1,4,7-Triazacyclononane-Based Chelators for the Complexation of [¹⁸⁶Re]Re- and [^99mTc]Tc-Tricarbonyl Cores

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This article references 22 other publications.

1. 1

   Aliev, R. A.; Kormazeva, E. S.; Furkina, E. B.; Moiseeva, A. N.; Zagryadskiy, V. A. Rhenium radioisotopes: Production, properties, and targeted delivery using nanostructures. Nanotechnol. Russ. 2020, 15 (7–8), 428– 436,  DOI: 10.1134/S1995078020040023

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   Rhenium Radioisotopes: Production, Properties, and Targeted Delivery Using Nanostructures

   Aliev, R. A.; Kormazeva, E. S.; Furkina, E. B.; Moiseeva, A. N.; Zagryadskiy, V. A.

   Nanotechnologies in Russia (2020), 15 (7-8), 428-436CODEN: NRAUAI; ISSN:1995-0799. (Pleiades Publishing, Ltd.)

   A review. Two rhenium isotopes (186Re and 188Re) are of interest for nuclear medicine. The multiplicity of oxidn. states and varying coordination chem. of rhenium provide opportunities for the synthesis of various bioconjugates, including those based on nanosized carriers (liposomes, dendrimers, and inorg. nanoparticles). This review analyzes nuclear reactions that lead to the formation of rhenium isotopes and methods of their isolation from targets, as well as different variants of the application of rhenium in nuclear medicine.
2. 2

   Alberto, R.; Schibli, R.; Egli, A.; Schubiger, A. P.; Abram, U.; Kaden, T. A. A novel organometallic aqua complex of technetium for the labeling of biomolecules: Synthesis of [^99mTc(OH₂)₃(CO)₃]⁺ from [^99mTcO₄]⁻ in aqueous solution and its reaction with a bifunctional ligand. J. Am. Chem. Soc. 1998, 120 (31), 7987– 7988,  DOI: 10.1021/ja980745t

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   A Novel Organometallic Aqua Complex of Technetium for the Labeling of Biomolecules: Synthesis of [99mTc(OH2)3(CO)3]+ from [99mTcO4]- in Aqueous Solution and Its Reaction with a Bifunctional Ligand

   Alberto, Roger; Schibli, Roger; Egli, Andre; Schubiger, August P.; Abram, Ulrich; Kaden, Thomas A.

   Journal of the American Chemical Society (1998), 120 (31), 7987-7988CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)

   [99mTc(CO)3(OH2)3]+, readily formed from [99Tc(CO)3Cl3]2- in H2O, was prepd. from 99mTcO4- and CO in THF and in saline soln. with small amts. of NaBH4 as reducing agent. [188ReO4]- reacted similarly. [99Tc(CO)3Cl3]2- or [99Tc(CO)3(OH2)3]+reacted with picolinamine-N,N-diacetic acid (HPADA) to give [99Tc(CO)3(PADA)] (I). I is orthorhombic, space group Pbca, a 13.225(1), b 14.660(1), c 14.942(2) Å, Z = 8, R = 0.0386, Rw = 0.1082. In H2O I is readily converted to [99Tc(CO)3(OH2)3]+. I is stable in serum.
3. 3

   Schibli, R.; Schwarzbach, R.; Alberto, R.; Ortner, K.; Schmalle, H.; Dumas, C.; Egli, A.; Schubiger, P. A. Steps toward high specific activity labeling of biomolecules for therapeutic application: Preparation of precursor [¹⁸⁸Re(H₂O)₃(CO)₃]⁺ and synthesis of tailor-made bifunctional ligand systems. Bioconjugate Chem. 2002, 13 (4), 750– 756,  DOI: 10.1021/bc015568r

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   Steps toward High Specific Activity Labeling of Biomolecules for Therapeutic Application: Preparation of Precursor [188Re(H2O)3(CO)3]+ and Synthesis of Tailor-Made Bifunctional Ligand Systems

   Schibli, Roger; Schwarzbach, Rolf; Alberto, Roger; Ortner, Kirstin; Schmalle, Helmut; Dumas, Cecile; Egli, Andre; Schubiger, P. August

   Bioconjugate Chemistry (2002), 13 (4), 750-756CODEN: BCCHES; ISSN:1043-1802. (American Chemical Society)

   Two kit prepns. of the organometallic precursor [188Re(H2O)3(CO)3]+ in aq. media are presented. Method A uses gaseous carbon monoxide and amine borane (BH3·NH3) as the reducing agent. In method B CO(g) is replaced by K2[H3BCO2] that releases carbon monoxide during hydrolysis. Both procedures afford the desired precursor in yields >85% after 10 min at 60 °C. HPLC and TLC analyses revealed 7 ± 3% of unreacted 188ReO4- and <5% of colloidal 188ReO2. Solns. of up to 14 GBq/mL Re-188 have been successfully carbonylated with these two methods. The syntheses of two tailor-made bifunctional ligand systems for the precursor [188Re(H2O)3(CO)3]+ are presented. The tridentate chelates consist of a bis[imidazol-2-yl]methylamine or an iminodiacetic acid moiety, resp. Both types of ligand systems have been prepd. with alkyl spacers of different length and a pendent primary amino or carboxylic acid functionality, enabling the amidic linkage to biomols. The tridentate coordination of the ligands to the rhenium-tricarbonyl core could be elucidated on the macroscopic level by X-ray structure analyses and 1D and 2D NMR expts. of two representative model complexes. On the nca level, the ligands allow labeling yields >95% with [188Re(H2O)3(CO)3]+ under mild reaction conditions (PBS buffer, 60 °C, 60 min) at ligand concns. between 5 × 10-4 M and 5 × 10-5 M. Thus, specific activities of 22-220 GBq per μmol of ligand could be achieved. Incubation of the corresponding Re-188 complexes in human serum at 37 °C revealed stabilities between 80 ± 4% and 45 ± 10% at 24 h, resp., and 63 ± 3% and 34 ± 3% at 48 h postincubation in human serum depending on the chelating system. Decompn. product was mainly 188ReO4-. The routine kit-prepn. of the precursor [188Re(H2O)3(CO)3]+ in combination with tailor-made ligand systems enables the organometallic labeling of biomols. with unprecedented high specific activities.
4. 4

   Alberto, R.; Egli, A.; Abram, U.; Hegetschweiler, K.; Gramlich, V.; Schubiger, P. A. Synthesis and reactivity of [NEt₄]₂[ReBr₃(CO)₃]. Formation and structural characterization of the clusters [NEt₄][Re₃(μ₃-OH)(μ-OH)₃(CO)₉] and [NEt₄][Re₂(μ-OH)₃(CO)₆] by alkaline treatment. J. Chem. Soc., Dalton Trans. 1994, 19, 2815– 2820,  DOI: 10.1039/DT9940002815

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   Abram, U.; Alberto, R. Technetium and rhenium - coordination chemistry and nuclear medical applications. J. Braz. Chem. Soc. 2006, 17 (8), 1486– 1500,  DOI: 10.1590/S0103-50532006000800004

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   Technetium and rhenium - coordination chemistry and nuclear medical applications

   Abram, Ulrich; Alberto, Roger

   Journal of the Brazilian Chemical Society (2006), 17 (8), 1486-1500CODEN: JOCSET; ISSN:0103-5053. (Sociedade Brasileira de Quimica)

   A review. Coordination compds. of the radioactive element technetium are well established in diagnostic nuclear medicine, and various complexes of the γ-emitting nuclide 99mTc are routinely used for organ imaging. Modern trends in the radiopharmaceutical chem. of technetium focus on the 'labeling' of biol. active mols. such as peptides, steroids or other receptor-seeking units. This requires more knowledge about the coordination chem. of the artificial transition metal, particularly with regard to stable or kinetically inert coordination spheres, which allow couplings to biomols. following a bioconjugate approach. The dominant role of technetium compds. in diagnostic procedures recommends the β--emitting rhenium isotopes 186Re and 188Re for applications in nuclear-medical therapy. 188Re is readily available from an 188W/188Re radionuclide generator system and general synthetic approaches can be adopted from the established technetium chem.
6. 6

   Melis, D. R.; Burgoyne, A. R.; Ooms, M.; Gasser, G. Bifunctional chelators for radiorhenium: Past, present and future outlook. RSC Med. Chem. 2022, 13 (3), 217– 245,  DOI: 10.1039/D1MD00364J

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   Bifunctional chelators for radiorhenium: past, present and future outlook

   Melis, Diana R.; Burgoyne, Andrew R.; Ooms, Maarten; Gasser, Gilles

   RSC Medicinal Chemistry (2022), 13 (3), 217-245CODEN: RMCSEZ; ISSN:2632-8682. (Royal Society of Chemistry)

   A review. Targeted radionuclide therapy (TRNT) is an ever-expanding field of nuclear medicine that provides a personalised approach to cancer treatment while limiting toxicity to normal tissues. It involves the radiolabelling of a biol. targeting vector with an appropriate therapeutic radionuclide, often facilitated by the use of a bifunctional chelator (BFC) to stably link the two entities. The radioisotopes of rhenium, 186Re (t1/2 = 90 h, 1.07 MeV β-, 137 keV γ (9%)) and 188Re (t1/2 = 16.9 h, 2.12 MeV β-, 155 keV γ (15%)), are particularly attractive for radiotherapy because of their convenient and high-abundance β--particle emissions as well as their imageable γ-emissions and chem. similarity to technetium. As a transition metal element with multiple oxidn. states and coordination nos. accessible for complexation, there is great opportunity available when it comes to developing novel BFCs for rhenium. The purpose of this review is to provide a recap on some of the past successes and failings, as well as show some more current efforts in the design of BFCs for 186/188Re. Future use of these radionuclides for radiotherapy depends on their cost-effective availability and this will also be discussed. Finally, bioconjugation strategies for radiolabelling biomols. with 186/188Re will be touched upon.
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   Boros, E.; Packard, A. B. Radioactive transition metals for imaging and therapy. Chem. Rev. (Washington, DC, U. S.) 2019, 119 (2), 870– 901,  DOI: 10.1021/acs.chemrev.8b00281

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   Radioactive Transition Metals for Imaging and Therapy

   Boros, Eszter; Packard, Alan B.

   Chemical Reviews (Washington, DC, United States) (2019), 119 (2), 870-901CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)

   Nuclear medicine is composed of two complementary areas, imaging and therapy. Positron emission tomog. (PET) and single-photon imaging, including single-photon emission computed tomog. (SPECT), comprise the imaging component of nuclear medicine. These areas are distinct in that they exploit different nuclear decay processes and also different imaging technologies. In PET, images are created from the 511 keV photons produced when the positron emitted by a radionuclide encounters an electron and is annihilated. In contrast, in single-photon imaging, images are created from the γ rays (and occasionally X-rays) directly emitted by the nucleus. Therapeutic nuclear medicine uses particulate radiation such as Auger or conversion electrons or β- or α particles. All three of these technologies are linked by the requirement that the radionuclide must be attached to a suitable vector that can deliver it to its target. It is imperative that the radionuclide remain attached to the vector before it is delivered to its target as well as after it reaches its target or else the resulting image (or therapeutic outcome) will not reflect the biol. process of interest. Radiochem. is at the core of this process, and radiometals offer radiopharmaceutical chemists a tremendous range of options with which to accomplish these goals. They also offer a wide range of options in terms of radionuclide half-lives and emission properties, providing the ability to carefully match the decay properties with the desired outcome. This Review provides an overview of some of the ways this can be accomplished as well as several historical examples of some of the limitations of earlier metalloradiopharmaceuticals and the ways that new technologies, primarily related to radionuclide prodn., have provided solns. to these problems.
8. 8

   Makris, G.; Radford, L. L.; Kuchuk, M.; Gallazzi, F.; Jurisson, S. S.; Smith, C. J.; Hennkens, H. M. NOTA and NODAGA [^99mTc]Tc- and [¹⁸⁶Re]Re-tricarbonyl complexes: Radiochemistry and first example of a [^99mTc]Tc-NODAGA somatostatin receptor-targeting bioconjugate. Bioconjugate Chem. 2018, 29 (12), 4040– 4049,  DOI: 10.1021/acs.bioconjchem.8b00670

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   NOTA and NODAGA [99mTc]Tc- and [186Re]Re-Tricarbonyl Complexes: Radiochemistry and First Example of a [99mTc]Tc-NODAGA Somatostatin Receptor-Targeting Bioconjugate

   Makris, George; Radford, Lauren L.; Kuchuk, Marina; Gallazzi, Fabio; Jurisson, Silvia S.; Smith, Charles J.; Hennkens, Heather M.

   Bioconjugate Chemistry (2018), 29 (12), 4040-4049CODEN: BCCHES; ISSN:1043-1802. (American Chemical Society)

   With the long-term goal of developing theranostic agents for applications in nuclear medicine, in this work we evaluated the well-known NOTA and NODAGA chelators as bifunctional chelators (BFCs) for the [99mTc/186Re]Tc/Re-tricarbonyl core. In particular, we report model complexes of the general formula fac-[M(L)(CO)3]+ (M = Re, 99mTc, 186Re) where L denotes NOTA-Pyr (1) or NODAGA-Pyr (2), which are derived from conjugation of NOTA/NODAGA with pyrrolidine (Pyr). Further, as proof-of-principle, we synthesized the peptide bioconjugate NODAGA-sst2-ANT (3) and explored its complexation with the fac-[Re(CO)3]+ and fac-[99mTc][Tc(CO)3]+ cores; sst2-ANT denotes the somatostatin receptor (SSTR) antagonist 4-NO2-Phe-c(DCys-Tyr-DTrp-Lys-Thr-Cys)-DTyr-NH2. Rhenium complexes Re-1 through Re-3 were synthesized and characterized spectroscopically, and receptor binding affinity was demonstrated for Re-3 in SSTR-expressing cells (AR42J, IC50 = 91 nM). Radiolabeled complexes [99mTc]Tc/[186Re]Re-1/2 and [99mTc]Tc-3 were prepd. in high radiochem. yield (>90%, detd. by radio-HPLC) by reacting [99mTc]/[186Re][Tc/Re(OH2)3(CO)3]+ with 1-3 and correlated well with the resp. Re-1 through Re-3 stds. in comparative HPLC studies. All radiotracers remained intact through 24 h (99mTc-labeled complexes) or 48 h (186Re-labeled complexes) against 1 mM L-histidine and 1 mM L-cysteine (pH 7.4, 37 °C). Similarly, rat serum stability studies displayed no decompn. and low nonspecific binding of 9-24% through 4 h. Biodistribution of [99mTc]Tc-3 in healthy CF-1 mice demonstrated a favorable pharmacokinetic profile. Rapid clearance was obsd. within 1 h post-injection, predominantly via the renal system (82% of the injected dose was excreted in urine by 1 h), with low kidney retention (% ID/g: 11 at 1 h, 5 at 4 h, and 1 at 24 h) and low nonspecific uptake in other organs/tissues. Our findings establish NOTA and NODAGA as outstanding BFCs for the fac-[M(CO)3]+ core in the design and development of organometallic radiopharmaceuticals. Future in vivo studies of [99mTc]Tc- and [186Re]Re-tricarbonyl complexes of NODAGA/NOTA-biomol. conjugates will further probe the potential of these chelates for nuclear medicine applications in diagnostic imaging and targeted radiotherapy, resp.
9. 9

   Joshi, T.; Kubeil, M.; Nsubuga, A.; Singh, G.; Gasser, G.; Stephan, H. Harnessing the coordination chemistry of 1,4,7-triazacyclononane for biomimicry and radiopharmaceutical applications. ChemPlusChem. 2018, 83 (7), 554– 564,  DOI: 10.1002/cplu.201800103

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   Harnessing the Coordination Chemistry of 1,4,7-Triazacyclononane for Biomimicry and Radiopharmaceutical Applications

   Joshi, Tanmaya; Kubeil, Manja; Nsubuga, Anne; Singh, Garima; Gasser, Gilles; Stephan, Holger

   ChemPlusChem (2018), 83 (7), 554-564CODEN: CHEMM5; ISSN:2192-6506. (Wiley-VCH Verlag GmbH & Co. KGaA)

   1,4,7-Triazacyclononane (TACN)-based mono- and poly-nuclear metal complexes have found extensive use as biol. mimics for understanding the structural and operational aspects of complex natural systems. Their coordination flexibility has also provided researchers access to a vast library of radiometal-binding motifs that display excellent thermodn. stability and kinetic inertness upon metal complexation. Synthetic modification of the TACN backbone has yielded ligands that can form metal complexes with coordination geometries well suited for these applications. In particular, Leone Spiccia's research has played a significant role in accelerating the progress in these two fields. With a focus on his contributions to the topics of biomimicry and radiopharmaceuticals, this Minireview uses relevant examples to put in perspective the utility of macrocyclic coordination chem. for biol. inorg. chem. applications.
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    Davey, P. R. W. J.; Paterson, B. M. Modern developments in bifunctional chelator design for gallium radiopharmaceuticals. Molecules 2023, 28 (1), 203,  DOI: 10.3390/molecules28010203

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    Modern Developments in Bifunctional Chelator Design for Gallium Radiopharmaceuticals

    Davey, Patrick R. W. J.; Paterson, Brett M.

    Molecules (2023), 28 (1), 203CODEN: MOLEFW; ISSN:1420-3049. (MDPI AG)

    A review. The positron-emitting radionuclide gallium-68 has become increasingly utilized in both preclin. and clin. settings with positron emission tomog. (PET). The synthesis of radiochem. pure gallium-68 radiopharmaceuticals relies on careful consideration of the coordination chem. The short half-life of 68 min necessitates rapid quant. radiolabelling (≤10 min). Desirable radiolabelling conditions include near-neutral pH, ambient temps., and low chelator concns. to achieve the desired apparent molar activity. This review presents a broad overview of the requirements of an efficient bifunctional chelator in relation to the aq. coordination chem. of gallium. Developments in bifunctional chelator design and application are then presented and grouped according to eight categories of bifunctional chelator: the macrocyclic chelators DOTA and TACN; the acyclic HBED, pyridinecarboxylates, siderophores, tris(hydroxypyridinones), and DTPA; and the mesocyclic diazepines.
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    Braband, H.; Imstepf, S.; Benz, M.; Spingler, B.; Alberto, R. Combining bifunctional chelator with (3 + 2)-cycloaddition approaches: Synthesis of dual-function technetium complexes. Inorg. Chem. 2012, 51 (7), 4051– 4057,  DOI: 10.1021/ic202212e

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    Combining Bifunctional Chelator with (3 + 2)-Cycloaddition Approaches: Synthesis of Dual-Function Technetium Complexes

    Braband, Henrik; Imstepf, Sebastian; Benz, Michael; Spingler, Bernhard; Alberto, Roger

    Inorganic Chemistry (2012), 51 (7), 4051-4057CODEN: INOCAJ; ISSN:0020-1669. (American Chemical Society)

    A new concept for the synthesis of dual-functionalized technetium (Tc) compds. is presented, on the basis of the reactivity of fac-{TcVIIO3}+ complexes. The concept combines the "classical" bifunctional chelator (BFC) approach with the new ligand centered labeling strategy of fac-{TcO3}+ complexes with alkenes ((3 + 2)-cycloaddn. approach). To evidence this concept, fac-{99TcO3}+ model complexes contg. functionalized 1,4,7-triazacyclononane (tacn) derivs. N-benzyl-2-(1,4,7-triazonan-1-yl)acetamide (tacn-ba) and 2,2',2''-(1,4,7-triazonane-1,4,7-triyl)triacetic acid (nota·3H) were synthesized and characterized. Whereas [99TcO3(tacn-ba)]+ [2]+ can be synthesized following a established oxidn. procedure starting from the TcV complex [99TcO(glyc)(tacn-ba)]+ [1]+, a new synthetic pathway for the synthesis of [99TcO3(nota)]2- [5]2- had to be developed, starting from [99Tc(nota·3H)(CO)3]+ [4]+ and using sodium perborate tetrahydrate (NaBO3·4H2O) as oxidizing reagent. While [99TcO3(nota)]2- [5]2- is a very attractive candidate for the development of trisubstituted novel multifunctional radioprobes, (3 + 2)-cycloaddn. reactions of [99TcO3(tacn-ba)]+ [2]+ with 4-vinylbenzenesulfonate (styrene-SO3-) demonstrated the suitability of monosubstituted tacn derivs. for the new mixed "BFC-(3 + 2)-cycloaddn." approach. Kinetic studies of this reaction indicated that the alteration of the electronic structure of the nitrogen donors by, e.g., alkylation can be used to tune the rate of the (3 + 2)-cycloaddn.
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    Makris, G.; Bandari, R. P.; Kuchuk, M.; Jurisson, S. S.; Smith, C. J.; Hennkens, H. M. Development and preclinical evaluation of ^99mTc- and ¹⁸⁶Re-labeled NOTA and NODAGA bioconjugates demonstrating matched pair targeting of GRPR-expressing tumors. Mol. Imaging Biol. 2021, 23 (1), 52– 61,  DOI: 10.1007/s11307-020-01537-1

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    Development and Preclinical Evaluation of 99mTc- and 186Re-Labeled NOTA and NODAGA Bioconjugates Demonstrating Matched Pair Targeting of GRPR-Expressing Tumors

    Makris, George; Bandari, Rajendra P.; Kuchuk, Marina; Jurisson, Silvia S.; Smith, Charles J.; Hennkens, Heather M.

    Molecular Imaging and Biology (2021), 23 (1), 52-61CODEN: MIBOCZ; ISSN:1860-2002. (Springer)

    Purpose: The goal of this work was to develop hydrophilic gastrin-releasing peptide receptor (GRPR)-targeting complexes of the general formula fac-[M(CO)3(L)]+ [M = natRe, 99mTc, 186Re; L: NOTA for 1, NODAGA for 2] conjugated to a powerful GRPR peptide antagonist (DPhe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH2) via a 6-aminohexanoic acid linker. Procedures: Metalated-peptides were prepd. employing the [M(OH2)3(CO)3]+ [M = Re, 99mTc, 186Re] precursors. Re-1/2 complexes were characterized with HR-MS. IC50 studies were performed for peptides 1/2 and their resp. Re-1/2 complexes in a binding assay utilizing GRPR-expressing human PC-3 prostate cancer cells and [125I]I-Tyr4-BBN as the competing ligand. The 99mTc/186Re-complexes were identified by HPLC co-injection with their Re-analogs. All tracers were challenged in vitro at 37 °C against cysteine/histidine (phosphate-buffered saline 10 mM, pH 7.4) and rat serum. Biodistribution and micro-SPECT/CT imaging of [99mTc]Tc-1/2 and [186Re]Re-2 were performed in PC-3 tumor-bearing ICR SCID mice. Results: High in vitro receptor affinity (IC50 2-3 nM) was demonstrated for all compds. The 99mTc/186Re-tracers were found to be hydrophilic (log D7.4 ≤ - 1.35) and highly stable. Biodistribution in PC-3 xenografted mice revealed good tumor uptake (%ID/g at 1 h: 4.3 ± 0.7 for [99mTc]Tc-1, 8.3 ± 0.9 for [99mTc]Tc-2 and 4.2 ± 0.8 for [186Re]Re-2) with moderate retention over 24 h. Rapid renal clearance was obsd. for [99mTc]Tc-2 and [186Re]Re-2 (> 84 % at 4 h), indicating favorable pharmacokinetics. Micro-SPECT/CT images for the 99mTc-tracers clearly visualized PC-3 tumors in agreement with the biodistribution data and with superior imaging properties found for [99mTc]Tc-2. Conclusions: [99mTc]Tc-2 shows promise for further development as a GRPR-imaging agent. [186Re]Re-2 demonstrated very similar in vivo behavior to [99mTc]Tc-2, and further studies are therefore justified to explore the theranostic potential of our approach for targeting of GRPR-pos. cancers.
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    Qiao, Z.; Xu, J.; Gonzalez, R.; Miao, Y. Novel [^99mTc]-tricarbonyl-NOTA-conjugated lactam-cyclized alpha-MSH peptide with enhanced melanoma uptake and reduced renal uptake. Mol. Pharmaceutics 2020, 17 (9), 3581– 3588,  DOI: 10.1021/acs.molpharmaceut.0c00606

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    Novel [99mTc]-Tricarbonyl-NOTA-Conjugated Lactam-Cyclized Alpha-MSH Peptide with Enhanced Melanoma Uptake and Reduced Renal Uptake

    Qiao, Zheng; Xu, Jingli; Gonzalez, Rene; Miao, Yubin

    Molecular Pharmaceutics (2020), 17 (9), 3581-3588CODEN: MPOHBP; ISSN:1543-8384. (American Chemical Society)

    The purpose of this study was to examine the melanoma targeting and imaging properties of 99mTc(CO)3-NOTA-GGNle-CycMSHhex {1,4,7-triazacyclononane-1,4,7-triyl-triacetic acid-GlyGlyNle-c[Asp-His-DPhe-Arg-Trp-Lys]-CONH2} and 99mTc(CO)3-NODAGA-GGNle-CycMSHhex {1,4,7-triazacyclononane,1-glutaric acid-4,7-acetic acid-GlyGlyNle-c[Asp-His-DPhe-Arg-Trp-Lys]-CONH2} on B16/F10 melanoma-bearing C57 mice to demonstrate the feasibility of NOTA/NODAGA as metal chelators for 99mTc(CO)3+ radiolabeling. NOTA/NODAGA-GGNle-CycMSHhex were synthesized using fluorenylmethoxycarbonyl (Fmoc) chem. The melanocortin-1 (MC1) receptor binding affinities of the peptides were detd. on B16/F10 melanoma cells. The biodistribution of 99mTc(CO)3-NOTA-GGNle-CycMSHhex and 99mTc(CO)3-NODAGA-GGNle-CycMSHhex were detd. on B16/F10 melanoma-bearing C57 mice at 2 h postinjection to select a lead peptide for further evaluation. The melanoma targeting and imaging properties of 99mTc(CO)3-NOTA-GGNle-CycMSHhex and 99mTc(CO)3-NODAGA-GGNle-CycMSHhex were detd. on B16/F10 melanoma-bearing C57 mice. The IC50 values of NOTA/NODAGA-GGNle-CycMSHhex were 0.8 ± 0.1 and 0.9 ± 0.1 nM on B16/F10 cells. 99mTc(CO)3-NOTA-GGNle-CycMSHhex and 99mTc(CO)3-NODAGA-GGNle-CycMSHhex were readily prepd. via the [99mTc(CO)3(OH2)3]+ intermediate and displayed MC1R-specific binding on B16/F10 cells. 99mTc(CO)3-NOTA-GGNle-CycMSHhex was further evaluated as a lead peptide because of its higher tumor uptake (19.76 ± 3.62% ID/g) and lower kidney uptake (1.59 ± 0.52% ID/g) at 2 h postinjection than 99mTc(CO)3-NODAGA-GGNle-CycMSHhex. The B16/F10 melanoma uptake of 99mTc(CO)3-NOTA-GGNle-CycMSHhex was 16.07 ± 4.47, 19.76 ± 3.62, 11.30 ± 2.81, and 3.16 ± 2.28% ID/g at 0.5, 2, 4, and 24 h postinjection, resp. 99mTc(CO)3-NOTA-GGNle-CycMSHhex showed high tumor to normal organ uptake ratios after 2 h postinjection. The B16/F10 melanoma lesions were clearly visualized by SPECT/CT using 99mTc(CO)3-NOTA-GGNle-CycMSHhex as an imaging probe at 2 h postinjection. High tumor uptake, low kidney uptake, and fast urinary clearance of 99mTc(CO)3-NOTA-GGNle-CycMSHhex highlighted its potential for melanoma imaging and facilitated the evaluation of 188Re(CO)3-NOTA-GGNle-CycMSHhex for melanoma therapy.
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    Makris, G.; Kuchuk, M.; Gallazzi, F.; Jurisson, S. S.; Smith, C. J.; Hennkens, H. M. Somatostatin receptor targeting with hydrophilic [^99mTc/¹⁸⁶Re]Tc/Re-tricarbonyl NODAGA and NOTA complexes. Nucl. Med. Biol. 2019, 71, 39– 46,  DOI: 10.1016/j.nucmedbio.2019.04.004

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    Somatostatin receptor targeting with hydrophilic [99mTc/186Re]Tc/Re-tricarbonyl NODAGA and NOTA complexes

    Makris, George; Kuchuk, Marina; Gallazzi, Fabio; Jurisson, Silvia S.; Smith, Charles J.; Hennkens, Heather M.

    Nuclear Medicine and Biology (2019), 71 (), 39-46CODEN: NMBIEO; ISSN:0969-8051. (Elsevier)

    The aim of this work was to develop diagnostic (99mTc) and therapeutic (186Re) agents for targeting somatostatin receptor (SSTR)-pos. neuroendocrine tumors (NETs). In this regard, we evaluated in vitro complexes of the general formula [M(CO)3(L-sst2-ANT)] (M = 99mTc, 186Re), where L denotes NODAGA or NOTA and sst2-ANT denotes the potent SSTR2 antagonist 4-NO2-Phe-c(DCys-Tyr-DTrp-Lys-Thr-Cys)-DTyr-NH2. Moreover, we assessed the in vivo properties of the 99mTc-complexes in an animal SSTR-tumor model. The [99mTc]/[186Re][Tc/Re(OH2)3(CO)3]+ precursors were utilized to prep. the 99mTc/186Re-complexes, which were identified by HPLC co-injection with their natRe analogs. The tracers were challenged in vitro at 37 °C against cysteine and histidine in phosphate-buffered saline (pH 7.4) and in rat serum. Biodistribution and micro-SPECT/CT imaging studies of the 99mTc-tracers were performed in AR42J tumor-bearing female ICR SCID mice. The 99mTc-complexes were prepd. in high radiochem. yield (RCY > 90%, by HPLC), with lower RCY (≤30%) obtained for 186Re-complexes. Tracers remained intact in vitro and displayed low non-specific binding (10-25%) to rat serum proteins. Biodistribution of [99mTc]Tc-NODAGA-sst2-ANT revealed low tumor uptake (2.78 ± 0.27 %ID/g) at 1 h, while high tumor uptake (16.70 ± 3.32 %ID/g) was found for [99mTc]Tc-NOTA-sst2-ANT. Moderate to low tumor retention was obsd. for both tracers after 4 and 24 h. Tumor uptake for [99mTc]Tc-NOTA-sst2-ANT was receptor-mediated, as demonstrated by parallel SSTR blocking studies. Rapid renal clearance was obsd. for both tracers, and SPECT/CT images clearly delineated the tumors, in agreement with the biodistribution data. The [99mTc]Tc-NOTA-sst2-ANT complex demonstrated high tumor uptake and rapid clearance in a SSTR-tumor mouse model, showing potential for further development. Preclin. data support the feasibility of the [99mTc]Tc/[186Re]Re-NOTA/NODAGA labeling strategy for use in the development of theranostic radiopharmaceuticals for translation into the human clinic for targeting of SSTR-expressing NETs.
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    Patinec, V.; Rolla, G. A.; Botta, M.; Tripier, R.; Esteban-Gomez, D.; Platas-Iglesias, C. Hyperfine coupling constants on inner-sphere water molecules of a triazacyclononane-based Mn(II) complex and related systems relevant as MRI contrast agents. Inorg. Chem. 2013, 52 (19), 11173– 11184,  DOI: 10.1021/ic4014366

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    Hyperfine Coupling Constants on Inner-Sphere Water Molecules of a Triazacyclononane-based Mn(II) Complex and Related Systems Relevant as MRI Contrast Agents

    Patinec, Veronique; Rolla, Gabriele A.; Botta, Mauro; Tripier, Raphael; Esteban-Gomez, David; Platas-Iglesias, Carlos

    Inorganic Chemistry (2013), 52 (19), 11173-11184CODEN: INOCAJ; ISSN:0020-1669. (American Chemical Society)

    The authors report the synthesis of the ligand H2MeNO2A (1,4-bis(carboxymethyl)-7-methyl-1,4,7-triazacyclononane) and a detailed exptl. and computational study of the hyperfine coupling consts. (HFCCs) on the inner-sphere H2O mols. of [Mn(MeNO2A)] and related Mn2+ complexes relevant as potential contrast agents in magnetic resonance imaging (MRI). Nuclear magnetic relaxation dispersion (NMRD) profiles, 17O NMR chem. shifts, and transverse relaxation rates of aq. solns. of [Mn(MeNO2A)] were recorded to det. the parameters governing the relaxivity in this complex and the 17O and 1H HFCCs. DFT calcns. (TPSSh model) performed in aq. soln. (PCM model) on the [Mn(MeNO2A)(H2O)]·xH2O and [Mn(EDTA)(H2O)]2-·xH2O (x = 0-4) systems were used to det. theor. the 17O and 1H HFCCs responsible for the 17O NMR chem. shifts and the scalar contributions to 17O and 1H NMR relaxation rates. The use of a mixed cluster/continuum approach with the explicit inclusion of a few second-sphere H2O mols. is crit. for an accurate calcn. of HFCCs of coordinated H2O mols. The impact of complex dynamics on the calcd. HFCCs was evaluated using mol. dynamics simulations within the atom-centered d. matrix propagation (ADMP) approach. The 17O and 1H HFCCs calcd. for these complexes and related systems show an excellent agreement with the exptl. data. Both the 1H and 17O HFCCs (Aiso values) are dominated by the spin delocalization mechanism. The Aiso values are significantly affected by the distance between the O atom of the coordinated H2O mol. and the Mn2+ ion, as well as by the orientation of the H2O mol. plane with respect to the Mn-O vector.
16. 16

    Cantorias, M. V.; Howell, R. C.; Todaro, L.; Cyr, J. E.; Berndorff, D.; Rogers, R. D.; Francesconi, L. C. MO tripeptide diastereomers (M = ^99/99mTc, Re): Models to identify the structure of ^99mTc peptide targeted radiopharmaceuticals. Inorg. Chem. 2007, 46 (18), 7326– 7340,  DOI: 10.1021/ic070077p

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    MO tripeptide diastereomers (M = 99/99mTc, Re): models to identify the structure of 99mTc peptide targeted radiopharmaceuticals

    Cantorias, Melchor V.; Howell, Robertha C.; Todaro, Louis; Cyr, John E.; Berndorff, Dietmar; Rogers, Robin D.; Francesconi, Lynn C.

    Inorganic Chemistry (2007), 46 (18), 7326-7340CODEN: INOCAJ; ISSN:0020-1669. (American Chemical Society)

    Biol. active mols., such as many peptides, serve as targeting vectors for radiopharmaceuticals based on 99mTc. Tripeptides can be suitable chelates and are easily and conveniently synthesized and linked to peptide targeting vectors through solid-phase peptide synthesis and form stable TcVO complexes. Upon complexation with [TcO]3+, two products form; these are syn and anti diastereomers, and they often have different biol. behavior. This is the case with the approved radiopharmaceutical [99mTcO]depreotide ([99mTcO]P829, NeoTect) that was used to image lung cancer. [99mTcO]depreotide indeed exhibits two product peaks in its HPLC profile, but assignment of the product peaks to the diastereomers proved to be difficult because the metal peptide complex is difficult to crystallize for structural anal. The authors isolated diastereomers of [99TcO] and [ReO] of several tripeptide ligands that model the metal chelator region of [99mTcO]depreotide. Using x-ray crystallog., the early eluting peak (A) corresponds to the anti diastereomer, where the Tc:O group is on the opposite side of the plane formed by the ligand backbone relative to the pendant groups of the tripeptide ligand, and the later eluting peak (B) corresponds to the syn diastereomer, where the Tc:O group is on the same side of the plane as the residues of the tripeptide. 1H NMR and CD spectroscopy report on the metal environment and prove to be diagnostic for syn or anti diastereomers, and the authors identified characteristic features from these techniques that can be used to assign the diastereomer profile in 99mTc peptide radiopharmaceuticals like [99mTcO]depreotide and in 188Re peptide radiotherapeutic agents. Crystallog., potentiometric titrn., and NMR results presented insights into the chem. occurring under physiol. conditions. The tripeptide complexes where lysine is the second amino acid crystd. in a deprotonated metallo-amide form, possessing a short N1-M bond. The pKa measurements of the N1 amine (pKa ∼5.6) suggested that this amine is rendered more acidic by both metal complexation and the presence of the lysine residue. Also, peptide chelators incorporating a lysine (like the chelator of [TcO]depreotide) likely exist in the deprotonated form in vivo, comprising a neutral metal center. Deprotonation possibly mediates the interconversion process between the syn and anti diastereomers. The N1 amine group on nonlysine-contg. metallopeptides is not as acidic (pKa ∼6.8) and does not deprotonate and crystallize as do the metallo-amide species. Three of the tripeptide ligands (FGC, FSC, and FKC) were radiolabeled with 99mTc, and the individual syn and anti isomers were isolated for biodistribution studies in normal female nude mice. The main organs of uptake were the liver, intestines, and kidneys, with the FGC compds. exhibiting the highest liver uptake. In comparing the diastereomers, the syn compds. had substantially higher organ uptake and slower blood clearance than the anti compds.
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    Radford, L. L.; Papagiannopoulou, D.; Gallazzi, F.; Berendzen, A.; Watkinson, L.; Carmack, T.; Lewis, M. R.; Jurisson, S. S.; Hennkens, H. M. Synthesis and evaluation of Re/^99mTc(I) complexes bearing a somatostatin receptor-targeting antagonist and labeled via a novel [N,S,O] clickable bifunctional chelating agent. Bioorg. Med. Chem. 2019, 27 (3), 492– 501,  DOI: 10.1016/j.bmc.2018.12.028

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    Synthesis and evaluation of Re/99mTc(I) complexes bearing a somatostatin receptor-targeting antagonist and labeled via a novel [N,S,O] clickable bifunctional chelating agent

    Radford, Lauren L.; Papagiannopoulou, Dionysia; Gallazzi, Fabio; Berendzen, Ashley; Watkinson, Lisa; Carmack, Terry; Lewis, Michael R.; Jurisson, Silvia S.; Hennkens, Heather M.

    Bioorganic & Medicinal Chemistry (2019), 27 (3), 492-501CODEN: BMECEP; ISSN:0968-0896. (Elsevier B.V.)

    The somatostatin receptor subtype 2 (SSTR2) is often highly expressed on neuroendocrine tumors (NETs), making it a popular in vivo target for diagnostic and therapeutic approaches aimed toward management of NETs. In this work, an antagonist peptide (sst2-ANT) with high affinity for SSTR2 was modified at the N-terminus with a novel [N,S,O] bifunctional chelator (2) designed for tridentate chelation of rhenium(I) and technetium(I) tricarbonyl cores, [Re(CO)3]+ and [99mTc][Tc(CO)3]+. The chelator-peptide conjugation was performed via a Cu(I)-assisted click reaction of the alkyne-bearing chelator (2) with an azide-functionalized sst2-ANT peptide (3), to yield NSO-sst2-ANT (4). Two synthetic methods were used to prep. Re-4 at the macroscopic scale, which differed based on the relative timing of the click conjugation to the [Re(CO)3]+ complexation by 2. The resulting products demonstrated the expected mol. mass and nanomolar in vitro SSTR2 affinity (IC50 values under 30 nM, AR42J cells, [125I]iodo-Tyr11-somatostatin-14 radioligand std.). However, a difference in their HPLC retention times suggested a difference in metal coordination modes, which was attributed to a competing N-triazole donor ligand formed during click conjugation. Surprisingly, the radiotracer scale reaction of [99mTc][Tc(OH2)3(CO)3]+ (99mTc; t1/2 = 6 h, 141 keV γ) with 4 formed a third product, distinct from the Re analogs, making this one of the unusual cases in which Re and Tc chemistries are not well matched. Nevertheless, the [99mTc]Tc-4 product demonstrated excellent in vitro stability to challenges by cysteine and histidine (≥98% intact through 24 h), along with 75% stability in mouse serum through 4 h. In vivo biodistribution and microSPECT/CT imaging studies performed in AR42J tumor-bearing mice revealed improved clearance of this radiotracer in comparison to a similar [99mTc][Tc(CO)3]-labeled sst2-ANT deriv. previously studied. Yet despite having adequate tumor uptake at 1 h (4.9% ID/g), tumor uptake was not blocked by co-administration of a receptor-satg. dose of SS-14. Aimed toward realignment of the Re and Tc product structures, future efforts should include distancing the alkyne group from the intended donor atoms of the chelator, to reduce the coordination options available to the [M(CO)3]+ core (M = Re, 99mTc) by disfavoring involvement of the N-triazole.
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    Sanders, V. A.; Iskhakov, D.; Abdel-Atti, D.; Devany, M.; Neary, M. C.; Czerwinski, K. R.; Francesconi, L. C. Synthesis, characterization and biological studies of rhenium, technetium-99m and rhenium-188 pentapeptides. Nucl. Med. Biol. 2019, 68–69, 1– 13,  DOI: 10.1016/j.nucmedbio.2018.11.001

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    Synthesis, characterization and biological studies of rhenium, technetium-99m and rhenium-188 pentapeptides

    Sanders, Vanessa A.; Iskhakov, David; Abdel-Atti, Dalya; Devany, Matthew; Neary, Michelle C.; Czerwinski, Ken R.; Francesconi, Lynn C.

    Nuclear Medicine and Biology (2019), 68-69 (), 1-13CODEN: NMBIEO; ISSN:0969-8051. (Elsevier)

    A pentapeptide macrocyclic ligand, KYCAR (lysyl-tyrosyl-cystyl-alanyl-arginine), has been designed as a potential chelating ligand for SPECT imaging and therapeutic in vivo agents. This study shows the synthesis and characterization of KYCAR complexes contg. nonradioactive rhenium, 99mTc, or 188Re. The metal complexes were also biol. evaluated to det. in vivo distribution in healthy mice. The overall goals of this project were (1) to synthesize the Tc/Re pentapeptide complexes, (2) to identify spectroscopic methods for characterization of syn vs. anti rhenium peptide complexes, (3) to analyze the ex vivo stability, and (4) to assess the biol. properties of the [99mTc]TcO-KYCAR and [188Re]ReO-KYCAR complexes in vivo. Details on these efforts are provided below.NatRe/99mTc/188ReO-KYCAR complexes were synthesized, and macroscopic species were characterized via HPLC, IR, NMR, and CD. These characterization data were compared to the crystallog. data of ReO-KYC to assist in the assignment of diastereomers and to aid in the detn. of the structure of the complex. The radiometal complexes were synthesized with high purity (greater than 95%). HPLC, IR, NMR and CD data on the macroscopic natReO-KYCAR complexes confirm the successful complexation as well as the presence of two diastereomers in syn and anticonformations. Tracer level complexes show favorable stabilities ex vivo for 2+ h. Macroscopic metal complexes form diastereomers with the KYCAR ligand; however, this phenomenon is not readily obsd. on the tracer level due to the rapid interconversion. It was detd. through pKa measurements that the macroscopic natReO-KYCAR complex is 0 at physiol. pH. The [99mTc]TcO-KYCAR is stable in vitro while the [188Re]ReO-KYCAR shows 50% decompn. in PBS and serum. Biol., the tracer level complexes clear through the hepatobiliary pathway. Some decompn. of both tracers is evident by uptake in the thyroid and stomach.
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    Drahos, B.; Kubicek, V.; Bonnet, C. S.; Hermann, P.; Lukes, I.; Toth, E. Dissociation kinetics of Mn²⁺ complexes of NOTA and DOTA. Dalton Trans. 2011, 40 (9), 1945– 1951,  DOI: 10.1039/c0dt01328e

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    Dissociation kinetics of Mn2+ complexes of NOTA and DOTA

    Drahos, Bohuslav; Kubicek, Vojtech; Bonnet, Celia S.; Hermann, Petr; Lukes, Ivan; Toth, Eva

    Dalton Transactions (2011), 40 (9), 1945-1951CODEN: DTARAF; ISSN:1477-9226. (Royal Society of Chemistry)

    The kinetics of transmetallation of [Mn(nota)]- and [Mn(dota)]2- was investigated in the presence of Zn2+ (5-50-fold excess) at variable pH (3.5-5.6) by 1H relaxometry. The dissocn. is much faster for [Mn(nota)]- than for [Mn(dota)]2- under both exptl. and physiol. relevant conditions (t1/2 = 74 h and 1037 h for [Mn(nota)]- and [Mn(dota)]2-, resp., at pH 7.4, c(Zn2+) = 10-5 M, 25 °C). The dissocn. of the complexes proceeds mainly via spontaneous ([Mn(nota)]-k0 = (2.6 ± 0.5) × 10-6 s-1; [Mn(dota)]2-k0 = (1.8 ± 0.6) × 10-7 s-1) and proton-assisted pathways ([Mn(nota)]-k1 = (7.8 ± 0.1) × 10-1 M-1 s-1; [Mn(dota)]2-k1 = (4.0 ± 0.6) × 10-2 M-1 s-1, k2 = (1.6 ± 0.1) × 103 M-2 s-1). The obsd. suppression of the reaction rates with increasing Zn2+ concn. is explained by the formation of a dinuclear Mn2+-L-Zn2+ complex which is about 20-times more stable for [Mn(dota)]2- than for [Mn(nota)]- (KMnLZn = 68 and 3.6, resp.), and which dissocs. very slowly (k3 ∼10-5 M-1 s-1). These data provide the first exptl. proof that not all Mn2+ complexes are kinetically labile. The absence of coordinated water makes both [Mn(nota)]- and [Mn(dota)]2- complexes inefficient for MRI applications. Nevertheless, the higher kinetic inertness of [Mn(dota)]2- indicates a promising direction in designing ligands for Mn2+ complexation.
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    Kubicek, V.; Havlickova, J.; Kotek, J.; Tircso, G.; Hermann, P.; Toth, E.; Lukes, I. Gallium(III) complexes of DOTA and DOTA-monoamide: Kinetic and thermodynamic studies. Inorg. Chem. 2010, 49 (23), 10960– 10969,  DOI: 10.1021/ic101378s

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    Gallium(III) Complexes of DOTA and DOTA-Monoamide: Kinetic and Thermodynamic Studies

    Kubicek, Vojtech; Havlickova, Jana; Kotek, Jan; Tircso, Gyula; Hermann, Petr; Toth, Eva; Lukes, Ivan

    Inorganic Chemistry (2010), 49 (23), 10960-10969CODEN: INOCAJ; ISSN:0020-1669. (American Chemical Society)

    Given the practical advantages of the 68Ga isotope in positron emission tomog. applications, gallium complexes are gaining increasing importance in biomedical imaging. However, the strong tendency of Ga3+ to hydrolyze and the slow formation and very high stability of macrocyclic complexes altogether render Ga3+ coordination chem. difficult and explain why stability and kinetic data on Ga3+ complexes are rather scarce. Here the authors report soln. and solid-state studies of Ga3+ complexes formed with the macrocyclic ligand 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, (DOTA)4-, and its mono(n-butylamide) deriv., (DO3AMBu)3-. Thermodn. stability consts., log K(GaDOTA) = 26.05 and log K(GaDO3AMBu) = 24.64, were detd. by out-of-cell pH-potentiometric titrns. Due to the very slow formation and dissocn. of the complexes, equilibration times of up to ∼4 wk were necessary. The kinetics of complex dissocn. were followed by 71Ga NMR under both acidic and alk. conditions. The GaDOTA complex is significantly more inert (τ1/2 ∼12.2 d at pH = 0 and τ1/2 ∼6.2 h at pH = 10) than the GaDO3AMBu analog (τ1/2 ∼2.7 d at pH = 0 and τ1/2 ∼0.7 h at pH = 10). Nevertheless, the kinetic inertness of both chelates is extremely high and approves the application of Ga3+ complexes of such DOTA-like ligands in mol. imaging. The solid-state structure of the GaDOTA complex, crystd. from a strongly acidic soln. (pH < 1), evidenced a diprotonated form with protons localized on the free carboxylate pendants.
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    Kankanamalage, P. H. A.; Hoerres, R.; Ho, K.-V.; Anderson, C. J.; Gallazzi, F.; Hennkens, H. M. p-NCS-Bn-NODAGA as a bifunctional chelator for radiolabeling with the ¹⁸⁶Re/^99mTc-tricarbonyl core: Radiochemistry with model complexes and a GRPR-targeting peptide. Nucl. Med. Biol. 2022, 108–109, 1– 9,  DOI: 10.1016/j.nucmedbio.2022.01.004

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    p-NCS-Bn-NODAGA as a bifunctional chelator for radiolabeling with the 186Re/99mTc-tricarbonyl core: Radiochemistry with model complexes and a GRPR-targeting peptide

    Kankanamalage, Pavithra H. A.; Hoerres, Rebecca; Ho, Khanh-Van; Anderson, Carolyn J.; Gallazzi, Fabio; Hennkens, Heather M.

    Nuclear Medicine and Biology (2022), 108-109 (), 1-9CODEN: NMBIEO; ISSN:0969-8051. (Elsevier Inc.)

    With the goal of developing theranostic agents for application in radiopharmaceutical chem., in this work, we studied p-NCS-Bn-NODAGA (1) as a bifunctional chelator for the fac-[M(CO)3]+ core (M = natRe, 186Re, 99mTc). Specifically, we studied complexes of the formula [M(CO)3(L)]+, where L denotes either Bn-NODAGA-Pyr (2) or Bn-NODAGA-Ser-Ser-RM2 (3). The model bioconjugate mol. 2 was synthesized by conjugating pyrrolidine with 1, while 3 was derived from the conjugation of the gastrin-releasing peptide receptor (GRPR)-targeting peptide Ser-Ser-RM2 with 1. Labeling of 2 and 3 was performed with [M(CO)3(OH2)3]+ (where M = natRe, 186Re, or 99mTc). The stability of the radioactive complexes was studied against L-histidine and L-cysteine (1 mM in PBS; pH 7.4, 37 °C). GRPR affinity of both peptide 3 and its metalated counterpart, Re-3, were detd. with in vitro competitive binding assays in GRPR-expressing PC-3 cells using [125I]I-Tyr4-BBN as the competitor. After a thorough radiolabeling optimization process, the [M(CO)3(2)]+ model complexes (M = 186Re and 99mTc) were synthesized with 94 ± 2radiochem. yield (RCY; estd. by radio-HPLC). In stability studies, [186Re]Re-2 remained intact through 7 d in L-cysteine and L-histidine. Similarly, stability studies in rat serum at 37 °C showed 99 ± 1intact [186Re]Re-2 through 4 h. Non-specific rat serum protein binding of [186Re]Re-2 was found to be 33 ± 4at 4 h. The [99mTc]Tc-2 complex was found to be stable in L-histidine and L-cysteine at 37 °C through 24 h. [99mTc]Tc-2 was also stable in rat serum, with 38 ± 3non-specific protein binding, at 4 h. The [M(CO)3(3)]+ peptide radiometal complex (M = 186Re and 99mTc) syntheses were also optimized, resulting in RCYs of 35for [186Re]Re-3 and 47for [99mTc]Tc-3 (estd. by radio-HPLC). [186Re]Re-3 showed 98 ± 2and 84 ± 5stability in L-histidine and L-cysteine, resp., through 48 h. Similarly, stability studies in rat serum at 37 °C showed 85 ± 3intact [186Re]Re-3 through 4 h, with 29 ± 7non-specific protein binding in rat serum. [99mTc]Tc-3 was found to be 84 ± 3and 82 ± 4stable in L-histidine and L-cysteine at 24 h, resp. [99mTc]Tc-3 in rat serum at 37 °C showed 88 ± 2stability through 4 h, with 25 ± 2non-specific protein binding. Both 3 and Re-3 demonstrated high GRPR affinity, with IC50 values of 3.1 nM and 3.9 nM, resp. The low nanomolar IC50 values obtained for 3 and Re-3 demonstrate high affinity of this novel [M(CO)3]-labeled bioconjugate for GRPR. The encouraging stability studies and receptor affinity results demonstrate promise for further development of these metal complexes as a theranostic matched pair for targeting GRPR.
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    Veerendra, B.; Sieckman, G. L.; Hoffman, T. J.; Rold, T.; Retzloff, L.; McCrate, J.; Prasanphanich, A.; Smith, C. J. Synthesis, radiolabeling and in vitro GRP receptor targeting studies of ^99mTc-triaza-X-BBN[7–14]NH₂ (X = serylserylserine, glycylglycylglycine, glycylserylglycine, or beta alanine). Synth. React. Inorg., Met.-Org., Nano-Met. Chem. 2006, 36 (6), 481– 491,  DOI: 10.1080/15533170600778075

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    Synthesis, radiolabeling and in vitro GRP receptor targeting studies of 99mTc-Triaza-X-BBN[7-14]NH2 (X = Serylserylserine, Glycylglycylglycine, Glycylserylglycine, or beta alanine)

    Veerendra, Bhadrasetty; Sieckman, Gary L.; Hoffman, Timothy J.; Rold, Tammy; Retzloff, Lauren; McCrate, Joseph; Prasanphanich, Adam; Smith, Charles J.

    Synthesis and Reactivity in Inorganic, Metal-Organic, and Nano-Metal Chemistry (2006), 36 (6), 481-491CODEN: SRIMDO; ISSN:1553-3174. (Taylor & Francis, Inc.)

    Gastrin-releasing peptide (GRP) receptors are over-expressed on several types of human cancer cells including prostate, breast, small cell lung and pancreatic cancers. Bombesin (BBN) is a 14 amino acid peptide that is an analog of human gastrin-releasing peptide, binding to GRP receptors (GRPr) with high affinity and specificity. The aim of these studies was to develop new 99mTc-labeled BBN analogs having high tumor uptake and optimal pharmacokinetics for specific targeting of human prostate cancers. A new tridentate bifunctional chelating agent, 2-(4,7-bis(tert-butoxycarbonyl)-1,4,7-triazanonan-1-yl)acetic acid, was synthesized by first reacting 2 equiv of BOC-ON with 1,4,7-triazacyclononane (Triaza) in CHCl3 at room temp. The product, N, N'-bis-(tert-butoxycarbonyl)-1,4,7-triazacyclononane, was alkylated using BrCH2COOH in acetonitrile. This new ligand framework was characterized by 1H and 13C NMR and electrospray ionization mass spectrometry (ESI-MS). Solid-phase peptide synthesis (SPPS) was used to produce Triaza-X-BBN[7-14]NH2 conjugates with the following structure: Triaza-X-Q-W-A-V-G-H-L-M-(NH2), where the spacer group X = SSS, GGG, GSG and β-Alanine (SSS = Serylserylserine, GGG = Glycylglycylglycine, and GSG = Glycylserylglycine). These conjugates were purified by reversed phase-HPLC (RP-HPLC) and characterized by ESI-MS. In vitro competitive binding assays, using 125I-Tyr4-BBN as the radiolabelling gold std., demonstrated IC50 values in the nanomolar range for all the new nonmetalated conjugates. For example, IC50s were 1.8 ± 0.4, 3.9. ± 0.4, 1.9 ± 0.3, and 1.3 ± 0.2 nM for X = SSS, GGG, GSG and β-Alanine, resp. The new BBN conjugates were radiolabeled with 99mTc in moderate yield via the Isolink radiolabeling kit available from Tyco Healthcare, St. Louis, MO. In vitro internalization and externalization analyses indicated receptor binding to be receptor specific in human, PC-3, prostate cancer cells. Future in vivo studies in tumor-bearing mouse models are justified.