Pretargeted imaging agents

Abstract

The present invention provides novel compounds, methods of preparing such compounds, methods of using such compounds for pretargeted imaging, and preparation of formulations for such uses.

Description

[0001] The present invention relates to the compound N-(4-(1,2,4,5-tetrazin-3-yl)benzyl)-2-fluoronicotinamide and the compound [ 18 F]-radiolabeled forms of these compounds, pharmaceutically acceptable salts of these compounds, intermediates for preparing these compounds, methods for pretargeted imaging using these compounds, compositions and formulations of these compounds for diagnostic imaging (such as pretargeted imaging), and methods for pretargeted imaging using these compounds, compositions and formulations.

[0002] The present invention also relates to the compound N-(4-(1,2,4,5-tetrazin-3-yl)benzyl)-6-fluoropicolinamide and the compound [ 18 F]-radiolabeled forms of these compounds, pharmaceutically acceptable salts of these compounds, intermediates for preparing these compounds, methods for pretargeted imaging using these compounds, compositions and formulations of these compounds for diagnostic imaging (such as pretargeted imaging), and methods for pretargeted imaging using these compounds, compositions and formulations.

[0003] In the past, PET imaging using macromolecules has been achieved by direct labeling of full-length antibodies. Antibodies have exquisite specificity and selectivity, but are generally hampered by their slow clearance and poor brain penetration. Imaging with antibodies utilizes long-lived radionuclides, wherein imaging is performed 7-10 days after injection of the radioimmunoconjugate to remove nonspecific background signals. This timeline is not easily incorporated into clinical practice, and patients are exposed to elevated radioactivity levels. In order to minimize interference with normal clinical practice and radioactive exposure to patients, an imaging system based on pretargeting has been developed. This pretargeting approach is a two-step method based on a biorthogonal inverse electron demand Diels-Alder (IEDDA) reaction between tetrazine and trans-cyclooctene (TCO) derivatives, which utilizes the specificity and selectivity of macromolecules and the rapid pharmacokinetics of small molecules with short-lived radionuclides. For peripheral targets, many preclinical examples of pretargeted imaging have been described in the literature (see J. Med. Chem. 2017, 60, 8201-8217 and J. Label Compd. Radiopharm 2014, 57285-290.).

[0004] For antibody-based CNS imaging agents, the blood-brain barrier (BBB) ​​presents an additional challenge. Professor and colleagues (Uppsala University) demonstrated that transferrin receptor (TfR)-mediated transport across the blood-brain barrier improves brain uptake of a bispecific antibody targeting Aβ fibrils. 124Subsequent PET imaging studies with I-labeled antibodies showed differential distribution between transgenic and wild-type mice. The distribution patterns in various brain regions correlated well with Aβ pathology, see Stina et al. Theranostics, 2017; 7(2): 308-318. See also S, Fang XT, R, Rokka J, Lannfelt L, Olberg DE, Eriksson J, Sehlin D. Fluorine-18-Labeled Antibody Ligands for PETImaging of Amyloid-β in Brain. ACS Chem. Neurosci. 2020, 11, 4460-4468.

[0005] In addition to brain-penetrating macromolecules, another prerequisite for any successful CNS pretargeting imaging study is the availability of brain-penetrating, rapidly cleared, reactive yet stable small molecule chasers containing reactive tetrazine groups. 11 C- and 18 F-labeled small molecule tetrazine tracers have been reported to have significant brain uptake (see Hannes Mikula et al. Bioconjugate Chem. 2016, 27, 7, 1707-1712; Hannes Mikula et al. Angew. Chem. Int. Ed. 2014, 53, 9655-9659). However, their application in CNS pretargeting imaging studies has not been reported.

[0006] In 2019, Brendon Cook and collaborators reported the first case of CNS pretargeting imaging to study the distribution of antisense oligonucleotides (ASOs) in the rat brain (2019 World Molecular Imaging Congress Conference, Poster 139). Rats were intrathecally administered 2.5 mM ASO-TCO conjugate in 30 μL saline solution, followed by intravenous injection of CNS-penetrating tetrazine 24 hours and 168 hours after ASO-TCO administration. 18 F]537-Tz. 18 Static PET-CT scans were performed 75-90 minutes after F537-Tz administration. Higher radiotracer uptake was observed in the brain and spine of animals receiving ASO-TCO relative to controls. Dynamic PET imaging has also been reported, 18F]537-Tz showed brain uptake in wild-type mice (1.7 ± 0.9% ID at 10 min post-injection). 18 F]537-Tz compound is believed to be 2-(4-(1,2,4,5-tetrazin-3-yl)phenyl)-N-(2-fluoroethyl)acetamide:

[0007]

[0008] In contrast, the [ 18 F] N-(4-(1,2,4,5-tetrazin-3-yl)benzyl)-2-fluoronicotinamide and [ 18 Dynamic PET imaging of [F]N-(4-(1,2,4,5-tetrazin-3-yl)benzyl)-6-fluoropicolinamide in CD-1 male mice showed that these compounds readily crossed the blood-brain barrier and reached peak brain uptake of 3.3±0.4% ID / g and 4.3±0.3% ID / g, respectively. These compounds subsequently showed steady clearance from the brain, clearing to near background levels (muscle) by 60 minutes post-injection. These agents with robust brain penetration followed by rapid and complete elution from the brain provide a larger window to achieve higher signal-to-background ratios, resulting in better image quality. Therefore, we expect that the compounds disclosed herein will provide advantages for pre-targeted CNS imaging.

[0009] The present embodiments provide novel compounds, compositions, formulations, and methods for pretargeted imaging. There is a need for improved technologies of this type that enhance patient imaging capabilities to expand the clinical benefits and impact of diagnostic imaging. The improved imaging agents will provide enhanced pretargeted images compared to currently known agents.

[0010] This embodiment also provides the compound N-(4-(1,2,4,5-tetrazin-3-yl)benzyl)-2-fluoronicotinamide, also referred to herein as "Compound 1," which can be structurally represented as a compound of Formula I:

[0011]

[0012] The compounds of formula I are shown above as free bases. The compounds of formula I can also be converted into pharmaceutically acceptable salts and used in this embodiment as such.

[0013] This embodiment provides compound: Compound 1 18 The F-form, which is also referred to herein as "Compound 2," can be structurally represented as a compound of Formula II:

[0014]

[0015] The compound of formula II is shown above as the free base. The compound of formula II can also be converted into a pharmaceutically acceptable salt and used in this embodiment as a salt.

[0016] This embodiment provides the use of a pharmaceutically acceptable salt of Formula I or Formula II or the use of said compound in free base form.

[0017] This embodiment further provides the use of a compound of Formula I and / or a compound of Formula II and / or a mixture thereof for preparing an imaging agent, such as a pretargeted imaging agent.

[0018] This embodiment provides the use of a compound of formula I or II for the preparation of a radiopharmaceutical for imaging in humans (pretargeted imaging).In another aspect, the present invention provides a process for the preparation of a compound of formula I or II.

[0019] In another aspect, the present embodiment provides a pharmaceutical composition comprising Compound 1 or Compound 2 or a pharmaceutically acceptable salt thereof formulated in ethanol (e.g., 10% EtOH (v / v)) and a buffer (which may be a PBS buffer), preferably for use in humans. It should also be noted that in some embodiments, the formulation does not contain ascorbate or ascorbic acid, as it has been found that the tetrazine moiety in Formula I and Formula II can be readily reduced by ascorbate formulations. Therefore, although ascorbate formulations are readily used in many imaging formulations, they may not be suitable for imaging using compounds of Formula I or Formula II.

[0020] The present invention also provides a pretargeted imaging method, which comprises introducing a detectable amount of Compound 1 or 2 or a pharmaceutically acceptable salt thereof or a composition thereof into a patient.

[0021] This embodiment also provides N-(4-(1,2,4,5-tetrazin-3-yl)benzyl)-6-fluoropicolinamide, also referred to herein as "Compound 3," which can be structurally represented as a compound of Formula III:

[0022]

[0023] The compound of formula III is shown as a free base. The compound of formula III can also be converted into a pharmaceutically acceptable salt and used in this embodiment as a salt.

[0024] This embodiment provides compound: Compound 3 18 The F-form, which is also referred to herein as "Compound 4," can be structurally represented as a compound of Formula IV:

[0025]

[0026] The compound of formula IV is shown as a free base. The compound of formula IV can also be converted into a pharmaceutically acceptable salt and used in this embodiment as a salt.

[0027] This embodiment provides the use of a pharmaceutically acceptable salt of Formula III or IV or the use of said compound in free base form.

[0028] This embodiment further provides the use of a compound of formula III and / or a compound of formula IV and / or a mixture thereof for preparing an imaging agent, such as a pretargeted imaging agent.

[0029] This embodiment provides the use of a compound of formula III or IV for the preparation of a radiopharmaceutical for imaging in humans (pretargeted imaging).In another aspect, the present invention provides a process for preparing a compound of formula III or IV.

[0030] In another aspect, the present embodiment provides a pharmaceutical composition comprising compound 3 or compound 4 or a pharmaceutically acceptable salt thereof formulated in ethanol (e.g., 10% EtOH (v / v)) and a buffer (which may be a PBS buffer), preferably for use in humans. It should also be noted that in some embodiments, the formulation does not contain ascorbate or ascorbic acid, as it has been found that the tetrazine moiety in Formula III and Formula IV can be readily reduced by ascorbate formulations. Therefore, although ascorbate formulations are readily used in many imaging formulations, they may not be suitable for imaging using compounds of Formula III or Formula IV.

[0031] The present invention also provides a pretargeted imaging method, which comprises introducing a detectable amount of Compound 3 or 4 or a pharmaceutically acceptable salt thereof or a composition thereof into a patient.

[0032] This embodiment provides a compound of formula V or a pharmaceutically acceptable salt thereof, which can be structurally represented as follows:

[0033]

[0034] Among them, X can be CF, C- 18 F or N

[0035] Y can be CF, C- 18 F or N

[0036] where one (but not both) of X and Y is N,

[0037] When X is N, Y is CF or C- 18 F;

[0038] and wherein when Y is N, X is CF or C- 18 F.

[0039] Those skilled in the art will recognize that Formula V represents a genus encompassing the above-mentioned compounds 1, 2, 3 and 4 (and pharmaceutically acceptable salts of these compounds). Therefore, the above disclosures regarding the embodiments of compounds 1, 2, 3 and 4 (or Formulas I-IV) and their related uses, compositions, formulations, etc., also apply to the compounds of Formula V.

[0040] This embodiment provides the use of a pharmaceutically acceptable salt of Formula V or the use of said compound in free base form.

[0041] This embodiment further provides the use of a compound of formula V and / or a mixture thereof for preparing an imaging agent, such as a pretargeted imaging agent.

[0042] This embodiment provides the use of a compound of formula V for the preparation of a radiopharmaceutical for imaging in humans (pretargeted imaging). In another aspect, the present invention provides a process for preparing a compound of formula V.

[0043] In another aspect, the present embodiment provides a pharmaceutical composition comprising a compound of Formula V or a pharmaceutically acceptable salt thereof formulated in ethanol (e.g., 10% EtOH (v / v)) and a buffer (which may be PBS buffer), preferably for use in humans. It should also be noted that in some embodiments, the formulation does not contain ascorbate or ascorbic acid, as it has been found that the tetrazine moiety in Formula V can be readily reduced by ascorbate formulations. Therefore, while ascorbate formulations are readily used in many imaging formulations, they may not be suitable for imaging using compounds of Formula V.

[0044] The present invention also provides a pretargeted imaging method comprising introducing a detectable amount of a compound of Formula V or a pharmaceutically acceptable salt thereof or a composition thereof into a patient.

[0045] The following preparations and examples are provided to better illustrate the practice of the present invention. Suitable reaction conditions for the steps of these schemes, preparations and examples are well known in the art, and appropriate modifications of the reaction conditions, including replacement of solvents and co-reagents, are within the capabilities of the skilled artisan.

[0046] In addition, the technical staff will recognize that, in some cases, the order of introduction of structural parts (moieties) is unimportant. As skilled chemists fully understand, the specific order of the required step of the compound of preparation formula I or formula II or formula III or formula IV depends on the relative instability (liability) of the specific compound, starting compound and substituted part of synthesis. These compounds can be protected or modified by methods well known in the art at a convenient point in the synthesis. If necessary, intermediates of the present invention and final products can be further purified by conventional techniques, as recrystallization or by the chromatography on solid supports such as silica gel or aluminum oxide.

[0047] The compounds of the present invention are preferably formulated as radiopharmaceutical compositions for administration by various routes. Preferably, such compositions are for intravenous use, preferably in humans. Such pharmaceutical compositions and methods for their preparation are well known in the art. See, for example, Remington: The Science and Practice of Pharmacy (PP Gerbino, 21st ed., Lippincott Williams & Wilkins, 2006).

[0048] A preferred formulation may be a formulation of compound 1 or compound 2. A preferred formulation may be a formulation of compound 3 or compound 4. Particularly preferred are compound 1 or compound 2 or compound 3 or compound 4 prepared according to the procedures described herein. Preferred formulations of compound 1 or compound 2 are formulated in ethanol, such as 10% EtOH (v / v). Such formulations may also include a buffer, such as PBS buffer. Other ingredients may also be used. Preferred formulations of compound 3 or compound 4 are formulated in ethanol, such as 10% EtOH (v / v). Such formulations may also include a buffer, such as PBS buffer. Other ingredients may also be used.

[0049] It has been found that compounds of Formula I and II and III and IV are surprisingly and unexpectedly useful for pretargeted imaging, preferably including human clinical imaging. In some embodiments, compounds of Formula I, II, III, IV or V can be used for pretargeted imaging. For example, compounds of Formula IV can be brain penetrants and therefore can be used as tracers for CNS (central nervous system) pretargeted imaging. In some embodiments, pretargeted imaging of CNS targets can be achieved in 3 or 4 steps:

[0050] 1. IV (intravenous) or IT (intrathecal) administration of a biologic-TCO conjugate (e.g., a shuttled bispecific antibody-TCO conjugate or an oligonucleotide-TCO conjugate);

[0051] 2. Waiting sufficient time (days) to allow for distribution and systemic clearance of the biologic-TCO conjugate;

[0052] 3. An optional step of intravenously injecting periphery-restricted tetrazines to mask the peripherally circulating biologic-TCO conjugate;

[0053] 4. Intravenous administration of a brain-penetrant compound of Formula I or Formula II or Formula III or Formula IV or Formula V followed by brain PET imaging.

[0054] Regarding the preparation of biologic-TCO conjugates, the use of this technology is disclosed in the literature (see below) and is known for use in oncology pretargeting imaging studies:

[0055] 1. Bioconjugate Chem. 2018, 29, 538-545 (TCO antibody conjugation; masking of circulating antibody-TCO using scavengers)

[0056] 2. Bioconjugate Chem. 2013, 24, 1210-1217 (TCO antibody conjugation)

[0057] J.Med.Chem.2017, 60, 8201-8217 (TCO antibody conjugation, pretargeting radioligand optimization)

[0058] For oligonucleotide-TCO conjugation, this type of conjugate (TCO-PEG4 Oligo Modification) is commercially available from Bio-Synthesis, Inc., Lewisville, Texas, USA. Thus, one skilled in the art will recognize how to achieve the preparation of biologic-TCO conjugates.

[0059] Some potential benefits of pretargeted imaging include the ability to use short-lived radionuclides by separating the presentation of radioactivity from the targeting vehicle, such as biologics, with high specificity and selectivity, and the ability to reduce patient exposure to radioactivity.

[0060] Figure 1 yes[ 18 Representative semi-preparative HPLC chromatogram of the purification of [F] N-(4-(1,2,4,5-tetrazin-3-yl)benzyl)-2-fluoronicotinamide;

[0061] Figure 2 It is formulated 18 Representative analytical HPLC chromatogram of F] N-(4-(1,2,4,5-tetrazin-3-yl)benzyl)-2-fluoronicotinamide;

[0062] Figure 3 yes[ 18 Representative semi-preparative HPLC chromatogram of the purification of F] N-(4-(1,2,4,5-tetrazin-3-yl)benzyl)-6-fluoropicolinamide;

[0063] Figure 4 It is formulated 18 Representative analytical HPLC chromatogram of F] N-(4-(1,2,4,5-tetrazin-3-yl)benzyl)-6-fluoropicolinamide;

[0064] Figure 5 yes[ 18 Representative semi-preparative HPLC chromatogram of the purification of [F] N-(4-(1,2,4,5-tetrazin-3-yl)benzyl)-6-fluoronicotinamide;

[0065] Figure 6 It is formulated 18 Representative analytical HPLC chromatogram of [F] N-(4-(1,2,4,5-tetrazin-3-yl)benzyl)-6-fluoronicotinamide;

[0066] Figure 7 Representative ESI mass spectra of the reaction of Malat1 ASO-TCO with tetrazine; and

[0067] Figure 8 .Tetrazine[ 18 F] N-(4-(1,2,4,5-tetrazin-3-yl)benzyl)-2-fluoropicolinamide, [ 18 Representative radiotracing of [F] N-(4-(1,2,4,5-tetrazin-3-yl)benzyl)-6-fluoropicolinamide and reaction with Malat1 ASO-TCO.

[0068] Certain abbreviations may be used below. The meanings of these abbreviations are as follows: "CAS#" refers to Chemical Abstracts Registry Number; "Ci" refers to Curie; "CT" refers to computed tomography; "δ" refers to chemical shift in nuclear magnetic resonance spectroscopy; "DMF" refers to N,N-dimethylformamide; "DMSO" refers to dimethyl sulfoxide; "DMSO-d6" refers to deuterated DMSO; "ES / MS" refers to electrospray mass spectrometry; "EtOH" refers to ethanol or ethyl alcohol; "HATU" refers to 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridine 3-oxide hexafluorophosphate; “HPLC” refers to high-performance liquid chromatography; “h” and “hr” refer to hours; “min” refers to minutes; “%ID / g” refers to % injected dose / gram; “mCi” refers to millicuries; “MHz” refers to megahertz; “μL” refers to microliter; “N” refers to the number of replicates or sample size in an experiment; “NMR” refers to nuclear magnetic resonance; “PET” refers to positron emission tomography; “OAc” refers to acetate; “ppm” refers to parts per million; “s” refers to singlet; “SEM” refers to standard error of the mean; “t R ” refers to retention time; “THF” refers to tetrahydrofuran.

[0069] General Chemistry and Preparation

[0070] The following preparations and examples further illustrate the present invention and represent typical syntheses of compounds of the present invention. Reagents and starting materials are readily available or can be readily synthesized by one of ordinary skill in the art. It should be understood that the preparations and examples are provided by way of example and not limitation, and various modifications may be made by one of ordinary skill in the art.

[0071] Used for 1 H and 19 F spectrum NMR spectroscopy at Bruker Avance TM III HD 400 MHz NMR spectrometer, obtained as CDCl3 or DMSO-d6 solutions reported in ppm, using the residual solvent resonance (CDCl3, 7.26 ppm; DMSO-d6, 2.50 ppm) as 1 H NMR reference standards. 19 F spectra, no reference standard was used. When reporting peak multiplicities, the following abbreviations may be used: s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), br s (broad singlet), dd (double of doublets), dt (double of triplets). When coupling constants (J) are reported, they are reported in Hertz (Hz).

[0072] ES / MS The UPLC system was connected to the Electrospray mass spectrometry measurements were performed on an Acquity QDa mass detector (acquired in positive and / or negative mode). Unless otherwise stated in the experimental section below, LC-MS conditions were as follows. Column: Waters Acquity BEH 2.1 × 30 mm, 1.7 μm; wavelength 250–650 nm. One of two gradients was used: Gradient 1: initial hold at 10% B for 0.5 min, 10–98% B over 3.5 min, hold at 98% B for 0.5 min, return to 10% B for 0.6 min; Gradient 2: initial hold at 10% B for 0.5 min, 10–98% B over 1.5 min, hold at 98% B for 0.5 min, return to 10% B to re-equilibrate; column temperature: 40°C ± 10°C; flow rate: 0.5 mL / min; solvent A: deionized water containing 0.1% HCOOH; solvent B: 100% acetonitrile.

[0073] Of course, other instruments and tools for detection and for ES / MS are known in the art and will be known to the skilled artisan.

[0074] The preparative and analytical HPLC conditions, when used, are detailed below.

[0075] Example 1

[0076] N-(4-(1,2,4,5-tetrazin-3-yl)benzyl)-2-fluoronicotinamide

[0077]

[0078] To a stirred solution of [4-(1,2,4,5-tetrazin-3-yl)phenyl]methanamine hydrochloride (115 mg, 0.51 mmol, ClickChemistry Tools, CAS #1416711-59-5) and 2-fluoropyridine-3-carboxylic acid (90 mg, 0.64 mmol) in dichloromethane (6 mL) was added HATU (260 mg, 0.67 mmol) and N,N-diisopropylethylamine (0.2 mL, 1.0 mmol). The reaction mixture was stirred at room temperature overnight. The reaction mixture was diluted with dichloromethane, and the organic layer was washed sequentially with saturated aqueous NaHCO3 solution, saturated aqueous NaCl solution, dried over Na2SO4, filtered, and concentrated under reduced pressure. The resulting residue was purified by column chromatography on silica gel eluting with a gradient of 0-50% dichloromethane / ethyl acetate to give the title compound (150 mg, 64% yield, containing hexafluorophosphate) as a purple solid after evaporation of the solvent of the desired chromatographic fraction. The title compound was further purified by dissolving in 20 mL of dichloromethane and washing with 3 x 0.5 M aqueous potassium acetate (75 mg, 0.16 mmol). The resulting organic layer was dried over Na2SO4, filtered and concentrated under reduced pressure to provide the purified title compound (51 mg) as a purple solid. 1 H NMR (400.13MHz, DMSO-d6) δppm: 4.63 (d, J=6.0Hz, 2H), 7.51-7.47 (m, 1H), 7.65 (d, J=8.6Hz, 2 H), 8.26-8.22(m, 1H), 8.36-8.39(m, 1H), 8.51-8.48(m, 2H), 9.17-9.12(m, 1H), 10.58(s, 1H). 19 F NMR (376.45MHz, DMSO-d6) δppm: -67.6. ES / MS (m / z): 311 (M+H).

[0079] Preparation 1

[0080] 2-[2-(3,5-Dimethoxyphenyl)-4-methyl-phenyl]thiopyridine-3-carboxylic acid

[0081]

[0082] A solution of methyl 2-chloropyridine-3-carboxylate (380 mg, 2.2 mmol) and 2-ethylhexyl 3-[2-(3,5-dimethoxyphenyl)-4-methyl-phenyl]thiopropionic acid (900 mg, 2.0 mmol), tris(dibenzylideneacetone)dipalladium(0) (180 mg, 0.20 mmol) and 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene (230 mg, 0.40 mmol) in 1,4-dioxane (8 mL) was sparged with N for 5 minutes, and a 1 M solution of potassium tert-butoxide in THF (2.20 mL, 2.20 mmol) was added. The reaction mixture was sealed and heated to 100° C. overnight. The reaction mixture was cooled to room temperature and a 1N NaOH aqueous solution (6 mL) was added. The resulting mixture was stirred at room temperature for 2-3 hours, diluted with water (30 mL) and extracted with ethyl acetate (2x30 mL). The aqueous layer was neutralized to pH ~ 1 with 1N HCl aqueous solution and extracted with ethyl acetate (2x30 mL). The organic layers were combined, washed with saturated NaCl aqueous solution, dried over Na2SO4, filtered and concentrated under reduced pressure. The resulting residue was purified by column chromatography on silica gel, eluting with a gradient of 0-100% hexane / ethyl acetate to provide the title compound (270 mg, 33% yield) as a yellow solid. ES / MS (m / z): 382 [M+H].

[0083] Preparation 2

[0084] 2-[2-(3,5-dimethoxyphenyl)-4-methyl-phenyl]sulfinyl-N-[[4-(1,2,4,5-tetrazin-3-yl)phenyl]methyl]pyridine-3-carboxamide

[0085]

[0086] To a stirred mixture of 2-[2-(3,5-dimethoxyphenyl)-4-methyl-phenyl]thiopyridine-3-carboxylic acid (100 mg, 0.26 mmol), [4-(1,2,4,5-tetrazin-3-yl)phenyl]methanamine hydrochloride (59 mg, 0.26 mmol, Click Chemistry Tools, CAS #1416711-59-5), and HATU (122 mg, 0.41 mmol) in dichloromethane (2.6 mL) was added N,N-diisopropylethylamine (120 μL, 0.67 mmol). The reaction mixture was stirred at room temperature overnight. Volatiles were removed under reduced pressure. The resulting residue was dissolved in ethyl acetate (50 mL) and washed sequentially with 0.5 M aqueous KHCO (2 x 30 mL) and saturated aqueous NaCl (30 mL). The organic extract was dried over NaSO, filtered, and concentrated under reduced pressure. The resulting residue was purified by chromatography on silica gel (0-100% hexanes / ethyl acetate) to afford 2-[2-(3,5-dimethoxyphenyl)-4-methyl-phenyl]sulfanyl-N-[[4-(1,2,4,5-tetrazin-3-yl)phenyl]methyl]pyridine-3-carboxamide (121 mg, 84% yield) as a pink foam.

[0087] To a stirred solution of 2-[2-(3,5-dimethoxyphenyl)-4-methyl-phenyl]sulfanyl-N-[[4-(1,2,4,5-tetrazin-3-yl)phenyl]methyl]pyridine-3-carboxamide (121 mg, 0.22 mmol) in dichloromethane (2.5 mL) was added 3-chloroperoxybenzoic acid (77% pure, 36 mg, 0.16 mmol). The mixture was stirred at room temperature for 30 minutes and concentrated under reduced pressure. The resulting residue was purified by chromatography on silica gel eluting with a gradient of 0-100% hexanes / ethyl acetate to provide 2-[2-(3,5-dimethoxyphenyl)-4-methyl-phenyl]sulfinyl-N-[[4-(1,2,4,5-tetrazin-3-yl)phenyl]methyl]pyridine-3-carboxamide (91 mg, 0.16 mmol, 73% yield) as a pink foam: 1 H NMR (400.13MHz, DMSO-d6) δppm: 2.39 (s, 3H), 3.69 (s, 6H), 4.60-4.42 (m, 2H), 6.50-6.46 (m, 3H), 7.19 (d, J=1.1Hz, 1H), 7. 36-7.33 (m, 1H), 7.59-7.56 (m, 5H), 8.10-8.07 (m, 1H), 8.43 (d, J=8.5, 2H), 8.69-8.67 (m, 1H), 9.08 (t, 1H), 10.56 (s, 1H). ES / MS(m / z): 567(M+H).

[0088] Preparation 3

[0089] 2-(2,4-dimethoxy-8-methyl-dibenzothiophene-5- -5-yl)-N-[[4-(1,2,4,5-tetrazin-3-yl)phenyl]methyl]pyridine-3-carboxamide trifluoromethanesulfonate

[0090]

[0091] To a stirred slurry of 2-[2-(3,5-dimethoxyphenyl)-4-methyl-phenyl]sulfinyl-N-[[4-(1,2,4,5-tetrazine-3-yl)phenyl]methyl]pyridine-3-carboxamide (25 mg, 0.04 mmol) and diethylaminopolystyrene (3.2 mmol / g, 100 mg, 0.32 mmol) in dichloromethane (3 mL) cooled to 0 ° C., a 1 M solution of trifluoromethanesulfonic anhydride in dichloromethane (150 μL, 0.15 mmol) was added. The reaction mixture was warmed to room temperature, stirred for 30 minutes, and the reaction was quenched by adding water droplets. The mixture was filtered and the solid was washed with dichloromethane. The combined filtrate was concentrated under reduced pressure, and the resulting residue was purified by chromatography on silica gel using a gradient of 0-15% methanol / dichloromethane to provide the title compound (10 mg, 0.014 mmol, 32% yield) as a purple solid: 1 H NMR (400.13MHz, DMSO-d6) δppm: 2.46 (s, 3H), 3.86 (s, 3H), 4.03 (s, 3H), 4.94-4. 81 (m, 2H), 6.43 (d, J = 2.1Hz, 1H), 7.45-7.42 (m, 1H), 7.67 (d, J = 2.1Hz, 1H), 7.80 (d, 8.5Hz, 2H), 7.91-7.88 (m, 1H), 8.18 (d, J=8.4Hz, 1H), 8.28 (s, 1H), 8.65 (d, J =8.4Hz, 2H), 8.60-8.59(m, 1H), 8.73-8.70(m, 1H), 10.30(t, 1H), 10.61(s, 1H). 19 F NMR (376.45MHz, DMSO-d6) δppm: -77.7.ES / MS (m / z): 549 (M + ).

[0092] Example 2

[0093] [ 18 F] N-(4-(1,2,4,5-tetrazin-3-yl)benzyl)-2-fluoronicotinamide

[0094]

[0095] Typical radiochemical yields for the radiosynthesis of the title compound were 3.6 ± 1.34% (N = 3) using 0.435 to 1.442 Ci starting activities and a synthesis time of 50 minutes.

[0096] [ 18 F] fluoride activity (0.435 to 1.442 Ci) was retained on a Sep-Pak Accell Plus QMA Plus Light Cartridge (130 mg, 37-55 μm, Waters Part # WAT023525; preconditioned with 5 mL of water for injection) and eluted onto a TRACERlab FX using 0.8 mL of an aqueous solution of tetraethylammonium bicarbonate [tetraethylammonium bicarbonate (3.5 mg) in water (0.2 mL) and acetonitrile (0.6 mL)]. F-N The eluted active was heated to 70 °C and dried under compressed nitrogen purge and vacuum for 5 minutes. The temperature was then raised to 100 °C and maintained under vacuum for 5 minutes to obtain [ 18 F] Tetraethylammonium fluoride. The reactor was cooled to 35°C and 2-(2,4-dimethoxy-8-methyl-dibenzothiophene-5- A solution of 4-[[4-(1,2,4,5-tetrazin-3-yl)phenyl]methyl]pyridine-3-carboxamide trifluoromethanesulfonate (1 mg, 1.43 μmol) in anhydrous DMSO (1 mL) was prepared. The temperature was raised to 40° C., and the reaction mixture was maintained at 40° C. for 5 minutes. The mixture was then diluted with 3.5 mL of 0.1% (v / v) trifluoroacetic acid / water and the resulting crude reaction mixture was loaded onto a semi-preparative HPLC column for purification (conditions listed in Figure 1 ). The purified [ 18 HPLC fractions of N-(4-(1,2,4,5-tetrazin-3-yl)benzyl)-2-fluoronicotinamide (F] Figure 1 ) were collected into a vial containing 40 mL of water and loaded into the Sep- Light1cc Vac C18 column (50 mg, 55-105 μm, Waters Part # WAT054955; pre-conditioned with 5 mL EtOH and 5 mL water in sequence). 18 F]N-(4-(1,2,4,5-tetrazin-3-yl)benzyl)-2-fluoronicotinamide was eluted with 0.5 mL of EtOH and reconstituted into 5 mL of 10% (v / v) EtOH / phosphate buffered saline. 18F]N-(4-(1,2,4,5-tetrazin-3-yl)benzyl)-2-fluoronicotinamide in an amount ranging from 0.014 to 0.033 Ci. 18 Representative analytical HPLC of [F]N-(4-(1,2,4,5-tetrazin-3-yl)benzyl)-2-fluoronicotinamide showed Figure 2 middle.

[0097] Example 3

[0098] N-(4-(1,2,4,5-tetrazin-3-yl)benzyl)-6-fluoropicolinamide

[0099]

[0100] To a mixture of [4-(1,2,4,5-tetrazine-3-yl)phenyl]methylamine hydrochloride (60 mg, 0.27 mmol, Click Chemistry Tools, CAS#1416711-59-5) and 6-fluoropyridine-2-carboxylic acid (45 mg, 0.32 mmol) in dichloromethane (4 mL) was added N, N-diisopropylethylamine (150 μL, 0.86 mmol) followed by HATU (135 mg, 0.35 mmol), and the reaction mixture was stirred at room temperature overnight. The resulting pink slurry was distributed between dichloromethane and 1 M aqueous citric acid solution. The layers were separated and the aqueous phase was extracted with dichloromethane (3x10 mL). The combined organic extracts were dried over sodium sulfate, filtered, and concentrated onto silica gel under reduced pressure. The resulting residue was purified by chromatography on silica gel using dichloromethane / ethyl acetate as an eluent gradient elution to obtain the crude title compound contaminated with fluorophosphate. The crude product was dissolved in dichloromethane (10 mL) and washed with 0.5 M aqueous potassium acetate (2 x 10 mL). The organic extract was dried over sodium sulfate, filtered and concentrated to afford the pure title compound as a magenta solid (64 mg, 77% yield): 1 H NMR (400.13MHz, DMSO-d6) δppm: 4.62 (d, J=6.2Hz, 2H), 7.45-7.43 (m, 1H), 7.61 (d, J=8.8H z, 2H), 8.02-7.99 (m, 1H), 8.20 (q, 1H), 8.46 (d, J=8.5Hz, 2H), 9.39 (t, 1H), 10.56 (s, 1H). 19 F NMR (376.45MHz, DMSO-d6) δppm: -68.0. ES / MS (m / z): 311 (M+H).

[0101] Preparation 4

[0102] 6-[2-(3,5-Dimethoxyphenyl)-4-methyl-phenyl]thiopyridine-2-carboxylic acid

[0103]

[0104] To a solution of tris(dibenzylideneacetone)dipalladium(0) (312 mg, 0.33 mmol) and bis(2-diphenylphosphinophenyl) ether (365 mg, 0.66 mmol) in N,N-dimethylacetamide (15 mL) was added methyl 6-bromopyridine-2-carboxylate (1.71 g, 8.0 mmol), 2-ethylhexyl 3-[2-(3,5-dimethoxyphenyl)-4-methyl-phenyl]thiopropanoate (3.0 g, 6.6 mmol) and potassium tert-butoxide (2.3 g, 19.8 mmol) in N,N-dimethylacetamide (20 mL). The mixture was stirred at 120° C. under N2 for 6 hours and combined with a smaller-scale reaction using 2-ethylhexyl 3-[2-(3,5-dimethoxyphenyl)-4-methyl-phenyl]thiopropanoate (100 mg, 0.22 mmol) and methyl 6-bromopyridine-2-carboxylate (58 mg, 0.27 mmol) in N,N-dimethylacetamide. The reaction mixture was treated with 2M aqueous NaOH solution (10 mL) and stirred at 40° C. for 1 hour. The mixture was diluted with water (70 mL), extracted with EtOAc (30 mL), and the organic layer was discarded. The aqueous phase was adjusted to pH~3 by adding 1N HCl aqueous solution. The acidified mixture was extracted with EtOAc (2x30 mL). The combined organic extracts were dried over Na25O4 and concentrated under reduced pressure. The resulting residue was purified by preparative HPLC (column: YMC-Triart C18, 250x50mm, 7μm) and eluted with a gradient of 40%-80% acetonitrile / water containing 0.225% formic acid over 25 minutes. The desired fractions were lyophilized to give the title compound (1.49 g, 53% yield) as a yellow solid. ES / MS (m / z): 382 [M+H]. ES / MS conditions: LC (low pH): ES / MS was performed on a Shimadzu LCMS 2020 liquid chromatography system. Electrospray mass spectrometry (acquired in positive mode) was performed on a Scan Mode quadrupole mass spectrometer connected to the LC system. LC-MS column: C182.1x30mm, 3μm, gradient: 10-80% B in 3min, 80% B hold for 0.5min, column temperature: 50℃, flow rate: 1.2mL / min, solvent A: deionized water containing 0.037% formic acid, solvent B: acetonitrile containing 0.018% formic acid, wavelength UV 220nm and 254nm.

[0105] Preparation 5

[0106] 6-[2-(3,5-dimethoxyphenyl)-4-methyl-phenyl]sulfinyl-N-[[4-(1,2,4,5-tetrazin-3-yl)phenyl]methyl]pyridine-2-carboxamide

[0107]

[0108] To a stirred solution of 6-[2-(3,5-dimethoxyphenyl)-4-methyl-phenyl]thiopicolinic acid (150 mg, 0.39 mmol) and [4-(1,2,4,5-tetrazin-3-yl)phenyl]methanamine hydrochloride (90 mg, 0.40 mmol, Click Chemistry Tools, CAS #1416711-59-5) in dichloromethane (4 mL) was added HATU (190 mg, 0.49 mmol) and N,N-diisopropylethylamine (0.14 mL, 0.80 mmol). The mixture was stirred at room temperature overnight. The reaction mixture was concentrated to partial volume under reduced pressure, diluted with 30 mL of ethyl acetate, and washed sequentially with 0.5 M aqueous KHCO (twice) and saturated aqueous NaCl (once), dried over NaSO, filtered, and concentrated under reduced pressure. The resulting residue was purified by column chromatography on silica gel eluting with a gradient of 0-90% ethyl acetate / hexanes to provide 6-[2-(3,5-dimethoxyphenyl)-4-methyl-phenyl]sulfanyl-N-[[4-(1,2,4,5-tetrazin-3-yl)phenyl]methyl]pyridine-2-carboxamide (180 mg, 85% yield) as a purple foamy solid. ES / MS (m / z): 551 (M+H).

[0109] To a stirred solution of 6-[2-(3,5-dimethoxyphenyl)-4-methyl-phenyl]sulfanyl-N-[[4-(1,2,4,5-tetrazin-3-yl)phenyl]methyl]pyridine-2-carboxamide (180 mg, 0.33 mmol) in dichloromethane (10 mL) was added 3-chloroperoxybenzoic acid (77% pure, 73 mg, 0.33 mmol). The resulting mixture was stirred at room temperature for 2-3 hours. The mixture was purified by column chromatography on silica gel eluting with a gradient of 0-100% ethyl acetate / hexanes to provide the title compound (167 mg, 99% yield) as a purple foamy solid. ES / MS (m / z): 567 (M+H).

[0110] Preparation 6

[0111] 6-(2,4-dimethoxy-8-methyl-dibenzothiophene-5- -5-yl)-N-[[4-(1,2,4,5-tetrazin-3-yl)phenyl]methyl]pyridine-2-carboxamide trifluoromethanesulfonate

[0112]

[0113] To a stirred suspension of 6-[2-(3,5-dimethoxyphenyl)-4-methyl-phenyl]sulfinyl-N-[[4-(1,2,4,5-tetrazine-3-yl)phenyl]methyl]pyridine-2-carboxamide (88 mg, 0.16 mmol) and polymer-supported triethylamine (0.4 g, 1 mmol, 3.2 mmol / g) in dichloromethane (8 mL) was added a 1 M solution of trifluoromethanesulfonic anhydride (0.42 mL, 0.42 mmol) in dichloromethane at 0 ° C. The mixture was stirred at room temperature for 30 minutes and quenched with water droplets. The resulting suspension was filtered, washed with dichloromethane, and concentrated under reduced pressure. The resulting residue was purified by column chromatography on silica gel, eluting with a gradient of 0-15% methanol / dichloromethane to provide the title compound (91 mg, 81% yield) as a purple solid. 1 H NMR (400MHz, DMSO-d6) δppm: 2.49 (s, 3H), 4.01-4.00 (m, 6H), 4.75-4.63 (m , 2H), 5.75 (s, 1H), 7.01 (d, J=2.1Hz, 1H), 7.62-7.59 (m, 3H), 7.76-7.72 (m, 2H), 8.23 ​​(t, J=7.8Hz, 1H), 8.30 (dd, J=0.9, 7.8Hz, 1H), 8.37-8.34 (m, 1H), 8.44 (d, J=8.3Hz, 1H), 8.55-8.52 (m, 2H), 9.12-9.08 (m, 1H), 10.61 (s, 1H). 19 F NMR (376MHz, DMSO-d6) δppm: -77.7.ES / MS (m / z): 549 (M + ).

[0114] Example 4

[0115] [ 18 F] N-(4-(1,2,4,5-tetrazin-3-yl)benzyl)-6-fluoropicolinamide

[0116]

[0117] Using 0.675 to 1.327 Ci starting activities and 45–50 min synthesis times, [ 18 The typical radiochemical yield of the radiosynthesis of [F]N-(4-(1,2,4,5-tetrazin-3-yl)benzyl)-6-fluoropicolinamide was 18.5±4.6% (N=6).

[0118] [ 18F] fluoride activity (0.195 to 1.475 Ci) was retained on a Sep-Pak Accell Plus QMA Plus Light Cartridge (130 mg, 37-55 μm, Waters Part # WAT023525; preconditioned with 5 mL of water for injection) and eluted onto a TRACERlab FX using 0.8 mL of aqueous tetraethylammonium bicarbonate [tetraethylammonium bicarbonate (3.5 mg) in water (0.2 mL) and acetonitrile (0.6 mL)]. F-N The eluted active was heated to 70 °C and dried under compressed nitrogen purge and vacuum for 5 minutes. The temperature was raised to 100 °C and maintained under vacuum for 5 minutes to obtain [ 18 F] Tetraethylammonium fluoride. Add 1 mL of anhydrous DMSO to the reactor and cool the reactor to 35 °C. 18 F] Tetraethylammonium fluoride solution was transferred to the solution containing 6-(2,4-dimethoxy-8-methyl-dibenzothiophene-5- A solution of 4-[[4-(1,2,4,5-tetrazin-3-yl)phenyl]methyl]pyridine-2-carboxamide trifluoromethanesulfonate (1 mg, 1.43 μmol) in anhydrous DMSO (0.2 mL) was placed in a TRACERlab syringe vial (RV2) and kept at room temperature for 5 minutes. The mixture was diluted with 3.5 mL of 0.1% (v / v) trifluoroacetic acid / water and the resulting crude reaction mixture was loaded onto a semi-preparative HPLC column for purification (conditions listed in Figure 3 ). The purified [ 18 HPLC fractions of N-(4-(1,2,4,5-tetrazin-3-yl)benzyl)-6-fluoropicolinamide (F] Figure 3 ) were collected into a vial containing 40 mL of water and loaded into the Sep- Light 1cc Vac C18 column (50 mg, 55-105 μm, Waters Part # WAT054955; pre-conditioned with 5 mL EtOH and 5 mL water). 18 F]N-(4-(1,2,4,5-tetrazin-3-yl)benzyl)-6-fluoropicolinamide was eluted with 0.5 mL of EtOH and reconstituted into 5 mL of 10% (v / v) EtOH / phosphate buffered saline. 18 F]N-(4-(1,2,4,5-tetrazin-3-yl)benzyl)-6-fluoropicolinamide in an amount ranging from 0.0085 to 0.066 Ci. 18 Representative analytical HPLC of [F] N-(4-(1,2,4,5-tetrazin-3-yl)benzyl)-6-fluoropicolinamide showed Figure 4 middle.

[0119] Comparative reference: Example 5

[0120] N-(4-(1,2,4,5-tetrazin-3-yl)benzyl)-6-fluoronicotinamide

[0121]

[0122] To a stirred solution of [4-(1,2,4,5-tetrazin-3-yl)phenyl]methanamine hydrochloride (60 mg, 0.27 mmol, Click Chemistry Tools, CAS #1416711-59-5) and 6-fluoropyridine-3-carboxylic acid (46 mg, 0.33 mmol) in dichloromethane (4 mL) was added HATU (135 mg, 0.35 mmol) and N,N-diisopropylethylamine (0.1 mL, 0.57 mmol). The reaction mixture was stirred at room temperature overnight. The reaction mixture was concentrated and diluted with ethyl acetate, and the organic layer was washed sequentially with saturated aqueous NaHCO3 solution, saturated aqueous NaCl solution, dried over Na2SO4, filtered, and concentrated under reduced pressure. The resulting residue was purified by column chromatography on silica gel eluting with a 0-50% dichloromethane / ethyl acetate gradient to afford the title compound (18 mg, 0.06 mmol) as a purple solid.

[0123] 1 H NMR (400.13MHz, CDCl3): 4.81 (d, J=5.9Hz, 2H), 6.61-6.56 (m, 1H), 7.06 (dd, J=2.1, 8 .4Hz, 1H), 7.62 (d, J=8.1Hz, 2H), 8.35-8.30 (m, 1H), 8.69-8.64 (m, 3H), 10.24 (s, 1H), 19 F NMR (376.45MHz, CDCl3) δppm: -62.8.ES / MS (m / z): 311[M+H)] + .

[0124] Preparation 7

[0125] 6-[2-(3,5-dimethoxyphenyl)-4-methyl-phenyl]thiopyridine-3-carboxylic acid

[0126]

[0127] To 6-chloropyridine-3-ethyl formate (280mg, 1.51mmol) and 3-[2-(3,5-dimethoxyphenyl)-4-methyl-phenyl] 2-ethylhexyl thiopropanoate (740mg, 1.66mmol) in 1,4-dioxane (10mL) is added 1M solution (1.8mL, 1.8mmol) of potassium tert-butoxide in THF. The solution is heated to 80 ℃ for 4-5 hours, then stirred at room temperature overnight. 2M lithium hydroxide aqueous solution (10mL, 20mmol) is added, and the mixture is stirred at 80 ℃ for 2-3 hours. The solution is cooled to room temperature, acidified with 2N HCl and extracted with ethyl acetate. The organic layer is then dried over Na2SO4, filtered and concentrated. The residue is purified by silica gel column (0-80% hexane / ethyl acetate) to provide the title compound (527mg, 1.31mmol) as a pale solid. ES / MS (m / z): 382 [M+H] + .

[0128] Preparation 8

[0129] N-(4-(1,2,4,5-tetrazin-3-yl)benzyl)-6-((3′,5′-dimethoxy-5-methyl-[1,1′-biphenyl]-2-yl)sulfinyl)nicotinamide

[0130]

[0131] To a stirred mixture of 6-[2-(3,5-dimethoxyphenyl)-4-methyl-phenyl]thiopyridine-3-carboxylic acid (94 mg, 0.25 mmol), [4-(1,2,4,5-tetrazin-3-yl)phenyl]methanamine hydrochloride (55 mg, 0.25 mmol), and HATU (115 mg, 0.30 mmol) in dichloromethane (2 mL) was added N,N-diisopropylethylamine (0.07 mL, 0.4 mmol). The reaction mixture was stirred at room temperature overnight. The resulting residue was diluted with dichloromethane and washed sequentially with saturated aqueous NaHCO3 solution and saturated aqueous NaCl solution. The organic extract was dried over Na2SO4, filtered, and concentrated under reduced pressure. The resulting residue was purified by chromatography on silica gel (0-80% hexanes / ethyl acetate) to afford N-(4-(1,2,4,5-tetrazin-3-yl)benzyl)-6-((3′,5′-dimethoxy-5-methyl-[1,1′-biphenyl]-2-yl)thio)nicotinamide (70 mg, 0.13 mmol) as a pink foam.

[0132] To a solution of N-(4-(1,2,4,5-tetrazin-3-yl)benzyl)-6-((3′,5′-dimethoxy-5-methyl-[1,1′-biphenyl]-2-yl)thio)nicotinamide (70 mg, 0.13 mmol) in dichloromethane (4 mL) was added 3-chloroperoxybenzoic acid (20 mg, 0.09 mmol, 77% pure). The mixture was stirred at room temperature for 2-3 hours. The mixture was then purified by silica gel column (12 g, 0-100% hexanes / ethyl acetate) to provide the title compound (24 mg) as a powder foam: ES / MS (m / z): 567 [M+H] + .

[0133] Preparation 9

[0134] 5-(5-((4-(1,2,4,5-tetrazin-3-yl)benzyl)carbamoyl)pyridin-2-yl)-2,4-dimethoxy-8-methyl-5H-dibenzo[b,d]thiophene-5-yl trifluoromethanesulfonate

[0135]

[0136] To a stirred slurry of N-(4-(1,2,4,5-tetrazin-3-yl)benzyl)-6-((3′,5′-dimethoxy-5-methyl-[1,1′-biphenyl]-2-yl)sulfinyl)nicotinamide (23 mg, 0.04 mmol) and diethylaminopolystyrene (3.2 mmol / g, 70 mg, 0.22 mmol) in dichloromethane (3 mL) cooled to 0° C. was added a 1 M solution of trifluoromethanesulfonic anhydride in dichloromethane (130 μL, 0.13 mmol). The reaction mixture was warmed to room temperature and stirred for 30 minutes. The reaction was quenched by the addition of water droplets. The mixture was filtered and the solids were washed with dichloromethane. The combined filtrates were concentrated under reduced pressure and the resulting residue was purified by chromatography on silica gel using a gradient of 0-20% methanol / dichloromethane to afford the title compound (12 mg, 0.017 mmol, 41% yield) as a purple solid: 1 H NMR (400.13MHz, DMSO-d6) δppm: 2.55 (s, 3H), 3.95 (s, 3H), 4.00 (s, 3H), 4.64-4.62 (m, 2H), 6.92 (d, J=2.1Hz, 1H), 7.62-7.57 (m, 3H), 7.69 (d, J=2.1Hz, 8.26(d, J=8.2Hz, 1H), 8.39-8.37(m, 2H), 8.48-8.44(m, 2H), 8.54-8.52(m, 1H), 8.92-8.91(m, 1H), 9.48-9.44(m, 1H), 10.58(s, 1H),19 F NMR (376.45MHz, DMSO-d6) δppm: -77.7.ES / MS (m / z): 549[M + ].

[0137] Comparative Reference Example 6

[0138] [ 18 F] N-(4-(1,2,4,5-tetrazin-3-yl)benzyl)-6-fluoronicotinamide

[0139]

[0140] Using 0.318 to 0.615 Ci starting activity and 50 min synthesis time, [ 18 The typical radiochemical yield of the radiosynthesis of [F]N-(4-(1,2,4,5-tetrazin-3-yl)benzyl)-6-fluoronicotinamide was 20±2.18% (N=3).

[0141] [ 18 F] fluoride activity (0.318 to 0.615 Ci) was retained on a Sep-Pak Accell Plus QMA Plus Light Cartridge (130 mg, 37-55 μm, Waters Part # WAT023525) and eluted onto a TRACERlab FX ELISA kit using 0.8 mL of aqueous tetraethylammonium bicarbonate solution [tetraethylammonium bicarbonate (3.6 mg) in water (0.2 mL) and acetonitrile (0.6 mL)]. F-N The eluted active was heated to 70 °C and dried under compressed nitrogen purge and vacuum for 5 minutes. The temperature was then raised to 100 °C and maintained under vacuum for 5 minutes to obtain tetraethylammonium [ 18 F] fluoride. The reactor was then cooled to 30°C and 1.2 mL of anhydrous DMSO was added to the reactor. Tetraethylammonium [ 18 F] The fluoride solution was transferred to a solution containing 5-(5-((4-(1,2,4,5-tetrazin-3-yl)benzyl)carbamoyl)pyridin-2-yl)-2,4-dimethoxy-8-methyl-5H-dibenzo[b,d]thiophene-5-yl)- The precursor solution of the triflate salt [(1 mg, 1.83 μmol) in 0.3 mL of anhydrous DMSO] was placed in a TRACERlab syringe vial (RV2) and kept at room temperature for 2 minutes. The mixture was then diluted with 3.5 mL of 1% (v / v) TFA / water and the resulting crude reaction mixture was loaded onto a semi-preparative HPLC column for purification (conditions listed in Figure 5 ). The purified [ 18HPLC fractions of N-(4-(1,2,4,5-tetrazin-3-yl)benzyl)-6-fluoronicotinamide (F] Figure 5 ) were collected into a vial containing 40 mL of 0.02% (v / v) TFA / water and loaded onto a Sep- Light C18 column (130 mg, 55-105 μm, Waters Part # WAT023501).

[0142] Reserved 18 F]N-(4-(1,2,4,5-tetrazin-3-yl)benzyl)-6-fluoronicotinamide was washed with 5 mL of 0.02% (v / v) TFA / water, eluted with 1 mL of EtOH and reconstituted into a 10 mL formulation of 10% (v / v) EtOH / PBS. 18 F]N-(4-(1,2,4,5-tetrazin-3-yl)benzyl)-6-fluoronicotinamide in an amount ranging from 0.046 to 0.125 Ci. 18 A representative analytical HPLC chromatogram of [F]N-(4-(1,2,4,5-tetrazin-3-yl)benzyl)-6-fluoronicotinamide is shown in FIG. Figure 6 middle.

[0143] [ 18 F] PET-CT imaging of N-(4-(1,2,4,5-tetrazin-3-yl)benzyl)-2-fluoronicotinamide (Example 2)

[0144] Siemens A multimodality scanner (Siemens, Germany) was used for microPET-CT imaging. Male CD-1 mice (6 weeks old, ∼30 g) were anesthetized with 3% isoflurane / 97% oxygen and placed on the scanner bed. Mice were administered [ 18 F]N-(4-(1,2,4,5-tetrazin-3-yl)benzyl)-2-fluoronicotinamide. A total of four dynamic PET scans were performed, followed by a short high-resolution CT scan for anatomical registration. PET images were generated for every minute of acquisition time. Tracer uptake in brain, muscle, and bone was determined by visually drawing regions of interest (ROIs) based on the fused PET / CT images and using The corresponding activity values ​​were determined using Research Workplace software. All values ​​were expressed as % injected dose / gram (% ID / g).

[0145] Analysis of four 60-minute PET scans showed [ 18F]N-(4-(1,2,4,5-tetrazin-3-yl)benzyl)-2-fluoronicotinamide readily crosses the blood-brain barrier. Peak brain uptake of 3.3% ID / g was observed at 4.5 minutes after injection, followed by steady clearance of the tracer to 1.2% ID / g at 59.5 minutes. [F]N-(4-(1,2,4,5-tetrazin-3-yl)benzyl)-2-fluoronicotinamide readily crosses the blood-brain barrier. Peak brain uptake of 3.3% ID / g was observed at 4.5 minutes after injection, followed by steady clearance of the tracer to 1.2% ID / g at 59.5 minutes. 18 F]N-(4-(1,2,4,5-tetrazin-3-yl)benzyl)-2-fluoronicotinamide uptake. Bone uptake remained low throughout the scan, consistent with the absence of defluorination. [ 18

[00145] Time Activity Curves for N-(4-(1,2,4,5-Tetrazin-3-yl)benzyl)-2-fluoronicotinamide. The data are presented below in a tabular format (Table 1).

[0146] Table 1 represents [ 18 F] 60 min PET time activity results of N-(4-(1,2,4,5-tetrazin-3-yl)benzyl)-2-fluoronicotinamide (brain, muscle and bone, N=4).

[0147] Table 1. 18 60-min PET Time Activity Table (Brain, Muscle & Bone) of N-(4-(1,2,4,5-Tetrazin-3-yl)benzyl)-2-fluoronicotinamide (Example 2)

[0148]

[0149]

[0150]

[0151] [ 18 PET-CT imaging of [F] N-(4-(1,2,4,5-tetrazin-3-yl)benzyl)-6-fluoropicolinamide (Example 4)

[0152] Siemens A multimodality scanner (Siemens, Germany) was used for microPET-CT imaging. Male CD-1 mice (6 weeks old, ∼30 g) were anesthetized with 3% isoflurane / 97% oxygen and placed on the scanner bed. Mice were administered [ 18F]N-(4-(1,2,4,5-tetrazin-3-yl)benzyl)-6-fluoropicolinamide. A total of four dynamic PET scans were performed, followed by a short high-resolution CT scan for anatomical registration. PET images were generated for every minute of acquisition time. Tracer uptake in brain, muscle, and bone was determined by visually drawing regions of interest (ROIs) based on the fused PET / CT images and using The corresponding activity values ​​were determined using ResearchWorkplace software. All values ​​were expressed as % injected dose / gram (% ID / g).

[0153] Analysis of four 60-minute PET scans showed [ 18 F]N-(4-(1,2,4,5-tetrazin-3-yl)benzyl)-6-fluoropicolinamide readily crosses the blood-brain barrier. Peak brain uptake of 4.3% ID / g was observed at 2.5 minutes after injection, followed by steady clearance of the tracer to 1.1% ID / g at 59.5 minutes. [F]N-(4-(1,2,4,5-tetrazin-3-yl)benzyl)-6-fluoropicolinamide was also observed in peripheral organs such as the liver, kidney, and heart. 18 F]N-(4-(1,2,4,5-tetrazin-3-yl)benzyl)-6-fluoropicolinamide uptake. Bone uptake remained low throughout the scan, consistent with the absence of defluorination. [ 18

[00145] Time Activity Curves for N-(4-(1,2,4,5-Tetrazin-3-yl)benzyl)-6-fluoropicolinamide. The data are presented below in a tabular format (Table 2).

[0154] Table 2 represents [ 18 F] 60 min PET temporal activity results of N-(4-(1,2,4,5-tetrazin-3-yl)benzyl)-6-fluoropicolinamide (brain, muscle and bone, N=4).

[0155] Table 2. 18 60-minute PET time activity table (brain, muscle and bone) of [F] N-(4-(1,2,4,5-tetrazin-3-yl)benzyl)-6-fluoropicolinamide (Example 4)

[0156]

[0157]

[0158] [ 18 F] PET-CT imaging of N-(4-(1,2,4,5-tetrazin-3-yl)benzyl)-6-fluoronicotinamide (Comparative Example 6)

[0159] Siemens A multimodality scanner (Siemens, Germany) was used for microPET / CT imaging. Male CD-1 mice (6 weeks old, 30-40 g) were anesthetized with 3% isoflurane / 97% oxygen and placed on the scanner bed. Mice were administered [ 18 F] N-(4-(1,2,4,5-tetrazin-3-yl)benzyl)-6-fluoronicotinamide. A total of two dynamic PET scans were performed, followed by a short high-resolution CT scan for anatomical registration. PET images were generated for every minute of acquisition time. Tracer uptake in brain, muscle, and bone was determined by visually drawing regions of interest (ROIs) based on the fused PET / CT images and using The corresponding activity values ​​were determined using Research Workplace software. All values ​​were expressed as % injected dose / gram (% ID / g).

[0160] Analysis of two PET scans showed [ 18 F]N-(4-(1,2,4,5-tetrazin-3-yl)benzyl)-6-fluoronicotinamide crosses the blood-brain barrier. Peak brain uptake of 2.6% ID / g was observed at 2.5 minutes after injection, followed by steady clearance of the tracer to 1.1% ID / g at 59.5 minutes. [F]N-(4-(1,2,4,5-tetrazin-3-yl)benzyl)-6-fluoronicotinamide crosses the blood-brain barrier. Peak brain uptake of 2.6% ID / g was observed at 2.5 minutes after injection, followed by steady clearance of the tracer to 1.1% ID / g at 59.5 minutes. 18 F]N-(4-(1,2,4,5-tetrazin-3-yl)benzyl)-6-fluoronicotinamide uptake. Bone uptake remained low throughout the scan, consistent with the absence of defluorination. [ 18

[00145] Time Activity Curves for N-(4-(1,2,4,5-Tetrazin-3-yl)benzyl)-6-fluoronicotinamide. The data are presented below in a tabular format (Table 3).

[0161] Table 3 represents [ 18 F] 60 min PET time activity results of N-(4-(1,2,4,5-tetrazin-3-yl)benzyl)-6-fluoronicotinamide (brain, muscle & bone, N=2).

[0162] Table 3. 18 60-minute PET activity table (brain, muscle & bone) of N-(4-(1,2,4,5-tetrazin-3-yl)benzyl)-6-fluoronicotinamide (Comparative Example 6)

[0163]

[0164]

[0165] The PET-CT data depicted in Tables 1 and 2 indicate that the PET-CT data of Examples 2 and 4, when tested in CD-1 mice, 18 The F-labeled compound crosses the blood-brain barrier. Peak brain uptake (%ID / g) was observed approximately 2 minutes after injection, followed by steady clearance of the tracer to baseline levels (defined by uptake in muscle) at 60 minutes after injection. Bone uptake remained low throughout the scan, consistent with the absence of in vivo defluorination. These data suggest that the 18 F-labeled compounds can be used as PET-CT imaging agents for CNS pretargeting imaging.

[0166] Analysis of two PET scans showed [ 18 [F] N-(4-(1,2,4,5-tetrazin-3-yl)benzyl)-6-fluoronicotinamide (Comparative Example 6) crossed the blood-brain barrier. A peak brain uptake of 2.6% ID / g was observed 2.5 minutes after injection, followed by a steady clearance of the tracer to 1.1% ID / g at 59.5 minutes.

[0167] In contrast, the [ 18 F] N-(4-(1,2,4,5-tetrazin-3-yl)benzyl)-2-fluoronicotinamide (Example 2) and [ 18 Dynamic PET imaging of [F]N-(4-(1,2,4,5-tetrazin-3-yl)benzyl)-6-fluoropicolinamide (Example 4) in CD-1 male mice showed that these compounds readily crossed the blood-brain barrier and reached peak brain uptake of 3.3±0.4% ID / g and 4.3±0.3% ID / g, respectively. These compounds subsequently showed steady clearance from the brain, reaching near background levels (muscle) by 60 minutes post-injection.

[0168] These agents have high brain penetration and are then rapidly and completely washed out of the brain, thus providing a larger window for achieving higher signal-to-background ratios and thus better image quality. 18 F] N-(4-(1,2,4,5-tetrazin-3-yl)benzyl)-2-fluoronicotinamide (Example 2) and [ 18 F]N-(4-(1,2,4,5-tetrazin-3-yl)benzyl)-6-fluoropicolinamide (Example 4) provides advantages over [ 18 F] Advantages of N-(4-(1,2,4,5-tetrazin-3-yl)benzyl)-6-fluoronicotinamide (Comparative Example 6).

[0169] In addition to its potential application in pre-targeted imaging studies, radiolabeled tetrazines have been shown in multiple publications to be useful prosthetic groups for labeling biomolecules. Steven Liang et al. have reported on the sortase-mediated modification of camelid single-domain antibody fragments and subsequent radiolabeling with 18F-labeled tetrazines (Angew. Chem. Int. Ed. 2016, 55, 528-533; WO 2017 / 059397 A1). and colleagues have reported radiolabeling of bispecific antibodies by functionalization with trans-cyclooctene (TCO) groups and subsequent conjugation with 18F-labeled tetrazines at ambient temperature (ACS Chem. Neurosci. 2020, 11, 24, 4460-4468). Michael Zalutsky and colleagues have disclosed radiolabeling of biomolecules such as nanobodies with radiolabeled tetrazines having A1F chelates or fluoropyridine functional groups (Bioconjugate Chem. 2018, 29, 4090-4103; WO 2020 / 242948 A1).

[0170] Based on literature precedent, it is expected that the tetrazines claimed herein can be used as prosthetic groups for labeling biomolecules. 19 F] and [ 18 F] The reactivity of tetrazine N-(4-(1,2,4,5-tetrazin-3-yl)benzyl)-2-fluoropicolinamide and N-(4-(1,2,4,5-tetrazin-3-yl)benzyl)-6-fluoropicolinamide with trans-cyclooctenes (TCOs) conjugated to the biomolecule Malat1 antisense oligonucleotide (ASO) was demonstrated in experiments (2019 World Molecular Imaging Congress Conference, Poster 139).

[0171] To a 2.5 mg / mL (2.67 μM) aqueous solution of Malat1 ASO-TCO was added 2 equivalents of tetrazine and the mixture was gently shaken at room temperature in the dark for 30 minutes. Xevo QTof mass spectrometer The LC-MS conditions were as follows: Column: ACQUITY PREMIER UPLColigonucleotide BEH C18( 1.7 μm, 2.1 mm × 50 mm); wavelength 250-650 nm; gradient: initial hold at 5% B for 3.5 minutes, 5-75% B in 1.5 minutes, hold at 75% B for 4 minutes, increase from 75% B to 98% B in 0.5 minutes, hold at 98% B for 1 minute, return to 5% B to re-equilibrate; column temperature: 70°C + / - 5°C; flow rate: 0.2 mL / min; solvent A: water containing 7 mM TEA and 100 mM HFIP; solvent B: 75% methanol / 25% acetonitrile containing 7 mM TEA and 100 mM HFIP.

[0172] Negative mode mass spectra (500-3000 m / z) were summed by peak of interest and processed in MaxEnt to generate zero charge mass results. The mass results before and after reaction with each tetrazine (N-(4-(1,2,4,5-tetrazin-3-yl)benzyl)-2-fluoropicolinamide and N-(4-(1,2,4,5-tetrazin-3-yl)benzyl)-6-fluoropicolinamide) showed a difference of 282 Da (from 7502 Da to 7784 Da) with no parent remaining, indicating that Malat1 ASO-TCO reacted completely with the tetrazine ( Figure 7 ).

[0173] calculate[ 18 F] N-(4-(1,2,4,5-tetrazin-3-yl)benzyl)-2-fluoropicolinamide and [ 18 The tetrazine concentration in the solution of [F] N-(4-(1,2,4,5-tetrazin-3-yl)benzyl)-6-fluoropicolinamide was determined, and 0.4 mL of each was reacted with two portions of Malat1-ASO-TCO. The reaction was gently shaken at room temperature in the dark for 15 minutes and then diluted 100-fold in water. The diluted sample was then analyzed on an Agilent 1290 Series UPLC UV coupled to a BGO coincidence detector. HPLC conditions were as follows: Column: ACQUITY PREMIER UPLC Oligonucleotide BEH C18( 1.7 μm, 2.1 mm X 50 mm); Gradient: initial hold at 5% B for 3 minutes, 5-75% B in 1.5 minutes, hold 75% B for 6 minutes, increase from 75% B to 98% B in 1 minute, hold 98% B for 1 minute, return to 5% B to re-equilibrate; Column temperature: 70°C + / - 5°C; Flow rate: 0.25 mL / min; Solvent for A: Water containing 7 mM TEA and 100 mM HFIP; Solvent for B: 75% methanol / 25% acetonitrile containing 7 mM TEA and 100 mM HFIP.

[0174] Each tetrazine ([ 18F] N-(4-(1,2,4,5-tetrazin-3-yl)benzyl)-2-fluoropicolinamide and [ 18 Radiotracing of [F]N-(4-(1,2,4,5-tetrazin-3-yl)benzyl)-6-fluoropicolinamide) before and after reaction with Malat1 ASO-TCO showed a shift from a single peak (RT = 9 min) consisting of unreacted tetrazine to two peaks, with the earlier eluting peak (RT = 3.4 min) representing the reacted species ( Figure 8 ).

[0175] Accordingly, the present embodiment discloses a method for radiolabeling a molecule. Specifically, in a first step, a biomolecule is modified to attach or include a TCO moiety to form a biomolecule-TCO conjugate. (An exemplary method for doing so is disclosed above in ACS Chem. Neurosci. 2020, 11, 24, 4460-4468). Then, in a second step, the biomolecule-TCO conjugate is reacted with 18F-labeled tetrazine to form biomolecule-TCO-tetrazine-18F via a cycloaddition reaction.

[0176] Specifically, the method may include a method of radiolabeling a biomolecule, comprising:

[0177] Attaching a TCO-moiety to a biomolecule, thereby forming a biomolecule-TCO conjugate;

[0178] The biomolecule-TCO conjugate is reacted with an 18F-labeled tetrazine to form a biomolecule-TCO-tetrazine- 18F molecule, wherein the 18F-labeled tetrazine is selected from the compound of Formula II and the compound of Formula IV.

Claims

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

2. The compound according to claim 1, which is 3. Compounds of the formula: or a pharmaceutically acceptable salt thereof.

4. The compound according to claim 3, which is 5. A pharmaceutical composition comprising a compound according to any one of claims 1 to 4 or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers, diluents or excipients.

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

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

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

9. The composition of claim 8, wherein the composition does not comprise ascorbate or ascorbic acid.

10. The following compounds or use of a pharmaceutically acceptable salt thereof in preparing a reagent for a method for pretargeting imaging, the method comprising: a. introducing into a mammal a detectable amount of the compound or a pharmaceutically acceptable salt thereof, and b. detecting the compound in PET imaging, wherein the compound is introduced after the biologic-TCO conjugate is introduced into the mammal.

11. The following compounds or use of a pharmaceutically acceptable salt thereof in preparing a reagent for a method for pretargeting imaging, the method comprising: a. introducing into a mammal a detectable amount of the compound or a pharmaceutically acceptable salt thereof, and b. detecting the compound in PET imaging, wherein the compound is introduced after the biologic-TCO conjugate is introduced into the mammal.

12. Use of the composition according to any one of claims 5 to 8 in the preparation of an agent for a method of pretargeted imaging, the method comprising: a. introducing a detectable amount of the composition into a mammal; b. Testing the composition.

13. Intermediates selected from:

14. A compound of the following formula or a pharmaceutically acceptable salt thereof: X is C— 18 F or N Y is C— 18 F or N The condition is that only one of X and Y is N. Wherein when X is N, Y is C— 18 F; and wherein when Y is N, X is C— 18 F.

15. The compound according to claim 14, which is 16. The compound according to claim 14, which is 17. A pharmaceutical composition comprising a compound according to any one of claims 14 to 16 or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers, diluents or excipients.

18. Use of a compound according to any one of claims 14 to 16 or a pharmaceutically acceptable salt thereof in the preparation of an agent for a method for pretargeted imaging, the method comprising: a. introducing into a mammal a detectable amount of the compound or a pharmaceutically acceptable salt thereof, and b. detecting the compound in PET imaging, wherein the compound is introduced after the biologic-TCO conjugate is introduced into the mammal.

19. A method for radiolabeling a biomolecule, comprising: attaching the TCO-moiety to a biomolecule, thereby forming a biomolecule-TCO conjugate; and The biomolecule-TCO conjugate is reacted with a 18F-labeled tetrazine to form a biomolecule-TCO-tetrazine-18F molecule, wherein the 18F-labeled tetrazine is selected from

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