Tracer compounds and methods for their preparation

Abstract

Disclosed are tracer compounds, or pharma- ceutically acceptable salts or solvates thereof, the tracer compounds having a structure comprising a tetrazine moiety, a zwitterionic moiety, and a linker moiety, the linker moiety connecting the tetrazine moiety and the zwitterionic moiety to each other, the linker moiety being composed of a specific moiety S1-Y-S2 as disclosed herein.Further disclosed are adducts of the tracer compounds with trans-cyclooctene (TCO) derivatized targeting moieties, and methods for making the tracer compounds and the adducts.

Description

[Technical field]

[0001] The present disclosure relates generally to radiolabeled radiopharmaceuticals, particularly, but not exclusively, to radiolabeled radiopharmaceuticals obtained by inverse electron demand Diels-Alder cycloaddition (IEDDA) of trans-cyclooctene with a tetrazine moiety. [Background technology]

[0002] This section illustrates useful background information without admitting that any of the art described herein is representative of the state of the art.

[0003] Bioorthogonal chemistry has demonstrated great potential in the synthesis of radiopharmaceuticals for diagnostic imaging. So far, the inverse electron-demand Diels-Alder cycloaddition (IEDDA) reaction between tetrazines and dienophiles has shown the fastest reaction rate in bioorthogonal chemistry and has been utilized for the radiosynthesis of a variety of fluorine-18 labeled radiopharmaceuticals, ranging from small molecules to macromolecules such as peptides and antibody fragments. The IEDDA reaction is 18 F, 68 Ga, 64 It is also used in pretargeted PET imaging using radionuclides such as Cu. Summary of the Invention

[0004] The inventors have surprisingly found that by synthesizing compounds containing a tetrazine moiety and a specific zwitterionic moiety and a linker moiety between them as described herein, tracer compounds with high modularity can be obtained. Furthermore, an inverse electron demand Diels-Alder reaction (IEDDA) between trans-cyclooctene and the tetrazine moiety of the tracer compound can provide an adduct containing the tracer compound and a trans-cyclooctene (TCO) derivatized targeting moiety. The modularity of the linker moiety in the tracer compound allows the use of the adduct and the tracer compound in targeting multiple different target entities in vivo and in vitro. The specific combination of moieties contained in the tracer compound and the adduct provides a tracer compound with good stability.

[0005] The object of the present disclosure is to provide a tracer compound that, as part of an adduct, exhibits high modularity in terms of targeting a number of different target entities in vivo and in vitro. Also, the object of the present disclosure is to provide a tracer compound that has performance and / or stability that allows its use in radiolabeling and radioimaging applications. Another object of the present disclosure is to provide a tracer compound that has improved properties when used to target target entities in vivo and in vitro. Yet another object of the present disclosure is to provide an adduct of a tracer compound and a TCO-derivatized targeting moiety that can be used to target entities in vivo and in vitro.

[0006] This application relates to the invention defined in the attached independent claims and to the embodiments thereof disclosed below. The attached claims define the scope of protection. Methods, processes, products or apparatus disclosed in the specification or drawings that are not claimed are not embodiments of the claimed invention, but are provided as examples that are useful for understanding the claimed invention.

[0007] Described herein are tracer compounds and adducts of tracer compounds with trans-cyclooctene (TCO) derivatized targeting moieties that can be used to target many diagnostic biomarkers in vivo and in vitro via radiolabeling and ultimately detected by radioimaging methods. The inventors have surprisingly found that the combination of a tetrazine moiety, a specific linker moiety and a zwitterionic moiety allows the synthesis of tracer compounds that have rapid synthesis and high modularity in terms of the targeting moiety being able to be conjugated to the tracer compounds via the TCO moiety, as shown in the examples below.

[0008] According to a first aspect, there is provided a tracer compound of formula (I) or a pharma- ceutically acceptable salt or solvate thereof: [ka] (In the formula, Each R1 is independently hydrogen (H) or a group of formula C n H 2n+1 where n is an integer selected from the range of 0 to 2; L is a linker moiety consisting of S1-Y-S2, where: Y is (-CH2-) m (wherein m is an integer selected from the range of 1 to 4), or Y is a polyethylene glycol linker-(PEG) X -(in the formula, (PEG) X contains x repeat units of polyethylene oxide -CH2-CH2-O- groups, where x is an integer selected from the range of 1 to 20; and S1 is -(CH2) Z -CO-NH-(CH2) Z - or S1 is -(CH2) Z -NH-CO-(CH2) Z -wherein each z is independently an integer selected from the range of 0 to 4; and S2 is -CH2- or S2 is -(CH2)f -CO-NH-(CH2) f - or S2 is -(CH2) f -NH-CO-(CH2) f where each f is independently an integer selected from the range of 0 to 4; and R2 is hydrogen (H) or a phenyl group substituent or a group of formula C S H 2S+1 (wherein s is an integer selected from the range of 0 to 2).

[0009] According to a second aspect, there is provided an adduct of a trans-cyclooctene (TCO) derivatised targeting moiety of the tracer compound of the first aspect, or a pharma-ceutically acceptable salt or solvate thereof, obtainable by inverse electron demand Diels-Alder reaction (IEDDA) between the TCO moiety of the TCO derivatised targeting moiety and the tetrazine moiety of the tracer compound.

[0010] According to a third aspect there is provided the adduct of the second aspect for use in detecting a target entity in a subject by radioimaging, preferably positron emission tomography.

[0011] According to a fourth aspect, there is provided a method for producing a tracer compound of the first aspect, the method comprising: a. dissolving a starting material in a polar aprotic solvent and reacting the starting material with 2-(iodomethyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane to provide an intermediate product; b. dissolving the intermediate product in a polar aprotic solvent and reacting the intermediate product with KHF2 in the presence of an acid such as HCl, water and an organic solvent to provide a tracer compound; Including, The starting material consists of a tetrazine moiety linked to a tertiary amine (-N(CH3)2) via a linker moiety; The tetrazine moiety is composed of a 1,2,4,5-tetrazine ring, a phenyl ring attached to C3 of the tetrazine ring, and R2 attached to C6 of the tetrazine ring, where R2 is a hydrogen (H) or a phenyl substituent or a group of formula C S H 2S+1 (wherein s is an integer selected from the range of 0 to 2); and The linker moiety is composed of S1-Y-S2, where: Y is (-CH2-) m (wherein m is an integer selected from the range of 1 to 4), or Y is a polyethylene glycol linker-(PEG) X -(in the formula, (PEG) X contains x repeat units of polyethylene oxide -CH2-CH2-O- groups, where x is an integer selected from the range of 1 to 20; and S1 is -(CH2) Z -CO-NH-(CH2) Z - or S1 is -(CH2) Z -NH-CO-(CH2) Z where each z is independently an integer selected from the range of 0 to 4; and S2 is -CH2- or S2 is -(CH2) f -CO-NH-(CH2) f - or S2 is -(CH2) f -NH-CO-(CH2) f -, where each f is independently an integer selected from the range of 0 to 4.

[0012] According to a fifth aspect, there is provided a method for preparing the adduct of the second aspect, or a pharma- ceutically acceptable salt or solvate thereof, the method comprising: providing a TCO-derivatized targeting moiety; providing a tracer compound of the first aspect; reacting the tetrazine moiety of the tracer compound with the TCO moiety of the TCO-derivatized targeting moiety; and Add at least one attachment 18radiolabeling with F to obtain the adduct; or Or the method may include: providing a TCO-derivatized targeting moiety; providing a tracer compound of the first aspect; The tracer compound of the first aspect is 18 radiolabeling with F; and reacting the tetrazine moiety of the radiolabeled tracer compound with the TCO moiety of the TCO-derivatized targeting moiety to obtain an adduct.

[0013] According to a sixth aspect there is provided the use of a tracer compound of the first aspect and / or an adduct of the second aspect in the detection of a target entity in a subject by radioactive imaging of the subject, wherein the target entity is targeted with a radiolabelled tracer compound and / or adduct.

[0014] According to a seventh aspect, there is provided a method for detecting a target entity in a subject by radioimaging, 18 A kit for the production of F-labelled adducts is provided, the kit comprising at least one compartment containing a tracer compound of the first aspect, at least one compartment containing at least one TCO-derivatised targeting moiety and a reagent for radiolabelling the tracer compound. 18 It comprises at least one compartment containing F and, optionally, aqueous and organic solvents for IEDDA conjugation and radiolabeling of tracer compounds and / or adducts.

[0015] According to a further aspect there is provided a diagnostic and / or therapeutic use of the tracer compound of the first aspect and / or the adduct of the second aspect in the detection of a target entity in a subject by radioactive imaging of the subject, wherein the target entity is targeted with a radiolabelled tracer compound and / or adduct.

[0016] According to a further aspect, there is provided a non-therapeutic use of the tracer compound of the first aspect and / or the adduct of the second aspect in detecting a target entity in a subject by radioimaging of the subject, wherein the target entity is targeted with a radiolabeled tracer compound and / or adduct. According to a further aspect, there is provided a non-diagnostic use of the tracer compound of the first aspect and / or the adduct of the second aspect in detecting a target entity in a subject by radioimaging of the subject, wherein the target entity is targeted with a radiolabeled tracer compound and / or adduct. In one embodiment, an example of a non-diagnostic and / or non-therapeutic use is in the evaluation of structures targeted for therapy.

[0017] The tracer compounds of the present invention have the advantage of being highly modular due to the modular linker moiety, which allows for optimized conjugation with multiple different targeting entities.The tracer compounds of the present invention in the first aspect have the advantage of low non-specific tissue uptake, even when no TCO-derivatized targeting moiety is conjugated thereto.The tracer compounds of the present invention in the first aspect have the advantage in pre-targeted PET imaging.

[0018] The adduct of the second aspect of the invention has the advantage that it allows for the rapid preparation of an easily usable adduct at room temperature.The adduct of the second aspect of the invention has the advantage that it is biocompatible for in vivo use.As shown in the examples provided below, the adduct of the second aspect has the performance and specificity to allow the visualization of target entities through the specific binding of the targeting moiety of the IEDDA cycloaddition product with a radiolabeled tracer compound.

[0019] The adduct of the present invention in the second aspect has the advantage of high target tissue specific uptake rate and low non-specific tissue uptake rate.The radiolabeled adduct of the present invention in the second aspect has the advantage of good metabolic stability and rapid clearance mainly via the kidney.The adduct of the present invention in the second aspect has the advantage of high modularity, and the pharmacokinetics of the adduct is flexible through the modification of the structural components of the tracer compound.

[0020] The adducts of the second aspect of the invention have the advantage of having high tumor-specific uptake rates, and the targeting moieties utilized in the adducts target biomolecules indicative of cancerous growth. [Brief description of the drawings]

[0021] Several illustrative embodiments will now be described with reference to the accompanying drawings.

[0022] [Figure 1] 1 shows the synthetic route and structural formula of tracer compound AmBF3-Tz(4) according to an exemplary embodiment. [Diagram 2] 1 shows the synthetic route and structural formula of tracer compound AmBF3-PEG4-TZ(8) according to an exemplary embodiment. [Diagram 3] 1 shows the synthetic route and structural formula of tracer compound AmBF3-PEG9-Tz (12) according to an exemplary embodiment. [Figure 4] 1 shows a synthetic route and structural formula of trans-cyclooctene aldehyde (TCO-CHO) (15), and the structural formula of TCO-PEG3-aldehyde (16), according to an exemplary embodiment. [Diagram 5] 1 shows a synthetic route and structural formula for PSMA-trans-cyclooctene (PSMA-TCO) (18) according to an exemplary embodiment. [Figure 6] 1 shows a synthetic route and structural formula of PSMA-Tranexamic acid-TCO (24) according to an exemplary embodiment. [Figure 7]1 shows the response to radiolabeling of AmBF3-Tz(4) prior to IEDDA conjugation, resulting in 18F-labeled AmBF3-Tz ([18F]4), according to an exemplary embodiment. [Figure 8a] FIG. 1 shows an alternative synthetic route according to an exemplary embodiment to obtain a radiolabeled adduct that is an IEDDA cycloaddition product of the tracer compound AmBF3(4) with a TCO-derivatized targeting moiety, where the targeting moiety is represented as a peptide. [Figure 8b] FIG. 1 shows an alternative synthetic route according to an exemplary embodiment to obtain a radiolabeled adduct that is an IEDDA cycloaddition product of the tracer compound AmBF3(4) with a TCO-derivatized targeting moiety, where the targeting moiety is represented as a peptide. [Figure 9] The % of total radioactivity observed at different time points as free, cell membrane-bound or internalized radioactivity-% after incubation of B16 / F10 melanoma cells with [18F]AmBF3-Tz ([18F]4) is shown. More than 99% of the total radioactivity was present in the free fraction at each time point, thereby suggesting low non-specific binding of [18F]4 to cell membranes. [Figure 10] Figure 1 shows the % of radioactivity in the intracellular compartment of AR42J cells from the total added radioactivity in the AR42J cell line, radioactivity detected as a function of time, indicating specific AR42J cell uptake of TOO-functionalized Tyr3-octreotide (TOC) conjugated with [18F]AmBF3-Tz ([18F]4) forming [18F]AmBF3-Tyr3-octreotide ([18F]25) at baseline conditions (internalization). Uptake at baseline conditions was inhibited by co-incubating cells with [18F]25 together with unmodified blocked octreotide (Blocked). [Figure 11] Addition PET / CT images of SCID mice 0-60 min after administration of [18F]AmBF3-Tz ([18F]4) are shown. B.=bladder, GB=gallbladder, K.=kidney, L.=liver. [Figure 12a]Quantifying radioactivity in the indicated tissues 270 ± 2 min after intravenous administration of [18F]AmBF3-Tz ([18F]4) in control SCID and C57BL / 6JRj mice (n = 2–3 / strain) as percentage of injected dose per gram of tissue (%ID / g) is shown, indicating clearance and excretion of the radiotracer compound. [Figure 12b] Quantifying radioactivity in urine and feces shown 270 ± 2 min after intravenous administration of [18F]AmBF3-Tz ([18F]4) in control SCID and C57BL / 6JRj mice (n = 2–3 / strain) as percentage of injected dose per gram of tissue (%ID / g), indicating clearance and excretion of the radiotracer compound. [Figure 13a] Standard uptake values ​​(SUVs) measured in the indicated tissues 60 min after administration of [18F]AmBF3-Tz ([18F]4) in male SCID mice (n=1) are shown, indicating the biodistribution and excretion of [18F]4. [Figure 13b] Standard uptake values ​​(SUVs) measured in urine shown 60 min after administration of [18F]AmBF3-Tz ([18F]4) in male SCID mice (n=1) are shown, indicating the biodistribution and excretion of [18F]4. [Figure 14] Figure 1 shows the percentage of injected dose per gram of tissue (%ID / g) of [18F]25 measured in different tissues of AR42J tumor-bearing Rj:NMRI-Foxn1 nu / nu mice (n = 2–4) after intravenous administration of the radiolabeled adduct [18F]25, suggesting its biodistribution at different time points. [Figure 15] Addition PET / CT images of Rj:NMRI-Foxn1 nu / nu mice bearing AR42J tumors are shown 20-80 min after administration of [18F]AmBF3-Tyr3-octreotide ([18F]25). The animal on the left was co-administered intravenously with unmodified blocked octreotide and [18F]25. The animal on the right was administered only [18F]25 without blocked octreotide to allow visualization of the subcutaneous AR42J tumor in the right shoulder. T=tumor. [Figure 16]Standard uptake values ​​(SUV) measured in AR42J tumor tissue as a function of time (min) are shown, where AR42J tumor-bearing mice (n=2 / group) were intravenously administered [18F]AmBF3-Tyr3-octreotide ([18F]25) alone (non-blocking) or co-administered [18F]25 and blocked octreotide (blocking). [Figure 17] Standard uptake values ​​(SUV) measured in the indicated tissues as a function of time (min) are shown, indicating the clearance of radioactivity following intravenous administration of [18F]AmBF3-Tyr3-octreotide ([18F]25) in tumor-bearing AR42J mice (n=1). [Figure 18] Maximum intensity projections of PET / CT are shown 15-90 min after injection of [18F]AmBF3-tranexamic acid-PSMA ([18F]29). The animal on the right was co-administered intravenously with 2-PMPA (blocking) and [18F]29. The animal on the left was administered [18F]29 alone without blocking 2-PMPA, allowing visualization of the subcutaneous C4-2 tumor in the left shoulder. T=tumor, G=gallbladder, K=kidneys, U=bladder / urine. [Figure 19] Figure 1 shows the percentage of injected dose per gram of tissue (%ID / g) of [18F]29 measured in different tissues of C4-2 tumor-bearing SCID mice (n=3) after intravenous administration of the radiolabeled adduct [18F]29, indicating its biodistribution 60 min after injection. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0023] As used herein, the term "tracer compound" or "tracer" refers to a compound that can be traced by a radiation detector. In an embodiment, the tracer compound comprises one or more atoms replaced by a radionuclide. In one embodiment, the tracer compound is a tracer compound according to the first aspect.

[0024] As used herein, the term "IEDDA" means inverse electron demand Diels-Alder reaction.

[0025] As used herein, the term "moiety" refers to a part of a molecule that can be functionally or structurally identified in the structure of the molecule, such as a tracer compound or an adduct as a whole. Thus, moieties can be named individually.

[0026] As used herein, the term "linker" or "linker moiety" refers to a modular region that connects two adjacent moieties in a tracer compound or adduct. In one embodiment, the linker moiety refers to a linker moiety according to the first aspect that links the tetrazine moiety and the zwitterionic moiety of the tracer compound to each other. In one embodiment, S2 of the linker moiety is attached to the N of the zwitterionic moiety and S1 of the linker moiety is attached to the phenyl of the tetrazine moiety of the tracer compound. In an alternative embodiment, "linker", or "linking moiety" or "linker moiety" refers to a linker moiety in a TCO-derivatized targeting moiety that links the targeting moiety and the TCO moiety to each other.

[0027] As used herein, the term "tetrazine" refers to a six-membered aromatic tetrazine ring containing four nitrogen atoms. As used herein, the term tetrazine refers to the 1,2,4,5-tetrazine isomer.

[0028] As used herein, the term "tetrazine moiety" refers to a moiety that includes a six-membered aromatic ring that includes four nitrogen atoms. The tetrazine structure in the tetrazine moiety is a 1,2,4,5-tetrazine isomer structure. The tetrazine moiety further includes a six-membered aromatic phenyl ring bonded to C3 of the tetrazine ring. The tetrazine moiety further includes R2 bonded to C6 of the tetrazine ring, where R2 is hydrogen (H) or a phenyl substituent or a group of formula C S H 2S+1where s is an integer selected from the range of 0 to 2. In one embodiment, the linker moiety L of the tracer compound according to formula (I) and the 1,2,4,5-tetrazine of the tetrazine moiety are attached to the phenyl ring of the tetrazine moiety in the para position relative to each other according to formula (I).

[0029] As used herein, the term "zwitterion" refers to a molecule having at least two functional groups and containing an equal number of positively and negatively charged functional groups. The overall charge of a zwitterionic molecule is zero. In one embodiment, a zwitterion is an organic trifluoroborate [ABF3] according to formula (I): - where A is [-CH2-N-(R1)2] + It is.

[0030] As used herein, the term "alkyl substituent" refers to a general (unspecified) alkane that is part of another molecule and is missing one hydrogen in relation to a chemical structure. The smallest "alkyl substituent" is the methyl group, which has the formula CH3-.

[0031] As used herein, the term "polyethylene glycol linker (-(PEG) X "-)" refers to a modular moiety that connects two other adjacent moieties within a tracer compound or adduct, where (PEG) X contains x repeat units of polyethylene oxide -CH2-CH2-O- groups, where x is an integer. X The moiety may be surrounded by separate linking moieties, referred to herein as S1 and S2, that connect the linker moiety to the remainder of the tracer compound or adduct.

[0032] As used herein, the term "integer" means a whole number that is not a fraction, which can be positive, negative, or zero.

[0033] As used herein, the term " 18"F" or "Fluorine-18" means a fluorine radioisotope that decays primarily by positron emission.

[0034] As used herein, the term "adduct" refers to an adduct product obtained by the addition of two or more separate molecules resulting in a single product. In one embodiment, the term "adduct" refers to an adduct of a tracer compound and a TCO-derivatized targeting moiety.

[0035] As used herein, the term "trans-cyclooctene (TCO)" refers to a trans isomer of a cycloalkene having a chain of eight carbons forming a cyclic hydrocarbon, in which the two C-C single bonds on either side of the C=C double bond are on opposite sides of the plane of the C=C double bond.

[0036] As used herein, the term "TCO moiety" refers to a TCO that is part of a molecule that includes at least one other moiety, such as a targeting moiety or a linking moiety, to which the at least one other moiety is linked. As used herein, the term "TCO moiety" refers to the trans isomer of the cyclic TCO of a TCO-derivatized targeting moiety prior to IEDDA conjugation.

[0037] As used herein, the term "TCO-derivatized targeting moiety" refers to a targeting moiety derivatized with a trans-cyclooctene (TCO) moiety. In one embodiment, the TCO-derivatized targeting moiety includes a linking moiety between the targeting moiety and the TCO moiety.

[0038] As used herein, the term "tertiary amine" refers to a compound that contains carbon, hydrogen, and nitrogen atoms, where three carbons are attached to the nitrogen of the amine to form three organic substituents. In one embodiment, the tertiary amine is N-(K)3, where each K is independently alkyl or aryl. In one embodiment, the tertiary amine is (-N(CH3)2), where the nitrogen is attached to a third carbon that is part of another part of the compound of which the tertiary amine is a part. In one embodiment, the tertiary amine is (-N(CH3)2), where the nitrogen is attached to a third carbon that is part of the linker part of the tracer compound.

[0039] As used herein, the term "targeting moiety" refers to a peptide, antibody, antibody fragment, or nanoparticle that targets a desired target entity via its sequence and / or 3D (surface) structure. In one embodiment, the targeting moiety is part of a larger structure or compound and can direct or co-locate said compound to the respective target entity in vitro as well as in vivo.

[0040] As used herein, the term "target entity" refers to a sequence and / or 3D (surface) structure that is targeted by and recognized by an adduct, i.e., a targeting moiety, in vitro and / or in vivo, thereby resulting in the co-location of the targeting moiety and the target entity. Examples of target entities are biomolecules.

[0041] As used herein, a "peptide" is an amino acid sequence that contains multiple consecutive polymerized amino acid residues. For purposes of this disclosure, a peptide is a molecule that contains up to 50 amino acid residues. A peptide may contain modified amino acid residues, naturally occurring amino acid residues that are not encoded by a codon, and non-naturally occurring amino acid residues.

[0042] As used herein, the term "antibody" means an immunoglobulin protein that recognizes and binds to an epitope of an antigen through its fragment antigen-binding (Fab) variable region.

[0043] As used herein, the term "radioimaging" refers to methods that utilize radioactive materials to visualize and measure internal physiological structures and activities of macro- or micro-organisms.

[0044] As used herein, the term "positron emission tomography (PET)" refers to an imaging technique that uses radioactive tracers to visualize and measure physiological structures and activities using medical scintillography techniques and detection of gamma rays by a gamma camera. As used herein, the term "positron emission tomography (PET)" also includes positron emission tomography-computed tomography (PET-CT), which further integrates an X-ray computed tomography (CT) scanner for sequential image acquisition, thereby forming a combined single superimposed (co-registered) image.

[0045] As used herein, the term "pre-targeted PET imaging" refers to a two-step PET labeling process, in which a target entity is first targeted and conjugated with a targeting moiety without having a tracer compound attached thereto.This is followed by a second step, in which a radiolabeled tracer compound is delivered into contact with the targeting moiety and is bound to the targeting moiety.In one embodiment, the targeting moiety is a TCO-derivatized targeting moiety, and the binding of the TCO-derivatized targeting moiety to the tracer compound is carried out via IEDDA conjugation.

[0046] As used herein, the term "target tissue-specific uptake" refers to the uptake or binding of a tracer compound or adduct to a target tissue of a subject via binding of a targeting moiety of the adduct to a specific target entity within the target tissue.

[0047] As used herein, the term "tumor-specific uptake" refers to the uptake or binding of a tracer compound or adduct to a target tissue of a subject via binding of a targeting moiety of the adduct to a target entity within the target tissue, where the target tissue is a cancerous tissue.

[0048] As used herein, the term "comprising" includes the broader terms "including," "containing," and "comprehending," as well as the narrower terms "consisting of" and "consisting only of."

[0049] The term "fluorination" refers to a chemical reaction in which fluorine is introduced into a compound. In one embodiment, the fluorine is introduced into a compound by the stable isotope fluorine-19( 19 In another embodiment, the fluorine is a radioisotope. 18 It's F.

[0050] As used herein, the term "organic trifluoroborate" refers to a compound having the general molecular formula [ABF3] - The "A" in the general formula of the organotrifluoroborate can be a positively charged functional group, A+, making the organotrifluoroborate moiety zwitterionic.

[0051] As used herein, the term “[ 18 "F]trifluoroborate" means an organic trifluoroborate in which at least one of the three fluoride atoms [F] is 18 Replaced by F-fluoride isotope.

[0052] As used herein, the term "biomolecule" refers to any medically, physiologically or scientifically important molecule, analogs or derivatives thereof, that may be compatible with living systems or that may or may not possess biological activity.

[0053] As used herein, the term "antibody fragment" means a piece of a whole antibody molecule, such as the antigen-binding fragment of the antibody molecule (Fab) or the crystallizable fragment of the antibody molecule (Fc, tail region).

[0054] As used herein, the term nanoparticle means a substance of any shape having dimensions between 1 and 300 nanometers (nm) in diameter. By way of example, a nanoparticle is an organic nanocrystal, an inorganic nanocrystal, or a liposome.

[0055] As used herein, -(CH2)0- means that there is no CH2 at the indicated position, -(CH2)1- means -CH2-, -(CH2)2- means -CH2-CH2-, -(CH2)3- means -CH2-CH2-CH2- and -(CH2)4- means -CH2-CH2-CH2-CH2-CH2-.

[0056] As used herein, the term “Tyr 3 "-Octreotide" means an octapeptide, i.e., an oligopeptide having eight amino acids, in which the phenylalanine at the third position of octreotide is replaced with tyrosine. Octreotide and Tyr 3 -Octreotide can both pharmacologically mimic natural somatostatin and bind to somatostatin receptors that are overexpressed in neuroendocrine tumors. 3 -Octreotide can be used as a targeting moiety or a model targeting moiety for the somatostatin receptor.

[0057] As used herein, the term "PSMA" refers to prostate specific membrane antigen, which is a type II membrane glycoprotein and is overexpressed in prostate cancer.In relation to compounds 28 and 29 disclosed in the present application, the term "PSMA" refers to PSMA targeting moiety, which can function as a ligand that binds to prostate specific membrane antigen (PSMA).Therefore, compounds 28 and 29 include targeting moiety or targeting moiety model for prostate specific membrane antigen.

[0058] As used herein, the term "tautomer" refers to any of at least two structural isomers of a compound which can exist simultaneously and are readily interchangeable by shifting of an atom or group within the molecule.

[0059] As used herein, the term "non-therapeutic use" refers to a use that is not directed to any therapeutic aspect of disease management. In one embodiment, the term "non-therapeutic use" can refer to a use for diagnostic purposes. As used herein, the term "non-diagnostic use" refers to a use that is not directed to the diagnosis of a disease.

[0060] In one embodiment, the tracer compound comprises a zwitterionic moiety, a linker moiety and a tetrazine moiety. In certain other embodiments, the tracer compound also comprises other moieties or side groups. In one embodiment, the linker moiety is positioned between the zwitterionic moiety and the tetrazine moiety.

[0061] In one embodiment, the zwitterionic portion of the tracer compound comprises an organic trifluoroborate. The organic trifluoroborate has a positive ( + ) bound to a charged (cationic) group (BF3) - More specifically, (BF3) - The group is positive ( + ) with a charged (cationic) group (N(R1)2) + is combined with

[0062] In one embodiment, each R of the tracer compound is independently represented by the formula C n H 2n+1wherein n is an integer selected from the range of 0 to 2. In one embodiment, each R1 is independently an alkyl substituent having a carbon chain length of C2 or less. In one embodiment, each R1 of the tracer compound is independently an alkyl substituent having the formula C1H3. In one embodiment, each R1 of the tracer compound is independently an alkyl substituent having the formula C2H5. In one embodiment, each R1 is independently hydrogen (H). In one embodiment, each R1 is independently a methyl group. In one embodiment, each R1 is independently an ethyl group.

[0063] In one embodiment, each alkyl substituent R1 individually enables nucleophilic attack on the carbon between the nitrogen (N) and the boron (B) in the zwitterionic moiety. In one embodiment, each alkyl substituent R1 is a non-interfering group with respect to the fluorination of boron (B). Non-interfering in this context means that R1 does not sufficiently or substantially prevent the fluorination of boron (B).

[0064] In one embodiment, the linker moiety is composed of the units S1-Y-S2, where Y represents the core unit structure of the linker, S1 and S2 represent the chains on either side of the core unit Y, and the linker moiety on the S1 side is attached to the tetrazine moiety and the linker moiety on the S2 side is attached to the zwitterionic moiety.

[0065] In one embodiment, Y of the tracer compound is (-CH2-). m wherein m is 1, 2, 3, or 4. In one embodiment, m is an integer less than 5.

[0066] In one embodiment, the linker moiety Y is (-CH-). m (wherein m is an integer selected from the range of 1 to 4), and S1 is -(CH2) Z -CO-NH-(CH2) Z - (wherein each z is independently an integer selected from the range of 0 to 4), and S2 is -CH2, and each R1 of the tracer compound is independently a group represented by the formula C n H 2n+1where n is an integer selected from the range of 0 to 2, or hydrogen (H), and R2 of the tracer compound is hydrogen (H) or a phenyl substituent or a group of formula C S H 2S+1 (wherein s is an integer selected from the range of 0 to 2).

[0067] In one embodiment, the linker moiety Y is (-CH-). m (wherein m is an integer selected from the range of 1 to 4), and S1 is -(CH2) Z -CO-NH-(CH2) Z - (wherein each z is independently an integer selected from the range of 0 to 4), and S2 is -(CH2) f -CO-NH-(CH2) f - (wherein each f is independently an integer selected from the range of 0 to 4), and each R1 of the tracer compound is independently H or a compound of the formula C n H 2n+1 where n is an integer selected from the range of 0 to 2, and R2 of the tracer compound is H or a phenyl substituent or a group of formula C S H 2S+1 (wherein s is an integer selected from the range of 0 to 2).

[0068] In one embodiment, the linker moiety Y is (-CH-). m (wherein m is an integer selected from the range of 1 to 4), and S1 is -(CH2) Z -CO-NH-(CH2) Z - (wherein each z is independently an integer selected from the range of 0 to 4), and S2 is -(CH2) f -NH-CO-(CH2) f - (wherein each f is independently an integer selected from the range of 0 to 4), and each R1 of the tracer compound is independently H or a compound of the formula C n H 2n+1where n is an integer selected from the range of 0 to 2, and R2 of the tracer compound is H or a phenyl substituent or a group of formula C S H 2S+1 (wherein s is an integer selected from the range of 0 to 2).

[0069] In one embodiment, the linker moiety Y is (-CH-). m (wherein m is an integer selected from the range of 1 to 4), and S1 is -(CH2) Z -NH-CO-(CH2) Z - (wherein each z is independently an integer selected from the range of 0 to 4), and S2 is -CH2-, and each R1 of the tracer compound is independently H or a group of formula C n H 2n+1 where n is an integer selected from the range of 0 to 2, and R2 of the tracer compound is H or a phenyl substituent or a group of formula C S H 2S+1 (wherein s is an integer selected from the range of 0 to 2).

[0070] In one embodiment, the linker moiety Y is (-CH-). m (wherein m is an integer selected from the range of 1 to 4), and S1 is -(CH2) Z -NH-CO-(CH2) Z - (wherein each z is independently an integer selected from the range of 0 to 4), and S2 is -(CH2) f -CO-NH-(CH2) f - (wherein each f is independently an integer selected from the range of 0 to 4), and each R1 of the tracer compound is independently H or a compound of the formula C n H 2n+1 where n is an integer selected from the range of 0 to 2, and R2 of the tracer compound is H or a phenyl substituent or a group of formula C S H 2S+1(wherein s is an integer selected from the range of 0 to 2).

[0071] In one embodiment, the linker moiety Y is (-CH-). m (wherein m is an integer selected from the range of 1 to 4), and S1 is -(CH2) Z -NH-CO-(CH2) Z - (wherein each z is independently an integer selected from the range of 0 to 4), and S2 is -(CH2) f -NH-CO-(CH2) f - (wherein each f is independently an integer selected from the range of 0 to 4), and each R1 of the tracer compound is independently H or a compound of the formula C n H 2n+1 where n is an integer selected from the range of 0 to 2, and R2 of the tracer compound is H or a phenyl substituent or a group of formula C S H 2S+1 (wherein s is an integer selected from the range of 0 to 2).

[0072] In one embodiment, the linker moiety Y is (-CH-). m(wherein m is an integer selected from the range of 1 to 4), S1 is -(CH2)1-CO-NH-(CH2)0-, and S2 is -CH2. In one embodiment, Y is (-CH2-)1, S1 is -(CH2)1-CO-NH-(CH2)0-, and S2 is -CH2. In one embodiment, Y is (-CH2-)2, S1 is -(CH2)1-CO-NH-(CH2)0-, and S2 is -CH2-. In one embodiment, Y is (-CH2-)3, S1 is -(CH2)1-CO-NH-(CH2)0-, and S2 is -CH2-. In one embodiment, Y is (-CH2-)4, S1 is -(CH2)1-CO-NH-(CH2)0-, and S2 is -CH2-. In one embodiment, Y of the linker moiety is (-CH2-)1, S1 is -(CH2)1-CO-NH-(CH2)0-, S2 is -CH2, both R1 are independently CH3 and R2 is H.

[0073] In one embodiment, the linker moiety Y is (-CH-). m (wherein m is an integer selected from the range of 1 to 4), S1 is -(CH2)1-NH-CO-(CH2)0-, and S2 is -CH2. In one embodiment, Y is (-CH2-)1, S1 is -(CH2)1-NH-CO-(CH2)0-, and S2 is -CH2. In one embodiment, Y is (-CH2-)2, S1 is -(CH2)1-NH-CO-(CH2)0-, and S2 is -CH2-. In one embodiment, Y is (-CH2-)3, S1 is -(CH2)1-NH-CO-(CH2)0-, and S2 is -CH2-. In one embodiment, Y is (-CH2-)4, S1 is -(CH2)1-NH-CO-(CH2)0-, and S2 is -CH2-.

[0074] In one embodiment, the linker moiety Y is -(PEG). X - (wherein x is an integer selected from the range of 1 to 20), and S1 is -(CH2) Z -CO-NH-(CH2) Z- (wherein each z is independently an integer selected from the range of 0 to 4), and S2 is -CH2-, and each R1 of the tracer compound is independently H or a group of formula C n H 2n+1 where n is an integer selected from the range of 0 to 2, and R2 of the tracer compound is H or a phenyl substituent or a group of formula C S H 2S+1 (wherein s is an integer selected from the range of 0 to 2).

[0075] In one embodiment, the linker moiety Y is -(PEG). X - (wherein x is an integer selected from the range of 1 to 20), and S1 is -(CH2) Z -CO-NH-(CH2) Z - (wherein each z is independently an integer selected from the range of 0 to 4), and S2 is -(CH2) f -CO-NH-(CH2) f (wherein each f is independently an integer selected from the range of 0 to 4), and each R1 of the tracer compound is independently H or a compound of the formula C n H 2n+1 where n is an integer selected from the range of 0 to 2, and R2 of the tracer compound is H or a phenyl substituent or a group of formula C S H 2S+1 (wherein s is an integer selected from the range of 0 to 2).

[0076] In one embodiment, the linker moiety Y is -(PEG). X - (wherein x is an integer selected from the range of 1 to 20), and S1 is -(CH2) Z -CO-NH-(CH2) Z - (wherein each z is independently an integer selected from the range of 0 to 4), and S2 is -(CH2) f -NH-CO-(CH2) f(wherein each f is independently an integer selected from the range of 0 to 4), and each R1 of the tracer compound is independently H or a compound of the formula C n H 2n+1 where n is an integer selected from the range of 0 to 2, and R2 of the tracer compound is H or a phenyl substituent or a group of formula C S H 2S+1 (wherein s is an integer selected from the range of 0 to 2).

[0077] In one embodiment, the linker moiety Y is -(PEG). X - (wherein x is an integer selected from the range of 1 to 20), and S1 is -(CH2) Z -NH-CO-(CH2) Z - (wherein each z is independently an integer selected from the range of 0 to 4), and S2 is -CH2-, and each R1 of the tracer compound is independently H or a group of formula C n H 2n+1 where n is an integer selected from the range of 0 to 2, and R2 of the tracer compound is H or a phenyl substituent or a group of formula C S H 2S+1 (wherein s is an integer selected from the range of 0 to 2).

[0078] In one embodiment, the linker moiety Y is -(PEG). X - (wherein x is an integer selected from the range of 1 to 20), and S1 is -(CH2) Z -NH-CO-(CH2) Z - (wherein each z is independently an integer selected from the range of 0 to 4), and S2 is -(CH2) f -CO-NH-(CH2) f (wherein each f is independently an integer selected from the range of 0 to 4), and each R1 of the tracer compound is independently H or a compound of the formula C n H 2n+1where n is an integer selected from the range of 0 to 2, and R2 of the tracer compound is H or a phenyl substituent or a group of formula C S H 2S+1 (wherein s is an integer selected from the range of 0 to 2).

[0079] In one embodiment, the linker moiety Y is -(PEG). X - (wherein x is an integer selected from the range of 1 to 20), and S1 is -(CH2) Z -NH-CO-(CH2) Z - (wherein each z is independently an integer selected from the range of 0 to 4), and S2 is -(CH2) f -NH-CO-(CH2) f (wherein each f is independently an integer selected from the range of 0 to 4), and each R1 of the tracer compound is independently H or a compound of the formula C n H 2n+1 where n is an integer selected from the range of 0 to 2, and R2 of the tracer compound is H or a phenyl substituent or a group of formula C S H 2S+1 (wherein s is an integer selected from the range of 0 to 2).

[0080] In one embodiment, a tracer compound according to formula (I), or a pharma- ceutically acceptable salt or solvate thereof, is provided, wherein: each R is independently hydrogen (H) or a group of formula C n H 2n+1 where n is an integer selected from the range of 0 to 2; L is a linker moiety composed of S1-Y-S2, where: Y is (-CH2-) m (wherein m is an integer selected from the range of 1 to 4), or Y is a polyethylene glycol linker-(PEG) X -(in the formula, (PEG) Xcontains x repeat units of polyethylene oxide -CH2-CH2-O- groups, where x is an integer selected from the range of 1 to 20; and S1 is -CH2-NH-CO-(CH2) Z - or -CH2-CO-NH-(CH2) Z - wherein each z is independently an integer selected from the range of 0 to 4; and S2 is -(CH2) f , or -(CH2) f -CO-NH-CH2-CH2-, or -(CH2) f -NH-CO-CH-CH-, where each f is independently an integer selected from the range of 0 to 4; and R2 is hydrogen (H) or a phenyl substituent or a group of formula C S H 2S+1 (wherein s is an integer selected from the range of 0 to 2).

[0081] In one embodiment there is provided a tracer compound according to formula (I) or a pharma- ceutically acceptable salt or solvate thereof, wherein: Each R1 is independently hydrogen (H) or a group of formula C n H 2n+1 where n is an integer selected from the range of 0 to 2; and L is a linker moiety consisting of S1-Y-S2, where: Y is (-CH2-) m (wherein m is an integer selected from the range of 1 to 4), S1 is -CH2-CO-NH-, or -CH2-NH-CO-, and S2 is -CH2-; or Y is a polyethylene glycol linker (PEG) X -(in the formula, (PEG) X contains x repeat units of polyethylene oxide -CH2-CH2-O- groups, where x is an integer selected from the range of 1 to 20; and S1 is -CH2-NH-CO-(CH2) Z- or -CH2-CO-NH-(CH2) Z - wherein each z is independently an integer selected from the range of 0 to 4; and S2 is -(CH2) f -CO-NH-CH2-CH2- or -(CH2) f -NH-CO-CH-CH-, where each f is independently an integer selected from the range of 0 to 4; and R2 is hydrogen (H) or a phenyl substituent or a group of formula C S H 2S+1 (wherein s is an integer selected from the range of 0 to 2).

[0082] In one embodiment, Y is a polyethylene glycol linker -(PEG). X -(in the formula, (PEG) X comprises x repeat units of polyethylene oxide -CH2-CH2-O- groups, where x is an integer selected from the range of 1 to 20. In one embodiment, the tracer compound -(PEG) X In -, x is an integer selected from the range of 1 to 15 or the range of 1 to 10. In one embodiment, -(PEG) X - in which x is an integer selected from the range of 1 to 9, or from the range of 1 to 8, or from the range of 1 to 7, or from the range of 1 to 6, or from the range of 1 to 5, or from the range of 1 to 4, or from the range of 1 to 3, or from the range of 1 to 2, or x is 1. In one embodiment, the tracer compound -(PEG) X In one embodiment, x in - is 4 or 9. In one embodiment, the tracer compound -(PEG) X -x is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

[0083] In one embodiment, Y is (-CH-). mwherein m is an integer selected from the range of 1 to 4, S1 is -CH2-CO-NH-, and S2 is -CH2-, and each R1 of the tracer compound is independently H or a group of formula C n H 2n+1 where n is an integer selected from the range of 0 to 2, and R2 of the tracer compound is H or a phenyl substituent or a group of formula C S H 2S+1 (wherein s is an integer selected from the range of 0 to 2).

[0084] In one embodiment, Y is (-CH-). m wherein m is an integer selected from the range of 1 to 4, S1 is -CH2-NH-CO-, and S2 is -CH2-, and each R1 of the tracer compound is independently H or a group of formula C n H 2n+1 where n is an integer selected from the range of 0 to 2, and R2 of the tracer compound is H or a phenyl substituent or a group of formula C S H 2S+1 (wherein s is an integer selected from the range of 0 to 2).

[0085] In one embodiment, Y is (-CH2-)1, S1 is -CH2-CO-NH- or -CH2-NH-CO-, S2 is -CH2-, and each R1 is independently H or a group of formula C n H 2n+1 wherein n is an integer selected from the range of 0 to 2, and R2 is a phenyl substituent.

[0086] In one embodiment, Y is (-CH2-)1, S1 is -CH2-CO-NH- or -CH2-NH-CO-, S2 is -CH2-, and each R1 is independently H or a group of formula C n H 2n+1 wherein n is an integer selected from the range of 0 to 2, and R2 is H.

[0087] In one embodiment, Y is (-CH2-)1, S1 is -CH2-CO-NH- or -CH2-NH-CO-, S2 is -CH2-, and each R1 is independently H or a group of formula C n H 2n+1 wherein n is an integer selected from the range of 0 to 2, and R2 is an alkyl substituent having the formula C1H3.

[0088] In one embodiment, Y is (-CH2-)1, S1 is -CH2-CO-NH- or -CH2-NH-CO-, S2 is -CH2-, and each R1 is independently H or a group of formula C n H 2n+1 where n is an integer selected from the range of 0 to 2, and R2 is an alkyl substituent having the formula C2H5.

[0089] In one embodiment, Y is (-CH-), S is -CH-CO-NH- or -CH-NH-CO-, S is -CH-, and each R of the tracer compound is independently H or a group of formula C n H 2n+1 wherein n is an integer selected from the range of 0 to 2, and R2 is a phenyl substituent.

[0090] In one embodiment, Y is (-CH-), S is -CH-CO-NH- or -CH-NH-CO-, S is -CH-, and each R of the tracer compound is independently H or a group of formula C n H 2n+1 wherein n is an integer selected from the range of 0 to 2, and R2 is H.

[0091] In one embodiment, Y is (-CH-), S is -CH-CO-NH- or -CH-NH-CO-, S is -CH-, and each R of the tracer compound is independently H or a group of formula C n H 2n+1wherein n is an integer selected from the range of 0 to 2, and R2 is an alkyl substituent having the formula C1H3.

[0092] In one embodiment, Y is (-CH-), S is -CH-CO-NH- or -CH-NH-CO-, S is -CH-, and each R of the tracer compound is independently H or a group of formula C n H 2n+1 where n is an integer selected from the range of 0 to 2, and R2 is an alkyl substituent having the formula C2H5.

[0093] In one embodiment, Y is (-CH2-)3, S1 is -CH2-CO-NH- or -CH2-NH-CO-, S2 is -CH2-, and each R1 of the tracer compound is independently H or a group of formula C n H 2n+1 wherein n is an integer selected from the range of 0 to 2, and R2 is a phenyl substituent.

[0094] In one embodiment, Y is (-CH2-)3, S1 is -CH2-CO-NH- or -CH2-NH-CO-, S2 is -CH2-, and each R1 of the tracer compound is independently H or a group of formula C n H 2n+1 wherein n is an integer selected from the range of 0 to 2, and R2 is H.

[0095] In one embodiment, Y is (-CH2-)3, S1 is -CH2-CO-NH- or -CH2-NH-CO-, S2 is -CH2-, and each R1 of the tracer compound is independently H or a group of formula C n H 2n+1 wherein n is an integer selected from the range of 0 to 2, and R2 is an alkyl substituent having the formula C1H3.

[0096] In one embodiment, Y is (-CH2-)3, S1 is -CH2-CO-NH- or -CH2-NH-CO-, S2 is -CH2-, and each R1 of the tracer compound is independently H or a group of formula C n H 2n+1 where n is an integer selected from the range of 0 to 2, and R2 is an alkyl substituent having the formula C2H5.

[0097] In one embodiment, Y is (-CH2-)4, S1 is -CH2-CO-NH- or -CH2-NH-CO-, S2 is -CH2-, and each R1 of the tracer compound is independently H or a group of formula C n H 2n+1 wherein n is an integer selected from the range of 0 to 2, and R2 is a phenyl substituent.

[0098] In one embodiment, Y is (-CH2-)4, S1 is -CH2-CO-NH- or -CH2-NH-CO-, S2 is -CH2-, and each R1 of the tracer compound is independently H or a group of formula C n H 2n+1 wherein n is an integer selected from the range of 0 to 2, and R2 is H.

[0099] In one embodiment, Y is (-CH2-)4, S1 is -CH2-CO-NH- or -CH2-NH-CO-, S2 is -CH2-, and each R1 of the tracer compound is independently H or a group of formula C n H 2n+1 wherein n is an integer selected from the range of 0 to 2, and R2 is an alkyl substituent having the formula C1H3.

[0100] In one embodiment, Y is (-CH2-)4, S1 is -CH2-CO-NH- or -CH2-NH-CO-, S2 is -CH2-, and each R1 of the tracer compound is independently H or a group of formula C n H 2n+1where n is an integer selected from the range of 0 to 2, and R2 is an alkyl substituent having the formula C2H5.

[0101] In one embodiment, each R1 of the tracer compound is independently C1H3, Y is (-CH2-)1, S1 is -CH2-CO-NH-, S2 is -CH2-, and R2 is H.

[0102] In one embodiment, Y is -(PEG) X -, S1 is -CH2-NH-CO-(CH2)0-, and S2 is -(CH2)0-CO-NH-CH2-CH2-. In one embodiment, Y is -(PEG) X -, S1 is -CH2-NH-CO-(CH2)1-, and S2 is -(CH2)0-CO-NH-CH2-CH2-. In one embodiment, Y is -(PEG) X -, S1 is -CH2-NH-CO-(CH2)2-, and S2 is -(CH2)0-CO-NH-CH2-CH2-. In one embodiment, Y is -(PEG) X -, S1 is -CH2-NH-CO-(CH2)3-, and S2 is -(CH2)0-CO-NH-CH2-CH2-. In one embodiment, Y is -(PEG) X -, S1 is -CH2-NH-CO-(CH2)4-, and S2 is -(CH2)0-CO-NH-CH2-CH2-.

[0103] In one embodiment, Y is -(PEG) X -, S1 is -CH2-NH-CO-(CH2)0-, and S2 is -(CH2)1-CO-NH-CH2-CH2-. In one embodiment, Y is -(PEG) X -, S1 is -CH2-NH-CO-(CH2)1-, and S2 is -(CH2)1-CO-NH-CH2-CH2-. In one embodiment, Y is -(PEG) X-, S1 is -CH2-NH-CO-(CH2)2-, and S2 is -(CH2)1-CO-NH-CH2-CH2-. In one embodiment, Y is -(PEG) X -, S1 is -CH2-NH-CO-(CH2)3-, and S2 is -(CH2)1-CO-NH-CH2-CH2-. In one embodiment, Y is -(PEG) X -, S1 is -CH2-NH-CO-(CH2)4-, and S2 is -(CH2)1-CO-NH-CH2-CH2-.

[0104] In one embodiment, Y is -(PEG) X -, S1 is -CH2-NH-CO-(CH2)0-, and S2 is -(CH2)2-CO-NH-CH2-CH2-. In one embodiment, Y is -(PEG) X -, S1 is -CH2-NH-CO-(CH2)1-, and S2 is -(CH2)2-CO-NH-CH2-CH2-. In one embodiment, Y is -(PEG) X -, S1 is -CH2-NH-CO-(CH2)2-, and S2 is -(CH2)2-CO-NH-CH2-CH2-. In one embodiment, Y is -(PEG) X -, S1 is -CH2-NH-CO-(CH2)3-, and S2 is -(CH2)2-CO-NH-CH2-CH2-. In one embodiment, Y is -(PEG) X -, S1 is -CH2-NH-CO-(CH2)4-, and S2 is -(CH2)2-CO-NH-CH2-CH2-.

[0105] In one embodiment, Y is -(PEG) X -, S1 is -CH2-NH-CO-(CH2)0-, and S2 is -(CH2)3-CO-NH-CH2-CH2-. In one embodiment, Y is -(PEG) X -, S1 is -CH2-NH-CO-(CH2)1-, and S2 is -(CH2)3-CO-NH-CH2-CH2-. In one embodiment, Y is -(PEG) X-, S1 is -CH2-NH-CO-(CH2)2-, and S2 is -(CH2)3-CO-NH-CH2-CH2-. In one embodiment, Y is -(PEG) X -, S1 is -CH2-NH-CO-(CH2)3-, and S2 is -(CH2)3-CO-NH-CH2-CH2-. In one embodiment, Y is -(PEG) X -, S1 is -CH2-NH-CO-(CH2)4-, and S2 is -(CH2)3-CO-NH-CH2-CH2-.

[0106] In one embodiment, Y is -(PEG) X -, S1 is -CH2-NH-CO-(CH2)0-, and S2 is -(CH2)4-CO-NH-CH2-CH2-. In one embodiment, Y is -(PEG) X -, S1 is -CH2-NH-CO-(CH2)1-, and S2 is -(CH2)4-CO-NH-CH2-CH2-. In one embodiment, Y is -(PEG) X -, S1 is -CH2-NH-CO-(CH2)2-, and S2 is -(CH2)4-CO-NH-CH2-CH2-. In one embodiment, Y is -(PEG) X -, S1 is -CH2-NH-CO-(CH2)3-, and S2 is -(CH2)4-CO-NH-CH2-CH2-. In one embodiment, Y is -(PEG) X -, S1 is -CH2-NH-CO-(CH2)4-, and S2 is -(CH2)4-CO-NH-CH2-CH2-.

[0107] In one embodiment, Y is -(PEG) X -, S1 is -CH2-NH-CO-(CH2)0-, and S2 is -(CH2)0-NH-CO-CH2-CH2-. In one embodiment, Y is -(PEG) X -, S1 is -CH2-NH-CO-(CH2)1-, and S2 is -(CH2)0-NH-CO-CH2-CH2-. In one embodiment, Y is -(PEG) X-, S1 is -CH2-NH-CO-(CH2)2-, and S2 is -(CH2)0-NH-CO-CH2-CH2-. In one embodiment, Y is -(PEG) X -, S1 is -CH2-NH-CO-(CH2)3-, and S2 is -(CH2)0-NH-CO-CH2-CH2-. In one embodiment, Y is -(PEG) X -, S1 is -CH2-NH-CO-(CH2)4-, and S2 is -(CH2)0-NH-CO-CH2-CH2-.

[0108] In one embodiment, Y is -(PEG) X -, S1 is -CH2-NH-CO-(CH2)0-, and S2 is -(CH2)1-NH-CO-CH2-CH2-. In one embodiment, Y is -(PEG) X -, S1 is -CH2-NH-CO-(CH2)1-, and S2 is -(CH2)1-NH-CO-CH2-CH2-. In one embodiment, Y is -(PEG) X -, S1 is -CH2-NH-CO-(CH2)2-, and S2 is -(CH2)1-NH-CO-CH2-CH2-. In one embodiment, Y is -(PEG) X -, S1 is -CH2-NH-CO-(CH2)3-, and S2 is -(CH2)1-NH-CO-CH2-CH2-. In one embodiment, Y is -(PEG) X -, S1 is -CH2-NH-CO-(CH2)4-, and S2 is -(CH2)1-NH-CO-CH2-CH2-.

[0109] In one embodiment, Y is -(PEG) X -, S1 is -CH2-NH-CO-(CH2)0-, and S2 is -(CH2)2-NH-CO-CH2-CH2-. In one embodiment, Y is -(PEG) X -, S1 is -CH2-NH-CO-(CH2)1-, and S2 is -(CH2)2-NH-CO-CH2-CH2-. In one embodiment, Y is -(PEG) X-, S1 is -CH2-NH-CO-(CH2)2-, and S2 is -(CH2)2-NH-CO-CH2-CH2-. In one embodiment, Y is -(PEG) X -, S1 is -CH2-NH-CO-(CH2)3-, and S2 is -(CH2)2-NH-CO-CH2-CH2-. In one embodiment, Y is -(PEG) X -, S1 is -CH2-NH-CO-(CH2)4-, and S2 is -(CH2)2-NH-CO-CH2-CH2-.

[0110] In one embodiment, Y is -(PEG) X -, S1 is -CH2-NH-CO-(CH2)0-, and S2 is -(CH2)3-NH-CO-CH2-CH2-. In one embodiment, Y is -(PEG) X -, S1 is -CH2-NH-CO-(CH2)1-, and S2 is -(CH2)3-NH-CO-CH2-CH2-. In one embodiment, Y is -(PEG) X -, S1 is -CH2-NH-CO-(CH2)2-, and S2 is -(CH2)3-NH-CO-CH2-CH2-. In one embodiment, Y is -(PEG) X -, S1 is -CH2-NH-CO-(CH2)3-, and S2 is -(CH2)3-NH-CO-CH2-CH2-. In one embodiment, Y is -(PEG) X -, S1 is -CH2-NH-CO-(CH2)4-, and S2 is -(CH2)3-NH-CO-CH2-CH2-.

[0111] In one embodiment, Y is -(PEG) X -, S1 is -CH2-NH-CO-(CH2)0-, and S2 is -(CH2)4-NH-CO-CH2-CH2-. In one embodiment, Y is -(PEG) X -, S1 is -CH2-NH-CO-(CH2)1-, and S2 is -(CH2)4-NH-CO-CH2-CH2-. In one embodiment, Y is -(PEG) X-, S1 is -CH2-NH-CO-(CH2)2-, and S2 is -(CH2)4-NH-CO-CH2-CH2-. In one embodiment, Y is -(PEG) X -, S1 is -CH2-NH-CO-(CH2)3-, and S2 is -(CH2)4-NH-CO-CH2-CH2-. In one embodiment, Y is -(PEG) X -, S1 is -CH2-NH-CO-(CH2)4-, and S2 is -(CH2)4-NH-CO-CH2-CH2-.

[0112] In one embodiment, Y is -(PEG) X -, S1 is -CH2-CO-NH-(CH2)0-, and S2 is -(CH2)0-CO-NH-CH2-CH2. In one embodiment, Y is -(PEG) X -, S1 is -CH2-CO-NH-(CH2)1-, and S2 is -(CH2)0-CO-NH-CH2-CH2. In one embodiment, Y is -(PEG) X -, S1 is -CH2-CO-NH-(CH2)2-, and S2 is -(CH2)0-CO-NH-CH2-CH2-. In one embodiment, Y is -(PEG) X -, S1 is -CH2-CO-NH-(CH2)3-, and S2 is -(CH2)0-CO-NH-CH2-CH2-. In one embodiment, Y is -(PEG) X -, S1 is -CH2-CO-NH-(CH2)4-, and S2 is -(CH2)0-CO-NH-CH2-CH2-.

[0113] In one embodiment, Y is -(PEG) X -, S1 is -CH2-CO-NH-(CH2)0-, and S2 is -(CH2)1-CO-NH-CH2-CH2. In one embodiment, Y is -(PEG) X -, S1 is -CH2-CO-NH-(CH2)1-, and S2 is -(CH2)1-CO-NH-CH2-CH2-. In one embodiment, Y is -(PEG) X-, S1 is -CH2-CO-NH-(CH2)2-, and S2 is -(CH2)1-CO-NH-CH2-CH2-. In one embodiment, Y is -(PEG) X -, S1 is -CH2-CO-NH-(CH2)3-, and S2 is -(CH2)1-CO-NH-CH2-CH2-. In one embodiment, Y is -(PEG) X -, S1 is -CH2-CO-NH-(CH2)4-, and S2 is -(CH2)1-CO-NH-CH2-CH2-.

[0114] In one embodiment, Y is -(PEG) X -, S1 is -CH2-CO-NH-(CH2)0-, and S2 is -(CH2)2-CO-NH-CH2-CH2-. In one embodiment, Y is -(PEG) X -, S1 is -CH2-CO-NH-(CH2)1-, and S2 is -(CH2)2-CO-NH-CH2-CH2-. In one embodiment, Y is -(PEG) X -, S1 is -CH2-CO-NH-(CH2)2-, and S2 is -(CH2)2-CO-NH-CH2-CH2-. In one embodiment, Y is -(PEG) X -, S1 is -CH2-CO-NH-(CH2)3-, and S2 is -(CH2)2-CO-NH-CH2-CH2-. In one embodiment, Y is -(PEG) X -, S1 is -CH2-CO-NH-(CH2)4-, and S2 is -(CH2)2-CO-NH-CH2-CH2-.

[0115] In one embodiment, Y is -(PEG) X -, S1 is -CH2-CO-NH-(CH2)0-, and S2 is -(CH2)3-CO-NH-CH2-CH2-. In one embodiment, Y is -(PEG) X -, S1 is -CH2-CO-NH-(CH2)1-, and S2 is -(CH2)3-CO-NH-CH2-CH2-. In one embodiment, Y is -(PEG) X-, S1 is -CH2-CO-NH-(CH2)2-, and S2 is -(CH2)3-CO-NH-CH2-CH2-. In one embodiment, Y is -(PEG) X -, S1 is -CH2-CO-NH-(CH2)3-, and S2 is -(CH2)3-CO-NH-CH2-CH2-. In one embodiment, Y is -(PEG) X -, S1 is -CH2-CO-NH-(CH2)4-, and S2 is -(CH2)3-CO-NH-CH2-CH2-.

[0116] In one embodiment, Y is -(PEG) X -, S1 is -CH2-CO-NH-(CH2)0-, and S2 is -(CH2)4-CO-NH-CH2-CH2-. In one embodiment, Y is -(PEG) X -, S1 is -CH2-CO-NH-(CH2)1-, and S2 is -(CH2)4-CO-NH-CH2-CH2-. In one embodiment, Y is -(PEG) X -, S1 is -CH2-CO-NH-(CH2)2-, and S2 is -(CH2)4-CO-NH-CH2-CH2-. In one embodiment, Y is -(PEG) X -, S1 is -CH2-CO-NH-(CH2)3-, and S2 is -(CH2)4-CO-NH-CH2-CH2-. In one embodiment, Y is -(PEG) X -, S1 is -CH2-CO-NH-(CH2)4-, and S2 is -(CH2)4-CO-NH-CH2-CH2-.

[0117] In one embodiment, Y is -(PEG) X -, S1 is -CH2-CO-NH-(CH2)0-, and S2 is -(CH2)0-NH-CO-CH2-CH2-. In one embodiment, Y is -(PEG) X -, S1 is -CH2-CO-NH-(CH2)1-, and S2 is -(CH2)0-NH-CO-CH2-CH2-. In one embodiment, Y is -(PEG) X-, S1 is -CH2-CO-NH-(CH2)2-, and S2 is -(CH2)0-NH-CO-CH2-CH2-. In one embodiment, Y is -(PEG) X -, S1 is -CH2-CO-NH-(CH2)3-, and S2 is -(CH2)0-NH-CO-CH2-CH2-. In one embodiment, Y is -(PEG) X -, S1 is -CH2-CO-NH-(CH2)4-, and S2 is -(CH2)0-NH-CO-CH2-CH2-.

[0118] In one embodiment, Y is -(PEG) X -, S1 is -CH2-CO-NH-(CH2)0-, and S2 is -(CH2)1-NH-CO-CH2-CH2-. In one embodiment, Y is -(PEG) X -, S1 is -CH2-CO-NH-(CH2)1-, and S2 is -(CH2)1-NH-CO-CH2-CH2-. In one embodiment, Y is -(PEG) X -, S1 is -CH2-CO-NH-(CH2)2-, and S2 is -(CH2)1-NH-CO-CH2-CH2-. In one embodiment, Y is -(PEG) X -, S1 is -CH2-CO-NH-(CH2)3-, and S2 is -(CH2)1-NH-CO-CH2-CH2-. In one embodiment, Y is -(PEG) X -, S1 is -CH2-CO-NH-(CH2)4-, and S2 is -(CH2)1-NH-CO-CH2-CH2-.

[0119] In one embodiment, Y is -(PEG) X -, S1 is -CH2-CO-NH-(CH2)0-, and S2 is -(CH2)2-NH-CO-CH2-CH2-. In one embodiment, Y is -(PEG) X -, S1 is -CH2-CO-NH-(CH2)1-, and S2 is -(CH2)2-NH-CO-CH2-CH2-. In one embodiment, Y is -(PEG) X-, S1 is -CH2-CO-NH-(CH2)2-, and S2 is -(CH2)2-NH-CO-CH2-CH2-. In one embodiment, Y is -(PEG) X -, S1 is -CH2-CO-NH-(CH2)3-, and S2 is -(CH2)2-NH-CO-CH2-CH2-. In one embodiment, Y is -(PEG) X -, S1 is -CH2-CO-NH-(CH2)4-, and S2 is -(CH2)2-NH-CO-CH2-CH2-.

[0120] In one embodiment, Y is -(PEG) X -, S1 is -CH2-CO-NH-(CH2)0-, and S2 is -(CH2)3-NH-CO-CH2-CH2-. In one embodiment, Y is -(PEG) X -, S1 is -CH2-CO-NH-(CH2)1-, and S2 is -(CH2)3-NH-CO-CH2-CH2-. In one embodiment, Y is -(PEG) X -, S1 is -CH2-CO-NH-(CH2)2-, and S2 is -(CH2)3-NH-CO-CH2-CH2-. In one embodiment, Y is -(PEG) X -, S1 is -CH2-CO-NH-(CH2)3-, and S2 is -(CH2)3-NH-CO-CH2-CH2-. In one embodiment, Y is -(PEG) X -, S1 is -CH2-CO-NH-(CH2)4-, and S2 is -(CH2)3-NH-CO-CH2-CH2-.

[0121] In one embodiment, Y is -(PEG) X -, S1 is -CH2-CO-NH-(CH2)0-, and S2 is -(CH2)4-NH-CO-CH2-CH2-. In one embodiment, Y is -(PEG) X -, S1 is -CH2-CO-NH-(CH2)1-, and S2 is -(CH2)4-NH-CO-CH2-CH2-. In one embodiment, Y is -(PEG) X-, S1 is -CH2-CO-NH-(CH2)2-, and S2 is -(CH2)4-NH-CO-CH2-CH2-. In one embodiment, Y is -(PEG) X -, S1 is -CH2-CO-NH-(CH2)3-, and S2 is -(CH2)4-NH-CO-CH2-CH2-. In one embodiment, Y is -(PEG) X -, S1 is -CH2-CO-NH-(CH2)4-, and S2 is -(CH2)4-NH-CO-CH2-CH2-.

[0122] In one embodiment, Y is a polyethylene glycol linker -(PEG). X where (PEG) X contains four repeating units of polyethylene oxide -CH2-CH2-O- groups, and S1 is -CH2-NH-CO-(CH2) Z - and S2 is -(CH2) f —CO—NH—CH2—CH2—, where z and f are each independently 0 or 2.

[0123] In one embodiment, Y is a polyethylene glycol linker -(PEG). X where (PEG) X contains four repeating units of polyethylene oxide -CH2-CH2-O- groups, and S1 is -CH2-NH-CO-(CH2) Z - and S2 is -(CH2) f —NH—CO—CH2—CH2—, where z and f are each independently 0 or 2.

[0124] In one embodiment, each R1 of the tracer compound is independently a methyl group, Y is a polyethylene glycol linker -(PEG)4- comprising four repeating units of polyethylene oxide -CH2-CH2-O- groups, S1 is -CH2-NH-CO-(CH2)0-, and S2 is -(CH2)2-CO-NH-CH2-CH2-, and R2 is H.

[0125] In one embodiment, each R1 of the tracer compound is independently a methyl group, Y is a polyethylene glycol linker -(PEG)4- comprising four repeating units of polyethylene oxide -CH2-CH2-O- groups, S1 is -CH2-NH-CO-(CH2)0-, and S2 is -(CH2)2-NH-CO-CH2-CH2-, and R2 is H.

[0126] In one embodiment, Y is -(PEG)4-, S1 is -CH2-NH-CO-(CH2)0-, and S2 is -(CH2)0-CO-NH-CH2-CH2-. In one embodiment, Y is -(PEG)4-, S1 is -CH2-NH-CO-(CH2)0-, and S2 is -(CH2)1-CO-NH-CH2-CH2-. In one embodiment, Y is -(PEG)4-, S1 is -CH2-NH-CO-(CH2)0-, and S2 is -(CH2)2-CO-NH-CH2-CH2-. In one embodiment, Y is -(PEG)4-, S1 is -CH2-NH-CO-(CH2)0-, and S2 is -(CH2)2-CO-NH-CH2-CH2-. In one embodiment, Y is -(PEG)4-, S1 is -CH2-NH-CO-(CH2)1-, and S2 is -(CH2)0-CO-NH-CH2-CH2-. In one embodiment, Y is -(PEG)4-, S1 is -CH2-NH-CO-(CH2)1-, and S2 is -(CH2)1-CO-NH-CH2-CH2-. In one embodiment, Y is -(PEG)4-, S1 is -CH2-NH-CO-(CH2)1-, and S2 is -(CH2)2-CO-NH-CH2-CH2-. In one embodiment, Y is -(PEG)4-, S1 is -CH2-NH-CO-(CH2)2-, and S2 is -(CH2)0-CO-NH-CH2-CH2-. In one embodiment, Y is -(PEG)4-, S1 is -CH2-NH-CO-(CH2)2-, and S2 is -(CH2)1-CO-NH-CH2-CH2-. In one embodiment, Y is -(PEG)-, S1 is -CH2-NH-CO-(CH2)2-, and S2 is -(CH2)2-CO-NH-CH2-CH2-.

[0127] In one embodiment, Y is -(PEG)4-, S1 is -CH2-NH-CO-(CH2)0-, and S2 is -(CH2)0-NH-CO-CH2-CH2-. In one embodiment, Y is -(PEG)4-, S1 is -CH2-NH-CO-(CH2)0-, and S2 is -(CH2)1-NH-CO-CH2-CH2-. In one embodiment, Y is -(PEG)4-, S1 is -CH2-NH-CO-(CH2)0-, and S2 is -(CH2)2-NH-CO-CH2-CH2-. In one embodiment, Y is -(PEG)4-, S1 is -CH2-NH-CO-(CH2)0-, and S2 is -(CH2)2-NH-CO-CH2-CH2-. In one embodiment, Y is -(PEG)4-, S1 is -CH2-NH-CO-(CH2)1-, and S2 is -(CH2)0-NH-CO-CH2-CH2-. In one embodiment, Y is -(PEG)4-, S1 is -CH2-NH-CO-(CH2)1-, and S2 is -(CH2)1-NH-CO-CH2-CH2-. In one embodiment, Y is -(PEG)4-, S1 is -CH2-NH-CO-(CH2)1-, and S2 is -(CH2)2-NH-CO-CH2-CH2-. In one embodiment, Y is -(PEG)4-, S1 is -CH2-NH-CO-(CH2)2-, and S2 is -(CH2)0-NH-CO-CH2-CH2-. In one embodiment, Y is -(PEG)4-, S1 is -CH2-NH-CO-(CH2)2-, and S2 is -(CH2)0-NH-CO-CH2-CH2-. In one embodiment, Y is -(PEG)4-, S1 is -CH2-NH-CO-(CH2)2-, and S2 is -(CH2)1-NH-CO-CH2-CH2-. In one embodiment, Y is -(PEG)-, S1 is -CH2-NH-CO-(CH2)2-, and S2 is -(CH2)2-NH-CO-CH2-CH2-.

[0128] In one embodiment, Y is -(PEG)4-, S1 is -CH2-CO-NH-(CH2)0-, and S2 is -(CH2)0-CO-NH-CH2-CH2-. In one embodiment, Y is -(PEG)4-, S1 is -CH2-CO-NH-(CH2)0-, and S2 is -(CH2)1-CO-NH-CH2-CH2-. In one embodiment, Y is -(PEG)4-, S1 is -CH2-CO-NH-(CH2)0-, and S2 is -(CH2)2-CO-NH-CH2-CH2-. In one embodiment, Y is -(PEG)4-, S1 is -CH2-CO-NH-(CH2)0-, and S2 is -(CH2)2-CO-NH-CH2-CH2-. In one embodiment, Y is -(PEG)4-, S1 is -CH2-CO-NH-(CH2)1-, and S2 is -(CH2)0-CO-NH-CH2-CH2-. In one embodiment, Y is -(PEG)4-, S1 is -CH2-CO-NH-(CH2)1-, and S2 is -(CH2)1-CO-NH-CH2-CH2-. In one embodiment, Y is -(PEG)4-, S1 is -CH2-CO-NH-(CH2)1-, and S2 is -(CH2)2-CO-NH-CH2-CH2-. In one embodiment, Y is -(PEG)4-, S1 is -CH2-CO-NH-(CH2)2-, and S2 is -(CH2)0-CO-NH-CH2-CH2-. In one embodiment, Y is -(PEG)4-, S1 is -CH2-CO-NH-(CH2)2-, and S2 is -(CH2)1-CO-NH-CH2-CH2-. In one embodiment, Y is -(PEG)-, S1 is -CH2-CO-NH-(CH2)2-, and S2 is -(CH2)2-CO-NH-CH2-CH2-.

[0129] In one embodiment, Y is -(PEG)4-, S1 is -CH2-CO-NH-(CH2)0-, and S2 is -(CH2)0-NH-CO-CH2-CH2-. In one embodiment, Y is -(PEG)4-, S1 is -CH2-CO-NH-(CH2)0-, and S2 is -(CH2)1-NH-CO-CH2-CH2-. In one embodiment, Y is -(PEG)4-, S1 is -CH2-CO-NH-(CH2)0-, and S2 is -(CH2)2-NH-CO-CH2-CH2-. In one embodiment, Y is -(PEG)4-, S1 is -CH2-CO-NH-(CH2)0-, and S2 is -(CH2)2-NH-CO-CH2-CH2-. In one embodiment, Y is -(PEG)4-, S1 is -CH2-CO-NH-(CH2)1-, and S2 is -(CH2)0-NH-CO-CH2-CH2-. In one embodiment, Y is -(PEG)4-, S1 is -CH2-CO-NH-(CH2)1-, and S2 is -(CH2)1-NH-CO-CH2-CH2-. In one embodiment, Y is -(PEG)4-, S1 is -CH2-CO-NH-(CH2)1-, and S2 is -(CH2)2-NH-CO-CH2-CH2-. In one embodiment, Y is -(PEG)4-, S1 is -CH2-CO-NH-(CH2)2-, and S2 is -(CH2)0-NH-CO-CH2-CH2-. In one embodiment, Y is -(PEG)4-, S1 is -CH2-CO-NH-(CH2)2-, and S2 is -(CH2)0-NH-CO-CH2-CH2-. In one embodiment, Y is -(PEG)4-, S1 is -CH2-CO-NH-(CH2)2-, and S2 is -(CH2)1-NH-CO-CH2-CH2-. In one embodiment, Y is -(PEG)-, S1 is -CH2-CO-NH-(CH2)2-, and S2 is -(CH2)2-NH-CO-CH2-CH2-.

[0130] In one embodiment, Y is a polyethylene glycol linker -(PEG). X where (PEG) X contains nine repeating units of polyethylene oxide -CH2-CH2-O- groups, and S1 is -CH2-NH-CO-(CH2) Z - and S2 is -(CH2) f —CO—NH—CH2—CH2—, where z and f are each independently 0 or 2.

[0131] In one embodiment, Y is a polyethylene glycol linker -(PEG). X where (PEG) X contains nine repeating units of polyethylene oxide -CH2-CH2-O- groups, and S1 is -CH2-NH-CO-(CH2) Z - and S2 is -(CH2) f —NH—CO—CH2—CH2—, where z and f are each independently 0 or 2.

[0132] In one embodiment, each R1 of the tracer compound is independently a methyl group, Y is a polyethylene glycol linker -(PEG)9- containing nine repeating units of polyethylene oxide -CH2-CH2-O- groups, S1 is -CH2-NH-CO-(CH2)0-, and S2 is -(CH2)2-CO-NO-CH2-CH2-, and R2 is H.

[0133] In one embodiment, each R1 of the tracer compound is independently a methyl group, Y is a polyethylene glycol linker -(PEG)9- containing nine repeating units of polyethylene oxide -CH2-CH2-O- groups, S1 is -CH2-NH-CO-(CH2)0-, and S2 is -(CH2)2-NH-CO-CH2-CH2-, and R2 is H.

[0134] In one embodiment, Y is -(PEG)9-, S1 is -CH2-NH-CO-(CH2)0-, and S2 is -(CH2)0-CO-NH-CH2-CH2-. In one embodiment, Y is -(PEG)9-, S1 is -CH2-NH-CO-(CH2)0-, and S2 is -(CH2)1-CO-NH-CH2-CH2-. In one embodiment, Y is -(PEG)9-, S1 is -CH2-NH-CO-(CH2)0-, and S2 is -(CH2)2-CO-NH-CH2-CH2-. In one embodiment, Y is -(PEG)9-, S1 is -CH2-NH-CO-(CH2)0-, and S2 is -(CH2)2-CO-NH-CH2-CH2-. In one embodiment, Y is -(PEG)9-, S1 is -CH2-NH-CO-(CH2)1-, and S2 is -(CH2)0-CO-NH-CH2-CH2-. In one embodiment, Y is -(PEG)9-, S1 is -CH2-NH-CO-(CH2)1-, and S2 is -(CH2)1-CO-NH-CH2-CH2-. In one embodiment, Y is -(PEG)9-, S1 is -CH2-NH-CO-(CH2)1-, and S2 is -(CH2)2-CO-NH-CH2-CH2-. In one embodiment, Y is -(PEG)9-, S1 is -CH2-NH-CO-(CH2)2-, and S2 is -(CH2)0-CO-NH-CH2-CH2-. In one embodiment, Y is -(PEG)9-, S1 is -CH2-NH-CO-(CH2)2-, and S2 is -(CH2)1-CO-NH-CH2-CH2-. In one embodiment, Y is -(PEG)9-, S1 is -CH2-NH-CO-(CH2)2-, and S2 is -(CH2)2-CO-NH-CH2-CH2-.

[0135] In one embodiment, Y is -(PEG)9-, S1 is -CH2-NH-CO-(CH2)0-, and S2 is -(CH2)0-NH-CO-CH2-CH2-. In one embodiment, Y is -(PEG)9-, S1 is -CH2-NH-CO-(CH2)0-, and S2 is -(CH2)1-NH-CO-CH2-CH2-. In one embodiment, Y is -(PEG)9-, S1 is -CH2-NH-CO-(CH2)0-, and S2 is -(CH2)2-NH-CO-CH2-CH2-. In one embodiment, Y is -(PEG)9-, S1 is -CH2-NH-CO-(CH2)0-, and S2 is -(CH2)2-NH-CO-CH2-CH2-. In one embodiment, Y is -(PEG)9-, S1 is -CH2-NH-CO-(CH2)1-, and S2 is -(CH2)0-NH-CO-CH2-CH2-. In one embodiment, Y is -(PEG)9-, S1 is -CH2-NH-CO-(CH2)1-, and S2 is -(CH2)1-NH-CO-CH2-CH2-. In one embodiment, Y is -(PEG)9-, S1 is -CH2-NH-CO-(CH2)1-, and S2 is -(CH2)2-NH-CO-CH2-CH2-. In one embodiment, Y is -(PEG)9-, S1 is -CH2-NH-CO-(CH2)2-, and S2 is -(CH2)0-NH-CO-CH2-CH2-. In one embodiment, Y is -(PEG)9-, S1 is -CH2-NH-CO-(CH2)2-, and S2 is -(CH2)1-NH-CO-CH2-CH2-. In one embodiment, Y is -(PEG)9-, S1 is -CH2-NH-CO-(CH2)2-, and S2 is -(CH2)2-NH-CO-CH2-CH2-.

[0136] In one embodiment, Y is -(PEG)9-, S1 is -CH2-CO-NH-(CH2)0-, and S2 is -(CH2)0-CO-NH-CH2-CH2-. In one embodiment, Y is -(PEG)9-, S1 is -CH2-CO-NH-(CH2)0-, and S2 is -(CH2)1-CO-NH-CH2-CH2-. In one embodiment, Y is -(PEG)9-, S1 is -CH2-CO-NH-(CH2)0-, and S2 is -(CH2)2-CO-NH-CH2-CH2-. In one embodiment, Y is -(PEG)9-, S1 is -CH2-CO-NH-(CH2)0-, and S2 is -(CH2)2-CO-NH-CH2-CH2-. In one embodiment, Y is -(PEG)9-, S1 is -CH2-CO-NH-(CH2)1-, and S2 is -(CH2)0-CO-NH-CH2-CH2-. In one embodiment, Y is -(PEG)9-, S1 is -CH2-CO-NH-(CH2)1-, and S2 is -(CH2)1-CO-NH-CH2-CH2-. In one embodiment, Y is -(PEG)9-, S1 is -CH2-CO-NH-(CH2)1-, and S2 is -(CH2)2-CO-NH-CH2-CH2-. In one embodiment, Y is -(PEG)9-, S1 is -CH2-CO-NH-(CH2)2-, and S2 is -(CH2)0-CO-NH-CH2-CH2-. In one embodiment, Y is -(PEG)9-, S1 is -CH2-CO-NH-(CH2)2-, and S2 is -(CH2)1-CO-NH-CH2-CH2-. In one embodiment, Y is -(PEG)9-, S1 is -CH2-CO-NH-(CH2)2-, and S2 is -(CH2)2-CO-NH-CH2-CH2-.

[0137] In one embodiment, Y is -(PEG)9-, S1 is -CH2-CO-NH-(CH2)0-, and S2 is -(CH2)0-NH-CO-CH2-CH2-. In one embodiment, Y is -(PEG)9-, S1 is -CH2-CO-NH-(CH2)0-, and S2 is -(CH2)1-NH-CO-CH2-CH2-. In one embodiment, Y is -(PEG)9-, S1 is -CH2-CO-NH-(CH2)0-, and S2 is -(CH2)2-NH-CO-CH2-CH2-. In one embodiment, Y is -(PEG)9-, S1 is -CH2-CO-NH-(CH2)0-, and S2 is -(CH2)2-NH-CO-CH2-CH2-. In one embodiment, Y is -(PEG)9-, S1 is -CH2-CO-NH-(CH2)1-, and S2 is -(CH2)0-NH-CO-CH2-CH2-. In one embodiment, Y is -(PEG)9-, S1 is -CH2-CO-NH-(CH2)1-, and S2 is -(CH2)1-NH-CO-CH2-CH2-. In one embodiment, Y is -(PEG)9-, S1 is -CH2-CO-NH-(CH2)1-, and S2 is -(CH2)2-NH-CO-CH2-CH2-. In one embodiment, Y is -(PEG)9-, S1 is -CH2-CO-NH-(CH2)2-, and S2 is -(CH2)0-NH-CO-CH2-CH2-. In one embodiment, Y is -(PEG)9-, S1 is -CH2-CO-NH-(CH2)2-, and S2 is -(CH2)1-NH-CO-CH2-CH2-. In one embodiment, Y is -(PEG)9-, S1 is -CH2-CO-NH-(CH2)2-, and S2 is -(CH2)2-NH-CO-CH2-CH2-.

[0138] In one embodiment, R2 of the tetrazine moiety of the tracer compound is a phenyl substituent. In one embodiment, R2 of the tetrazine moiety of the tracer compound is a phenyl substituent. n H 2n+1where n is an integer selected from the range of 0 to 2. In one embodiment, R2 of the tetrazine moiety of the tracer compound is an alkyl substituent having the formula C1H3. In one embodiment, R2 of the tetrazine moiety of the tracer compound is an alkyl substituent having the formula C2H5. In one embodiment, R2 of the tetrazine moiety of the tracer compound is hydrogen (H).

[0139] In one embodiment, the tetrazine portion of the tracer compound that includes the R2 substituent is 3-phenyl-1,2,4,5-tetrazine. In one embodiment, the tetrazine portion of the tracer compound that includes the R2 substituent is 3-phenyl-6-methyl-1,2,4,5-tetrazine. In one embodiment, the tetrazine portion of the tracer compound that includes the R2 substituent is 3-phenyl-6-ethyl-1,2,4,5-tetrazine. In one embodiment, the tetrazine portion of the tracer compound that includes the R2 substituent is 3-phenyl-6-phenyl-1,2,4,5-tetrazine.

[0140] In one embodiment, the substituent R2 is a non-interfering group with respect to the reactivity of IEDDA conjugation between the tracer compound and the TCO-derivatized targeting moiety. Non-interfering in this context means that R2 does not sufficiently or substantially prevent IEDDA conjugation.

[0141] In one embodiment, the tracer compound (BF3) - At least one F in the part 18 F. In one embodiment, the tracer compound is (BF3) - One F in the part is 18 F and the remaining two 19 It's F.

[0142] In one embodiment, the adduct is obtained by inverse electron demand Diels-Alder reaction (IEDDA) between a trans-cyclooctene derivatized target moiety and the tetrazine moiety of a tracer compound.

[0143] In one embodiment, the tetrazine ring of the tetrazine moiety of the tracer compound is chemically bound to the TCO moiety of the TCO-derivatized targeting moiety in an adduct.

[0144] In one embodiment, the targeting moiety of the adduct is a protein, peptide, antibody, antibody fragment, or nanoparticle. The targeting moiety of the adduct targets a specific biomolecule in vitro and in vivo and is conjugated to it via its sequence and / or 3D (surface) structure.

[0145] In one embodiment, the adduct (BF3) - At least one F in the part 18 F. In one embodiment, the adduct (BF3) - One F in the part is 18 F and the remaining two 19 It's F.

[0146] In one embodiment, the TCO moiety of the TCO derivatized targeting moiety is directly bound to the targeting moiety. In certain other embodiments, the TCO moiety of the TCO derivatized targeting moiety is indirectly bound to the targeting moiety via a linking moiety such as a PEGX chain or a polylysine chain. In one embodiment, the polylysine chain is an α-polylysine chain. In one embodiment, the polylysine chain is an ε-polylysine chain. In one embodiment, the polylysine chain is a poly-I-lysine chain.

[0147] In one embodiment, the adduct or a pharma- ceutically acceptable salt or solvate thereof has a structure according to formula (II): [ka] wherein each R1 is independently hydrogen (H) or a group of formula C n H 2n+1 where n is an integer selected from the range of 0 to 2; and L is a linker moiety consisting of S1-Y-S2, where: Y is (-CH2-) m(wherein m is an integer selected from the range of 1 to 4), or Y is a polyethylene glycol linker-(PEG) X -(in the formula, (PEG) X contains x repeat units of polyethylene oxide -CH2-CH2-O- groups, where x is an integer selected from the range of 1 to 20; and S1 is -(CH2) Z -CO-NH-(CH2) Z or S1 is -(CH2) Z -NH-CO-(CH2) Z where each z is independently an integer selected from the range of 0 to 4; and S2 is -CH2- or S2 is -(CH2) f -CO-NH-(CH2) f or S2 is -(CH2) f -NH-CO-(CH2) f where each f is independently an integer selected from the range of 0 to 4; and R2 is hydrogen (H) or a phenyl substituent or a group of formula C S H 2S+1 (wherein s is an integer selected from the range of 0 to 2), T comprises the targeting moiety of the adduct and, optionally, the linking moiety, where the linking moiety is (PEG) X -chain or polylysine chain.

[0148] In one embodiment, the adduct is an IEDDA cycloaddition product of the TCO moiety of the TCO-derivatized targeting moiety and the tetrazine ring of the tracer compound, and has a structure according to formula (II).

[0149] In one embodiment, in formula (II), the groups R1, R2, and L have the same meanings as defined herein for formula (I).

[0150] In one embodiment, the TCO derivatized targeting moiety includes a trans-cyclooctene moiety and a targeting moiety. In one embodiment, the TCO derivatized targeting moiety includes other moieties or side groups / chains in addition to the trans-cyclooctene moiety and the targeting moiety. In one embodiment, more than one linking moiety is disposed between the TCO moiety and the targeting moiety, thereby linking the TCO moiety and the targeting moiety to one another.

[0151] In one embodiment, the linking moiety between the TCO moiety and the targeting moiety is a polyethylene glycol linker comprising repeating units of polyethylene oxide-CH2-CH2-O- groups. In one embodiment, the TCO derivatized targeting moiety is TCO-(PEG). X -aldehyde, where x is an integer selected from the range of 0 to 10. In one embodiment, the TCO derivatized targeting moiety comprises TCO-(PEG)0-aldehyde. In one embodiment, the TCO derivatized targeting moiety comprises TCO-(PEG)3-aldehyde. In one embodiment, the TCO derivatized targeting moiety comprises TCO-(PEG)4-aldehyde. In one embodiment, the TCO derivatized targeting moiety comprises TCO-(PEG)7-aldehyde.

[0152] In one embodiment, the TCO derivatized targeting moiety is TCO-(PEG). X In one embodiment, the TCO derivatized targeting moiety comprises a TCO-(PEG)0 targeting moiety. In one embodiment, the TCO derivatized targeting moiety comprises a TCO-(PEG)4 targeting moiety. In one embodiment, the TCO derivatized targeting moiety comprises a TCO-(PEG)7 targeting moiety.

[0153] In one embodiment, the TCO derivatized targeting moiety comprises a TCO moiety and Tyr as the targeting moiety. 3 -octreotide (TOC). In one embodiment, the TCO-derivatized targeting moiety comprises TCO-(PEG). X-TOC, where x is an integer selected from the range of 0 to 10. In one embodiment, the TCO derivatized targeting moiety comprises TCO-(PEG)4-TOC. In one embodiment, the TCO derivatized targeting moiety comprises TCO-(PEG)7-TOC. In one embodiment, the -(PEG) X The linking moiety has the advantage of reducing the lipophilicity of the construct resulting from the TCO moiety and the IEDDA cycloaddition product (Tz+TCO). In one embodiment, a polyethylene glycol linker (PEG) between the TCO moiety and the targeting moiety is used. X -The linking moiety is useful for adjusting the pharmacokinetics and metabolic stability of the final adduct. In one embodiment, the linking moiety between the TCO moiety and the targeting moiety is a polylysine linker, which is a biocompatible and biodegradable linker. In one embodiment, the polylysine linking moiety between the TCO moiety and the targeting moiety is also useful for adjusting the pharmacokinetics and metabolic stability of the final adduct.

[0154] In one embodiment, the structure and length of the linker portion (L) of the tracer compound is optimized to ensure optimal pharmacokinetics after conjugation of the radiolabeled adduct with a specific targeting entity. The optimal structure and length of the linker portion of the tracer compound allows the adduct of the tracer compound and the TCO derivatized targeting moiety to obtain an optimal target-specific configuration once combined, allowing the specific binding of the targeting moiety to the targeting entity. Thus, the modular structure and length of the linker portion of the tracer compound promotes a high target-to-non-targeted uptake ratio of the adduct in cells and tissues in vitro and in vivo. In one embodiment, the modular structure and length of the linker portion of the tracer compound allows PET imaging with good signal-to-noise ratio.

[0155] In one embodiment, clearance of the tracer compound or adduct occurs primarily via the kidney, hi one embodiment, the linker moiety or adduct is primarily directed towards renal excretion in vivo.

[0156] In one embodiment, a method for producing a tracer compound is disclosed, wherein the linker moiety of the starting material is composed of S1-Y-S2, where: Y is (-CH2-) m (wherein m is an integer selected from the range of 1 to 4), S1 is -CH2-CO-NH-, or -CH2-NH-CO-, and S2 is -CH2-; or Y is a polyethylene glycol linker (PEG) X -(in the formula, (PEG) X contains x repeat units of polyethylene oxide -CH-CH-O- groups, where x is an integer selected from the range of 1 to 20; S1 is -CH2-NH-CO-(CH2) Z - or -CH2-CO-NH-(CH2) Z -wherein each z is independently an integer selected from the range of 0 to 4; S2 is -(CH2) f -CO-NH-CH2-CH2- or -(CH2) f -NH-CO-CH2-CH2- (wherein each f is independently an integer selected from the range of 0 to 4).

[0157] In one embodiment, the IEDDA reaction rate depends on the IEDDA reaction partners conjugated together, and the reaction conditions, which are determined by at least the concentrations of the reagents, the reaction temperature, and the reaction pH. In one embodiment, the IEDDA conjugation of the tetrazine moiety of the tracer compound with the TCO moiety of the TCO derivatized target moiety takes 30 minutes or less, preferably 20 minutes or less, more preferably 15 minutes or less, even more preferably 10 minutes or less, and even more preferably 5 minutes or less. In the most preferred embodiment, the IEDDA conjugation of the tetrazine moiety of the tracer compound with the TCO moiety of the TCO derivatized target moiety takes less than 1 minute, preferably less than 30 seconds, and more preferably less than 15 seconds, since IEDDA between the tetrazine ring and the TCO moiety is likely to occur within a few seconds. Nevertheless, in one embodiment, the IEDDA conjugation of the tetrazine moiety of the tracer compound with the TCO moiety of the TCO derivatized target moiety is carried out at a temperature above +20° C. for 20 to 30 minutes to improve the tautomeric uniformity and stability of the resulting adduct.

[0158] In one embodiment, the IEDDA conjugation between the tetrazine moiety of the tracer compound and the TCO moiety of the TCO derivatized targeting moiety is carried out at ambient temperature (room temperature). In one embodiment, the IEDDA conjugation between the tetrazine moiety of the tracer compound and the TCO moiety of the TCO derivatized targeting moiety is carried out efficiently at any temperature between +20°C and +80°C.

[0159] In one embodiment, the method for producing the adduct comprises reacting the tetrazine moiety of the radiolabeled tracer compound with the TCO moiety of the TCO derivatized targeting moiety at a temperature between 20-80° C., preferably between 40-70° C., more preferably between 55-65° C., and most preferably at 60° C. In one embodiment, the maximum reaction temperature for IEDDA conjugation of the tetrazine moiety of the tracer compound with the TCO moiety of the TCO derivatized targeting moiety is 80° C. In embodiments where pre-targeted PET imaging is utilized to image the adduct, the reaction temperature of the IEDDA conjugation is lower, and the temperature is determined by the (body) temperature of the subject.

[0160] In one embodiment, the tautomeric homogeneity of the adduct is improved and the amount of intermediate tautomers is reduced when the adduct is heated at a temperature above +20°C. In one embodiment, the tautomeric homogeneity of the adduct is improved when the adduct is heated at 20-80°C, preferably 40-70°C, more preferably 55-65°C, and most preferably 60°C. In one embodiment, heating the adduct at said temperatures is performed for at least 5 minutes, preferably at least 10 minutes, or at least 15 minutes. In one embodiment, heating the adduct increases the tautomeric homogeneity of the adduct and irreversibly reduces the amount of intermediate tautomers of the adduct. In one embodiment, heating the adduct prevents the back conversion of the adduct to its intermediate tautomer after heating and during storage at ambient room temperature of +20°C. In one embodiment, the above treatment by heating is utilized simultaneously with IEDDA conjugation of the tetrazine moiety of the tracer compound with the TCO moiety of the TCO derivatized target moiety. In one embodiment, the above-mentioned treatment with heat is utilized after IEDDA conjugation of the tetrazine moiety of the tracer compound with the TCO moiety of the TCO-derivatized targeting moiety.

[0161] In one embodiment, the solvent in the IEDDA conjugation of a tracer compound with a TCO-derivatized targeting moiety is water, an aqueous medium, an aqueous buffer, or a mixture of an organic solution and an aqueous solution, where the percentage of organic solvent is less than 50%, preferably less than 10%, and the organic solvent comprises, for example, DMSO, ethanol, acetonitrile (MeCN), or methanol.

[0162] In one embodiment, the tetrazine moiety of the tracer compound reacts with the TCO moiety of the TCO derivatized targeting moiety under acidic conditions to obtain an IEDDA conjugate adduct. In one embodiment, the tetrazine moiety of the tracer compound is conjugated with the TCO moiety of the TCO derivatized targeting moiety under conditions of pH 2-7, preferably 2-4, most preferably 2-3.

[0163] In one embodiment, 18The temperature for the radiolabeling reaction of the tracer compound with the F radioisotope is between 80 and 100° C., preferably between 80 and 90° C., and more preferably between 85 and 90° C. In one embodiment, 18 The reaction temperature for radiolabeling of the tracer compound with F radioisotope is 85°C.

[0164] In one embodiment, 18 Radiolabeling of tracer compounds with F radioisotopes is carried out at a pH between 2.0 and 3.0.

[0165] In one embodiment, 18 The radiolabeling reaction of the tracer compound with the F radioisotope is carried out in an acidic pH-controlled buffer containing a sufficient percentage of an organic solvent to dissolve the tracer, the organic solvent being, for example, MeCN or DMF. In one embodiment, at least one 18 The radiolabeling reaction of the tracer compound with the F radioisotope is carried out in a pyridazine HCl buffer containing an organic solvent such as MeCN or DMF.

[0166] In one embodiment, 18 Radiolabeling of the adduct with the F radioisotope is carried out at a temperature between 80 and 100° C., preferably between 80 and 90° C., more preferably between 85 and 90° C. In one embodiment, 18 Radiolabeling of the adduct with the F radioisotope is carried out at a temperature of 85°C.

[0167] In one embodiment, 18 Radiolabeling of the adduct with the F radioisotope is carried out at a pH between 2.0 and 3.0.

[0168] In one embodiment, 18 The radiolabeling reaction of the adduct with the F radioisotope is carried out in an acidic pH controlled buffer containing a sufficient percentage of an organic solvent to dissolve the tracer, the organic solvent being, for example, MeCN or DMF. 18The radiolabeling reaction of the adduct with the F radioisotope is carried out in a pyridazine HCl buffer containing an organic solvent such as MeCN or DMF.

[0169] In one embodiment, the adduct is radiolabeled after IEDDA conjugation of the tracer compound with the TCO-derivatized targeting moiety. 18 The replacement of fluoride F with F radioisotope is first carried out after IEDDA conjugation of the tracer compound to the TCO-derivatized targeting moiety. The synthesis of the entire adduct before radiolabeling allows the adduct to be radiolabeled immediately before use, thereby minimizing decay of the radiolabel. In certain embodiments, by-products are created during IEDDA conjugation that are laborious or impossible to remove from the reaction mixture. Thus, radiolabeling the adduct after IEDDA conjugation is beneficial to ensure the chemical purity of the resulting adduct.

[0170] In another embodiment, the zwitterionic portion of the tracer compound is 18 F radioisotope. Therefore, at least one of the three fluorides (F) attached to the boron (B) of the zwitterion of the tracer compound is 18 F radioisotope. In one embodiment, 18 Replacement of fluoride F by F radioisotope is carried out prior to IEDDA conjugation of the tracer compound to the TCO-derivatized targeting moiety. 18 In such an embodiment, the tracer compound is sensitive to the conditions required for F radiolabeling. 18 F radiolabeling is performed prior to IEDDA conjugation with the TCO-derivatized targeting moiety. Thus, in embodiments in which the adduct comprises a sensitive and / or fragile targeting moiety, the tracer molecule 18F radiolabeling is carried out prior to IEDDA conjugation. In one embodiment, such sensitive and / or fragile targeting moieties are antibodies or enzymes. This is beneficial to ensure the integrity of the targeting moiety in the resulting adduct, since the process sequence allows the fragile targeting moiety to survive the radiolabeling process conditions.

[0171] In one embodiment, when IEDDA conjugation between the tracer compound and the TCO derivatized targeting moiety occurs in vivo or in vitro in a subject, the radiolabeling of the tracer compound before IEDDA conjugation is utilized.In this case, the tracer compound is first contacted with the TCO derivatized targeting moiety in the subject after the TCO derivatized targeting moiety reaches its target site.The radioimaging of the radiolabeled tracer compound utilizing this methodology is a pre-targeted PET imaging method.

[0172] In one embodiment, there is provided a use of the tracer compound and / or adduct of the first aspect in detecting a target entity in a subject by radioimaging of the subject, wherein the target entity is targeted with a radiolabeled tracer compound and / or adduct. In one embodiment, there is provided a method of detecting a target entity in a subject by radioimaging of the subject, wherein the target entity is targeted with a radiolabeled tracer compound and / or adduct.

[0173] In one embodiment, the tracer compound and / or adduct are used in radioimaging in vivo via systemic administration of the adduct to a subject. In one embodiment, the tracer compound and / or adduct are used in radioimaging, more specifically detection of target entities in a subject via imaging of radiolabeled target entities, by positron emission tomography. In one embodiment, imaging of target entities refers to imaging of radiolabeled tracer compounds and / or adducts capable of binding to selected target entities in vitro and in vivo.

[0174] In one embodiment, the tracer compound and / or adduct are administered in an imaging effective amount for positron emission of the tracer compound and / or adduct to a subject undergoing radioimaging.

[0175] In one embodiment, the tracer compounds and / or adducts are used for in vitro radioimaging of tissues and / or cells. In one embodiment, the tracer compounds and / or adducts are used for labeling of target entities in vitro and in vivo.

[0176] In one embodiment, the targeting moiety of the adduct is capable of binding to a targeting entity in vitro. In one embodiment, the targeting moiety of the adduct is capable of binding to a targeting entity in vivo. In one embodiment, the targeting entity is a biomolecule, such as a receptor, an enzyme, or a nanoparticle. In one embodiment, the targeting entity to which the targeting moiety of the adduct can bind is indicative of a particular physiological disease, such as cancer, neurodegeneration, inflammation, or infectious disease.

[0177] In one embodiment, an adduct that does not include a targeting moiety or in which binding of the targeting moiety to the target entity is blocked has low or insignificant binding to the target entity or biomolecule in vitro, In one embodiment, an adduct that does not include a targeting moiety or in which binding of the targeting moiety to the target entity is blocked has low or insignificant binding to the target entity or biomolecule in vivo.

[0178] In one embodiment, the tracer compounds and adducts have good stability in plasma in vitro and in vivo. In one embodiment, the bone uptake rate of the tracer compounds and adducts is low, which suggests that the radiolabel of the tracer compounds and adducts has good metabolic stability. In one embodiment, the tracer compounds and adducts show relatively low accumulation in non-target tissues.

[0179] In one embodiment, the radiolabeled tracer compound can be further processed and used for radioimaging up to 8 hours, preferably up to 5 hours, and most preferably up to 2 hours after the tracer compound is radiolabeled. In one embodiment, the radiolabeled adduct that is the IEDDA cycloaddition product of the tracer compound and the TCO-derivatized targeting moiety can be further processed and used for radioimaging up to 8 hours, preferably up to 5 hours, and most preferably up to 2 hours after the adduct is radiolabeled.

[0180] In one embodiment, a kit for detecting a target entity in a subject by radioimaging comprises at least one compartment containing a tracer compound, at least one compartment containing at least one TCO-derivatized targeting moiety, and a reagent for radiolabeling the tracer compound. 18 and at least one compartment containing F. In one embodiment, the kit also includes an aqueous solvent and an organic solvent for IEDDA conjugation and radiolabeling of the tracer compound and / or adduct. In one embodiment, the kit provides the materials necessary for radiolabeling the tracer compound prior to IEDDA conjugation of the adduct. In one embodiment, the kit provides the materials necessary for radiolabeling the adduct after IEDDA conjugation of the tracer compound with a TCO-derivatized targeting moiety. In one embodiment, the kit is configured for use in a pre-targeted PET imaging method. In one embodiment, the kit provides all the components necessary for preparing the tracer compound and / or adduct for detection of a target entity in a subject. In one embodiment, the kit provides most of the materials necessary for preparing the tracer compound and / or adduct for detection of a target entity in a subject. EXAMPLES

[0181] Various embodiments have been presented. It should be understood that as used herein, the words "comprise," "include," and "contain" are each used as open-ended expressions without any intended exclusivity.

[0182] Summary All reagents were purchased from commercial suppliers and used as received without further purification. Tetrazines were purchased from Conju-Probe, BroadPharm, or Jena Biosciences, and iodoboron pinacol esters were purchased from Enamine. Sep-Pak C18-Light cartridges were purchased from Waters, and PS-HCO3 cartridges (Macherey-Nagel™ Chromafix™) were purchased from Fisher Scientific. No carrier added. 18 F fluoride was produced in-house using an IBA 10 / 5 medical cyclotron from Hyox-18 18O enriched water purchased from Rotem Industries Limited (Arava, Israel). The synthesized precursors were analyzed by high-resolution mass spectrometry (HRMS) and nuclear magnetic resonance spectroscopy (NMR) analysis. Radiolabeled tracers were analyzed by radio-high performance liquid chromatography (radio-HPLC). Excised organs were weighed and measured in a Wizard gamma counter. Molecubes PET (β-CUBE) was coupled to CT (γ-CUBE) to acquire 60-minute dynamic positron emission tomography (PET) scans with computed tomography (CT).

[0183] Example 1. Synthesis of AmBF3-Tz (Compound 4 in Figure 1) 2-[4-(1,2,4,5-Tetrazine-3-yl)phenyl]-N-[2-(dimethylamino)ethyl]acetamide (2). N,N-Dimethylethylenediamine (13 μL, 0.12 mmol) was dissolved in 2 mL of DCM under argon, then tetrazine NHS-ester (1) (25 mg, 0.08 mmol) was added in 3 mL of DCM, which was added dropwise to the clear solution. After stirring the mixture at room temperature for 1.5 h, the crude reaction mixture was evaporated to dryness, resuspended in 1 mL of ultrapure water (Milli-Q), and purified on SEP-Pak silica (eluted with MeOH:DCM 1:9) to give a pink solid. The yield was 68±26% (n=3) (11.5 mg, 0.04 mmol). 1 H NMR (300 MHz, acetonitrile-d3) δ 10.26 (s, 1H), 8.50 (d, J = 8.4 Hz, 2H), 7.56 (d, J = 8.2 Hz, 2H), 3.60 (s, 2H), 3.26 (s, 2H), 2.40 (s, 2H), 2.21 (s, 6H).

[0184] 2-(2-(4-(1,2,4,5-tetrazin-3-yl)phenyl)acetamido)-N,N-dimethyl-N-((4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)methyl)ethane-1-aminium (3). Compound (2) (11.5 mg, 0.04 mmol) was dissolved in 1 mL of dry acetonitrile under argon, and then 2-(iodomethyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (10.8 mg, 0.04 mmol) was dissolved in 300 μL of dry acetonitrile. The reaction mixture was stirred overnight and evaporated to dryness. The yield was 58±31% (n=3) (11.5 mg, 0.04 mmol). 1H NMR (300 MHz, acetonitrile-d3) δ 10.28 (s, 1H), 8.52 (d, J = 8.3 Hz, 2H), 7.58 (d, J = 8.2 Hz, 2H), 3.68 (s, 2H), 3.58 (s, 2H), 3.48 (s, 2H), 3.13 (s, 6H), 2.14 (s, 2H), 1.28 (s, 12H).

[0185] {[(2-{2-[4-(1,2,4,5-tetrazin-3-yl)phenyl]acetamido}ethyl)dimethylammonio]methyl}trifluoroborate (4). Compound (3) (0.043 mmol, 18 mg) was dissolved in a 15 mL Falcon (LDPE) tube with 1153 μL of DMF, followed by the addition of 387 μL of Milli-Q water, 577 μL of 4 M HCl, and 577 μL of 3 M KHF2. The Falcon tube was closed and the reaction mixture was heated at 70 °C for 30 min, and the fluorination reaction was closely monitored by HPLC (PDA detector 534 nm, 2.5 mL / min t) of an isocratic mixture of 0.1% TFA-ACN:0.1% TFA Milli-Q water (80:20). R (AmBF3-Tz) = 10.3 min), avoiding decomposition of the tetrazine. The reaction gave quantitative conversion of compound (3) to compound (4). The reaction mixture was diluted with 6 mL of Milli-Q water and applied to two parallel SPE C18 PLUS cartridges, each preconditioned with 5 mL of ACN and 10 mL of Milli-Q water. The C18 cartridges were washed with 20 mL of Milli-Q water, air-dried, and eluted with 1 mL of ACN to give 13.9 mg of 4. 1 H NMR (400 MHz, CD3CN) δ 10.30 (s, 1H), 8.54 (d, J = 8.5 Hz, 2H), 7.58 (d, J = 8.6 Hz, 2H), 3.68 - 3.56 (m, 4H), 3.34 (t, J = 6.7 Hz, 2H), 3.01 (s, 6H), 2.38 (s, 2H). 11 B NMR (128 MHz, CD3CN) δ 2.19, 1.80, 1.43, 1.03. 19F NMR (376 MHz, CD3CN) δ -138.77, -138.89, -139.04, -139.17. 13 C NMR (101 MHz, CD3CN) δ 171.47, 167.25, 158.98, 141.95, 131.82, 131.42, 129.05, 118.30, 65.43, 54.32, 43.42, 34.75, 1.32. HRMS Calculated value, C 15 H 21 BF3N6O + [M+H] + 369.18165m / z, measured value, C 15 H 21 BF3N6O + [M+H] + 369.18134m / z (mass error -0.85ppm).

[0186] Example 2. Synthesis of AmBF3-PEG4-Tz (Compound 8 in Figure 2) N-(4-(1,2,4,5-tetrazin-3-yl)benzyl)-1-(3-(dimethylamino)propanamide)-3,6,9,12-tetraoxapentadecan-15-amide (6). To 3-(dimethylamino)propanoic acid (4.8 mg, 31 μmol) in 0.3 mL of DMF under argon atmosphere was added HATU (8.5 mg, 23 μmol) in 0.1 mL of DMF and stirred at room temperature for 10 min. N-(4-(1,2,4,5-tetrazin-3-yl)benzyl)-1-amino-3,6,9,12-tetraoxapentadecan-15-amide (10 mg, 21 μmol) (5) and DIPEA (30 μL) were added and the reaction was stirred at room temperature for 2 h. The solvent was evaporated and analysis by LC-MS showed a purity of >95%. LC-MS(+) calculated value, 534 m / z [M+H] for C25H40N7O5 + , Measured value, m / z(%)=534(100)[M+H] + , t R =8.5 minutes.

[0187] 1-(4-(1,2,4,5-tetrazin-3-yl)phenyl)-N,N-dimethyl-3,19-dioxo-N-((4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)methyl)-6,9,12,15-tetraoxa-2,18-diazahenicosan-21-aminium (7). Compound (6) (2 mg, 3.56 μmol) was dissolved in 200 μL of dry acetonitrile under argon, and then 2-(iodomethyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (1.01 mg, 3.7 μmol) was dissolved in 100 μL of dry acetonitrile. The reaction mixture was stirred for 20 min and evaporated to dryness.

[0188] 24-(4-(1,2,4,5-tetrazin-3-yl)phenyl)-1,1,1-trifluoro-3,3-dimethyl-6,22-dioxo-10,13,16,19-tetraoxa-3,7,23-triaza-1-boratetracosan-3-ium-1-uide (8). Without further purification, compound (7) (3.56 μmol) was dissolved in a 15 mL Falcon (LDPE) tube with 14.94 μL of DMF, followed by the addition of 4.93 L of Milli-Q water, 7.47 μL of 4 M HCl and 7.47 μL of 3 M KHF2. The Falcon tube was closed and the reaction mixture was heated at 85° C. for 10 min. The reaction mixture was diluted with 6 mL of Milli-Q and added to two parallel SPE C18 PLUS cartridges, each preconditioned with 5 mL of ACN and 10 mL of Milli-Q. The C18 cartridge was washed with 20 mL of Milli-Q, air-dried, and eluted with 1 mL of ACN to give 1.2 mg (1.95 μmol) of compound (8). The solvent was evaporated and analysis by LC-MS showed a purity of >95%. LC-MS (+) calculated, C 26 H 41 596m / z[MF] for BF2N7O6 + , Measured value, m / z(%)=596(100)[MF] + , t R=11.5 minutes. 1H NMR (400 MHz, acetone-d6) δ 10.43 (s, 1H), 8.54 (s, 2H), 7.63 (s, 2H), 5.35 (s, 2H), 4.58 (s, 2H), 3.78 (s, 3H), 3.60 (s, 16H), 3.35 (s, 2H), 3.10 (s, 6H), 2.51 (s, 3H), 2.33 (s, 3H), 2.21 (s, 2H). 19F NMR (376 MHz, acetonitrile-d3) δ -138.98, -139.12, -139.25.

[0189] Example 3. Synthesis of AmBF3-PEG9-Tz (Compound 12 in Figure 3). N 1 -(4-(1,2,4,5-tetrazin-3-yl)benzyl)-N 31 -(2-(dimethylamino)ethyl)-4,7,10,13,16,19,22,25,28-nonaoxahentriacontanediamide (10). Dimethylethylenediamine (0.677 mg, 7.7 μmol) was dissolved in 400 μL of DCM under argon, followed by the addition of tetrazine-PEG9-NHS-ester (9) (5 mg, 6.4 μmol) in 600 μL of DCM, which was added dropwise to the clear solution. The reaction was monitored by TLC (RP-TLC, ACN: MilliQ water (80:20), R f = tetrazine 0.83, R f = amine 0.00, R f = tetrazine amine 0.28). After stirring the mixture at room temperature for 20 min, the crude reaction mixture was loaded onto 3×C18 cartridges, air dried, and eluted with 3 mL of ACN in 4 fractions. Pure fractions were combined and evaporated to dryness to give a pink solid. Yield ≧98% (1.3 mg, 0.0016 mmol). 1H NMR (400 MHz, acetonitrile-d3) δ 10.28 (s, 1H), 8.53 (d, J = 8.5 Hz, 2H), 7.57 (d, J = 8.7 Hz, 2H), 5.36 (s, 1H), 4.50 (d, J = 6.2 Hz, 2H), 3.74 (t, J = 6.0 Hz, 2H), 3.67 (t, J = 6.0 Hz, 2H), 3.59 (d, J = 0.9 Hz, 30H), 3.47 (q, J = 5.7 Hz, 2H), 3.29 (s, 1H), 3.04 (s, 2H), 2.73 (s, 6H), 2.48 (t, J = 6.0 Hz, 3H), 2.39 (t, J = 6.0 Hz, 3H).

[0190] 1-(4-(1,2,4,5-tetrazin-3-yl)phenyl)-N,N-dimethyl-3,33-dioxo-N-((4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)methyl)-6,9,12,15,18,21,24,27,30-nonaoxa-2,34-diazahexatriacontan-36-aminium (11). 2-(iodomethyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (0.0017 mmol, 0.46 mg) was dissolved in dry acetonitrile and added dropwise to a stirred solution of compound (10) (0.0017 mmol, 1.3 mg) in ACN under argon atmosphere overnight. The reaction was monitored by HPLC (PDA detector, 534 nm). The reaction mixture was evaporated to dryness and used as is in the subsequent fluorination reaction.

[0191] 39-(4-(1,2,4,5-tetrazin-3-yl)phenyl)-1,1,1-trifluoro-3,3-dimethyl-7,37-dioxo-10,13,16,19,22,25,28,31,34-nonaoxa-3,6,38-triaza-1-boranonateriacontan-3-ium-1-uide (12). Compound (11) (0.0017 mmol, 1.74 mg) was dissolved in a 15 mL Falcon (LDPE) tube with 45.6 μL of DMF, followed by the addition of 15.5 μL of Milli-Q water, 22.8 μL of 4 M HCl, and 22.8 μL of 3 M KHF2. The Falcon tube was closed and the reaction mixture was heated at 70° C. for 30 min and the fluorination reaction was closely monitored by HPLC (PDA detector 534 nm) to avoid decomposition of the tetrazine. The reaction gave complete conversion of compound (11) to compound (12). The reaction mixture was diluted with 1 mL of Milli-Q water and applied to an SPE C18 Light cartridge (preconditioning: 5 mL ACN and 10 mL Milli-Q water). The C18 cartridge was washed with 10 mL Milli-Q, air-dried and eluted with 200 μL ACN to give 1.9 mg of compound (12). 1 H NMR (400 MHz, CD3CN) δ 10.29 (s, 1H), 8.56-8.54 (d, 2H), 7.60-7.58 (d, 2H), 7.22 (s, broad, 1H), 6.88 (s, broad, 1H), 4.53-4.51 (d, 2H), 3.75 (t, 2H), 3.67 (t, 2H), 3.62-3.56 (m, 32H), 3.33 (t, 2H), 3.03 (s, 6H), 2.49 (t, 2H), 2.38 (t, 2H). 19 F NMR (376 MHz, CD3CN) δ -138.80, -138.97, -139.08. 13 C-NMR (101 MHz, CD3CN). HRMS calculated value, C 36 H 62 BF3N7O 11 + [M+H] + 836.45470m / z, measured value, C 36 H62 BF3N7O 11 + [M+H] + 836.45538m / z (mass error 0.82ppm).

[0192] Example 4. Synthesis of trans-cyclooctene aldehyde (TCO-CHO) (compound 15 in FIG. 4). 15.6 mg (91 nmol, 1.5 equiv.) of compound (13) as presented in FIG. 4 was dissolved in 500 μL of THF and 150 μL of DMSO under argon, then 9.7 mg of pyridine (122 nmol, 2.0 equiv.) in 100 μL of THF was added and the solution was mixed for 10 min. 16.3 mg (61 nmol, 1.0 equiv.) of compound (14) was added dropwise and the solution was stirred at room temperature overnight. The reaction was monitored by normal phase TLC using ethyl acetate:cyclohexane (1:1) as the mobile phase and stained with KMnO4 stain solution (t R (pyridine) = 0.00, t R (1)=0.00, t R (2)=0.90, R T (3)=0.80). To remove pyridine and unreacted compound (14), the crude mixture was purified on a Sep-Pak SPE-Sil cartridge (preconditioned with 50 mL of ultrapure water). The mixture was pushed onto the SPE-Sil cartridge (fraction 1) and eluted with 1 mL of DCM (fraction 2). The collected fractions were further purified by semi-preparative HPLC (Phenomenex Alltima C18 column, isocratic 3 mL / min of 80% ACN+0.1% TFA), where compound (15) was isolated by t R = 6 min. LC-MS (+) m / z (%) = 288.36 (27) [M+H] + , 310.30(19)[M+Na] + t R =9.3 minutes. 1 H NMR (400 MHz, CDCl3) δ ppm, 10.00, 7.86, 7.84, 7.45, 7.43, 5.53, 4.99, 4.41, 2.35, 1.97, 1.75, 1.57, 1.27, 1.26. 13C NMR (101 MHz, CDCl3) δ ppm, 191.81, 145.79, 135.64, 134.89, 133.01, 130.13, 127.77, 81.14, 44.70, 41.14, 38.67, 34.27, 32.50, 30.96.

[0193] Example 5. Aminooxy-functionalized peptides (α-MSH-ONH2, Exendin-4-ONH2, Tyr 3 General procedure for functionalizing -octreotide-ONH2) with trans-cyclooctene. The aminooxy-functionalized custom synthesized peptide (1 equiv.) was dissolved in 600 μL of 0.3 M anilinium acetate buffer (pH 4.6). Commercially available trans-cyclooctene-PEG3-aldehyde (Figure 4, compound 16) (1.5 equiv.) was dissolved in 17 μL of chloroform and added dropwise to the stirred peptide solution. The reaction was monitored by HPLC (PDA detector, 280 nm). The functionalized peptide was purified by HPLC (MeCN(B)-HO(A)+0.1% TFA; 20-30-20% B, 30 min). t R = α-MSH-TCO; 23 min, t R TOC-TCO; 25 min, t R =Exendin-4-TCO; 15.5 min. MeCN in the fractions collected from the HPLC was evaporated with compressed air and the fractions containing mainly water were frozen (-80°C). The frozen fractions were lyophilized and then used as is. When necessary, the fractions were used as is and mixed with the selected tetrazine immediately after collection from the HPLC. In such cases, the solution was diluted to contain 95% or more water during the IEDDA cycloaddition.

[0194] Example 6. Synthesis of PSMA-trans-cyclooctene (compound 18, FIG. 5). To TCO-NHS (4.5 mg, 17 μmol) and DIPEA (3.2 mg, 25 μmol) in dry DMF (300 μL) under argon atmosphere was added PSMA-amine (17) (5 mg, 15.7 μmol) in dry DMF (250 μL) dropwise and stirred overnight. PSMA-TCO (18) was purified by HPLC to give 5.3 mg (71%). LC-MS (+) m / z (%) = 472.5 (100) [M+H] + , 320.3(96)[M-TCO-formate] + t R =10.3 minutes. 1 H NMR (400 MHz, CD3OD) δ(ppm) = 5.59 (m, 1H), 5.50 (m, 1H), 4.31 (m, 1H), 4.25 (s, 1H), 3.31 (s, 2H), 3.06 (m, 2H), 2.41 (m, 2H), 2.33 (m, 2H), 2.33 (m, 2H), 2.15 (m, 2H), 1.94 (m, 6H), 1.68 (m, 4H), 1.40 (m, 4H), 1.29 (m, 2H), 13C NMR (101 MHz, CD3OD) δ (ppm) = 136.10, 133.76, 81.59, 54.07, 53.59, 42.23, 39.67, 35.18, 33.49, 33.25, 32.11, 31.15, 30.55, 29.03, 23.86.

[0195] Example 7. Synthesis of PSMA-tranexamic acid-trans-cyclooctene (Compound 24, FIG. 6) Di-tert-butyl((6-(4-(((((9H-fluoren-9-yl)methoxy)carbonyl)amino)methyl)cyclohexane-1-carboxamido)-1-(tert-butoxy)-1-oxohexan-2-yl)carbamoyl)glutamate (21). To HBTU (76.6 mg, 201.5 μmol) and DIPEA (26.4 mg, 206 μmol) in dry DMF (400 μl) was added Fmoc-tranexamic acid (19) (78 mg, 206 μmol) in dry DMF (600 μL) and stirred under argon for 10 min. Di-tert-butyl((6-amino-1-(tert-butoxy)-1-oxohexan-2-yl)carbamoyl)glutamate (20) (25 mg, 51.5 μmol) was added to dry DMF (400 μL) and stirred under argon for 2 h. LC-MS (+) m / z (%) = 850.1 (4) [M+H] + , 872.1(3)[M+Na] + t R =18.5 minutes.

[0196] Di-tert-butyl ((6-(4-(aminomethyl)cyclohexane-1-carboxamido)-1-(tert-butoxy)-1-oxohexan-2-yl)carbamoyl)glutamate (22). Without further purification, 1.4 mL of piperidine was added to (21) and stirred at room temperature for more than 10 min. The solvent was evaporated and the product was extracted with 5 mL of ethyl acetate and 3×2 mL of brine solution. LC-MS (+) m / z (%)=628.0 (100) [M+H] + t R =11.3 minutes.

[0197] ((5-(4-(aminomethyl)cyclohexane-1-carboxamido)-1-carboxypentyl)carbamoyl)glutamic acid (23). Without further purification, (22) was dissolved in 3 mL of CH2Cl2 / TFA (1:1) and stirred at room temperature for 90 min. The product was purified by HPLC (t R = 7.2 min) to give 10.2 mg (43%). LC-MS (+) m / z (%) = 459 (100) [M+H] + t R=2.6 minutes.

[0198] (E)-((1-Carboxy-5-(4-(((cyclooct-4-en-1-yloxy)carbonyl)amino)methyl)cyclohexane-1-carboxamido)pentyl)carbamoyl)glutamic acid (24). TCO-NHS (13 mg, 49 μmol) and DIPEA (8.9 mg, 70 μmol) (23) in dry DMF (600 μL) were added dropwise to 250 μL of dry DMF and stirred overnight under argon atmosphere. The product was purified by HPLC (t R = 4.5 min) to give 5.63 mg (41%). LC-MS (+) m / z (%) = 611 (100) [M+H] + t R =11.4 minutes. 1 H NMR (400 MHz, CD3OD) δ (ppm) = 5.61 (m, 1H), 5.52 (m, 1H), 4.32 (m, 2H), 4.26 (m, 1H), 3.17 (m, 2H), 3.01 (s, 1H), 2.92 (m, 2H), 2.88 (s, 1H), 2.43 (m, 2H), 2.34 (m, 2H), 2.15 (m, 2H), 1.98 (m, 4H), 1.80 (m, 5H), 1.70 (m, 4H), 1.51 (m, 2H), 1.44 (m, 5H), 0.98 (m, 2H), 13C NMR (101 MHz, CD3OD) δ (ppm) = 136.10, 133.77, 53.94, 53.50, 46.47, 42.24, 39.92, 39.65, 39.09, 35.18, 33.50, 33.19, 32.11, 30.95, 30.24, 29.97, 28.93, 26.45, 23.89.

[0199] Example 8. Procedure a) [ 18 F]4(AmBF3-Tz), [ 18 F]8 and [ 18 Radiolabeling of [F]12 [ 18 [F] fluoride, 150 μL of 0.9% NaCl 18The tetrazine was eluted as F-NaF into a reaction vial and concentrated at 125 °C for 10 min under a stream of argon gas to reach a reaction volume of 10–25 μL. Tetrazine (100 nmol) in 5 μL of acetonitrile was added to a polypropylene tube containing 10 μL of pyridazine HCl buffer (pH 2.0). The reaction mixture was heated at 83 °C for an additional 10 min and quenched with 600 μL of MilliQ:EtOH (50:50). Alternatively, [ 18 [F] fluoride was captured on a PS-HCO3 cartridge and eluted into a tube containing tetrazine (100 μL, pyridazine HCl buffer, pH 2.0). The mixture was concentrated at 85 °C under a stream of argon until a volume of ~10-20 μL was reached (t = 15 min), quenched with ultrapure water (600 μL), and purified on a Sep-Pak C18 cartridge to obtain the tracer. Compound 4 (AmBF3-Tz) was 18 Radiolabeled with F radioisotope, resulting in [ 18 The procedures leading to F]4 are presented in Figure 7. An example of procedure a) is shown in Figure 8a.

[0200] Example 9. Procedure b) Pre-IEDDA conjugation prior to radiolabeling of AmBF3-Tz To tetrazine-AmBF4, 8 or 12 (1.85 μmol) in 20 μL of dry acetonitrile was added an equimolar amount of TCO-functionalized peptide 18 or 24 in Milli-Q water (800 μL). The reaction was heated to 60° C. for 20 min. The product was purified by HPLC to give (28) (49%) (t R =7.9min)LC-MS(+)m / z(%)=811(100)[M+H] + t R = 8.8 minutes or (29)(48%)(t R =9.5min)LC-MS(+)m / z(%)=950(100)[M+H] + t R = 9.5 min. An example of a synthetic route according to procedure b) is shown in Figure 8b.

[0201] Example 10. TCO-functionalized peptide Tyr 3-octreotide (25), α-MSH, exendin-4, PSMA (18), and PSMA-tranexamic acid (24), and [ 18 F]AmBF3-Tz([ 18 F]4) or its PEGylated derivative [ 18 F]8 or [ 18 F]12 to give the product [ 18 F]AmBF3-Tyr 3 -Octreotide ([ 18 F]25), [ 18 F]AmBF3-α-MSH([ 18 F]26), [ 18 F]AmBF3-exendin-4([ 18 F]27) and [ 18 F]AmBF3-PEG9-exendin-4 ([ 18 F]30), [ 18 F]AmBF3-PSMA([ 18 F]28) and [ 18 F]AmBF3-PSMA-tranexamic acid ([ 18 F]29) production. Trans-cyclooctene-based peptides (α-MSH-ONH2, exendin-4-ONH2, Tyr 3 Functionalization of trans-cyclooctene-functionalized peptide (20-50 μL, 50 nmol in Milli-Q water) was carried out as described in Example 5. The trans-cyclooctene-functionalized peptide (20-50 μL, 50 nmol in Milli-Q water) was added to the reaction mixture of radiolabeled tetrazine (20 μL) and heated at 60 °C for 15 min. The reaction mixture was purified on two C18 cartridges by diluting with Milli-Q water, washing with ultrapure Milli-Q water (45 mL), and eluting with 150 μL ethanol and 200 μL 0.01 M PBS. The purified peptide solution was diluted with 0.01 M PBS to contain less than 5% ethanol for intravenous administration. The crude mixture was analyzed using HPLC: MeCN (B)-H2O (A) + 0.1% TFA 20-30-20% B for 30 min. Retention times on HPLC: Compound [ 18 F]26([ 18 F]AmBF3-α-MSH) R ;14.7 min, compound [ 18F]25([ 18 F]AmBF3-Tyr 3 -Octreotide) R ;17.5 min, compound [ 18 F]27([ 18 F]AmBF3-Exendin-4) R ;15.0~16.0 min, compound [ 18 F]30([ 18 F]AmBF3-PEG9-Exendin-4) R , 15.8-16.5 min. HRMS(E / Z)-[ 18 The actual value measured by F25 is [M+H+Na] 2+ 1048.49255 (-0.0855 ppm). These results demonstrate that the use of tracer compounds allows for the rapid radiolabeling of a variety of different peptides under mild conditions.

[0202] [ka]

[0203] Example 11. Radiolabeling of pre-IEDDA products Fluorine-18 (1.8 GBq) was eluted from the PS-HCO3 cartridge with 100 μL of 0.9% NaCl solution or pyridazine HCl buffer (pH 2, 100 μL) and evaporated to a volume of 10–15 μL at 100°C (0.9% NaCl) or 80–85°C when pyridazine HCl buffer was used. 28 or 29 (100 nmol) in 10 μL of pyridazine buffer (pH = 2) was added via an external line and the resulting solution was heated at 85°C for 10 min. After dilution with 10 mL of Milli-Q water, the activity was loaded onto a preconditioned C18 cartridge and, after washing with an additional 40 mL of Milli-Q water, eluted with 400 μL of 50% EtOH / PBS with a specific activity of (9.2 ± 3.8 GBq / μmol) [100 nmol]. 18 F]28 (RCY: 5.2 ± 1%) and a specific activity of (16.3 ± 4.3 GBq / μmol) 18F]29 (RCY: 11.8±3.1%) was obtained.

[0204] Example 12. Cells and cell culture The rat pancreatic tumor cell line AR42J expressing SSTR was obtained from the American Type Culture Collection (Manassas, VA). C4-2 cells (ATCC® CRL-3314™) were cultured in DMEM medium (Gibco) supplemented with 18% F12 medium (Sigma), 10% FBS (Gibco), and 1% T medium. Both cell lines were grown at 37°C in a humidified incubator containing 5% CO2. Cells grown to 80%-90% confluence were used for in vitro or in vivo experiments. Mouse skin melanoma B16 / F10 cells were cultured in CO2-independent medium (Life Technologies Gibco, catalog no. 18045054) supplemented with GlutaMax (1x final concentration, 10% FBS and Pen-Strep) at 37°C in a humidified incubator. The viability of B16 / F10 cells was 97%. C4-2 cells (ATCC® CRL-3314™) were cultured in DMEM medium (Gibco) supplemented with 18% F12 medium (Sigma), 10% FBS (Gibco), and 1% T medium. Both cell lines were grown at 37°C in a humidified incubator with 5% CO2. Cells grown to 80%-90% confluence were used for in vitro or in vivo experiments.

[0205] Example 13. 18 F]AmBF3-Tz([ 18 F]4) nonspecific B16 / F10 melanoma cell uptake. 500,000 cells / well were seeded overnight on a 6-well plate. The growth medium was removed and the radioactive tracer [ 18Reaction medium containing [F]4 was added. To determine the amount of radioactive tracer in the free fraction, at the indicated time points (15, 30, 60 and 120 min), the reaction medium was removed and collected in a microtube, after which the cells were washed with 1 mL of cold 1x PBS and the supernatant was collected in the same microtube. The membrane-bound fraction was collected by adding cold glycine buffer (1 mL) to the cells, incubating on ice for 5 min, removing the supernatant, repeating the procedure, washing the cells with cold 1x PBS and collecting all the supernatant in the same microtube. To determine the internalized fraction, 1 M NaOH was added to the cells and incubated at ambient temperature for 10 min. The supernatant was removed, the cells were washed twice with cold 1x PBS and the supernatant was collected in the same microtube. The supernatants collected separately for each phase were measured in a gamma counter to determine the radioactivity ratio (%) of each fraction. Based on the determined radioactivity distribution among the free, membrane-bound and internalized fractions, [F]4 was measured in a gamma counter to determine the radioactivity ratio (%) of each fraction. 18 It was clearly shown that F]4 did not demonstrate nonspecific uptake in B16 / F10 cells but remained in the extracellular free fraction throughout the study (99.3±0.09% at 15 min to 99.3±0.08% at 240 min, n=3). 18 The non-specific cellular uptake of [F]4 demonstrates that the tracer compound or adduct used to radiolabel the targeting moiety does not bind to an entity on the cell membrane and is not internalized into the cell in the absence of a targeting moiety such as a peptide. The distribution of radioactivity among the aforementioned fractions is shown in Figure 9.

[0206] Example 14. 18 TCO-functionalized Tyr conjugated with [F]AmBF3-Tz 3 -Octreotide ([ 18 F]AmBF3-Tyr 3 -Octreotide, [ 18 F]25) AR42J cell uptake. One million cells / well were seeded overnight on a 6-well plate. The growth medium was removed and the radioactive tracer [ 18Reaction medium containing [F]25 was added. To study the specificity of cellular uptake, one set of cells was co-incubated in the presence of a 1 μM solution of unmodified octreotide. The unmodified octreotide used as blocking octreotide contained only the octreotide peptide, which was not conjugated to a TCO moiety or a tracer compound and was therefore not radioactively labeled. To determine the amount of radioactive tracer in the free fraction, at the indicated time points (15, 30, 60 and 120 min), the reaction medium was removed and collected in a microtube, after which the cells were washed with 1 mL of cold 1×PBS and the supernatant was collected in the same microtube. The membrane-bound fraction was collected by adding cold glycine buffer (1 mL) to the cells, incubating on ice for 5 min, removing the supernatant, repeating the procedure, washing the cells with cold 1×PBS and collecting all the supernatant in the same microtube. To determine the internalized fraction, 1 M NaOH was added to the cells and allowed to incubate at ambient temperature for 10 min. The supernatant was removed, the cells were washed twice with cold 1x PBS, and the supernatant was collected in the same microtube. The supernatants collected separately for each phase were measured in a gamma counter to determine the % radioactivity in each fraction. Based on the determined distribution of radioactivity among the free, membrane-bound, and internalized fractions, [ 18 It was clear that cellular uptake of [F]25 was specific. Uptake (internalization) (from 3.21±0.06% at 15 min to 6.12±0.63% at 240 min, n=3) was efficiently blocked by excess unmodified octreotide (blockade: 0.58±0.11% at 15 min to 0.73±0.04% at 240 min, n=3). 18 Blockade of cellular uptake of [F]25 was efficient throughout the study, whereas uptake in unblocked conditions continued to increase with time. The distribution of radioactivity among the aforementioned fractions in unblocked (internalized) and blocked conditions is shown in Figure 10. AR42J cells and compound [F]25 were 18 These results with [F]25 demonstrate that internalization of the radiolabeled adduct is target specific (in this example to the somatostatin receptor) and can be prevented by blocking access of the targeting moiety of the adduct to the respective target entity.

[0207] Example 15. In control SCID mice 18 F]AmBF3-Tz([ 18 F]4) PET / CT scan. [ 18 F]AmBF3-Tz([ 18 F]4) was formulated in 10% ethanol in 0.01M PBS and administered intravenously to SCID mice. PET / CT images were acquired using an Inveon PET / CT and a Molecubes PET and CT. In the PEC / CT (Figure 11) and biodistribution studies (Figures 12a and 12b), the tracer ([ 18 [F]4) demonstrated excellent stability evidenced by the lack of bone uptake. The primary excretion route for the tracer was via the kidney, with minor accumulation in the liver and gallbladder (Figure 11), indicating an optimal prosthetic group profile.

[0208] Example 16. [ 18 F]AmBF3-Tz([ 18 F]4) after intravenous administration and 18 F]AmBF3-Tz([ 18 Excretion of radioactivity in urine following intravenous administration of [F]4). [ 18 F]AmBF3-Tz([ 18 F]4) was formulated in 10% ethanol in 0.01M PBS and administered intravenously to SCID mice. Standard uptake values ​​(SUVs) (Figures 13a and 13b), which provide the elimination profile, were determined from PET images by drawing regions of interest (ROIs) around selected organs (heart, liver, kidneys, lungs, muscle, bladder) and measuring the ratio of radioactivity per unit volume of the ROI and normalized to the injected dose. 18[F]4) demonstrated excellent stability evidenced by the lack of bone uptake. The primary excretion route for the tracer was via the kidney, with only trace accumulation in the liver and gallbladder, indicating an optimal prosthetic group profile. Excretion of radioactivity as a function of time in mouse tissues (Figures 13a and 13b) suggests that the adduct is rapidly excreted primarily via the kidney.

[0209] Example 17. 18 Biodistribution of [F]25 [ 18 F]AmBF3-Tyr 3 -Octreotide ([ 18 F]25, 0.2 nmol, 150 μL, ~1 MBq) was formulated in 4% ethanol in 0.01 M PBS and administered intravenously to Rj:NMRI-Foxn1 nu / nu mice bearing AR42J tumors. At the designated time points (t=30, 60, 120 and 240 min) after administration, selected organs were extracted, washed with water, blotted dry and then gamma counted. Based on the gamma counted data, the percentage of injected dose (ID) per gram of tissue (ID% / g) values ​​were calculated using the formula [(observed gamma counts / ID)×100] / weight of tissue (g). The resulting values ​​were plotted in the biodistribution graph presented in Figure 14. Tracer [ 18 [F]25 demonstrated tumor accumulation and extended blood circulation time, with excretion primarily via the kidney, but also some liver uptake. Bone uptake was significantly lower, suggesting that the tracer is extremely stable to defluorination in vivo.

[0210] Example 18. AR42J tumor-bearing mice 18 F]AmBF3-Tyr 3 -Octreotide ([ 18 F]25) PET / CT. [ 18 F]AmBF3-Tyr 3 -Octreotide ([ 18[F]25, 0.2 nmol, 150 μL, ~1 MBq) was formulated in 4% ethanol in 0.01 M PBS and administered intravenously to Rj:NMRI-Foxn1 nu / nu mice bearing AR42J tumors. To investigate the specificity of uptake in AR42J tumors, mice were co-administered with the blocker octreotide (44 nmol) to block accumulation of radioactivity. PET / CT images were acquired using Molecubes PET and CT. In the PET / CT (Figure 15), the tracer ([ 18 The PET-treated rat was injected with octreotide and radiolabeled [F]25) demonstrated excellent stability evidenced by a lack of bone uptake. The primary excretion pathway for the tracer was through the kidneys into the urine, although trace accumulation was detected in the liver, gallbladder, and intestine, which accounted for some background radioactivity levels in the PET images. The first animal (left animal in Figure 15) received the blockade octreotide and radiolabeled [F]26. 18 The second animal (the animal on the right in Figure 15) was given [F]25 intravenously without the blockade of octreotide (45 μg, 44 nmol). 18 The mouse received only [F]25, allowing visualization of a subcutaneous tumor in the right shoulder. 18 Blockade of [F]25 was successful, demonstrating specific uptake of radioactivity in the tumor (T=tumor) as seen in the PET image comparison in FIG. 15.

[0211] Example 19. AR42J Tumor-Bearing Mice 18 F]AmBF3-Tyr 3 -Octreotide ([ 18 Excretion of radioactivity after intravenous administration of [F]25) [ 18 F]AmBF3-Tyr 3 -Octreotide ([ 18F]25, 0.2 nmol, 150 μL, ∼1 MBq) was formulated in 4% ethanol in 0.01 M PBS and administered intravenously to Rj:NMRI-Foxn1 nu / nu mice bearing AR42J tumors. Standardized uptake values ​​(SLIV) (Figures 16 and 17) presenting the elimination profile were determined from PET images (Figure 15) with ROIs around selected organs (heart, liver, kidney, lung, muscle, bladder, tumor) and SUV was calculated as disclosed in Example 16. Mice were administered intravenously [ 18 F]25 alone (non-blocking) or after administration of 18 In AR42J tumor-bearing mice (n = 2 / group) [F]25 and octreotide (blockade) were coadministered by comparing the SUVs in the tumor at various time points. 18 The binding specificity of [F]25 was determined. The results showed that [ 18 The results demonstrate that the binding of the tracer ([F]25) is specific and can be blocked by coadministration of the blockade octreotide (blockade) (Figure 16). 18 [F]25) was primarily excreted via the kidneys with only minor accumulation in the liver that decreased over time (Figure 17). Radioactivity in the tumor peaked at approximately 40 minutes and remained relatively stable thereafter. Successful blockade of radioactivity in the tumor was demonstrated by SUV comparison data (Figure 17), demonstrating that tumor uptake was specific to the target area.

[0212] Example 20. 18 F]AmBF3-PSMA([ 18 F]28) and [ 18 F]AmBF3-tranexamic acid-PSMA ([ 18 F]29) in vitro cellular internalization C4-2 cells or LNCaP cells were cultured in 6-well plates (6 × 10 5 The cells were seeded in a 100-well plate (100 / well) and the medium was changed to a CO2-independent medium 30 minutes before the experiment. 18 F]28 or [ 18F]29 (15-20 GBq / mmol) in 1 mL of a 250 nM solution in CO2-independent medium. Specific cellular uptake was determined by blocking with 2-(phosphonomethyl)pentanedioic acid (2-PMPA) (final concentration, 400 μM, Sigma). All experiments were performed at 37 °C. Incubations were terminated after 30, 60 and 120 min by washing twice with 1 mL of ice-cold phosphate-buffered saline. Subsequently, cells were incubated twice for 5 min each with 1 mL of glycine HCl buffer (50 mM; pH 2.8) to remove the surface-bound fraction and the supernatant was collected. After a further washing step with 1 mL of ice-cold phosphate-buffered saline, cells were lysed with 0.5 mL of NaOH (1 N), collected and radioactivity was measured in a γ-counter ... 5 The radioactivity was calculated as the percentage of the initially added radioactivity bound to the cells (%IA / 10 5 All experiments were performed in triplicate. Based on the results of this experiment, the tracer [ 18 F]28 and [ 18 F]29 uptake was significantly blocked at 60 min when challenged with 2-PMPA blockade ([ 18 F]28 vs. 2.05 ± 0.28% vs. 0.58 ± 0.09% and [ 18 F]29 (1.05 ± 0.09% vs. 0.31 ± 0.03%) and is therefore specific.

[0213] Example 21. 18 F]AmBF3-PSMA([ 18 F]28) and [ 18 F]AmBF3-tranexamic acid-PSMA ([ 18 F]29) PET / CT. [ 18 F]28 or [ 18 PET / CT imaging of [F]29 was performed in C4-2 tumor-bearing mice (n=3-4) and the specificity of uptake was challenged by blocking with 2-PMPA. 18 F]28 or [ 18Mice were injected with 0.4 mM 2-PMPA (100 μL; 40 nmol) via tail vein injection 30 min prior to injection of the radioactive tracer, [F]29. 18 F]28 or [ 18 F]29 was administered via tail vein injection as a 0.01 mM solution (100 μL; 1 nmol). PET / CT images were acquired using an Inveon PET / CT and a Molecubes PET and CT. PET / CT showed that both tracers ([ 18 F]28 and [ 18 F29) demonstrated good to excellent stability evidenced by a lack of bone uptake. The primary excretion pathway for the tracer was through the kidneys into the urine. 18 As shown in FIG. 18 for F]29, specific tumor uptake was demonstrated by blocking uptake with 2-PMPA where tracer uptake was significantly reduced in the tumor (T).

[0214] Example 22. 18 F]AmBF3-PSMA([ 18 F]28) and [ 18 F]AmBF3-tranexamic acid-PSMA ([ 18 F]29) distribution in the body. [ 18 F]28 or [ 18 The biodistribution of [F]29 was determined in C4-2 tumor-bearing mice and the specificity of uptake was challenged by blocking with 2-PMPA. Each experiment was performed in triplicate. 18 F]28 or [ 18 SCID mice received 0.4 mM 2-PMPA (100 μL; 40 nmol) via tail vein injection 30 min prior to injection of [F]29.

[0215] Radioactive tracers, [ 18 F]28 or [ 18[F]29 was administered as a 0.01 mM solution (100 μL; 1 nmol) via tail vein injection. One hour after injection, animals were sacrificed (CO2 asphyxiation) and organs of interest were dissected, blotted dry, and weighed. Radioactivity was measured in a gamma counter (1480 Wizard, PerkinElmer) and calculated as a percentage of the injected dose per gram (%ID / g). The tracer [ 18 F]28 or [ 18 Tumor-associated uptake of F]29 (Figure 19) was blocked by pre-injection of 2-PMPA ([ 18 F] 7.51 ± 0.69% vs. 1.18 ± 0.19% and [ 18 F]29 (12.87±4.83% vs. 1.90±0.66%). Other organs that showed high uptake values ​​were the kidney, spleen, and gallbladder, where application of a blocking agent also reduced uptake.

[0216] Example 23. 18 F]AmBF3-PSMA([ 18 F]28), [ 18 F]AmBF3-tranexamic acid-PSMA ([ 18 F]29) shelf life and plasma stability. Formulated in 1x PBS [ 18 F]28 or [ 18 To determine the shelf-life stability of [F]29, 5 μL samples were analyzed by radio-TLC at 0.5, 1, 2, 3, 4, 5, and 6 hours after storage at room temperature (n=3). 18 F]28 and [ 18 Stability (>95%) in PBS for up to 6 hours was demonstrated for [F]29. For enzyme stability measurements, 400 μL of human plasma was added to 400 μL of [ 18 F]28 or [ 18 F]29 formulation at 37°C (n=3). After 0.5, 1, 2, 3, and 4 hours, 100 mL samples were removed from the mixture, and proteins were precipitated by adding 50 μL of acetonitrile and separated from the supernatant by centrifugation at 13,000 rpm. The supernatant was analyzed by radioactive TLC.18 F]28 and [ 18 Plasma stability (>95%) was demonstrated for up to 4 hours for compound [F]29. 18 F]28 and [ 18 [F]29 showed no significant degradation, both in the formulation solution and in human plasma, over the entire observation period of the experiment.

[0217] The foregoing description provides a complete and informative description of the best mode currently contemplated by the inventor for carrying out the invention, as non-limiting examples of specific implementations and embodiments. However, it will be apparent to those skilled in the art that the invention is not limited to the details of the embodiments presented above, but can be carried out in other embodiments or different combinations of embodiments using equivalent means without departing from the characteristics of the invention.

[0218] Moreover, some of the features of the exemplary embodiments disclosed above may be used to advantage without the corresponding use of other features. As such, the foregoing description should be considered as merely illustrative of the principles of the invention, and not in limitation thereof. The scope of the invention is therefore limited only by the appended claims.

[0219] Various non-binding exemplary aspects and embodiments have been described above. The foregoing embodiments are used only to describe selected aspects or steps that may be utilized in various implementations. Some embodiments may be presented with reference to only certain exemplary aspects. It should be understood that the corresponding embodiments may be applied to other exemplary aspects as well.

Claims

1. A tracer compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof 【Chemistry 1】 (In the formula, Each R1 is independently hydrogen (H), or a group of formula C n H 2n+1 wherein n is an integer selected from the range of 1 to 2; L is a linker moiety composed of S1-Y-S2, where: Y is (-CH 2 -) m wherein m is an integer selected from the range of 1 to 4, or Y is a polyethylene glycol linker - (PEG) X - (in the formula, (PEG) X is polyethylene oxide-CH 2 -CH 2 x repeating units of —O— group, where x is an integer selected from the range of 1 to 20; and S1 is -(CH 2 ) Z -CO-NH-(CH 2 ) Z - or S1 is -(CH 2 ) Z -NH-CO-(CH 2 ) Z -wherein each z is independently an integer selected from the range of 0 to 4; and S2 is -CH 2 - or S2 is -(CH 2 ) f -CO-NH-(CH 2 ) f - or S2 is -(CH 2 ) f -NH-CO-(CH 2 ) f -wherein each f is independently an integer selected from the range of 0 to 4; and R2 is hydrogen (H), or a phenyl substituent or a group of formula C S H 2S+1 wherein s is an integer selected from the range of 1 to 2.

2. Each R is independently hydrogen (H), or a group of formula C n H 2n+1 wherein n is an integer selected from the range of 1 to 2; L is a linker moiety composed of S1-Y-S2, where: Y is (-CH 2 -) m (wherein m is an integer selected from the range of 1 to 4), and S1 is -CH 2 —CO—NH—, or —CH 2 -NH-CO- and S2 is -CH 2 - or; or Y is a polyethylene glycol linker (PEG) X - (in the formula, (PEG) X is polyethylene oxide-CH 2 -CH 2 x repeating units of —O— group, where x is an integer selected from the range of 1 to 20; and S1 is -CH 2 -NH-CO-(CH 2 ) Z - or -CH 2 -CO-NH-(CH 2 ) Z -wherein each z is independently an integer selected from the range of 0 to 4; and S2 is -(CH 2 ) f —CO—NH—CH 2 -CH 2 - or -(CH 2 ) f —NH—CO—CH 2 -CH 2 -wherein each f is independently an integer selected from the range of 0 to 4; and R2 is hydrogen (H), or a phenyl substituent or a group of formula C S H 2S+1 2. The tracer compound of claim 1, or a pharmaceutically acceptable salt or solvate thereof, wherein the alkyl substituent has:

3. -(PEG) X 3. The tracer compound according to claim 1, wherein x is an integer selected from the range of 1 to 15 or the range of 1 to 10, or a pharmaceutically acceptable salt or solvate thereof.

4. (BF 3 ) - At least one F in the moiety 18 The tracer compound according to any one of claims 1 to 3, wherein F is F, or a pharmaceutically acceptable salt or solvate thereof.

5. An adduct of a trans-cyclooctene (TCO)-derivatized target moiety and a tracer compound described in any one of claims 1 to 4, or a pharmaceutically acceptable salt or solvate thereof, obtained by inverse electron demand Diels-Alder reaction (IEDDA) between the TCO portion of the TCO-derivatized target moiety and the tetrazine portion of the tracer compound described in any one of claims 1 to 4.

6. 6. The adduct of claim 5, or a pharmaceutically acceptable salt or solvate thereof, wherein the tetrazine ring of the tetrazine moiety of the tracer compound is chemically bonded to the TCO moiety of the TCO-derivatized targeting moiety.

7. 7. The adduct of claim 5 or 6, or a pharmaceutically acceptable salt or solvate thereof, wherein the targeting moiety is a protein, peptide, antibody, antibody fragment, or nanoparticle.

8. (BF 3 ) - At least one F in the moiety 18 The adduct of any one of claims 5 to 7, or a pharmaceutically acceptable salt or solvate thereof, wherein

9. 9. The adduct of any one of claims 5 to 8, or a pharmaceutically acceptable salt or solvate thereof, for use in detecting a target entity in a subject by radioimaging.

10. The adduct of claim 9, or a pharmaceutically acceptable salt or solvate thereof, wherein the radioimaging is positron emission tomography.

11. A method for producing a tracer compound according to any one of claims 1 to 3, comprising the steps of: a. dissolving a starting material in a polar aprotic solvent and reacting said starting material with 2-(iodomethyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane to provide an intermediate product; b. Dissolving the intermediate product in a polar aprotic solvent, and oxidizing the intermediate product with KHF in the presence of an acid, water, and an organic solvent. 2 to provide said tracer compound; The starting material is linked to a tertiary amine (—N(CH 3 ) 2 ) linked to a tetrazine moiety; The tetrazine moiety is composed of a 1,2,4,5-tetrazine ring, a phenyl ring attached to C3 of the tetrazine ring, and R2 attached to C6 of the tetrazine ring, wherein R2 is hydrogen (H), or a phenyl substituent or a group of formula C S H 2S+1 wherein s is an integer selected from the range of 1 to 2; and The linker moiety is composed of S1-Y-S2, where: Y is (-CH 2 -) m wherein m is an integer selected from the range of 1 to 4, or Y is a polyethylene glycol linker (PEG) X - (in the formula, (PEG) X is polyethylene oxide-CH 2 -CH 2 x repeating units of the —O— group, where x is an integer selected from the range of 1 to 20; and S1 is -(CH 2 ) Z -CO-NH-(CH 2 ) Z - or S1 is -(CH 2 ) Z -NH-CO-(CH 2 ) Z -wherein each z is independently an integer selected from the range of 0 to 4; and S2 is -CH 2 - or S2 is -(CH 2 ) f -CO-NH-(CH 2 ) f - or S2 is -(CH 2 ) f -NH-CO-(CH 2 ) f -wherein each f is independently an integer selected from the range of 0 to 4.

12. The linker portion of the starting material is made up of S1-Y-S2, where: Y is (-CH 2 -) m (wherein m is an integer selected from the range of 1 to 4), and S1 is -CH 2 —CO—NH—, or —CH 2 -NH-CO- and S2 is -CH 2 - or; or Y is a polyethylene glycol linker (PEG) X - (in the formula, (PEG) X is polyethylene oxide-CH 2 -CH 2 x repeating units of —O— group, where x is an integer selected from the range of 1 to 20; and S1 is -CH 2 -NH-CO-(CH 2 ) Z - or -CH 2 -CO-NH-(CH 2 ) Z -wherein each z is independently an integer selected from the range of 0 to 4; and S2 is -(CH 2 ) f —CO—NH—CH 2 -CH 2 - or -(CH 2 ) f —NH—CO—CH 2 -CH 2 -wherein each f is independently an integer selected from the range of 0 to 4.

13. A method for preparing the adduct of any one of claims 5 to 10, or a pharmaceutically acceptable salt or solvate thereof, said method comprising: providing a TCO-derivatized targeting moiety; Providing a tracer compound according to any one of claims 1 to 3; reacting the tetrazine moiety of the tracer compound with the TCO moiety of the TCO-derivatized targeting moiety; and The additive is at least one 18 radiolabeling with F to obtain the adduct; Alternatively, the method comprises: providing said TCO-derivatized targeting moiety; Providing the tracer compound according to any one of claims 1 to 3; The tracer compound according to any one of claims 1 to 3 is 18 radiolabeling with F; and reacting the tetrazine moiety of the radiolabeled tracer compound with the TCO moiety of the TCO-derivatized targeting moiety to obtain the adduct.

14. 14. The method of claim 13, comprising reacting the tetrazine moiety of the radiolabeled tracer compound with the TCO moiety of the TCO-derivatized targeting moiety at a temperature between 20 and 80°C.

15. 10. Use of a tracer compound according to any one of claims 1 to 4, or a pharmaceutically acceptable salt or solvate thereof, in the detection of a target entity in a subject by radioactive imaging of the subject, wherein the target entity is targeted by the radiolabeled tracer compound, or a pharmaceutically acceptable salt or solvate thereof.

16. For detecting target entities in a subject by radioimaging 18 A kit for the production of F-labeled adducts, comprising at least one compartment containing a tracer compound according to any one of claims 1 to 3, at least one compartment containing at least one TCO-derivatized targeting moiety, and a reagent for radiolabeling said tracer compound. 18 A kit comprising at least one compartment containing F and, optionally, aqueous and organic solvents for the inverse electron demand Diels-Alder reaction (IEDDA) and for radiolabeling of the tracer compound and / or the adduct.

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