, 1 mL / min, [ 155 Tb][Tb(nonenunpaX-Ahx-Tyr 3 -TATE)](t R =9.99 minutes, 99%), [ 155 Tb][Tb(nonenunpaX-PEG2-Tyr 3 -TATE)](t R = 9.78 min, 96%) (Figures 35 and 36). A small aliquot of each radiolabeled tracer was taken for quantification of radioactivity using gamma spectroscopy. No further purification was performed prior to administration of either radiotracer. Each of the radiolabeled bioconjugates was split into two different stocks and diluted in PBS to provide appropriate doses for SPECT / CT imaging (10.2-13.5 MBq per subject) or biodistribution studies (approximately 800 kBq per subject).

[0154] Tumor implantation was performed at the BCCRC under a protocol (A20-0113) approved by the Animal Care Committee (ACC) of the University of British Columbia. Female NRG mice were anesthetized by inhalation of 2% isoflurane in 2.0 L / min oxygen and inoculated subcutaneously in the left shoulder with the AR42J exocrine pancreatic tumor cell line. After tumor growth reached approximately 8-10 mm in diameter (2-3 weeks post-inoculation), in vivo imaging and biodistribution studies were performed.

[0155] Animal studies were performed in accordance with the Canadian Council on Animal Care (CCAC) using a protocol (A20-0132) approved by the Animal Care Committee (ACC) at the University of British Columbia. Female NRG mice bearing AR42J exocrine pancreatic tumor xenografts were anesthetized with 5% isoflurane in an induction chamber and restrained in a tail vein restraining device (Braintree Scientific) under a continuous flow of 1-1.5% isoflurane. Mice were administered 100 µL of [ 155Tb][Tb(noneunpaX-Ahx-Tyr 3 -TATE (10.2MBq, 23.6MBq / nmol) or [ 155 Tb][Tb(noneunpaX-PEG2-Tyr 3 Either 100- or 150-TATE (13.5MBq, 22.5MBq / nmol) was administered via the lateral tail vein. Animal subjects were maintained at constant body temperature using a blanket on a heated bed under a continuous flow of 1.5-2% isoflurane, and respiration rate was monitored throughout the duration of each scan. Immediately after injection, whole-body dynamic SPECT / CT imaging scans were acquired over the first 60 min using a multimodal VECTor / CT system (MILabs, The Netherlands) equipped with an ultra-high sensitivity (XUHS) 2 mm pinhole collimator. Dynamic imaging consisting of 6 frames of 10 min was acquired over the first 60 min, centered on the mouse whole-body region with a 14 mm axial field of view, and then static SPECT / CT scans were recorded 2 and 4 h after injection using a single frame of 20 min acquisition. 155 Energy windows centered on the 44, 85, and 106 keV photopeaks of Tb were applied with a spectral width of 25%. For quantitative analysis, SPECT images were scanned at 0.4 mm 3 The SPECT images were reconstructed using a pixel-based ordered subset expectation-maximization (POSEM) reconstruction algorithm using a voxel size of 16 subsets (96MLEM equivalents) with 6 iterations. SPECT images were attenuation corrected and attenuation coefficients were applied based on the CT acquisition at each time point. 53 The calibration factor for radioactivity concentration (counts / voxel) is: 155The mean standard uptake value (SUV) was defined according to the following formula: SUV (g / mL) = radioactivity concentration (MBq / mL) / [administered dose (MBq) / body weight (g)]. Gaussian filtering (FWHM = 3 mm) and image rendering were performed after reconstruction for data visualization purposes only.

[0156] [ 155 Tb][Tb(noneunpaX-Ahx-Tyr 3 -TATE)] and [ 155 Tb][Tb(noneunpaX-PEG2-Tyr 3 Biodistribution studies using the 5'-TATE) were performed in NRG mice bearing AR42J exocrine pancreatic tumor xenografts. Prior to administration of each radiotracer (~800 kBq, 0.033 nmol) in PBS (~100 μL), mice were anesthetized by inhalation of 2% isoflurane and restrained using a tail vein restrainer (Braintree Scientific). Intravenous administration of each radiotracer was performed via the lateral tail vein. After injection, mice were allowed to roam freely in their cages and were sacrificed by CO2 asphyxiation under 2% isoflurane anesthesia 2 or 4 hours after injection. Immediately after sacrifice, blood was collected by cardiac puncture and organs of interest were harvested, rinsed with PBS, and blotted dry. Each organ was weighed and radioactivity was measured using a calibrated gamma counter (Packard Cobra II Auto-gamma counter, Perkin Elmer, Waltham, MA, USA) with an acquisition time of 1 min per sample. All radioactivity measurements were decay-corrected for injection time, and the injected dose per gram of tissue (%ID / g) was calculated according to literature values. 54 Calculations were based on measured organ weights, except for blood, bone, and muscle, which were scaled according to

[0157] General Methods and Materials All solvents and reagents were purchased from commercial suppliers (Sigma-Aldrich, AK Scientific, Alfa Aesar) and used directly without further purification. Analytical thin-layer chromatography (TLC) sheets were purchased from Merck (TLC silica gel 60 F254, aluminum sheets). Deionized water (18.2 MΩ / cm at 25 °C) was obtained from a PURELAB Ultra water purification system, ELGA LabWater. Flash column chromatography was performed using Siliaflash F60 silica gel (60 Å, particle size 40–63 μm, 230–400 mesh) from Silicycle Inc. Automated column chromatography was performed using a Teledyne Isco (Lincoln, NE) CombiFlash Rf autopurification system equipped with RediSep Rf Gold HP prepacked reusable silica and neutral alumina column cartridges. Low-resolution mass spectrometry (LR-MS) was performed using a Waters 2965 ZQ spectrometer equipped with an electrospray / chemical ionization (ESI / CI) source. High resolution mass spectrometry (HR-MS) was performed using a Waters Micromass LCT TOF instrument. Elemental analysis (CHN) was performed using a Thermoflash 2000 elemental analyzer. 1 H and 13 C{ 1 H}NMR spectra were recorded using Bruker AV300 and AV400 spectrometers; all spectra are reported on the delta scale referenced to the residual solvent peak. Analytical and semi-preparative high performance liquid chromatography (HPLC) were performed using a Waters600 system equipped with a Waters 2487 dual wavelength absorbance detector monitoring at 254 and 210 nm and a Phenomenex Synergi 250 mm × 21.2 mm 4 μm hydro-RP 80 Å column (10 mL / min). All HPLC methods used a H2O / MeCN biphasic solvent system buffered with 0.1% TFA. HPLC solvent system 1: (A: H2O (0.1% TFA), B: MeCN (0.1% TFA), HPLC solvent system 2: (A: H2O (0.01% TFA), B: MeCN). [44 Sc]Sc 3+ (t 1 / 2 = 3.97 hours) as previously reported. 28 , 12.8MeV protons nat Produced at TRIUMF by proton bombardment of a Ca target. 111 In]In 3+ (t 1 / 2 =2.83 days) (purchased from BWX Technologies) 111 Cd(p,n) 111 In was generated by proton irradiation (Advanced Cyclotron Systems, model TR30) via the In reaction and provided as a 0.05 M HCl solution. 155 Tb]Tb 3+ (t 1 / 2 = 5.58 days) was produced at TRIUMF by irradiation of a tantalum target with 500 MeV protons, followed by directed on-line isotope separation (ISOL) and injection into NH4Cl layered aluminum disks. 43 was carried out. 132 / 135 La]La 3+ (t 1 / 2 = 19.5 hours) was reported by Aluicio-Sarduy et al. 44 Using a similar approach to 12.8 MeV protons nat It was produced by irradiation of a Ba target. 177 Lu]Lu 3+ (t 1 / 2 = 6.67 days) was obtained from ITM Medical Isotopes GmbH Germany as a 0.05 M HCl solution. 213 Bi][Bi 3+ is an established methodology 45 Based on the results, an in-house constructed AG-MP-50 cation exchange resin was used. 225 Ac / 213 Obtained from a Bi generator. 225 Ac]Ac 3+ (t 1 / 2 =10.0 days) is due to 500 MeV protons 232 Produced at TRIUMF by spallation of Th targets and reported previously 46The radiolabeling of the compounds was evaluated by instant thin-layer chromatography (iTLC) using silicic acid (SA)-impregnated paper TLC plates supplied by Agilent technologies. TLC imaging was performed using an AR-2000 imaging scanner equipped with P-10 gas, followed by analysis of radiochemical conversion (RCC) using WinScan V3_14 software. Radio-HPLC was performed using an Agilent 1200 instrument equipped with a Phenomenex Synergi 4 μm 250 mm × 4.6 mm hydro-RP 80 Å column. Radioactivity was quantified using a calibrated high-purity germanium (HPGe) detector (Mirion Technologies (Canberra) Inc.) with Genie 2000 software. All work with radionuclides at TRIUMF was performed in a shielded fume hood to minimize dose to the experimenters (special precautions were used to prevent contamination). Peptides were prepared using an AAPPTec Focus Xi semi-automated solid-phase peptide synthesizer. SPECT / CT studies were performed using a multimode VECTor / CT system (MILabs, The Netherlands) in combination with an ultra-high sensitivity (XUHS) 2 mm pinhole collimator. Image analysis was performed using the AMIDE (v.1.0.4) software. 47 The experiment was carried out using

[0158] conclusion To investigate the effect of donor group placement on the metal-binding properties within the "NON framework", a novel nonadentate chelating ligand, H4noneunpaX, was synthesized. Characterization of the metal complexation of H4noneunpaX by NMR spectroscopy, mass spectrometry, radiolabeling, solution thermodynamic stability studies, and DFT calculations indicates excellent compatibility with a wide range of large trivalent metal cations, with particular preference for the rigid lanthanide ions. 44 Sc]Sc 3+ , [ 111 In]In 3+ , [ 132 / 235 La]La 3+ , [ 155 Tb]Tb 3+ , [177 Lu]Lu 3+ , [ 213 Bi]Bi 3+ , and [ 225 Ac]Ac 3+ Radiolabeled identification of H4noneunpaX was evaluated using ELISA kits, which showed quantitative RCC within 10 min at ambient temperature and [ 111 In]In 3+ (54GBq / μmol), [ 155 Tb]Tb 3+ (1.0GBq / μmol), [ 177 Lu]Lu 3+ (2.0 GBq / μmol), and [ 225 Ac]Ac 3+ A remarkably high molar activity was achieved with 134 MBq / μmol. The reactivity trends were found to be highly comparable to the structurally related chelating ligand, H4noneunpa, which achieved similar RCY and molar activity. Serum challenge studies were used to determine the kinetic inactivity of each metal complex, which was [ 225 While Ac][Ac(noneunpaX) maintained high radiochemical integrity (>99% RCP over 7 days), it showed a decrease in integrity over time (approximately 10% decomposition)[ 225 With the exception of [Ac][Ac(noneunpa)], they exhibited excellent in vivo stability (>95% RCP over 5-7 days), thereby indicating a small effect between chelate structural arrangements on the stability of the complexes. Solution thermodynamic stability studies indicated the formation of highly stable metal complexes with H4noneunpaX, whereby in complexation with the lanthanide series, a single predominant species was observed over a wide pH range (pH = 2.0-11.5). High stability constants were determined for each of the metal complexes, indicating highly effective metal capture capabilities.

[0159] A bifunctional analogue of H4noneunpaX was prepared using an efficient and versatile / adaptable synthetic approach and its complexation properties were re-evaluated, showing comparable characteristics to the unmodified chelator. 177 Lu]Lu 3+Dose escalation studies of bifunctional H4noneunpaX-Bn-NH2 with β-aminobutyric acid showed highly competitive radiolabeling with remarkably high molar activity (250 GBq / μmol). A bifunctional derivative of H4noneunpaX was developed using two different Tyr-linked β-aminobutyric acid (Tyr-linked β-aminobutyric acid) for targeting SSTR2 in NER tumors. 3 -conjugated to the octreotate analogue.

[0160] Preliminary in vivo SPECT / CT imaging, pharmacokinetics and biodistribution studies were performed using 155 Tb]Tb 3+ -Radiolabeled H4noneunpaX-Ahx-Tyr 3 -TATE and H4noneunpaX-PEG2-Tyr 3 A study was performed in NRG mice bearing AR42J exocrine pancreatic tumor xenografts using -TATE. Both radiolabeled bioconjugates demonstrated good in vivo performance, with no apparent degradation over the course of the study and good tumor uptake (13.3 ± 0.18% ID / g at 2 h). The biodistribution profile of each tracer was characterized by the SSTR2-targeting tracer (i.e., [ 177 Lu][Lu(DOTATATE)]) showed a typical expected clearance profile, being primarily eliminated via the renal route, taken up by the kidney and bladder, and subsequently excreted in the urine. 177 Lower accumulation was seen in non-target organs expressing SSTR2 (adrenal glands, lungs, pancreas) compared to [Lu][Lu(DOTATATE)]. Ultimately, these studies support the use of H4noneunpaX in combination with other suitable radionuclides for diagnostic imaging, e.g., in targeted alpha therapy. 225 Ac]Ac 3+ The mild conditions required for radiolabeling of H4noneunpaX make it particularly well suited for combination with heat-sensitive antibody-based targeting vectors, and it can therefore be reasonably predicted that H4noneunpaX will likely have good utility as a chelator for targeted in vivo delivery of radiometals.

[0161] The objective of this study was to investigate the effect of donor group substitution on the common backbone and to study the effect of inverted group substitution on metal ion affinity. H4noneunpaX was developed to this effect and studied in direct comparison with H4noneunpa. The inverted arrangement of the donor pendant arm [ 213 Bi]Bi 3+ With the exception of 111 In]In 3+ , [ 155 Tb]Tb 3+ , [ 177 Lu]Lu 3+ , and [ 225 Ac]Ac 3+ Using this method, high radiochemical yields (RCYs) and molar activities for H4noneunpaX were achieved under mild conditions (room temperature, 10 min), in addition to demonstrating high kinetic inactivity when challenged in human serum. Low-temperature complexation studies and DFT simulations indicate highly versatile metal ion chelation with H4noneunpaX, with chelation of trivalent lanthanide ions being particularly favored.

[0162] To further evaluate the utility of this approach, we prepared a bifunctional analogue of H4noneunpaX and 44 Sc]Sc 3+ and 177 Lu]Lu 3+ Radiolabeling studies with H4noneunpaX were performed. Structural modifications on the pendant donor groups retained the metal chelating properties of the original chelating ligand. Given the availability of synthesis of bifunctional chelating ligands featuring this inverted donor group arrangement (as opposed to symmetrically derivatized bifunctional chelators), further investigation of H4noneunpaX in vivo is of great interest. The versatile coordination properties and mild labeling conditions of H4noneunpaX make it suitable for imaging radionuclides ([ 135 La]La 3+ , [ 155 Tb]Tb3+ , [ 111 In]In 3+ , [ 44 Sc]Sc 3+ etc.) combined with [ 225 Ac]Ac 3+ The compound may be particularly well suited for application in targeted alpha therapy (TAT), including

[0163] While several exemplary aspects and embodiments have been described above, those skilled in the art will recognize certain modifications, permutations, additions, and subcombinations thereof. Accordingly, it is intended that the following appended claims and any claims hereafter introduced be interpreted as including all such modifications, permutations, additions, and subcombinations consistent with the broadest interpretation of the specification as a whole.

[0164] References The following references are of interest with respect to the subject matter described herein, each of which is incorporated herein by reference in its entirety.

[0165] [Table 6-1]

[0166] [Table 6-2]

[0167] [Table 6-3]

[0168] [Table 6-4]

[0169] [Table 6-5]

Claims

1. A chelating agent having the structure (I), wherein each R １ are independently OH, NH, or SH, and X is O, S, or NR ３ and R ３ is H or CH ２ C(=O)R １ A chelating agent which is: 【Chemistry 1】 or A chelator having the structure (III), wherein each R 1 is independently OH, NH, or SH, or a functional group; each R 2 is independently H or a functional group; each R 4 is independently H or a functional group, or both R 4 s taken together form a cyclohexyl moiety; each R 5 is independently H or a functional group, or both R 5 s taken together form a cyclohexyl moiety; X is O, S, or NR 3 , wherein one or more of the R 1 , R 2 , R 4 , or R 5 groups is a functional group that enables the chelator to be attached to a biological targeting moiety, and R 3 is H or CH 2 C(═O)R 1 . 【Chemistry 2】

2. 2. The chelating agent of claim 1 having the structure: 【Chemistry 3】

3. having the structure (II), １ is independently OH, NH, or SH, or a functional group; ２ are independently H or a functional group, and X is O, S, or NR ３ and R １ Or R ２ One or more of the groups is a functional group that allows the chelator to be attached to a biological targeting moiety, R ３ is H or CH ２ C(=O)R １ and optionally the functional group is a carboxyl, ester, amide, imide, thioamide, thioester, or guanidinium group. 【Chemistry 4】

4. A chelating compound comprising a radioactive metal and the chelating agent according to any one of claims 1 to 3.

5. A biological targeting moiety and a chelator according to claim 1, comprising one R １ group, one R ２ group, one R ４ group, or one R ５ and a chelator group attached to said biological targeting moiety, said in vivo targeting construct optionally comprising a linker interposed between said chelator and said biological targeting moiety, said linker comprising a C 1 -C 10 hydrocarbon linker, optionally substituted with one or more heteroatoms or having one or more substituents, an aromatic linker, a cationic linker, an anionic linker, an amino acid linker having 1 to 10 amino acids, a cyclized amino acid linker, a PEG linker, a cyclized ring linker, an aromatic linker, or a click chemistry linker.

6. further comprising a radiometal chelated by said chelating agent; The radioactive metal is selected from the group consisting of 225 Ac, 227 Th, 226 Th, 211 At, 44 Sc, 90 Y, 89 Zr, 177 Lu, 111 In, 86 / 89 / 90 Y, 211 At, 211 Fr, 212 / 213 Bi, 153 Sm, 161 / 166 Ho, 165 / 166 Dy, 161 / 155 Tb, 140 La, 142 / 143 / 145 Pr, 159 Gd, 169 / 175 Yb, 167 / 170 Tm, 169 Er, 149 Pm, 150 Eu, 68 Ga, Contains 137 Cs or 141 Ce, the radiometal comprises 227 Th, 225 Ac, 155 Tb, 177 Lu, 111 In, 132 La, 235 La, 90 Y, 68 Ga, 44 Sc, 203 Pb, or 212 Pb; the radiometal comprises 225 Ac, 155 Tb, 177 Lu, 111 In, 132 La, 235 La, or 44 Sc; or 6. The in vivo targeting construct of claim 5, wherein the radiometal comprises 225 Ac.

7. 6. The in vivo targeting construct of claim 5, wherein the targeting moiety comprises a hapten, an antigen, an aptamer, an affibody, an enzyme, a protein, a peptide, an antibody, an antigen-binding fragment of an antibody, a peptidomimetic, a receptor ligand, a steroid, a hormone, a growth factor, a cytokine, a molecule that recognizes a cell surface receptor, a lipid, a lipophilic group, or a carbohydrate, and the antigen-binding fragment of an antibody optionally comprises a Fab fragment, a F(ab')2 fragment, an Fv fragment, an scFv fragment, a minibody, or a diabody.

8. The biological targeting moiety may be A33 antibody, dihydrotestosterone (DHT), HuMab-5B1, girentuximab, AMG211 bispecific T cell engager, IAB22M2C minibody, rituximab, obinutuzumab, U36 antibody, plerixafor, pentixafor, NFB, ipilimumab, erlotinib, PD153035, afatinib, cetuximab, panitumumab, ABY-025 affibody, HER2-nanobody, trastuzumab, pertuzumab, GSK2849330, ramletuzumab, 4FMFES, FAPI-04, FAPI-21, FAPI-46, galactose, CB-TE2A-AR06 peptide (with H instead of DOTA), ４ noneunpaX), BAY864367 peptide (H instead of 18F labeling) ４ noneunpaX-linked ligand label), RM2 peptide (with H instead of DOTA) ４ noneunpaX), SB3 peptide (with H instead of DOTA ４ noneunpaX), RM26 peptide, BBN-RGD peptide, Aca-BBN peptide, NeoBOMB1 peptide (with H instead of DOTA) ４ noneunpaX), exendin-4 peptide, glucose, codrituzumab, EF5, MISO, AZA, HX4, ASTM, LLP2A, peptidomimetics, galacto-RGD peptide, FPP(RGD)2 peptide, RGD-K5 peptide, fluciculatide, alfatide I, alfatide II, PRGD2 peptide, αvβ6-BP peptide, CycMSHhex targeting peptide, MMOT053 0A antibody, SP peptide, neurotensin, PARPi, PSMA peptidomimetic, DCFPyL, DCFBC, HuJ591 antibody, durvalumab, nivolumab, pembrolizumab, BMS-986192 adnectin, atezolizumab, MSTP2109A antibody, TATE peptide (octreotate), TOC peptide, NOC peptide, JR11, thymidine, fresolimumab, or bevacizumab; The biological target targeted by the in vivo targeting construct is selected from the group consisting of tumor associated antigens, A33 transmembrane glycoprotein, androgen receptor (AR), CA19.9, carbonic anhydrase 9 (CA-IX), carcinoembryonic antigen, CD8, CD20, CD44v6, C-X-C chemokine receptor type 4 (CXCR4), cytotoxic T lymphocyte associated protein 4 (CTLA-4), epidermal growth factor receptor (EGFR), epidermal growth factor receptor 2 (ERBB2), epidermal growth factor receptor 3 (ERBB3), estrogen receptor (ER), fibroblast activation protein alpha, gastrin releasing peptide receptor (GRPR), glucagon-like peptide 1 receptor (GLP-1R), glypican 3, and integument.

6. The in vivo targeting construct of claim 5, comprising a targeting molecule selected from the group consisting of integrin α4β1, integrin αvβ3, integrin αvβ6, melanocortin-1 receptor (MC1R), mesothelin, neurokinin 1 receptor (NK1R), neurotensin 1 receptor (NTS1R), poly(ADP-ribose) polymerase 1 (PARP1), prostate specific membrane antigen (PSMA), programmed cell death protein (PD-1), programmed death ligand 1 (PD-L1), six transmembrane epithelial antigen of the prostate-1 (STEAP1), somatostatin receptor 2 (SSTR2), thymidine kinase, transforming growth factor-β (TGF-β), or vascular endothelial growth factor receptor (VEGFR).

9. The in vivo targeting construct of claim 5 , wherein the biological targeting moiety comprises octreotate (TATE).

10. An in vivo targeting construct for use in imaging or therapy within a subject, optionally wherein the subject is a human, as described in claim 5.

11. An in vivo targeting construct as described in claim 10, wherein the use includes a step in which the targeting portion of the in vivo targeting construct enhances accumulation of a radioisotope at a selected location in the body compared to other locations in the body, enabling selective delivery of radiation to the selected location.

12. The method of claim 1, further comprising forming a chelating compound comprising a radioisotope and the in vivo targeting construct, the forming of the chelating compound comprising combining the in vivo targeting construct with the radioisotope at a temperature of about 10° C. to about 65° C. for an incubation period; Optionally, During said incubation period, the temperature is about 15° C. to about 25° C.; said incubation period is from about 5 minutes to about 30 minutes; the combining step is carried out at a pH in the range of about 5.0 to about 7.4; and / or 11. The in vivo targeting construct of claim 10, wherein the combining step is carried out in an aqueous solution that is substantially free of alcohol.

13. The in vivo targeting construct of claim 10, wherein the use includes imaging processing to evaluate localization of the in vivo targeting construct within the body, optionally including positron emission tomography (PET) imaging or single photon emission computed tomography (SPECT) imaging.

14. An in vivo targeting construct as described in claim 5 for use in the treatment of cancer.

15. The in vivo targeting construct of claim 14, wherein the in vivo targeting construct is used to bind to a radioisotope, the radioisotope emitting radiation, the radiation including alpha or beta rays.

Images