Synthesis and Chemical Analysis

The NOTA-like starting material was purchased from Macrocyclics (Plano, TX). HPLC-grade acetonitrile and all other chemical reagents were purchased from Sigma Aldrich (St. Louis, MO). NMR spectra were recorded on a Bruker (Karlsruhe, Germany) NMR spectrometer (500 and 600 MHz for 1H, 126 and 150 MHz for 13C). 1H-NMR chemical shifts were measured relative to tetramethylsilane (TMS). The organic compounds were purified using column chromatography on silica gel. The final compounds (tricarboxylic acid) were purified using an Agilent 1220 HPLC system (Santa Clara, CA). The solvent systems were used: solvent A (0.1% TFA in water) and solvent B (0.1% TFA in acetonitrile). The flow rates were 1 mL/min for analytical HPLC (4.6 mm diameter column) and 4 mL/min for semipreparative HPLC (9.2 mm diameter column) at the indicated method. Electro-spray ionization mass spectra (ESI–MS) were acquired using a Waters (Milford, MA) ESI ion trap spectrometer using positive and negative ion detection.

Synthetic Procedures

Tert-butyl 2-(4-ethoxyphenyl)acetate (2) To a solution of 2-(4-ethoxyphenyl)acetic acid (2 g), in dichloromethane (DCM) added tBuOH (1.2 equiv) and DMAP was dissolved at 0 ºC. The reaction was allowed to come to room temperature and stirred overnight. After completion of reaction monitored with TLC, the reaction mixture was filtered, and the filtrate was workup with H2O and extracted with DCM solvent. The organic layer (DCM) was evaporated using vacuum and purified with column chromatography using silica gel. The product yield 92%. 1H NMR (500 MHz, CDCl3) δ 7.17 (d, J = 8.6 Hz, 2H), 6.84 (d, J = 8.7 Hz, 2H), 4.01 (q, J = 7.0 Hz, 2H), 3.45 (s, 2H), 1.43 (s, 9H), 1.40 (t, J = 7.0 Hz, 3H). 13C NMR (126 MHz, CDCl3) δ 171.32, 157.85, 130.17, 126.66, 114.43, 80.63, 63.38, 41.73, 28.04, 14.86.

tert-butyl 2-bromo- 2-(4-ethoxyphenyl)acetate (3) To a solution of 2 in CCl4 (20 mL) added NBS (1.5 equiv.) and AIBN (0.1 equiv.) the reaction was stirred at 80 ºC for 3–4 h. After completion of reaction monitored with TLC, the reaction mixture transferred into room temperature and quenched with H2O. After workup with H2O and DCM solvent the organic layer was evaporated using rotavapor and purified with column chromatography using silica gel. 1H NMR (500 MHz, CDCl3) δ 7.46 (d, J = 8.8 Hz, 2H), 6.86 (d, J = 8.8 Hz, 2H), 5.24 (s, 1H), 4.03 (q, J = 7.0 Hz, 2H), 1.46 (s, 9H), 1.42 (t, J = 6.8 Hz, 3H). 13C NMR (126 MHz, CDCl3) δ 167.39, 159.50, 130.03, 129.25, 128.06, 114.59, 82.90, 63.51, 48.40, 27.74, 14.77.

di-tert-butyl 2,2'-(7-(2-(tert-butoxy)-1-(4-ethoxyphenyl)-2-oxoethyl)-1,4,7-triazonane-1,4-diyl)diacetate (5) To a solution of di-tert-butyl 2,2'-(1,4,7-triazonane-1,4-diyl)diacetate (4) and 3 in CH3CN (20 mL) solvent added K2CO3 (5 equiv.). The reaction was allowed at 60 ºC for 3–5 h. after completion of reaction monitored with TLC the solvent was evaporated using rotavapor and workup done with ethyl acetate and H2O. The organic layer was concentrated using rotavapor. The yellow liquid product was obtained with a yield of 62%. 1H NMR (500 MHz, CDCl3) δ 7.29 (d, J = 8.7 Hz, 2H), 6.82 (d, J = 8.7 Hz, 2H), 4.35 (s, 1H), 4.01 (q, J = 7.0 Hz, 2H), 3.27 (d, J = 3.2 Hz, 4H), 3.01 (d, J = 11.4 Hz, 2H), 2.89–2.74 (m, 10H), 1.44 (s, 9H), 1.43 (s, 18H), 1.40 (t, J = 7.0 Hz, 3H). 13C NMR (126 MHz, CDCl3) δ 172.41, 171.51, 158.29, 130.07, 130.00, 114.07, 81.00, 80.59, 72.24, 63.31, 59.50, 55.38, 55.03, 53.40, 28.19, 28.14, 14.85. HRMS calculated for C32H54N3O7, 592.3962 [M + H +], found: 592.4023 [M + H +]. Enantiomeric resolution of 5a and 5b, A CHIRALPAK IC column (9.2 × 250 mm, 5 μ) was used for the resolution, solvent was hexane/isopropanol (96/4), with a flow rate of 4 mL/min. 5a had a retention time of 13.4 min and 5b had a retention time of 18.1 min, respectively. The collected compounds were dried under vacuum with rotavapor. The optical rotation of 5a is [a] D20 = + 0.8 deg·mL·g−1·dm−1 and that of 5b is [a] D20 =—0.7 deg·mL·g−1·dm−1.

2,2'-(7-(carboxy(4-ethoxyphenyl)methyl)-1,4,7-triazonane-1,4-diyl)diacetic acid (6) To a solution of 5, 5a or 5b, added 4 M HCl in 1,4-dioxane (10 equiv). The reaction was allowed at room temperature for 3–5 h, monitored with HPLC. After completion of reaction, the product was purified using a C18 HPLC column (mobile phase water and acetonitrile). The compounds 6, 6a and 6b were concentrated using a lyophilizer. Yield was 30% ± 15%. 1H NMR (600 MHz, DMSO) δ 7.29 (d, J = 8.7 Hz, 2H), 6.90 (d, J = 8.7 Hz, 2H), 4.79 (s, 1H), 4.08–3.94 (m, 4H), 3.88 (d, J = 17.7 Hz, 2H), 3.75 (s, 3H), 3.42 (d, J = 6.0 Hz, 2H), 3.10 (td, J = 13.9, 6.7 Hz, 3H), 2.98 (dt, J = 31.0, 6.8 Hz, 5H), 2.84–2.74 (m, 3H), 1.32 (t, J = 6.9 Hz, 3H). 13C NMR (150 MHz, DMSO) δ 173.6, 171.1, 170.7, 158.7, 130.9, 128.1, 117.9, 116.0, 114.7, 67.4, 63.5, 55.1, 54.4, 51.0, 50.5, 49.3, 48.5, 47.3, 15.0. HRMS calculated for C20H30N3O7, 424.2078, [M + H+], found: 424.2133 [M + H+].

Radiochemistry

[64Cu]Cu2+ (130–160 MBq) was provided in 0.1 M HCl. After HCl was evaporated at 120 ºC, [64Cu]Cu2+ was dissolved in 50 µL of 1.0 M acetate buffer (pH 5.0–5.5). Then 6a or 6b (5 µg (0.011 µmol)/5 µL of water) was added and incubated for 30 min at 37 ºC with constant shaking on an Eppendorf Thermomixer. Labeling yield was found to be 92% and 94% for 6a and 6b, respectively, determined by radiochemical thin layer chromatography (radio-TLC). TLC analyses were performed with silica plates (Silica gel 60 mesh, Merck, Darmstadt, Germany) with 1 M aqueous ammonium acetate – methanol (1:1) as the developing solvent. [64Cu]Cu-6a and [64Cu]Cu-6b were purified using PD- 10 columns with phosphate-buffered saline (PBS) as the mobile phase. The radioactive fractions containing [64Cu]Cu-6a and [64Cu]Cu-6b were collected and passed through a 0.2 µm syringe filter for in vivo experiments. Higher than 90% yield (decay-corrected) was achieved after purification. The radiochemical purities of [64Cu]Cu-6a and [64Cu]Cu-6b were determined by RP-HPLC and TLC being higher than 99%. Reversed-phase HPLC (RPHPLC) analyses were performed with a C18 column eluted with a linear gradient of a 10–90% mixture of acetonitrile and 0.1% aqueous TFA. Specific activity of the [64Cu]Cu-6a and [64Cu]Cu-6b were 13.2 ± 4.6 GBq/µmol (n = 4). The stability of [64Cu]Cu-6a and [64Cu]Cu-6b was assessed by sitting the final dose in PBS (pH 7.4) at room temperature for 1, 3, 6 and 18 h, the samples were analyzed by HPLC to measure the amount of free 64Cu dissociated from [64Cu]Cu-6a and [64Cu]Cu-6b.

In Vivo PET/MRI

Images were acquired on a Bruker BioSpec 70/30 7T MRI with a PET insert for simultaneous PET/MRI, using Paravision 360v3.2 and v3.5. Mice were either wild-type (WT) mice (FVB, n = 2, ages 40 weeks, BALB/c, n = 4, ages 60 weeks, all female), or liver humanized mice (n = 6, ages 22–40 weeks, 4 female, 2 male) (used under license from Taconic). DCE-MRI was acquired using a 40 mm volume transmit/receive coil using a retrospectively respiration-gated sequence, IgFLASH with: TR/TE 80/2.1 ms, 6 coronal slices with 0.25 × 0.2x1 mm resolution, 6 (WT) or 8 oversamples (liver humanized), flip angle 20°, temporal resolution 1–1.25 min. PET images were corrected for decay and scatter and reconstructed using a maximum-a-posteriori (MAP) algorithm at 0.5 mm voxel size, with 12, 5-min bins. We injected each mouse with 0.025 mmol/kg of Gd-EOB-DTPA mixed with ~ 75 ± 31 µCi (0.094 ± 0.039 µg) of [64Cu]Cu-EOB-NOTA through a tail vein catheter after 5–6 min of baseline scans. To demonstrate the difference between transporter-mediated uptake and blood-pool uptake, one liver humanized mouse was scanned in a 72 mm transmit/receive coil with a gradient echo T1 weighted FLASH sequence to allow for whole body MRI, with: TR/TE 12.5/2 ms, 1 coronal slice with 0.5 × 0.5x2 mm voxel size, 30° flip angle, 60 averages, 1 min 15 s temporal resolution. Additionally, one liver humanized mouse was scanned in the 40 mm volume coil as described above, with co-injection of [64Cu]Cu-EOB-NOTA (A) and 0.025 mmol/kg Gd-DO3A-butrol (Gadobutrol), a clinically approved blood pool contrast agent.

Image Analysis

All images were segmented in PMOD 4.2. For DCE-MRI images, circular ROIs with 1.5–2 mm radius were placed in the liver and the signal over time was measured. The data was then exported to Microsoft Excel where the percent enhancement was calculated (S(t)-S(0–5))/S(0–5)* 100. The total injected dose was measured from the whole-body PET image as the maximum total activity in the first 3 frames of the dynamic scan. ROIs from the DCE-MRI measurements were transferred to the PET images co-registered to those scans to measure the TAC, which was then expressed as % injected dose/mL.

Analysis of Waste Clearance

4 mice (FVB enantiomer A or B, liver humanized mice enantiomer A or B) were singly housed in metabolic cages (Tecniplast, Italy) immediately after their PET/MRI scan. Feces and urine were collected after ~ 20 h and activity was measured in a dose calibrator (CRC- 55 tR, Capintec).

In Vitro Cellular Transport Assays

For in vitro transport assays, HEK293T cells stably overexpressing mouse OATP1B2 or human OATP1B3 were used. To each well of a 6-well plate, 1 × 104 cells were seeded and incubated at 37 °C and 5% CO2 overnight, one plate for each cell line. The next day, 20 μCi of [64Cu]Cu-EOB-NOTA -A or -B was added to each well (N = 3 for each cell line) and incubated at 37°C and 5% CO2 for 2 h at gentle shaking. Next, the cell culture supernatant was removed, and cells were washed thrice with PBS. To lyse the cell monolayer, 1 mL of 1 M NaOH was added and incubated for 10 min rocking at 700 rpm. The whole cell lysates were collected, and activity was counted on a Gamma Counter (Wizard2, Perkin Elmer). Protein concentration was measured using a NanoDrop spectrophotometer (Thermo Scientific) at an A280 setting (280 nm wavelength). The total activity for each cell lysate was normalized with the protein concentration to calculate activity per microgram of protein.