Rate-tunable metal-mediated amide bond cleavage for the controlled release of drugs

Abstract

Drug release compositions based on metal chelate complexes conjugated to a therapeutic agent via an amino acid linker comprising a complex-adjacent amide bond are provided. Also provided are methods of treating a patient in need of treatment with the therapeutic agent using the drug release compositions. In the drug release compositions, the metal chelate complex includes a trivalent Lewis acidic metal ion chelated by 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA). For imaging or radiopharmaceutical applications, the metal ion may be a radionuclide. For targeted therapeutic agent delivery, the NOTA also may be conjugated to a biological targeting vector.

Description

Atty. Dkt. No. 00300-0450-PCTRATE-TUNABLE METAL-MEDIATED AMIDE BOND CLEAVAGE FOR THE CONTROLLED RELEASE OF DRUGS CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims priority to U.S. provisional patent application number 63 / 775,609 that was filed March 21, 2025, the entire contents of which are incorporated herein by reference.REFERENCE TO GOVERNMENT RIGHTS

[0002] This invention was made with government support under EB032349 awarded by the National Institutes of Health. The government has certain rights in the invention.BACKGROUND

[0003] Prodrug activation enables spatiotemporally controlled release of active therapeutic agents within target tissues. To this end, stimuli-activated prodrugs incorporating transition metals have attracted considerable interest owing to their distinctive features, such as tunable redox states, varied coordination geometries, and catalytic activity.

[0004] For instance, numerous Pt(IV)-based prodrugs, such as satraplatin and miriplatin, have been developed to mitigate the off-target toxicity of platinum based chemotherapeutic agents, such as cisplatin and oxaliplatin, leveraging their six-coordinate octahedral geometry' to prevent unfavorable interactions with DNA. Reductive triggers, including exogenous X-ray irradiation or photons, can be employed to trigger conversion of Pt(IV) prodrugs to the biologically active Pt(II) species. The reduction-triggered release of the axial ligands can provide optical turn-on by Anorogenic release of a fiuorescent probe or uncage small molecule ligands with chemotherapeutic action. Beyond platinum-based systems, exogeneous transition metal-mediated prodrug activation strategies have been developed. For instance, Bemardes and cow orkers pioneered a [AuCU] ‘-induced amide bond cleavage strategy to achieve the release of dual-functional therapeutic agents. (Unnikrishnan, V. B. et al., Journal of the American Chemical Society 2024, 146 (33), 23240-23251.)

[0005] Despite the attractive features of transition metal platforms for prodrug activation, their reliance on external triggers or biocompatible catalysts that can colocalize at sufficientlyAtty. Dkt. No. 00300-0450-PCThigh concentrations with the prodrug at a site of interest remains a critical barrier to clinical translation.

[0006] Some progress has been made toward developing single-molecule, modular metallo-prodrug activation using endogenous activation triggers. Groves and coworkers demonstrated that a small-molecule Co(ITI) chelate system could promote amide bond hydrolysis by coordination of a ternary hydroxide which acts as a nucleophile to hydrolyze the amide bond. (Groves, J. T.; Baron, L. A. Journal of the American Chemical Society’ 1989, 111 (14). 5442-5448.)

[0007] In a related study, Burstyn showed that a nontethered small Cu2+complex could hydrolyze both the inactivated dipeptide Gly-Gly and bovine serum albumin under physiological conditions. (Hegg, E. L.; Burstyn. J. N. Journal of the American Chemical Society’ 1995, 117 (26), 7015-7016.)

[0008] Related work by Bal and coworkers demonstrated that Ni(II) aqua ions could selectively hydrolyze amide bonds adjacent to serine and threonine residues at pH > 8.5. (Krozel, A. et al., Journal of the American Chemical Society 2010, 132 (10), 3355-3366; and Wezynfeld, N. E. et al., Metallomics 2020, 12 (5). 649-653.).

[0009] However, these systems all had significant shortcomings, preventing their application to functional metallodrugs in vivo: the metal ions, even if chelated, were coordinatively undersaturated and therefore labile and incompatible with more complex biological environments. In a further development, a metal-mediated autolytic amide bond cleavage (MMAAC) approach has recently been developed in which Lewis acidic metal ions chelated by (7-amido)-I.4.7-triazonane-1.4-diyl)diacetic acid, trigger the rearrangement and cleavage of a serine residue adjacent to the metal complex via an N,0 acyl shift, followed by ester hydrolysis. (Smilowicz, D. et al., Journal of the American Chemical Society 2023, 145 (29), 16261-16270)BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Illustrative embodiments of the invention will hereafter be described with reference to the accompanying drawings, wherein like numerals denote like elements.Atty. Dkt. No. 00300-0450-PCT

[0011] FIG. 1. Illustration of a tunable metal-mediated amide bond cleavage (TMAC) utilizing endogenous metal ions to mediate amide bond cleavage, with kinetics readily tunable by either adjacent amino acids or the identity of the metal centers (M).

[0012] FIG. 2. Synthesis pathways for tripeptides.

[0013] FIG. 3. Synthesis pathways for model tripeptides.

[0014] FIGS. 4A-4D. (FIG.4A) Stepwise complexation of natural gallium (na*Ga) and radiochemical labeling of68Ga with an alanine-based model tripeptide, alongside the reaction scheme for monitoring autolytic amide bond cleavage. (FIG. 4B) Stacked HPLC chromatograms tracking the progression of amide bond cleavage at 80 °C and pH 7.4. (FIG.4C) Stacked radio-HPLC chromatograms monitoring the progression of radiolabeled amide bond cleavage at 80 °C and pH 7.4. (FIG. 4D) Comparative heatmap illustrating timedependentnatGa-driven autolytic amide bond cleavage kinetics across tested model tripeptide systems.

[0015] FIGS. 5A-5B. (FIG.5A) Schematic description of preparation and direct complexation procedure of [natGa(16)]+. (FIG. 5B) Quantification of the pH-dependent cleavage at 80 °C and pH 7.4.

[0016] FIGS. 6A-6B. (FIG.6A) Schematic description of preparation and direct complexation procedure of [natGa(12)]+. (FIG. 6B) Quantification of the pH-dependent cleavage at 80 °C and pH 7.4.

[0017] FIGS. 7A-7B. (FIG. 7A) Schematic description of preparation and direct complexation procedure of [natGa(15)]+. ((FIG. 7B) Quantification of the pH-dependent cleavage at 80 °C and pH 7.4.

[0018] FIG. 8A-8D. Schematic description of preparation and direct complexation procedure of [nalGa(17)]+upper panels and quantification of the pH-dependent cleavage (lower panels) at 80 °C and pH 4.5 (FIG. 8A), 80 °C and pH 6.5 (FIG. 8B), 80 °C and pH 7.4 (FIG. 8C), and 80 °C and pH 8.5 (FIG.8D).

[0019] FIG. 9A-8D. Schematic description of preparation and direct complexation procedure of [natGa(18)]+upper panels and quantification of the pH-dependent cleavageAtty. Dkt. No. 00300-0450-PCT(lower panels) at 80 °C and pH 4.5 (FIG. 9A), 80 °C and pH 6.5 (FIG. 9B), 80 °C and pH 7.4 (FIG. 9C), and 80 °C and pH 8.5 (FIG.9D).

[0020] FIG. 10. Comparative heatmap illustrating time-dependent68Ga-driven autolytic amide bond cleavage kinetics across tested model tripeptide systems.

[0021] FIGS. 11A-11B. (FIG. 11A) Schematic description of preparation and direct radiolabeling procedure ofX IcGly-containing radiopharmaceutical. (FIG. 11B) Quantification of the temperature-dependent release of radiopharmaceutical from [68Ga][Ga(NO2A)]+-NMeG-G-W-CONH2at 80 °C.

[0022] FIGS. 12A-12B. (FIG. 12A) Schematic description of preparation and direct radiolabeling procedure ofNMeSer-containing radiopharmaceutical. (FIG. 12B) Quantification of the temperature-dependent release of radiopharmaceutical from [68Ga][Ga(NO2A)]+-NMeS-G-W-CONH2at 80 °C.

[0023] FIGS. 13A-13B. (FIG. 13A) Schematic description of preparation and direct radiolabeling procedure ofx lcMet-containing radiopharmaceutical (FIG. 13B) Quantification of the temperature-dependent release of radiopharmaceutical from [68Ga][Ga(NO2A)]+-NMeM-G-W-CONH2at 80 °C.

[0024] FIG. 14. Comparative heatmap illustrating time-dependent various metal-driven autolytic amide bond cleavage kinetics of a Gly conjugate (10).

[0025] FIG. 15. Comparative heatmap illustrating time-dependent various metal-driven autolytic amide bond cleavage kinetics of aNMeGly conjugate (17).

[0026] FIGS. 16A-16E. (FIG. 16A) Reaction mechanic pathways for pH-dependent amide bond cleavage: (Left) Cleavage mechanism for non-methylated model tripeptides under acidic (via N3O3isomer-driven faster kinetics) and basic conditions (via N4O2isomer-driven slower kinetics), facilitated by direct amide bond hydrolysis. (Right) Cleavage mechanism for methylated model tripeptides across all pH ranges, mediated by direct amide bond hydrolysis. (FIG. 16B) ' H NMR spectra (400 MHz in H2O with 0.1 M KC1 and 0.01 M HC1, pH 3.95-7.57) of [Ga(NO2A)]+-Ala-Gly-Trp (FIG. 16C) ' H NMR spectra (400 MHz in + NMeH2O with 0.1 M KC1 and 0.01 M HC1. pH 3.95-7.57) of [Ga(NO2A)] - Gly-Gly-Trp, referenced to trimethylsilyl propanoic acid (TSP). (FIG. 16D) pH-dependent speciation plotAtty. Dkt. No. 00300-0450-PCTshowing equilibrium transformation between N3O3and N4O2coordination geometries of the [Ga(NO2A)]+-Ala-Gly-Trp complex. (FIG. 16E) pH-dependent autolytic cleavage profiles (pH 4.5, pH 6.5, pH 7.4, pH 8.5) for model tripeptides containing Gly, Ala, Met,NMeGly, and NMc. ,Met.

[0027] FIG. 17A and 17B. Synthesis pathways for DUPA-Targeting Peptides.

[0028] FIGS. 18A-18B. (FIG. 18A) mechanism of action for self-cleaving PSMA-targeted conjugates labeled with68Ga, with rapidly cleaving conjugates clearing faster renally, whereas slow / noncleaving conjugates are retained in circulation before the target binding event. (FIG. 18B) 30 min post injection PET imaging of Gly, Ser,VXIeGly andNMeSer conjugates showing accelerated renal clearance for the latter conjugates, which also possess faster cleavage in vivo. Labeled, shaded arrows indicate tumor, white arrows indicate liver and heart uptake due to enhanced blood retention and labeled, shaded arrows indicate kidneys. (FIG. 18C) Urine metabolite analysis with intact conjugate (labeled, shaded box) and cleaved [68Ga]Ga(NOTA) (labeled, shaded box).

[0029] FIG. 19. Synthetic route for PD1. The first step involves the amide coupling reaction of NOTA-bis (t-Bu ester) with MMAE to make SI, which was deprotected in the second step to form PD1.

[0030] FIGS. 20A-20E. (FIG. 20A) Schematic representation of different metal ion-triggered uncaging of monomethyl auristatin E (MMAE) in cells, utilizing substrates PD1, Ga-PDl, Fe-PDl, and Zn-PDl. (FIG. 20B) Cytotoxicity assessment in HeLa cells exposed to increasing concentrations of MMAE and Ga-PDl for 24 hours and 72 hours. (FIG. 20C) Cytotoxicity assessment in HeLa cells exposed to increasing concentrations of MMAE and Fe-PDl for 24 hours and 72 hours. (FIG. 20D) Cytotoxicity assessment in HeLa cells exposed to increasing concentrations of MMAE and Zn-PDl for 24 hours and 72 hours. (FIG. 20E) Cytotoxicity assessment in HeLa cells exposed to increasing concentrations of MMAE and PD1 for 24 hours and 72 hours.

[0031] FIGS. 21A-21C. (FIG. 21A) Schematic depiction of targeted MMAE conjugate design. (FIG. 21B) Cytotoxicity assessment in PC3-PIP cells exposed to increasing concentrations of MMAE, PD2, Ga-PDl, and Ga-PD2 for 72 hours. (FIG. 21C) CoronalAtty. Dkt. No. 00300-0450-PCTPET-CT images 30 minutes postinjection of mice injected with 50 pCi of [68Ga][Ga-PD2], As indicated by the arrows, the regions correspond to PSMA-positive tumors.

[0032] FIG. 22A and 22B. Synthetic route for PD2. The first step involves the thiourea formation of p-SCN-NOTA with hex-EuE (t-Bu) to make S2. The second step involves the amide coupling reaction of S2 with MMAE to make S3 (FIG. 22A), which was deprotected in the last step to form PD2 (FIG. 22B).DETAILED DESCRIPTION

[0033] Drug release compositions based on metal chelate complexes conjugated to a therapeutic agent via an amino acid linker with a complex- adjacent amide bond are provided. Also provided are methods of treating a subject in need of treatment with the therapeutic agent using the drug release compositions.

[0034] In the drug release compositions, the metal chelate complex includes a trivalent Lewis acidic metal ion chelated by l,4,7-triazacyclononane-l,4,7-triacetic acid (NOTA), or another ligand that chelates the trivalent Lewis acidic metal ion. For imaging or radiopharmaceutical applications, the chelated metal ion may be a radionuclide (e.g.. a positron-emitting radionuclide). For targeted therapeutic agent delivery, the chelating ligand also may be conjugated to a biological targeting vector. The targeting vector may also be conjugated to the chelating ligand via an amino acid linker with a complex-adjacent amide bond.

[0035] Under physiologically relevant conditions (e.g., aqueous solution and biological temperature and pH), the metal ion mediates the autolytic cleavage of the amide bond to release the therapeutic agent (and / or the biological targeting vector) without the need for an external trigger. In aqueous solution, the metal chelate complex polarizes the amide bond to form two coordinative isomers, N3O3 and N4O2. (The N3O3 isomer is characterized by a metal center coordinated by the carbonyl oxygen, whereas the N4O2 isomer forms a coordinative bond with the corresponding amidate, as shown in FIG. 16A.) An external nucleophile (e.g., -OH) in the solution then hydrolyzes the amide bond, cleaving the therapeutic agent from the metal chelate complex. Notably, because the amide bond cleavage does not rely on an N, O acyl shift mechanism, the cleavage can be carried out with amino acid linkers in which theAtty. Dkt. No. 00300-0450-PCTamino acid residues of the linker and, in particular, the metal chelate complex-adjacent amino acid residue lacks a nucleophilic side chain or group, such as an -OH group.

[0036] The rate of the hydrolysis can be tailored through the selection of the amino acid linker and / or the metal of the metal ion of the chelate complex, enabling the optimization of the biological half-life of the radionuclide and / or the therapeutic agent activation in vivo. Therefore, the drug release compositions are useful in a wide range of controlled drug and pro-drug release applications. The autolytic cleavage of a radionuclide facilitates the rapid clearing of the radionuclide and. therefore, reduces the radioactive dose burden on the patient. This is significant, since many radiopharmaceuticals face challenges wherein prolonged bodily exposure to radiation induces systemic toxicity, including hematologic, renal, and secondary' carcinogenic effects.

[0037] As illustrated in the Example (see, e.g., FIGS.4D and 10), the rate of the hydrolysis may be such that at least 15% of the metal chelate complex- adjacent amide bonds in the drug release compositions remain intact after 20 minutes in an aqueous composition under physiological pH and temperature (or in vivo). However, faster or slower amide bond hydrolysis rates can be achieved. By way of illustration only, at least 20% or at least 50% of the metal chelate complex-adjacent amide bonds in the drug release compositions may¬ remain intact amide bond after 20 minutes in an aqueous composition under physiological pH and temperature (or in vivo). Complete or near-complete (> 80%) hydrolysis of the metal chelate complex-adjacent amide bonds in the drug release compositions can be achieved within, for example, 1 hour, 2 hours, 6 hours, or 10 hours.

[0038] In some embodiments of the drug release compositions, the metal chelate complex- adjacent amino acid residue is bonded to the chelating ligand (e g., NOTA) via a tertiary amide bond. In such embodiments, the metal chelate complex- adjacent amino acid residue has a bulky leaving group, such as an N-methyl group, that favors the N3O3 coordinative isomer over the N4O2 coordinative isomer. This is advantageous if a rapid release of a therapeutic agent is desired, as the N3O3 isomer tends to produce a faster basecatalyzed amide bond hydrolysis reaction. Alternatively, if a slower release is desired, an amino acid linker in which the metal chelate complex-adjacent amino acid residue lacks a bulky leaving group may be preferable.Atty. Dkt. No. 00300-0450-PCT

[0039] The amino acid linker includes or consists of a peptide having at least two amino acid residues and typically includes or consists of a peptide having three or more amino acid residues. Non-limiting examples of amino acid residues that include a N-methyl group include N-methyl glycine (NMeGly), N-methyl methionine (NMeMet), and N-methyl serine (NMeSer). (In some embodiments of the amino acid linkers, the metal chelate complex-adjacent amino acid residue is not a serine.) Non-limiting examples of amino acid residues that do not include a bulky leaving group or a nucleophilic side chain include alanine (Al) and methionine (Met).

[0040] Illustrative examples of trivalent (3+) metal ions that can be used in the drug release compositions include Ga3+, Fe3+, and Co3+. However, other trivalent metals can be used, including other first-row transition metals and metalloids. For imaging applications, the radionuclide gallium-68 (68Ga) is an example of a useful metal isotope that can be included in a metal chelate complex, due to its short half-life (ti / 2 = 68 min). Longer-lived therapeutic and imaging isotopes, such as lutetium-177 (177Lu) and terbium-161 (161Tb) can also be used. Still other examples include AL18F, Mn-52, Sc-44, and Y-86 / 90.

[0041] The trivalent metals and radionuclides can be combined in aqueous solution with the chelating ligands to form the metal chelate complexes, as illustrated in the Example.

[0042] The therapeutic agents, also referred to as ‘payloads’, are chemicals (e.g., molecules, including biomolecules) that are used to prevent, inhibit, treat, and / or study a disease, such as cancer. Antibody-drug conjugates, such as monomethyl auristatin E, are examples of therapeutic agents. Other anti-cancer therapeutic agents that can be used in the drug release compositions include campthothecins, including derivatives thereof, such as exatecan, irinotecan, and topotecan. Other suitable antibody-drug conjugates are described in Fu, Zhiwen, et al., Signal transduction and targeted therapy 7.1 (2022): 93 and Tsuchikama, Kyoji, et al., Nature Reviews Clinical Oncology 21.3 (2024): 203-22.)

[0043] As noted above, for use in targeted diagnostic, imaging, or medical treatment applications it is desirable for the metal chelate complex to be conjugated to a biological targeting vector to facilitate the delivery of the therapeutic agent to a desired location in the subject being treated. Therefore, the chelating ligand, such as the NOTA, is desirably covalently linked to a biological targeting vector, either through the formation of a direct covalent bond to the biological targeting vector or through the formation of a covalent bondAtty. Dkt. No. 00300-0450-PCTto a molecular linker attached to the biological targeting molecule. The targeting vector can be linked to the chelating ligand via a amino acid linkage having a complex- adjacent amide bond, as described herein. However, other types of linkers can be used if the cleavage of the targeting vector is not desired.

[0044] The biological targeting vectors are vectors that specifically bind to a particular type of cell, such as a cancer cell, and direct the chelates to a region of the body to be treated and / or imaged using the radionuclides. For example, in diagnostics and treatment the biological targeting vectors may have a specific affinity to a cellular disease marker, such as a receptor protein overexpressed in cancer, to deliver the therapeutic agent to the location of the cancer.

[0045] Biological targeting vectors include hormones, signaling molecules, binding moieties, antibodies, antibody fragments (e.g., an antigen-binding fragment), binding proteins, binding peptides, binding polypeptides (such as a selective targeting oligopeptide containing up to 50 amino acids), binding proteins, enzymes, nucleobase-containing moieties (such as oligonucleotide, DNA or RNA vectors, or aptamers), or lectins.

[0046] Illustrative examples of targeting vector antibodies include belimumab.Mogamulizumab, Blinatumomab, Ibritumomab tiuxetan, Obinutuzumab, Ofatumumab, Rituximab, Inotuzumab ozogamicin, Moxetumomab pasudotox, Brentuximab vedotin, Daratumumab, Ipilimumab, Cetuximab, Necitumumab, Panitumumab, Dinutuximab, Pertuzumab, Trastuzumab. Trastuzumab emtansine. Siltuximab, Cemiplimab, Nivolumab, Pembrolizumab, Olaratumab, Atezolizumab, Avelumab, Durvalumab, Capromab pendetide, Elotuzumab, Denosumab, Ziv-aflibercept, Bevacizumab, Ramucirumab, Tositumomab, Gemtuzumab ozogamicin, Alemtuzumab, Cixutumumab, Girentuximab, Nimotuzumab, Catumaxomab. Etaracizumab, and fragments thereof.

[0047] Illustrative examples of targeting vector peptides include a prostate specific membrane antigen ("PSM A") binding peptide, a somatostatin receptor agonist, a bombesin receptor agonist, and a seprase (fibroblast activation protein) binding compound.

[0048] Illustrative antigens that can be targeted include CD20, CD147. CD22, CD37, HLA-DR, CD38, HK2, PSMA, CD46, CD33, CD45, CEA, PD-L1, A33, HER2, Ep-CAM, TAG-72, CSA-p, HK2, EGFR, PSCA, CEA / HSG, CEA / hapten, SSTR, GRPR, Integrins, GRPR / PSMA, Integrin / GRPR, PSMA / GRPR, GnRH-R, NK-1R, VEGF / Integrins, MMP-1,Atty. Dkt. No. 00300-0450-PCTCCK2R, CXCR4, Neurotensin, Upar, MC1R, and GLP-1R, description of which can be found in Zhang, Taotao, et al., Cell Death Discovery 10.1 (2024): 16.

[0049] The drug release compositions can be used to treat a subject in need of treatment by administering a therapeutically effective amount of the drug release composition to the subject. The subject being treated may be, for example, a mammal, such as a human, a veterinary animal (e.g., a pet), an animal kept as livestock, or a research animal.

[0050] A therapeutically effective amount of a drug release composition refers to an amount that is effective to prevent, inhibit, treat, diagnose, and / or study a particular disease, such as a cancer, or to alleviate a symptom thereof. A therapeutically effective amount may vary depending on the size, age, and / or gender of the subject, and / or a desired therapeutic outcome.

[0051] The drug release compositions may be administered to a subject by direct injection into the bloodstream or a tumor. However, other forms of administration, including intra-muscular, oral, subcutaneous, intrarectal, inhalation, or topical may be used. For administration via injection, the drug release composition may be formulated as an aqueous solution or suspension. For other means of administration, such as oral administration, the drug release compositions may be formulated a solid powder, a tablet, a pill, a capsule, a gel, or a syrup.

[0052] In addition to the drug release composition, the formulations may include pharmaceutically acceptable carriers, where pharmaceutically acceptable indicates approval by a regulatory agency of the Federal or a state government or listed in the U.S.Pharmacopeia or other generally recognized pharmacopeia for use in animals. For purposes of illustration, pharmaceutically acceptable carriers include diluents, absorption delaying agents, antioxidants, binders, buffering agents, bulking agents or fillers, coatings, dispersants, emulsifiers, isotonic agents, lubricants, preservatives, binders, salts, solvents, stabilizers, and surfactants.

[0053] The drug release compositions have applications in vitro, such as in research or dosimetry, and in the in vivo treatment of subjects in need of treatment by the therapeutic agents. For in vivo applications, the amide cleavage takes place at physiological conditions (pH 7.4 and 37 °C). However, for in vitro applications, the drug release compositions can beAtty. Dkt. No. 00300-0450-PCTdesigned such that the amide cleavage takes place outside of physiological conditions (e.g., at pH in the range from 6.5 to 8.5 and / or at temperatures in the range from 30 °C to 90 °C).

[0054] While the drug release compositions can be used without an external trigger, variations on methods of using the drug release compositions can include an external trigger, such as external radiation. By way of illustration only, Co2+is the cobalt ion that is stable at pH 7.4 and 80 °C and does not form a complex with NOTA. However, upon addition of an oxidizing agent (e.g., H2O2 or X-ray irradiation) to induce conversion to Co3+, the metal ion can be chelated by NOTA. enabling metal-mediated amide bond cleavage via a redox approach.EXAMPLE

[0055] This Example demonstrates a tunable metal-mediated amide bond cleavage (TMAC) strategy (FIG. 1), wherein the cleavage kinetics can be precisely modulated via varying the identity of the Lewis acidic metal ion and / or the complex- adjacent amino acid residues. Using a model tripeptide sequence, reactivity' rate and mechanism were characterized. Subsequently, an isomer-driven amide bond cleavage mechanism was elucidated, distinct from previously reported N,0 acyl shift mechanisms. Finally, the use of TMAC was demonstrated for tailoring the pharmacokinetics of radiopharmaceuticals and developing a monomethyl auristatin E (MMAE)-based prodrug platform.

[0056] Results and Discussion

[0057] Complex Adjacent Amino Acid Screening and Scope.

[0058] To investigate the impact of the complex adjacent amino acid, a study was initiated by synthesis of a model tripeptide sequence which was capped at the N-terminus by an aza-macrocyclic metal chelator. l,4,7-triazacyclononane-l,4,7-triacetic acid (NOTA) was conjugated to the N-terminus of H-X-G-W-NH2 (where X represents the variable metal chelate complex-adjacent amino acid residue). Tryptophan (W) was incorporated as a spectroscopic handle for HPLC monitoring due to its characteristic absorbance at 280 nm, while glycine (G) was included as a short spacer amino acid to minimize the steric influence of W on the metal chelate. Subsequently, chelation with Ga3+yielded a model tripeptide metal complex, [Ga(NO2A)]+-X-G-W-NH2. The Ga3+ion was selected as the gold-standard metal ion since it effectively triggered bond cleavage in the Ser model tripeptide in priorAtty. Dkt. No. 00300-0450-PCTstudies. (Smilowicz, D. et al., Journal of the American Chemical Society 2023, 145 (29), 16261-16270)

[0059] Synthesis of Model Tripeptides: The synthesis of model tripeptides was carried out following a general procedure, typically on 0.13 mmol scale using a Rink amide (RA) resin with Boc-protected tryptophan (200 mg, 0.64 mmol / g). The RA resin was swollen in DCM (2 mL) and DMF (2 mL) for 1 min three times each. Fmoc-Gly-OH (152 mg, 0.51 mmol) was loaded onto the resin using benzotriazole-l-yloxy-tris-pyrrolidinophosphonium hexafluorophosphate (PyBOP) (133 mg. 0.26 mmol) as coupling reagent in the presence of N, N-diisopropylethylamine (DIEA) (89 pL, 0.51 mmol) within 12h. The Fmoc group was subsequently removed by treatment of the resin with 20% piperidine in DMF (2 mL) for 30 min. Subsequent amino acids were coupled in the same manner until the full sequences were assembled. Finally, the Fmoc group on N-terminus was deprotected and NOTA-bis (t-Bu ester) (106 mg, 0.26 mmol) was coupled using PyBOP (100 mg, 0.19 mmol) as coupling reagent in the presence of DIEA (89 pL, 0.51 mmol) within 12 h. Eventually, the products were washed and dried. The final model tripeptides from the resins were released by treating with a mixture of TFA / TIS / H2O (95% / 2.5% / 2.5%). (FIGS. 2 and 3)

[0060] The 1:1 metal complexation was performed at pH 4.5 in a 10 mM sodium acetate (NaOAc) buffer. Subsequently, the pH and reaction temperature of the metal complex solution were adjusted to pH 7.4 and 80 °C. The reaction progress was monitored using high-performance liquid chromatography and mass spectrometry (HPLC-MS). In the absence of a metal ion, the model tripeptide remained intact at pH 7.4 and 80 °C. Amide bond hydrolysis was observed for serine (Ser) and threonine (Thr) owing to the presence of nucleophilic hydroxyl groups in their side chains, in accordance with a mechanism that the amide bond hydrolysis proceeds through N,0 acyl shift. In the liquid chromatograph, a second peak with a longer retention time, with the same mass as the reactant, was consistent with an intramolecular rearrangement or isomerization process prior to the subsequent amide bond cleavage (FIGS.4A-4C). In contrast, amide bond cleavage was not observed for glycine and (3-alanine, as their side chains contain only a hydrogen substituent, preventing the necessary intramolecular rearrangement to trigger an N, O acyl shift. However, isomer peaks were also detected in the liquid chromatograph, indicating a second mechanism of complex isomerization of model tripeptide complexes at pH 7.4.Atty. Dkt. No. 00300-0450-PCT

[0061] Further evidence for a competing bond cleavage mechanism and relevance of structural isomers was provided by reactivity profiles of non-canonical. methylamide derived amino acid containing sequences. It was contemplated that the amide bond hydrolysis could be accelerated significantly by designing the peptide sequences that act as a bulkier leaving group. To confirm this, a tertiary amide bond conjugate model sequence containing N-methyl serine (NMeSer) was synthesized, which efficiently promoted amide bond cleavage (FIGS. 5A-5B)

[0062] Additional peptide sequences containing complex-adjacent alanine (Ala) and methionine (Met) readily produced hydrolysis products (FIGS. 6A-6B, 7A-7B). Similarly, the tertiary amide conjugates,NMeGly andNN'IeMet also exhibited efficient amide bond cleavage under the same conditions with rates exceeding those of the non-methylated canonical amino acids (FIGS. 8A-8D, 9A-9D). This observation was at odds with the amide bond hydrolysis requiring N, O acyl shift for efficient amide bond cleavage.

[0063] Reaction rates were revealed to be variable, ranging from comparatively slow (Ala, 20% cleavage after 6 h) to moderate (Thr, 50% cleavage after 6 h) to fast (NMeSer, 100% cleaved after 1 h). (FIG. 4D)

[0064] Application of self-cleaving metal complexes bears relevance in the design of rapidly clearing radiopharmaceuticals to reduce radioactive dose burden. As such, experiments were conducted with the positron emitting radioisotope68Ga (ti / 2 = 68 min). For the radioisotope studies, 1:1 metal complexation was carried out at pH 4.5 in 1 M NaOAc buffer. Subsequently, the pH and reaction temperature of the metal complex solution were adjusted to pH 7.4 and 80 °C. Cleavage rates were reproduced under radiochemical tracer conditions employing68Ga-radiolabled complexes, indicating that cleavage kinetics were governed by the complex’s reactivity (FIG. 10) and were not influenced by the relative hydroxide concentration. In all instances, side-reactions were not detected, with only one scissile bond and formation of inert products. Observations made with sequences that were not compatible with the N, O acyl shift mechanism, as shown in FIGS. 11A-11B, 12A-12B, and 13A-13B, showed the tertiary amide conjugates having the methylated amino acids efficiently promoted amide bond cleavage.

[0065] To assess the ability of different metal centers to induce TMAC, cleavage experiments were conducted using Gly andNMeGly conjugates. Cu2+, Zn2+complexes wereAtty. Dkt. No. 00300-0450-PCTinert toward direct amide bond hydrolysis at pH 7.4 and 80 °C (FIGS. 14 and 15). In contrast, the trivalent metal centers Fe3+and In3+exhibited faster cleavage than Ga3+for both conjugates (FIGS. 14 and 15). These experiments demonstrate that TMAC is compatible with other Lewis acidic, trivalent metal centers, whereas divalent ions show no reactivity.

[0066] Mechanism studies

[0067] To demonstrate the role of nucleophiles in amide bond hydrolysis, the effect of an external nucleophile (putatively OH") on cleavage kinetics was explored. pH-dependent cleavage experiments were conducted at pH 4.5 (10 mM NaOAc), 6.5 (10 mM 3-(N-morpholino)propanesulfonic acid (MOPS)), 8.5 (10 mM 3-[4-(2-hydroxyethyl)piperazin-l-yl]propane-l -sulfonic acid (EPPS)), and 80 °C. Notably, tertiary amide model tripeptides exhibited faster cleavage rates with increasing pH, indicating that the OH" concentration played a significant role in hydrolyzing amide bonds. In contrast, non-tertiary model tripeptides showed slower cleavage rates as the reaction pH was increased, suggesting the involvement of other factors (FIG. 16E).

[0068] Following the observation of a putative structural isomer by HPLC-MS, it was confirmed that the formation of pH dependent coordinative isomers could affect cleavage kinetics. To this end, the solution structures of two sample constructs were characterized by nuclear magnetic resonance (NMR) spectroscopy. Diagnostic chemical shifts in immediate vicinity of the metal center offered valuable insight into the pH-dependent behavior of the corresponding isomers.

[0069] Indeed, the 'H NMR spectra of the [Ga(NO2A)]+-Ala-Gly-Trp. as shown in FIG 16B, revealed the presence of two distinct coordination isomers, N CL and N4O2. The N3O.3 isomer is characterized by a metal center coordinated by the carbonyl oxygen, whereas the N4O2 isomer forms a coordinative bond with the corresponding amidate. Methylene signal peaks were identified at 4.30 ppm and 4.22 ppm as diagnostic of N3O3 isomer at low pH. whereas, at higher pH, an upfield shift to 3.76 ppm and 3.71 ppm indicated the formation of the N4O2 isomer (FIG. 16B). Acquisition of pH dependent data allowed observation of gradual interconversion of the 4.30 ppm and 4.22 ppm signals to those appearing at 3.76 ppm and 3.71 ppm, providing strong evidence of the isomer transformation from N3O3 to N4O2 (FIG. 16B). The isomer transformation can be quantified by integrating these two shifting peaks, providing an approximation of the pKa value of pH 6.33 (FIGS. 16A and 16D). TheAtty. Dkt. No. 00300-0450-PCT'H NMR spectra of the [Ga(N02A)]+-Gly-Gly-Trp and |Ga(N02A) | -Met-Gly-Trp complexes exhibited a comparable pH dependent pattern, further supporting the identification of analogous coordination isomers.

[0070] In the absence of Bronsted acids with correlating p / G on the molecular scaffold, it was contemplated that the immediate vicinity of the amide proton to the Ga3+metal center lowered the p a by several orders of magnitude. The corresponding deprotonation event resulted in transformation of the coordinative donor environment from N3O3 to N4O2. The1H NMR spectra of the [Ga(NO2A)]+-Gly-Gly-Trp and [Ga(NO2A)]+-Met-Gly-Trp complexes exhibited comparable pA'a values (5.72 and 6.40, respectively), further supporting consistent formation of the proposed coordination isomers.

[0071] In contrast, the ’H spectra of the [Ga(NO2A)]+-NMeGly-Gly-Trp showed no peak shift as pH increased, indicating that methylation of the amide nitrogen maintained coordination pattern to the N3O3 species (FIG. 16C). Integrating findings from pH-dependent cleavage and NMR experiments confirmed that the N3O3 species mediated a rapid basecatalyzed amide bond hydrolysis reaction, whereas the N4O2 species resulted in slowed rate of release (FIG. 16A). N-methylation of the amide resulted in locked N3O3 configuration, which efficiently retained rapid amide bond hydrolysis rates at elevated pH (FIG. 16E). Accordingly, the nature of the complex-adjacent amino acid allowed the selective tuning of the cleavage rate.

[0072] TMAC in vivo

[0073] Given the compatibility of TMAC under biocompatible conditions (pH 7.4 and 37 °C), it was contemplated that the hydrolysis of the metal complex could modulate the pharmacokinetics of radiopharmaceuticals. To demonstrate this, a targeted derivative of [Ga(NO2A)]+-R (where R represents a variable amino acid linker) was designed, conjugated to a human serum albumin (HSA) binder, 3-iodo-tyrosine and a prostate-specific membrane antigen (PSMA) targeting vector, hexKuE. (Jin. W et al., International Journal of Pharmaceutics 2016, 513 (1), 138-147.) The amino acid 3-iodo-tyrosine has been demonstrated to bind efficiently to murine and human serum albumin, thereby prolonging the biological half-life of (radio)pharmaceuticals. (Sleep, D. et al, Biochim Biophys Acta 2013, 1830 (12), 5526-5534.)Atty. Dkt. No. 00300-0450-PCT

[0074] Synthesis pathways for DUPA-Targeting Peptides: The synthesis of targeted derivatives was achieved by solid-phase peptide synthesis (FIGS. 17A and 17B). The synthesis of the DUPA-targeting peptide (Glu-urea-Lys-NOTA) was carried out on a 0.16 mmol scale using a Wang resin (200 mg, 0.8 mmol / g). The starting material, Glu-urea-Lys was synthesized according to our previously established protocol. (Smilowicz, Dariusz, et al. "Evaluation of a radio-IMmunoStimulant (RIMS) in a syngeneic model of murine prostate cancer and immunoPET analysis of T-cell distribution. " Molecular Pharmaceutics 19.9 (2022): 3217-3227) The Fmoc group from N-terminus was removed by shaking resin with 20% piperidine in DMF for 30 min. The Fmoc-Tyr(3-I)-OH was coupled to the peptide using PyBOP (167 mg, 0.32 mmol) as the coupling reagent in the presence of DIEA (111 uL, 0.64 mmol) within 12 h. After that, the peptide was elongated by coupling NOTA-bis (t-Bu) (133 mg, 0.32 mmol) using PyBOP (125 mg, 0.24 mmol) in the presence of DIEA (111 uL, 0.64 mmol) within 12 h. After coupling, resin was washed and dried. The final product was cleaved from the resin by treating it with a solution of TFA / TIS / H2O (95%:2.5%:2.5%) for 6 h.

[0075] Derivatives were selected with a range of cleavage rates from non-cleavable (Gly), to slow cleaving (Met < Ser < ^“Gly) to rapidly cleaving (NMcMet <NMcSer).

[0076] First, it was evaluated whether the amide bond hydrolysis rate of the functionalized derivatives was agnostic to the cargo. It was found that the targeted constructs displayed half-lives of amide bond hydrolysis consistent with those of the corresponding model tripeptides (Table 1). Furthermore, experiments conducted at 37 °C and in the presence of mouse plasma confirmed previously determined trends in reactivity', including a vastly accelerated cleavage rate for A-methylated amino acid derivatives. HSA binding affinity measurements confirmed comparable binding affinity for all conjugates, indicating that the change of the amino acid linker did not have a significant impact on the conjugate’s ability' to bind serum albumin (Table 2). In tables 1 and 2, the numbers in parentheses are the compound numbers for the conjugates, the full names and synthesis of which can be found in below in Additional Experimental Details - Peptide Intermediates and Final Products.

[0077] Table 1. Characterization Parameters for Select TMAC Conjugates Including Cleavage Half-Life, Cleaved Metabolite, and Tumor Uptake.Atty. Dkt. No. 00300-0450-PCTAmino Acid Half-Life of Half-Life of % Cleaved Tumor Uptake Linker R Model PSMA- Complex in 2 h at 2 h p.i. (%Tripeptide (h) Conjugate (h) p.i. Urine ID / g)MetaboliteGly (20) not observed not observed 4.0 ± 0.8 8.3 ± 0.1Ser (21) 365a / 182b533a / 408b10.8 ± 1.9 10.2 ± 2.7 Met (22) 408a / 239b462a / 462b6.0 7.5NMeGlv (23) 35.9a / 49.5b38.3a / 32.3b7.4 ± 1.3 15.2 ± 6.9NMeSer (24) 3.65a / 6.12b2.52a / 2.49b32.3 ± 2.1 19.6 ± 3.8NMeMet (25) 28.1a / 151b27.0a / 22.4b5.9 3.5aComparative amide bond cleavage half-lives of67Ga-radiolabeled model tripeptide complexes and corresponding functionalized conjugates performed in 0.25 M pH 7.4 HEPES buffer at 37 °C.bPerformed in a 1 : 1 mixture of HEPES buffer and mouse plasma at 37 °C. The half-lives of Ser (21) and Met (22) conjugates are estimated based on pseudo first-order kinetics.

[0078] Table 2. Decay -corrected biodistribution of [67Ga][Ga(20)], [67Ga][Ga(23)], and [67Ga] [Ga(24)] 12 hour post-injection (n=3) in NU / J mice models.[67Ga] [Ga(19) 24 h [67Ga] [Ga(22) 24 h [67Ga] [Ga(23)24 h(n=3) (n=3) (n=3) blood 0.0040 ± 0.00180 0.0038 ± 0.00055 0.0029 ± 0.00041 heart 0.0074 ± 0.00133 0.0087 ± 0.00104 0.0103 ± 0.00033 lungs 0.0149 ± 0.00325 0.0175 ± 0.00276 0.0172 ± 0.00145 liver 0.0240 ± 0.00546 0.0290 ± 0.00740 0.0548 ± 0.00766 spleen 0.0493 ± 0.01927 0.0336 ± 0.00399 0.0349 ± 0.00139 kidneys 0.7929 ± 0.30325 0.1665 ± 0.05120 0.1037 ± 0.00616 stomach 0.0241 ± 0.01783 0.0412 ± 0.03715 0.0125 ± 0.00238 Small intestine 0.0105 ± 0.00113 0.0126 ± 0.00278 0.0097 ± 0.00037 large intestine 0.2923 ± 0.16900 0.1166 ± 0.02485 0.0381 ± 0.02243 muscle 0.0070 ± 0.00193 0.0089 ± 0.00244 0.0110 ± 0.00524 bone 0.0190 ± 0.00242 0.0230 ± 0.00606 0.0231 ± 0.00374 brain 0.0150 ± 0.00428 0.0030 ± 0.00058 0.0033 ± 0.00030 tumor + 8.2616 ± 1.40654 1.6540 ± 0.31890 0.5134 ± 0.09621 tumor - 0.0272 ± 0.00317 0.0284 ± 0.00148 0.0300 ± 0.00115 tail 0.0634 ± 0.05417 0.1581 ± 0.05180 0.0813 ± 0.05256

[0079] With the constructs validated in vitro, the impact was probed of different rates of cleavage on the pharmacokinetic behavior of the corresponding68Ga-radiolabeled conjugates. To this end, the conjugates were first radiolabeled with68Ga isotope at a consistent molar activity of 10 nmol / mCi. A bilateral tumor model with a PSMA-expressing tumor on the right flank and a PSMA-negative tumor on the left flank was employed. Each cohort was imagedAtty. Dkt. No. 00300-0450-PCTby positron emission tomography-computed tomography (PETCT) at 30, 60, and 90 min post injection, followed by terminal biodistribution and urine metabolite analysis at 120 min.

[0080] Taking into consideration the characterized cleavage rates, it was contemplated that the conjugates with cleavable linkers. Ser (21),NMeGly (23), andNMeSer (24), would exhibit shorter biological half-lives and reduced off-target activity, whereas the construct with a non-cleavable linker, Gly (20), would show longer biological half-life and prolonged retention in blood pool and liver (FIG. 18A). Indeed, PET-CT images revealed that both [68Ga]Ga-20 (Gly). and the slow-cleaving conjugate [68Ga]Ga-21 (Ser), exhibited prolonged blood circulation, evidenced by the enhanced heart and liver uptake visible in PET images at all three time points (FIG. 18B). This was in line with expectations, with previous work on serum albumin binding radiopharmaceuticals demonstrating elevated liver and blood / heart uptake at early time points. (Deberle, L. M. et al., Molecules 2020, 25 (11). 2542; Li. L. et al., Eur. J. Nucl. Med. Mol.Imaging 2024, 51 (9), 2794-2805.) In contrast, the more rapidly cleaving [68Ga]Ga-23 (NMeGly) and [68Ga]Ga-24 (NMeSer) demonstrated decrease in liver uptake and lowered blood pool retention, paired with efficient renal clearance (FIG. 18B). Further affirmation for in vivo TMAC activity was provided by analysis of the urine metabolites (FIG. 18C): the cleavage product [68Ga]Ga(NOTA) was readily observed for cleavable constructs [68Ga]Ga-21 (Ser), [68Ga]Ga-23 (NMeGly) and [68Ga]Ga-24 (NMeSer).

[0081] TMAC-based prodrug release

[0082] It was contemplated that TMAC could serve as a platform for developing an in vivo prodrug release system. Several anticancer drugs employed as antibody-drug conjugates in the clinic contain terminal amino acid residues that are TMAC-compatible. (Lahnif, H. et al., International Journal of Molecular Sciences 2023, 24 (10), 8543; Moquist, P. N. et al., Molecular Cancer Therapeutics 2021. 20 (2), 320-328) Monomethyl auristatin E (MMAE), a synthetic tubulin polymerization inhibitor with a terminal N-methyl valine, was identified as a suitable candidate.

[0083] It was contemplated that incorporation of the [M(N02A)]n+complex to the N-terminus could modulate the drug’s potency, with TMAC serving as a slow release cleavable linker.

[0084] The synthesis of a corresponding conjugate, PD1, was achieved by solution-phase amidation using an orthogonally protected version of l,4,7-triazacyclononane-l,4,7-triaceticAtty. Dkt. No. 00300-0450-PCTacid, followed by tert-butyl deprotection (FIG. 19). The resulting construct PD1 was then complexed with Ga3+, Fe3+, and Zn2to form Ga-PDl, Fe-PDl. and Zn-PDl. respectively. First, the amide bond cleavage rate of NMe-V aline (NMeVal) model tripeptides was evaluated with various metal ions at pH 7.4 and 80 °C in vitro. The Ga complex exhibited the fastest cleavage rate with half-life of 8.05 hours, whereas the Fe?+complex showed a slower rate of cleavage with the half-life of 8.42 hours. In contrast, the Zn2+complex remained intact under the same conditions.

[0085] To evaluate the cytotoxicity (ICso) of the nontargeted therapeutic conjugates Ga-PDl, Fe-PDl and native MMAE were tested in human HeLa cervical cancer cells using a standard MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) assay (FIG.20A). (Supino, R. MTT Assays. In In Vitro Toxicity Testing Protocols , O’Hare, S., Atterwill, C. K. Eds.; Humana Press, 1995; pp 137-149) As a result of the structural modification, the cytotoxicity of the conjugates decreased, but it remained on the nanomolar scale: the Ga conjugate showed a decrease of about 20-fold in toxicity (ICso = 20.50 nM at 72-hour incubation) compared with MMAE (ICso = 1.15 nM at 72-hour incubation) for the tested concentrations (FIG. 20B). Control experiments showed that the Ga(NOTA) complex provided no additional toxicity, affirming that the released MMAE was responsible for the cytotoxic effect of the Ga-PDl conjugate. Notably, the Ga conjugate exhibited increased cytotoxicity with prolonged incubation time, suggesting the time-dependent release of MMAE (FIG. 20B). In comparison, Fe-PDl demonstrated lower cytotoxicity (ICso = 196.10 nM at 72-hour incubation) than Ga-PDl, consistent with the observation in cleavage experiments (FIG. 20C). The Zn2+conjugate, Zn-PDl, lacking the ability to induce TMAC, displayed low toxicity at higher concentrations comparable with the free conjugate PD1 (ICso = 421 nM at 72-hour incubation) (FIGS. 20D, 20E). While these results were encouraging, it was noted that in vivo applications require the targeting of the construct to a cancer subtype to prevent systemic toxicity characteristic of non-functionalized MMAE.

[0086] To this end, a prostate cancer-targeting peptide, hex-EuE, was incorporated to the backbone of l,4,7-triazacyclononane-l,4,7-triacetic acid in an non-cleavable fashion (FIGS.21A , 22A and 22B). The cytotoxicity' of targeted conjugate PD2, Ga-PD2 w as then probed in direct comparison with Ga-PDl in PSMA-expressing PC3-PIP cells for 72 hours. Both conjugates showed comparable ICso values of 24.19 nM (Ga-PDl) and 24.05 nM (Ga-PD2) respectively, demonstrating that the Ga-PD2 retained TMAC properties even when furtherAtty. Dkt. No. 00300-0450-PCTfunctionalized (FIG.21B). In addition to in vitro cell viability assessments, the in vivo targeting specificity of PD2 was further evaluated. PD2 was radiolabeled with68Ga, and PET-CT imaging was performed at 30, 60, and 90 min post injection, followed by a terminal biodistribution study (120 min). The PET-CT imaging revealed that [68Ga]Ga-PD2 exhibited selective accumulation in PSMA-ov erexpressing tumors (FIG.21C). Biodistribution analysis at 120 min p.i. further confirmed the enhanced specificity of the PD2-targeted conjugate compared to the nontargeted [68Ga] Ga-PDl. The synthetic route for PD2 synthesis is shown in FIGS.22A and 22B

[0087] Additional Experimental Details

[0088] Experimental procedures

[0089] Materials

[0090] All starting materials were purchased from Acros Organics, Alfa Aesar, Millipore Sigma or TCI America and used without further purification. Fmoc-protected amino acids were purchased from Bachem. NOTA-bis(t-Bu ester) and p-SCN-Bn-NOTA compounds were obtained from Macrocyclics. Rink Amide resin and Wang resin resins were purchased from Millipore Sigma.68GaCh was obtained from a68Ge / 68Ga generator (Eckhard & Ziegler, 10 mCi).

[0091] General Methods for Characterization

[0092] NMR spectra ('ll. COSY, HSQC and HMBC) were collected at University of Wisconsin Madison a 400 and 500 MHz III Bruker instrument at 25 °C and processed using TopSpin 4.0.9. and at the University of Wisconsin-Madison Department of Chemistry Paul Bender Chemical Instrumentation Center (CIC) using a Bruker Avance III 500 with a DCH liquid He ciyoprobe (Bender Fund), a Bruker Avance Neo 500 with a 5mm Prodigy-BBO liquid N2 ciy oprobe (NSF CHE-2017891), and a Bruker Avance III 600 with a TCI-F liquid He cryoprobe (NIH S10 OD012245). Data were processed using MestReNova 14.3.3-33362. Chemical shifts (8) were reported in parts per million (ppm) relative to tetramethylsilane. ’H.13C{JH}, COSY, HSQC and HMBC NMR spectra were referenced to residual solvent signals,45Sc NMR spectra were referenced in MestReNova using a 'l l NMR spectra as an absolute reference.Atty. Dkt. No. 00300-0450-PCT

[0093] High resolution ESI mass spectrometry was carried out at the Stony Brook University Center for Advanced Study of Drug Action (CASDA) with a Bruker Impact II UHR QTOF MS system, and at the University of Wisconsin-Madison Department of Chemistry Paul Bender Chemical Instrumentation Center (CIC) using a Thermo Scientific Q Exactive Focus Orbitrap MS system, and at the University' of Wisconsin-Madison School of Pharmacy Analytical Instrumentation Cluster using a Bruker MaXis Ultra-High Resolution Quadrupole Time-of-Flight MS system.

[0094] Ultraviolet-visible spectra were collected with the NanoDrop 1C instrument (AZY1706045). Spectra were recorded from 190 to 850 nm in a quartz cuvette with 1 cm path length. Copper titration and molar extinction measurements were made in sodium acetate buffer (10 mM, pH 5.5).

[0095] ICP-OES and MP -AES analyses were carried out using an Agilent 5110 inductively coupled plasma optical emission spectrometer and an Agilent 4210 microwave plasma atomic emission spectrometer, respectively. For both techniques, a 6-point standard curve (1-100 ppm) with respect to scandium or copper was used and fits were found to be R2> 0.99.

[0096] Analytical- and radio-HPLC methods were carried out using a Shimadzu HPLC-20AR equipped with a binary gradient pump, UV-vis detector, autoinjector, or an Agilent 1260 Infinity II system. Both instruments were set to detect UV absorption was recorded at 220 nm and 254 nm and were coupled to an in-line LabLogic Dual Scan-RAM detector and were controlled using the LabLogic Laura software package. RadioHPLC analyses utilized a LabLogic 1” Nal photomultiplier tube detector with 2” lead shielding and radioTLC analyses utilized a LabLogic plastic photomultiplier tube detector.

[0097] Semipreparative HPLC was carried out using a Shimadzu HPLC-20AR equipped with a binary gradient pump, UV-vis detector, and manual injector. UV absorption was recorded at 220 and 254 nm. Flash chromatography was carried out using a Combi Flash Rf+ system with UV detection at 220 and 254 nm.

[0098] Liquid chromatography mass spectrometry (LCMS) was carried out on a Phenomenex Luna C18 column (5 pm, 150 mm x 3 mm, 100 A, AXIA packed) at a flow rate of 0.8 mL / min using a single quadrupole Agilent 1200 Infinity II LC / MSD system equipped with a binary gradient pump, UV- vis detector, automatic injector, and an atmosphericAtty. Dkt. No. 00300-0450-PCTpressure electrospray ionization (AP-ESI) source. Ultraviolet absorption was recorded at 220 nm and 254 nm, and positive and negative mass spectra were collected from m / z = 100-800.

[0099] Chromatography Solvent Systems:

[0100] Analytical HPLC - Method A: binary' solvent sy stem (A: water + 0.1% TFA; B: MeCN + 0.1% TFA); gradient (0-2 min: 5% B; 2-14 min: 5-95% B; 14-16 min: 95% B; 16-16.5 min: 95-5% B; 16.5-20 min 5% B); flow rate: 0.8 mL / min; column: Phenomenex Luna Cl 8 column (5 pm, 150 mm x 3 mm, 100 A, AXIA packed).

[0101] Analytical HPLC - Method B: binary solvent system (A: water + 0.1% TFA; B: MeCN + 0.1% TFA); gradient (0-2 mm: 0% B; 2-14 min: 0-90% B; 14-16 mm: 90% B; 16-16.5 min: 90-0% B; 16.5-20 min 0% B); flow rate: 1.0 mL / min; temperature: 50 °C; column: Restek Ultra AQ Cl 8 column (5 pm, 250 mm x 3 mm).

[0102] Analytical HPLC - Method C: binary solvent system (A: 10 mM ammonium formate pH 9.0; B: MeCN); gradient (0-2 min: 0% B; 2-14 min: 0-90% B; 14-16 min: 90% B; 16-16.5 min: 90-0% B; 16.5-20 min 0% B); flow rate: 1.0 mL / min; temperature: 50 °C; column: Restek Ultra AQ Cl 8 column (5 pm, 250 mm x 3 mm).

[0103] Analytical HPLC - Method D: binary solvent system (A: 10 mM ammonium formate pH 4.0; B: MeCN); gradient (0-2 min: 0% B; 2-14 min: 0-90% B; 14-16 min: 90% B; 16-16.5 min: 90-0% B; 16.5-20 min 0% B); flow rate: 1.0 mL / min; temperature: 50 °C; column: Restek Ultra AQ Cl 8 column (5 pm. 250 mm x 3 mm).

[0104] Semipreparative HPLC - Method E: binary’ solvent system (A: yvater + 0.1% TFA; B: MeCN + 0.1% TFA); gradient: (0-1 min: 5% B; 1-14 mm: 5-50% B; 14-23 mm: 50-95% B; 23-26 min: 95% B; 26-27 min: 95-5% B; 27-30 min: 5% B); flow rate: 15 mL / min; column: Phenomenex Luna C18 column (250 mm x 21.2 mm, 100 A, AXIA packed).

[0105] Flash chromatography - Method F: Binary- solvent system (A: yvater + 0.1% TFA; B: MeCN + 0.1% TFA); floyv rate: 60 mL / min; column: RediSep C18 column (100 g HP gold).

[0106] LCMS - Method G: binary solvent system (A: water + 0.1% FA; B: MeCN + 0.1% FA); gradient (0-3 min: 5% B; 3-10 min: 5-95% B; 10-13 min: 95% B; 13-13.5 min: 95-5% B; 13.5-16 min: 5% B); flow rate: 0.8 mL / min; column: Phenomenex Luna C18 column (5 pm, 150 mm x 3 mm, 100 A, AXIA packed).Atty. Dkt. No. 00300-0450-PCT

[0107] Peptide Intermediates and Final Products

[0108] (R)-2-(2-(2-aminoacetamido) acetamido) -3-( lH-indol-3-yl) propenamide, H-G-G- W-CONH2. Compound 1 (200 mg, 0.128 mmol) was synthesized using the general coupling strategy outlined in FIG. 2 and the section titled "Synthesis of Model Tripeptides" above from compound Fmoc-Trp(Boc)-RA.

[0109] (R)-3 -amino-N-(2-((l-amino-3-(lH-indol-3-yl)-l-oxopropan-2-yl)amino)-2-oxoethyl)propanamide, H-[RAla-G-W-CONH2. Compound 2 (200 mg, 0.128 mmol) was synthesized using the general coupling strategy7outlined in FIG. 2 and the section titled "Synthesis of Model Tripeptides" above from compound Fmoc-Trp(Boc)-RA.

[0110] 2-amino-N-(2-(((R)-l-amino-3-(lH-indol-3-yl)-l-oxopropan-2-yl)amino)-2-oxoethyl)propanamtde H-A-G-W-CONH2. Compound 3 (200 mg, 0.128 mmol) was synthesized using the general coupling strategy outlined in FIG. 2 and the section titled "Synthesis of Model Tripeptides" above from compound Fmoc-Trp(Boc)-RA.

[0111] (S)-2-amino-N-(2-(((R)-l-amino-3-(lH-indol-3-yl)-l-oxopropan-2-yl)ammo)-2-oxoethyl) hydroxypropanamide, H-S-G-W-CONH2. Compound 4 (200 mg,0.128 mmol) was synthesized using the general coupling strategy outlined in FIG. 2 and the section titled "Synthesis of Model Tripeptides" above from compound Fmoc-Trp(Boc)-RA.

[0112] (2S)-2 -amino-N-(2-(((S)-l-amino-3-(lH-mdol-3-yl)-l-oxopropan-2-yl)amino)-2-oxoethyl)-3-hydroxybutanamide. H-T-G-W-CONH2. Compound 5 (200 mg.0.128 mmol) was synthesized using the general coupling strategy outlined in FIG. 2 and the section titled "Synthesis of Model Tripeptides" above from compound Fmoc-Trp(Boc)-RA.

[0113] (S)-2 -ammo-N-(2-(((R)-l-amino-3-(lH-indol-3-yl)-l-oxopropan-2-yl) amino)-2-oxoethyl)-4-(methylthio)butanamide, H-M-G-W-CONH2. Compound 6 (200 mg, 0.128 mmol) was synthesized using the general coupling strategy outlined in FIG. 2 and the section titled "Synthesis of Model Tripeptides" above from compound Fmoc-Trp(Boc)-RA.

[0114] (R)-N-(2-(((S)-l-amino-3-(lH-mdol-3-yl)-l-oxopropan-2-yl)amino)-2-oxoethyl)-3-hydroxy-2-(methylamino)propanamide, H-NMeS-G-W-CONH2. Compound 7 (200 mg, 0.128 mmol) was synthesized using the general coupling strategy' outlined in FIG. 2 and the section titled "Synthesis of Model Tripeptides" above from compound Fmoc-Trp(Boc)-RA.Atty. Dkt. No. 00300-0450-PCT

[0115] (R)-3-(lH-indol-3-yl)-2-(2-(2-(methylamino)acetamido)acetamido)propanamide, H-NKIeG-G-W-CONH2. Compound 8 (200 mg, 0.128 mmol) was synthesized using the general coupling strategy outlined in FIG. 2 and the section titled "Synthesis of Model Tripeptides" above from compound Fmoc-Trp(Boc)-RA.

[0116] (S)-2 -amino-N-(2-(((R)-l-amino-3-(lH-indol-3-yl)-l-oxopropan-2-yl) amino)-2-oxoethyl)-4-(methylthio)hutanamide, H-NAIeM-G-W-CONH2. Compound 9 (200 mg, 0.128 mmol) was synthesized using the general coupling strategy outlined in FIG. 2 and the section titled "Synthesis of Model Tripeptides" above from compound Fmoc-Trp(Boc)-RA.

[0117] (R)-2,2'-(7-(2-((2-((2-((l-amino-3-(lH-indol-3-yl)-l-oxopropan-2-yl)amino)-2-oxoethyl)amino)-2-oxoethyl)amino)-2-oxoethyl)-l, 4, 7-triazonane-l,4-diyl)diacetic acid, NOTA-G-G-W-CONH2. Compound 10 (157 mg, 0.100 mmol) was synthesized using the general coupling strategy' outlined in FIG. 3 and the section titled "Synthesis of Model Tripeptides" above from compound Fmoc-Trp(Boc)-RA.

[0118] (S)-2,2'-(7-(2-((3-((2-((l-amino-3-(lH-indol-3-yl)-l-oxopropan-2-yl)amino)-2-oxoethyl)amino)-3-oxopropyl)amino)-2-oxoethyl)-1.4.7 -triazonane- 1.4-diy I) diacetic acid, NOTA-PA-G-W-CONH2. Compound 11 (157 mg, 0.100 mmol) was synthesized using the general coupling strategy' outlined in FIG. 3 and the section titled "Synthesis of Model Tripeptides" above from compound Fmoc-Trp(Boc)-RA.

[0119] 2,2'-(7-(2-(((R)-l-((2-(((R)-l-amino-3-(lH-indol-3-yl)-l-oxopropan-2-yl)amino)- 2- oxoethyl)amino)-l-oxopropan-2-yl)amino)-2-oxoethyl)-l, 4, 7 -triazonane- 1, 4-diyl)diacetic acid, NOTA-A-G-W-CONH2. Compound 12 (157 mg, 0.100 mmol) was synthesized using the general coupling strategy outlined in FIG. 3 and the section titled "Synthesis of Model Tripeptides" above from compound Fmoc-Trp(Boc)-RA.

[0120] 2,2'-(7-(2-(((R)-l-((2-(((S)-l-amino-3-(lH-indol-3-yl)-l-oxopropan-2-yl)amino)- 2-oxoethyl)amino)-3-hydroxy-l-oxopropan-2-yl)amino)-2-oxoethyl)-l,4, 7 -triazonane- 1,4-diyl)diacetic acid, NOTA-S-G-W-CONH2. Compound 13 was synthesized using the general coupling strategy outlined in FIG. 3 and the section titled "Synthesis of Model Tripeptides" above from compound Fmoc-Trp(Boc)-RA.

[0121] 2,2'-(7-(2-(((R)-l-((2-(((S)-l-amino-3-(lH-indol-3-yl)-l-oxopropan-2-yl)amino)- 2-oxoethyl)amino)-3-hydroxy-l-oxopropan-2-yl)amino)-2-oxoethyl)-l,4, 7 -triazonane- 1,4-Atty. Dkt. No. 00300-0450-PCTdiyl)diacetic acid, NOTA-T-G-W-CONH2. Compound 14 was synthesized using the general coupling strategy outlined in FIG. 3 and the section titled "Synthesis of Model Tripeptides" above from compound Fmoc-Trp(Boc)-RA.

[0122] 2.2'-(7-(2-(((R)-l-((2-(((S)-l-amino-3-(lH-indol-3-yl)-l-oxopropan-2-yl)amino)- 2-oxoethyl)amino)-3-hydroxy-l-oxopropan-2-yl)amino)-2-oxoethyl)-l,4, 7 -triazonane- 1,4-diyl)diacetic acid, NOTA-M-G-W-CONH2. Compound 15 was synthesized using the general coupling strategy outlined in FIG. 3 and the section titled "Synthesis of Model Tripeptides" above from compound Fmoc-Trp(Boc)-RA.

[0123] 2,2'-(7-(2-(((R)-l-((2-(((S)-l-amino-3-(lH-indol-3-yl)-l-oxopropan-2-yl)amino)- 2-oxoethyl)amino)-3-hydroxy-l-oxopropan-2-yl)amino)-2-oxoethyl)-l,4, 7 -triazonane- 1,4-diyl)diacetic acid, NOTA-NMeS-G-W-CONH2. Compound 16 was synthesized using the general coupling strategy' outlined in FIG. 3 and the section titled "Synthesis of Model Tripeptides" above from compound Fmoc-Trp(Boc)-RA.

[0124] 2,2'-(7-(2-(((R)-l-((2-(((S)-l-amino-3-(lH-indol-3-yl)-l-oxopropan-2-yl)amino)- 2-oxoethyl)amino)-3-hydroxy-l-oxopropan-2-yl)amino)-2-oxoethyl)-l ,4, 7 -triazonane- 1,4-diyl)diacetic acid, NOTA-NMeG-G-W-CONH2. Compound 17 was synthesized using the general coupling strategy' outlined in FIG. 3 and the section titled "Synthesis of Model Tripeptides" above from compound Fmoc-Trp(Boc)-RA.

[0125] 2, 2 '-(7-(2-( ( (R)-l-( ( 2-( ((S)-l-amino-3-( lH-indol-3-yl)-l-oxopropan-2-yl)amino)- 2-oxoethyl)amino)-3-hydroxy-l-oxopropan-2-yl)amino)-2-oxoethyl)-l,4, 7 -triazonane -1,4-diyl)diacetic acid, NOTA-NMeM-G-W-CONH2. Compound 18 was synthesized using the general coupling strategy' outlined in FIG. 3 and the section titled "Synthesis of Model Tripeptides" above from compound Fmoc-Trp(Boc)-RA.

[0126] (7R,21S,25S)-l-(4, 7-bis(carboxymethyl)-l,4,7-triazonan-l-yl)-7-(4-hydroxy-3-iodobenzyl)-2, 5, 8, 15, 23-pentaoxo-3, 6, 9, 16, 22, 24-hexaazaheptacosane-21, 25, 27 -tricarboxylic acid, NO2A-G-T-Ahx-KuE. Compound 19 (200 mg, 0.128 mmol) was synthesized using the general coupling strategy’ outlined in FIGS. 17A and 17B and the section titled "Synthesis pathways for DUPA-Targeting Peptides" above.

[0127] (4S.7R21S.25S)-l-(4, 7-bis(carboxymethyl)-l,4, 7-triazonan-l-yl)-7-(4-hydroxy-3-iodobenzyl)-4-(hydroxymethyl)-2, 5, 8,15, 23-pentaoxo-3, 6, 9, 16, 22, 24-hexaazaheptacosane-Atty. Dkt. No. 00300-0450-PCT21.25.27-tricarboxylic acid, N02A-S-T-Ahx-KuE. Compound 20 (200 mg, 0.128 mmol) was synthesized using the general coupling strategy outlined in FIGS. 17A and 17B and the section titled "Synthesis pathways for DUPA-Targeting Peptides" above.

[0128] (5S,8R,22S,26S)-5-(2-(4, 7-bis(carboxymethyl)-l,4, 7-triazonan-l-yl)acetamido)-8- (4-hydroxy-3-iodobenzyl)-6,9, 16,24-tetraoxo-2-thia-7. 10, 17,23,25-pentaazaoctacosane- 22.26.28-tricarboxylic acid, NO2A-M-T-Ahx-KuE. Compound 21 (200 mg, 0.128 mmol) was synthesized using the general coupling strategy outlined in FIGS. 17A and 17B and the section titled "Synthesis pathways for DUPA-Targeting Peptides" above.

[0129] (7R.21S,25S)-l-(4, 7-bis(carboxymethyl)-l .4.7-triazonan-l-yl)-7-(4-hydroxy-3-iodobenzyl)-3-methyl-2, 5, 8,15.23-pentaoxo-3.6.9,16, 22,24-hexaazaheptacosane-21 ,25.27-tricarboxylic acid, NO2A-NMeG-T-Ahx-KuE. Compound 22 (200 mg, 0.128 mmol) was synthesized using the general coupling strategy outlined in FIGS. 17A and 17B and the section titled "Synthesis pathways for DUPA-Targeting Peptides" above.

[0130] (7R,21S,25S)-l-(4, 7-bis(carboxymethyl)-l ,4, 7-triazonan-l-yl)-7-(4-hydroxy-3-iodobenzyl)-3-methyl-2, 5, 8, 15, 23-pentaoxo-3, 6, 9, 16, 22.24-hexaazaheptacosane-21 , 25.27-tricarboxylic acid, NO2A-NMeS-T-Ahx-KuE. Compound 23 (200 mg.0.128 mmol) was synthesized using the general coupling strategy outlined in FIGS. 17A and 17B and the section titled "Synthesis pathways for DUPA-Targeting Peptides" above.

[0131] (5S,8R,22S,26S)-5-(2-(4, 7-bis(carboxymethyl)-l .4, 7-triazonan-l-yl)-N-methylacetamido)-8-( 4-hydroxy-3-iodobenzyl)-6, 9, 16, 24-tetraoxo-2-thia- 7,10,17, 23, 25-pentaazaoctacosane-22,26,28-tricarboxylic acid, NO2A-NMeM-T-Ahx-KuE. Compound 24 (200 mg.0.128 mmol) was synthesized using the general coupling strategy outlined in FIGS.17A and 17B and the section titled "Synthesis pathways for DUPA-Targeting Peptides" above.

[0132] General Complexation Protocol

[0133] A 5 mg aliquot of the peptide was dissolved in 500 pL of room-temperature distilled water. 2.5 equivalents of thenatGa3+,natFe3+,natZn2+, ornatCo2+salts were added to the solution. The reactions proceed for 1 hour. Full complexation was confirmed by LCMS. The complex was then purified via Sep-Pak Cl 8 short cartridge. The complexes were characterized by NMR and HRESI.Atty. Dkt. No. 00300-0450-PCT

[0134] Cleavage of the cold model complexes was observed under 80 °C and pH values of 7.4. Aqueous buffer solutions were prepared using 10 mM NaOAc (pH 4.5). 10 mM MOPS (pH 6.5), 10 mM HEPES (pH 7.4), and 10 mM EPPS (pH 8.5) which were subsequently adjusted to the desired pH using NaOH and / or HC1. The complex was formed under room temperature and pH 4.5 10 mM NaOAc buffer. An aliquot of the complex was placed in an aqueous solution of the desired pH and incubated for 24 hours at 80 °C. The reaction was monitored via analytical HPLC.

[0135] General radiolabeling protocol

[0136] Radiolabeling with68Ga.

[0137] 68Ga-chloride was obtained from the University of Wisconsin Radiopharmaceutical Production Facility, at an average specific activity of 740.0 MBq / mL. For radio-labeling of model tripeptides and NOTA-HAS-DUPA conjugates, a 35 pL aliquot containing 25.9 MBq of68GaCl was added to the ligand (10 nmol, 265 pL) in 0.067 M of sodium acetate (NaOAc) buffer pH 4.5. Radiolabeling was completed after 10 minutes at room temperature. The pH of the solution was then adjusted to 7.4 by pH 7.40.2 M HEPES buffer. The radiolabeled conjugate was cleaved at 80 °C for 5 hours and characterized with radio-HPLC.

[0138] Radiolabeling with67Ga.

[0139] 67Ga-citrate was purchased from Jubilant Radiopharma at an average activity of 206.0 MBq / mL. The67Ga-citrate solution was first converted to67GaCh using an established solid-phase extraction protocol. The resulting average specific activity of the obtained67Ga-chloride solution used for solution radio-labeling was 80.5 MBq / mL. For radio-labeling of NOTA-HSA-DUPA conjugates, a 50 pL aliquot containing 40.5 MBq of67GaCh was added to the conjugate (10 nmol, 300 pL) in 0.067 M solium acetate (NaOAc) pH 4.5 buffer.

[0140] Synthesis of68Ga- radiolabeled complexes.

[0141] 6SGallium (S)-2,2'-(7-(2-((2-((2-((l-amino-3-(lH-indol-3-yl)-l-oxopropan-2-yl)amino)-2-oxoethyl)amino)-2-oxoethyl)amino)-2-oxoethyl)-l.4.7 -triazonane- 1.4-diyl)diacetic acid, [68Ga][Ga(NO2A)]+-G-G-W-CONH2. Compound was synthesized using the general radiolabeling protocol from compound 10.Atty. Dkt. No. 00300-0450-PCT

[0142] 68Gallium (S)-2,2'-(7-(2-((3-((2-((l-amino-3-(lH-indol-3-yl)-l-oxopropan-2-yl)amino)-2-oxoethyl)amino)-3-oxopropyl)amino)-2-oxoethyl)-l, 4, 7 -triazonane- 1, 4-diyl) diacetic acid, [68Ga][Ga(NO2A)]+-pA-G-W-CONH2. Compound was synthesized using the general radiolabeling protocol from compound 11.

[0143] 68Gallium 2, 2 '-(7-( 2-(((R)-l-((2-(((S)-l -amino- 3-( 1 H-indol-3-yl)-l -oxopropan-2-yl)amino)-2-oxoethyl)amino)-l-oxopropan-2-yl)amino)-2-oxoethyl)-l,4, 7-triazonane-l,4-diyl)diacetic acid, [68Ga][Ga(NO2A)]+-A-G-W-CONH2. Compound was synthesized using the general radiolabeling protocol from compound 12.

[0144] 68Gallium 2,2'-(7-(2-(((R)-l-((2-(((S)-l-amino-3-( lH-indol-3-yl)-l-oxopropan-2-yl)amino)-2-oxoethyl)amino)-3-hydroxy-l-oxopropan-2-yl)amino)-2-oxoethyl)-1.4, 7-triazonane-1 ,4-diyl)diacetic acid, [68Ga][Ga(NO2A)]+-S-G-W-CONH2. Compound was synthesized using the general radiolabeling protocol from compound 13.

[0145] 68Gallium 2,2'-(7-(2-(((2R3R)-l-((2-(((S)-l-ammo-3-(lH-indol-3-yl)-l-oxopropan-2-yl)amino)-2-oxoethyl)amino)-3-hydroxy-l-oxobutan-2-yl)amino)-2-oxoethyl)-1 ,4, 7-triazonane-l ,4-diyl)diacetic acid, [68Ga][Ga(NO2A)]+-T-G-W-CONH2. Compound was synthesized using the general radiolabeling protocol from compound 14.

[0146] 6SGallium 2,2'-(7-(2-(((R)-l-((2-(((S)-l-amino-3-(lH-indol-3-yl)-l-oxopropan-2-yl)amino)-2-oxoethyl)amino)-4-(methylthio)-l-oxobutan-2-yl)amino)-2-oxoethyl)-l,4, 7-triazonane-l,4-diyl)diacetic acid, [68Ga][Ga(NO2A)]+-M-G-W-CONH2. Compound was synthesized using the general radiolabeling protocol from compound 15.

[0147] 68Gallium 2, 2 '-(7-(2-( ((R)-l-((2-( ((S)-l-amino-3-( lH-indol-3-yl)-l -oxopropan-2-yl)amino)-2-oxoethyl)amino)-3-hydroxy-l-oxopropan-2-yl)(methyl)amino)-2-oxoethyl)-l,4, 7-triazonane-l,4-diyl)diacetic acid, [68Ga][Ga(NO2A)]+-NMeS-G-W-CONH2. Compound was synthesized using the general radiolabeling protocol from compound 16.

[0148] 68Gallium (S)-2,2'-(7-(2-((2-((2-((l-amino-3-(lH-indol-3-yl)-l-oxopropan-2-yl)amino)-2-oxoethyl)amino)-2-oxoethyl)(methyl)amino)-2-oxoethyl)-l ,4, 7 -triazonane- 1 ,4-diyl) diacetic acid, [68Ga][Ga(NO2A)]+-NMeG-G-W-CONH2. Compound was synthesized using the general radiolabeling protocol from compound 17.

[0149] 68Gallium 2,2'-(7-(2-(((R)-l-((2-(((S)-l-amino-3-(lH-indol-3-yl)-l-oxopropan-2-yl)amino)-2-oxoethyl)amino)-4-(melhyllhio)-l-oxobulan-2-yl)(methyl)amino)-2-oxoelhyl)-Atty. Dkt. No. 00300-0450-PCT1,4, 7-triazonane-l,4-diyl)diacetic acid, [68Ga][Ga(NO2A)]+-NMeM-G-W-CONH2. Compound was synthesized using the general radiolabeling protocol from compound 17.

[0150] Metal Complex Speciation

[0151] NMR (!H) spectroscopic titrations were performed on a Bruker Avance 400 instrument at 25 °C. Each NMR sample was prepared by adding TMSP-d4 (10 pL, 10 pM) to 600 pL of the pH-adjusted sample in water. For metal-ligand complex speciation, pH-adjusted aliquots were prepared from stock solutions ([Ligand] = 0.1 mM; [Ga3+] = 0.1 mM; 0.1 M KC1; 0.01 M HC1). A water suppression method was employed in the absence of the deuterated solvent. 'H spectra were referenced to TMSP-d4 (0.00 pm).

[0152] In vitro and In Vivo Experiments

[0153] All animal experiments were conducted with the approval of the University of Wisconsin-Madison Institutional Animal Care and Use Committee (IACUC). All studies were conducted in accordance with the relevant guidelines and regulations and approved under protocol number M006738 (PI: Boros) and conducted at UW-Madison School of Medicine and Public Health, at the Small Animal Imaging and Radiotherapy Facility (SAIRF).

[0154] HAS Binding Assay

[0155] To measure HSA binding of the complexes, a 0.05 mM solution (determined by ICP-OES) of the correspondingnatGa complex in 4.5%w / v HSA was prepared and pipetted into an Amicon Ultra-0.5 Centrifugal Filter Unit (50 kDa cutoff, Millipore. UFC500396). The mixture was incubated at 37 °C for 15 min and subsequently centrifuged at 14000 rpm for 15 min. Binding was determined by measurement of Ga content in the filtrate by ICP-OES and compared to non-specific binding to the filter in absence of HSA. Experiments were conducted in accordance with published procedures.

[0156] In Vivo Biodistribution and Pharmacokinetics in NU / J Mice

[0157] All animal experiments were conducted with the approval of the University of Wisconsin-Madison Institutional Animal Care and Use Committee (IACUC). All studies were conducted in accordance with the relevant guidelines and regulations and approved under protocol number M006738 (PI: Boros) and conducted at UW-Madison School of Medicine and Public Health, at the Small Animal Imaging and Radiotherapy FacilityAtty. Dkt. No. 00300-0450-PCT(SAIRF). Male NU / J mice (5 weeks) were purchased from Jackson Laboratory.[68Ga][Ga(20)], [68Ga][Ga(21)], [68Ga][Ga(22)]. [68Ga][Ga(23)], [68Ga][Ga(24)], and [68Ga][Ga(2)] were intravenously injected via tail vein catheter in tumor-implanted mice. Mice were imaged by PET / CT scanner at 30-minute, 60-minute, and 90-minute post injection time points and sacrificed at 120-minute post-injection time point, and select organs (blood, heart, liver, kidneys, spleen, stomach, small intestines, large intestines, muscle, bone, tumor, brain, and tail) were harvested. Radioactivity was counted by using a gamma counter, and the radioactivity associated with each organ was expressed as percent injected dose per gram tissue (%ID / g).

[0158] Tumor Xenograft Model

[0159] Tumor xenograft model PC3-Pip and PC3-flu cells were maintained in DMEM with 5% FBS at 37 °C and 5% CO2. Male NU / J mice (7 weeks, Jackson Laboratory) were implanted subcutaneously on the right shoulder with 1 x 106 PC3-Pip cells and on the left shoulder with 1 x io6PC3-flu cells suspended in Matrigel (1:1). When tumors reached 500 mm3, the mice were randomized based on tumor volumes into 4 groups (4 mice per group) for single dose compound administration.

[0160] Positron emission tomography / computed tomography (PET / CT) imaging:PET / CT imaging was performed using a Siemens Inveon Hybrid MicroPET / CT Scanner (Siemens Medical Solutions USA, Inc., Knoxville, TN). Mice were anesthetized with 4% isoflurane gas and anesthesia was maintained during scans at 2% isoflurane in oxygen. CT scans were acquired prior to PET scans for anatomical coregistration as well as attenuation correction. CT scan parameters were as follows: 220 rotation degrees, 120 rotation steps, binning factor of 4, exposure time of 250 ms, x-ray energy of 80 kVp, 1 mA current, and 105 pm resolution. PET scans were acquired with 40 million coincidence events per mouse, an energy window of 350-650 keV, and a timing window of 3.432 ns. Quantification of PET / CT images was performed in an Inveon Research Workstation and data is expressed as percent injected dose per gram of tissue (% ID / cc).

[0161] 68Ga Tracer Preparation Procedure

[0162] To an aqueous solution of sodium acetate (20 pL, IM, pH 4.5) was added 80 pL H2O and ligand stock solution (2-10 pL, 10 nmol) of known concentration as determined UV-vis spectroscopy, followed by an aliquot of the [68Ga]GaC13 stock (30 - 35 pL. -600 pCi).Atty. Dkt. No. 00300-0450-PCTTotal reaction volume was 150 pL. The mixtures were incubated at room temperature for 15 min. The radiolabeled compounds were purified via radio-HPLC to achieve a radiochemical purity of > 98%.

[0163] Metabolite Analysis

[0164] The 100 pL mouse urine was collected and analyzed by radio-HPLC during biodistribution studies. If mouse urine was less than 100 pL, the urine was diluted by 1 x PBS to total volume of 100 pL. If the total activity was below the sensitivity of the radio detector, fractions were collected every 30 seconds. The metabolite traces were reconstructed by quantifying each fraction using a gamma counter.

[0165] General PD1 and PD2 Complexation Protocol

[0166] To a ligand stock solution of known concentration, as determined by UV-vis spectroscopy, 1 equivalence ofZn2+, Ga3+, Fe3+stock of known concentrations, as determined by ICP, was added. Total reaction volume was 150 pL. The reactions proceeded at room temperature (~23 °C) for 1 hour. The complexation was confirmed by LC-MS (method).

[0167] Cell viability studies

[0168] MTT assays were carried out for MMAE NO2A conjugates to assess toxicity towards HeLa cells. 96-well plates were seeded with 3000 cells per well (100 uL) and incubated for 24 hours in DMEM supplemented with 10% FBS and 5% streptomycin. A dilution series of different metal complexes in PBS was added and cells were incubated for 24, 48, and 72 hours. The cell media was aspirated and replenished in each well with 100 pL fresh media. MTT dye in PBS (5 mg / mL, 10 pL) was added to each well. Cells were incubated for 3 hours. All liquids were removed and 100 pL DMSO was added to each well. The absorbance at 540 nm was measured. Each 96-well plate contained 3 replicates for each concentration.

[0169] The word "illustrative" is used herein to mean serving as an example, instance, or illustration. Any aspect or design described herein as "illustrative" is not necessarily to be construed as preferred or advantageous over other aspects or designs. Further, for the purposes of this disclosure and unless otherwise specified, "a" or "an" means "one or more.”

[0170] The foregoing description of illustrative embodiments of the invention has been presented for purposes of illustration and of description. It is not intended to be exhaustive orAtty. Dkt. No. 00300-0450-PCTto limit the invention to the precise form disclosed, and modifications and variations are possible in light of the above teachings or may be acquired from practice of the invention. The embodiments were chosen and described in order to explain the principles of the invent on and as practical applications of the invention to enable one skilled in the art to utilize the invention in various embodiments and with various modifications as suited to the particular use contemplated. It is intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

[0171] If not already included all numeric values of parameters in the present disclosure are proceeded by the term ’‘about” which means approximately. This encompasses those variations inherent to the measurement of the relevant parameter as understood by those of ordinary7skill in the art. This also encompasses the exact value of the disclosed numeric value and values that round to the disclosed numeric value.

Claims

Atty. Dkt. No. 00300-0450-PCTWHAT IS CLAIMED IS:

1. A drug release composition comprising:a metal chelate complex comprising a trivalent Lewis acidic metal chelated by 1,4,7-triazacyclononane-l,4,7-triacetic acid (NOTA);a biological targeting vector conjugated to the 1.4.7-triazacyclononane-l,4,7-triacetic acid (NOTA); anda therapeutic agent conjugated to the l,4,7-triazacyclononane-l,4,7-triacetic acid (NOTA) via an amino acid linker comprising a metal chelate complex- adjacent amide bond;wherein the metal chelate complex forms an N3O3 coordination isomer or an N4O2 coordination isomer in aqueous solution at a pH in the range from 6.5 to 8.5 and a temperature in the range from 30 °C to 90 °C.

2. The drug release composition of claim 1, wherein the amino acid linker comprises a metal chelate complex-adjacent amino acid residue that does not have a hydroxyl group.

3. The drug release composition of claim 1, wherein the metal chelate complex forms the N3O3 coordination isomer at the pH in the range from 6.5 to 8.5 and the temperature in the range from 30 °C to 90 °C.

4. The drug release composition of claim 3, wherein the amide of the amide bond is a tertiary amide.

5. The drug release composition of claim 4, wherein the amino acid linker comprises a metal chelate complex-adjacent amino acid residue that is an N-methylated.

6. The drug release composition of claim 5, wherein the N-methylated, metal chelate complex-adjacent amino acid residue is aNMeGly residue.

7. The drug release composition of claim 5, wherein the N-methylated, metal chelate complex-adjacent amino acid residue is aNMeMet residue.

8. The drug release composition of claim 5. wherein the N-methylated, metal chelate complex-adjacent amino acid residue is aNMeSer residue.Atty. Dkt. No. 00300-0450-PCT9. The drug release composition of claim 1, wherein the metal chelate complex-adjacent amino acid residue of the amino acid linker is an Ala residue.

10. The drug release composition of claim 1, wherein the metal chelate complex-adjacent amino acid residue of the amino acid linker is an Met residue.

11. The drug release composition of claim 1 , wherein the trivalent Lewis acidic metal is a radionuclide.

12. The drug release composition of claim 11, wherein the radionuclide is68Ga.

13. The drug release composition of claim 11, wherein the radionuclide is177Lu161Tb,18F,52Mn,44Sc, or86 / 90Y.

14. The drug release composition of claim 1 , wherein the therapeutic agent is an antibody-drug conjugate or a campthothecin or campthothecin derivative.

15. The drug release composition of claim 1, wherein the biological targeting vector comprises a hormone, signaling molecule, antibody, antibody fragment, binding protein, binding peptide, binding polypeptide, enzyme, nucleobase-containing moiety, or lectin.

16. The drug release composition of claim 5, wherein the trivalent Lewis acidic metal is a radionuclide.

17. The drug release composition of claim 16, wherein the N-methylated amino acid residue is aNMeGly residue, a ^“Met residue, or aNKfcSer residue and the radionuclide is68Ga.

18. The drug release composition of claims 17, wherein the biological targeting vector comprises a hormone, signaling molecule, antibody, antibody fragment, binding protein, binding peptide, binding polypeptide, enzyme, nucleobase-containing moiety, or lectin.

19. The drug release composition of claim 18, wherein the therapeutic agent is an antibody-drug conjugate or a campthothecin or campthothecin derivative.Atty. Dkt. No. 00300-0450-PCT20. A method of treating a subject in need of treatment, the method comprising administering to the subject a therapeutically effective amount of drug release composition of claim 1.

21. The method of claim 20, wherein the amino acid linker comprises a metal chelate complex-adjacent amino acid residue that is an N-methylated.

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