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ArticleSeptember 11, 2024

[¹⁸⁶Re]Re- and [^99mTc]Tc-Tricarbonyl Metal Complexes with 1,4,7-Triazacyclononane-Based Chelators Bearing Amide, Alcohol, or Ketone Pendent Groups
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Rebecca Hoerres
Rebecca Hoerres
Department of Chemistry, University of Missouri, 601 South College Avenue, Columbia, Missouri 65211, United States
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Ritin Kamboj
Ritin Kamboj
Department of Chemistry, University of Missouri, 601 South College Avenue, Columbia, Missouri 65211, United States
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Nora Pryor
Nora Pryor
Department of Chemistry, University of Missouri, 601 South College Avenue, Columbia, Missouri 65211, United States
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Steven P. Kelley
Steven P. Kelley
Department of Chemistry, University of Missouri, 601 South College Avenue, Columbia, Missouri 65211, United States
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https://orcid.org/0000-0001-6755-4495
Heather M. Hennkens*
Heather M. Hennkens
Department of Chemistry, University of Missouri, 601 South College Avenue, Columbia, Missouri 65211, United States
Research Reactor Center, University of Missouri, 1513 Research Park Drive, Columbia, Missouri 65211, United States
*Email: [email protected]
More by Heather M. Hennkens
https://orcid.org/0000-0002-3283-8751

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ACS Omega

Cite this: ACS Omega 2024, 9, 38, 39925–39935

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https://doi.org/10.1021/acsomega.4c05699

Published September 11, 2024

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Abstract

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1,4,7-Triazacyclononane (TACN)-based chelators, such as NOTA and NODAGA, have shown great promise as bifunctional chelators for [M(CO)₃]⁺ cores (M = ^99mTc and ¹⁸⁶Re) in radiopharmaceutical development. Previous investigations of TACN-based chelators bearing pendent acid and ester arms demonstrated the important role the pendent arms have in successful coordination of the [M(CO)₃]⁺ core with the TACN backbone nitrogens. In this work, we introduce three TACN-based bifunctional chelators bearing amide, alcohol, and ketone pendent arms and evaluate their (radio)labeling efficiency with the [M(CO)₃]⁺ core as well as the in vitro stability and hydrophilicity of the resulting radiometal complexes. Following their synthesis and characterization, the amide (2) and alcohol (3) chelators were successfully labeled with the [M(CO)₃]⁺ cores (M = ^natRe, ^99mTc, and ¹⁸⁶Re), while the ketone (4) was not successfully labeled. Radiometal complexes M-2 and M-3 demonstrated hydrophilic character in logD_7.4 studies as well as excellent stability in phosphate-buffered saline (pH 7.4), l-histidine, l-cysteine, and rat serum at 37 °C through 24 h. While the hydrophilicity and stability of these radiocomplexes are attractive, future TACN chelator design modifications to increase radiolabeling yields under milder reaction conditions would improve their potential for use in development of [M(CO)₃]⁺ radiopharmaceuticals.

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Subjects

1. Introduction

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The field of radiopharmaceutical development is growing exponentially as researchers and clinicians continue to demonstrate the effectiveness of using radiolabeled drugs for both diagnosis and treatment of diseases, such as cancer. (1−3) Targeted radionuclide therapy (TRT) is of particular interest for the treatment of cancer because of its superior specificity for diseased cells over other treatments like chemotherapy and external beam radiation therapy. (4,5) TRT commonly involves either the direct radiolabeling of a biological targeting vector, (6) such as a peptide or antibody, or the conjugation of the biological targeting vector to a bifunctional chelator that is then radiolabeled with a suitable radionuclide. (7) One example of the TRT approach is [¹⁷⁷Lu]Lu-DOTATATE (Lutathera), which incorporates a radionuclide (¹⁷⁷Lu), bifunctional chelator (DOTA), and targeting peptide (Tyr³-octreotate). Lutathera has been approved by both the Food and Drug Administration and the European Medicines Agency (8) and has shown great success in the clinic for patients with gastroenteropancreatic neuroendocrine tumors. New developments in TRT are imperative to bring this success to patients with other types of cancer.

The use of bifunctional chelators allows for development of theranostic radiopharmaceuticals. Theranostic radiopharmaceuticals, as the name suggests, can be used for both therapy and diagnosis (imaging) of a disease. An identical pharmaceutical scaffold can be radiolabeled with either a radionuclide that decays by particle emission (e.g., alpha or beta minus decay) for therapeutic applications or a radionuclide that decays with photon emission (e.g., isomeric transition or positron decay) for diagnostic applications. The radionuclide(s) can be a single radionuclide that decays with both particle and photon emissions (e.g., ⁶⁴Cu, positron and beta minus emissions), a pair of isotopes of the same element (e.g., ¹⁵⁵Tb, imageable gamma emission, and ¹⁶¹Tb, beta minus emission), or a matched pair of radionuclides of different elements (e.g., ^99mTc, imageable gamma emission, and ^186/188Re, beta minus emission). Ideally, if isotopes of two different elements are used as a theranostic matched pair, they will have similar chemical properties allowing for coordination to the same bifunctional chelator and a similar pharmacokinetic profile of the theranostic radiopharmaceuticals in vivo.

An example of a potential theranostic matched pair of radionuclides is ^99mTc and ¹⁸⁶Re/¹⁸⁸Re. In 2019, it was reported that of the 30 million nuclear medicine diagnostic scans conducted worldwide, 85% were performed using ^99mTc radiopharmaceuticals. (9) Technetium-99m (t_1/2 = 6.0 h) is extremely popular for diagnostic radiopharmaceutical applications because it is readily available in the form of a ⁹⁹Mo/^99mTc generator, and it decays primarily by isomeric transition with the emission of a 140 keV gamma-ray that is ideal for single photon emission computed tomography (SPECT) imaging. Technetium has no nonradioactive isotopes or radioisotopes with useful therapeutic decay properties. However, rhenium is a congener of technetium with similar chemical and physical properties, making it the ideal candidate as a therapeutic counterpart. Two beta-minus emitting radioisotopes of rhenium, ¹⁸⁶Re (t_1/2 = 3.7 d) and ¹⁸⁸Re (t_1/2 = 17 h), have previously been explored as theranostic matched radionuclide partners to ^99mTc with promising results demonstrating similar pharmacokinetic profiles in vivo. (10,11)

Rhenium and technetium have rich redox chemistry, allowing them to bind to a variety of different bifunctional chelators. The +1 oxidation state of these metals can be accessed and stabilized through the synthesis of a [M(CO)₃(OH₂)₃]⁺ precursor (M = ^99mTc, ^natRe, ¹⁸⁶Re, and ¹⁸⁸Re) from the permetallate or metal pentacarbonyl bromide chemical forms. The syntheses of these precursors are well established in literature. (12−14) The [M(CO)₃(OH₂)₃]⁺ precursors can be reacted with bifunctional chelators, during which the labile water ligands are replaced to form metal complexes. The tridentate NOTA chelator (2,2′,2″-(1,4,7-triazacyclononane-1,4,7-triyl)triacetic acid) has proven to be an excellent bifunctional chelator for coordination of the [M(CO)₃]⁺ core (M = ^99mTc, ^natRe, and ¹⁸⁶Re) due, in part, to the ionizable pendent acid arms that aid in (radio)labeling and increase the hydrophilicity of the overall metal complex. (15,16) These [M(CO)₃NOTA]⁻ metal complexes, both as model complexes and as peptide bioconjugates, have demonstrated excellent in vitro stability with moderate to high (radio)labeling yields. (10,15,16)

Our current work is investigating how changing the pendent arms on the 1,4,7-triazacyclononane (TACN) backbone impacts the chelator’s interaction with the [M(CO)₃]⁺ core (M = ^99mTc, ^natRe, and ¹⁸⁶Re). Modified TACN chelators have been evaluated with radionuclides including ⁶⁴Cu and ⁶⁸Ga, (17−19) but there are very few examples of these chelators (radio)labeled with the [M(CO)₃]⁺ cores. (15,20) Previously, we evaluated TACN derivative chelators bearing no pendent arms, acid arms (NOTA derivative), ester arms, and mixed acid/ester arms. (16) In those studies, we demonstrated that although the TACN pendent arms did not participate in coordination to the metal in the final complex, they played a significant role in facilitating (radio)labeling. Only the TACN chelator with no pendent arms and the chelators bearing at least one acid arm were successfully (radio)labeled with the [M(CO)₃]⁺ cores. We hypothesized that there is an electrostatic attraction between the ionized pendent arms and the positively charged metal center, as well as a steric component, contributing to the success of the (radio)labeling. Further investigations into the interactions between the [M(CO)₃]⁺ core and TACN-based chelators may provide insights to improve upon the design of these chelators for radiopharmaceutical applications.

With that in mind, this work presents three TACN-based chelators, namely, those bearing amide, alcohol, and ketone pendent arms, to evaluate how effectively they can be (radio)labeled with the [M(CO)₃]⁺ cores. In this fundamental chemistry study, the amide and alcohol chelators were selected because they are not ionized in aqueous solutions at physiological pH; however, the polar functional groups they bear carry a partial negative charge that may directly or indirectly (e.g., via hydrogen bond formation with anions in solution) facilitate metal coordination. The ketone chelator was chosen as a comparator to the ester derivatives studied previously, as it does not contain a terminal polar group, but it is less sterically hindering than an ester group. TACN-based chelators bearing amide (21) and alcohol (22) pendent arms have been evaluated in the literature with gallium and zinc, respectively, with some success, but these chelators have not been evaluated with [M(CO)₃]⁺ cores.

2. Materials and Methods

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2.1. General

All chemicals were purchased from Sigma-Aldrich (St. Louis, Missouri) or Fisher Scientific (Pittsburgh, Pennsylvania) and were of reagent grade. The 1,4,7-triazacyclononane and di-tert-butyl 2,2′-(1,4,7-triazacyclononane-1–4-diyl)diacetate (NO2AtBu) were purchased from CheMatech (Dijon, France). Microwave syntheses were performed in a CEM Discover microwave synthesizer at a fixed power of 200 W.

Shimadzu Nexera or Prominence HPLCs with photodiode array (PDA) detectors and in-line NaI(Tl) detectors were used for HPLC analyses. The HPLC-grade solvents were filtered and degassed prior to use. All HPLC analyses were carried out on Thermo Fisher Scientific BetaBasic C18 columns (150 mm × 4.6 mm, 5 μm) with a flow rate of 1 mL/min and analyzed at 210 nm (TACN chelators) or 254 nm (Re-labeled chelators) wavelengths. Compound purifications were carried out on a Shimadzu Nexera semipreparative HPLC using a Phenomenex Luna C18 column (250 × 10 mm, 10 μm) with a flow rate of 4 mL/min. Binary linear gradients were used with pump A containing water (0.1% trifluoroacetic acid (TFA)) and pump B containing methanol (0.1% TFA). The following gradients were used for HPLC analyses and purifications: 5 to 95% B in A over 18 min (method 1), 30 to 55% B in A over 10 min (method 2), 30 to 40% B in A over 10 min (method 3), 30 to 80% B in A over 20 min (method 4), and 50 to 70% B in A over 10 min (method 5). Radio thin layer chromatography (radio-TLC) experiments were conducted on A81-24 saturation pads (Analtech, Newark, Delaware) and developed in 75% acetonitrile in saline. The radio thin layer chromatograms were read and analyzed on an Eckert and Ziegler AR-2000 radio-TLC Imager Scanner (Hopkinton, Massachusetts).

High-resolution electrospray ionization mass spectrometry (HS-ESI-MS) analyses were conducted on an LTQ Orbitrap XL or a Bruker timTOF-Pro2 mass spectrometer and analyzed with Xcalibur Qual Browser version 2.2 or CompassDA software. Liquid chromatography electrospray ionization mass spectrometry (LC-ESI-MS) analyses were conducted using a HPLC Gold System (Beckman Coulter, Fullerton, California) coupled to an ion trap mass spectrometer (LCQ Fleet from Thermo Fisher). The column, a Thermo Fisher BetaBasic C18 column (150 mm × 4.6 mm, 5 μm), was used with a flow rate of 1 mL/min and a binary linear gradient with pump A containing water (0.1% TFA) and pump B containing acetonitrile (0.1% TFA). The following gradient was used for LCMS analyses: 10 to 50% B in A over 30 min (method 6).

¹H and ¹³C NMR spectra were recorded on a Bruker Avance III 500 or 600 MHz spectrometer and analyzed with Bruker TopSpin version 4.0.9. A Thermo Scientific Nicolet Summit Pro FTIR spectrometer was used for infrared spectroscopy analysis. Wavelengths between 500 and 4000 cm^–1 were recorded. Combustion analysis was used to determine the elemental composition of the ^natRe-labeled complexes (Atlantic Microlab, Norcross, Georgia).

[^99mTc]TcO₄^– in saline was obtained in high specific activity from ⁹⁹Mo/^99mTc generators (Curium, St. Louis, Missouri) that were generously donated by Mid-America Isotopes, Inc. (Ashland, Missouri). The ^99mTc activity was measured by a Capintec CRC-55tR dose calibrator (Ramsey, New Jersey) or an ORTEC 4890 NaI(Tl) well detector. Low specific activity [¹⁸⁶Re]ReO₄^– (44–56 GBq/mg, 1.2–1.5 Ci/mg at the end of irradiation) was produced by neutron irradiation of enriched [¹⁸⁵Re]Al(ReO₄)₃ targets at the University of Missouri Research Reactor (MURR). The ¹⁸⁶Re activity was measured by a Capintec dose calibrator or an ORTEC HPGe GEM20–70 high-purity germanium detector (Oak Ridge, Tennessee) coupled to a Canberra multichannel analyzer (Meriden, Connecticut). All HPGe spectroscopy data were analyzed using the Canberra Genie 2000 (3.3) software.

2.2. N-Benzyl-2-(1,4,7-triazonan-1-yl)acetamide (1)

Compound 1 was synthesized using two different methods (Scheme 1 and Scheme S1 in the Supporting Information). Details for the alternative synthetic method and synthesis of N-benzyl-2-bromoacetamide were reported previously (16,23) and can be found in the Supporting Information. Commercially available TACN (100 mg, 0.77 mmol) and potassium carbonate (160 mg, 1.16 mmol) were dissolved in 5 mL of acetonitrile. In another vial, N-benzyl-2-bromoacetamide (175 mg, 0.77 mmol) was dissolved in 2.5 mL of acetonitrile and then added dropwise to the TACN solution over 20 min. After complete addition of the N-benzyl-2-bromoacetamide, the reaction was stirred at room temperature for 24 h. The solvent was removed under reduced pressure, and the crude residue was purified by semipreparative HPLC in small batches (method 1, t_R = 10.4 min). Isolated yield: 85 mg (40%). The product was characterized by LC-ESI-MS and ¹H NMR. LC-ESI-MS (m/z) calculated for C₁₅H₂₄N₄O [M + H]⁺ 277.28, found 277.31 (method 6, t_R = 7.9 min). ¹H NMR (D₂O, 300 MHz): δ_H 7.23–7.15 (m, 5H), 4.35 (s, 2H), 3.51 (s, 2H), 3.24 (t, 8H, J = 5.7 Hz), 2.99 (t, 4H, J = 5.5 Hz) and matches values previously reported in literature. (23)

Scheme 1. Synthesis of TACN-Based Chelators 2–4^a

2.3. 2,2′-(7-(2-(Benzylamino)-2-oxoethyl)-1,4,7-triazonane-1,4-diyl)diacetamide (2)

The synthesis of model chelators 2–4 is shown in Scheme 1. Compound 1 (110 mg, 0.4 mmol) was dissolved in 3 mL of anhydrous acetonitrile. Triethylamine (166 μL, 1.2 mmol) and 2-bromoacetamide (110 mg, 0.8 mmol) were added, and the resulting solution was heated at 80 °C for 3 h in an oil bath. Reaction progress was monitored by analytical HPLC (method 1, t_R = 8.0 min). Upon reaction completion, the solvent was removed under reduced pressure, and the product was purified by semipreparative HPLC in small batches (method 1, t_R = 8.2 min). Isolated yield: 115 mg (74%). The product was characterized by HR-ESI-MS, ¹H NMR, and ¹³C NMR. HR-ESI-MS (m/z) calculated for C₁₉H₃₀N₆O₃ [M + H]⁺ 391.2452, found 391.2444. ¹H NMR (CD₃CN, 600 MHz): δ_H 7.66 (t, 1H, J = 5.8 Hz), 7.39–7.29 (m, 5H), 6.90 (s, 2H), 6.35 (s, 2H), 4.41 (d, 2H, J = 5.7 Hz), 3.73 (s, 2H), 3.68 (s, 2H), 3.13 (s, 12H). ¹³C NMR (CD₃CN, 600 MHz): δ_C 171.1, 168.8, 138.4, 128.5, 127.6, 127.2, 58.0, 57.2, 50.4, 50.2, 43.0. Values were compared to and matched those reported in the literature. (24)

2.4. N-Benzyl-2-(4,7-bis(2-hydroxyethyl)-1,4,7,-triazonan-1-yl)acetamide (3)

Compound 1 (130 mg, 0.5 mmol) was dissolved in 3 mL of anhydrous acetonitrile. Triethylamine (216 μL, 1.5 mmol) and 2-bromoethanol (68 μL, 1 mmol) were added, and the resulting solution was heated at 80 °C for 9 h in an oil bath. The reaction progress was monitored by analytical HPLC (method 1, t_R = 7.8 min). More 2-bromoethanol (68 μL, 1 mmol) was added to the reaction every 3 h, as necessary, until the reaction reached completion (i.e., all of compound 1 had reacted). The crude product was purified by semipreparative HPLC in small batches (method 3, t_R = 7.3 min). Isolated yield: 92 mg (54%). The product was characterized by HR-ESI-MS, ¹H NMR, and ¹³C NMR. HR-ESI-MS (m/z) calculated for C₁₉H₃₂N₄O₃ [M + H]⁺ 365.2547, found 365.2536. ¹H NMR (D₂O, 600 MHz): δ_H 7.40–7.30 (m, 5H), 4.40 (s, 2H), 3.87–3.86 (m, 8H), 3.64 (s, 2H), 3.51–3.01 (m, 12H). ¹³C NMR (CD₃CN, 600 MHz): δ_C 173.2, 138.1, 128.5, 127.7, 127.3, 59.4, 56.9, 56.8, 55.9, 51.4, 50.4, 49.1, 43.2.

2.5. N-Benzyl-2-(4,7-bis(2-oxopropyl)-1,4,7-triazonan-1-yl)acetamide (4)

Compound 1 (20 mg, 0.07 mmol) was dissolved in 2.5 mL of anhydrous acetonitrile. Triethylamine (30 μL, 0.2 mmol) and 2-chloroacetone (12 μL, 0.14 mmol) were added, and the resulting solution was heated at 80 °C for 3 h. The reaction progress was monitored by analytical HPLC (method 1, t_R = 9.4 min). The solvent was removed under reduced pressure, and the crude product was purified by semipreparative HPLC in small batches (method 4, t_R = 13.6 min). Isolated yield: 26 mg (93%). The product was characterized by HR-ESI-MS, ¹H NMR, and ¹³C NMR. HR-ESI-MS (m/z) calculated for C₂₁H₃₂N₄O₃ [M + H]⁺ 389.2547, found 389.2531. ¹H NMR ((CD₃)₂SO, 600 MHz): δ_H 8.62 (t, 1H, J = 5.7 Hz), 7.35–7.25 (m, 5H), 4.33 (d, 2H, J = 5.5 Hz), 3.90 (s, 4H), 3.71 (s, 2H), 3.19–2.75 (m, 12H), 2.05 (s, 6H). ¹³C NMR ((CD₃)₂SO, 600 MHz): δ_C 206.1, 168.5, 139.4, 128.8, 127.8, 127.4, 63.4, 57.0, 50.2, 49.9, 49.6, 42.6, 27.7.

2.6. 2,2′-(7-(2-(Benzylamino)-2-oxoethyl)-1,4,7-triazonane-1,4-diyl)diacetic Acid (5)

The synthesis of chelator 5 is shown in Scheme 2 and was adapted from a previously reported procedure. (25) Commercially available NO2AtBu (18 mg, 0.05 mmol) was dissolved in 1.5 mL of acetonitrile with potassium carbonate (11.9 mg, 0.09 mmol). N-Benzyl-2-bromoacetamide (12 mg, 0.05 mmol) was added, and the reaction was stirred at room temperature for 48 h. The reaction mixture was then filtered, and the solvent was removed under reduced pressure. The crude residue was reconstituted in a mixture of TFA, triisopropylsilane, and water (8.5:1:0.5, 1 mL) and stirred for 6 h at room temperature. The reaction was dried under nitrogen and reconstituted in 3 mL of diethyl ether with <5% methanol, resulting in the formation of a white precipitate. The precipitate was recovered by filtration and washed with dichloromethane. The final product was purified by semipreparative HPLC (t_R = 8.0 min, method 2). Isolated yield: 35 mg (64%). The product was characterized by HR-ESI-MS, ¹H NMR, and ¹³C NMR. HR-ESI-MS (m/z) calculated for C₁₉H₂₈N₄O₅ [M + H]⁺ 393.2133, found 393.2105. ¹H NMR ((CD₃)₂SO, 600 MHz): δ_H 8.78 (t, 1H, J = 5.4 Hz), 7.34–7.25 (m, 5H), 4.34 (d, 2H, J = 5.8 Hz), 3.84 (s, 2H), 3.67 (s, 4H), 3.15–3.00 (m, 12H). ¹³C NMR ((CD₃)₂SO, 600 MHz): δ_C 171.9, 167.3, 139.2, 128.8, 127.8, 127.4, 57.0, 55.1, 50.9, 49.7, 48.7, 42.7.

Scheme 2. Synthesis of TACN-Based Chelator 5^a

2.7. Labeling of TACN-Based Chelators with the [^natRe(CO)₃]⁺ Core

The [^natRe(CO)₃(OH₂)₃](NO₃) precursor was synthesized from (NEt₄)₂[Re(CO)₃Br₃], as previously described. (14) Details for the synthesis can be found in the Supporting Information. The [^natRe(CO)₃(OH₂)₃](NO₃) precursor was reacted with the TACN-based chelators in a ∼2:1 molar ratio. The optimized reaction conditions for the individual chelators are as follows.

2.7.1. Re-2

Chelator 2 (11.5 mg, 0.029 mmol) and [^natRe(CO)₃(OH₂)₃](NO₃) (23 mg, 0.059 mmol) were combined in 900 μL of phosphate-buffered saline (PBS; 1 mM, pH 9) and heated at 95 °C for 3 h in a thermomixer. The product was purified by semipreparative HPLC (method 1, t_R = 13.8 min) and characterized by HRMS, IR, ¹H NMR, ¹³C NMR, and elemental analyses. Isolated yield: 9.0 mg (47%). HR-ESI-MS (m/z) calculated for C₂₂H₂₉N₆O₆¹⁸⁵Re⁺ [M]⁺ 661.1780, found 661.1808. IR (solid, cm^–1): 3312, 3195, 2033, 1905, 1664, 1134. ¹H NMR (CD₃CN, 600 MHz): δ_H 7.38–7.32 (m, 5H), 7.22 (t, 1H, J = 5.6 Hz), 6.47 (s, 2H), 6.00 (s, 2H), 4.40 (d, 2H, J = 5.3 Hz), 4.23 (s, 6H), 3.64–3.58 (m, 12H). ¹³C NMR (CD₃CN, 600 MHz): δ_C 194.5, 169.4, 167.3, 138.4, 128.5, 127.5, 127.3, 65.33, 65.30, 57.6, 57.41, 57.39, 42.4. Elemental analysis: anal. calcd. for C₂₂H₂₉N₆O₆Re⁺(TFA)₂(H₂O)((CH₃)₂CO): C, 35.47; H, 4.21; N, 8.56. Found C, 35.14; H, 3.81; N, 8.42.

2.7.2. Re-3

Chelator 3 (15 mg, 0.041 mmol) and [^natRe(CO)₃(OH₂)₃](NO₃) (25 mg, 0.065 mmol) were combined in 500 μL of aqueous sodium bicarbonate (0.1 M, pH 8.2) and heated at 95 °C for 3 h in a thermomixer. The product was purified by semipreparative HPLC (method 5, t_R = 7.4 min) and characterized by HRMS, IR, ¹H NMR, ¹³C NMR, and elemental analyses. Isolated yield: 9.6 mg (37%). HR-ESI-MS (m/z) calculated for C₂₂H₃₁N₄O₆¹⁸⁵Re⁺ [M]⁺ 635.1875, found 635.1899. IR (solid, cm^–1): 3100–3600, 2917, 2850, 2029, 1903, 1671, 1176, 1128. ¹H NMR (CD₃CN, 600 MHz): δ_H 7.39–7.29 (m, 5H), 7.22 (s, 1H), 4.67 (t, 1H, J = 4.7 Hz), 4.40 (d, 2H, J = 6.0 Hz), 4.25 (m, 2H), 3.86–3.89 (m, 4H), 3.69–3.38 (m, 12H), 3.32–3.26 (m, 4H). ¹³C NMR (CD₃CN, 600 MHz): δ_C 195.5, 167.4, 138.5, 128.5, 127.5, 127.2, 68.1, 66.2, 58.5, 57.6, 57.0, 56.9, 42.5. Elemental analysis: anal. calcd. for C₂₂H₃₁N₄O₆Re⁺(TFA)₂: C, 36.20; H, 3.97; N, 6.49. Found C, 35.96; H, 3.85; N, 6.20.

2.7.3. Re-4

Chelator 4 (1 mg, 2.6 μmol) and [^natRe(CO)₃(OH₂)₃](NO₃) (2 mg, 5.2 μmol) were combined in 500 μL of 0.2 M MES (2-(N-morpholino)ethanesulfonic acid) buffer pH 4–6, 0.1 M sodium acetate pH 4–5, 0.1 M sodium bicarbonate pH 7–9, 0.1 M sodium carbonate pH 10–12, 1 mM PBS pH 7–12, or organic solvent (methanol or dimethyl sulfoxide) and heated at 95 °C for 3 h in a thermomixer. Labeling was unsuccessful under all tested conditions.

2.7.4. Re-5

Chelator 5 (10 mg, 0.025 mmol) and [^natRe(CO)₃(OH₂)₃](NO₃) (20 mg, 0.052 mmol) were combined in 500 μL of MES buffer (0.2 M, pH 5) and heated at 95 °C for 1 h in a thermomixer. The product was purified by semipreparative HPLC (method 5, t_R = 7.8 min) and characterized by HRMS, IR, ¹H NMR, ¹³C NMR, and elemental analyses. Isolated yield: 8.7 mg (53%). HR-ESI-MS (m/z) calculated for C₂₂H₂₇N₄O₈¹⁸⁵Re⁺ [M]⁺ 663.1460, found 663.1445. IR (solid, cm^–1): 2032, 1903, 1725, 1660. ¹H NMR (CD₃CN, 600 MHz): δ_H 7.45–7.28 (m, 5H), 4.40 (d, 2H, J = 5.6 Hz), 4.34 (s, 4H), 4.24 (s, 2H), 3.68–3.51 (m, 12H). ¹³C NMR (CD₃CN, 600 MHz): δ_C 194.3, 169.4, 167.4, 138.4, 128.5, 127.5, 127.3, 66.1, 64.9, 57.6, 57.3, 42.6. Elemental analysis: anal. calcd. for C₂₂H₂₇N₄O₈Re⁺(TFA)₂: C, 35.06; H, 3.39; N, 6.29. Found C, 35.29; H, 3.51; N, 6.53.

2.8. Radiolabeling of TACN-Based Chelators with the [^99mTc][Tc(CO)₃]⁺ Core

High specific activity [^99mTc]TcO₄^–, eluted in saline from a ⁹⁹Mo/^99mTc generator, was used to synthesize the [^99mTc][Tc(CO)₃(OH₂)₃]⁺ precursor, as previously reported. (13) The optimized reaction conditions for the individual chelators are as follows.

2.8.1. [^99mTc]Tc-2

Chelator 2 (35 μg, 0.09 μmol) and [^99mTc][Tc(CO)₃(OH₂)₃]⁺ (3.7–55.5 MBq, 0.1–1.5 mCi) were combined in 300 μL of sodium bicarbonate (0.1 M, pH 7) and heated at 95 °C for 1 h.

2.8.2. [^99mTc]Tc-3

Chelator 3 (33 μg, 0.09 μmol) and [^99mTc][Tc(CO)₃(OH₂)₃]⁺ (3.7–55.5 MBq, 0.1–1.5 mCi) were combined in 300 μL of sodium bicarbonate (0.1 M, pH 9) and heated at 95 °C for 1 h.

2.8.3. [^99mTc]Tc-4

Chelator 4 was not successfully reacted with [^99mTc][Tc(CO)₃(OH₂)₃]⁺ under all tested conditions.

2.8.4. [^99mTc]Tc-5

Chelator 5 (35 μg, 0.09 μmol) and [^99mTc][Tc(CO)₃(OH₂)₃]⁺ (3.7–55.5 MBq, 0.1–1.5 mCi) were combined in 300 μL of MES buffer (0.2 M, pH 5) and heated at 95 °C for 1 h.

Reaction progress was monitored by radio-HPLC, and the formation of colloidal ^99mTcO₂ was monitored by radio-TLC (colloid R_f = 0; other solution components near R_f = 1, Supporting Information Figures S19–S21). All ^99mTc-labeled complexes were characterized by HPLC coinjection with the fully characterized ^natRe-labeled analogues.

2.9. Radiolabeling of TACN-Based Chelators with the [¹⁸⁶Re][Re(CO)₃]⁺ Core

Low specific activity [¹⁸⁶Re]ReO₄^–, produced by neutron irradiation of an enriched ¹⁸⁵Re target, was used to synthesize the [¹⁸⁶Re][Re(CO)₃(OH₂)₃]⁺ precursor as previously reported. (16) The optimized reaction conditions for the individual chelators are as follows.

2.9.1. [¹⁸⁶Re]Re-2

Chelator 2 (195 μg, 0.5 μmol) and [¹⁸⁶Re][Re(CO)₃(OH₂)₃]⁺ (0.7–7.4 MBq, 20–200 μCi) were combined in 500 μL of sodium bicarbonate (0.1 M, pH 8) and heated at 95 °C for 1 h.

2.9.2. [¹⁸⁶Re]Re-3

Chelator 3 (182 μg, 0.5 μmol) and [¹⁸⁶Re][Re(CO)₃(OH₂)₃]⁺ (0.7–1.9 MBq, 20–50 μCi) were combined in 500 μL of sodium bicarbonate (0.1 M, pH 9) and heated at 95 °C for 1 h.

2.9.3. [¹⁸⁶Re]Re-4

Chelator 4 was not successfully reacted with [¹⁸⁶Re][Re(CO)₃(OH₂)₃]⁺.

2.9.4. [¹⁸⁶Re]Re-5

Chelator 5 (59 μg, 0.15 μmol) and [¹⁸⁶Re][Re(CO)₃(OH₂)₃]⁺ (0.7–37 MBq, 0.02–1 mCi) were combined in 500 μL of MES (0.2 M, pH 5) and heated at 95 °C for 1 h.

Reaction progress was monitored by radio-HPLC, and the formation of colloidal ¹⁸⁶ReO₂ was monitored by radio-TLC (colloid R_f = 0; other solution components near R_f = 1, Supporting Information Figures S22–S24). All ¹⁸⁶Re-labeled complexes were characterized by HPLC coinjection with the fully characterized ^natRe-labeled analogues.

2.10. In Vitro Stability and logD_7.4 Studies

The crude radiocomplexes were isolated by radio-HPLC, and the HPLC eluent was evaporated under a nitrogen gas stream until the volume was reduced to 50–100 μL. The purified radiocomplex was then added to a solution of PBS buffer (350–400 μL, 1 mM, pH 7.4), l-cysteine (1 mM in 350–400 μL PBS), l-histidine (1 mM in 350–400 μL PBS), or rat serum (400–450 μL, Innovative grade US Origin Sprague–Dawley Rat Serum, Innovative Research, Novi, Michigan). Ascorbic acid (50 μL, 1 mg/mL in PBS) was added as a radioprotectant, and the solutions were incubated at 37 °C. Aliquots of each solution (PBS, l-cysteine, and l-histidine) were taken at 24 h, and the stability of the radiocomplexes was evaluated by radio-HPLC and radio-TLC. Aliquots (100–500 μL) of rat serum were taken at 24 h and combined with a 4× volume of acetonitrile to precipitate the serum proteins. The resulting mixture was vortexed (5 min) and centrifuged (10 min, 1900 × g), and the supernatant was carefully removed. The precipitated proteins were washed with an additional 100–500 μL of acetonitrile, vortexed (5 min), and centrifuged (10 min, 1900 × g), and the supernatant was carefully removed. The combined supernatants were used to determine radiocomplex stability by radio-HPLC and radio-TLC analyses. The supernatant and pellet activities were used to determine the percent protein binding of the radiocomplex.

The distribution coefficients were determined with octanol/water partitioning. The HPLC-purified radiocomplexes were diluted to 500 μL in PBS (1 mM, pH 7.4) and added to a mixture of 5 mL of octanol and 4.5 mL of PBS. The resulting solutions were vortexed (3 min) and centrifuged (10 min, 1900 × g). The octanol and PBS layers were separated and aliquoted into four 1 mL fractions. The activity in each fraction was counted on either an HPGe detector (¹⁸⁶Re-complexes) or NaI(Tl) well detector (^99mTc-complexes). Each logD_7.4 value was calculated by taking the log of the average counts in the organic layer divided by the average counts in the aqueous layer. This experiment was conducted in triplicate for each radiocomplex. Each reported logD_7.4 value represents an average of the three experiments.

2.11. Single-Crystal X-ray Diffraction Analysis

Attempts to grow X-ray quality crystals of ^natRe-2/3/5 were unsuccessful. However, X-ray quality crystals of a related [^natRe(CO)₃]⁺-labeled TACN-based chelator bearing one N-benzyl-acetamide arm and two ethyl ester arms (the synthesis of which was previously reported (16)) were successfully grown by the slow evaporation of a 50% ethanol in water solution of the compound over the course of 3 weeks. Single-crystal X-ray diffraction (SCXRD) data were measured on a Bruker D8 Venture diffractometer with a Photon II CMOS area detector using Mo Kα radiation from a microfocus source (Bruker AXS, Madison, Wisconsin, USA). The crystal was cooled to 173.0 K under a cold stream of N₂ using a Cryostream 800 cryostat (Oxford Cryosystems, Oxford, UK). A hemisphere of unique data was collected using strategies of scans about the phi and omega axes. The Apex4 software suite was used for data collection, unit cell determination, data reduction, absorption correction and scaling, and space group determination. (26)

The structure was solved in the monoclinic space group P2₁/n by direct methods, as implemented in SHELXS (27) and refined by full matrix least-squares refinement using SHELXL v.2017. (28) Olex2 was used as an interface for structure visualization and model building. (29) Non-hydrogen atoms were located from the difference map and refined anisotropically. Carbon atoms in the heterocyclic core were found to be disordered and refined over two positions located from the difference map with their occupancies set to values that gave similar isotropic thermal parameters (30%/70% for one group and 40%/60% for the other). The amide N–H hydrogen atom was located from the difference map, and its coordinates were refined with the N–H distance restrained to 0.92(1) Å. All other hydrogen atoms were placed in calculated positions, and their coordinates and thermal parameters were constrained to ride on the carrier atoms. The final model has a significant difference map peak near the Re atom, which has the same y coordinates as the Re atom generated by a glide operation and is attributed to a packing fault where chains of molecules related by the glide operation substitute for each other. The [CF₃COO]⁻ ion shows evidence for positional disorder of the entire molecule, but the minor part failed to refine to a realistic geometry consistent with hydrogen bonding to the amide. Refining this group anisotropically at full occupancy gave the best model but with some anomalously short C–O and C–F distances likely due to the imprecision of these atomic positions. Crystal data, including selected bond lengths and angles, can be found in the Supporting Information.

3. Results and Discussion

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3.1. Synthesis of Modified TACN Chelators

The synthesis of the modified TACN chelators is shown in Schemes 1 and 2. Chelators 2–4 were synthesized from commercially available TACN following two synthetic methods. In the first method, TACN was reacted directly with N-benzyl-2-bromoacetamide in a 1:1 molar ratio. This reaction resulted in the formation of three products in a 6:3:1 ratio: the desired monosubstituted N-benzyl acetamide product (1), a disubstituted N-benzyl acetamide chelator, and a trisubstituted chelator. The three products were easily separated by HPLC isolation, resulting in a 40% isolated yield for the desired product, 1. Given the multiple side products formed that affected the overall yield of the desired product, an alternative synthetic method was used (Scheme S1 in the Supporting Information). This method began with the reaction of TACN with N,N-dimethylformamide dimethyl acetal to form TACN-orthoamide in near quantitative yields. The orthoamide intermediate allowed for preferential formation of the monosubstituted product upon reaction with N-benzyl-2-bromoacetamide, (30) which was then reacted with sodium hydroxide to convert the orthoamide back to an unrestricted TACN ring and form chelator 1. The overall isolated yield for the orthoamide approach was 50–60%, with loss in yield attributed to incomplete addition of the N-benzyl acetamide arm, minor formation of the di- and trisubstituted products (<10%), and/or hydrolysis of the amide bond by the sodium hydroxide (as confirmed by LC-ESI-MS analysis).

Chelators 2–4 were formed by heating chelator 1 with 2-bromoacetamide, 2-bromoethanol, or 2-chloroacetone, respectively, in acetonitrile with triethylamine as the base at 80 °C for 3–9 h. The progress of these reactions was monitored by HPLC and LC-ESI-MS analyses, revealing a very clean synthesis with minimal byproduct formation. The chelators were purified via semipreparative HPLC with isolated yields between 50 and 90%. These reactions were easily scaled up from 5 to 10 mg to 100–200 mg with minimal impact on yield. The purified chelators were isolated as yellow or colorless oils and were fully characterized prior to (radio)labeling studies. Chelators 2 (diamide) and 3 (dialcohol) were soluble in water, methanol, acetonitrile, and dimethyl sulfoxide and remained stable when stored at 4 °C for several weeks. Chelator 4 (diketone) was soluble in dimethyl sulfoxide and remained stable at 4 °C for several weeks.

Chelator 5 (diacid) was used as a comparator for these experiments, since high (radio)labeling yields and excellent stability of NOTA derivative complexes with the [M(CO)₃]⁺ cores is well established in the literature. (15,16,20) Chelator 5 was synthesized in high yield from commercially available NO2AtBu following a literature procedure (Scheme 2). (25) Prior to (radio)labeling studies, the chelator was isolated by HPLC as a colorless oil and fully characterized. The chelator was soluble in water, methanol, acetonitrile, and dimethyl sulfoxide and remained stable at room temperature for several months.

3.2. Chelator Labeling with the [^natRe(CO)₃]⁺ Core

Since the synthesis of radiometal complexes is performed at extremely small scales (nmol scale or lower in these studies), traditional characterization techniques cannot be used to confirm their chemical structures. Thus, chelators 2–5 were labeled with the [^natRe(CO)₃]⁺ core, and the resulting products were fully characterized for subsequent use as nonradioactive metal complex standards for comparison against the radiocomplexes via HPLC coinjection. These ^natRe-labeled complexes served as standards for both the ¹⁸⁶Re- and ^99mTc-labeled complexes because there are no nonradioactive isotopes of technetium. Rhenium and technetium belong to the same group of the periodic table, resulting in them having similar chemical and physical properties. As such, rhenium and technetium were expected to coordinate to the TACN chelators in a matching manner. (15,16)

Chelator 2 (diamide) was reacted with the [^natRe(CO)₃(OH₂)₃]⁺ core in PBS buffer (pH 7–9) at 95 °C for 3 h in a thermomixer, with pH 9 resulting in the highest yields. The reaction progress was monitored by HPLC (t_R = 11.0 min, method 1). After 3 h, only ∼50% of the chelator had been successfully labeled (by HPLC), leading to an isolated yield of 40%. Due to decomposition of the [^natRe(CO)₃(OH₂)₃]⁺ precursor, further heating was not expected to increase the labeling yield. The reaction was also tested in MES buffer (0.2 M, pH 3–6), PBS (1 mM, pH 3–7 and 10–12), sodium bicarbonate (0.1 M, pH 7–9), and sodium carbonate (0.1 M, pH 10–12). The labeling was unsuccessful at a pH less than 7 or greater than 9, regardless of the buffer. In sodium bicarbonate, two products were formed: the desired ^natRe-2 product and a byproduct with a mass to charge ratio corresponding to 1 Da greater than the ^natRe-2 complex (LC-ESI-MS, t_R = 18.5 min, method 6). The formation of this byproduct was attributed to the base hydrolysis of the amide group, converting it to a carboxylic acid. (31) The formation of the hydrolysis product was limited by performing the labeling reaction in sodium bicarbonate under microwave heating at 95 °C for 30 min. Microwave heating had the added benefit of a shorter reaction time while resulting in a yield similar to that achieved from 3 h of conventional heating in PBS buffer (∼40%). Performing the microwave reaction in PBS did not significantly increase the yield.

Chelator 3 (dialcohol) was reacted with [^natRe(CO)₃(OH₂)₃]⁺ in sodium bicarbonate (0.1 M, pH 7–9) at 95 °C for 3 h in a thermomixer. The yield (by HPLC) was near quantitative throughout that pH range with minimal byproduct formation observed. The labeling reaction was successful at pH 10–12 in sodium carbonate (0.1 M); however, these reaction conditions were not used for labeling of more than 1 mg of chelator due to the instability of [^natRe(CO)₃(OH₂)₃]⁺ at very high pH. (32) Sodium bicarbonate was ultimately chosen as the optimal buffer.

Chelator 4 (diketone) proved more difficult to label. Chelator 4 was reacted with [^natRe(CO)₃(OH₂)₃]⁺ in sodium acetate (0.1 M, pH 4–5), MES buffer (0.2 M, pH 4–6), sodium bicarbonate (0.1 M, pH 7–9), PBS (1 mM, pH 7–12), sodium carbonate (0.1 M, pH 10–12), and organic solvent (30% methanol in water or dimethyl sulfoxide) with heating at 95 °C for 3 h. Under all tested reaction conditions, successful labeling was not achieved. Alternative routes to synthesize ^natRe-4 from ^natRe-1 (nonfunctionalized) or ^natRe-5 (diacid) were explored but deemed impractical due to long reaction times and the excessive reagents required to achieve the chemical conversions.

Previous labeling investigations of TACN-based chelators with the [M(CO)₃]⁺ core found an association between chelators with ionizable pendent group(s) and increased labeling efficiency. (16) Successful labeling was only demonstrated with chelators that were not sterically hindered (i.e., had no pendent arms, 1) or that bore pendent arms that were ionized under the labeling conditions (e.g., the diacid, 5, and mixed acid/ester pendent arms). The TACN-based chelators bearing exclusively ester pendent arms were not successfully labeled and had to be indirectly synthesized by esterification of the [^natRe(CO)₃]⁺-labeled diacid chelator (^natRe-5). The inability to directly label the diester chelators was postulated to be due, in part, to the lack of an electrostatic attraction between the pendent arms and the metal center.

Consistent with those findings, chelator 4 (diketone), which bears nonpolar pendent arms that do not ionize under the labeling conditions, was not successfully labeled with the [^natRe(CO)₃]⁺ core, even despite the smaller size (steric bulk) of its pendent arms as compared with the previously studied diester derivatives. The pendent arms on the modified TACN chelators clearly impact the efficiency and the extent of labeling, yet they do not participate in coordination of the metal center in the final complex. The latter is evidenced by both the X-ray crystal structure shown in Figure 1 for the [^natRe(CO)₃]⁺-labeled diethyl ester TACN derivative chelator as well as the previously reported X-ray crystal structures of the [^natRe(CO)₃]⁺- and [^99gTc(CO)₃]⁺-labeled NOTA complexes reported by Braband et al. (23)

Figure 1. Crystal structure of the [^natRe(CO)₃]⁺-labeled TACN-based chelator bearing one N-benzyl-acetamide arm and two ethyl ester arms. The crystal structure demonstrates the distorted octahedral coordination of the rhenium metal center with three *fac*-coordinated carbon monoxide ligands and the three nitrogens in the TACN backbone. The pendent arms do not participate in binding to the metal center in the final metal complex. The pendent amide arm is hydrogen bonded with a trifluoroacetate anion. Labeled 50% probability ellipsoid plot of asymmetric unit; disorder omitted for clarity.

The amide and alcohol pendent arms of chelators 2 and 3, respectively, are also not ionized under the labeling conditions; however, their polar functional groups bear partial negative charges, which appear to be sufficient for electrostatic attraction of the positively charged metal center to facilitate coordination. Such an attraction could occur directly between the partial negative terminal polar group and the positive metal center. Additionally, as supported by the crystal structure in Figure 1, indirect interactions may also occur, wherein polar pendent groups can form strong hydrogen bonds with anions in solution (e.g., trifluoroacetate or bicarbonate anions). The hydrogen bonded, noncoordinating anion thereby creates an anionic region around the chelator that could aid in electrostatically attracting the positively charged metal center to facilitate its coordination with the TACN backbone. The noncoordinating anions may also be positioned near enough to the metal center where they may engage in stabilizing outer sphere Coulombic interactions. As also depicted in the crystal structure, nonpolar pendent arms (e.g., ester or ketone) do not form hydrogen bonds with anions in solution, consistent with diminished attraction of the metal center.

As previously reported, chelator 5 (diacid) was reacted with [^natRe(CO)₃(OH₂)₃]⁺ in MES buffer (0.2 M, pH 3–5) at 95 °C for 1 h in a thermomixer. No significant difference in yield was observed across that pH range. (16) The yield (by HPLC) was near quantitative with no formation of byproducts observed.

All ^natRe-X complexes were soluble in water and acetonitrile and stable for several months at room temperature. The ^natRe-X complexes were isolated as hygroscopic white powders and characterized by HR-ESI-MS, ¹H NMR, ¹³C NMR, IR, and elemental analyses. While attempts to grow X-ray quality crystals of ^natRe-2/3/5 have thus far been unsuccessful, the ^natRe-X complexes reported in this work are expected to have similar coordination properties to the [^natRe(CO)₃]⁺-labeled diethyl ester complex shown in Figure 1. HR-ESI-MS analyses confirmed the anticipated mass to charge ratio and isotopic distribution for each of the ^natRe-X complexes. ¹H NMR analyses confirmed the anticipated structures and provided evidence that the pendent arms did not coordinate to the metal center. For example, coordination of the [^natRe(CO)₃]⁺ core results in a distinctive downfield shift for the hydrogen atoms of the TACN ring (3.1 to 3.6 ppm for Re-2 and 3.4 to 3.6 ppm for Re-3). The metal coordination sphere is filled by the three TACN backbone nitrogens and the three carbon monoxide ligands. The presence of the carbon monoxide ligands was confirmed with both ¹³C NMR spectra (chemical shifts at 195 ppm) and IR spectra (∼2030 cm^–1 symmetrical stretching and ∼1900 cm^–1 overlapping antisymmetric stretching (14)). Elemental analysis confirmed the anticipated elemental composition of the ^natRe-X complexes as TFA salts.

3.3. Chelator Radiolabeling with the [^99mTc][Tc(CO)₃]⁺ and [¹⁸⁶Re][Re(CO)₃]⁺ Cores

For radiolabeling studies, high specific activity [^99mTc]TcO₄^– was eluted from a ⁹⁹Mo/^99mTc generator in saline. The [^99mTc]TcO₄^– was converted to the [^99mTc][Tc(CO)₃(OH₂)₃]⁺ precursor following an established literature procedure. (13) The pH of the precursor (∼10 upon successful synthesis) was not adjusted, except for radiolabeling of chelator 5, in which case 6 M HCl was added to reduce the pH to ∼5. After combining the precursor, buffer, and chelator, the reaction pH for labeling of chelators 2 and 3 was adjusted, as needed, with 1 M HCl prior to heating. The conditions for radiolabeling were optimized for each chelator in terms of buffer, buffer pH, temperature, and ligand concentration. Characterization of the radiocomplexes was conducted by HPLC coinjection with the ^natRe-X counterparts (Figure 2; coinjection data for M-5 were reported previously (16)).

Figure 2. HPLC coinjections (method 1) of the radiocomplexes (lighter colors; NaI(Tl) detector) with their fully characterized ^natRe-X counterparts (darker colors; UV detector, 254 nm). Differences in retention times between the [¹⁸⁶**Re]Re-X** and [^99mTc]Tc-X complexes are attributed to different HPLCs being used for analysis.

Radiochemical yields (RCYs) for the radiocomplexes, obtained by HPLC peak area integration coupled with radio-TLC analysis of colloid formation, are listed in Table 1. Chelators 2 and 3 were reacted with the [^99mTc][Tc(CO)₃(OH₂)₃]⁺ precursor in sodium bicarbonate (0.1 M, pH 7–9) at 95 °C for 1 h, with pH 7 giving the highest RCY of 46 ± 8% for [^99mTc]Tc-2 and pH 9 giving the highest RCY of 35 ± 6% for [^99mTc]Tc-3. The RCY of [^99mTc]Tc-3 was increased to ∼45% using the same reaction conditions, except for heating the reaction in the microwave at 95 °C for 20 min. As observed with the ^natRe-labeling reactions, neutral to basic conditions were required for the labeling to occur. The reactions were unsuccessful at a pH of less than 7 (in MES buffer or PBS) or greater than 10 (in 0.1 M sodium carbonate). Increasing the reaction time had no effect on yield, since the [^99mTc][Tc(CO)₃(OH₂)₃]⁺ precursor had either reacted with the chelator or oxidized to [^99mTc]TcO₄^– (as evidenced by HPLC) in 1 h. No colloidal [^99mTc]TcO₂, however, was observed by radio-TLC during the labeling reactions.

Table 1. Radiochemical Yields, logD_7.4 Values, Percent Stability, and Percent Protein Binding for the Radiolabeled Chelators^a

M = [^99mTc]Tc	M-2 (diamide)	M-3 (dialcohol)	M-5 (diacid)^c
RCY (%)	46 ± 8	35 ± 6	96 ± 1
logD_7.4	–1.15 ± 0.01	–0.27 ± 0.01	–2.2 ± 0.3
stability in PBS^b(%)	100 ± 0	100 ± 0	100 ± 0
stability inl-cysteine^b(%)	100 ± 0	100 ± 0	100 ± 0
stability inl-histidine^b(%)	100 ± 0	100 ± 0	100 ± 0
rat serum protein binding^b(%)	1 ± 1	3 ± 1	8 ± 2

M = [¹⁸⁶Re]Re	M-2 (diamide)	M-3 (dialcohol)	M-5 (diacid)^c
RCY (%)	8 ± 1	32 ± 3	96 ± 1
logD_7.4	–1.04 ± 0.02	–0.19 ± 0.06	–1.9 ± 0.2
stability in PBS^b(%)	100 ± 0	100 ± 0	100 ± 0
stability inl-cysteine^b(%)	100 ± 0	100 ± 0	100 ± 0
stability inl-histidine^b(%)	100 ± 0	100 ± 0	100 ± 0
rat serum protein binding^b(%)	3 ± 1	3 ± 1	5 ± 4

^{^a}

Values are reported as mean ± SD (n = 3).

^{^b}

Following incubation at 37 °C for 24 h.

^{^c}

A high ligand concentration of 1 mM was required for radiolabeling to achieve the highest RCY. Although this concentration is higher than typically used for radiopharmaceutical development, it is consistent with previously reported [M(CO)₃]⁺-labeled TACN derivative complexes. (16,33) Due to the lipophilic character of the [M(CO)₃]⁺ core, radiolabeling of the TACN-based chelators reduces the overall hydrophilicity of the complexes and results in a significant shift to a later HPLC retention time for the labeled complex. This conveniently allows for a relatively simple separation of the radiolabeled product from the excess unlabeled chelator, with a corresponding increase in apparent molar activity of the radiocomplex. However, the need for a postradiosynthesis purification step prior to dose formulation is not ideal and may limit the potential for application of such radiopharmaceuticals in the clinical setting. The high temperature of 95 °C used to achieve the maximum RCY is also a disadvantage, for example by precluding the use of temperature-sensitive biological targeting vectors (e.g., antibodies) or by requiring labeling at lower temperatures with longer reaction times and concomitant lower RCY.

The yield of [^99mTc]Tc-2 was ∼20% higher in sodium bicarbonate than in PBS buffer, which differed from the results obtained for ^natRe-2. Formation of the amide hydrolysis product was also seen with [^99mTc]Tc-2; however, the yield of this byproduct was negligible (<10%) compared to the yield of the [^99mTc]Tc-2 product. [^99mTc]Tc-3 was labeled under the same conditions used for ^natRe-3, and similar to ^natRe-3, minimal byproduct formation was observed during radiolabeling. Loss of yield was attributed to oxidation of the [^99mTc][Tc(CO)₃(OH₂)₃]⁺ precursor to [^99mTc]TcO₄^– for both the [^99mTc]Tc-2 and [^99mTc]Tc-3 complexes. Both complexes were successfully isolated by radio-HPLC for characterization and for conducting in vitro stability and logD_7.4 studies.

Chelator 4 was not successfully reacted with the [^99mTc][Tc(CO)₃(OH₂)₃]⁺ precursor. Alternative syntheses for [^99mTc]Tc-4 from [^99mTc]Tc-5 were investigated but deemed impractical due to the long reaction times required for the acid to ketone conversion, which would exceed several half-lives of ^99mTc.

As reported previously, (16) chelator 5 was reacted with [^99mTc][Tc(CO)₃(OH₂)₃]⁺ in MES buffer (0.2 M, pH 5) at 95 °C for 30 min, resulting in near quantitative yields by radio-HPLC. A ligand concentration of 0.3 mM was typically used for radiolabeling but could be decreased to 0.1 mM without significant impact on RCY. No colloidal [^99mTc]TcO₂ formation was observed by radio-TLC.

Low specific activity [¹⁸⁶Re]ReO₄^– in saline, produced by neutron irradiation of enriched [¹⁸⁵Re]Al(ReO₄)₃ targets, was used for radiolabeling studies. Due to the slower reaction kinetics and higher reduction potential of rhenium compared to technetium, a decrease in RCY was expected for the [¹⁸⁶Re]Re-X complexes. Indeed, chelators 2 and 3 proved more difficult to label with the [¹⁸⁶Re][Re(CO)₃(OH₂)₃]⁺ precursor. Prior to labeling studies, sodium hydroxide or sodium bicarbonate was added to the precursor to raise the pH to ∼8–9. Chelator 2 (diamide) and [¹⁸⁶Re][Re(CO)₃(OH₂)₃]⁺ were combined in sodium bicarbonate (0.1 M, pH 8) and heated at 95 °C for 1 h, resulting in a maximum RCY of 8 ± 1%, for which a ligand concentration of 1 mM was required. Loss of yield was attributed to both the oxidation of the metal to the permetallate form (no colloidal [¹⁸⁶Re]ReO₂ was observed) along with the simultaneous formation of the labeled amide hydrolysis product in a ∼1:1 ratio. After ∼1 h of reaction time at 95 °C in the presence of base, the [¹⁸⁶Re][Re(CO)₃(OH₂)₃]⁺ precursor was completely oxidized to the permetallate form and thus no longer able to react with the chelator. To isolate sufficient activity for in vitro stability, logD_7.4, and characterization studies of the radiocomplex, the radiolabeling reaction was run for 3 h, with additional aliquots of the [¹⁸⁶Re][Re(CO)₃(OH₂)₃]⁺ precursor (7.4 MBq, 200 μCi) added every 30 min, in an attempt to extend the presence of the [¹⁸⁶Re][Re(CO)₃(OH₂)₃]⁺ precursor in solution for reaction with the chelator. Using this approach, the radiolabeling was successfully scaled up to activities of 44 MBq (1.2 mCi).

Chelator 3 (dialcohol) and [¹⁸⁶Re][Re(CO)₃(OH₂)₃]⁺ were combined in sodium bicarbonate (0.1 M, pH 9) and heated at 95 °C for 1 h, resulting in a maximum RCY of 32 ± 3%. A ligand concentration of 1 mM was required to achieve this yield. The RCY was highly dependent on the activity concentration. Attempts to scale up the labeling reaction from ∼1.9 MBq (50 μCi) to ∼7.4 MBq (200 μCi), by increasing the volume of precursor without changing the total volume of the reaction, led to formation of several byproducts with similar retention times (±0.4 min) to the desired product by radio-HPLC analysis. To minimize the formation of these byproducts, the reactions were diluted in sodium bicarbonate (0.1 M, pH 9) so that the volume of [¹⁸⁶Re][Re(CO)₃(OH₂)₃]⁺ added did not exceed 10% of the total volume. This byproduct formation was not observed with either the ^natRe-3 complex or the [^99mTc]Tc-3 complex. No colloidal [¹⁸⁶Re]ReO₂ was observed by radio-TLC. All loss of yield was attributed to oxidation of the metal, with minimal byproduct formation under the optimized conditions.

Due to the inability to label chelator 4 (diketone) with both the [^natRe(CO)₃]⁺ and [^99mTc][Tc(CO)₃]⁺ cores, reaction of chelator 4 with the [¹⁸⁶Re][Re(CO)₃(OH₂)₃]⁺ precursor was not attempted.

Chelator 5 (diacid), as expected, reacted in high yields with the [¹⁸⁶Re][Re(CO)₃(OH₂)₃]⁺ precursor in MES buffer (0.2 M, pH 5) at 95 °C for 1 h. (16) The RCY was near quantitative, and the reaction was easily scaled to higher activity concentrations. Ligand concentrations of 0.1 to 0.3 mM were used without a significant impact on the RCY. No colloidal [¹⁸⁶Re]ReO₂ was observed by radio-TLC.

3.4. In Vitro Stability and logD_7.4 Values of the Radiocomplexes

The in vitro stability of the HPLC-purified radiocomplexes was tested in solutions of PBS, l-cysteine, and l-histidine as well as in rat serum at 37 °C through 24 h. The stability for the standard M-5 complexes was reported previously. (16) In short, both the [^99mTc]Tc-5 and [¹⁸⁶Re]Re-5 complexes remained completely intact under all tested conditions at 37 °C through 24 h, as monitored by radio-HPLC and radio-TLC.

Similarly, the [^99mTc]Tc-2/3 and [¹⁸⁶Re]Re-2/3 complexes remained completely stable in PBS, l-cysteine, and l-histidine challenge experiments at 24 h at 37 °C (Table 1). The complexes also demonstrated complete stability in rat serum at 24 h at 37 °C (Figure 3) with nonspecific protein binding of <10% (Table 1). No oxidation to the permetallate or colloidal (MO₂) chemical forms was observed by radio-HPLC (method 1) or radio-TLC, respectively, nor was decomposition of the metal complex observed. The stability of these complexes is attractive for radiopharmaceutical development and provides further evidence that the [M(CO)₃]⁺ cores form highly stable complexes with TACN-based chelators.

Figure 3. HPLC analysis (method 1; NaI(Tl) detector) of radiocomplex stability after 24 h in rat serum at 37 °C.

All successfully synthesized radiocomplexes demonstrated hydrophilic character. The M-5 (diacid) complexes, which carry an overall −1 charge at physiological pH, demonstrated the greatest hydrophilic character, with logD_7.4 values of around −2 (Table 1). Complexes M-2 (diamide), carrying an overall +1 charge at physiological pH, demonstrated the next highest hydrophilicity, with logD_7.4 values of around −1. Finally, complexes M-3 (dialcohol), also carrying a +1 charge, demonstrated the least hydrophilic character, with logD_7.4 values of around −0.3. For radiopharmaceutical development, hydrophilic complexes are typically preferred for their rapid clearance via the renal-urinary pathway. This results in shorter residence times of the radiopharmaceutical in the clearance organs leading to less exposure of healthy tissues to radiation and higher tumor to background ratios for activity accumulation, for improved nuclear imaging. Given their excellent stability and hydrophilic character, either chelator 2 or 3 could be conjugated to a biological targeting vector, labeled with the [M(CO)₃]⁺ cores, and evaluated as theranostic radiopharmaceuticals. However, improvement of the RCYs would be necessary, which may be aided in future efforts via incorporation of density functional theory (DFT) computational studies.

4. Conclusions

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TACN-based chelators bearing amide, alcohol, and ketone functional groups on their pendent arms were successfully synthesized and fully characterized. These chelators were reacted with [M(CO)₃]⁺ cores (M = ^natRe, ¹⁸⁶Re, ^99mTc). Only chelator 4 (ketone derivative) was not successfully labeled with the [M(CO)₃]⁺ cores, in alignment with our previous investigations of TACN-based chelators. (16) Chelator 2 (amide derivative) was labeled with the [^natRe(CO)₃]⁺ and [^99mTc][Tc(CO)₃]⁺ cores in moderate yields, with low yield observed for radiolabeling with the [¹⁸⁶Re][Re(CO)₃]⁺ core. Chelator 3 (alcohol derivative) was labeled with the [^natRe(CO)₃]⁺ core in excellent yields and radiolabeled with the [^99mTc][Tc(CO)₃]⁺ and [¹⁸⁶Re][Re(CO)₃]⁺ cores in moderate yields. The M-2 and M-3 complexes demonstrated hydrophilic character and were highly stable in PBS, l-cysteine, l-histidine, and rat serum through 24 h. The radiolabeling of these chelators presented some challenges, which may limit their potential for radiopharmaceutical development. Nevertheless, valuable insights were gained about the impact of the TACN pendent arms on chelator radiolabeling with the [M(CO)₃]⁺ cores, which will inform optimization of the TACN chelator design. Future studies will include the evaluation of TACN-based chelators bearing polar and ionizable functional groups on their pendent arms, such as sulfonate and phosphonic acid pendent arms.

Supporting Information

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The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.4c05699.

Synthesis protocols; ¹H NMR and ¹³C NMR spectra for the chelators; ¹H NMR, ¹³C NMR, and IR spectra for the ^natRe-complexes; TLC for the radiocomplexes; and crystal data (PDF)

ao4c05699_si_001.pdf (2.25 MB)

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Author Information

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Corresponding Author
- Heather M. Hennkens - Department of Chemistry, University of Missouri, 601 South College Avenue, Columbia, Missouri 65211, United States; Research Reactor Center, University of Missouri, 1513 Research Park Drive, Columbia, Missouri 65211, United States; https://orcid.org/0000-0002-3283-8751; Email: [email protected]
Authors
- Rebecca Hoerres - Department of Chemistry, University of Missouri, 601 South College Avenue, Columbia, Missouri 65211, United States; Present Address: Oak Ridge National Laboratory, 1 Bethel Valley Road, Oak Ridge, Tennessee, 37830, United States
- Ritin Kamboj - Department of Chemistry, University of Missouri, 601 South College Avenue, Columbia, Missouri 65211, United States; Present Address: Curia Global, 21 Corporate Circle, Albany, New York, 12203, United States
- Nora Pryor - Department of Chemistry, University of Missouri, 601 South College Avenue, Columbia, Missouri 65211, United States
- Steven P. Kelley - Department of Chemistry, University of Missouri, 601 South College Avenue, Columbia, Missouri 65211, United States; https://orcid.org/0000-0001-6755-4495
Author Contributions
R.H. and R.K. contributed equally to this paper.
Notes
The authors declare no competing financial interest.

Acknowledgments

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This work was funded by the Nuclear Regulatory Commission (grant number 31310021M0040) as well as by University of Missouri (MU) departmental support (Department of Chemistry and MU Research Reactor). We acknowledge the generous donation of ⁹⁹Mo/^99mTc generators from Mid-America Isotopes, Inc. We also acknowledge Mary Embree (¹⁸⁶Re production, MU Research Reactor), Dr. Brian P. Mooney (HR-ESI-MS analyses, MU Charles W. Gehrke Proteomics Center), and Dr. Fabio Gallazzi (LC-ESI-MS analyses, MU Molecular Interactions Core and MU Department of Chemistry) for their technical expertise.

References

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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.
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Hoerres, R.; Hennkens, H. M. 1,4,7-Triazacyclononane-based chelators for the complexation of [¹⁸⁶Re]Re- and [^99mTc]Tc-tricarbonyl cores. Inorg. Chem. 2023, 62 (50), 20688– 20698, DOI: 10.1021/acs.inorgchem.3c01934
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Matching chelators to radiometals for radiopharmaceuticals
Price, Eric W.; Orvig, Chris
Chemical Society Reviews (2014), 43 (1), 260-290CODEN: CSRVBR; ISSN:0306-0012. (Royal Society of Chemistry)
A review. Radiometals comprise many useful radioactive isotopes of various metallic elements. When properly harnessed, these have valuable emission properties that can be used for diagnostic imaging techniques, such as single photon emission computed tomog. (SPECT, e.g.67Ga, 99mTc, 111In, 177Lu) and positron emission tomog. (PET, e.g.68Ga, 64Cu, 44Sc, 86Y, 89Zr), as well as therapeutic applications (e.g.47Sc, 114mIn, 177Lu, 90Y, 212/213Bi, 212Pb, 225Ac, 186/188Re). A fundamental crit. component of a radiometal-based radiopharmaceutical is the chelator, the ligand system that binds the radiometal ion in a tight stable coordination complex so that it can be properly directed to a desirable mol. target in vivo. This article is a guide for selecting the optimal match between chelator and radiometal for use in these systems. The article briefly introduces a selection of relevant and high impact radiometals, and their potential utility to the fields of radiochem., nuclear medicine, and mol. imaging. A description of radiometal-based radiopharmaceuticals is provided, and several key design considerations are discussed. The exptl. methods by which chelators are assessed for their suitability with a variety of radiometal ions is explained, and a large selection of the most common and most promising chelators are evaluated and discussed for their potential use with a variety of radiometals. Comprehensive tables have been assembled to provide a convenient and accessible overview of the field of radiometal chelating agents.
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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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Chang, C.-C.; Chang, C.-H.; Lo, Y.-H.; Lin, M.-H.; Shen, C.-C.; Liu, R.-S.; Wang, H.-E.; Chen, C.-L. Preparation and characterization of a novel Al¹⁸F-NOTA-BZA conjugate for melanin-targeted imaging of malignant melanoma. Bioorg. Med. Chem. Lett. 2016, 26 (16), 4133– 4139, DOI: 10.1016/j.bmcl.2016.06.022
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Qiao, Z.; Xu, J.; Gonzalez, R.; Miao, Y. Effects of polyethylene glycol and 8-aminooctanoic acid linkers on melanoma uptake of [^99mTc]Tc-tricarbonyl-NOTA-conjugated lactam-cyclized α-MSH peptides. Bioconjugate Chem. 2023, 34 (5), 934– 940, DOI: 10.1021/acs.bioconjchem.3c00164
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Aljammaz, I.; Al-Otaibi, B.; Al-Hokbany, N.; Amer, S.; Okarvi, S. Development and pre-clinical evaluation of new ⁶⁸Ga-NOTA-folate conjugates for PET imaging of folate receptor-positive tumors. Anticancer Res. 2014, 34 (11), 6547– 6556
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Development and pre-clinical evaluation of new 68Ga-NOTA-folate conjugates for PET imaging of folate receptor-positive tumors
Aljammaz, Ibrahim; Al-Otaibi, Basim; Al-Hokbany, Nourah; Amer, Suad; Okarvi, Subhani
Anticancer Research (2014), 34 (11), 6547-6556CODEN: ANTRD4; ISSN:0250-7005. (International Institute of Anticancer Research)
In an attempt to develop new folate radiotracers with favorable biochem. properties for detecting folate receptor-pos. cancers, we synthesized 68Ga-NOTA- and 68Ga-NOTAM-folate conjugates using a straightforward and a one-step simple reaction. Radiochem. yields were greater than 95% (decay-cor.) with total synthesis time of less than 20 min. Radiochem. purities were always greater than 98% without high-performance liq. chromatog. (HPLC) purifn. These synthetic approaches hold considerable promise as a rapid and simple method for 68Ga-folate conjugate prepn. with high radiochem. yield in a short synthesis time. In vitro tests on the KB cell line showed that significant amts. of the radioconjugates were assocd. with cell fractions. Biodistribution studies in nude mice bearing human KB xenografts, demonstrated a significant tumor uptake and favorable biodistribution profile for 68Ga-NOTA-folate over the 68Ga-NOTAM-folate conjugate. The uptake in the tumors was blocked by excess injection of folic acid, suggesting a receptor-mediated process. These results demonstrate that the 68Ga-NOTA-folate conjugate may be useful as a mol. probe for detection and staging of folate receptor-pos. cancers, such as ovarian cancer and their metastasis, as well as monitoring tumor response to treatment.
22
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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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Sheldrick, George M.
Acta Crystallographica, Section C: Structural Chemistry (2015), 71 (1), 3-8CODEN: ACSCGG; ISSN:2053-2296. (International Union of Crystallography)
The improvements in the crystal structure refinement program SHELXL have been closely coupled with the development and increasing importance of the CIF (Crystallog. Information Framework) format for validating and archiving crystal structures. An important simplification is that now only one file in CIF format (for convenience, referred to simply as 'a CIF') contg. embedded reflection data and SHELXL instructions is needed for a complete structure archive; the program SHREDCIF can be used to ext. the and files required for further refinement with SHELXL. Recent developments in SHELXL facilitate refinement against neutron diffraction data, the treatment of H atoms, the detn. of abs. structure, the input of partial structure factors and the refinement of twinned and disordered structures. SHELXL is available free to academics for the Windows, Linux and Mac OS X operating systems, and is particularly suitable for multiple-core processors.
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Harnessing the Coordination Chemistry of 1,4,7-Triazacyclononane for Biomimicry and Radiopharmaceutical Applications
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Alberto, R.; Schibli, R.; Waibel, R.; Abram, U.; Schubiger, A. P. Basic aqueous chemistry of [M(OH₂)₃(CO)₃]⁺ (M = Re, Tc) directed towards radiopharmaceutical application. Coord. Chem. Rev. 1999, 190–192, 901– 919, DOI: 10.1016/S0010-8545(99)00128-9
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Basic aqueous chemistry of [M(OH2)3(CO)3]+ (M=Re, Tc) directed towards radiopharmaceutical application
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Coordination Chemistry Reviews (1999), 190-192 (), 901-919CODEN: CCHRAM; ISSN:0010-8545. (Elsevier Science S.A.)
A review with 20 refs. A review on the synthesis and properties of the organometallic aqua-ion [M(OH2)3(CO)3]+ (M=Re, 99Tc, 99mTc), as relevant for radiopharmaceutical application, is presented. These important starting compds. can be prepd. quant., (a) on the no carrier added (n.c.a.) level (99mTc) in water, or (b) in org. solvents (Re, 99Tc) at atm. pressure in a short time and from [MO4]-. The main characteristics of these carbonyl complexes are the high substitution stability of the three CO ligands and the substitution lability of the coordinated water mols. [M(OH2)3(CO)3]+ can be considered as a 'semi aquo-ion'. On the macroscopic level, upon titrn. with OH-, hydroxo-bridged oligomers have been isolated and characterized. The formation of hydroxo-bridged complexes is a consequence of the considerable Bronstedt acidity of [M(OH2)3(CO)3]+, whereas on the n.c.a. level no such behavior was obsd. Conditions and products of the water exchange by imidazole (i.m.) and derivs. thereof (histamine, histidine) will be presented. The different mononuclear complexes with these ligands are of extraordinary inertness, which is the basis for potential applications in biol. and nuclear medicine. Finally, as a basis for bioorganometallic chem., the adoption of the results from basic coordination chem. to the labeling of biomols. with an organometallic moiety will be exemplified with a selected penta-peptide and a recombinant single chain fragment.
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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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Abstract
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Scheme 1
Scheme 1. Synthesis of TACN-Based Chelators 2–4^a
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^a(i) Potassium carbonate (1.5 equiv), acetonitrile, RT, 24 h, 40%. (ii) 2-Bromoacetamide (2 equiv), triethylamine (3 equiv), acetonitrile, 80 °C, 3 h, 74%. (iii) 2-Bromoethanol (2 equiv), triethylamine (3 equiv), acetonitrile, 80 °C, 9 h, 54%. (iv) 2-Chloroacetone (2 equiv), triethylamine (3 equiv), acetonitrile, 80 °C, 3 h, 93%.
Scheme 2
Scheme 2. Synthesis of TACN-Based Chelator 5^a
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^a(i) N-Benzyl-2-bromoacetamide (1 equiv), potassium carbonate (1.8 equiv), acetonitrile, RT, 48 h. (ii) Trifluoroacetic acid (850 μL), triisopropylsilane (100 μL), water (50 μL), RT, 6 h. Overall isolated yield: 64%.
Figure 1
Figure 1. Crystal structure of the [^natRe(CO)₃]⁺-labeled TACN-based chelator bearing one N-benzyl-acetamide arm and two ethyl ester arms. The crystal structure demonstrates the distorted octahedral coordination of the rhenium metal center with three fac-coordinated carbon monoxide ligands and the three nitrogens in the TACN backbone. The pendent arms do not participate in binding to the metal center in the final metal complex. The pendent amide arm is hydrogen bonded with a trifluoroacetate anion. Labeled 50% probability ellipsoid plot of asymmetric unit; disorder omitted for clarity.
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Figure 2
Figure 2. HPLC coinjections (method 1) of the radiocomplexes (lighter colors; NaI(Tl) detector) with their fully characterized ^natRe-X counterparts (darker colors; UV detector, 254 nm). Differences in retention times between the [¹⁸⁶Re]Re-X and [^99mTc]Tc-X complexes are attributed to different HPLCs being used for analysis.
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Figure 3
Figure 3. HPLC analysis (method 1; NaI(Tl) detector) of radiocomplex stability after 24 h in rat serum at 37 °C.
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  Jackson Isaac M; Scott Peter J H; Thompson Stephen
  Seminars in nuclear medicine (2017), 47 (5), 493-523 ISSN:.
  Radiolabeled peptides are a valuable class of radiotracer that occupies the space between small molecules and large biologics, and are able to exploit the advantages of both classes of compound. To date, radiolabeled peptides have mainly been utilized in oncology, where the same peptide can often be exploited for diagnostic imaging and targeted radiotherapy by simply varying the radionuclide. In this review, we introduce the main strategies used for synthesis of radiolabeled peptides, and highlight the state of the art for clinical imaging (and therapy) in oncology using the main classes of radiolabeled peptides that have been translated to date. Beyond oncology, radiolabeled peptides are also increasingly being used in other PET applications such as diabetes and cardiac imaging, and we review progress for the new applications.
3. 3
  Kwekkeboom, D. J.; Mueller-Brand, J.; Paganelli, G.; Anthony, L. B.; Pauwels, S.; Kvols, L. K.; O’Dorisio, T. M.; Valkema, R.; Bodei, L.; Chinol, M. Overview of results of peptide receptor radionuclide therapy with 3 radiolabeled somatostatin analogs. J. Nucl. Med. 2005, 46 (Suppl 1), 62S– 66S
  There is no corresponding record for this reference.
4. 4
  Oun, R.; Moussa, Y. E.; Wheate, N. J. The side effects of platinum-based chemotherapy drugs: A review for chemists. Dalton Trans. 2018, 47 (19), 6645– 6653, DOI: 10.1039/C8DT00838H
  4
  The side effects of platinum-based chemotherapy drugs: a review for chemists
  Oun, Rabbab; Moussa, Yvonne E.; Wheate, Nial J.
  Dalton Transactions (2018), 47 (19), 6645-6653CODEN: DTARAF; ISSN:1477-9226. (Royal Society of Chemistry)
  The platinum-based drugs cisplatin, carboplatin and oxaliplatin are regularly prescribed in the treatment of cancer and while they are effective, their use is limited by their severe, dose-limiting side effects (also referred to as adverse effects/events). In total, a cancer patient can experience any combination of around 40 specific side effects. The dose-limiting side effect for cisplatin is nephrotoxicity, for carboplatin it is myelosuppression, and for oxaliplatin it is neurotoxicity. Other common side effects include anaphylaxis, cytopenias (including leukopenia and neutropenia, thrombocytopenia, and anemia), hepatotoxicity, ototoxicity, cardiotoxicity, nausea and vomiting, diarrhea, mucositis, stomatitis, pain, alopecia, anorexia, cachexia, and asthenia. The side effects may require patients to be prescribed dose redns. in their platinum drugs of between 25 and 100%. Furthermore, patients require extensive monitoring of their biochemistries, kidney and liver function, and depending on the drug, hearing tests. Finally, patients are commonly co-prescribed addnl. non-chemotherapy based drugs to treat the side effects which can include antiemetics, antibiotics and myeloid growth factors, mannitol, propafenone, saline hyperhydration, magnesium supplements, monoclonal antibody cytokine blockers and antioxidants.
5. 5
  Asadian, S.; Mirzaei, H.; Kalantari, B. A.; Davarpanah, M. R.; Mohamadi, M.; Shpichka, A.; Nasehi, L.; Es, H. A.; Timashev, P.; Najimi, M. β-radiating radionuclides in cancer treatment, novel insight into promising approach. Pharmacol. Res. 2020, 160, 105070 DOI: 10.1016/j.phrs.2020.105070
  There is no corresponding record for this reference.
6. 6
  De Decker, M.; Bacher, K.; Thierens, H.; Slegers, G.; Dierckx, R. A.; De Vos, F. In vitro and in vivo evaluation of direct rhenium-188-labeled anti-CD52 monoclonal antibody alemtuzumab for radioimmunotherapy of B-cell chronic lymphocytic leukemia. Nucl. Med. Biol. 2008, 35 (5), 599– 604, DOI: 10.1016/j.nucmedbio.2008.03.001
  There is no corresponding record for this reference.
7. 7
  Sarko, D.; Eisenhut, M.; Haberkorn, U.; Mier, W. Bifunctional chelators in the design and application of radiopharmaceuticals for oncological diseases. Curr. Med. Chem. 2012, 19 (17), 2667– 2688, DOI: 10.2174/092986712800609751
  7
  Bifunctional chelators in the design and application of radiopharmaceuticals for oncological diseases
  Sarko, D.; Eisenhut, M.; Haberkorn, U.; Mier, W.
  Current Medicinal Chemistry (2012), 19 (17), 2667-2688CODEN: CMCHE7; ISSN:0929-8673. (Bentham Science Publishers Ltd.)
  A review. Radiopharmaceuticals constitute diagnostic and therapeutic tools for both clin. and preclin. applications. They are a blend of a tracer moiety that mediates a site specific accumulation and an effector: a radioisotope whose decay enables either mol. imaging or exhibits cytotoxic effects. Radioactive halogens and lanthanides are the most commonly used isotopes for radiopharmaceuticals. Due to their ready availability and the facile labeling metallic radionuclides offer ideal characteristics for applications in nuclear medicine. A stable link between the radionuclide and the carrier mol. is the primary prerequisite for in vivo applications. The radionuclide is selected according to its phys. and chem. properties i.e. half-life, the type of decay, the energy emitted and its availability. Bifunctional chelating agents are used to stably link the radiometal to the carrier moiety of the radiopharmaceutical. The design of the bifunctional chelator has to consider the impact of the radiometal chelate on the biol. properties of the target-specific pharmaceutical. Here, with an emphasis on oncol., we review applications of radiopharmaceuticals that contain bifunctional chelators, while highlighting successes and identifying the key challenges that need to be addressed for the successful translation of target binding mols. into tracers for mol. imaging and endoradiotherapy.
8. 8
  Hennrich, U.; Kopka, K. Lutathera: The first FDA- and EMA-approved radiopharmaceutical for peptide receptor radionuclide therapy. Pharmaceuticals 2019, 12 (3), 114, DOI: 10.3390/ph12030114
  8
  Lutathera: the first FDA- and EMA-approved radiopharmaceutical for peptide receptor radionuclide therapy
  Hennrich, Ute; Kopka, Klaus
  Pharmaceuticals (2019), 12 (3), 114CODEN: PHARH2; ISSN:1424-8247. (MDPI AG)
  A review. As the first radiopharmaceutical for Peptide Receptor Radionuclide Therapy (PRRT), Lutathera was approved by the EMA in 2017 and the FDA in 2018 for the treatment of somatostatin receptor (SSTR) pos. gastroenteropancreatic neuroendocrine tumors. Using the concept of PRRT, Lutathera combines the radionuclide 177Lu with the somatostatin analog DOTA-TATE, thus delivering ionizing radiation specifically to tumor cells expressing somatostatin receptors. As a result, DNA single- and double-strand breaks are provoked, in case of double-strand breaks leading to cell death of the tumor and its SSTR-pos. lesions.
9. 9
  OECD/NEA. The supply of medical isotopes ; 2019. https://doi.org/10.1787/9b326195-en
  There is no corresponding record for this reference.
10. 10
  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
  10
  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.
11. 11
  Dias, C. R.; Jeger, S.; Osso, J. A., Jr.; Mueller, C.; De Pasquale, C.; Hohn, A.; Waibel, R.; Schibli, R. Radiolabeling of rituximab with ¹⁸⁸Re and ^99mTc using the tricarbonyl technology. Nucl. Med. Biol. 2011, 38 (1), 19– 28, DOI: 10.1016/j.nucmedbio.2010.05.010
  There is no corresponding record for this reference.
12. 12
  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.
13. 13
  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
  13
  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.
14. 14
  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
  There is no corresponding record for this reference.
15. 15
  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
  15
  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.
16. 16
  Hoerres, R.; Hennkens, H. M. 1,4,7-Triazacyclononane-based chelators for the complexation of [¹⁸⁶Re]Re- and [^99mTc]Tc-tricarbonyl cores. Inorg. Chem. 2023, 62 (50), 20688– 20698, DOI: 10.1021/acs.inorgchem.3c01934
  There is no corresponding record for this reference.
17. 17
  Price, E. W.; Orvig, C. Matching chelators to radiometals for radiopharmaceuticals. Chem. Soc. Rev. 2014, 43 (1), 260– 290, DOI: 10.1039/C3CS60304K
  17
  Matching chelators to radiometals for radiopharmaceuticals
  Price, Eric W.; Orvig, Chris
  Chemical Society Reviews (2014), 43 (1), 260-290CODEN: CSRVBR; ISSN:0306-0012. (Royal Society of Chemistry)
  A review. Radiometals comprise many useful radioactive isotopes of various metallic elements. When properly harnessed, these have valuable emission properties that can be used for diagnostic imaging techniques, such as single photon emission computed tomog. (SPECT, e.g.67Ga, 99mTc, 111In, 177Lu) and positron emission tomog. (PET, e.g.68Ga, 64Cu, 44Sc, 86Y, 89Zr), as well as therapeutic applications (e.g.47Sc, 114mIn, 177Lu, 90Y, 212/213Bi, 212Pb, 225Ac, 186/188Re). A fundamental crit. component of a radiometal-based radiopharmaceutical is the chelator, the ligand system that binds the radiometal ion in a tight stable coordination complex so that it can be properly directed to a desirable mol. target in vivo. This article is a guide for selecting the optimal match between chelator and radiometal for use in these systems. The article briefly introduces a selection of relevant and high impact radiometals, and their potential utility to the fields of radiochem., nuclear medicine, and mol. imaging. A description of radiometal-based radiopharmaceuticals is provided, and several key design considerations are discussed. The exptl. methods by which chelators are assessed for their suitability with a variety of radiometal ions is explained, and a large selection of the most common and most promising chelators are evaluated and discussed for their potential use with a variety of radiometals. Comprehensive tables have been assembled to provide a convenient and accessible overview of the field of radiometal chelating agents.
18. 18
  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
  18
  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.
19. 19
  Chang, C.-C.; Chang, C.-H.; Lo, Y.-H.; Lin, M.-H.; Shen, C.-C.; Liu, R.-S.; Wang, H.-E.; Chen, C.-L. Preparation and characterization of a novel Al¹⁸F-NOTA-BZA conjugate for melanin-targeted imaging of malignant melanoma. Bioorg. Med. Chem. Lett. 2016, 26 (16), 4133– 4139, DOI: 10.1016/j.bmcl.2016.06.022
  There is no corresponding record for this reference.
20. 20
  Qiao, Z.; Xu, J.; Gonzalez, R.; Miao, Y. Effects of polyethylene glycol and 8-aminooctanoic acid linkers on melanoma uptake of [^99mTc]Tc-tricarbonyl-NOTA-conjugated lactam-cyclized α-MSH peptides. Bioconjugate Chem. 2023, 34 (5), 934– 940, DOI: 10.1021/acs.bioconjchem.3c00164
  There is no corresponding record for this reference.
21. 21
  Aljammaz, I.; Al-Otaibi, B.; Al-Hokbany, N.; Amer, S.; Okarvi, S. Development and pre-clinical evaluation of new ⁶⁸Ga-NOTA-folate conjugates for PET imaging of folate receptor-positive tumors. Anticancer Res. 2014, 34 (11), 6547– 6556
  21
  Development and pre-clinical evaluation of new 68Ga-NOTA-folate conjugates for PET imaging of folate receptor-positive tumors
  Aljammaz, Ibrahim; Al-Otaibi, Basim; Al-Hokbany, Nourah; Amer, Suad; Okarvi, Subhani
  Anticancer Research (2014), 34 (11), 6547-6556CODEN: ANTRD4; ISSN:0250-7005. (International Institute of Anticancer Research)
  In an attempt to develop new folate radiotracers with favorable biochem. properties for detecting folate receptor-pos. cancers, we synthesized 68Ga-NOTA- and 68Ga-NOTAM-folate conjugates using a straightforward and a one-step simple reaction. Radiochem. yields were greater than 95% (decay-cor.) with total synthesis time of less than 20 min. Radiochem. purities were always greater than 98% without high-performance liq. chromatog. (HPLC) purifn. These synthetic approaches hold considerable promise as a rapid and simple method for 68Ga-folate conjugate prepn. with high radiochem. yield in a short synthesis time. In vitro tests on the KB cell line showed that significant amts. of the radioconjugates were assocd. with cell fractions. Biodistribution studies in nude mice bearing human KB xenografts, demonstrated a significant tumor uptake and favorable biodistribution profile for 68Ga-NOTA-folate over the 68Ga-NOTAM-folate conjugate. The uptake in the tumors was blocked by excess injection of folic acid, suggesting a receptor-mediated process. These results demonstrate that the 68Ga-NOTA-folate conjugate may be useful as a mol. probe for detection and staging of folate receptor-pos. cancers, such as ovarian cancer and their metastasis, as well as monitoring tumor response to treatment.
22. 22
  Creaser, S. P.; Pyke, S. M.; Lincoln, S. F. Complexation of zinc(II) and cadmium(II) by hydroxyethyl- and bis(hydroxyethyl)-1,4,7-triazacyclononane in water. Aust. J. Chem. 2003, 56 (1), 61– 64, DOI: 10.1071/CH02202
  There is no corresponding record for this reference.
23. 23
  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
  23
  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.
24. 24
  Badertscher, M.; Buhlmann, P.; Pretsch, E. Structure determination of organic compounds: Tables of spectral data; Springer: Berlin Heidelberg, 2009.
  There is no corresponding record for this reference.
25. 25
  Shetty, D.; Choi, S. Y.; Jeong, J. M.; Hoigebazar, L.; Lee, Y.-S.; Lee, D. S.; Chung, J.-K.; Lee, M. C.; Chung, Y. K. Formation and characterization of gallium(III) complexes with monoamide derivatives of 1,4,7-triazacyclononane-1,4,7-triacetic acid: A study of the dependency of structure on reaction pH. Eur. J. Inorg. Chem. 2010, 2010 (34), 5432– 5438, DOI: 10.1002/ejic.201000748
  There is no corresponding record for this reference.
26. 26
  Apex4; AXScale; SAINT. version 2022.1, Bruker AXS, Inc.: Madison, WI, 2022.
  There is no corresponding record for this reference.
27. 27
  Sheldrick, G. M. SHELXS, v.2013–1 , 2013.
  There is no corresponding record for this reference.
28. 28
  Sheldrick, G. M. Crystal structure refinement with SHELXL. Acta Crystallogr., Sect. C Struct. Chem. 2015, 71 (1), 3– 8, DOI: 10.1107/S2053229614024218
  28
  Crystal structure refinement with SHELXL
  Sheldrick, George M.
  Acta Crystallographica, Section C: Structural Chemistry (2015), 71 (1), 3-8CODEN: ACSCGG; ISSN:2053-2296. (International Union of Crystallography)
  The improvements in the crystal structure refinement program SHELXL have been closely coupled with the development and increasing importance of the CIF (Crystallog. Information Framework) format for validating and archiving crystal structures. An important simplification is that now only one file in CIF format (for convenience, referred to simply as 'a CIF') contg. embedded reflection data and SHELXL instructions is needed for a complete structure archive; the program SHREDCIF can be used to ext. the and files required for further refinement with SHELXL. Recent developments in SHELXL facilitate refinement against neutron diffraction data, the treatment of H atoms, the detn. of abs. structure, the input of partial structure factors and the refinement of twinned and disordered structures. SHELXL is available free to academics for the Windows, Linux and Mac OS X operating systems, and is particularly suitable for multiple-core processors.
29. 29
  Dolomanov, O. V.; Bourhis, L. J.; Gildea, R. J.; Howard, J. A. K.; Puschmann, H. OLEX2: A complete structure solution, refinement and analysis program. J. Appl. Crystallogr. 2009, 42 (2), 339– 341, DOI: 10.1107/S0021889808042726
  29
  OLEX2: a complete structure solution, refinement and analysis program
  Dolomanov, Oleg V.; Bourhis, Luc J.; Gildea, Richard J.; Howard, Judith A. K.; Puschmann, Horst
  Journal of Applied Crystallography (2009), 42 (2), 339-341CODEN: JACGAR; ISSN:0021-8898. (International Union of Crystallography)
  New software, OLEX2, was developed for the detn., visualization and anal. of mol. crystal structures. The software has a portable mouse-driven workflow-oriented and fully comprehensive graphical user interface for structure soln., refinement and report generation, as well as novel tools for structure anal. OLEX2 seamlessly links all aspects of the structure soln., refinement and publication process and presents them in a single workflow-driven package, with the ultimate goal of producing an application which will be useful to both chemists and crystallographers.
30. 30
  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
  30
  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.
31. 31
  Zahn, D. On the role of water in amide hydrolysis. Eur. J. Org. Chem. 2004, 2004 (19), 4020– 4023, DOI: 10.1002/ejoc.200400316
  There is no corresponding record for this reference.
32. 32
  Alberto, R.; Schibli, R.; Waibel, R.; Abram, U.; Schubiger, A. P. Basic aqueous chemistry of [M(OH₂)₃(CO)₃]⁺ (M = Re, Tc) directed towards radiopharmaceutical application. Coord. Chem. Rev. 1999, 190–192, 901– 919, DOI: 10.1016/S0010-8545(99)00128-9
  32
  Basic aqueous chemistry of [M(OH2)3(CO)3]+ (M=Re, Tc) directed towards radiopharmaceutical application
  Alberto, R.; Schibli, R.; Waibel, R.; Abram, U.; Schubiger, A. P.
  Coordination Chemistry Reviews (1999), 190-192 (), 901-919CODEN: CCHRAM; ISSN:0010-8545. (Elsevier Science S.A.)
  A review with 20 refs. A review on the synthesis and properties of the organometallic aqua-ion [M(OH2)3(CO)3]+ (M=Re, 99Tc, 99mTc), as relevant for radiopharmaceutical application, is presented. These important starting compds. can be prepd. quant., (a) on the no carrier added (n.c.a.) level (99mTc) in water, or (b) in org. solvents (Re, 99Tc) at atm. pressure in a short time and from [MO4]-. The main characteristics of these carbonyl complexes are the high substitution stability of the three CO ligands and the substitution lability of the coordinated water mols. [M(OH2)3(CO)3]+ can be considered as a 'semi aquo-ion'. On the macroscopic level, upon titrn. with OH-, hydroxo-bridged oligomers have been isolated and characterized. The formation of hydroxo-bridged complexes is a consequence of the considerable Bronstedt acidity of [M(OH2)3(CO)3]+, whereas on the n.c.a. level no such behavior was obsd. Conditions and products of the water exchange by imidazole (i.m.) and derivs. thereof (histamine, histidine) will be presented. The different mononuclear complexes with these ligands are of extraordinary inertness, which is the basis for potential applications in biol. and nuclear medicine. Finally, as a basis for bioorganometallic chem., the adoption of the results from basic coordination chem. to the labeling of biomols. with an organometallic moiety will be exemplified with a selected penta-peptide and a recombinant single chain fragment.
33. 33
  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
  33
  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.
Supporting Information
Supporting Information
The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.4c05699.
- Synthesis protocols; ¹H NMR and ¹³C NMR spectra for the chelators; ¹H NMR, ¹³C NMR, and IR spectra for the ^natRe-complexes; TLC for the radiocomplexes; and crystal data (PDF)
- ao4c05699_si_001.pdf (2.25 MB)
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[186Re]Re- and [99mTc]Tc-Tricarbonyl Metal Complexes with 1,4,7-Triazacyclononane-Based Chelators Bearing Amide, Alcohol, or Ketone Pendent GroupsClick to copy article linkArticle link copied!

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1. Introduction

2. Materials and Methods

2.1. General

2.2. N-Benzyl-2-(1,4,7-triazonan-1-yl)acetamide (1)

Scheme 1

2.3. 2,2′-(7-(2-(Benzylamino)-2-oxoethyl)-1,4,7-triazonane-1,4-diyl)diacetamide (2)

2.4. N-Benzyl-2-(4,7-bis(2-hydroxyethyl)-1,4,7,-triazonan-1-yl)acetamide (3)

2.5. N-Benzyl-2-(4,7-bis(2-oxopropyl)-1,4,7-triazonan-1-yl)acetamide (4)

2.6. 2,2′-(7-(2-(Benzylamino)-2-oxoethyl)-1,4,7-triazonane-1,4-diyl)diacetic Acid (5)

Scheme 2

2.7. Labeling of TACN-Based Chelators with the [natRe(CO)3]+ Core

2.7.1. Re-2

2.7.2. Re-3

2.7.3. Re-4

2.7.4. Re-5

2.8. Radiolabeling of TACN-Based Chelators with the [99mTc][Tc(CO)3]+ Core

2.8.1. [99mTc]Tc-2

2.8.2. [99mTc]Tc-3

2.8.3. [99mTc]Tc-4

2.8.4. [99mTc]Tc-5

2.9. Radiolabeling of TACN-Based Chelators with the [186Re][Re(CO)3]+ Core

2.9.1. [186Re]Re-2

2.9.2. [186Re]Re-3

2.9.3. [186Re]Re-4

2.9.4. [186Re]Re-5

2.10. In Vitro Stability and logD7.4 Studies

2.11. Single-Crystal X-ray Diffraction Analysis

3. Results and Discussion

3.1. Synthesis of Modified TACN Chelators

3.2. Chelator Labeling with the [natRe(CO)3]+ Core

Figure 1

3.3. Chelator Radiolabeling with the [99mTc][Tc(CO)3]+ and [186Re][Re(CO)3]+ Cores

Figure 2

3.4. In Vitro Stability and logD7.4 Values of the Radiocomplexes

Figure 3

4. Conclusions

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Abstract

Scheme 1

Scheme 2

Figure 1

Figure 2

Figure 3

References

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[¹⁸⁶Re]Re- and [^99mTc]Tc-Tricarbonyl Metal Complexes with 1,4,7-Triazacyclononane-Based Chelators Bearing Amide, Alcohol, or Ketone Pendent Groups
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2.7. Labeling of TACN-Based Chelators with the [^natRe(CO)₃]⁺ Core

2.8. Radiolabeling of TACN-Based Chelators with the [^99mTc][Tc(CO)₃]⁺ Core

2.8.1. [^99mTc]Tc-2

2.8.2. [^99mTc]Tc-3

2.8.3. [^99mTc]Tc-4

2.8.4. [^99mTc]Tc-5

2.9. Radiolabeling of TACN-Based Chelators with the [¹⁸⁶Re][Re(CO)₃]⁺ Core

2.9.1. [¹⁸⁶Re]Re-2

2.9.2. [¹⁸⁶Re]Re-3

2.9.3. [¹⁸⁶Re]Re-4

2.9.4. [¹⁸⁶Re]Re-5

2.10. In Vitro Stability and logD_7.4 Studies

3.2. Chelator Labeling with the [^natRe(CO)₃]⁺ Core

3.3. Chelator Radiolabeling with the [^99mTc][Tc(CO)₃]⁺ and [¹⁸⁶Re][Re(CO)₃]⁺ Cores

3.4. In Vitro Stability and logD_7.4 Values of the Radiocomplexes