PRE-TARGETING IMAGING AGENTS

Abstract

The present invention provides a novel compound of the formula: methods of making this compound, methods of using this compound for pretargeted imaging, and preparations of such formulations.

##STR00001##

Claims

1. A compound of the formula: ##STR00006## or pharmaceutically acceptable salt thereof.

2. The compound according to claim 1, which is ##STR00007##

3. A compound of the formula: ##STR00008## or pharmaceutically acceptable salt thereof.

4. The compound according to claim 3, which is ##STR00009##

5. A pharmaceutical composition comprising a compound or a pharmaceutically acceptable salt thereof according to claim 1, with one or more pharmaceutically acceptable carriers, diluents or excipients.

6. A composition comprising: ##STR00010## or a pharmaceutically acceptable salt thereof, and ethanol and PBS buffer.

7. The composition of claim 6, wherein the composition does not include ascorbate or ascorbic acid.

8. A method of pretargeted imaging comprising: a. introducing into a mammal a detectable quantity of the compound: ##STR00011##  or a pharmaceutically acceptable salt thereof, and b. detecting said compound in PET imaging, wherein the compound is introduced after a biologics-TCO conjugate was introduced into the mammal.

9. A method of pretargeted imaging comprising: a. introducing into a mammal a detectable quantity of the composition of claim 5; and b. detecting the composition.

10. A pharmaceutical composition comprising a compound or a pharmaceutically acceptable salt thereof according to claim 3, with one or more pharmaceutically acceptable carriers, diluents or excipients.

11. A method of pretargeted imaging comprising: a. introducing into a mammal a detectable quantity of the composition of claim 6; and b. detecting the composition.

Description

EXAMPLE 1

N-(4-(1,2,4,5-tetrazin-3-yl)benzyl)-4-fluorobenzamide

[0038] ##STR00004##

[0039] To a stirred suspension of [4-(1,2,4,5-tetrazin-3-yl)phenyl]methanamine hydrochloride (90 mg, 0.4 mmol, commercially available from Click Chemistry Tools, CAS #1416711-59-5), Polymer Supported triethylamine (400 mg, 1.2 mmol) in dichloromethane (10 mL) is added dropwise a solution of 4-fluorobenzoyl chloride (70 mg, 0.44 mmol, commercially available from Sigma-Aldrich, CAS #403-43-0) in dichloromethane (2 mL). The reaction mixture is stirred at room temperature for 30 min, filtered, and concentrated under reduced pressure. The resulting residue is purified by chromatography on silica gel, eluting with a gradient of 0-60% dichloromethane/ethyl acetate, to afford the title compound as a purple solid (60 mg, 48% yield) after evaporation of the desired chromatographic fractions. .sup.1H NMR (400.13 MHz, DMSO-d.sub.6) δ ppm: 4.62 (d, J=6.0 Hz, 2H), 7.35-7.30 (m, 2H), 7.62 (d, J=8.7 Hz, 2H), 8.02-7.98 (m, 2H), 8.49-8.46 (m, 2H), 9.16 (t, J=6.0 Hz, 1H), 10.57 (s, 1H). .sup.19F NMR (376.45 MHz, DMSO-d.sub.6) δ ppm: −109.3. ES/MS m/z 310 (M+H).

EXAMPLE 2

[0040] [.sup.18F]—N-(4-(1,2,4,5-tetrazin-3-yl)benzyl)-4-fluorobenzamide

##STR00005##

[0041] A typical radiochemical yield of the title compound is 3% (using 0.7-3.7 Ci starting activity) with a total synthesis time of 120-130 min. The intermediate N-succinimidyl 4-[.sup.18F]fluorobenzoate is prepared according to literature methods (see Wüst, F., Hultsch, C., Bergmann, R., Johannsen, B., Henle, T., 2003; Radiolabelling of isopeptide N-ε-(γ-glutamyl)-L-lysine by conjugation with N-succinimidyl-4-[18F]fluorobenzoate. Appl. Radiat. Isot. 59, 43-48) adapted onto a TRACERlab FX.sub.F-N. [.sup.18F]fluorobenzoate (0.15-0.51 Ci) in acetonitrile (1 mL) is dried under a stream of nitrogen at 60° C. and to the resulting residue, a solution of [4-(1,2,4,5-tetrazin-3-yl)phenyl]methanamine hydrochloride (5 mg, 26.5 μmol) and triethylamine (29.1 mg, 287 μmol) in anhydrous DMF (1 mL) are added and the resulting solution is kept at room temperature for 15 min with occasional shaking. The crude reaction is diluted with water (2 mL) and purified by semi-preparative HPLC (Agilent ZORBAX Eclipse XDB-C18, 4×250 mm, 5 μm, 55% 20 mM NH.sub.4OAc in water/45% acetonitrile, Flow rate 4 mL/min, 280 nm). The fraction containing the purified title compound is diluted with water (40 mL) and reconstituted into a formulation of 10% (v/v) EtOH in 0.9% saline (10 mL) for subsequent use. The amount of specific radioactivity of the obtained title compound ranges from 0.015 to 0.044 Ci.

PET-CT imaging of [.sup.18F]—N-(4-(1,2,4,5-tetrazin-3-yl)benzyl)-4-fluorobenzamide

[0042] A Siemens Inveon® Multimodality Scanner (Siemens, Germany) is used for micro PET/CT imaging. Male CD-1 (6-week-old, ˜30 g) mice are anesthetized with 3% isoflurane/97% oxygen and placed on the bed of the scanner. The mice are administered [.sup.18F]—N-(4-(1,2,4,5-tetrazin-3-yl)benzyl)-4-fluorobenzamide via a bolus intravenous tail vein injection (˜300 Ci in a total volume of 200 μL saline). A total of six (four 60 min and two 90 min) dynamic PET scans are conducted, followed by a short high-resolution CT scan for anatomical registration. PET images are generated for each minute of the acquisition time. Uptake of the tracer in the brain, muscle, and bone are determined by visually drawing regions of interest (ROIs) based on the fused PET/CT images and the corresponding activity values are determined using the Inveon® Research Workplace software. All values are represented as % injected dose per gram (% ID/g).

[0043] Analysis of the four 60-minute PET scans indicate that [.sup.18F]—N-(4-(1,2,4,5-tetrazin-3-yl)benzyl)-4-fluorobenzamide readily crosses the blood-brain-barrier. Peak brain uptake of 4.1% ID/g is observed at 2.5 min post injection followed by a steady clearance of tracer to 0.8% ID/g at 59.5 min. Uptake of [.sup.1′F]—N-(4-(1,2,4,5-tetrazin-3-yl)benzyl)-4-fluorobenzamide is also observed throughout the body in organs such as the liver, kidneys and heart. Bone uptake throughout the scan period remained low, consistent with an absence of defluorination for this tracer. Results of the 90 min scans are similar to the 60 min scan results with peak brain uptake observed at 3.4% ID/g at 2.5 min post injection followed by an immediate wash out to 0.6% ID/g by 59.5 min. Residual uptake is also observed throughout the body of the mouse. Time activity curves are generated for [.sup.18F]—N-(4-(1,2,4,5-tetrazin-3-yl)benzyl)-4-fluorobenzamide in CD-1 male mice (n=4). Data is shown in graphical format in FIGS. 1A and 1B and in tabular format below.

[0044] FIG. 1A is a graph of 60 minute PET time activity curves for [.sup.18F]—N-(4-(1,2,4,5-tetrazin-3-yl)benzyl)-4-fluorobenzamide (Brain, Muscle, & Bone, N=4)

[0045] FIG. 1B is a graph of 90 minute PET time activity curves for [.sup.18F]—N-(4-(1,2,4,5-tetrazin-3-yl)benzyl)-4-fluorobenzamide (Brain, Muscle, & Bone, N=2)

TABLE-US-00001 TABLE 1A 60 minute PET time activity table for [.sup.18F]-N-(4-(1,2,4,5-tetrazin-3- yl)benzyl)-4-fluorobenzamide (Brain, Muscle, & Bone) Time Brain Muscle Bone (min) Mean SEM N Mean SEM N Mean SEM N 0 0 0 4 0 0 4 0 0 4 0.5 0.32 0.32 4 0.02 0.02 4 0.08 0.08 4 1.5 3.67 0.37 4 0.84 0.24 4 0.97 0.12 4 2.5 4.06 0.28 4 1.14 0.20 4 1.28 0.17 4 3.5 3.70 0.25 4 1.13 0.21 4 1.09 0.16 4 4.5 3.36 0.21 4 1.15 0.22 4 1.04 0.13 4 5.5 3.20 0.18 4 1.17 0.21 4 1.01 0.17 4 6.5 3.01 0.18 4 1.10 0.21 4 1.07 0.17 4 7.5 2.81 0.17 4 1.11 0.20 4 1.12 0.18 4 8.5 2.62 0.15 4 1.15 0.19 4 1.04 0.19 4 9.5 2.46 0.14 4 1.09 0.19 4 0.97 0.14 4 10.5 2.32 0.14 4 1.09 0.16 4 1.03 0.12 4 11.5 2.17 0.12 4 1.08 0.17 4 1.01 0.14 4 12.5 2.07 0.13 4 1.06 0.17 4 0.93 0.19 4 13.5 1.97 0.13 4 1.06 0.15 4 0.96 0.11 4 14.5 1.88 0.12 4 1.00 0.13 4 0.78 0.13 4 15.5 1.79 0.12 4 1.02 0.14 4 0.89 0.11 4 16.5 1.70 0.11 4 1.02 0.15 4 0.96 0.10 4 17.5 1.64 0.11 4 1.03 0.15 4 0.87 0.09 4 18.5 1.58 0.11 4 1.05 0.14 4 0.92 0.12 4 19.5 1.52 0.11 4 0.94 0.12 4 0.93 0.10 4 20.5 1.47 0.11 4 0.95 0.11 4 1.01 0.13 4 21.5 1.42 0.10 4 1.06 0.14 4 0.88 0.12 4 22.5 1.38 0.11 4 1.04 0.15 4 0.99 0.11 4 23.5 1.33 0.10 4 0.92 0.13 4 0.85 0.16 4 24.5 1.29 0.10 4 0.96 0.13 4 0.88 0.10 4 25.5 1.26 0.10 4 0.97 0.15 4 1.02 0.08 4 26.5 1.23 0.10 4 0.92 0.12 4 0.88 0.08 4 27.5 1.18 0.10 4 0.89 0.13 4 0.83 0.20 4 28.5 1.17 0.10 4 0.91 0.12 4 0.92 0.15 4 29.5 1.14 0.10 4 0.94 0.12 4 0.93 0.07 4 30.5 1.11 0.10 4 0.93 0.12 4 0.92 0.05 4 31.5 1.09 0.09 4 0.99 0.14 4 0.96 0.09 4 32.5 1.07 0.10 4 0.89 0.12 4 0.85 0.16 4 33.5 1.04 0.09 4 0.93 0.12 4 0.95 0.07 4 34.5 1.04 0.10 4 0.92 0.11 4 0.87 0.10 4 35.5 1.03 0.10 4 0.91 0.16 4 0.84 0.07 4 36.5 1.00 0.10 4 0.93 0.14 4 0.84 0.11 4 37.5 0.98 0.09 4 0.91 0.15 4 0.90 0.09 4 38.5 0.97 0.09 4 0.93 0.15 4 0.87 0.13 4 39.5 0.97 0.10 4 0.97 0.14 4 0.85 0.11 4 40.5 0.95 0.09 4 0.93 0.14 4 0.89 0.12 4 41.5 0.93 0.09 4 0.89 0.14 4 0.93 0.14 4 42.5 0.93 0.09 4 0.95 0.17 4 0.88 0.10 4 43.5 0.91 0.09 4 0.88 0.11 4 0.75 0.14 4 44.5 0.90 0.09 4 0.91 0.12 4 0.87 0.10 4 45.5 0.89 0.09 4 0.95 0.16 4 0.86 0.07 4 46.5 0.87 0.09 4 0.92 0.13 4 0.79 0.09 4 47.5 0.87 0.09 4 0.95 0.15 4 0.87 0.15 4 48.5 0.86 0.09 4 0.86 0.12 4 0.86 0.05 4 49.5 0.86 0.09 4 0.99 0.17 4 0.87 0.12 4 50.5 0.85 0.09 4 0.91 0.13 4 0.90 0.11 4 51.5 0.85 0.09 4 0.88 0.12 4 0.92 0.06 4 52.5 0.84 0.08 4 0.81 0.10 4 0.85 0.12 4 53.5 0.85 0.09 4 0.96 0.13 4 0.85 0.05 4 54.5 0.84 0.09 4 0.91 0.09 4 0.80 0.11 4 55.5 0.83 0.09 4 0.87 0.10 4 0.85 0.13 4 56.5 0.82 0.09 4 0.91 0.11 4 0.93 0.13 4 57.5 0.82 0.09 4 0.82 0.14 4 0.84 0.13 4 58.5 0.81 0.09 4 0.83 0.11 4 0.89 0.10 4 59.5 0.82 0.09 4 0.88 0.12 4 0.88 0.12 4

TABLE-US-00002 TABLE 1B 90-minute PET time activity table for [.sup.18F]-N-(4-(1,2,4,5-tetrazin-3- yl)benzyl)-4-fluorobenzamide (Brain, Muscle, & Bone, % ID/g ± SEM) Time Brain Muscle Bone (min) Mean SEM N Mean SEM N Mean SEM N 0 0 0 2 0 0 2 0 0 2 0.5 0 0 2 0 0 2 0 0 2 1.5 2.02 0.66 2 0.29 0.05 2 0.48 0.10 2 2.5 3.41 0.74 2 0.63 0.02 2 0.90 0.29 2 3.5 3.06 0.66 2 0.66 0.05 2 0.90 0.15 2 4.5 2.76 0.58 2 0.63 0.08 2 0.90 0.30 2 5.5 2.50 0.50 2 0.60 0.13 2 0.87 0.02 2 6.5 2.38 0.49 2 0.68 0.09 2 0.97 0.30 2 7.5 2.19 0.44 2 0.62 0.13 2 0.94 0.29 2 8.5 2.02 0.40 2 0.68 0.17 2 0.93 0.17 2 9.5 1.87 0.37 2 0.71 0.17 2 1.01 0.35 2 10.5 1.74 0.34 2 0.65 0.12 2 0.70 0.38 2 11.5 1.63 0.33 2 0.66 0.19 2 0.82 0.28 2 12.5 1.55 0.32 2 0.67 0.14 2 0.84 0.17 2 13.5 1.44 0.29 2 0.65 0.14 2 0.83 0.45 2 14.5 1.36 0.27 2 0.64 0.19 2 0.63 0.21 2 15.5 1.29 0.27 2 0.64 0.21 2 0.62 0.36 2 16.5 1.23 0.25 2 0.60 0.20 2 0.71 0.42 2 17.5 1.16 0.24 2 0.62 0.20 2 0.51 0.19 2 18.5 1.11 0.23 2 0.63 0.22 2 0.71 0.15 2 19.5 1.04 0.21 2 0.63 0.22 2 0.74 0.34 2 20.5 1.02 0.22 2 0.66 0.21 2 0.63 0.36 2 21.5 0.97 0.20 2 0.61 0.13 2 0.70 0.35 2 22.5 0.95 0.22 2 0.61 0.21 2 0.69 0.34 2 23.5 0.91 0.20 2 0.65 0.18 2 0.78 0.33 2 24.5 0.88 0.20 2 0.64 0.25 2 0.73 0.34 2 25.5 0.85 0.19 2 0.62 0.19 2 0.95 0.49 2 26.5 0.83 0.18 2 0.58 0.21 2 0.64 0.28 2 27.5 0.82 0.19 2 0.60 0.21 2 0.69 0.20 2 28.5 0.81 0.19 2 0.62 0.21 2 0.60 0.21 2 29.5 0.78 0.18 2 0.63 0.23 2 0.76 0.42 2 30.5 0.76 0.18 2 0.64 0.24 2 0.76 0.39 2 31.5 0.75 0.18 2 0.66 0.19 2 0.60 0.35 2 32.5 0.74 0.20 2 0.63 0.24 2 0.70 0.41 2 33.5 0.72 0.18 2 0.62 0.25 2 0.67 0.29 2 34.5 0.70 0.17 2 0.60 0.24 2 0.74 0.48 2 35.5 0.70 0.17 2 0.65 0.25 2 0.69 0.24 2 36.5 0.70 0.18 2 0.65 0.25 2 0.79 0.43 2 37.5 0.68 0.17 2 0.59 0.27 2 0.62 0.40 2 38.5 0.68 0.17 2 0.63 0.24 2 0.87 0.16 2 39.5 0.66 0.17 2 0.61 0.23 2 0.60 0.29 2 40.5 0.66 0.17 2 0.61 0.27 2 0.82 0.44 2 41.5 0.66 0.17 2 0.59 0.25 2 0.77 0.40 2 42.5 0.65 0.18 2 0.63 0.20 2 0.61 0.23 2 43.5 0.64 0.17 2 0.64 0.22 2 0.65 0.44 2 44.5 0.64 0.17 2 0.66 0.20 2 0.61 0.39 2 45.5 0.64 0.17 2 0.63 0.20 2 0.66 0.30 2 46.5 0.64 0.17 2 0.64 0.22 2 0.67 0.32 2 47.5 0.64 0.17 2 0.65 0.27 2 0.69 0.43 2 48.5 0.62 0.16 2 0.61 0.25 2 0.76 0.42 2 49.5 0.62 0.16 2 0.62 0.20 2 0.91 0.40 2 50.5 0.61 0.16 2 0.60 0.22 2 0.58 0.28 2 51.5 0.63 0.17 2 0.59 0.20 2 0.70 0.43 2 52.5 0.63 0.16 2 0.65 0.18 2 0.51 0.27 2 53.5 0.62 0.17 2 0.64 0.24 2 0.68 0.27 2 54.5 0.63 0.16 2 0.69 0.27 2 0.55 0.28 2 55.5 0.61 0.16 2 0.63 0.20 2 0.68 0.42 2 56.5 0.62 0.17 2 0.67 0.22 2 0.65 0.44 2 57.5 0.63 0.17 2 0.63 0.29 2 0.62 0.32 2 58.5 0.63 0.17 2 0.67 0.25 2 0.67 0.31 2 59.5 0.62 0.16 2 0.63 0.17 2 0.58 0.25 2 60.5 0.62 0.16 2 0.63 0.24 2 0.70 0.35 2 61.5 0.62 0.17 2 0.65 0.22 2 0.76 0.26 2 62.5 0.62 0.16 2 0.65 0.24 2 0.79 0.41 2 63.5 0.62 0.17 2 0.69 0.28 2 0.79 0.27 2 64.5 0.62 0.17 2 0.57 0.18 2 0.65 0.20 2 65.5 0.62 0.17 2 0.63 0.21 2 0.66 0.12 2 66.5 0.62 0.17 2 0.69 0.30 2 0.79 0.50 2 67.5 0.63 0.18 2 0.70 0.26 2 0.89 0.47 2 68.5 0.64 0.17 2 0.64 0.21 2 0.71 0.51 2 69.5 0.64 0.17 2 0.63 0.24 2 0.68 0.25 2 70.5 0.63 0.17 2 0.61 0.28 2 0.53 0.13 2 71.5 0.63 0.17 2 0.67 0.20 2 0.82 0.36 2 72.5 0.64 0.18 2 0.65 0.18 2 0.74 0.23 2 73.5 0.64 0.17 2 0.68 0.26 2 0.73 0.26 2 74.5 0.65 0.17 2 0.68 0.25 2 0.72 0.25 2 75.5 0.66 0.18 2 0.63 0.25 2 0.67 0.20 2 76.5 0.65 0.18 2 0.68 0.18 2 0.58 0.24 2 77.5 0.65 0.18 2 0.66 0.21 2 0.63 0.28 2 78.5 0.64 0.17 2 0.63 0.16 2 0.71 0.44 2 79.5 0.65 0.17 2 0.69 0.15 2 0.51 0.27 2 80.5 0.65 0.17 2 0.66 0.19 2 0.70 0.29 2 81.5 0.66 0.17 2 0.66 0.21 2 0.56 0.09 2 82.5 0.65 0.17 2 0.68 0.21 2 0.76 0.53 2 83.5 0.65 0.18 2 0.68 0.23 2 0.77 0.33 2 84.5 0.66 0.17 2 0.70 0.21 2 0.70 0.22 2 85.5 0.65 0.17 2 0.67 0.17 2 0.57 0.12 2 86.5 0.66 0.18 2 0.68 0.26 2 0.59 0.37 2 87.5 0.66 0.16 2 0.71 0.22 2 0.76 0.24 2 88.5 0.66 0.17 2 0.66 0.24 2 0.81 0.38 2 89.4 0.66 0.17 2 0.70 0.23 2 0.76 0.26 2