Novel hexadentate and pentadentate chelators for ⁶⁴Cu-based targeted PET imaging

Abstract

A series of new hexadentate and pentadentate chelators were designed and synthesized as chelators of ⁶⁴Cu. The new pentadentate and hexadentate chelators contain different types of donor groups and are expected to form neutral complexes with Cu(II). The new chelators were evaluated for complex kinetics and stability with ⁶⁴Cu. The new chelators instantly bound to ⁶⁴Cu with high labeling efficiency and maximum specific activity. All ⁶⁴Cu-radiolabeled complexes in human serum remained intact for 2 days. The ⁶⁴Cu-radiolabeled complexes were further challenged by EDTA in a 100-fold molar excess. Among the ⁶⁴Cu-radiolabeled complexes evaluated, ⁶⁴Cu-complex of the new chelator E was well tolerated with a minimal transfer of ⁶⁴Cu to EDTA. ⁶⁴Cu-radiolabeled complex of the new chelator E was further evaluated for biodistribution studies using mice and displayed rapid blood clearance and low organ uptake. ⁶⁴Cu-chelator E produced a favorable in vitro and in vivo complex stability profiles comparable to ⁶⁴Cu complex of the known hexadentate NOTA chelator. The in vitro and in vivo data highlight strong potential of the new chelator E for targeted PET imaging application.

1. Introduction

⁶⁴Cu (t_1/2 = 12.7 h, E_max^β+ = 656 keV; E_max^β− = 573 keV; E_max^γ = 511 keV) is one of the most useful radioisotopes for positron emission tomography (PET) imaging.^1–2 Various ⁶⁴Cu-radiolabeled antibody or peptide conjugates have been employed for PET imaging of cancers in preclinical and clinical settings.^1–2 A bifunctional chelator that can rapidly complex Cu(II) with high kinetic inertness and thermodynamic stability is a critical component of clinically viable ⁶⁴Cu-based radiopharmaceuticals for targeted PET imaging. Many antibodies and proteins are not tolerant of heating and radiolysis, and effective radiolabeling of a bifunctional chelator with a short-lived ⁶⁴Cu under mild condition is required to minimize radiolytic damage of an antibody conjugated with the chelator. Bifunctional DTPA (diethylene triamine pentaacetic acid), TETA (1,4,8,11-tetraazacyclotetradecane-N,N′,N″,N′-tetraacetic acid), DOTA (1,4,7,10-tetraazacyclododecane-N,N′, N″, N′″-tetraacetic acid), and CB-TE2A (1,4,8,11-tetraazabicyclo[6.6.2]hexadecane-4,11-diacetic acid) have been investigated as chelators of ⁶⁴Cu.² Although acyclic DTPA is known to rapidly complex with Cu(II), ⁶⁴Cu-DTPA was reported be kinetically labile and dissociated in serum.² The macrocyclic chelators, DOTA (log K = 22.3) and TETA (log K = 21.7) form fairly stable complexes with Cu(II) using the four nitrogens in the macrocyclic ring and two pendant carboxylate oxygens as demonstrated by X-ray crystallography.³ In particular, TETA conjugated to an antibody was found to display high binding selectivity to Cu(II) in the presence of other metals with an oxidation state of +2 including Zn(II), Mg(II), and Ca(II).⁴ The ⁶⁴Cu complexes of DOTA and TETA remained intact with a minimal loss of ⁶⁴Cu in serum.² However, in vivo biodistribution data suggested that ⁶⁴Cu-DOTA and ⁶⁴Cu-TETA analogues conjugated to a biomolecule including octreotide were dissociated in mice, and high liver uptake was observed with the ⁶⁴Cu-radiolabeled complexes.^{5–7 64}Cu complexes of cross-bridged cyclam (CB-TE2A) analogues were shown to possess improved kinetic in vivo stability compared to ⁶⁴Cu-DOTA and ⁶⁴Cu-TETA complexes.^7–8 However, radiolabeling of CB-TE2A with ⁶⁴Cu required a complicated and harsh condition due to slow complexation kinetics of the chelator with ⁶⁴Cu.⁸ NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid) is a hexadentate macrocyclic chelator containing aminocarboxylate donors and binds to Cu(II) with high thermodynamic stability (log K = 21.6).^{3 64}Cu-radiolabeled NOTA complexes have produced encouraging in vitro and in vivo profiles.^6,9–10 We previously reported that ⁶⁴Cu-NOTA complex remained stable in both serum and mice and displayed fast blood clearance and low uptake in normal organs.¹⁰

In our continued effort on improved chelation chemistry for development of radiopharmaceuticals,^10–14 we synthesized and evaluated new hexadentate and pentadentate NOTA analogues (Figure 1) as potential chelators of ⁶⁴Cu. The new chelators were evaluated for radiolabeling with ⁶⁴Cu to determine the maximum specific activity and radiolabeling kinetics with ⁶⁴Cu under mild condition. ⁶⁴Cu-radiolabeled complexes of the promising new chelator were evaluated for complex stability in human serum and EDTA (ethylenediaminetetraacetic acid) challenge and in vivo biodistribution using mice.

Figure 1.

Potential Chelators for PET imaging using ⁶⁴Cu

2. Material and Methods

Instruments and reagents

¹H, ¹³C, and DEPT NMR spectra were obtained using a Bruker 300 NMR instrument, and chemical shifts are reported in ppm on the δ scale relative to TMS. Electro spray ionization (ESI) high resolution mass spectra (HRMS) were obtained on JEOL double sector JMS-AX505HA mass spectrometer (University of Notre Dame, IN). ⁶⁴Cu was prepared on CS-15 cyclotron at Washington University Medical School, St. Louis, MO according to the previous reported method.¹⁰ Radioactivity was counted with a Beckman Gamma 8000 counter containing a NaI crystal (Beckman Instruments, Inc., Irvine, CA). Analytical and semi-prep HPLC were performed on Agilent 1200 (Agilent, Santa Clara, CA) equipped with a diode array detector (λ= 254 and 280 nm), thermostat set at 35 °C and a Zorbax Eclipse XDB-C18 column (4.6 × 150 mm, 80Å, Agilent, Santa Clara, CA). The mobile phase of a binary gradient (0–100%B/40 min; solvent A = 0.1% TFA in water; solvent B = 0.1% TFA in acetonitrile for method 1; 0–100%B/15 min; solvent A: H₂O, solvent B: MeOH for method 2) at a flow rate of 1 mL/min was used for analytical HPLC. The mobile phase of a binary gradient (0–100%B/40 min; solvent A: H₂O, solvent B: MeOH, flow rate: 3 mL/min) was used for semi-prep HPLC (method 3).

tert-butyl 2-{4,7-bis[2-(tert-butoxy)-2-oxoethyl]-1,4,7-triazanonan-1-yl}-4-(4-nitrophenyl)-butanoate (3)

To a solution of 1¹² (272 mg, 0.761mmol) in CH₃CN (1 mL) was added portionwise compound 2¹⁵ (230 mg, 0.761 mmol) and DIPEA (295 mg, 2.284 mmol) in CH₃CN (1 mL). The resulting mixture was stirred for 7 days at room temperature. The reaction mixture was concentrated to dryness in vacuo. 0.1M HCl aqueous solution (20 mL) was added to the residue, and the resulting mixture was extracted with CH₂Cl₂ (2 × 20 mL). The combined organic layers were dried over MgSO₄, filtered, and concentrated to the dryness in vacuo. The residue was purified via column chromatography on silica gel (220–440 mesh) eluting with 15% CH₃OH in CH₂Cl₂ to afford pure product 3 (240 mg, 51%). ¹H NMR (CDCl₃, 300 MHz) δ 1.42 (s, 18H), 1.96–1.99 (m, 2H), 2.67–2.98 (m, 15H), 3.23–3.29 (m, 5H), 3.65 (s, 3H), 7.36 (d, J = 8.5 Hz, 2H), 8.12 (d, J = 8.5 Hz, 2H); ¹³C NMR (CDCl₃, 75 MHz) δ 28.2 (q), 31.8 (t), 32.7 (t), 51.2 (q), 53.5 (t), 55.5 (t), 56.0 (t), 59.4 (t), 66.4 (d), 80.8 (s), 123.7 (d), 129.4 (d), 146.4 (s), 149.7 (s), 171.4 (s), 173.7 (s). HRMS (positive ion ESI) Calcd for C₂₉H₄₇N₄O₈ [M + H]⁺ m/z 521.3388. Found: [M + H]⁺ m/z 521.3391.

2-[4,7-bis(carboxymethyl)-1,4,7-triazanonan-1-yl]-4-(4-nitrophenyl)butanoic acid (A)¹⁶

Compound 3 (20 mg, 0.032 mmol) was treated with 6M HCl solution (2 mL), and the resulting solution was refluxed for 3 h. The reaction mixture was cooled to room temperature, and the resulting solution was filtered, and the filtrate was concentrated in vacuo to provide chelator A (16 mg, 89%) as a yellow solid. ¹H NMR (D₂O, 300 MHz) δ 1.90–1.92 (m, 1H), 2.06–2.10 (m, 1H), 2.59–2.84 (m, 2H), 2.95–3.18 (m, 12H), 3.49–3.53 (m, 1H), 3.79 (s, 4H), 7.30 (d, J = 8.4 Hz, 2H), 7.99 (d, J = 8.4 Hz, 2H); ¹³C NMR (D₂O, 75 MHz) δ 29.7 (t), 32.1 (t), 45.7 (t), 49.2 (t), 50.6 (t), 55.3 (t), 63.7 (d), 123.7 (d), 129.4 (d), 146.4 (s), 149.7 (s), 172.2 (s), 175.6 (s). The data of ¹H and ¹³C NMR data were essentially identical to those previously reported.¹⁶ Analytical HPLC (t_R = 7.6 min, method 1).

tert-butyl 2-{4-[2-(tert-butoxy)-2-oxoethyl]-7-[3-(4-nitrophenyl)propyl]-1,4,7-triazanonan- 1-yl}acetate (5)

To a solution of 1¹² (72 mg, 0.29 mmol) in CH₃CN (2 mL) at 0 °C was added dropwise 4¹¹ (105 mg, 0.29 mmol) in CH₃CN (1 mL) and DIPEA (112 mg, 0.87 mmol). The resulting mixture was stirred for 61 h at room temperature. The resulting mixture was concentrated to dryness in vacuo. Water (10 mL) and 0.1M HCl aqueous solution (1 mL) were added to the residue, and the resulting mixture was extracted with CHCl₃ (3 × 10 mL). The combined organic layers were dried over MgSO₄, filtered, and concentrated to the dryness in vacuo The residue was purified via column chromatography on silica gel (60–220 mesh) eluting with 20% MeOH in CH₂Cl₂ to afford 5 (81 mg, 54%) as an oil. ¹H NMR (CDCl₃, 300 MHz) δ 1.41 (s, 18H), 2.21 (m, 2H), 2.65–2.90 (m, 8H), 3.01–3.18 (m, 5H), 3.37–3.70 (m, 8H), 7.41 (d, J = 6.0 Hz, 2H), 8.12 (d, J = 6.0 Hz, 2H); ¹³C NMR (CDCl₃, 75 MHz) 5 25.9 (t), 28.1 (q), 32.8 (t), 49.8 (t), 52.3 (t), 53.5 (t), 55.3 (t), 58.2 (t), 81.7 (s), 123.9 (d), 129.4 (d), 146.7 (s), 147.9 (s), 170.7 (s). HRMS (positive ion ESI) Calcd for C₂₇H₄₅N₄O₆ [M + H]⁺ m/z 521.3334. Found: [M + H]⁺ m/z 521.3309.

2-[4-(carboxymethyl)-7-[3-(4-nitrophenyl)propyl]-1,4,7-triazanonan-1-yl]acetic acid (B)

Compound 5 (20 mg, 0.038 mmol) at 0~5 oC was treated dropwise with 4M HCl (g) in 1,4-dioxane (2 mL) over 10 min. The resulting mixture was gradually warmed to room temperature and stirred for 24 h. Diethyl ether (20 mL) was added to the reaction mixture which was stirred for 10 min. The resulting mixture was capped and placed in the freezer for 1 h. The solid formed was filtered, washed with ether, and quickly dissolved in deionized water. The resulting aqueous solution was concentrated in vacuo to provide chelator B (14 mg, 78%). ¹H NMR (D₂O, 300 MHz) δ 2.02–2.17 (m, 2H), 2.71–2.79 (m, 2H), 3.18 (m, 14H), 3.75–3.82 (m, 4H), 7.37 (d, J = 8.4 Hz, 2H), 8.07 (d, J = 8.7 Hz, 2H); ¹³C NMR (D₂O, 75 MHz) 5 24.9 (t), 31.7 (t), 49.6 (t), 50.7 (t), 51.0 (t), 56.9 (t), 57.3 (t), 123.9 (d), 129.5 (d), 146.4 (s), 148.7 (s), 172.9 (s). HRMS (positive ion ESI) Calcd for C₁₉H₂₉N₄O₆ [M + H]⁺ m/z 409.2082. Found: [M + H]⁺ m/z 409.2087. Analytical HPLC (t_R = 7.3 min, method 1).

Ethyl 2-[(4-nitrophenyl)methyl]-3-oxobutanoate (6)¹⁷

Ethyl acetoacetate (10 g, 76.84 mmol) was added dropwise to NaH (1.84 g, 76.84 mmol) in the THF (220 ml). p-nitro benzyl bromide (16.6 g, 76.84 mmol) was added portionwise over 1 h. The reaction mixture was stirred for 1.5 h. After evaporation of the solvent, the residue was treated with H₂O (100 mL) and extracted with ethyl acetate (2 × 100 mL). The combined organic layers were dried over MgSO₄, filtered, and concentrated to the dryness in vacuo. The residue was recrystallized with ethanol to provide pure product 6 (10 g, 49%). ¹H NMR (CDCl₃, 300 MHz) δ 1.21 (t, 3H, J = 7.2 Hz), δ 2.23 (s, 3H), δ 3.23–3.27 (m, 2H), δ 3.79 (t, 3H, J = 7.5 Hz), δ 4.15–4.17 (m, 2H), δ 7.36 (d, 2H, J = 8.4 Hz), δ 8.13 (d, 2H, J = 8.4 Hz). ¹³C NMR (CDCl₃, 75 MHz) δ 14.0(q), 29.5 (q), 33.4 (t), 60.6 (d), 61.9 (d), 123.8 (d), 129.8 (d), 146.1 (s), 148.9 (s), 168.5 (s), 201.2 (s).

4-(4-nitrophenyl)butan-2-one (7)¹⁸

Compound 6 (10 g, 37.7 mmol) was dissolved in the mixture of acetic acid (85 mL) and conc. HCl solution (30 mL), and the resulting solution was refluxed for 24 h after which the reaction mixture was allowed to room temperature and concentrated to dryness in vacuo. The residue was treated with H₂O (100 ml) and extracted with ethyl acetate (2 × 100 mL). The combined organic layers were dried over MgSO₄, filtered, and concentrated in vacuo to provide pure 7 (7.3 g, 100%). ¹H NMR (CDCl₃, 300 MHz) δ 2.09 (s, 3H), 2.77 (t, 2H, J = 9 Hz), 2.92 (t, 2H, J = 9 Hz), 7.29 (d, 2H, J = 9 Hz), 8.01 (d, 2H, J = 9 Hz); ¹³C NMR (CDCl₃, 75 MHz) δ 29.3 (t), 30.1 (q), 44.2 (t), 123.7 (d), 129.3 (d), 146.5 (s), 149.0 (s), 206.8 (s). The data of ¹H and ¹³C NMR data were essentially identical to those of 7 as previously reported.¹⁶

1-bromo-4-(4-nitrophenyl)butan-2-one (8)

To a solution of 7 (5.0 g, 25.88 mmol) in anhydrous methanol (10 mL) was added dropwise bromine (4.14 mg, 25.88 mmol) in anhydrous methanol (10 mL) at 7~9 °C over 1 h. The mixture was stirred for 34 h in same temperature. DI water (30 mL) was added to the mixture which was warmed to room temperature and continuously stirred for 12 h. The reaction mixture was extracted with CH₂Cl₂ (2 × 30 mL). The combined organic layers were dried over MgSO₄, filtered, and the filtrate was concentrated in vacuo. The residue was purified via column chromatography on silica gel (60–220 mesh) and eluted with 10% ethyl acetate in hexanes to afford 8 (1.26 g, 18%) as a solid. ¹H NMR (CDCl₃, 300 MHz) δ 3.05 (s, 4H), 3.87 (s, 2H), 7.35 (d, 2H, J = 7.8 Hz), 8.12 (d, 2H, J = 8.1 Hz); ¹³C NMR (CDCl₃, 75 MHz) δ 29.4 (t), 33.9 (t), 40.4 (t), 123.8 (d), 129.3 (d), 146.6 (s), 148.2 (s), 200.4 (s). HRMS (positive ion ESI) Calcd for C₁₀H₁₁BrNO₃ [M + H]⁺ m/z 271.9917. Found: [M + H]⁺ m/z 271.9924.

tert-butyl 2-{4-[2-(tert-butoxy)-2-oxoethyl]-7-[4-(4-nitrophenyl)-2-oxobutyl]-1,4,7-triazanonan-1-yl}acetate (9)

Compound 8 (114 mg, 0.420 mmol) was added portionwise to a solution of 1¹² (150 mg, 0.420 mmol) in CH₃CN (5 mL) at 0 °C. DIPEA (163 mg, 1.259 mmol) in CH₃CN (2 mL) was added portionwise, and the resulting mixture was allowed to room temperature and stirred for 24 h. The reaction mixture was concentrated to dryness in vacuo. The residue was treated with deionized water (10 mL) and extracted with CHCl₃ (2 × 10 mL). The combined organic layer was concentrated in vacuo. Then the resulting mixture was dissolved with 0.1M HCl solution (10 mL) and washed with CHCl₃ (2 × 10 mL). The aqueous layer was neutralized using 0.1M NaOH (10 mL) and extracted with CHCl₃ (3 × 20 mL). The combined organic layers were dried over MgSO₄, filtered, and concentrated to the dryness in vacuo. The residue was purified via column chromatography on silica gel (60–220mesh) eluting with 30% CH₃OH in dichloromethane containing Et₃N to provide pure 9 (96.8 mg, 42%). ¹H NMR (CDCl₃, 300 MHz) δ 1.38 (s, 18H), 2.89–3.57 (m, 21H), 4.47 (s, 1H), 7.31 (d, 2H, J = 7.5 Hz), 8.05 (d, 2H, J = 7.5 Hz).(This is Yongliang’s proton data.) ¹³C NMR (CDCl₃, 75 MHz) δ 28.1 (q), 28.8 (t), 40.8 (t), 48.4 (t), 50.3 (t), 51.5 (t), 55.8 (t), 62.9 (t), 82.0 (s), 123.7 (d), 129.3 (d), 146.5 (s), 148.4 (s), 169.4 (s), 203.4 (s) HRMS (positive ion ESI) Calcd for C₂₈H₄₄N₄O₇ [M + H]+ m/z 549.6716. Found: [M + H]+ m/z 549.3403.

tert-butyl 2-{4-[2-(tert-butoxy)-2-oxoethyl]-7-[2-hydroxy-4-(4-nitrophenyl)butyl]-1,4,7-triazanonan-1-yl}acetate (10)

A solution of 9 (33 mg, 0.060 mmol) in anhydrous methanol (1 mL) at 0 °C was added portionwise NaBH₄ (10 mg, 0.264 mmol) over 1 h. The mixture was then warmed to room temperature and stirred for 3 h. The reaction mixture was concentrated to dryness and treated with H₂O (10 mL) and extracted with ethyl acetate (2 × 15 mL). The combined organic layers were dried over MgSO₄, filtered, and the filtrated was concentrated in vacuo to provide pure 10 (26.5 mg, 81%). ¹H NMR (CDCl₃, 300 MHz) δ 1.43 (s, 18H), 1.59–1.70 (m, 2H), 2.36 (m, 1H), 2.69–3.05 (m, 15H), 3.29 (s, 4H), 3.51–3.69 (m, 1H), 7.35 (d, 2H, J = 8.4 Hz), 8.11 (d, 2H, J = 8.4 Hz). ¹³C NMR (D₂O, 75 MHz) δ 28.2 (q), 32.2 (t), 35.8 (t), 55.8(t), 56.0 (t), 56.4 (t), 58.9 (t), 63.8 (t), 68.0 (d), 80.8 (s), 123.6 (d), 129.3 (d), 146.3 (s), 150.6 (s), 171.5 (s). HRMS (positive ion ESI) Calcd for C₂₈H₄₇N₄O₇ [M + H]⁺ m/z 551.3439. Found: [M + H]⁺ m/z 551.3463.

2-[4-(carboxymethyl)-7-[4-(4-nitrophenyl)-2-oxobutyl]-1,4,7-triazanonan-1-yl]acetic acid (C)

TFA (800 µl) was added dropwise to compound 9 (22.6 mg 0.041 mmol) at 0 °C, and the resulting solution was stirred for 2 h at 0 °C. The resulting mixture was allowed to room temperature and stirred for additional 1 h. The reaction mixture was concentrated to dryness in vacuo and treated with ether (2 mL), and the ether layer was decanted. The residue was dissolved in H₂O (2 mL) and washed by CHCl₃ (2 × 5 mL). The aqueous layer was concentrated to dryness in vacuo to provide pure product C (16.4 mg, 91%). ¹H NMR (D₂O, 300 MHz) δ 2.85 (m, 4H), 3.24–3.28 (m, 12H), 3.82 (s, 4H), 4.20 (s, 2H), 7.30 (d, 2H, J = 9.0 Hz), 8.00 (d, 2H, J = 9.0 Hz); ¹³C NMR (D₂O, 75 MHz) δ 28.3 (t), 39.8 (t), 50.0 (t), 50.4 (t), 51.3 (t), 56.3 (t), 64.2 (t), 123.6 (d), 129.3 (d), 146.0 (s), 148.8 (s), 171.8 (s), 205.8 (s). HRMS (positive ion ESI) Calcd for C₂₀H₂₉N₄O₇ [M + H]⁺ m/z 437.2031. Found: [M + H]⁺ m/z 437.2041. Analytical HPLC (t_R = 7.8 min, method 1)

2-[4-(carboxymethyl)-7-[2-hydroxy-4-(4-nitrophenyl)butyl]-1,4,7-triazanonan-1-yl]acetic acid (D)

TFA (1 mL) was added dropwise to compound 10 (24 µg 0.045 mmol) at 0 °C, the resulting mixture was stirred for 3 h at 0 °C. The resulting mixture was allowed to room temperature and stirred for additional 1 h. The reaction mixture was concentrated to dryness and treated with ether (3 mL), and the ether layer was decanted. The residue was dissolved in H₂O (2 mL) and washed by CHCl₃ (2 × 5 mL). The aqueous layer was concentrated to dryness in vacuo, and the residue was purified by semi-prep HPLC (method 3, t_R = ~21 min) to provide pure product D (7.2 mg, 37%). ¹H NMR (D₂O, 300 MHz) δ 1.69 (m, 2H), 2.66–2.78 (m, 2H), 3.01–3.49 (m, 18H), 3.86 (m, 1H), 7.31 (d, 2H, J = 8.1 Hz), 8.01 (d, 2H, J = 8.4 Hz). ¹³C NMR (D₂O, 75 MHz) δ 30.7 (t), 35.1 (t), 49.8 (t), 50.5 (t), 58.5(t), 62.3 (t), 65.5 (d), 123.7 (d), 129.4 (d), 145.9 (s), 150.0 (s), 175.5 (s). HRMS (positive ion ESI) Calcd for C₁₀H₃₁N₄O₇ [M + H]⁺ m/z 439.2187. Found: [M + H]⁺ m/z 439.2203. Analytical HPLC (t_R = 8.0 min, method 2)

tert-butyl 2-{4-[2-(tert-butoxy)-2-oxoethyl]-7-(pyridin-2-ylmethyl)-1,4,7-triazanonan-1-yl}acetate (11)

To a solution of 1¹² (50 mg, 0.140 mmol) in 1,2-dichloroethane (1 mL) was added 2-pyridinecarboxaldehyde (15 mg, 0.140 mmol). The resulted solution was stirred for 10 min and then added with sodium triacetoxyborohydride (44.5 mg, 0.210 mmol) portionwise over 10 min. The mixture was stirred at room temperature for overnight. The reaction mixture was quenched by adding saturated NaHCO₃ (15 mL), and the resulting solution was extracted with ethyl acetate (3 × 15 mL). The combined organic layers were concentrated to dryness in vacuo. The residue was dissolved in 0.1M HCl solution (10 mL) and washed with CHCl₃ (2 × 10 mL). The aqueous layer was treated with saturated NaHCO₃ (10 mL) and extracted with ethyl acetate (3 × 20 mL). The combined organic layers were dried over MgSO₄, filtered, and concentrated in vacuo to provide pure 11 (60 mg, 96%) as a yellowish oil. ¹H NMR (CDCl₃, 300 MHz) δ 1.44 (s, 18H), 2.75–2.90 (m, 12H), 3.31–3.36 (m, 4H), 3.87 (s, 2H), 7.13 (t, J = 5.4 Hz, 1H), 7.54 (d, J = 7.8 Hz, 1H), 7.61–7.67 (m, 1H), 8.50 (d, J = 3.9 Hz, 1H); ¹³C NMR (CDCl₃, 75 MHz) δ 28.2 (q), 55.3 (t), 55.6 (t, 2C), 59.8 (t), 64.1 (t), 80.6 (s), 121.7 (d), 123.1 (d), 136.2 (d), 148.8 (d), 160.6 (s), 171.5 (s). HRMS (positive ion ESI) Calcd for C₂₄H₄₁N₄O₄ [M + H]⁺ m/z 449.3122. Found: [M + H]⁺ m/z 449.3118.

2-[4-(carboxymethyl)-7-(pyridin-2-ylmethyl)-1,4,7-triazanonan-1-yl]acetic acid (E)

Compound 11 (16 mg, 0.036 mmol) was treated with 6M HCl solution (3 mL), and the resulting solution was refluxed for 5 h. The reaction mixture was gradually cooled to room temperature, filtered, and concentrated to dryness in vacuo to provide chelator E (15 mg, 86%) as a yellow solid. ¹H NMR (D₂O, 300 MHz) δ 2.59–2.80 (m, 4H), 3.01 (m, 4H), 3.08 (s, 4H), 3.72 (s, 4H), 4.19 (s, 2H), 7.84 (t, J = 7.2 Hz, 2H), 7.98 (d, J = 7.8 Hz, 1H), 8.39 (dt, J = 7.5 Hz, J = 1.8 Hz 1H), 8.51 (d, J = 5.4 Hz, 1H). ¹³C NMR (D₂O, 75 MHz) δ 47.5 (t), 48.9 (t), 50.5 (t), 55.4 (t), 56.4 (t), 126.4 (d), 127.9 (d), 141.4 (d), 147.3 (d), 153.0 (s), 173.1 (s). HRMS (positive ion ESI) Calcd for C₁₆H₂₅N₄O₄ [M + H]⁺ m/z 337.1870. Found: [M + H]⁺ m/z 337.1863. Analytical HPLC (t_R = 4.2 min, method 1)

tert-butyl 2-{4-[2-(tert-butoxy)-2-oxoethyl]-7-(thiophen-2-ylmethyl)-1,4,7-triazanonan-1-yl}acetate (12)

To a solution of 1¹² (50.0 mg, 0.140 mmol) in 1,2-dichloroethane (1 mL) was added 2-thiophenecarboxaldehyde (15.7 mg, 0.140 mmol). The resulted solution was stirred for 10 min and treated with sodium triacetoxyborohydride (44.5 mg, 0.210 mmol) portionwise over 10 min. The reaction mixture was stirred at room temperature for 3 d. The reaction mixture was quenched by adding saturated NaHCO₃ (15 mL), and the resulting solution was extracted with ethyl acetate (3 × 15 mL). The combined organic layer was concentrated in vacuo. The residue was treated with 0.1M HCl solution (10 mL) and extracted with CHCl₃ (3 × 10 mL). The combined organic layers were dried over MgSO₄, filtered, and the filtrate was concentrated to the dryness in vacuo. The residue was purified via column chromatography on silica gel (60–220 mesh) eluting with 15% CH₃OH in CH₂Cl₂ to afford pure 12 (46 mg, 73%). ¹H NMR (CDCl₃, 300 MHz) δ 1.45 (s, 18H), 2.82–2.96 (m, 12H), 3.31 (s, 4H), 3.87 (s, 2H), 6.89–6.93 (m, 2H), 7.19 (dd, J = 1.2 Hz, J = 4.8 Hz, 1H); ¹³C NMR (CDCl₃, 75 MHz) δ 28.2 (q), 55.0 (t), 55.2 (t), 55.3 (t), 56.8 (t), 59.9 (t), 80.7 (s), 124.6 (d), 125.3 (d), 126.3 (d), 160.6 (s), 171.5 (s). HRMS (positive ion ESI) Calcd for C₂₃H₄₀N₃O₄S [M + H]⁺ m/z 454.2734. Found: [M + H]⁺ m/z 454.2723.

2-[4-(carboxymethyl)-7-(thiophen-2-ylmethyl)-1,4,7-triazanonan-1-yl]acetic acid (F)

Compound 12 (17 mg, 0.037 mmol) at 0–5 °C was treaeted dropwise with 4M HCl (g) in 1,4-dioxane (2.5 mL) over 10 min. The resulting mixture was warmed to room temperature and stirred for 24 h. Diethyl ether (40 mL) was added to the mixture which was continued to stir for 10 min. The solid formed was filtered, washed with ether, and quickly dissolved in DI water. The aqueous solution was concentrated in vacuo to provide chelator F (13.5 mg, 80%) as a yellow solid. ¹H NMR (D₂O, 300 MHz) δ 2.90–3.61 (m, 18H), 6.91–7.08 (m, 1H), 7.27 (d, J = 2.4 Hz, 1H), 7.49 (d, J = 4.8 Hz, 1H). ¹³C NMR (D₂O, 75 MHz) δ 49.0 (t), 50.2 (t), 50.3 (t), 54.3 (t), 56.9 (t), 128.0 (d), 129.8 (s), 130.1 (d), 132.7 (d), 173.95 (s). HRMS (positive ion ESI) Calcd for C₁₅H₂₄N₃O₄S [M + H]⁺ m/z 342.1482. Found: [M + H]⁺ m/z 342.1469. Analytical HPLC (t_R = 5.8 min, method 1)

Preparation and characterization of Cu(II) complexes

Cu(II)-complexes of the chelators A-F were prepared by reaction of each chelator (5 µl, 10 mM) with CuCl₂ (5 µl, 10 mM) in 0.25M NH₄OAc buffer (pH 5.5) for 24 h at room temperature and 300 rpm. Each of Cu(II)-complex was purified by semi-prep HPLC (solvent A: 0.1% TFA in H₂O, solvent B: 0.1% TFA in CH₃CN, 0–60% B/40 min, flow rate: 3 mL/min). The purified Cu(II)-complex was characterized by analytical HPLC (method 1, Supporting Information)

Determination of maximum specific activity (MSA)

Whatman C18 silica gel TLC plates (KC18F, 60 Å) were purchased from Fisher Scientific (Pittsburgh, PA). Radio-TLCs were developed with 10% NH₄OAc:MeOH (3:7) and analyzed using a Bioscan 200 imaging scanner (Bioscan, Inc., Washington, DC). ⁶⁴CuCl₂ was diluted with a 10-fold excess of 0.1M NH₄OAc (pH 5.5) for radiolabeling. The maximum specific activities were determined experimentally via titrating ⁶⁴CuCl₂ in 0.1M NH₄OAc (pH 5.5) with the chelators. Briefly, for each chelator, six reaction vials were prepared in 0.1M NH₄OAc (pH 5.5) via dilution to give final chelator masses in the range 0.001 to 0.1 µg. 3.7 MBq (100 µCi) of ⁶⁴Cu in 0.1M NH₄OAc (pH ~5.5) was added to each vial and adjusted the final volume to 100 µL (final pH 5.5) and vortexed for 10–15 seconds. The reactions were incubated on a rotator at 37 °C for 1 h. After incubation, 1 µL aliquots were withdrawn from reaction vials an alyzed by TLC (C-18) with a mixture of 10% NH₄OAc/MeOH (3:7) as a mobile phase for labeling percentage. All reactions were done in triplicate. The data were plotted as % labeling vs. amount of chelator used in the reaction and fitted using sigmoidal dose response equation in GraphPad Prism (La Jolla, CA). Based on this, the amount of mass required to achieve 50% labeling was determined, and this mass was multiplied by 2 to obtain the minimal mass for 100% labeling to determine the maximum specific activity.

Complexation formation kinetics of chelators with ⁶⁴Cu

All HCl solutions were prepared from ultra-pure HCl (Fisher Scientific, #A466–500). For metal-free radiolabeling, plasticware including pipette tips, tubes, and caps was soaked in 0.1M HCl (aq) overnight and washed thoroughly with Milli-Q (18 MΩ) water, and air-dried overnight. 0.25M NH₄OAc buffer solution (pH 5.5) was prepared using ultra-pure ammonium acetate and pH of the solution was adjusted using 0.1M and 1M HCl solution. The resulting buffer solution was treated with Chelex-100 resin (Biorad, #142–2842), shaken overnight at room temperature, and filtered through 0.22 µm filter (Corning, #430320) prior to use. TLC plates (6.6 × 1 cm or 6.6 × 2 cm, Silica gel 60 F₂₅₄, EMD Chemicals Inc., #5554–7) with the origin line drawn at 0.6 cm from the bottom were prepared. To a buffer solution (11~18 µL, 0.25M NH₄OAc, pH 5.5) in a capped microcentrifuge tube (1.5 mL, Fisher Scientific, #05–408–129) was sequentially added a solution of the chelator (6.7 µg, 1–7 µL) in the buffer and ⁶⁴Cu in 0.05M HCl (20 µCi, 1~3 µL). The total volume of the resulting solution was 20 µL, and the concentration of each chelator in the reaction mixture was 0.6 mM. The reaction mixture was agitated on the thermomixerset at 1,000 rpm at room temperature for 30 min. The labeling efficiency was determined by ITLC eluted with 20mM EDTA in 0.15M NH₄OAc as the mobile phase. A solution of radiolabeled complexes (2.0 µL) was withdrawn at the designated time points (1 min, 10 min, and 30 min), spotted on a TLC plate, and then eluted with the mobile phase. After completion of elution, the TLC plate was warmed and dried on the surface of a hot plate maintained at 35 °C and scanned using TLC scanner (Bioscan). Unbound and bound radioisotope appeared 30~35 mm (R_f = 0.6) and 50~55 mm (R_f = 1.0) from the bottom of the TLC plate, respectively.

In vitro serum stability of ⁶⁴Cu-radiolabeled complexes

Human serum was purchased from Gemini Bioproducts (#100110). ⁶⁴Cu-radiolabeled complexes were prepared by reaction of the bifunctional chelators (30 µg) with ⁶⁴Cu (100 µCi) in 0.25M NH₄OAc buffer (pH 5.5) for 1 h at room temperature, and labeling efficiency of the radiolabeled complexes were found to be ~100% as determined by ITLC (20mM EDTA in 0.15M NH₄OAc). The freshly prepared radiolabeled complexes of chelators were directly used for serum stability studies without further purification. ⁶⁴Cu-radiolabeled complex (85 µCi, 10 µL) was added to human serum (90 µL) in a microcentrifuge tube, and concentration of chelator in the resulting mixture was 0.5 mM. The stability of ⁶⁴Cu-radiolabeled complexes in human serum was evaluated at 37 °C for 2 days. A solution of the radiolabeled complex in serum was withdrawn at the designated time points and evaluated by ITLC as described above.

EDTA Challenge¹⁹

⁶⁴Cu-radiolabeled complexes were prepared by reaction of each chelator (20 µg) with ⁶⁴Cu (60 µCi) in 0.25M NH₄OAc buffer (40 µL, pH 5.5) for 2 h at room temperature. The labeling efficiency of the ⁶⁴Cu-radiolabeled complexes was found to be ~100% as determined by ITLC (20mM EDTA in 0.15M NH₄OAc). The ⁶⁴Cu-radiolabeled complexes were directly used for EDTA challenge experiments. A solution of the ⁶⁴Cu-radiolabeled complex containing each chelator (~1 mM) in 0.25M NH₄OAc buffer (40 µL) was mixed with a solution of EDTA (~ 100 mM, 40 µL, H₂O, pH 5.0) at a 100-fold molar excess. The resulting mixture was incubated for 24 h at 37 °C. The stability of ⁶⁴Cu-radiolabeled complexes in the solution was evaluated using ITLC (20 mM EDTA in 0.15M NH₄OAc). A solution of the radiolabeled complex in serum (3~20 µL) was withdrawn at the designated time points and evaluated by ITLC as described above. Stability of the complexes was also evaluated at 25 h time point by HPLC (solvent A: 0.1% TFA in H₂O, solvent B: 0.1% TFA in CH₃CN, 0–100% B/15 min, flow rate: 1 mL/min). ⁶⁴Cu-EDTA complex was eluted early (t_R = ~ 2.5 min), while ⁶⁴Cu-radiolabeled complexes of chelators A-D, E, and F have the respective retention time (t_R = 7~8 min, t_R = 4.6 min, and t_R = 6.2 min).

In vivo biodistribution

All animal studies were performed in compliance with the Guidelines for Care and Use of Research Animals established by Washington University’s Animal Studies Committee. Biodistribution studies were carried out in female CD-1 mice (Charles River Labs). The tissue uptakes of ⁶⁴Cu-chelator A and ⁶⁴Cu-chelator E were evaluated in mice (n = 4) that were injected via the tail vain with 0.74 MBq (20 µCi) per animal (100 µL). At 1, 4, and 24 h post-injection, mice were anesthetized with 1–2% isoflurane and sacrificed by cervical dislocation. Subsequently, the tissues of interest were harvested, weighed, and measured in a gamma counter. Samples were corrected for radioactive decay to calculate percent injected-dose per gram (%ID/g) of tissue.

3. Results and Discussion

3.1. Design of new chelators

The new chelators (Figure 1) were designed based on various factors including donor number (denticity), donor type, charge of the complex, compatibility between metal ion and donor group that can influence coordination chemistry of a metal. Cu(II) has a relatively small ionic radius of 73 ppm for coordination number 6 and is known to display a high affinity for nitrogen, oxygen, and sulfur donors.¹ A bifunctional version of the known parent NOTA chelator (A)¹⁶ was prepared and evaluated for comparison to the new NOTA analogues. A new bifunctional chelator B contains five donor groups and the p-NO₂-Bn group that is linked to the macrocyclic backbone by a relatively long propyl chain. Hexadentate chelators C and D contain a carbonyl donor group and a hydroxyl group in addition to the donor groups attached to the macrocyclic backbone, respectively. It would be interesting to evaluate if P-carbonyl or less flexible secondary hydroxyl group can efficiently cooperate with the other donor for rapid and tight complexation with Cu(II). The new hexadentate chelators E and F differ from NOTA wherein one of the aminocarboxylate donors is replaced with a pyridyl (E) or a thiophenyl (F) group. The hexadentate NOTA chelator A can form an anionic complex with Cu(II), while all new pentadentate and hexadentate chelators B-F are expected to form a neutral complex that would have an advantage of less protein interaction and a potentially more favorable in vivo tissue distribution over charged complexes.

3.2. Synthesis of new chelators and their Cu(II) complexes

Synthesis of chelators A and B is shown in Scheme 1. Bisubstituted 1,4,7-triazacyclononane (TACN) analogue 1¹² was reacted with an alkylating agent 2¹⁵ and 4¹¹ to provide the key precursor molecules 3 and 5, respectively. Removal of tert-butyl groups in 3 and 5 was accomplished by treatment of 3 and 5 with HCl (aq) to afford the respective bifunctional chelators A¹⁶ and B. Synthesis of chelators C and D is outlined in Scheme 2. Hydrolysis followed by decarboxylation of 6¹⁷ under acidic condition provided compound 7¹⁸ which was subjected to α-bromination using Br₂ to produce 8. Base-promoted reaction of 8 with 1¹² at room temperature for 24 h provided substitution product 9 which was further treated with TFA to provide chelator C. Compound 9 containing the carbonyl group was reduced to alcohol 10 using NaBH₄. tert-butyl groups in 10 was removed by treatment of 10 with TFA in CHCl₃ to furnish chelator D. The chelators E and F containing the heteroaromatic rings were synthesized as outlined in Scheme 3. Reductive amination of 2-pyridyl aldehyde and 2-thiophenyl aldehyde with bisubstituted TACN analogue 1¹² provided compounds 11 and 12, respectively. Compounds 11 and 12 were treated with 6M HCl (aq) and heated to reflux for 5 h to afford the desired chelators E and F, respectively.

Scheme 1.

Synthesis of TACN-Based Chalators A and B

Scheme 2.

Synthesis of Chelators C and D

Scheme 3.

Synthesis of TACN-Based Chalators E and F Containing a Heteroaromatic Ring

Cold Cu(II) complexes of the chelators A-F were prepared and characterized by HPLC. A solution of each chelator was reacted with CuCl₂ in an equal molar concentration at room temperature for 24 h to provide the corresponding Cu(II) complexes. The Cu(II) complexes were purified using semi-prep HPLC and characterized by analytical HPLC (Supporting Information). The Cu(II) complexes of chelator E and F containing pyridyl and thiophenyl ring were eluted earlier with +/− 2 min window (t_R = 4 min and t_R = 6 min, respectively) as compared to Cu(II) complexes of chelators A-D (t_R = 7~8 min).

3.3. Maximum specific activity

The new chelators were evaluated for radiolabeling with ⁶⁴Cu to determine the maximum specific activity. The specific activity was determined by titrating chelators with ⁶⁴Cu, and the result is shown in Figure 2. The chelators in different concentrations (0.0001 µg to 0.1 µg) were labeled with ⁶⁴Cu (0.1 M NH₄OAc, pH 5.5, 37 °C). All chelators studied bound to ⁶⁴Cu with high labeling efficiency (> 98%, 1 h). The respective maximum specific activity (Ci/µmol) of 4.87, 51.73, 27.45, 1.18, 14.69, and 1.82 was determined for chelators A-F. It is noteworthy that the pentadentate chelator B bound to ⁶⁴Cu with the highest maximum specific activity (51.73 Ci/µmol). A TACN analogue substituted with two N-carboxymethyl groups were known to effectively complex with Cu(II).³ Introduction of a functional linker to the TACN backbone appears to have little impact on complexation of the donor groups with Cu(II). The relatively lower maximum specific activity was observed with the hexadentate chelators D (1.18 Ci/µmol) and F (1.82 Ci/µmol) containing a hydroxyl group and a thiophenyl group. As compared to the known NOTA bifunctional chelator A, significantly higher specific activity was observed with chelators B, C, and E

Figure 2.

Radiolabeling (%) of chelators in different concentration (0.0001 to 0.1 µg chelator) with ⁶⁴Cu (100 µCi).

3.4. Radiolabeling reaction kinetics

The new chelators were evaluated for radiolabeling reaction kinetics with ⁶⁴Cu at room temperature (Table 1 and Supporting Information). Each chelator (6.7 µg) in 0.25M NH₄OAc (pH 5.5) was radiolabeled with ⁶⁴Cu (20 µCi) at room temperature. During the reaction time (30 min), the components were withdrawn at the designated time points (1 min, 10 min, and 30 min), and the radiolabeling efficiency (%) was determined using ITLC (20 mM EDTA in 0.15M NH₄OAc). The bifunctional NOTA chelator (A) was employed for comparison and displayed rapid complexation with ⁶⁴Cu as expected.¹⁰ All new chelators instantly bound to ⁶⁴Cu with excellent radiolabeling efficiency (>99%) at room temperature. Radiolabeling of the chelators with ⁶⁴Cu was nearly complete within 1 min as determined by ITLC. ⁶⁴Cu-EDTA migrated with the solvent front on TLC (R_f = 1.0), while ⁶⁴Cu-radiolabeled chelator complexes travel slower on the TLC (R_f = 0.6). The ⁶⁴Cu-radiolabeled complexes of the chelators and ⁶⁴Cu-EDTA were well separated on the ITLC. All ⁶⁴Cu-radiolabeled complexes were shown to be stable against EDTA present in the eluent of TLC.

Table 1.

Radiolabeling kinetics of chelators with ⁶⁴Cu (0.25M NH₄OAC, pH 5.5, RT)^#

Time (min)	Bound complex (%)
	A	B	C	D	E	F
1	99.8 ± 0.1	99.7 ± 0.1	99.3 ± 0.3	99.0 ± 0.0	99.9 ± 0.1	99.8 ± 0.2
10	99.8 ± 0.2	99.6 ± 0.4	99.2 ± 0.9	99.6 ± 0.2	99.9 ± 0.1	99.9 ± 0.2
30	99.6 ± 0.3	100 ± 0.0	99.5 ± 0.5	99.5 ± 0.4	99.9 ± 0.1	99.8 ± 0.2

^#Radiolabeling efficiency (mean ± standard deviation%) was measured in triplicate using ITLC (eluent: 20mM EDTA in 0.15M NH₄OAc).

In vitro serum stability

In vitro serum stability of the radiolabeled complexes was performed to determine if the chelators radiolabeled with ⁶⁴Cu remained stable without loss of the radioactivity in human serum. This was assessed by measuring the transfer of ⁶⁴Cu from the complex to human serum proteins using ITLC (20 mM EDTA in 0.15M NH₄OAc, Table 2 and Supporting Information). ⁶⁴Cu-radioabeled chelators were readily prepared from the reactions of the chelators with ⁶⁴Cu at room temperature. Essentially no unbound ⁶⁴Cu was detected in the reaction mixture at 2 h time point after the reaction at room temperature as determined by ITLC. The ⁶⁴Cu-radioabeled chelators were directly used for serum stability studies (pH 7, 37 °C) without further purification. All ⁶⁴Cu-radiolabeled complexes of chelator A-F remained intact in human serum for 2 days as evidenced by ITLC analysis (Supporting Information). ⁶⁴Cu-chelator C was found to be least stable in serum, and ~3% of ⁶⁴Cu was dissociated from the complex containing the carbonyl group over 2 days. No measurable radioactivity was released from ⁶⁴Cu-chelator E over 2 days. A tiny amount of ⁶⁴Cu (<0.3%) was detected from other ⁶⁴Cu-complexes of chelators B, D-F

Table 2.

Complex stability of ⁶⁴Cu-radiolabeled complexes in human serum (pH 7, 37 °C)^#

Time (day)	Bound complex (%)
	A	B	C	D	E	F
0	99.9 ± 0.0	99.9 ± 0.1	99.9 ± 0.1	100 ± 0.1	99.9 ± 0.0	99.9 ± 0.1
1	100.0 ± 0.0	100 ± 0.1	98.7 ± 0.5	100 ± 0.0	100.0 ± 0.0	99.9 ± 0.1
2	99.9 ± 0.1	99.7 ± 0.4	97.8 ± 1.1	99.9 ± 0.1	100.0 ± 0.0	99.9 ± 0.0

^#Bound complex (mean ± standard deviation%) was measured in triplicate using ITLC.

3.5. Stability of ⁶⁴Cu-radiolabeled complexes in EDTA solution

⁶⁴Cu-radiolabeled complexes were further evaluated for complex stability based on EDTA challenge. ⁶⁴Cu-radiolabeled complexes were freshly prepared and treated with a solution of EDTA at a 100-fold molar excess, and the resulting solution (pH 5.5) was incubated at 37 °C for 24 h. A sample was withdrawn at different time points (0 h, 1 h, 4 h, and 24 h) and analyzed using both ITLC and HPLC (Table 3 and Supporting Information). ⁶⁴Cu-radiolabeled complexes of chelators A and E remained intact against EDTA challenge, and a small portion of the activity (~5%) was transferred from the complexes to EDTA at 24 h time point (ITLC). Among the complexes tested, ⁶⁴Cu-radiolabeled pentadentate chelator B was least tolerant of EDTA treated, and most of ⁶⁴Cu was dissociated from the complex (~80%) at 24 h time point. ⁶⁴Cu-radiolabeled chelator C containing the carbonyl donor group was found dissociated rapidly in the presence of the excess EDTA, and 50% of the activity was transchelated by EDTA at 1 h time point. ⁶⁴Cu-radiolabeled chelators D and F with the respective hydroxyl and thiophenyl donor group were slower in dissociation than ⁶⁴Cu-radiolabeled chelators B and C and released >45% of ⁶⁴Cu at 24 h time point. Dissociation of the activity from the ⁶⁴Cu-radiolabeled chelators was also measured using radio-HPLC at 25 h time point (Figure 3). The peak related to ⁶⁴C u-EdTA (t_R = 2.5 min) was clearly separated from bound ⁶⁴Cu complex of the chelators. ⁶⁴Cu-radiolabeled complex of polar chelators E and F have the respective retention time at 4.8 min and 6.2 min, while other ⁶⁴Cu-radiolabeled complexes have similar retention time (t_R = 7~8 min). The ⁶⁴Cu-radiolabeled chelators C and D gave a small peak (t_R = ~3 min and t_R = ~7 min, respectively). It is speculated that the less stable complexes interact with mobile phase during HPLC to give the minor unbound peaks.

Table 3.

Stability of ⁶⁴Cu-radiolabeld complexes in EDTA solution (37 °C, pH 5.5)^#

Time (h)	Bound complex (%)
	A	B	C	D	E	F
0	99.9 ± 0.1	99.9 ± 0.1	97.7 ± 0.2	99.5 ± 0.3	99.7 ± 0.1	100 ± 0.1
1	98.6 ± 0.6	90.4 ± 0.1	50.4 ± 0.4	86.8 ± 0.7	98.2 ± 0.3	95.7 ± 0.5
4	96.8 ± 0.0	69.2 ± 0.3	45.8 ± 1.5	79.1 ± 0.4	92.6 ± 0.3	87.5 ± 0.4
19	95.3 ± 0.5	22.9 ± 2.1	41.0 ± 0.4	59.3 ± 0.0	93.6 ± 0.1	59.5 ± 0.6
24	94.9 ± 0.0	19.4 ± 1.1	39.0 ± 0.2	55.0 ± 0.6	94.3 ± 0.6	53.4 ± 1.7
25*	90.3 ± 0.3	11.9 ± 0.6	21.7 ± 1.3	41.0 ± 2.6	93.1 ± 0.1	42.3 ± 0.3

^#Bound complex (mean ± standard deviation%) was measured in duplicate using ITLC.

^*Bound complex (mean ± standard deviation%) was measured in duplicate using radio-HPLC.

Figure 3.

HPLC chromatograms of ⁶⁴Cu-radiolabeled complex against EDTA challenged for 25 h (37 °C, pH 5.5).

In summary, the in vitro complexation kinetic and stability data indicate that substitution of the N-carboxymethyl group in the NOTA chelating backbone with a different donor group including heteroaromatic ring, carbonyl group, or hydroxyl group gave no measurable effect on radiolabeling efficiency of the chelators and stability of the corresponding complexes in serum. It appears that the donor type in the chelators has little impact on complexation with Cu(II) since the chelators possess the adequate macrocyclic cavity and denticity for effective complexation of Cu(II). All new chelators were found to be highly effective in binding ⁶⁴Cu. It is noteworthy that pentadentate chelator B rapidly bound to ⁶⁴Cu and the complex remained intact in serum. However, when rigorously challenged by EDTA at a 100-fold molar excess, ⁶⁴Cu-radioabeled complexes produced different complex stability profiles. The bifunctional NOTA chelator (A) and pyridyl-containing chelator (E) were well tolerant of EDTA challenge, and only a small amount of the activity was transferred to EDTA. Other chelators B-D and F radiolabeled with ⁶⁴Cu were slowly or rapidly dissociated to produce ⁶⁴Cu transchelated to EDTA, although the complexes remained stable in human serum at 37 oC over 2 days. The results of EDTA challenge experiment indicate that among the new chelators B-F tested, the chelator E containing the pyridyl group was the best in forming a stable complex with ⁶⁴Cu that was comparable to the ⁶⁴Cu complex of the known chelator A. The pentadentate chelator B was less effective in tightly binding ⁶⁴Cu than other hexadentate chelators C - F.

3.6. Biodistribution of ⁶⁴Cu-radiolabeled complexes

⁶⁴Cu-radiolabeled complexes of chelators A and E which produced excellent in vitro complexation kinetic and stability data were further evaluated for a biodistribution study using non-tumor bearing mice (iv injection, n = 4). The ⁶⁴Cu-radiolabeled complexes were independently prepared from reaction of the chelators with ⁶⁴Cu at 37 °C for 1 h for in vivo biodistribution studies. ⁶⁴Cu–radiolabeled complexes were evaluated for in vivo stability by measuring radioactivity that accumulated in selected organs and cleared from the blood of mice at three time points, 1 h, 4 h, and 24 h post-injection (n = 4). The results of the biodistribution studies for ⁶⁴Cu-chelator A and ⁶⁴Cu-chelator E are shown in Figures 4 and 5, respectively. Both ⁶⁴Cu-chelator A and ⁶⁴Cu-chelator E displayed a rapid blood clearance and low uptake in the normal organs. ⁶⁴Cu-chelator A and ⁶⁴Cu-chelator E exhibited the highest radioactivity level in the blood at 1 h (0.13 ± 0.03 %ID/g and 0.25 ± 0.05 %ID/g, respectively) which decreased over the time points. The radioactivity accumulated in the blood at 24 h was negligible for both ⁶⁴Cu-chelator A (0.06 ± 0.00% ID/g) and ⁶⁴Cu-chelator E (0.10 ± 0.01% ID/g). The ⁶⁴Cu-radiolabeled complexes exhibited low uptake in the lung, muscle, bone, and pancreas (<0.57% ID/g) which peaked at 1 h and decreased at 24 h. A very low radioactivity level in the spleen over the time points was observed with ⁶⁴Cu-chelator A (~0.2% ID/g) and ⁶⁴Cu-chelator E (~0.1% ID/g). ⁶⁴Cu-chelator E displayed higher renal and liver uptake as compared to ⁶⁴Cu-chelator A over the course of the study. ⁶⁴Cu-chelator A exhibited the highest radioactivity level in the liver (2.20 ± 0.62% ID/g) and kidney (1.25 ± 0.35% ID/g) at 1 h that declined to (0.52 ± 0.07% ID/g) and (0.40 ± 0.04% ID/g) at 24 h. A higher radioactivity level at the liver and kidney relative to other normal organs was measured with ⁶⁴Cu-chelator E. Renal and liver uptake of ⁶⁴Cu-chelator E peaked at 1 h (3.20 ± 0.21% ID/g and 3.11 ± 0.39% ID/g, respectively) and decreased over the time points (0.67 ±0.12% ID/g and 0.98 ± 0.12% ID/g at 24 h, respectively). The in vivo biodistribution data suggest that ⁶⁴Cu-chelator E displayed favorable in vivo stability as evidenced by rapid blood clearance and low organ uptake and produced a biodistribution profile comparable to the ⁶⁴Cu complex of the known NOTA-based chelator A.

Figure 4.

Biodistribution of ⁶⁴Cu-radiolabeled chelator A in female CD-1 mice following iv injection.

Figure 5.

Biodistribution of ⁶⁴Cu-radiolabeled chelator E in female CD-1 mice following iv injection.

4. Conclusion

The novel pentadentate or hexadentate NOTA analogues with different donor groups were prepared and evaluated as chelators of ⁶⁴Cu. The radiolabeling efficiency data indicate that all new chelators instantly and almost completely bound to ⁶⁴Cu at room temperature. All chelators were efficiently radiolabeled with ⁶⁴Cu in a broad range of maximum specific activity. The corresponding ⁶⁴Cu-radiolabeled complexes remained intact in human serum for 2 days. No obvious effect of donor atom and denticity on complexation kinetics and serum stability with ⁶⁴Cu was observed with the chelators studied. However, the ⁶⁴Cu-radiolabeled chelators B-D and F when challenged by EDTA lost a significant amount of ⁶⁴Cu, while ⁶⁴Cu-radiolabeled complexes of two hexadentate chelators (A and E) were quite inert against the rigorous EDTA challenge and released a minimal amount of the activity for 24 h. The results of EDTA challenge experiments indicate that the pyridyl donor (E) in the chelating backbone is more effective in tightly holding ⁶⁴Cu than the thiophenyl, carbonyl, and hydroxyl group. The pentadentate chelator B was shown to form a thermodynamically less stable complex with ⁶⁴Cu and not tolerant of the EDTA challenge. The ⁶⁴Cu-chelator E containing a pyridyl group produced a biodistribution profile comparable to the known NOTA chelator A and remained stable in mice and displayed low radioactivity level in the blood and the normal organs. The results of the in vitro and in vivo evaluations indicate that the new chelator E is effective in binding ⁶⁴Cu and will be further modified for use in targeted PET imaging.