Method for obtaining labeled, single isomeric chemical moiety targeting vectors based on the use of isomer-free dienophiles

Abstract

The present disclosure relates to a method for obtaining a labeled, single isomeric chemical moiety targeting vector suitable for obtaining a targeting vector. The method applies a specific combination between a diene and a dienophile with complementary inverse electron demand Diels-Alder cycloaddition reactivity, which upon ligation and subsequent oxidation forms a single isomeric compound. The labeled, single isomeric chemical moiety targeting vector is for use in therapy, radiotherapy, theranostics, diagnostics, and imaging. The method applies click chemistry, where one chemical moiety conjugated to a label is clicked with a second chemical moiety with complementary inverse electron demand Diels-Alder cycloaddition reactivity that is conjugated to the targeting vector and then rapidly oxidized to form a single isomeric compound.

Description

[Technical field]

[0001] The present invention relates to methods for obtaining labeled single isomeric chemical entity targeting vectors, the obtained targeting vectors, and uses of the targeting vectors. The labeled single isomeric chemical entity targeting vectors can be used in treatments such as radiotherapy, diagnosis, imaging, and other photochemical methods. [Background technology]

[0002] Labeled targeting vectors based on click chemistry between dienes and dienophiles are used for both imaging purposes and therapy, such as diagnostics and other photochemical imaging methods. Such targeting vectors are labeled with radiolabels, for example, which can be applied in diagnostics and / or therapy. Since different radionuclides serve different purposes, the specific use depends on the identity of the radiolabel used. The specific application further depends on the specific target that the vector is intended for. Currently, several combinations of radiolabels and vectors are applied in diagnostics, therapy, theranostics and imaging. There are different chemical moieties that connect the radiolabeled entity(ies) with the targeting moiety, and the present invention is based on click chemistry, in which dienes and the corresponding dienophiles are ligated, thereby bridging the radiolabel and the targeting moiety.

[0003] The term "click chemistry" refers to a class of reactions that are fast, easy to use, versatile, chemoselective, and give high product yields. These reactions have found applications in a wide variety of research fields, including materials science, polymer chemistry, and pharmaceutical science. Radiochemistry is one area that has shown the true potential of click chemistry, as disclosed, for example, in Zeng et al, Journal of Nuclear Medicine, 54, 829-832, 2013. In essence, the selectivity, ease, rapidity, and modularity of click ligation make it almost ideal for the construction of radioactive tracers, a process that typically involves working with biomolecules in aqueous conditions using a rapidly decaying radioisotope. Among the various click chemistries, one of the most suitable and utilized for radiolabeling is tetrazine ligation.

[0004] Tetrazine ligation is a click reaction characterized by the formation of a covalent bond between 1,2,4,5-tetrazine (Tz) and, typically, trans-cyclooctene (TCO). The reaction is initiated by an inverse electron-demand Diels-Alder reaction, followed by a retro Diels-Alder reaction driven by N2 emission. Tetrazine ligation is one of the fastest known chemical ligations, with a second-order rate constant of 10 in acetonitrile at 25 °C. 6 M -1 s -1 This is the Staudinger ligation (0.003M -1 s -1 ) and strain-promoted azide-alkyne cycloaddition (SPAAC) (0.1M -1 s -1), which is in contrast to other known click reactions such as . For this reason and because of its specificity, Tz ligation is ideally suited for synthon-based labeling. One major drawback of conventional ligation of Tz with TCO is that the ligation is performed without any position specificity. Furthermore, conventional ligation of Tz with TCO results in several tautomeric forms. This means that conventional ligation of Tz with TCO typically results in a complex reaction mixture of at least 16 unique isomeric products (Scheme 1). Such a mixture of isomeric products cannot be directly applied for pharmaceutical purposes, since the specific isomer of the compound affects the pharmacokinetics of the therapeutic agent and the potential toxic effects of any of the individual isomers cannot be distinguished. Although the multiple dihydropyridazines resulting from conventional ligation can slowly autooxidize to the corresponding pyridazines over a period of hours to days, thereby slowly reducing the number of tautomeric forms of the product (WO2012 / 121746), the number of regioisomeric and stereoisomeric products is not reduced by this slow oxidative transformation. Although means to speed up the conversion of dihydropyridines to pyridazines have been described (Karaki Fumika et al, Tetrahedron, 97, 132411, 2021; Keinanen et al., ACS Medicinal Chemistry Letters, 7, 62-66, 2015), none of the reported cases completed the oxidation within the 2 hours required for the use of pyridazines as radiopharmaceuticals. WO2017 / 059397, WO2020 / 242948, Syvanen et al., ACS Chemical Neuroscience, 11, 4460-4468, 2020, and WO2012 / 121746 disclose ligations between tetrazines and TCO, which inevitably result in several isomeric chemical moieties.

[0005] Alternatively, pyridazines can be prepared by ligation of Tz to strained cyclic alkynes, but this reaction suffers from a slow second-order rate constant.

[0006] Radiopharmaceuticals are increasingly being used in theranostics, especially within oncology, for both diagnostic imaging and targeted radionuclide therapy. Positron emission tomography (PET) is the gold standard in nuclear imaging, with better resolution and quantification than other modalities. 2,200,800 clinical PET scans were performed in 2019 in the United States alone. Targeted radionuclide therapy is more effective in treating cancer than many state-of-the-art chemotherapy. It also has an advantage over external beam radiotherapy (e.g., "Gamma Knife") in that it offers a way to limit the delivered dose to the tumor and its immediate surrounding areas, which makes particular sense in the radiotherapy of micrometastatic disease. The combination of both diagnostic imaging and targeted radiotherapy can be used in "theranostics," a concept with powerful applications in personalized medicine, for patient selection, dose setting, and treatment response monitoring. A theranostic pair is two radionuclides that do not change the pharmacokinetics of the radiopharmaceutical but can be substituted for one another, shifting their application between diagnostic imaging and radionuclide therapy.

[0007] The two most widely used imaging modalities are nuclear-based PET and SPECT. Both methods rely on the combination of a radionuclide with a vector that specifically targets cancer cells. In imaging, such radiolabeled vectors are called "radiotracers". The radiotracer accumulates in tumor lesions, whose location can then be visualized by detecting the emitted radiation. PET is strongly favored in oncology, while SPECT predominates in cardiology and in the production of bone scans and certain other specialized organ scans. Worldwide, the ratio of SPECT to PET cameras used in hospitals is about 5:1.

[0008] Single photon emission computed tomography (SPECT) is the older of the two methods. SPECT imaging uses radionuclides that emit gamma photons, typically in the 100-200 keV range. A series of 2D projection images of the distribution of the radiotracer in the body are acquired by one of the additional gamma cameras from multiple angles. These projection images are then assembled to produce a 3D image.

[0009] Positron emission tomography (PET) is currently considered the most advanced form of nuclear imaging. An important use of PET in oncology is the diagnosis and treatment monitoring, especially of metastatic cancer. Compared to previous modalities, especially SPECT, PET provides images with improved resolution and sensitivity, as well as generally higher image quality. These properties are particularly relevant for the detection and subsequent treatment of very small metastases. New innovations, especially whole-body PET, which dramatically increase sensitivity and detect more metastases, are expected to further increase PET's favorable position in the modern clinic. PET relies on the use of radionuclides that emit positrons upon decay. These positrons travel a limited distance and are then annihilated by electrons in the surrounding medium. This produces two annihilation photons, each of 511 keV, emitted in opposite directions. These photons can be detected by a PET scanner.

[0010] The radionuclide of choice for PET is fluorine-18( 18 F). The decay half-life is 110 minutes, and 97% of the positrons are released each time it decays. 18 F is nearly ideal for clinical PET applications. This is especially true for small molecule and peptide-based radiopharmaceuticals, which make up the majority of relevant PET tracers. Equally important, 18 F can be practically produced in huge quantities (>300 doses per production) in standard biomedical cyclotrons, which are readily available in most of the world, of which there are over 200 in Europe alone. Thus, 18 F is produced by its closest competitor, the radioactive metal gallium-68 (68 Ga) and does not share the concerns about adequate supplies. 18 The lower positron energy of F allows for higher resolution images. Compared to the most widely used current diagnostic radionuclides, the SPECT radionuclide technetium-99m, 18 F provides the highest quality images through its status as a PET radionuclide. Therefore, 18 F is suitable as an important diagnostic radionuclide of the future.

[0011] Radioactive iodine is widely used in SPECT imaging. Traditionally, iodine-131 ( 131 I was used in the past, but today, clinical SPECT scans typically use iodine-123 ( 123 I) is used. 123 This is due to the relatively short half-life of I (T1 / 2 = 13.2 hours), which, combined with the lack of beta-minus emission, 131 Its gamma photon energy of 159 keV is well suited for clinical SPECT imaging. Due to the inherent accumulation of iodine in the thyroid gland, its free form 123 I is widely used in imaging thyroid disease. As a component of a SPECT radiotracer, 123 I is used, for example, in the contrast agents MIBG (oncology) and isoflupane (CNS). 123 I is a clinically used beta-negative emitting therapeutic radionuclide 131 I and investigational Auger electron radiotherapy agents 125 It forms a theranostic pair with I. Iodine-123 is produced in a cyclotron by proton bombardment of xenon in capsules and is commercially available.

[0012] Iodine-124( 124 I) can be used for PET imaging. It is usually produced in a cyclotron by bombardment with enriched tellurium-124. However, 124The imaging properties of I are not ideal: it has a complex decay scheme with many high-energy gamma emissions, only 23% of which decay into positron emission.

[0013] Astatine-211( 211 At) is a therapeutic nuclide that primarily emits alpha particles upon decay. Alpha particles are absorbed in tissues at just 100 μm and are undetectable by external scanners. However, 211 One of the attenuation branches of At also produces X-rays in the 70-90 keV range that can be imaged with a gamma camera or SPECT scanner. 211 The distribution of At-labeled radiopharmaceuticals can be assessed clinically by SPECT imaging.

[0014] However, incorporating the above radionuclides into molecules presents challenges that limit the practical use of radionuclide-based therapy, diagnostics, and imaging.

[0015] Most of the PET, SPECT and therapeutic radionuclides mentioned above are radiometals. The traditional method of introducing radiometals into vector molecules is the use of chelator groups that form coordinate bonds with radiometal atoms. Radiolabeling procedures typically involve mixing a radiolabeled precursor (a vector with a chelator group attached) with a radiometal ion and heating the mixture to allow the chelation reaction to proceed. Although radiometal chelation is conceptually simple, it has several drawbacks, namely: -The radiolabeled product often cannot be separated from the unlabeled precursor because the differences in physicochemical properties are not significant; - Chelation reactions are sensitive to trace metal impurities in the solutions used for radiolabeling, making scale-up problematic; -The heating required to overcome the activation barrier of the chelation reaction may degrade temperature-sensitive vectors There is.

[0016] Compared to its radioactive metal alternatives, 18F is a halogen and requires covalent attachment to the targeting vector. This is in contrast to the chelator-based labeling techniques utilized for radiometals. The covalent bond is currently typically formed by direct nucleophilic displacement of a leaving group such as a triflate. The conditions for such chemistry are harsh, lengthy, poorly scalable, and therefore incompatible with many vectors of growing importance, especially the peptide class.

[0017] Small molecule radiopharmaceuticals containing radioactive iodine are typically prepared using either electrophilic decyclization or iodine-iodine exchange radiochemistry. The former is a mild, versatile, and practical reaction in which a radioactive iodide is oxidized to a positively charged iodine species, which then displaces a leaving group, typically a stannyl, in an aromatic substitution reaction. This reaction occurs at room temperature, often in quantitative yields. Iodine-iodine isotope exchange is used when high molar activity is not a concern and when the substrate can tolerate harsh conditions. The exchange occurs at high temperatures with acid and copper as a catalyst.

[0018] Similar to fluorine and iodine, astatine-211 is a halogen and can be attached to targeting vectors via covalent bonds. The aliphatic astatine-carbon bond does not provide sufficient in vivo stability, thereby 211 At is typically introduced onto an aryl ring to form an astatoaryl moiety. Unlike iodine, astatine cannot stably couple to tyrosine residues in proteins. Instead of binding to tyrosine, 211 At has been found to form weak bonds with the sulfhydryl groups of cysteine. 211 At labeling requires the synthesis of dedicated precursors that display a suitable leaving group, such as a trialkylstannyl, attached to the aryl ring. 211 At labeling protocols use oxidizing agents such as chloramine-T or N-chlorosuccinimide, which can potentially degrade biomolecules used as targeting vectors.

[0019] 18F has long been established as the best-in-class radionuclide for diagnostic PET imaging, 211 At is the most promising therapeutic radionuclide for alpha therapy. Iodine radioisotopes 123 I is SPECT imaging, 124 I is PET imaging, 125 I is Auger therapy, 131 I are each useful for beta therapy. Moreover, all three elements can be introduced into the aryl ring to form fluoro / iodo / astatoaryl moieties with maximum structural similarity, which is essential for theranostic pairs. Nevertheless, 18 F and 211 At or radioactive iodine 123 I, 124 I, 125 I, 131 There are no reports of theranostic use in combination with any of the I.

[0020] 18 F / 211 At and 18 F / 123 I, 124 I, 125 I, 131 One obstacle to the development of theranostic pairs of I is the poor labeling chemistry, as mentioned above, which prevents direct site-selective labeling of biomolecular targeting vectors with these radionuclides. Another obstacle is that not all targeting vectors contain aryl moieties, so these must be introduced as prosthetic groups, also known as synthons.

[0021] Due to these challenges, synthon-based methods have been investigated for the preparation of radiopharmaceuticals. These methods involve the direct labeling of a separate intermediate compound ("synthon"), which is then conjugated to the vector under mild conditions. Thus, the vector is not exposed to the harsh conditions of direct radiolabeling, even though it does not need to be modified by a chemical tag complementary to the radiolabeled synthon. What becomes important then is the chemistry used to conjugate the synthon with the vector. This chemistry must be specific, compatible with pharma-ceutical relevant aqueous media, and high-yielding, to avoid loss of radioactivity and minimize the need for subsequent purification. Importantly, it must also be extremely rapid, since the procedures for preparing radiopharmaceuticals must be carried out at nano- to micromolar concentrations and proceed to completion or near completion within minutes due to the decay of the radionuclide. In light of this, click chemistry has emerged as a strategic approach to radiolabel a range of targeting vectors, such as mAbs, nanomedicines, peptides or small molecules.

[0022] Targeted radionuclide therapy (TRT) can be based on beta emitters, Auger electron emitters, and alpha emitters.

[0023] Beta particle-emitting radionuclides (e.g. 90 Y, 177 Lu, 131I) decays via the emission of high energy electrons (beta particles) that travel a distance of tissue up to about 12 mm. The decay energy is deposited within the largest volume of tissue for the three treatment types described in this application. Therefore, beta emitters are suitable for the treatment of medium-sized tumors where most of the dose is absorbed by cancer cells. However, in micrometastases or heterogeneous tumors, even if the concentration of the radionuclide in and around the tumor is perfect, most of the radiation dose is absorbed by the surrounding healthy cells. Therefore, beta emitters are not optimal for the treatment of micrometastases or heterogeneous tumors. This is an important drawback of beta emitters, since micrometastases are one of the main causes of cancer recurrence and cancer death.

[0024] Alpha emitter ( 225 Ac, 211 At, 212 Pb) decay with the emission of alpha particles. Alpha particles are much heavier than beta particles, and their trajectories are linear and on the order of 30-100 μm, the diameter of a few mammalian cells. Thus, all the energy from the decay is delivered to very few adjacent cells. Alpha radiation has greater cytotoxicity compared to beta radiation and can be delivered to micrometastases in a highly concentrated manner.

[0025] A striking example of the benefits of alpha therapy is the most important beta therapy. 177 Patients with metastatic prostate cancer resistant to Lu-PSMA were treated with alpha therapy 225 A complete remission was achieved after three cycles of Ac-PSMA.

[0026] Auger electron radiotherapy (AeRT) uses radionuclides that, upon decay by electron capture (EC) or internal conversion (IC), emit a shower of extremely short-range electrons. Advanced drug delivery techniques allow these specialized radionuclides to be delivered to the nuclei of cancer cells, where the emitted Auger electrons disrupt DNA and kill the cancer cells. Notably, the short range of Auger electrons ensures that their energy is deposited primarily within the target cells, allowing for extremely localized treatment.123 I and 125 I has high Auger electron yields and is suitable for AeRT.

[0027] Iodine-131( 131 I) is a beta particle emitter that is widely used in clinical radionuclide therapy. It has a decay half-life of 8.0 days and a primary emission of beta particles with a maximum energy of 606 keV at 90% abundance. These beta particles have a maximum range in tissue of about 2 mm and 131 It allows I to treat small to medium sized tumor lesions. It is widely used in thyroid ablation due to its inherent accumulation in thyroid tissue. In addition, 131 A therapeutic variant of MIBG radiolabeled with I is available, 131 I is used in radioimmunotherapy. 123 I(SPECT) and 124 It forms a theranostic pair with I(PET). Both iodine-123 and iodine-125 have a substantial emission of approximately 10 and 20 Auger electrons, respectively. This makes them suitable for Auger electron radiotherapy, a form of radionuclide therapy currently under investigation.

[0028] Astatine-211 is an alpha-emitting radionuclide with a half-life of 7.2 hours. Unlike most other alpha emitters used in targeted alpha therapy, 211 At produces one alpha particle per decay chain, which offers several interpretative advantages. First, toxic side effects from radioactive daughter nuclides released from the targeting vector as a result of the initial decay are limited. Second, radiation dosimetry calculations are simplified. Furthermore, (for therapeutic nuclides) 211 The relatively short half-life of At allows for improved control of the radiation dose delivered to the patient.

[0029] Tumor targeting 211Phase I / II clinical trials using At-labeled radiopharmaceuticals have demonstrated low acute toxicity and the absence of radiation side effects. Preclinical studies have demonstrated that At-labeled radiopharmaceuticals are effective in animal models. 211 We demonstrated efficient tumor eradication by At. 211 At is a very promising therapeutic radionuclide.

[0030] However, for click chemistry such as Tz-TCO ligation to result in a viable final product in regulatory and commercial contexts, it is important that only a single ligation product is generated. This is not possible using available methods, as ligation traditionally results in a large number of isomeric products that cannot be separated, preventing clinical translation due to toxicity concerns and unpredictable pharmacokinetics. The present invention provides a method in which a specific combination of chemical moieties with complementary inverse electron demand Diels-Alder cycloaddition reactivity allows for the formation of a single compound by ligation followed by rapid oxidation. This means that only one isomeric product is obtained and therefore separation of the isomeric products is not required. This method is advantageously applicable to, for example, 18 F, 123 I, 124 I, 125 I or 131 I, and 211 With At, radiolabeling of any tracer is possible with unparalleled efficiency and practicality. Summary of the Invention

[0031] The present invention provides a method for obtaining a labeled, single isomeric chemical moiety targeting vector. The method applies click chemistry, where one chemical moiety conjugated to a label is clicked with a second chemical moiety with complementary inverse electron demand Diels-Alder cycloaddition reactivity that is conjugated to a targeting vector and then rapidly oxidized to form a single isomeric compound. The advantage of this method is that it yields a single isomeric final product within a minimal period of time, thereby reducing clinical translation and manufacturing costs.

[0032] The method for obtaining a labeled, single isomeric chemical moiety targeting vector comprises the following steps: a) labeling a first chemical moiety having inverse electron demand Diels-Alder cycloaddition reactivity and conjugated to a drug, imaging agent, or therapeutic agent with a labeling agent, wherein the first chemical moiety is selected from the group consisting of symmetrically substituted dienes, unsymmetrically substituted dienes, and isomer-free dienophiles, at least one of whose planes of symmetry passes through the nitrogen-nitrogen bond of at least one tetrazine ring; b) ligating the labeled first chemical moiety obtained in step a) with a second chemical moiety having complementary inverse electron demand Diels-Alder cycloaddition reactivity and conjugated to a targeting vector, wherein the second chemical moiety is selected from the group consisting of symmetrically substituted dienes, unsymmetrically substituted dienes, and isomer-free dienophiles, in which at least one of said planes of symmetry passes through the nitrogen-nitrogen bond of at least one tetrazine ring; Here, the kinetics of the inverse electron-demand Diels-Alder cycloaddition between a first chemical moiety and a second chemical moiety are determined by stopped-flow spectrophotometry at 500 M in phosphate-buffered saline (PBS) at 25 °C. -1 s -1 has a minimum second order rate constant of However, the labeling agent in step a) 94 Tc, 99m Tc, 211 At, 223 Ra or 225When Ac, the labeling agent is conjugated to an asymmetrically substituted diene; and c) oxidizing the ligated, labeled targeting vector obtained from step b) by adding 1 to 100 equivalents of chloranil, fluoranil, DDQ or NaNO2 at a temperature ranging from 15°C to 50°C for up to 60 minutes.

[0033] The labeling agent can be any agent that is useful as a marker, imaging agent, therapeutic agent or theranostic agent, including radionuclides and fluorescent moieties.

[0034] The targeting vector can be any suitable vector directed to a particular target, including antibodies, nanobodies, polymers, nanomedicines, cells, proteins, peptides, and small molecules.

[0035] Some examples of symmetrically substituted dienes, asymmetrically substituted dienes, and isomer-free dienophiles suitable for this method are disclosed herein. Suitable dienes include, for example, tetrazine. Suitable dienophiles include, for example, trans-cycloheptene (TCH'), trans-cyclooctene (TCO') and trans-cyclononene (TCN').

[0036] The methods of the present invention also include embodiments in which the first chemical moiety and / or the second chemical moiety are derived from specific precursors, including precursors to symmetrically substituted dienes, precursors to asymmetrically substituted dienes, and precursors to isomer-free dienophiles, respectively.

[0037] The invention further provides for the use of the labeled, single isomeric chemical moiety targeting vectors obtained by the method in theranostics, therapy, radiotherapy, diagnostics, and imaging. [Brief description of the drawings]

[0038] [Figure 1] 1 is a scheme showing the synthesis of symmetrical tetrazines. [Diagram 2] 1 is a scheme showing an alternative synthesis of symmetrical tetrazines. [Diagram 3] Radio-HPLC of [18F]I after deprotection. [Figure 4] The UV trace of [18F]XX is shown. [Diagram 5] The radioactive trace of [18F]XX is shown. [Figure 6] UV and radioactive traces of [18F]XX-analytical HPLC are shown. [Figure 7] UV trace of [18F]X-Semi-Prep HPLC. [Figure 8] Radioactive trace of [18F]X - semi-preparative HPLC. [Figure 9] UV and radioactive traces of [18F]X-analytical HPLC. [Figure 10] UV trace of [18F]XI-Semi-Prep HPLC. [Figure 11] Radioactive trace of [18F]XI - semi-preparative HPLC. [Figure 12] UV and radioactive traces of [18F]XI-analytical HPLC. [Figure 13] Radio-HPLC of crude [125I]XVII is shown. [Figure 14] UV and radiotraces of [211At]XIV-semi-preparative HPLC. [Figure 15] Radioactive trace-analysis HPLC of purified [211At]XIV. [Figure 16] 1 is a table showing the results of a click experiment using radiolabeled tetrazines from Example 11. [Figure 17] 1 is a scheme of click reactions performed with 27 and a table of data obtained from click reactions performed with 27. [Figure 18] 1 shows a scheme of click reactions performed with Tz and two different TCOs and a table of data from these click reactions. [Figure 19]HPLC analysis of the tetrazine-TCO pyridazine tested in Example 12 after oxidation. [Figure 20] 1 shows a scheme of click reactions performed with [18F]Tz and four different TCOs and a table of data obtained from these click reactions. [Figure 21] The structure of the vector tested in Example 13. [Figure 22] 1 is a table showing the results of oxidation of the vector from Example 13. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0039] In a first aspect, the present invention provides a method for obtaining labeled, single isomeric chemical moiety targeting vectors by applying a specific combination between a diene and a dienophile with complementary inverse electron demand Diels-Alder cycloaddition reactivity, which upon ligation and subsequent oxidation forms a single isomeric compound.

[0040] Either the diene or dienophile is conjugated to an agent of interest, such as a drug, imaging agent, or therapeutic agent, and labeled with a labeling agent. A compatible diene or dienophile, respectively, is conjugated to a targeting vector of interest.

[0041] Ligation between dienes and dienophiles is based on inverse electron demand Diels-Alder cycloaddition reactivity, therefore, dienes and dienophiles to be ligated must have complementary inverse electron demand Diels-Alder cycloaddition reactivity. Furthermore, ligation between dienes and dienophiles is based on inverse electron demand Diels-Alder cycloaddition reactivity, as determined by stopped-flow spectrophotometry, for relevance to the present method. -1 s -1 The reaction kinetics should have a minimum second-order rate constant of 500 M in phosphate buffered saline (PBS) at 25 °C. -1 s -1Ligations with second order rate constants below 100 s take too long to yield a labeled targeted vector, as the radiolabeled agent often has limited time to be used for imaging and / or therapy after the ligation step.

[0042] The second-order rate constant can be measured by various means, but is typically measured by stopped-flow spectrometry, for example as described in (Chance, Rev. Sci. Instrum. 1951, 22, 619-627). In this application, the method described in Battisti et al. J. Med. Chem. 2021, 64, 20, 15297-15312 was applied.

[0043] In order to obtain a labeled, chemical moiety targeting vector that results in only a single isomer when dienes and dienophiles are ligated with complementary Diels-Alder cycloaddition reactivity, a combination of two requirements is necessary. The first requirement concerns the selection of the structure of the diene and dienophile to be ligated. Here, it was found that two ligation options are available to combine dienes and dienophiles to obtain a composite (i.e., clicked) product that upon oxidation yields only one isomeric form of the targeting vector: the combination option i) ligation between an unsymmetrically substituted diene and an isomer-free dienophile; the ligation combination option ii) ligation between a symmetrically substituted diene and an isomer-free dienophile.

[0044] The second requirement concerns the oxidation step. It has been shown in the present application that not all oxidizing agents are suitable for this method, either because the oxidation is not efficient enough or gives by-products when working within the required time frame available, or because the oxidation affects the structure of the targeting vector, thereby potentially preventing the labeled targeting vector from binding to its target. The oxidation efficiency of this oxidation step should be at least 90%, i.e., at least 90% of the labeled and clicked targeting vector should be in the form of a single isomer after the oxidation step. Oxidation conditions that obtain less than 90% of the product are in the form of a single isomer, which is not pure enough for use in therapy / imaging / diagnosis, and which requires additional toxicological studies.

[0045] It has been shown herein that the addition of 1 to 100 equivalents of an oxidizing agent selected from the group including chloranil, fluoranil, DDQ, and NaNO2 results in suitable oxidation at temperatures ranging from 15°C to 50°C for up to 60 minutes.

[0046] Thus, a method for obtaining a labeled, single isomeric chemical moiety targeting vector includes: a) labeling a first chemical moiety having inverse electron demand Diels-Alder cycloaddition reactivity and conjugated to a drug, imaging agent, or therapeutic agent with a labeling agent, wherein the first chemical moiety is selected from the group consisting of symmetrically substituted dienes, unsymmetrically substituted dienes, or isomer-free dienophiles, at least one of whose planes of symmetry passes through the nitrogen-nitrogen bond of at least one tetrazine ring; b) ligating the labeled first chemical moiety obtained in step a) with a second chemical moiety having complementary inverse electron demand Diels-Alder cycloaddition reactivity and conjugated to a targeting vector, wherein the second chemical moiety is selected from the group consisting of symmetrically substituted dienes, unsymmetrically substituted dienes, or isomer-free dienophiles, at least one of whose planes of symmetry passes through the nitrogen-nitrogen bond of at least one tetrazine ring; Here, the kinetics of the inverse electron demand Diels-Alder cycloaddition between a first chemical moiety and a second chemical moiety are determined spectrophotometrically at 500 μM in PBS at 25 °C. -1 s -1 has a minimum rate constant of However, the labeling agent in step a) 94 Tc, 99m Tc, 211 At, 223 Ra or 225 When Ac, the labeling agent is conjugated to an asymmetrically substituted diene; and c) oxidizing the ligated, labeled targeting vector obtained from step b) by adding 1 to 100 equivalents of a quinone oxidizing agent, such as chloranil, fluoranil, DDQ or NaNO2, at a temperature ranging from 15°C to 50°C for up to 60 minutes.

[0047] The drug, imaging agent or therapeutic agent to which the first chemical moiety is conjugated is in some embodiments the same as the labeling agent, such as may be the case when the labeling agent is an agent that can be applied both as a label and as a therapeutic or imaging agent.

[0048] In some embodiments, the labeling agent is a radionuclide.Some radionuclides can be applied to both imaging, diagnosis and / or treatment, and in this example, the same radionuclide is applied as labeling agent and imaging agent or treatment agent.Labeling dienes or dienophiles with radionuclides usually does not bring about 100% labeling efficiency with radionuclides, and some of the labeled products are necessarily labeled with the stable isotope of the corresponding radionuclide element.

[0049] When using symmetrically substituted dienes as the starting point for the radiolabeled moiety, it is only possible to obtain a radiolabeled, single isomeric chemical moiety targeting vector if the radiolabel exists in both radioactive and stable forms, since the symmetrically substituted diene contains two targets for the radionuclide / stable isotope.

[0050] However, some of the radionuclides of interest in therapy and imaging are not available in stable forms. Therefore, the corresponding elements cannot label symmetric positions of the labeled target, which inevitably results in two or more isomeric forms of the final product. Therefore, when the labeling agent is 211 At, 223 Ra or 225 Ac, symmetrically substituted dienes cannot be obtained, and therefore, if these radionuclides are to be applied as labels and / or diagnostic / therapeutic agents, option ii) of the ligation combination is to be applied, where the asymmetrically substituted diene is ligated with an isomer-free dienophile. Thus, this method is advantageous in that the labeling agent in step a) is 94 Tc, 99m Tc, 211 At, 223 Ra, or 225 When Ac is used, it should be provided that the labeling agent is conjugated to an asymmetrically substituted diene. This method can be implemented, for example, as follows: 18 F, radioactive iodine ( 123 I, 124 I, 125 I, or 131 I) and 211 It allows the labeling, such as radiolabeling, of any targeting vector with At with unparalleled efficiency and practicality. The groundbreaking nature of the method is the possibility to form a single final product within 60 minutes, often much less than 60 minutes, e.g., within 1-20 minutes, thereby facilitating clinical interpretation. In contrast, conventional tetrazine ligation leads to multiple products, thus requiring large and cumbersome toxicological packages or laborious and time-consuming separations.

[0051] definition Symmetrical tetrazine means, in the context of the present invention, any tetrazine that displays one or more planes of symmetry in its chemical structure, either as a "cold" reference or after radiolabeling / deprotection, one of which passes through the nitrogen-nitrogen bond of the tetrazine ring.

[0052] By cold reference, in the context of the present invention, is meant a compound that is labeled with a non-radioactive isotope of an atom, where a radioactive isotope of the same atom is required to obtain a radiolabeled version of the same compound. The term cold reference, in the context of the present invention, further includes compounds that contain one or more protecting groups that are replaced by a labeling agent upon labeling.

[0053] By asymmetric tetrazine is meant in the context of the present invention, as a "cold" reference or after radiolabeling / deprotection, any tetrazine that does not have a plane of symmetry through the nitrogen-nitrogen bond of the tetrazine ring.

[0054] Isomer-free dienophile or isomer-free TCH / TCO / TCN means in the context of the present invention any dienophile or any TCH, TCO and TCN, respectively, which leads to the formation of only one isomer / enantiomer after reaction with the corresponding tetrazine and oxidation.

[0055] TCH, in the context of the present invention, means any seven-membered ring with at least one double bond in the trans configuration capable of reacting as a dienophile in an inverse electron demand Diels-Alder cycloaddition.

[0056] By TCO is meant in the context of the present invention any eight-membered ring having at least one double bond in the trans configuration capable of reacting as a dienophile in an inverse electron demand Diels-Alder cycloaddition.

[0057] TCN means, in the context of the present invention, any nine-membered ring with at least one double bond in the trans configuration capable of reacting as a dienophile in an inverse electron demand Diels-Alder cycloaddition.

[0058] It is well known within click chemistry to use a diene such as tetrazine that is clicked with a dienophile such as trans-cycloheptene (TCH'), trans-cyclooctene (TCO') or trans-cyclononene (TCN') that has complementary inverse electron demand Diels-Alder cycloaddition reactivity. In a preferred embodiment, the method to obtain a labeled, single isomeric chemical moiety targeting vector is one in which the diene is tetrazine and the dienophile is trans-cycloheptene (TCH), trans-cyclooctene (TCO) or trans-cyclononene (TCN).

[0059] The labeled, single isomeric chemical moiety targeting vector obtainable by the method according to the present invention can be applied for various purposes depending on the characteristics of the agent applied as a label. Labeling agents suitable for this method include radioactive and fluorescent labels.

[0060] In a preferred embodiment, the labeling agent applied in step a) of the method for obtaining the labeled, single isomeric chemical moiety targeting vector is a radionuclide or a stable isotope of the corresponding element.The characteristics of different radionuclides that are commonly applied and their corresponding uses are well known in the art.

[0061] Radionuclide labeling agents and the corresponding stable isotopes of elements suitable for use as labeling agents in step a) of the method for obtaining a labeled, single isomeric chemical moiety targeting vector are 1 H, 2 H, 3 H, 11 C. 12 C. 13 C. 14 C. 13 N, 14 N, 15 N 18 F, 19 F, 123 I, 124 I, 125 I, 127 I, 131 I, 15 O.16 O、 17 O、 18 O、 43 Scr、 44 Scr、 45 Scr、 45 To, 46 To, 47 To, 48 To, 49 To, 50 To, 55 Co、 58 mCo、 59 Co、 60 Cu、 61 Cu、 63 Cu、 64 Cu、 65 Cu、 67 Cu、 67 Ga、 68 Ga、 69 Ga、 71 Ga、 76 Br、 77 Br、 79 Br、 80 mBr、 81 Br、 72 As、 75 As、 86 Y、 89 Y、 90 Y、 89 Zr、 90 Zr、 91 Zr、 92 Zr、 94 Zr、 149 Tb、 152 Tb、 159 Tb、 161 Tb、 111 My, 113 My, 114 mIn、 115 mIn、 175 Lu、 177 Lu、 185 King、 186 King、 188 King、 201 Tl、 203 Tl、 205 Tl、 206 Pb、 207 Pb、 208 Pb、 212 Pb、 209 Wind、 212 Wind、 213Bi、 31 P、 32 P、 33 P、 32 S、 35 S、 45 Sc、 47 Sc、 84 Sr、 86 Sr、 87 Sr、 88 Sr、 89 Sr、 165 Your, 166 Your, 156 Dy、 158 Dy、 160 Dy、 161 Dy、 162 Dy、 163 Dy、 164 Dy、 165 Dy、 227 Th、 232 Th、 51 Cr、 52 Cr、 53 Cr、 54 Cr、 73 The、 74 The、 75 The、 76 The、 77 The、 78 The、 80 The、 82 The、 94 Tc、 99m Tc、 103 Rh、 103 mRh、 119 Sb、 121 Sb、 123 Sb、 135 The、 138 The、 139 The、 162 Er、 164 Er、 165 Er、 166 Er、 167 Er、 168 Er、 170 Er、 193 mPt、 195 mPt、 192 Pt、 194 Pt、 195 Pt、 196 Pt、 198 Pt、 211 And、 223 Does,225 Contains Ac.

[0062] In a preferred embodiment, the radionuclide labeling agent is 11 C. 13 N, 15 O. 18 F, 43 Sc, 44 Sc, 45 Ti, 55 Co, 60 Cu, 61 Cu, 64 Cu, 68 Ga, 76 Br, 72 As, 86 Y, 89 Zr, 90 Y, 149 Tb, 152 Tb and the corresponding stable isotope of the element is selected from the group consisting of 12 C. 13 C. 14 N, 15 N, 16 O. 17 O. 18 O. 19 F, 45 Sc, 46 Ti, 47 Ti, 48 Ti, 49 Ti, 50 Ti, 59 Co, 63 Cu, 65 Cu, 69 Ga, 71 Ga, 75 As, 79 Br, 81 Br, 89 Y, 90 Zr, 91 Zr, 92 Zr, 94 Zr, 159 Tb These radionuclides and their stable isotopes of the corresponding elements are particularly useful in positron emission tomography (PET).

[0063] In another preferred embodiment, the radionuclide labeling agent is 64 Cu, 67Cu, 67 Ga, 111 In, 131 I, 177 Lu, 186 Re, 201 Tl, 212 Pb, 213 Bi, and the corresponding stable isotope of the element is 63 Cu, 65 Cu, 69 Ga, 71 Ga, 113 In, 127 I, 175 Lu, 185 Re, 203 Tl, 205 Tl, 206 Pb, 207 Pb, 208 Pb, 209 Bi. These radionuclides and their stable isotopes of the corresponding elements are particularly useful in single photon emission computed tomography (SPECT).

[0064] In another preferred embodiment, the radionuclide labeling agent is 32 P, 33 P, 47 Sc, 64 Cu, 67 Cu, 89 Sr, 90 Y, 166 Ho, 161 Tb, 165 Dy, 177 Lu, 186 Re, 188 Re, and the corresponding stable isotope of the element is 31 P, 45 Sc, 63 Cu, 65 Cu, 84 Sr, 86 Sr, 87 Sr, 88 Sr, 89 Y, 165 Ho, 159 Tb, 156 Dy, 158 Dy, 160 Dy, 161 Dy, 162Dy, 163 Dy, 164 Dy, 175 Lu, 185 These radionuclides are beta particle emitters and, together with their stable isotopes of the corresponding elements, have therapeutic applications, for example in connection with the treatment of various oncological diseases.

[0065] In another preferred embodiment, the radionuclide labeling agent is 149 Tb, 212 Pb, 212 Bi, 213 Bi, 227 and the corresponding stable isotope of the element is selected from the group consisting of: 159 Tb, 206 Pb, 207 Pb, 208 Pb, 209 Bi, 232 These radionuclides are alpha particle emitters and these radionuclides, together with their stable isotopes of the corresponding elements, have therapeutic applications, for example in connection with the treatment of various oncological diseases.

[0066] In another preferred embodiment, the radionuclide labeling agent is 51 Cr, 58 mCo, 64 Cu, 67 Ga, 73 Se, 75 Se, 77 Br, 80 mBr, 94 Tc, 99m Tc, 103 mRh, 111 In, 114 mIn, 115 mIn, 119 Sb, 123 I, 124 I, 125 I, 135 La, 165 Er, 193 mPt, 195 mPt, and the corresponding stable isotope of the element is selected from the group consisting of: 52 Cr, 53 Cr,54 Cr, 59 Co, 63 Cu, 65 Cu, 69 Ga, 71 Ga, 74 Se, 76 Se, 77 Se, 78 Se, 80 Se, 82 Se, 79 Br, 81 Br, 103 Rh, 113 In, 121 Sb, 123 Sb, 127 I, 138 La, 139 La, 162 Er, 164 Er, 166 Er, 167 Er, 168 Er, 170 Er, 192 Pt, 194 Pt, 195 Pt, 196 Pt, 198 Pt. These radionuclides release electrons by the Auger effect at low kinetic energy. These radionuclides, together with their stable isotopes of the corresponding elements, are applied in Auger therapy, for example in connection with the highly targeted treatment of various oncological diseases.

[0067] In another preferred embodiment, the radionuclide labeling agent is 3 H, 14 C and 35 S, and the corresponding stable isotope of the element is 1 H, 2 H, 12 C. 13 C. 32 S. These radionuclides have application in in vitro studies, such as substitution and tritiation assays.

[0068] In another preferred embodiment, the radionuclide labeling agent is 11 C. 13 N, 18F, 123 I, 125 I, 131 I, or 211 At, and the corresponding stable isotopes of the elements (if stable isotopes of such elements are available). 12 C. 14 N, 19 F, 127 I. These radionuclides, together with their stable isotopes of the corresponding elements when available, are currently among the most frequently used radionuclides in therapy and imaging.

[0069] The targeting vector that is conjugated to either diene or dienophile mentioned in step b) to obtain the targeting vector of the labeled, single isomeric chemical moiety can be any kind of targeting vector suitable for use in therapy, imaging or diagnosis.Such commonly used targeting vectors that are suitable in this method include antibodies, nanobodies, polymers, nanomedicines, cells, proteins, peptides and small molecules.

[0070] Commonly applicable targeting vectors suitable for this method include peptides such as octreotide, octreotate, AE105, small molecules such as FAPI derivatives and PSMA derivatives.

[0071] In a preferred embodiment, the targeting vector applied in the method for obtaining a labeled, single isomeric chemical moiety targeting vector is selected from the group including octreotide, octreotate, AE105, FAPI derivatives and PSMA derivatives.

[0072] The oxidation step c) in the method for obtaining the labeled, single isomeric chemical moiety targeting vector is carried out at a certain temperature and time by adding a specific oxidizing agent to the ligated compound obtained in step b).These conditions ensure that the efficiency of the oxidation step is 90% or more, thereby meeting the required speed for the use of the labeled, single isomeric chemical moiety targeting vector in therapy, diagnosis or imaging.

[0073] The time required to obtain an oxidation efficiency of 90% or more depends on the particular compound being oxidized, the temperature, the oxidant equivalent, and the oxidant. Under the conditions applied in the present method, an oxidation efficiency of 90% or more is obtained within 60 minutes, such as 0-50 minutes, 0-40 minutes, 0-30 minutes, 0-20 minutes, 0-10 minutes, or 0-5 minutes. In a preferred embodiment, the oxidation efficiency obtained is 90% or more in 0-20 minutes.

[0074] The temperature of the oxidation step is between 15° C. and 50° C., for example between 15° C. and 45° C., between 15° C. and 40° C., between 15° C. and 35° C., between 20° C. and 30° C., or between about 20° C. and −25° C. A preferred temperature is room temperature, such as between 20° C. and −25° C.

[0075] The oxidizing agent should be a quinone oxidizing agent selected from chloranil, fluoranil, DDQ or NaNO2. Surprisingly, it has been found in the present application that the use of other types of oxidizing agents does not provide the desired single isomer of the labeled, chemical moiety targeting vector or adversely affects the structure of the targeting vector. The oxidizing agent is added to the ligated labeled compound obtained in step b) in a method for obtaining a labeled, single isomer chemical moiety targeting vector in an amount of 1 to 100 equivalents of the product obtained in step b), for example, 10 to 90, 20 to 80, 30 to 70, 40 to 60 or 50 equivalents of the product obtained in step b). Preferably, 1 to 10, for example 1 equivalent, of the oxidizing agent is added to the labeled compound obtained from step b).

[0076] In one embodiment, the oxidizing agent is supported on a solid phase. Any commonly available solid support, such as alumina, silica gel, polymer, montmorillonite, zeolite or nanomaterial supported oxidizing agent, may be applicable. The advantages of using a solid supported oxidizing agent generally include easy removal from the reaction by filtration, the reaction can be completed without using excess reagents to make purification difficult, easy handling, and economical and efficient recycling of recovered reagents.

[0077] Conventional ligation between tetrazine and TCO leads to several different tautomers, regioisomers and enantiomers, as shown generally in Scheme 1. [ka]

[0078] Scheme 1. Overview of all isomeric forms following conventional tetrazine ligation, including tautomers, enantiomers and positional isomers.

[0079] Scheme 2 below is an example of the current state of the art tetrazine ligation. Reactions with excess isomeric products: [ka]

[0080] Scheme 2. A clear example of the current state of the art tetrazine ligation. (All isomers omitted, see Scheme 1 for an overview of isomers.)

[0081] This reaction can form multiple isomers (at least 12 isomers), all of which have very similar polarities and are therefore very difficult to separate, for example by HPLC.

[0082] In this application, we describe a method that allows the use of a labeled, first chemical moiety, such as a radiolabeled tetrazine-based synthon, in the radiolabeling of isomerically free dienophiles, e.g., isomerically free trans-cycloheptene (TCH), isomerically free trans-cyclooctene (TCO) and isomerically free trans-cyclononene (TCN) functionalized vectors, and vice versa, with subsequent chemical oxidation to yield a single final compound within a short period of time, e.g., 0-60 min (Figure 17). The method involves two steps, a ligation step followed by an oxidation step.

[0083] In a preferred embodiment, the method for obtaining a labeled, single isomeric chemical moiety targeting vector comprises: a) labeling a first chemical moiety having inverse electron demand Diels-Alder cycloaddition reactivity and conjugated to a drug, imaging agent, or therapeutic agent with a labeling agent, wherein the first chemical moiety is selected from the group consisting of symmetrical tetrazines, asymmetrical tetrazines, isomer-free trans-cycloheptene (TCH), isomer-free trans-cyclooctene (TCO) and isomer-free trans-cyclononene (TCN), wherein at least one of the planes of symmetry is through the nitrogen-nitrogen bond of at least one tetrazine ring; b) ligating the labeled first chemical moiety obtained in step a) with a second chemical moiety having complementary inverse electron demand Diels-Alder cycloaddition reactivity and conjugated to a targeting vector, wherein the second chemical moiety is selected from the group consisting of symmetrical tetrazines, in which at least one of the planes of symmetry passes through the nitrogen-nitrogen bond of at least one tetrazine ring, asymmetrical tetrazines, isomer-free trans-cycloheptene (TCH), isomer-free trans-cyclooctene (TCO), isomer-free trans-cyclononene (TCN) and pre-meso trans-cycloheptene (TCH); Here, the kinetics of the inverse electron-demand Diels-Alder cycloaddition between a first chemical moiety and a second chemical moiety are determined by stopped-flow spectrophotometry at 500 μM in PBS at 25 °C. -1s -1 has a minimum rate constant of However, the labeling agent in step a) 94 Tc, 99m Tc, 211 At, 223 Ra or 225 When Ac, the labeling agent is conjugated to an asymmetrically substituted diene; and c) oxidizing the ligated, labeled targeting vector obtained from step b) by adding 1 to 100 equivalents of chloranil, fluoranil, DDQ or NaNO2 at a temperature ranging from 20°C to 30°C for up to 20 minutes.

[0084] The starting entities to be ligated are isomer-free dienophiles such as isomer-free TCH, TCO or TCN and dienes such as tetrazines. The dienes can be either asymmetrically substituted dienes such as asymmetrically substituted tetrazines, or symmetrically substituted tetrazines where at least one of the planes of symmetry passes through the nitrogen-nitrogen bond of at least one tetrazine ring, such as symmetrical tetrazines. The use of isomer-free dienophiles such as isomer-free TCH, TCO or TCN reduces the number of click products formed by eliminating all enantiomeric and regioisomeric products. The tautomers formed, such as dihydropyridazines, are then oxidized to the corresponding single isomers, such as pyridazines, resulting in a single product. The "R" substituents of dienes such as tetrazines used in this method are typically: 18 F, 123 I, 124 I, 125 I, 131 I or 211 At is functionalized with a substituted aryl.

[0085] Schemes 2 and 3 below are illustrations of examples of ligation according to the methods of the invention, illustrating the use of asymmetric tetrazines and TCOs conjugated to targeting vectors. [ka]

[0086] Scheme 3. [ka]

[0087] Scheme 4. The targeting vector is an antibody, a nanobody, a polymer, a nanomedicine, a cell, a protein, a peptide or a small molecule.

[0088] The labeling agent in step a) 211 At, 223 Ra or 225 When Ac, the labeling agent must be conjugated to an asymmetrically substituted diene.

[0089] The following dienes are examples of symmetrical tetrazines of formula Tz1 suitable for ligation in step b) of the method for obtaining a labeled, single isomeric chemical moiety targeting vector: [ka] In the formula, R and R1 are [ka] where the curvilinear symbol indicates the link to the tetrazine; where R2 is -H or (i) an isotopic labeling agent attached directly to the aromatic ring, or (ii) an isotopic labeling agent attached to the aromatic ring via a linker, said linker being (CH2) n , -LO(CH2) n , -LNH(CH2) n , -LCONH(CH2) n , -LNHCO(CH2) n wherein L is selected from the group consisting of -(CH2) m or -O(CH2CH2O) mwherein n and m are independently selected from 1 to 25; or (iii) 1,4,7,10-tetraazacyclododecane-N,N',N',N''-tetraacetic acid (DOTA), N,N'-bis(2-hydroxy-5-(carboxyethyl)benzyl)ethylenediamine-N,N'-diacetic acid (HBED-CC), 14,7-triazacyclononane-1,4,7-triacetic acid (NOTA), 2-(4,7-bis(carboxymethyl)-1,4,7-triazonan-1-yl)pentaacetate (HBE-CC), 1,4,7-triazanocyclododecane-N,N',N',N''-tetraacetic acid ... pentane diacid (NODAGA), 2-(4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl)pentanedioic acid (DOTAGA), 14,7-triazacyclononanephosphinic acid (TRAP), 14,7-triazacyclononane-1-methyl(2-carboxyethyl)phosphinic acid-4,7-bis(methyl(2-hydroxymethyl)phosphinic acid (NOPO), 3,6,9,15-tetraazabicyclopentadec-1(15-phenyl)-1,3,4,5,6 ... ), 11,13-triene-3,6,9-triacetic acid (PCTA), N'-(5-acetyl(hydroxy)aminopentyl-N-(5-(4-(5-aminopentyl)(hydroxy)amino-4-oxobutanoyl)amino)pentyl-N-hydroxysuccinamide (DFO), diethylenetriaminepentaacetic acid (DTPA), trans-cyclohexyl-diethylenetriaminepentaacetic acid (CHX-DTPA), 1-oxa-4,7,10-triazacyclododecane-4,7,10-triacetate (DTPA), isotopic labeling agent chelated through a chelator selected from acetic acid (OXO-Do3A), p-isothiocyanatobenzyl-DTPA (SCN-BZ-DTPA), 1-(p-isothiocyanatobenzyl)-3-methyl-DTPA (1B3M), 2-(p-isothiocyanatobenzyl)-4-methyl-DTPA (1M3B), and 1-(2)-methyl-4-isocyanatobenzyl-DTPA (MX-DTPA) attached to an aromatic ring via a linker, the linker being -(CH2) n , -LO(CH2) n , -LNH(CH2) n ,-LCONH(CH2) n , -LNHCO(CH2) n wherein L is -(CH2) mor -O(CH2CH2O) m where n and m are independently selected from 1 to 25; Wherein, when R2 is either (i) or (ii), the isotopic labeling agent is selected from the group consisting of: 1 H, 2 H, 3 H, 11 C. 12 C. 13 C. 13 N, 14 N, 15 N 18 F, 19 F, 123 I, 124 I, 125 I, 127 I, 131 I, 211 At, 15 O. 16 O. 17 O. 18 O. 32 S, 35 S, 43 Sc, 44 Sc, 45 Sc, 45 Ti, 46 Ti, 47 Ti, 48 Ti, 49 Ti, 50 Ti, 55 Co, 58 mCo, 59 Co, 60 Cu, 61 Cu, 63 Cu, 64 Cu, 65 Cu, 67 Cu, 67 Ga, 68 Ga, 69 Ga, 71 Ga, 76 Br, 77 Br, 79 Br, 80 mBr, 81 Br, 72 As, 75 As, 86 Y, 89 Y, 90 Y, 89Zr、 90 Zr、 91 Zr、 92 Zr、 94 Zr、 149 Tb、 152 Tb、 159 Tb、 161 Tb、 111 In、 113 In、 114 mIn、 115 mIn、 175 Ridiculous, 177 Ridiculous, 185 Too, 186 Too, 188 Too, 201 Tl、 203 Tl、 205 Tl、 206 Pb、 207 Pb, 208 Pb, 212 Pb、 209 Bi、 212 Bi、 213 Bi、 31 P、 32 P、 33 P、 45 Sc、 47 Sc、 84 Sr、 86 Sr、 87 Sr、 88 Sr、 89 Sr、 165 Your, 166 Your, 156 Dy、 158 Dy、 160 Dy、 161 Dy、 162 Dy、 163 Dy、 164 Dy、 165 Dy、 227 Th、 232 Th、 51 Cr、 52 Cr、 53 Cr、 54 Cr、 73 The、 74 The、 75 The、 76 The、 77 The、 78 The、 80 The、 82 The、 94 Tc、99m Tc, 103 Rh, 103 mRh, 119 Sb, 121 Sb, 123 Sb, 135 La, 138 La, 139 La, 162 Er, 164 Er, 165 Er, 166 Er, 167 Er, 168 Er, 170 Er, 193 mPt, 195 mPt, 192 Pt, 194 Pt, 195 Pt, 196 Pt, 198 Pt, wherein X and Y are independently selected from -CH- and -N-; In the formula, R3 is H or a hydroxy group, a sulfonamide, a carboxyl group, a sulfonyl group, an amine, a substituted amine having 1 to 5 polyethylene glycol units, -(O-CH2-CH2) 1~5and optionally substituted arylalkyl, ... C10)cycloalkyl, (C3-C10)heterocycloalkyl, (C3-10)cycloalkylene, (C3-C10)heterocycloalkylene, (C1-C10)haloalkyl, (C1-C10)perhaloalkyl, (C2-C10)-alkenyloxy, (C3-C10)-alkynyloxy, aryloxy, arylalkyloxy, heteroaryloxy, heteroarylalkyloxy, (C1-C6)alkyloxy-(C1-C4)alkyl, optionally substituted aryl, optionally substituted heteroaryl, and optionally substituted arylalkyl; optionally substituted means one or more substituents selected from halogen, hydroxyl group, sulfonamide, carboxyl group, sulfonyl group, amine, substituted amine having 1 to 5 polyethylene glycol units, -(O-CH2-CH2) 1~5 means one or more substituents selected from -OCH2-COOH, H, methyl, ethyl, propyl, optionally substituted heteroaryl, and optionally substituted arylalkyl; optionally substituted in relation to said substituted amine means one or more substituents selected from halogen, hydroxyl, sulfonamide, carboxyl, sulfonyl, and amine; In the formula, R and R1 are the same or differ only in the isotope number of the labeling agent.

[0090] The following tetrazines are preferred tetrazines of formula Tz1 for use in the ligation in step b) of the method for obtaining a labeled, single isomeric chemical moiety targeting vector: [ka]

[0091] The asymmetric tetrazine of formula Tz2 is a further example of a preferred diene suitable for ligation in step b) of the method for obtaining a labeled, single isomeric chemical moiety targeting vector: [ka] wherein R4 is -H or (i) an isotopic labeling agent attached directly to the aromatic ring, or (ii) an isotopic labeling agent attached to the aromatic ring via a linker, the linker being -(CH2) n , -LO(CH2) n , -LNH(CH2) n , -LCONH(CH2) n , -LNHCO(CH2) n wherein L is selected from the group consisting of -(CH2) m or -O(CH2CH2O) mwherein n and m are independently selected from 1 to 25; or (iii) 1,4,7,10-tetraazacyclododecane-N,N',N',N''-tetraacetic acid (DOTA), N,N'-bis(2-hydroxy-5-(carboxyethyl)benzyl)ethylenediamine-N,N'-diacetic acid (HBED-CC), 14,7-triazacyclononane-1,4,7-triacetic acid (NOTA), 2-(4,7-bis(carboxymethyl)-1,4,7-triazonan-1-yl)pentaacetate (C1H-1H,C1H-2H- ... pentane diacid (NODAGA), 2-(4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl)pentanedioic acid (DOTAGA), 14,7-triazacyclononanephosphinic acid (TRAP), 14,7-triazacyclononane-1-methyl(2-carboxyethyl)phosphinic acid-4,7-bis(methyl(2-hydroxymethyl)phosphinic acid (NOPO), 3,6,9,15-tetraazabicyclopentadec-1(15)-1,3-dimethyl-1,4-dihydro-1,5-tri ... ,11,13-triene-3,6,9-triacetic acid (PCTA), N'-(5-acetyl(hydroxy)aminopentyl-N-(5-(4-(5-aminopentyl)(hydroxy)amino-4-oxobutanoyl)amino)pentyl-N-hydroxysuccinamide (DFO), diethylenetriaminepentaacetic acid (DTPA), trans-cyclohexyl-diethylenetriaminepentaacetic acid (CHX-DTPA), 1-oxa-4,7,10-triazacyclododecane-4,7,10-triacetic acid isotopic labeling agent chelated through a chelator selected from -(OXO-Do3A), p-isothiocyanatobenzyl-DTPA (SCN-BZ-DTPA), 1-(p-isothiocyanatobenzyl)-3-methyl-DTPA (1B3M), 2-(p-isothiocyanatobenzyl)-4-methyl-DTPA (1M3B), and 1-(2)-methyl-4-isocyanatobenzyl-DTPA (MX-DTPA) and attached to the aromatic ring via a linker, said linker being -(CH2) n , -LO(CH2) n , -LNH(CH2) n , -LCONH(CH2) n , -LNHCO(CH2) n wherein L is selected from the group consisting of -O(CH2)m or -(CH2CH2O) m wherein n and m are independently selected from 1 to 25; when R4 is (i), (ii) or (iii), the isotope labeling agent is 1 H, 2 H, 3 H, 11 C. 12 C. 13 C. 13 N, 14 N, 15 N, 18 F, 19 F, 123 I, 124 I, 125 I, 127 I, 131 I, 211 At, 15 O. 16 O. 17 O. 18 O. 32 S, 35 S, 43 Sc, 44 Sc, 45 Sc, 45 Ti, 46 Ti, 47 Ti, 48 Ti, 49 Ti, 50 Ti, 55 Co, 58 mCo, 59 Co, 60 Cu, 61 Cu, 63 Cu, 64 Cu, 65 Cu, 67 Cu, 67 Ga, 68 Ga, 69 Ga, 71 Ga, 76 Br, 77 Br, 79 Br, 80 mBr, 81 Br, 72 As, 75 As, 86 Y, 89 Y, 90 Y, 89 Zr, 90 Zr, 91Zr、 92 Zr、 94 Zr、 149 Tb、 152 Tb、 159 Tb、 161 Tb、 111 In、 113 In、 114 mIn、 115 mIn、 175 Ridiculous, 177 Ridiculous, 185 Too, 186 Too, 188 Too, 201 Tl、 203 Tl、 205 Tl、 206 Pb、 207 Pb, 208 Pb, 212 Pb、 209 Bi、 212 Bi、 213 Bi、 31 P、 32 P、 33 P、 45 Sc、 47 Sc、 84 Sr、 86 Sr、 87 Sr、 88 Sr、 89 Sr、 165 Your, 166 Your, 156 Dy、 158 Dy、 160 Dy、 161 Dy、 162 Dy、 163 Dy、 164 Dy、 165 Dy、 227 Th、 232 Th、 51 Cr、 52 Cr、 53 Cr、 54 Cr、 73 The、 74 The、 75 The、 76 The、 77 The、 78 The、 80 The、 82 The、 94 Tc、 99m Tc、 103 Rh,103 mRh, 119 Sb, 121 Sb, 123 Sb, 135 La, 138 La, 139 La, 162 Er, 164 Er, 165 Er, 166 Er, 167 Er, 168 Er, 170 Er, 193 mPt, 195 mPt, 192 Pt, 194 Pt, 195 Pt, 196 Pt, 198 Pt, 211 At, 223 Ra, 225 Ac, wherein X and Y are independently selected from -CH__ and -N-; In the formula, R6 is H or [ka] where Q and Z are independently selected from -CH- and -N-, and the curved symbol indicates the linkage to the tetrazine; In the formula, R5 and R7 are H or a hydroxy group, a sulfonamide, a carboxyl group, a sulfonyl group, an amine, a substituted amine having 1 to 5 polyethylene glycol units, -(O-CH2-CH2) 1~5and optionally substituted arylalkyl, ... C10)cycloalkyl, (C3-C10)heterocycloalkyl, (C3-10)cycloalkylene, (C3-C10)heterocycloalkylene, (C1-C10)haloalkyl, (C1-C10)perhaloalkyl, (C2-C10)-alkenyloxy, (C3-C10)-alkynyloxy, aryloxy, arylalkyloxy, heteroaryloxy, heteroarylalkyloxy, (C1-C6)alkyloxy-(C1-C4)alkyl, optionally substituted aryl, optionally substituted heteroaryl, and optionally substituted arylalkyl; optionally substituted means one or more substituents selected from halogen, hydroxyl group, sulfonamide, carboxyl group, sulfonyl group, amine, substituted amine having 1 to 5 polyethylene glycol units, -(O-CH2-CH2) 1~5 means one or more substituents selected from -OCH2-COOH, H, methyl, ethyl, propyl, optionally substituted heteroaryl, and optionally substituted arylalkyl; optionally substituted in relation to said substituted amine means one or more substituents selected from halogen, hydroxyl, sulfonamide, carboxyl, sulfonyl, and amine.

[0092] The following tetrazines are preferred tetrazines of formula Tz2 for use in the ligation in step b) of the method for obtaining a labeled, single isomeric chemical moiety targeting vector: [ka]

[0093] The following trans-cycloheptene (TCH'), trans-cyclooctene (TCO'), and trans-cyclononene (TCN) are preferred isomer-free dienophiles used for ligation in step b) of the method to obtain a labeled, single isomeric chemical moiety targeting vector: [ka] wherein X is N, NO, or CR8; Y is N, NO, or CR8; and R8 is selected from the group consisting of: -H, -F, -OH, -NH2, -COOH, -COOCH3, CF3, -Cl, -CONH2, CONHCH3, -CON(CH3)2, -CH2CH2OH, -CH2CH2NH2, -CHCH2N(CH3)2. The linker is -(CH2) n -(CH2) n NH, (CH2) n CO, (CH2) n O, (CH2CH2O) n , (CH2CH2O) n CH2CH2NH, (CH2CH2O) n CH2CH2CO, -CO(CH)2-CO(CH2) n NH, CO(CH2) n CO, CO(CH2) n O, CO(CH2CH2O) n CO(CH2CH2O) n CH2CH2NH, CO(CH2CH2O) n CH2CH2CO, COO(CH)2-COO(CH2) n NH, COO(CH2) n CO, COO(CH2) n O, COO(CH2CH2O) n COO(CH2CH2O) n CH2CH2NH, COO(CH2CH2O) n CH2CH2CO, CONH(CH)2-CONH(CH2) nNH, CONH(CH2) n CO, CONH(CH2) n O, CONH(CH2CH2O) n , CONH(CH2CH2O) n CH2CH2NH, CONH(CH2CH2O) n CH2CH2CO, -CONHPhCO, -COOPhCO, -COPhCO, CONHCHMCO, (CH2) n NHCHMCO, (CH2) n OCONHCHMCO, (CH2) n NHCHMCO, (CH2) n NHCOCHMNH, (CH2)OCOCHMNH, (CH2CH2O) n CH2CH2NHCHMCO, (CH2CH2O) n CH2CH2CONHCHMCO, (CH2CH2O) n CH2CH2NHCHMCO, (CH2CH2O) n CH2CH2NHCOCHMNH, (CH2CH2O) n COCHMNH, where n is 0-25, where M is a side chain selected from the group consisting of the side chains of natural amino acids which are H, CH3, CH2SH, CH2COOH, CH2CH2COOH, CH2C6H5, CH2C3H3N2, CH(CH3)CH2CH3, (CH2)4NH2, CH2CH(CH3)2, CH2CH2SCH3, CH2CONH2, (CH2)4NHCOC4H5NCH3, CH2CH2CH2, CH2CH2CONH2, (CH2)3NH-C(NH)NH2, CH2OH, CH(OH)CH3, CH2SeH, CH(CH3)2, CH2C8H6N, CH2C6H4OH; The targeting vector is an antibody, a nanobody, a polymer, a nanomedicine, a cell, a protein, a peptide or a small molecule.

[0094] Examples of asymmetric Tz suitable for ligation in step b) of the method for obtaining a labeled, single isomeric chemical moiety targeting vector: [ka]

[0095] Examples of monocyclic isomer-free TCH and TCN suitable for ligation in step b) of the method for obtaining a labeled, single isomer chemical moiety targeting vector: [ka] The linker is -(CH2) n -(CH2) n NH, (CH2) n CO, (CH2) n O, (CH2CH2O) n , (CH2CH2O) n CH2CH2NH, (CH2CH2O) n CH2CH2CO, -CO(CH)2-CO(CH2) n NH, CO(CH2) n CO, CO(CH2) n O, CO(CH2CH2O) n CO(CH2CH2O) n CH2CH2NH, CO(CH2CH2O) n CH2CH2CO, COO(CH)2-COO(CH2) n NH, COO(CH2) n CO, COO(CH2) n O, COO(CH2CH2O) n COO(CH2CH2O) n CH2CH2NH, COO(CH2CH2O) n CH2CH2CO, CONH(CH)2-CONH(CH2) n NH, CONH(CH2) n CO, CONH(CH2) n O, CONH(CH2CH2O) n , CONH(CH2CH2O) n CH2CH2NH, CONH(CH2CH2O) n CH2CH2CO, -CONHPhCO, -COOPhCO, -COPhCO, CONHCHMCO, (CH2) n NHCHMCO, (CH2) n OCONHCHMCO, (CH2) nNHCHMCO, (CH2) n NHCOCHMNH, (CH2)OCOCHMNH, (CH2CH2O) n CH2CH2NHCHMCO, (CH2CH2O) n CH2CH2CONHCHMCO, (CH2CH2O) n CH2CH2NHCHMCO, (CH2CH2O) n CH2CH2NHCOCHMNH, (CH2CH2O) n COCHMNH, where n is 0-25 and where M is a side chain selected from the group consisting of the side chains of natural amino acids which are H, CH3, CH2SH, CH2COOH, CH2CH2COOH, CH2C6H5, CH2C3H3N2, CH(CH3)CH2CH3, (CH2)4NH2, CH2CH(CH3)2, CH2CH2SCH3, CH2CONH2, (CH2)4NHCOC4H5NCH3, CH2CH2CH2, CH2CH2CONH2, (CH2)3NH-C(NH)NH2, CH2OH, CH(OH)CH3, CH2SeH, CH(CH3)2, CH2C8H6N, CH2C6H4OH; the targeting vector is an antibody, nanobody, polymer, nanomedicine, cell, protein, peptide or small molecule.

[0096] Examples of bicyclic isomer-free TCOs suitable for ligation in step b) of the method for obtaining a labeled, single isomer chemical moiety targeting vector: [ka] R8 is -H, -F, -OH, -NH2, -CH3, -COOH, -COOCH3, CF3, -Cl, -CONH2, CONHCH3, -CON(CH3)2, -CH2CH2OH, -CH2CH2NH2, -CHCH2N(CH3)2, and the linker is -(CH2) n -(CH2) n NH, (CH2) n CO, (CH2) n O, (CH2CH2O) n , (CH2CH2O) nCH2CH2NH、(CH2CH2O) n CH2CH2CO、-CO(CH)2-CO(CH2) n NH、CO(CH2) n CO、CO(CH2) n O、CO(CH2CH2O) n CO(CH2CH2O) n CH2CH2NH、CO(CH2CH2O) n CH2CH2CO、COO(CH)2-COO(CH2) n NH、COO(CH2) n CO、COO(CH2) n O、COO(CH2CH2O) n COO(CH2CH2O) n CH2CH2NH、COO(CH2CH2O) n CH2CH2CO、CONH(CH)2-CONH(CH2) n NH、CONH(CH2) n CO,CONH(CH2) n O,CONH(CH2CH2O) n 、CONH(CH2CH2O) n CH2CH2NH、CONH(CH2CH2O) n CH2CH2CO、-CONHPhCO、-COOPhCO、-COPhCO、CONHCHMCO、(CH2) n NHCHMCO、(CH2) n OCONHCHMCO、(CH2) n NHCHMCO、(CH2) n NHCOCHMNH、(CH2)OCOCHMNH、(CH2CH2O) n CH2CH2NHCHMCO、(CH2CH2O) n CH2CH2CONHCHMCO、(CH2CH2O) n CH2CH2NHCHMCO、(CH2CH2O) n CH2CH2NHCOCHMNH、(CH2CH2O) nCOCHMNH, where n is 0-25 and where M is a side chain selected from the group consisting of the side chains of natural amino acids which are H, CH3, CH2SH, CH2COOH, CH2CH2COOH, CH2C6H5, CH2C3H3N2, CH(CH3)CH2CH3, (CH2)4NH2, CH2CH(CH3)2, CH2CH2SCH3, CH2CONH2, (CH2)4NHCOC4H5NCH3, CH2CH2CH2, CH2CH2CONH2, (CH2)3NH-C(NH)NH2, CH2OH, CH(OH)CH3, CH2SeH, CH(CH3)2, CH2C8H6N, CH2C6H4OH; the targeting vector is an antibody, nanobody, polymer, nanomedicine, cell, protein, peptide or small molecule.

[0097] Examples of tricyclic isomer-free TCOs suitable for ligation in step b) of the method for obtaining a labeled, single isomer chemical moiety targeting vector: [ka] wherein X is N, NO or CR8; Y is N, NO or CR8; and R8 is selected from the group consisting of: -H, -F, -OH, -NH2, -COOH, -COOCH3, CF3, -Cl, -CONH2, CONHCH3, -CON(CH3)2, -CH2CH2OH, -CH2CH2NH2, -CHCH2N(CH3)2. The linker is -(CH2) n -(CH2) n NH, (CH2) n CO, (CH2) n O, (CH2CH2O) n , (CH2CH2O) n CH2CH2NH, (CH2CH2O) n CH2CH2CO, -CO(CH)2-CO(CH2) n NH, CO(CH2) n CO, CO(CH2) n O, CO(CH2CH2O) n CO(CH2CH2O) nCH2CH2NH、CO(CH2CH2O) n CH2CH2CO、COO(CH)2-COO(CH2) n NH、COO(CH2) n CO、COO(CH2) n O、COO(CH2CH2O) n COO(CH2CH2O) n CH2CH2NH、COO(CH2CH2O) n CH2CH2CO、CONH(CH)2-CONH(CH2) n NH、CONH(CH2) n CO、CONH(CH2) n O、CONH(CH2CH2O) n 、CONH(CH2CH2O) n CH2CH2NH、CONH(CH2CH2O) n CH2CH2CO、-CONHPhCO、-COOPhCO、-COPhCO、CONHCHMCO、(CH2) n NHCHMCO、(CH2) n OCONHCHMCO、(CH2) n NHCHMCO、(CH2) n NHCOCHMNH、(CH2)OCOCHMNH、(CH2CH2O) n CH2CH2NHCHMCO、(CH2CH2O) n CH2CH2CONHCHMCO、(CH2CH2O) n CH2CH2NHCHMCO、(CH2CH2O) n CH2CH2NHCOCHMNH、(CH2CH2O) nCOCHMNH, where n is 0-25, and where M is a side chain selected from the group consisting of the side chains of natural amino acids which are H, CH3, CH2SH, CH2COOH, CH2CH2COOH, CH2C6H5, CH2C3H3N2, CH(CH3)CH2CH3, (CH2)4NH2, CH2CH(CH3)2, CH2CH2SCH3, CH2CONH2, (CH2)4NHCOC4H5NCH3, CH2CH2CH2, CH2CH2CONH2, (CH2)3NH-C(NH)NH2, CH2OH, CH(OH)CH3, CH2SeH, CH(CH3)2, CH2C8H6N, CH2C6H4OH; The targeting vector is an antibody, a nanobody, a polymer, a nanomedicine, a cell, a protein, a peptide or a small molecule.

[0098] Examples of tetracyclic isomer-free TCOs suitable for ligation in step b) of the method for obtaining a labeled, single isomer chemical moiety targeting vector: [ka] X is N, NO or CR8; Y is N, NO or CR8; R8 is selected from the group consisting of: -H, -F, -OH, -NH2, -COOH, -COOCH3, CF3, -Cl, -CONH2, CONHCH3, -CON(CH3)2, -CH2CH2OH, -CH2CH2NH2, -CHCH2N(CH3)2, and the linker is -(CH2) n -(CH2) n NH, (CH2) n CO, (CH2) n O, (CH2CH2O) n , (CH2CH2O) n CH2CH2NH, (CH2CH2O) n CH2CH2CO, -CO(CH)2-CO(CH2) n NH, CO(CH2) n CO, CO(CH2) n O, CO(CH2CH2O) n CO(CH2CH2O) nCH2CH2NH、CO(CH2CH2O) n CH2CH2CO、COO(CH)2-COO(CH2) n NH、COO(CH2) n CO、COO(CH2) n O、COO(CH2CH2O) n COO(CH2CH2O) n CH2CH2NH、COO(CH2CH2O) n CH2CH2CO、CONH(CH)2-CONH(CH2) n NH、CONH(CH2) n CO、CONH(CH2) n O、CONH(CH2CH2O) n 、CONH(CH2CH2O) n CH2CH2NH、CONH(CH2CH2O) n CH2CH2CO、-CONHPhCO、-COOPhCO、-COPhCO、CONHCHMCO、(CH2) n NHCHMCO、(CH2) n OCONHCHMCO、(CH2) n NHCHMCO、(CH2) n NHCOCHMNH、(CH2)OCOCHMNH、(CH2CH2O) n CH2CH2NHCHMCO、(CH2CH2O) n CH2CH2CONHCHMCO、(CH2CH2O) n CH2CH2NHCHMCO、(CH2CH2O) n CH2CH2NHCOCHMNH、(CH2CH2O) nCOCHMNH, where n is 0-25, where M is a side chain selected from the group consisting of the side chains of natural amino acids which are H, CH3, CH2SH, CH2COOH, CH2CH2COOH, CH2C6H5, CH2C3H3N2, CH(CH3)CH2CH3, (CH2)4NH2, CH2CH(CH3)2, CH2CH2SCH3, CH2CONH2, (CH2)4NHCOC4H5NCH3, CH2CH2CH2, CH2CH2CONH2, (CH2)3NH-C(NH)NH2, CH2OH, CH(OH)CH3, CH2SeH, CH(CH3)2, CH2C8H6N, CH2C6H4OH; The targeting vector is an antibody, a nanobody, a polymer, a nanomedicine, a cell, a protein, a peptide or a small molecule.

[0099] Step c) in the method for obtaining a labeled, single isomeric chemical moiety targeting vector is an oxidation step. Although auto-oxidation of the ligated moiety targeting vector, such as pyridazine obtained in step b) of the method, occurs spontaneously, this process is very slow and can last from hours to days. Step c) of the method provides a rapid method of oxidizing pyridazine compounds, obtaining only a single isomeric form within at least 60 minutes, for example within 1 to 20 minutes. To facilitate this process, the dihydropyridazine is oxidized by either a standard or solid-supported oxidizing agent, preferably a solid-supported oxidizing agent. The oxidation step can be carried out at a temperature ranging from 15°C to 50°C, for example 20°C to 30°C, preferably at room temperature, for about 10 minutes to 60 minutes, preferably less than 20 minutes. To facilitate the oxidation, 1 to 100 equivalents, preferably 1 equivalent, of an oxidizing agent is added to the ligated compound obtained from the ligation step. The oxidizing agent must be selective for the oxidation of dihydropyrazine to pyridazine (95% efficiency). The targeting vector must not be chemically modified by the oxidizing agent. The oxidizing agent is a quinone oxidizing agent selected from the group including chloranil, fluoranil, DDQ, NaNO2.

[0100] In a preferred embodiment, the diene has at least one plane of symmetry: [ka] [ka] a symmetrically substituted diene through the nitrogen-nitrogen bond of at least one tetrazine ring obtained from a precursor selected from In the formula, X is CH or N.

[0101] In another preferred embodiment, the diene is an asymmetrically substituted diene obtained from a precursor selected from: [ka]

[0102] In another preferred embodiment, the isomer-free dienophile is [ka] from a precursor selected from The targeting vector is an antibody, a nanobody, a polymer, a nanomedicine, a cell, a protein, a peptide or a small molecule.

[0103] The labeled, single isomeric chemical moiety targeting vectors provided by the methods for obtaining labeled, single isomeric chemical moiety targeting vectors can be used in therapy, radiotherapy, theranostics, diagnosis or imaging depending on the labeling agent, or pharmaceutical, or imaging or therapeutic agent, and depending on the targeting vector.

[0104] Preferably, the targeting vector is coupled to the linker via a nitrogen on the targeting vector. Alternatively, the targeting vector is preferably coupled to the linker via a carbonyl on the targeting vector.

[0105] In a preferred embodiment, the labeled, single isomeric chemical moiety targeting vector obtained by the method for obtaining a labeled, single isomeric chemical moiety targeting vector is used in therapy.

[0106] In another preferred embodiment, the labeled, single isomeric chemical moiety targeting vector obtained by the method for obtaining a labeled, single isomeric chemical moiety targeting vector is used in radiotherapy.

[0107] In another preferred embodiment, the labeled, single isomeric chemical moiety targeting vector obtained by the method for obtaining a labeled, single isomeric chemical moiety targeting vector is used in theranostics.

[0108] In another preferred embodiment, the labeled, single isomeric chemical moiety targeting vector obtained by the method for obtaining a labeled, single isomeric chemical moiety targeting vector is used for diagnosis.

[0109] In another preferred embodiment, the labeled, single isomeric chemical moiety targeting vector obtained by the method for obtaining a labeled, single isomeric chemical moiety targeting vector is used for imaging.

[0110] The following examples describe (1) the synthesis of representative tetrazines and TCOs used in steps a) and b) of the method to obtain labeled, single isomeric chemical moiety targeting vectors, and (2) the click reaction between such compounds and oxidation to yield single isomeric pyridazines.

[0111] example General matters All reagents and solvents were dried before use according to standard methods. Commercially available reagents were used without further purification. Analytical TLC was performed using silica gel 60 F254 (Merck) and detected by UV absorption and / or charring after immersion in a 7% ethanolic solution of sulfuric acid or KMnO4 solution (1.5 g KMnO4, 10 g K2CO3, and 1.25 mL 10% NaOH in 200 mL water). Purification of compounds was performed by column chromatography on silica gel (40-60 μm, 60 Å) or using a CombiFlash NextGen 300+ (Teledyne ISCO). 1 H and 13 C NMR spectra were recorded on a Brucker (400 MHz and 600 MHz instruments) using chloroform-d, methanol-d4 or DMSO-d6 as deuterated solvents with residual solvent as internal reference. In all NMR experiments, the deuterated solvent signal was used as an internal lock. Chemical shifts are reported in δ parts per million (ppm). Coupling constants (J values) are given in Hertz (Hz). 1H NMR signal multiplicities are reported as follows: s, singlet; d, doublet; dd, doublet of doublet; ddd, doublet of doublet of doublet; dt, triplet of triplet; t, triplet; q, quartet; m, multiplet; br, broad signal. NMR spectra of all compounds were reprocessed with MestReNova software (version 12.0.22023) from the original FID files. Mass spectral analysis was performed using MS-Acquity-A: Waters Acquity UPLC with QDa-detector. Purification by preparative HPLC was performed on an Agilent 1260 infinity system, column SymmetryPrep-C18, 17 mL / min HO-MeCN gradient 50–100% in 0.1% trifluoroacetic acid for 15 min. All final compounds were >95% pure as determined by analytical HPLC. Analytical HPLC method: C-18 column (Luna® 5u C18(2) 100 Å, 150×4.6 mm), eluent: A: HO with 0.1% TFA, B: MeCN with 0.1% TFA (Thermo Fisher® UltiMate 3000). Gradient 100% A → 100% B in 15 min, back to 100% A in 4 min, flow rate 1.5 mL / min. Detection by UV absorbance at λ=254 nm on a UVD 170U detector.

[0112] Example 1 Synthesis of symmetrical tetrazines and their precursors. Compounds I and XXXIV Figure 1 shows the reaction scheme for the synthesis of symmetrical tetrazines. Reagents and conditions: i) NH2(CH2)2R, MeCN, 12 h, rt; ii) Boc2O, Et3N, DCM, 12 h, rt; iii) Zn(OTf)2, NH2NH2, H2O, EtOH, 65 °C, 24 h; iv) HCl, dioxane, rt, 4 h; v) t-butyl bromoacetate, Et3N, DMF, 50 °C, 12 h; vi) TFA, DCM, rt, 2 h; vii) MsCl, Et3N, DMAP, DCM, rt, 12 h.

[0113] Figure 2. Reagents and conditions: i) NH2(CH2)2OH, MeCN, 12 h, rt; ii) Boc2O, Et3N, DCM, 12 h, rt; iii) Zn(OTf)2, NH2NH2·H2O, EtOH, 65 °C, 24 h; iv) HCl, dioxane, rt, 4 h; v) t-Butyl bromoacetate, Et3N, DMF, 50 °C, 12 h; vi) DAST, DCM, -78 °C to rt, 4 h; vii) TFA, DCM, rt, 2 h; viii) MsCl, Et3N, DMAP, DCM, rt, 12 h.

[0114] Synthesis of 4-(((2-hydroxyethyl)amino)methyl)benzonitrile (3) To a solution of ethanolamine in DCM (60 mL) was added dropwise a solution of 4-(bromomethyl)benzonitrile (4 gr, 20.20 mmol) in DCM (20 mL). The reaction was stirred at room temperature for 1 h. The organic phase was then washed with water (3×30 mL), dried and concentrated under reduced pressure to give 3.55 g (99%) of the desired product as a white solid. Rf=0.21 (DCM / MeOH 95 / 5);

number

[0115] Synthesis of tert-butyl (4-cyanobenzyl)(2-hydroxyethyl)carbamate (4) To a solution of 4-((((2-hydroxyethyl)amino)methyl)benzonitrile (1.62 g, 9.19 mmol) and Et3N (2.56 mL, 18.39 mmol) in DCM (30 mL) was added Boc2O (2.10 gr, 9.65 mmol). The reaction was stirred at room temperature for 12 h. The solution was then washed with water (50 mL) and K2CO3 saturated solution (50 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give 2.36 g (93%) of the desired product as crude product (mixture of rotamers). Rf.=0.4 (heptane / EtOAc 50 / 50);

number

[0116] Synthesis of 4-(((2-fluoroethyl)amino)methyl)benzonitrile (5) To a solution of 4-(bromomethyl)benzonitrile (0.78 g, 4.00 mmol) in CH3CN (40 mL) was added K2CO3 (0.33 g, 24.0 mmol) and 2-fluoroethylamine hydrochloride (0.16 g, 16.0 mmol). The mixture was stirred at room temperature overnight. The solvent was removed under reduced pressure and the residue was diluted with water (20 mL) and extracted with EtOAc. The combined organic layers were washed with brine, dried over MgSO4, filtered and concentrated under reduced pressure. The crude product was purified by flash column chromatography using EtOAc in heptane (heptane / EtOAc 50 / 50) to give 0.54 g (76%) of the desired product as a colorless oil. Rf=0.24 (heptane / EtOAc 40 / 60).

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[0117] Synthesis of tert-butyl 4-cyanobenzyl(2-fluoroethyl)carbamate (6) To a solution of 4-((((2-fluoroethyl)amino)methyl)benzonitrile (540 mg, 3.03 mmol) and EtN (1.27 mL, 9.09 mmol) in CHCl (40 mL) was added BocO (790 mg, 3.63 mmol) and the mixture was stirred at room temperature for 12 h. The solution was washed with water and saturated KCO solution, dried over NaSO, filtered and concentrated under reduced pressure. The crude product was purified by flash column chromatography using (heptane / EtOAc 70 / 30) to give 0.710 g (84%) of the desired product as a colorless oil (mixture of rotamers). Rf=0.42 (heptane / EtOAc 80 / 20).

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[0118] Synthesis of di-tert-butyl (((1,2,4,5-tetrazine-3,6-diyl)bis(4,1-phenylene))bis(methylene))bis((2-fluoroethyl)carbamate) (7) and tert-butyl (4-(6-(4-(((tert-butoxycarbonyl)(2-fluoroethyl)amino)methyl)phenyl)-1,2,4,5-tetrazine-3-yl)benzyl)(2-hydroxyethyl)carbamate (8) To a suspension of tert-butyl 4-cyanobenzyl(2-fluoroethyl)carbamate (1.1gr, 3.95mmol), tert-butyl(4-cyanobenzyl)(2-hydroxyethyl)carbamate (0.27gr, 0.99mmol) and Zn(OTf)2 (0.72gr, 1.98mmol) in EtOH (30mL) was added hydrazine monohydrate (3.83mL, 79mmol). The mixture was stirred at 70°C for 22 hours and, once the reaction was complete, cooled at room temperature. The volatiles were removed under reduced pressure and the residue was dissolved in EtOH (40mL). An aqueous solution (20mL) of NaNO2 (5.52g, 80.00mmol) was added to the crude reaction, followed by the dropwise addition of HCl (2M) until gas evolution ceased and a pH of 2-3 was achieved, producing a red mixture. The crude reaction was extracted with DCM (3 x 40 mL) and washed with brine (3 x 20 mL). The organic phase was collected, dried over MgSO4, filtered and concentrated under reduced pressure. Purification by flash chromatography (DCM / MeOH 98 / 2) gave 0.300 g (26%) of di-tert-butyl (((1,2,4,5-tetrazine-3,6-diyl)bis(4,1-phenylene))bis(methylene))bis((2-fluoroethyl)carbamate) as a red oil (mixture of rotamers). Rf=0.45 (DCM / MeOH 98 / 2);

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[0119] Synthesis of N,N'-(((1,2,4,5-tetrazine-3,6-diyl)bis(4,1-phenylene))bis(methylene))bis(2-fluoroethan-1-amine) (9) Di-tert-butyl (((1,2,4,5-tetrazine-3,6-diyl)bis(4,1-phenylene))bis(methylene))bis((2-fluoroethyl)carbamate) (0.15 gr, 0.25 mmol) was treated with a solution of HCl (4 M) in dioxane (1 mL). A precipitate formed. Filtration gave 0.1 gr (83%) of the desired product as the hydrochloride salt.

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[0120] Synthesis of di-tert-butyl 2,2'-((((1,2,4,5-tetrazine-3,6-diyl)bis(4,1-phenylene))bis(methylene))bis((2-fluoroethyl)azanediyl)) diacetate (10) To a suspension of the hydrochloride salt of N,N'-(((1,2,4,5-tetrazine-3,6-diyl)bis(4,1-phenylene))bis(methylene))bis(2-fluoroethan-1-amine) (0.09 gr, 0.20 mmol) in anhydrous DMF (3 mL) was added Et3N (0.13 mL, 1.00 mmol). The reaction was stirred until all precipitate was dissolved. tert-Butyl bromoacetate (0.12 mL, 0.80 mmol) was added and the reaction was stirred at 50°C for 6 h. The mixture was cooled to room temperature and diluted with EtOAc (30 mL). The organic phase was washed with water (2 x 15 mL) and brine (2 x 15 mL). The organic phase was collected, dried over MgSO4, filtered and concentrated under reduced pressure to give 0.11 gr (91%) of the desired compound as a red oil. Rf=0.48 (heptane / EtOAc 70 / 30);

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[0121] Synthesis of 2,2'-((((1,2,4,5-tetrazine-3,6-diyl)bis(4,1-phenylene))bis(methylene))bis((2-fluoroethyl)azanediyl))diacetic acid (I) To a solution of di-tert-butyl 2,2'-((((1,2,4,5-tetrazine-3,6-diyl)bis(4,1-phenylene))bis(methylene))bis((2-fluoroethyl)azanediyl))diacetate (0.12 g, 0.19 mmol) in 5 mL of CHCl was added 2 mL of TFA. The mixture was stirred at room temperature for 4 h. The solvent was then removed under reduced pressure. Purification by preparative HPLC afforded 0.70 g (50%) of the desired compound (TFA salt) as a red solid.

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[0122] Synthesis of 2-((4-(6-(4-(((2-fluoroethyl)amino)methyl)phenyl)-1,2,4,5-tetrazin-3-yl)benzyl)amino)ethan-1-ol (11) (4-(6-(4-(((tert-butoxycarbonyl)(2-fluoroethyl)amino)methyl)phenyl)-1,2,4,5-tetrazin-3-yl)benzyl)(2-hydroxyethyl)carbamate (0.06 gr, 0.1 mmol) was treated with a solution of HCl (4 M) in dioxane (1 mL). A precipitate formed. Filtration gave 0.035 gr (75%) of the desired product as the hydrochloride salt.

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[0123] Synthesis of tert-butyl N-(4-(6-(4-(((2-(tert-butoxy)-2-oxoethyl)(2-fluoroethyl)amino)methyl)phenyl)-1,2,4,5-tetrazin-3-yl)benzyl)-N-(2-hydroxyethyl)glycinate (12) To a suspension of the hydrochloride salt of 2-((4-(6-(4-(((2-fluoroethyl)amino)methyl)phenyl)-1,2,4,5-tetrazin-3-yl)benzyl)amino)ethan-1-ol (0.03 gr, 0.077 mmol) in anhydrous DMF (3 mL) was added Et3N (0.05 mL, 0.38 mmol). The reaction was stirred until all precipitate had dissolved. tert-Butyl bromoacetate (0.04 mL, 0.3 mmol) was added and the reaction was stirred at 50°C for 6 h. The mixture was cooled to room temperature and diluted with EtOAc (30 mL). The organic phase was washed with water (2 x 15 mL) and brine (2 x 15 mL). The organic phase was collected, dried over MgSO4, filtered and concentrated under reduced pressure to give 0.03 gr (64%) of the desired compound as a red oil. Rf=0.25 (heptane / EtOAc 70 / 30);

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[0124] Synthesis of tert-butyl N-(4-(6-(4-(((2-(tert-butoxy)-2-oxoethyl)(2-chloroethyl)amino)methyl)phenyl)-1,2,4,5-tetrazin-3-yl)benzyl)-N-(2-fluoroethyl)glycinate (XXXIV) To a solution of compound tert-butyl N-(4-(6-(4-(((2-(tert-butoxy)-2-oxoethyl)(2-fluoroethyl)amino)methyl)phenyl)-1,2,4,5-tetrazin-3-yl)benzyl)-N-(2-hydroxyethyl)glycinate (0.04 g, 0.065 mmol) and DIPEA (0.034 mL, 0.19 mmol) in CHCl (10 mL) was added mesyl chloride (0.01 g, 0.019 mmol) and DMAP (0.001 g, 0.01 mmol). The reaction was stirred at room temperature for 12 h. The solvent was removed under reduced pressure. Purification by flash chromatography (80 / 20 heptane / EtOAc) afforded 0.020 (48%) of the desired product as a red solid. Rf=0.55 (heptane / EtOAc 70 / 30);

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[0125] Synthesis of di-tert-butyl 2,2'-((((1,2,4,5-tetrazine-3,6-diyl)bis(4,1-phenylene))bis(methylene))bis((2-hydroxyethyl)azanediyl))diacetate (13) To a suspension of tert-butyl (4-cyanobenzyl) (2-hydroxyethyl) carbamate (4 gr, 14.47 mmol) and Zn(OTf)2 (2.63 gr, 7.23 mmol) in EtOH (40 mL) was added hydrazine monohydrate (14.04 mL, 289 mmol). The mixture was stirred at 70 °C for 22 h and, once the reaction was complete, cooled at room temperature. The volatiles were removed under reduced pressure and the residue was dissolved in EtOH (80 mL). An aqueous solution (50 mL) of NaNO2 (19.97 g, 289.00 mmol) was added to the crude reaction, followed by dropwise addition of HCl (2 M) until gas evolution ceased and a pH of 2-3 was achieved to produce a red mixture. The crude reaction was extracted with DCM (3 × 60 mL) and washed with brine (3 × 50 mL). The organic phase was collected, dried over MgSO4, filtered and concentrated under reduced pressure. Purification by flash chromatography (DCM / MeOH 95 / 5) afforded 1.2 g (28%) of the desired product as a red solid (mixture of rotamers). Rf=0.21 (DCM / MeOH 95 / 5);

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[0126] Synthesis of di-tert-butyl 2,2'-((((1,2,4,5-tetrazine-3,6-diyl)bis(4,1-phenylene))bis(methylene))bis((2-hydroxyethyl)azanediyl)) diacetate (14) (4-(6-(4-(((tert-butoxycarbonyl)(2-fluoroethyl)amino)methyl)phenyl)-1,2,4,5-tetrazin-3-yl)benzyl)(2-hydroxyethyl)carbamate (0.9 gr, 1.55 mmol) was treated with a solution of HCl (4 M) in dioxane (15 mL). A precipitate formed. Filtration gave 0.68 gr (97%) of the desired product as the hydrochloride salt.

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[0127] Synthesis of di-tert-butyl 2,2'-((((1,2,4,5-tetrazine-3,6-diyl)bis(4,1-phenylene))bis(methylene))bis((2-hydroxyethyl)azanediyl)) diacetate (15) To a suspension of the hydrochloride salt of di-tert-butyl 2,2'-((((1,2,4,5-tetrazine-3,6-diyl)bis(4,1-phenylene))bis(methylene))bis((2-hydroxyethyl)azanediyl))diacetate (0.25 gr, 0.55 mmol) in anhydrous DMF (10 mL) was added Et3N (0.38 mL, 2.75 mmol). The reaction was stirred until all precipitate was dissolved. tert-Butyl bromoacetate (0.32 mL, 2.20 mmol) was added and the reaction was stirred at 50 °C for 6 h. The mixture was cooled to room temperature and diluted with EtOAc (40 mL). The organic phase was washed with water (2 x 15 mL) and brine (2 x 15 mL). The organic phase was collected, dried over MgSO4, filtered and concentrated under reduced pressure to give 0.31 gr (91%) of the desired compound as a red oil. Rf=0.23 (heptane / EtOAc 60 / 40);

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[0128] Synthesis of di-tert-butyl 2,2'-((((1,2,4,5-tetrazine-3,6-diyl)bis(4,1-phenylene))bis(methylene))bis((2-fluoroethyl)azanediyl)) diacetate (10) and di-tert-butyl 2,2'-((((1,2,4,5-tetrazine-3,6-diyl)bis(4,1-phenylene))bis(methylene))bis((2-hydroxyethyl)azanediyl)) diacetate (12) To a solution of di-tert-butyl 2,2'-((((1,2,4,5-tetrazine-3,6-diyl)bis(4,1-phenylene))bis(methylene))bis((2-hydroxyethyl)azanediyl))diacetate (0.24 g, 0.39 mmol) in anhydrous THF (15 mL) at -78 °C was added DAST (0.055 mL, 0.39 mmol). The resulting mixture was stirred at -78 °C for 1 h and at room temperature for another 3 h. The reaction was then quenched with saturated NaHCO3 solution (10 mL) and stirred for 30 min. The reaction mixture was extracted with DCM (3 x 30 mL) and washed with brine (3 x 30 mL). The organic phase was collected, dried over MgSO4, filtered and concentrated under reduced pressure. Purification by flash chromatography (DCM / MeOH 95 / 5) gave 0.12 g (50%) of di-tert-butyl 2,2'-((((1,2,4,5-tetrazine-3,6-diyl)bis(4,1-phenylene))bis(methylene))bis((2-fluoroethyl)azanediyl))diacetate (NMR see example x). A more polar fraction was isolated in 0.04 g (14%) corresponding to di-tert-butyl 2,2'-((((1,2,4,5-tetrazine-3,6-diyl)bis(4,1-phenylene))bis(methylene))bis((2-hydroxyethyl)azanediyl))diacetate.

[0129] Example 2 Synthesis of unsymmetrical tetrazines and their precursors. Compounds XII, pretargeted bioorthogonal PET imaging.Chem.Sci.2021,12,11668-11675 and Battisti,UM;Bratteby,K.;Jorgensen,JT;Hvass,L.;Shalgunov,V.;Mikula,H.;Kjaer,A.;Herth,MMDevelopment of the First Aliphatic 18F-Labeled Tetrazine Suitable for Pretargeted PET Imaging-Expanding the Bioorthogonal Tool Box. J. Med. Chem. 2021, 64, 15297-15312.

[0130] Example 2.1 Compound XLVIII [ka] Scheme 5. Synthesis of precursor XLVIII. Reagents and conditions: i) NBS, AIBN, CH3CN, reflux, 24 h, 49%; ii) a) glycine tert-butyl ester hydrochloride, K2CO3, CH3CN, 25 °C, 24 h, b) Boc2O, CH2Cl2, room temperature, 12 h, 91%; iii) a) CH2Cl2, S8, NH2NH2H2O, EtOH, 50 °C, 24 h, b) NaNO2, AcOH, room temperature, 30 min, 27%; iv) (Me3Sn)2, Pd(PPh3)4, THF, 65 °C, MW, 3 h, 47%.

[0131] 3-(Bromomethyl)-5-iodobenzonitrile (23) To a solution of 3-iodo-5-methylbenzonitrile (2.50 g, 10.28 mmol) and N-bromosuccinimide (2.28 g, 12.86 mmol) in CH3CN (40 mL) was added AIBN (0.67 g, 4.11 mmol). The reaction was refluxed for 24 h. The solvent was removed under high vacuum and the crude product was purified by flash chromatography (95 / 5 n-heptane / EtOAc) to give 1.61 g (49%) of the desired product as a white solid. Rf=0.28 (95 / 5 n-heptane / EtOAc);

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[0132] tert-Butyl 2-((tert-butoxycarbonyl)(3-cyano-5-iodobenzyl)amino)acetate (24) To a solution of 3-(bromomethyl)-5-iodobenzonitrile (2.00 g, 6.21 mmol) in CH3CN (40 mL) was added K2CO3 (4.29 g, 31.07 mmol) and glycine tert-butyl ester hydrochloride (3.12 g, 18.63 mmol). The reaction mixture was stirred at room temperature overnight. The solvent was removed under reduced pressure and the resulting mixture was diluted with water (20 mL), extracted with EtOAc (2 x 25 mL), washed with brine (30 mL), dried over MgSO4, filtered and concentrated. Purification by flash chromatography (80 / 20 n-heptane / EtOAC); 1.91 g (83%) of the desired product was obtained as a colorless oil. Rf = 0.23 (80 / 20 n-heptane / EtOAC);

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[0133] To a solution of this amine (1.9 g, 5.10 mmol) and Et3N (1.71 mL, 12.25 mmol) in CHCl2 (40 mL) was added Boc2O (1.41 g, 6.12 mmol). The reaction was stirred at room temperature for 12 h. The solution was then washed with water (50 mL) and saturated K2CO3 solution (50 mL), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to give 2.5 g of crude product. Purification by flash chromatography (80 / 20 n-heptane / EtOAc) afforded 2.2 g (91%) of the desired product as a colorless oil (60 / 40 mixture of unassigned rotamers). Rf=0.38 (80 / 20 n-heptane / EtOAc);

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[0134] tert-Butyl 2-((tert-butoxycarbonyl)(3-iodo-5-(1,2,4,5-tetrazin-3yl)benzyl)amino)acetate (25) To a Biotage microwave vial (10-20 mL) fitted with a stir bar was added CHCl (0.17 mL, 2.67 mmol), sulfur (0.17 g, 0.67 mmol), hydrazine monohydrate (1.1 mL, 21.39 mmol) and ethanol (4.0 mL) along with tert-butyl 2-((tert-butoxycarbonyl)(3-cyano-5-iodobenzyl)amino)acetate (2.10 g, 4.44 mmol). The vessel was sealed and the reaction mixture was heated to 50 °C for 24 h, then cooled to room temperature and unsealed. 3 ml of CHCl and NaNO (1.84 g, 26.73 mmol) in water (30 ml) was then added to this now yellow mixture, followed by dropwise addition of acetic acid (10 mL) resulting in a red mixture. The reaction mixture was extracted with CH2Cl2, washed with brine, dried over MgSO4, filtered, and concentrated in vacuo. The crude product was purified using flash chromatography (95 / 5 n-heptane / EtOAc) to give 0.64 g (27%) of the desired product as a red solid (60 / 40 mixture of unassigned rotamers). Rf=0.31 (80 / 20 n-heptane / EtOAc);

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[0135] tert-Butyl N-(3-(1,2,4,5-tetrazin-3-yl)-5-(trimethylstannyl)benzyl)-N-(tert-butoxycarbonyl)glycinate (XLVIII) Pd(PPh3)4 (19.4 mg, 10%) and hexamethylditin (87 μL, 0.42 mmol) were added sequentially to a microwave vial equipped with a stir bar, which was then sealed and purged with N2. A solution of tert-butyl 2-((tert-butoxycarbonyl)(3-iodo-5-(1,2,4,5-tetrazin-3yl)benzyl)amino)acetate (0.08 g, 0.17 mmol) in dry THF (2.5 mL) was added via syringe, and the reaction was stirred in a microwave at 65 °C for 3 h. The reaction was cooled to room temperature, opened, and then quenched with saturated aqueous KF solution (1 mL). The solution was extracted with CHCl2 (3 × 10 mL), washed with brine (10 mL), dried over MgSO4, filtered, and concentrated under reduced pressure. The crude product was purified using flash chromatography (95 / 5 n-heptane / EtOAc) to give 0.055 g (66%) of the desired product as a purple oil (60 / 40 mixture of unassigned rotamers): Rf=0.38 (80 / 20 n-heptane / EtOAc);

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[0136] Example 3 Synthesis of isomer-free TCO [ka] Scheme 6. Synthesis of isomer-free TCO: i) mCPBA, THF, H2O, 0 °C → RT, 17 h quantitative; ii) HClO4, THF, H2O, RT, 24 h (48%); iii) AgNO3, hV, RT, 8 h, 60%; iv) oxalyl chloride, DMSO, Et3N, DCM, -78 °C, 3 h (62%); v) 2-aminoaniline, MeOH. RT, 0.2 h, quantitative.

[0137] (Z)-9-Oxabicyclo[6.1.0]non-4-ene (26). Cyclooctadiene (1.00 g, 9.24 mmol) was dissolved in dry DCM (20 mL) and cooled to 0 °C, to which mCBPA (2.07 g, 9.24 mmol) was added portionwise. After addition, the reaction was stirred at 0 °C for 30 min, after which the reaction was allowed to warm to room temperature. The reaction was stirred for 17 h. The reaction was quenched with 2 M K2CO3 (20 mL) and the crude product was extracted 3 x DCM (15 mL). The combined organic phase was washed with K2CO3 (25 mL) and brine (25 mL). The solvent was evaporated and the crude product was used in the next reaction without purification.

[0138] (Z)-Cyclooct-5-ene-1,2-diol (27). 25 (1.15 g, 9.24 mmol) was dissolved in a mixture of THF / water (3:2 v / v, 50 mL) containing HClO4 (250 μL, 2.90 mmol). The reaction was stirred at room temperature for 24 h. Water (10 mL) was added to the reaction mixture and the product was extracted with 3×Et2O (20 mL). The combined ether fractions were washed with 2×2M NaHCO3 (25 mL). The crude product was purified by CombiFlash to give a clear viscous oil. (624 mg, 4.40 mmol, 48%).

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[0139] (E)-Cyclooct-5-ene-1,2-diol (28) The compounds were obtained as described in Royzen, M.; Yap, GPA; Fox, JMA Photochemical Synthesis of Functionalized trans-Cyclooctenes Driven by Metal Complexation. Journal of the American Chemical Society 2008, 130, 3760-3761.

[0140] A flash cartridge (220 g, threaded top, Luer lock end fittings, catalog number FCSTLL-220-6) was packed with 8 cm of silica (15-40 μm) at the bottom and silver nitrate impregnated silica to the top. The column was flushed with 9:1 diethyl ether / n-heptane (500 mL) and the column was protected from light with aluminum foil. The cooling fence and UV lamp were turned on and after 10 min, no detectable silver leakage was observed. Methyl benzoate (1 mL), 27 (1 g) and an additional 50 mL of the 9:1 diethyl ether / n-heptane solution were added to the round bottom flask. The mixture was then added to the quartz flask. The pump was turned on (flow rate = 100 mL / min), the photoreactor was turned on and the photoisomerization was carried out for 8 h. After 8 h, the photoreactor was turned off and the column was dried with a stream of air. The silica was removed from the column and washed with 400 mL of ammonia and 400 mL of DCM. The mixture was stirred for 30 min, filtered, and the organic layer was collected. The organic layer was washed with brine, dried over MgSO4, filtered, and concentrated to give 0.60 g (60%) of the product as a yellowish oil (mixture of axial and equatorial isomers).

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[0141] (E)-Cyclooct-5-ene-1,2-dione (29) A solution of DMSO (1.05 mL, 14.77 mmol) in dry DCM (30 mL) was added dropwise to a solution of (COCl)2 (736 μL, 8.44 mmol) in dry DCM (15 mL) at -78 °C under an argon atmosphere. After addition, the reaction mixture is stirred at -78 °C for 30 min. A solution of 28 (500 mg, 3.52 mmol) in anhydrous DCM (10 mL) is added dropwise and the reaction mixture is stirred at -78 °C for another 30 min. Et3N (4.90 mL, 35.16 mmol) is added dropwise and the reaction mixture is kept at -78 °C for another 60 min. The reaction mixture is allowed to warm to room temperature and stirred for an additional 1 h. The reaction mixture is diluted with water (50 mL), 2 × 0.5 M HCl, 10 mL of ethyl acetate, 10 mL of ethyl acetate, and 10 mL of ethyl acetate. (水溶液)(50 mL) and brine (30 mL). The organic phase was dried over MgSO4 and evaporated to give the desired compound as a clear liquid. (302 mg, 2.19 mmol, 62%).

[0142] (E)-6,7,10,11-Tetrahydrocycloocta[b]quinoxaline (30) The compounds were obtained following the procedure reported in Delpivo, C.; Micheletti, G.; Boga, CA Green Synthesis of Quinoxalines and 2,3-Dihydropyrazines. Synthesis 2013, 45, 1546-1552. A 50 mL round-bottom flask was charged with a solution of the appropriate 29 (138 mg, 1 mmol) in MeOH (3 mL). To this solution was added the respective 2-aminoaniline (110 mg, 1 mmol) and the mixture was stirred at room temperature. After 30 min, the crude mixture was extracted with CHCl (3×10 mL). The combined organic layers were dried (MgSO), filtered and the solvent was removed to give the desired product 0.2 g (97%).

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[0143] Example 3.1 [ka] Scheme 7. Synthesis of isomer-free TCO i) sulfuric acid, nitric acid, 80 °C, 0.5 h (56%); ii) urea, 150 °C, 6 h (87%); iii) tin(II) chloride, EtOH, reflux, 15 h (quantitative); iv) Cs2CO3, tert-butyl bromoacetate or methyl bromoacetate, DMF, 120 °C, 4 h (33–49%); v) Method A: tert-butyl 2-(5,6-diamino-1,3-dioxoisoindolin-2-yl)acetate, TFA, DCM, rt, 4 h (99%); Method B: methyl 2-(5,6-diamino-1,3-dioxoisoindolin-2-yl)acetate, concentrated HCl (水溶液), dioxane, 70 °C, 24 h (99%); vi) AcOH, (E)-cyclooct-5-ene-1,2-dione, DMF, rt, 6 h (27%); vii) LiOH, THF, HO, rt, 3 h (89%); viii) N-hydroxysuccinimide, DCC, DMF, rt, 18 h (72%).

[0144] 5-Chloro-6-nitroisoindoline-1,3-dione (31) The compound was synthesized according to the literature. 1 A mixture of concentrated sulfuric acid (33.1 mL) and fuming nitric acid (2.26 mL) was added dropwise to 4-chlorophthalimide (5.00 g, 27.53 mmol). The reaction mixture was heated at 80° C. for 0.5 h. After cooling to room temperature, the dark red solution was poured onto ice. The resulting yellow precipitate was collected by filtration, washed with water, and dried in vacuum to give a pale yellow solid, which was recrystallized from EtOH to give 3.5 g (56% yield) of the desired product. Rf=0.45 (n-heptane / EtOAc 60 / 40);

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[0145] 5-Amino-6-nitroisoindoline-1,3-dione (32) The compound was synthesized according to the literature. 1 A mixture of 5-chloro-6-nitroisoindoline-1,3-dione (2.4 g, 10.59) and urea (6.36 g, 105.92 mmol) was stirred and heated to 150 °C under argon for 6 h. After cooling to room temperature, the solid was suspended in hot water (80 °C), filtered and washed with hot water (3 × 50 mL). The solid was recrystallized from EtOH to give 1.92 g (87%) of a yellow solid. Rf = 0.28 (n-heptane / EtOAc 60 / 40);

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[0146] 5,6-Diaminoisoindoline-1,3-dione (33) The compound was synthesized according to the literature. 1 A suspension of 4-amino-5-nitro-phthalimide (1.0 g, 4.27 mmol) and tin(II) chloride (4.57 g, 24.14 mmol) in ethanol (30 mL) was refluxed under argon for 15 h. The resulting bright red suspension was cooled to room temperature and the red solid was collected by filtration, washed with ethanol and dried in vacuum to give a red-orange solid. Rf=0.15 (95 / 5 DCM / MeOH);

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[0147] tert-Butyl 2-(5,6-diamino-1,3-dioxoisoindolin-2-yl)acetate (34) To a suspension of Cs2CO3 (1.93 g, 5.92 mmol) and 5,6-diaminoisoindoline-1,3-dione (1.0 g, 5.64 mmol) in anhydrous DMF (5 mL) was added tert-butyl bromoacetate (0.87 mL, 5.92 mmol). The reaction was heated at 120 °C under argon for 4 h. The mixture was cooled to room temperature and water (20 mL) was added. The reaction mixture was extracted with DCM (3 x 50 mL). The organic layer was dried over anhydrous MgSO4, filtered and concentrated under reduced pressure. The resulting residue was purified using flash chromatography (98 / 2 DCM / MeOH) to give 0.8 g (49%) of the desired compound as a yellow solid. Rf = 0.42 (DCM / MeOH 93 / 7);

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[0148] Methyl 2-(5,6-diamino-1,3-dioxoisoindolin-2-yl)acetate (35) To a suspension of Cs2CO3 (1.93 g, 5.92 mmol) and 5,6-diaminoisoindoline-1,3-dione (1.0 g, 5.64 mmol) in anhydrous DMF (5 mL) was added methyl bromoacetate (0.63 mL, 5.92 mmol). The reaction was heated at 120 °C under argon for 4 h. The mixture was cooled to room temperature and water (20 mL) was added. The reaction mixture was extracted with DCM (3 x 50 mL). The organic layer was dried over anhydrous MgSO4, filtered and concentrated under reduced pressure. The resulting residue was purified using flash chromatography (98 / 2 DCM / MeOH) to give 0.45 g (33%) of the desired compound as a yellow solid. Rf = 0.40 (DCM / MeOH 93 / 7);

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[0149] 2-(5,6-Diamino-1,3-dioxoisoindolin-2-yl)acetic acid (36) Method A To a solution of tert-butyl 2-(5,6-diamino-1,3-dioxoisoindolin-2-yl)acetate (0.4 g, 1.37 mmol) in DCM (6 mL) was added TFA (2 mL). The reaction was stirred at room temperature for 4 h and then concentrated under reduced pressure to give 0.32 g (99%) of the desired compound as a yellow solid. Rf=0.29 (DCM / MeOH 99 / 1+0.1% AcOH);

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[0150] Method B: To a solution of methyl 2-(5,6-diamino-1,3-dioxoisoindolin-2-yl)acetate (0.25 g, 1.01 mmol) in dioxane (6 mL) was added concentrated HCl (1 mL). The reaction was stirred at 70° C. for 24 h and then concentrated under reduced pressure to give 0.23 g (99%) of the desired compound as a yellow solid. Rf=0.29 (DCM / MeOH 99 / 1+0.1% AcOH);

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[0151] tert-Butyl (E)-2-(1,3-dioxo-1,3,6,7,10,11 hexahydro-2H-cycloocta[b]pyrrolo[3,4g]quinoxalin-2-yl)acetate (37) A solution of tert-butyl 2-(5,6-diamino-1,3-dioxoisoindolin-2-yl)acetate (0.4 g, 1.40 mmol) and acetic acid (0.8 mL, 14.03 mmol) in DMF (5 mL) and water (1 mL) was added to (E)-cyclooct-5-ene-1,2-dione (0.21 g, 1.54 mmol). The reaction was stirred at room temperature in the dark for 6 h. The solution was diluted with 10 mL of water and purified directly by preparative HPLC to give 0.09 g (17%) of the desired compound as a yellow solid. Rf=0.36 (heptane / EtOAc 75 / 25);

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[0152] Methyl (E)-2-(1,3-dioxo-1,3,6,7,10,11 hexahydro-2H-cycloocta[b]pyrrolo[3,4g]quinoxalin-2-yl)acetate (38) A solution of methyl 2-(5,6-diamino-1,3-dioxoisoindolin-2-yl)acetate (0.38 g, 1.40 mmol) and acetic acid (0.8 mL, 14.03 mmol) in DMF (5 mL) and water (1 mL) was added to (E)-cyclooct-5-ene-1,2-dione (0.21 g, 1.54 mmol). The reaction was stirred at room temperature in the dark for 6 h. The solution was diluted with 10 mL of water and purified directly by preparative HPLC to give 0.15 g (26%) of the desired compound as a yellow solid. Rf=0.31 (heptane / EtOAc 75 / 25);

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[0153] (E)-2-(1,3-dioxo-1,3,6,7,10,11 hexahydro-2H-cycloocta[b]pyrrolo[3,4-g]quinoxalin-2-yl)acetic acid (39) A solution of 2-(5,6-diamino-1,3-dioxoisoindolin-2-yl)acetic acid (0.33 g, 1.40 mmol) and acetic acid (0.8 mL, 14.03 mmol) in DMF (5 mL) and water (1 mL) was added to (E)-cyclooct-5-ene-1,2-dione (0.21 g, 1.54 mmol). The reaction was stirred at room temperature in the dark for 6 h. The solution was diluted with 10 mL of water and purified directly by preparative HPLC to give 0.13 g (27%) of the desired compound as a yellow solid. Rf=0.45 (DCM / MeOH 99 / 1 + 0.1% AcOH);

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[0154] 2,5-Dioxopyrrolidin-1-yl (E)-2-(1,3-dioxo-1,3,6,7,10,11 hexahydro-2H-cycloocta[b]pyrrolo[3,4-g]quinoxalin-2-yl)acetate (40) To a solution of (E)-2-(1,3-dioxo-1,3,6,7,10,11-hexahydro-2H-cycloocta[b]pyrrolo[3,4-g]quinoxalin-2-yl)acetic acid (0.06 g, 0.18 mmol) in anhydrous DMF (3 mL) under argon was added DCC (0.04 g, 0.19 mmol) followed by N-hydroxysuccinimide (0.04 g, 0.35 mmol). The reaction was stirred overnight at room temperature under argon. EtOAc (10 mL) was added and the organic layer was washed with water (2×10 mL) and brine (2×10 mL). The organic layer was dried over anhydrous MgSO4, filtered and concentrated under reduced pressure. The residue was dissolved in dry cold MECN (4 mL), filtered and concentrated to give a yellow solid. The solid was triturated in diethyl ether and filtered to give 0.056 g (72%) of the desired compound as a yellow solid. Rf=0.41 (n-heptane / EtOAc 50 / 50);

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[0155] Example 3.2 [ka] Scheme 8. Synthesis of isomer-free TCO i) AcOH, glycine methyl ester HCl, reflux, 16 h (58%); ii) NaN3, MeOH, rt, 17 h (89%); iii) Pd / C (10%), H2, EtOH, rt, 24 h (95%); iv) AcOH, (E)-cyclooct-5-ene-1,2-dione, DMF, rt, 6 h (24%).

[0156] Methyl 2-(3,4-dichloro-2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetate (41) To a solution of anhydrous dichloromethane (5.0 g, 29.95 mmol) in acetic acid (100 mL) was added glycine methyl ester hydrochloride (4.51 g, 35.94 mmol). The mixture was heated to reflux for 16 h. The solvent was removed under reduced pressure. Water (100 mL) was added and the resulting suspension was stirred for 1 h. The solid was filtered to give 4.15 g (58%) of the desired product as a beige solid.

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[0157] Methyl 2-(3,4-diazido-2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetate (42) A solution of 2-(3,4-dichloro-2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetate (4.15 g, 17.43 mmol) and sodium azide (2.72 g, 41.84 mmol) in methanol (70 mL) was stirred overnight. The solvent was removed under vacuum, the residue was dissolved in dichloromethane (50 mL), washed with water (2×30 mL) and brine (2×30 mL), dried over anhydrous sodium sulfate, and the solvent was removed by vacuum to give the compound (3.89 g, 89%) as a yellow solid.

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[0158] Methyl 2-(3,4-diazido-2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetate (43) To a solution of 2-(3,4-diazido-2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetate (1.6 g, 6.37 mmol) in EtOH (40 mL) was added 10% Pd / C (0.34 g, 0.31 mmol). The mixture was stirred at room temperature and placed under H2 atmosphere for 24 h. The catalyst was then filtered through a pad of Celite and the solvent was removed to give 1.2 g (95%) of the desired compound as a red oil. NMR was normal.

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[0159] Methyl (E)-2-(1,3-dioxo-1,3,5,6,9,10 hexahydro-2H-cycloocta[b]pyrrolo[3,4-e]pyrazin-2-yl)acetate (45) A solution of methyl 2-(3,4-diazido-2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetate (0.28 g, 1.40 mmol) and acetic acid (0.8 mL, 14.03 mmol) in DMF (5 mL) and water (1 mL) was added to (E)-cyclooct-5-ene-1,2-dione (0.21 g, 1.54 mmol). The reaction was stirred at room temperature in the dark for 6 h. The solution was diluted with 10 mL of water and purified directly by preparative HPLC to give 0.1 g (24%) of the desired compound as a yellow solid. Rf=0.28 (heptane / EtOAc 75 / 25);

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[0160] Example 3.3 [ka] Scheme 9. Synthesis of isomer-free TCO. i) NH4CO3, methyl-4-oxobutanoate, MeOH, rt, 16 h (63%), ii) LiOH, H2O, THF, rt, 3 h (76%).

[0161] Methyl (E)-3-(4,5,8,9-tetrahydro-1H-cycloocta[d]imidazol-2-yl)propanoate (46) In a 15 mL screw neck, ammonium bicarbonate (384 mg, 4.86 mmol) and methyl-4-oxobutanoate (85%, 331 mg, 2.43 mmol) were stirred in 2 mL of MeOH at room temperature for 15 min. (E)-Cyclooct-5-ene-1,2-dione (305 mg, 2.21 mmol) in 3 mL of MeOH was added to the suspension and stirred for 16 h. The reaction mixture was diluted with 0.1% TFA. (水溶液) and preparative HPLC afforded methyl (E)-3-(4,5,8,9-tetrahydro-1H-cycloocta[d]imidazol-2-yl)propanoate as a beige viscous liquid (325 mg, 1.39 mmol, 63%).

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[0162] (E)-3-(4,5,8,9-tetrahydro-1H-cycloocta[d]imidazol-2-yl)propanoic acid (47) In a 5 mL screw-necked vial, 1 M LiOH solution (861 μL, 861 μmol) was added to methyl (E)-3-(4,5,8,9-tetrahydro-1H-cycloocta[d]imidazol-2-yl)propanoate (100 mg, 287 μmol) in 2.5 mL of THF / water (3:2 v / v). The suspension was stirred at room temperature for 3 h. The reaction was diluted with 1 M TFA. (水溶液)Dilution with 100% water and preparative HPLC afforded (E)-3-(4,5,8,9-tetrahydro-1H-cycloocta[d]imidazol-2-yl)propanoic acid as a pale yellow amorphous solid (73 mg, 218 μmol, 76%).

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[0163] Scheme 10. Synthesis of E,E-Cyclooctadiene v) AcOOH, Na2CO3, DCM, 0 °C → RT, 48 h (65%); vi) nBuLi, PH(Ph)2, AcOH, H2O2, THF, -78 °C to RT, 12 h, 52%; vii) NaH, THF, -78 °C to RT, 12 h, 25%.

[0164] cis,cis-5,10-Dioxatricyclo[7.1.0.04,6]decane (48) Compounds were synthesized as described in Stockmann, H.; Neves, AA; Day, HA; Stairs, S.; Brindle, KM; Leeper, FJ (E,E)-1,5-Cyclooctadiene: a small and fast click-chemistry multitalent. Chemical Communications 2011, 47, 7203-7205.

[0165] Sodium carbonate (20.5 g, 50.84 mmol, 1.1 equiv) was added to a stirred solution of (Z,Z)-1,5-cyclooctadiene (5 g, 46.2 mmol, 1.0 equiv) in DCM (25 mL). The suspension was cooled to 0 °C and peracetic acid (36%, 17.9 mL, 97.1 mmol, 2.1 equiv) was added. The mixture was warmed to room temperature and stirred until the reaction was complete after 48 h. The reaction was quenched with saturated Na2CO3 solution followed by extraction with EtOAc (4 x 25 mL). The combined organic layers were dried over MgSO4, concentrated and distilled to give cis,cis-5,10-dioxatricyclo[7.1.0.04,6]decane (4.19 g, 29.89 mmol, 65%).

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[0166] ((trans,trans)-2,6-dihydroxycyclooctane-1,5-diyl)bis(diphenylphosphine oxide) (49) Compounds were synthesized as described in Stockmann, H.; Neves, AA; Day, HA; Stairs, S.; Brindle, KM; Leeper, FJ (E,E)-1,5-Cyclooctadiene: a small and fast click-chemistry multitalent. Chemical Communications 2011, 47, 7203-7205.

[0167] To a solution of diphenylphosphine (10.43 mL, 11.16 g, 59.92 mmol, 2.1 equiv) and cis,cis-5,10-dioxatricyclo-[7.1.0.04,6]decane (4.0 g, 28.5 mmol, 1.0 equiv) in THF (120 mL) was added BuLi (2 M solution in hexanes, 29.96 mL, 59.92 mmol, 2.1 equiv) dropwise at -78 °C. The mixture was stirred for 1 h, warmed to room temperature, and stirred for 12 h. The brown solution was cooled to 0 °C, diluted with THF (86 mL), and quenched by the addition of AcOH (4.9 mL, 5.14 g, 85.60 mmol, 3 equiv) and H2O2 (30%, 8.7 mL, 85.60 mmol, 3 equiv). The mixture became clear, followed by the formation of a white precipitate. The mixture was removed from the ice bath and stirred vigorously at 25 °C for 2 h. The suspension was then transferred to a separatory funnel, EtOAc (150 mL) and HO (50 mL) were added, the aqueous layer was removed, and the organic layer was washed with brine (2 × 20 mL). The precipitate was filtered off to give ((trans,trans)-5,8-dihydroxycyclooctane-1,4-diyl)bis(diphenylphosphine oxide). The combined organic layers were dried over MgSO4, filtered, and concentrated to give a white solid. The remaining AcOH was azeotropically removed with toluene to give an isomeric mixture of phosphine oxides (8.14 g, 14.9 mmol, 52%). Spectroscopic data were consistent with previously published data.

[0168] (E,E)-1,5-Cyclooctadiene (50) Compounds were synthesized as described in Stockmann, H.; Neves, AA; Day, HA; Stairs, S.; Brindle, KM; Leeper, FJ (E,E)-1,5-Cyclooctadiene: a small and fast click-chemistry multitalent. Chemical Communications 2011, 47, 7203-7205.

[0169] A mixture of phosphine oxides (6.00 g, 11.0 mmol, 1.0 equiv) was dissolved in DMF (30 mL) with stirring under an Ar atmosphere. The clear solution turned cloudy and NaH (60% in mineral oil, 1.44 g, 34.2 mmol, 3.1 equiv) was added portionwise over 5 min at room temperature under a positive Ar stream. The reaction mixture was stirred at room temperature for 12 h, cooled to 0 °C, diluted with pentane (50 mL) and quenched with half-saturated NH4Cl (22 mL). The aqueous layer was extracted with pentane (50 mL) and the combined organic layers were washed with water (5 × 22 mL) and brine (1 × 22 mL) to give (E,E)-1,5-cyclooctadiene (25% yield). Spectroscopic data were consistent with previously published data. [ka]

[0170] Scheme 11. Synthesis of isomer-free TCO X. i) CHCl3, reflux, 2 days, 63%) ii) 33, CH2Cl2, room temperature, 18 hours, (50%).

[0171] Dimethyl 6-benzyl-5,7-dioxo-6,7-dihydro-5H-pyrrolo[3,4-d]pyridazine-1,4-dicarboxylate (51) Dimethyl 1,2,4,5-tetrazine-3,6-dicarboxylate (50 mg, 0.25 mmol) was dissolved in CHCl3 (1 mL) to which 1-phenyl-1H-pyrrole-2,5-dione (53 mg, 0.30 mmol) was added and the reaction was stirred at reflux for 2 days. Upon reaction, the dihydropyridazine spontaneously oxidized to the corresponding pyridazine. Once dimethyl 1,2,4,5-tetrazine-3,6-dicarboxylate was completely consumed, the reaction was cooled to room temperature. The product was purified by CombiFlash to give a pale solid (54 mg, 0.16 mmol, 63%).

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[0172] Dimethyl (E)-1,3-dioxo-2-phenyl-2,3,5,6,9,10-hexahydro-1H-cycloocta[f]isoindole-4,11-dicarboxylate (52) To a solution of 35 (20 mg, 0.06 mmol) in CH2Cl2 was added compound 33 in pentane (0.06 mmol). The solution was stirred at room temperature for 18 h. The volatiles were then removed under reduced pressure. Purification by flash chromatography afforded 12 mg (50%) of the desired product as a white solid.

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[0173] Example 4 Ligation between unsymmetrical tetrazines and isomer-free TCOs [ka] Scheme 12. 1-(4-Fluorophenyl)-5,6,13,14-tetrahydropyridazino[4',5':5,6]cycloocta[1,2-b]quinoxaline (53) 3-(4-Fluorophenyl)-1,2,4,5-tetrazine was obtained as described in Kjaer, A.; Petersen Ida, N.; Herth Matthias, M.; Kristensen Jesper, L. NOVEL TETRAZINE COMPOUNDS FOR IN VIVO IMAGING. WO 2020 / 108720 A1, 2019 / 11 / 29, 2020.

[0174] Compound 30 (12 mg, 0.057 mmol) was dissolved in a mixture of MeCN / HO (4 / 1 v / v, 2.5 mL) to which was added 3-(4-fluorophenyl)-1,2,4,5-tetrazine (10 mg, 0.057 mmol). The reaction was stirred at room temperature for 30 min. p-Chloranil (17 mg, 0.068 mmol) was added and the reaction was stirred for 10 min. The reaction was then diluted with HO (5 mL) and filtered. The solution was concentrated under reduced pressure and purified by flash chromatography to give 10 mg (0.028 mmol) of the desired product.

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[0175] Scheme 13. 2-(1-(4-(2-fluoroethoxy)phenyl)-9,11-dioxo-5,6,9,11,14,15-hexahydro-10H-pyridazino[4',5':5,6]cycloocta[1,2-b]pyrrolo[3,4-g]quinoxalin-10-yl)acetic acid (54) 3-(4-(2-Fluoroethoxy)phenyl)-1,2,4,5-tetrazine was obtained as described in Garcia Vazquez et al. Pharmaceuticals 2022,15(2),245. 3-(4-(2-Fluoroethoxy)phenyl)-1,2,4,5-tetrazine (3.2 mg, 0.0148 mmol) was dissolved in MeCN (1 mL) and added to a stirred solution of methyl (E)-2-(1,3-dioxo-1,3,6,7,10,11 hexahydro-2H-cycloocta[b]pyrrolo[3,4-g]quinoxalin-2-yl)acetate (5 mg, 0.0148 mmol) in HO (2 mL). p-Chloranil (18 mg, 0.074 mmol) was added and the reaction was stirred for 15 min. The reaction was then diluted with HO (15 mL) and filtered. The solution was concentrated under reduced pressure and purified by preparative HPLC, and all fractions containing pure product were lyophilized to afford the product as a white solid (5 mg, 0.0095 mmol, 64%).

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[0176] Scheme 14. Methyl 3-(6-(4-(2-fluoroethoxy)phenyl)-4,5,10,11-tetrahydro-1H-imidazo[4',5':5,6]cycloocta[1,2-d]pyridazin-2-yl)propanoate 2,2,2-trifluoroacetate (55) 3-(4-(2-Fluoroethoxy)phenyl)-1,2,4,5-tetrazine was obtained as described in Garcia Vazquez et al. Pharmaceuticals 2022,15(2),245. 3-(4-(2-Fluoroethoxy)phenyl)-1,2,4,5-tetrazine (9 mg, 0.043 mmol) was dissolved in MeCN (1 mL) and added to a stirred solution of methyl (E)-3-(4,5,8,9-tetrahydro-1H-cycloocta[d]imidazol-2-yl)propanoate (15 mg, 0.043 mmol) in MeCN / HO (50 v / v%, 1 mL). p-Chloranil (53 mg, 0.215 mmol) was added and the reaction was stirred for 25 min. The reaction was then diluted with HO (25 mL) and filtered. The solution was concentrated under reduced pressure and purified by preparative HPLC, and all fractions containing pure product were lyophilized to afford the product as a yellow solid (5 mg, 0.012 mmol, 27%).

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[0177] Scheme 15. Methyl 3-(6-(4-iodophenyl)-4,5,10,11-tetrahydro-1H-imidazo[4',5':5,6]cycloocta[1,2-d]pyridazin-2-yl)propanoate 2,2,2-trifluoroacetate (56) 3-(4-Iodophenyl)-1,2,4,5-tetrazine was obtained as described by Garcia Vazquez et al. Pharmaceuticals 2022,15(2),245. 3-(4-Iodophenyl)-1,2,4,5-tetrazine (16 mg, 0.057 mmol) was dissolved in MeCN (1 mL) and added to a stirred solution of methyl (E)-3-(4,5,8,9-tetrahydro-1H-cycloocta[d]imidazol-2-yl)propanoate (20 mg, 0.057 mmol) in MeCN / HO (50 v / v%, 1 mL). p-Chloranil (71 mg, 0.287 mmol) was added and the reaction was stirred for 15 min. The reaction was then diluted with HO (20 mL) and filtered. The solution was concentrated under reduced pressure and purified by preparative HPLC, and all fractions containing pure product were lyophilized to afford the product as a yellow solid (21 mg, 0.034 mmol, 61%).

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[0178] Example 5 Ligation between symmetric tetrazines and isomer-free TCOs [ka] Scheme 16. 1,4-Di(pyridin-2-yl)-5,6,13,14-tetrahydropyridazino[4',5':5,6]cycloocta[1,2-b]quinoxaline (57) 30 (50 mg, 0.24 mmol) was dissolved in a mixture of MeCN / HO (4 / 1 v / v, 5 mL) to which was added 3,6-di(pyridin-2-yl)-1,2,4,5-tetrazine (56 mg, 0.24 mmol). The reaction was stirred at room temperature for 30 min. p-Chloranil (117 mg, 0.47 mmol) was added and the reaction was stirred for 10 min. The reaction was then diluted with HO (50 mL) and filtered. The solution was concentrated under reduced pressure and purified by flash chromatography to give 80 mg (0.19 mmol) of the desired product.

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[0179] radioactive label Common methods: All reagents and solvents were purchased from ABX, Sigma Aldrich, Fluorochem, and VWR, unless otherwise stated, and used as received without further purification. Dry THF and DCM were obtained from an SG water solvent purification system, and dry dimethyl sulfoxide (DMSO), MeCN, pyridine, and methanol (MeOH) were purchased from commercial suppliers. Room temperature corresponds to a temperature interval of 18-21 °C. Reactions requiring anhydrous conditions were carried out under an inert atmosphere (nitrogen) using oven-dried glassware (152 °C). Thin-layer chromatography (TLC) was performed on silica-plated aluminum sheets (silica gel 60 F254) from Merck, and spots were visualized by ultraviolet light at 254 nm, by anisaldehyde, and / or by potassium permanganate staining.

[0180] Example 6 Symmetrical tetrazines 18 F radioactive label [ka] Scheme 17. Radiolabeling of symmetric tetrazines. I) [ 18 F]Bu4NF / Bu4NOMs PO4 3- ,t-BuOH / DMSO, 100°C, 5 min; ii) TFA, CH3CN, 80°C, 10 min.

[0181] Symmetric 18 F-labeled tetrazine [ 18 F]I was prepared as follows: Received from Cyclotron 18The [F] fluoride solution was passed through a preconditioned anion exchange resin (Sep-Pak Light QMA cartridge). The QMA was preconditioned by washing with 10 mL of 0.5 M K3PO4, followed by rinsing with 10 mL of HO. 18 F]F - was eluted from the QMA into a 4 mL v-shaped vial containing 1 mL of Bu4NOMs dissolved in MeOH. The eluate was dried under a stream of N2 at 100 °C for 5 min. Precursor XXXIV (9.3 μmol, 6 mg) was dissolved in 167 μL of DMSO and then diluted with 833 μL of tBuOH. The solution was dried [ 18 The [F] fluoride solution was added and reacted at 100 °C for 5 min. The reaction was cooled to 50 °C with air, after which 3 mL of H2O was added. The radiochemical conversion (RCC) measured by radio-HPLC after the first step was 54%. The crude mixture was applied to a Sep-pak+C18 solid phase extraction (SPE) cartridge, which was preconditioned by flushing with 10 mL of EtOH followed by 10 mL of H2O. The SPE was flushed with an additional 5 mL of H2O and dried with N2. The product was eluted from the SPE with 2 mL of MeCN into a 7 mL v-vial containing 600 μL of TFA. The mixture was reacted at 80 °C for 10 min. The [RCC] measured by radio-HPLC was 54%. 18 The RCC of F]I was 95% (Fig. 3 ).

[0182] Radio-HPLC was performed on a Luna 5 μm C18(2) column (150 × 4.6 mm) using a gradient of acetonitrile (CH3CN) in water with 0.1% TFA. Gradient conditions: 0 min - 0% CH3CN, 0-10 min - linear increase in CH3CN content to 100%, 10-12 min - 100% CH3CN, 12-13 min - linear decrease in CH3CN content to 0%, 13-15 min - 0% CH3CN, elution rate 2 mL / min.

[0183] Figure 3 shows the [ 18 Radio-HPLC of [F]I is shown.

[0184] Example 7 Asymmetric tetrazine18 F radioactive label Asymmetric synthesis of 18F-labeled tetraazine from the nosyl precursor XXXI via nucleophilic substitution as disclosed in Battisti, U.M.; Bratteby, K.; Jorgensen, J.T.; Hvass, L.; Shalgunov, V.; Mikula, H.; Kjaer, A.; Herth, M.M. Development of the First Aliphatic 18F-Labeled Tetrazine Suitable for Pretargeted PET Imaging-Expanding the Bioorthogonal Tool Box. J. Med. Chem. 2021, 64, 15297-15312. 18 F-labeled tetrazine [18F]XIII was prepared.

[0185] Example 8 Asymmetric tetrazine 18 F-radiolabel 18 F-labeled tetrazines were prepared using the general method described in Garcia-Vazquez et al. (doi:10.3390 / ph15020245) with minor modifications. [ka]

[0186] Scheme 18. Received from Cyclotron 18 The [F] fluoride solution was passed through a Sep-Pak Light QMA cartridge preconditioned with 10 mL of 0.5 M K3PO4. 18[F]F- was eluted from the QMA into a 4 mL v-shaped vial using a Bu4NOMs solution (20 mM in MeOH, 1 mL). The eluate was dried at 100 °C for 5 min under a stream of nitrogen or helium. After the MeOH was evaporated, acetonitrile (0.5 mL) was added to the same vial and evaporated under the same conditions to remove traces of water. Two additions of acetonitrile were performed. The nosyl precursor LIX (1.5 mg) was dissolved in anhydrous DMSO (0.2 mL), diluted with tBuOH (0.8 mL) and dried [ 18 [F] fluoride residue was added. After reacting at 100 °C for 5 min, the reaction was cooled to 80 °C with ambient air flow, diluted with water (2 mL) and purified by semi-preparative HPLC: Luna 5 μm C18(2) 100 Å, 250 mm × 10 mm column, isocratic elution with 66% acetonitrile in 20 mM citrate buffer pH 6.1, elution rate 6 mL / min. The product peak (retention time 10.2 min) was collected, diluted with water (50–100 mL) and passed through a Sep-pak Plus C18 Short solid phase extraction cartridge (Waters, USA), which was preconditioned by flushing with an EtOH / water mixture (1 / 1 v / v, 10 mL). The Sep-pak cartridge was then flushed with additional water (5 mL), sparged with nitrogen, and eluted with 1–2 mL of organic solvent (MeCN or EtOH), which was diluted with water to obtain the required concentration of organic solvent for the click / oxidation experiment.

[0187] [ 18 F]XX was obtained in a radiochemical yield of 39 ± 13% (n = 4) and with a radiochemical purity of >95%.

[0188] Figure 4 shows 18 The UV trace of [F]XX is shown.

[0189] Figure 5 shows 18 Radioactive trace of [F]XX is shown.

[0190] Figure 6 shows 18 UV and radioactive traces of [F]XX-analytical HPLC are shown.

[0191] HPLC conditions: Luna 5 μm C18(2) column (150 × 4.6 mm) eluted with a gradient of acetonitrile (CH3CN) in water with 0.1% TFA. Gradient conditions: 0-1 min - 25% CH3CN, 1-8 min - linear increase in CH3CN content to 95%, 8-9 min - 95% CH3CN, 9-9.5 min - linear decrease in CH3CN content to 25%, 9.5-10 min - 25% CH3CN, elution rate 1.5 mL / min. [ka]

[0192] Scheme 19. Received from Cyclotron 18 The [F] fluoride solution was passed through a Sep-Pak Light QMA cartridge preconditioned with 10 mL of 0.5 M K3PO4. 18 [F]F- was eluted from the QMA cartridge into a 4 mL v-shaped vial using a Bu4NOTf solution (20 mM in MeOH, 1 mL). The eluate was dried at 100 °C for 5 min under a stream of nitrogen or helium. After the MeOH was evaporated, acetonitrile (0.5 mL) was added to the same vial and evaporated under the same conditions to remove traces of water. The nosyl precursor XLVI (1.5 mg) was dissolved in anhydrous acetonitrile (0.2 mL), diluted with tBuOH (0.8 mL) and dried [ 18[F] fluoride residue was added. After reacting at 100 °C for 5 min, the reaction was cooled to 80 °C with ambient air flow, diluted with water (2 mL) and purified by semi-preparative HPLC: Luna 5 μm C18(2) 100 Å, 250 mm × 10 mm column, isocratic elution with 55% acetonitrile in 20 mM citrate buffer pH 6.1, elution rate 5 mL / min. The product peak (retention time 11.5 min) was collected, diluted with water (50–100 mL) and passed through a Sep-pak Plus C18 Short solid phase extraction cartridge (Waters, USA), which was preconditioned by flushing with an EtOH / water mixture (1 / 1 v / v, 10 mL). The Sep-pak cartridge was then flushed with additional water (5 mL), sparged with nitrogen, and eluted with 1–2 mL of organic solvent (MeCN or EtOH), which was diluted with water to obtain the required concentration of organic solvent for the click / oxidation experiment.

[0193] [ 18 F]X was obtained in a radiochemical yield of 11 ± 5% (n = 3) and a radiochemical purity of >95%.

[0194] Figure 7 shows 18 4 shows the UV trace of F]X-semi-preparative HPLC.

[0195] Figure 8 shows 18 Radioactive trace of F]X-semi-preparative HPLC.

[0196] Figure 9 shows 18 4 shows the UV and radioactive traces of [F]X-analytical HPLC.

[0197] HPLC conditions: Luna 5 μm C18(2) column (150 × 4.6 mm) eluted with a gradient of acetonitrile (CH3CN) in water with 0.1% TFA. Gradient conditions: 0-1 min - 15% CH3CN, 1-8 min - linear increase in CH3CN content to 85%, 8-9 min - 85% CH3CN, 9-9.5 min - linear decrease in CH3CN content to 15%, 9.5-10 min - 15% CH3CN, elution rate 1.5 mL / min. [ka]

[0198] Scheme 20. Received from Cyclotron 18 The aqueous solution of [F] fluoride was passed through a 45 mg PS anion exchange cartridge (Synthra) preconditioned with 5 mL of 0.5 M K3PO4. A solution of Bu4NOTf (20 mM in MeOH, 0.6 mL) was used to determine the 18 [F]F- was eluted from the PS cartridge into a 4 mL v-shaped vial. The eluate was dried at 100 °C for 5 min under a stream of nitrogen or helium. After the MeOH was evaporated, acetonitrile (0.5 mL) was added to the same vial and evaporated under the same conditions to remove traces of water. Two additions of acetonitrile were performed. The nosyl precursor XLVII (1.5 mg) was dissolved in anhydrous acetonitrile (0.3 mL), diluted with tBuOH (0.7 mL) and dried [ 18 [F] was added to the fluoride residue. After reacting at 100 °C for 5 min, the reaction was cooled to 80 °C with ambient air flow, diluted with water (2 mL) and purified by semi-preparative HPLC: Discovery HS F5 5 μm, 250 mm × 10 mm column, isocratic elution with 50% acetonitrile in 20 mM citrate buffer pH 6.1, elution rate 5 mL / min. The product peak (retention time 9.0 min) was collected, diluted with water (50–100 mL) and passed through a Sep-pak Plus C18 Short solid phase extraction cartridge (Waters, USA), which was preconditioned by flushing with an EtOH / water mixture (1 / 1 v / v, 10 mL). The Sep-pak cartridge was then flushed with additional water (5 mL), sparged with nitrogen and eluted with 1–2 mL of organic solvent (MeCN or EtOH) and diluted with water to obtain the required concentration of organic solvent for the click / oxidation experiment.

[0199] [ 18 F]XI was obtained in a radiochemical yield of 16 ± 10% ( n = 4) and with a radiochemical purity of >95%.

[0200] Figure 10 shows 18 F]XI UV trace-semi-preparative HPLC.

[0201] Figure 11 shows the 18 Radioactive trace of [F]XI-semi-preparative HPLC.

[0202] Figure 12 shows the 18 1 shows the UV and radioactive traces of [F]XI--analytical HPLC.

[0203] HPLC conditions: Luna 5 μm C18(2) column (150 × 4.6 mm) eluted with a gradient of acetonitrile (CH3CN) in water with 0.1% TFA. Gradient conditions: 0-1 min - 25% CH3CN, 1-8 min - linear increase in CH3CN content to 60%, 8-9 min - 60% CH3CN, 9-9.5 min - linear decrease in CH3CN content to 25%, 9.5-10 min - 25% CH3CN, elution rate 1.5 mL / min.

[0204] Example 9 Asymmetric tetrazine 125 I-Radiolabel Battisti Umberto, M.; Herth Matthias, M.; Kjaer, A.; Garcia, R. NUCLIDE LABELLED H-TETRAZINES AND USE THEREOF FOR PET AND SPECT PRETARGETED IMAGING AND RADIONUCLIDE THERAPY. WO 2021 / 228992 A1, 2021 / 05 / 12, 2021. 125 I-labeled tetrazine [ 125 I]XVII was prepared.

[0205] Figure 13 shows the rough 125 I]XVII is shown as a radio-HPLC product.

[0206] Example 10 Asymmetric tetrazine 211 AT-radiolabeled Battisti Umberto, M.; Herth Matthias, M.; Kjaer, A.; Garcia, R. NUCLIDE LABELLED H-TETRAZINES AND USE THEREOF FOR PET AND SPECT PRETARGETED IMAGING AND RADIONUCLIDE THERAPY. WO 2021 / 228992 A1, 2021 / 05 / 12, 2021. 211 At-labeled tetrazine [ 211 At]XIV was prepared.

[0207] Figure 14 shows the 211 4 shows the UV and radioactive traces of At]XIV-semi-preparative HPLC.

[0208] FIG. 15 shows the purified 211 4 shows the radioactive trace-analysis HPLC of At]XIV.

[0209] Example 11 Click experiments with radiolabeled tetrazines In click experiments, excess TCO is added to the non-carrier-added radioactively labeled 18 The half-life of a bimolecular reaction is determined by the concentration of excess reagent. Thus, in a click experiment, the observed conversion time was determined by the TCO concentration until all of the 18F-Tz was consumed.

[0210] TCO stock solutions (20-2000 μM) were prepared as follows: 18 The TCO stock was then rapidly mixed with the formulated 18F-Tz solution in a volume / volume ratio of 1:3 to 1:9 to achieve the desired TCO concentration (5-200 µM). The total volume of the mixture was 100-2000 µL. The mixture was left at room temperature for a period of time, and then a sample was injected into a radio-HPLC to evaluate the click conversion. The results are shown in Figure 16.

[0211] Example 12 Screening of oxidants for the oxidation of dihydropyridazine to pyridazine, producing a single final product The top of Figure 17 and Figure 18 show the reaction between tetrazine and TCO dissolved in 1:1 HO / EtOH (% v / v). The cycloaddition is completed within 5 min to give several isomers. An oxidizing agent is then added to give the final single isomeric product. Each oxidizing agent (5 equivalents) was added to the mixture and the reaction was analyzed by HPLC-MS after 60 min. These screening tests surprisingly showed that not all oxidizing agents could be applied to give a single isomeric tetrazine-TCO pyridazine. For all quinone-based oxidizing agents, the corresponding pyridine is formed. No inorganic-based example is able to convert the click product (dihydropyridazine) to the corresponding pyridine within 60 min. Figure 19 shows the HPLC analysis after oxidation of the tested tetrazine-TCO pyridazines.

[0212] Oxidation of radiolabeled compounds The upper part of Figure 20 shows the 1:1 H2O / EtOH (%v / v) solution. 18 The reaction between F-tetrazine and TCO is shown. As described in the Click section 18 The result was obtained by mixing F-Tz and TCO solutions. 18 Crude solutions of F-click products (50–100 μL) were used for oxidation experiments. The oxidation protocol consisted of the introduction of an oxidizing agent. After the oxidation mixture was left in a sealed vial at room temperature for the desired time, a sample was removed and analyzed by HPLC.

[0213] Example 13 Compatibility of targeting vectors with oxidative conditions: To test whether conditions leading to oxidation of the click product would not result in degradation of typical targeting vectors, we subjected a series of vectors relevant for the development of theranostic radiopharmaceuticals to oxidative conditions previously shown to result in efficient conversion of dihydropyridazines to the monoproduct pyridazines.

[0214] The structures of the vectors tested are shown in FIG.

[0215] Vector oxidation test procedure: A solution of targeted vector (70 μM) and oxidant (350 μM, 5 equiv.) in an EtOH / water mixture (89-94% EtOH, v / v) was stirred at 25 °C for 10 min and subsequently analyzed by analytical HPLC and LC / ESI-MS. None of the tested compounds showed oxidation and / or decomposition, meaning that they do not react with the oxidant. The results are shown in Figure 22 and indicate that these vectors are compatible with the oxidants tested.

[0216] Example 14 Measurement of second-order rate constants Second-order rate constants of all click reactions carried out during the previous examples were measured by stopped-flow spectroscopy at 25 °C in phosphate-buffered saline (PBS) according to the method described by Battisti et al. J. Med. Chem. 2021, 64, 20, 15297-15312 (see page 15310 for experimental details and influencing factors). Briefly, stopped-flow measurements were performed using an SX20-LED stopped-flow spectrophotometer (Applied Photophysics) equipped with a 535 nm LED (path length 10 mm, also full width at half maximum 34 nm) to monitor the characteristic tetrazine visible light absorbance (520-540 nm). Reagent syringes were filled with a solution of axial-TCO-PEG4 and the instrument was primed. Subsequent data were collected in triplicate for each tetrazine. Reactions were carried out at 25 °C in PBS and automatically recorded upon acquisition. Data sets were analyzed by fitting exponential decays using Prism 6 (GraphPad) to calculate the observed pseudo-first-order rate constants, which were converted to second-order rate constants by dividing by the concentration of excess TCO compound.

[0217] 500 M in phosphate buffered saline at 25°C -1 s -1Only reactions that exhibited a minimum second-order rate constant of 0.1 to 0.25 were considered suitable for obtaining sufficient kinetics; therefore, reactions with lower reaction kinetics were ignored for the purposes of the method according to the invention.

Claims

1. 1. A method for obtaining a labeled, single isomeric chemical moiety targeting vector, comprising: a) labeling a first chemical moiety having inverse electron demand Diels-Alder cycloaddition reactivity and conjugated to a drug, imaging agent, or therapeutic agent with a labeling agent, said first chemical moiety being selected from the group consisting of symmetrically substituted dienes, unsymmetrically substituted dienes, and isomer-free dienophiles, at least one of whose planes of symmetry passes through the nitrogen-nitrogen bond of at least one tetrazine ring; b) ligating the labeled first chemical moiety obtained in step a) with a second chemical moiety having complementary inverse electron demand Diels-Alder cycloaddition reactivity and conjugated to a targeting vector, wherein the second chemical moiety is selected from the group consisting of symmetrically substituted dienes, unsymmetrically substituted dienes, and isomer-free dienophiles, in which at least one of the planes of symmetry passes through the nitrogen-nitrogen bond of at least one tetrazine ring; wherein the kinetics of the inverse electron demand Diels-Alder cycloaddition between the first chemical moiety and the second chemical moiety are determined by stopped-flow spectrophotometry at 500 M in phosphate buffered saline (PBS) at 25° C. －１ s －１ has a minimum rate constant of However, the labeling agent in step a) ９４ Tc, ９９ｍ Tc, ２１１ At, ２２３ Ra, or ２２５ When Ac, the labeling agent is conjugated to an asymmetrically substituted diene; and c) 1 to 100 equivalents of chloranil, fluoranil, DDQ or NaNO ２ oxidizing the ligated, labeled targeting vector resulting from step b) by adding at a temperature ranging from 15° C. to 50° C. for up to 60 minutes.

2. 2. The method of claim 1, wherein the symmetrically substituted diene is a tetrazine, with at least one of the planes of symmetry passing through the nitrogen-nitrogen bond of at least one tetrazine ring, and the dienophile is a trans-cycloheptene (TCH), trans-cyclooctene (TCO) or trans-cyclononene (TCN) derivative.

3. 3. The method according to claim 1 or 2, wherein the labeling agent in step a) is a radionuclide or a stable isotope of the corresponding element.

4. The labeling agent is １ H, ２ H, ３ H, １１ C, １２ C, １３ C, １４ C, １３ N, １４ N, １５ N, １８ F, １９ F, １２３ I, １２４ I, １２５ I, １２７ I, １３１ I, １５ O, １６ O, １７ O, １８ O, ４３ Sc, ４４ Sc, ４５ Sc, ４５ Ti, ４６ Ti, ４７ Ti, ４８ Ti, ４９ Ti, ５０ Ti, ５５ Co, ５８ mCo, ５９ Co, ６０ Cu, ６１ Cu, ６３ Cu, ６４ Cu, ６５ Cu, ６７ Cu, ６７ Ga, ６８ Ga, ６９ Ga, ７１ Ga, ７６ Br, ７７ Br, ７９ Br, ８０ mBr, ８１ Br, ７２ As, ７５ As, ８６ Y, ８９ Y, ９０ Y, ８９ Zr, ９０ Zr, ９１ Zr, ９２ Zr, ９４ Zr, １４９ Tb, １５２ Tb, １５９ Tb, １６１ Tb, １１１ In, １１３ 9n、 １１４ mｩn、 １１５ mｩn、 １７５ Lu、 １７７ Lu、 １８５ e、 １８６ e、 １８８ e、 ２０１ 4l、 ２０３ 4l、 ２０５ 4l、 ２０６ Pb、 ２０７ Pb, ２０８ Pb, ２１２ Pb、 ２０９ Bi、 ２１２ Bi、 ２１３ Bi、 ３１ P、 ３２ P、 ３３ P、 ３２ 、 ３５ 、 ４５ ｳc、 ４７ ｳc、 ８４ 3r、 ８６ 3r、 ８７ 3r、 ８８ 3r、 ８９ 3r、 １６５ ｨo、 １６６ ｨo、 １５６ Dy、 １５８ Dy、 １６０ Dy、 １６１ Dy、 １６２ Dy、 １６３ Dy、 １６４ Dy、 １６５ Dy、 ２２７ Th、 ２３２ Th、 ５１ Cr、 ５２ Cr、 ５３ Cr、 ５４ Cr、 ７３ ｳe、 ７４ ｳe、 ７５ ｳe、 ７６ ｳe、 ７７ ｳe、 ７８ ｳe、 ８０ ｳe、 ８２ ｳe、 ９４ Tc、 ９９ｍ Tc、 １０３ h, １０３ mｲh、 １１９ ｳb、 １２１ ｳb、 １２３ ｳb、 １３５ La、 １３８ La, １３９ La, １６２ Er, １６４ Er, １６５ Er, １６６ Er, １６７ Er, １６８ Er, １７０ Er, １９３ mPt, １９５ mPt, １９２ Pt, １９４ Pt, １９５ Pt, １９６ Pt, １９８ Pt, ２１１ At, ２２３ Ra, ２２５ The method of claim 3, wherein said at least one aryl group is selected from the group consisting of aryl, ... and aryl.

5. The method according to any one of claims 1 to 4, wherein the targeting vector is an antibody, a nanobody, a polymer, a nanomedicine, a cell, a protein, a peptide or a small molecule.

6. The method according to any one of claims 1 to 5, wherein the oxidizing agent is supported on a solid phase.

7. The diene is a symmetrical tetrazine of formula Tz1: 【Chemistry 1】 In the formula, R and R １ teeth, 【Chemistry 2】 where the curved line symbol indicates the link to the tetrazine; ２ is -H or (i) an isotopic labeling agent attached directly to the aromatic ring, or (ii) an isotopic labeling agent attached to the aromatic ring via a linker, the linker being -(CH ２ ) ｎ , LO(CH ２ ) ｎ , -LNH(CH ２ ) ｎ , -LCONH(CH ２ ) ｎ , -LNHCO(CH ２ ) ｎ wherein L is selected from the group consisting of -(CH ２ ) ｍ Or - (CH ２ CH ２ O) ｍ wherein n and m are independently selected from 1 to 25; or (iii) 1,4,7,10-tetraazacyclododecane-N,N',N',N''-tetraacetic acid (DOTA), N,N'-bis(2-hydroxy-5-(carboxyethyl)benzyl)ethylenediamine-N,N'-diacetic acid (HBED-CC), 14,7-triazacyclononane-1,4,7-triacetic acid (NOTA), 2-(4,7-bis(carboxymethyl)-1,4,7-triazonan-1-yl)pentane diacid (NODAGA), 2-(4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl)pentanedioic acid (DOTAGA), 14,7-triazacyclononanephosphinic acid (TRAP), 14,7-triazacyclononane-1-methyl(2-carboxyethyl)phosphinic acid-4,7-bis(methyl(2-hydroxymethyl)phosphinic acid (NOPO), 3,6,9,15-tetraazabicyclopentadec-1-yl (15-pentadecyl) phosphinic acid ... ,11,13-triene-3,6,9-triacetic acid (PCTA), N'-(5-acetyl(hydroxy)aminopentyl-N-(5-(4-(5-aminopentyl)(hydroxy)amino-4-oxobutanoyl)amino)pentyl-N-hydroxysuccinamide (DFO), diethylenetriaminepentaacetic acid (DTPA), trans-cyclohexyl-diethylenetriaminepentaacetic acid (CHX-DTPA), 1-oxa-4,7,10-triazacyclododecane-4,7,10-triacetic acid isotopic labeling agent chelated through a chelator selected from acid (OXO-Do3A), p-isothiocyanatobenzyl-DTPA (SCN-BZ-DTPA), 1-(p-isothiocyanatobenzyl)-3-methyl-DTPA (1B3M), 2-(p-isothiocyanatobenzyl)-4-methyl-DTPA (1M3B), and 1-(2)-methyl-4-isocyanatobenzyl-DTPA (MX-DTPA) attached to the aromatic ring via a linker, the linker being -(CH ２ ) ｎ , -LO(CH ２ ) ｎ , -LNH(CH ２ ) ｎ ,-LCONH(CH ２ ) ｎ , -LNHCO(CH ２ ) ｎ wherein L is selected from the group consisting of -(CH ２ ) ｍ Or -- (CH ２ CH ２ O) ｍ where n and m are independently selected from 1 to 25; In the formula, R ２ is either (i) or (ii), the isotopic labeling agent is １ 8、 ２ 8、 ３ 8、 １１ 3、 １２ 3、 １３ 3、 １４ 3、 １３ 8、 １４ 8、 １５ 8、 １８ 6、 １９ 6、 １２３ 9、 １２４ 9、 １２５ 9、 １２７ 9、 １３１ 9、 ２１１ At、 １５ 9、 １６ 9、 １７ 9、 １８ 9、 ４３ ｳc、 ４４ ｳc、 ４５ ｳc、 ４５ 4i、 ４６ 4i、 ４７ 4i、 ４８ 4i、 ４９ 4i、 ５０ 4i、 ５５ Co、 ５８ｍ Co、 ５９ Co、 ６０ Cu、 ６１ Cu、 ６３ Cu、 ６４ Cu、 ６５ Cu、 ６７ Cu、 ６７ Ga、 ６８ Ga、 ６９ Ga、 ７１ Ga、 ７６ 、、 ７７ 、、 ７９ 、、 ８０ｍ 、、 ８１ 、、 ７２ As、 ７５ As、 ８６ 9、 ８９ 9、 ９０ 9、 ８９ Zr、 ９０ Zr、 ９１ Zr、 ９２ Zr、 ９４ Zr、 １４９ 4b、 １５２ 4b、 １５９ 4b、 １６１ 4b、 １１１ 9n、 １１３ 9n、 １１４ mｩn、 １１５ mｩn、 １７５ Lu、 １７７ Lu、 １８５ e、 １８６ e、 １８８ e、 ２０１ 4l、 ２０３ 4l、 ２０５ 4l、 ２０６ Pb、 ２０７ Pb, ２０８ Pb, ２１２ Pb、 ２０９ Bi、 ２１２ Bi、 ２１３ Bi、 ３１ P、 ３２ P、 ３３ P、 ３２ 、 ３５ 、 ４５ ｳc、 ４７ ｳc、 ８４ 3r、 ８６ 3r、 ８７ 3r、 ８８ 3r、 ８９ 3r、 １６５ ｨo、 １６６ ｨo、 １５６ Dy、 １５８ Dy、 １６０ Dy、 １６１ Dy、 １６２ Dy、 １６３ Dy、 １６４ Dy、 １６５ Dy、 ２２７ Th、 ２３２ Th、 ５１ Cr、 ５２ Cr、 ５３ Cr、 ５４ Cr、 ７３ ｳe、 ７４ ｳe、 ７５ ｳe、 ７６ ｳe、 ７７ ｳe、 ７８ ｳe、 ８０ ｳe、 ８２ ｳe、 ９４ Tc、 ９９ｍ Tc、 １０３ h, １０３ mｲh、 １１９ ｳb、 １２１ ｳb、 １２３ ｳb、 １３５ La、 １３８ La, １３９ La, １６２ Er, １６４ Er, １６５ Er, １６６ Er, １６７ Er, １６８ Er, １７０ Er, １９３ mPt, １９５ mPt, １９２ Pt, １９４ Pt, １９５ Pt, １９６ Pt, １９８ Pt; wherein X and Y are independently selected from -CH and -N-; In the formula, R ３ is H, or a hydroxy group, a sulfonamide, a carboxyl group, a sulfonyl group, an amine, a substituted amine having 1 to 5 polyethylene glycol units, -(O-CH ２ -CH ２ ) ｎ -OCH ２ -COOH, n being selected from 1 to 5; or one or more substituents selected from methyl, ethyl, propyl, optionally substituted heteroaryl, and optionally substituted arylalkyl; with respect to the substituted amine, optionally substituted means halogen, hydroxyl, sulfonamide, carboxyl, sulfonyl, amine, (C1-C10) alkyl, (C2-C10) alkenyl, (C2-C10) alkynyl, (C1-C10) alkylene, (C1-C10) alkoxy, (C2-C10) dialkylamino, (C1-C10) alkylthio, (C2-C10) heteroalkyl, (C2-C10) heteroalkylene, (C3-30) aryl, (C4-30) aryl, (C5-30) aryl, (C6-30) aryl, (C7-30) aryl, (C8-30) aryl, (C9-30) aryl, (C10) aryl, (C11-30) aryl, (C12-30) aryl, (C13-30) aryl, (C14-30) aryl, (C15-30) aryl, (C16-30) aryl, (C17-30) aryl, (C18-30) aryl, (C19-30) aryl, (C20-30) aryl, (C21-30) aryl, (C22-30) aryl, (C23-30) aryl, (C24-30) aryl, (C25-30) aryl, (C26-30) aryl, (C27-30) aryl, (C28-30) aryl, (C29-30) aryl, (C30) aryl, (C31-30) aryl, (C32-30) aryl, (C33-30) aryl, C10)cycloalkyl, (C3-C10)heterocycloalkyl, (C3-10)cycloalkylene, (C3-C10)heterocycloalkylene, (C1-C10)haloalkyl, (C1-C10)perhaloalkyl, (C2-C10)-alkenyloxy, (C3-C10)-alkynyloxy, aryloxy, arylalkyloxy, heteroaryloxy, heteroarylalkyloxy, (C1-C6)alkyloxy-(C1-C4)alkyl, optionally substituted aryl, optionally substituted heteroaryl, and optionally substituted arylalkyl; optionally substituted means halogen, hydroxy group, sulfonamide, carboxyl group, sulfonyl group, amine, substituted amine having 1 to 5 polyethylene glycol units, -(O-CH ２ -CH ２ ) ｎ -OCH ２ -COOH, n being selected from 1 to 5; or H, methyl, ethyl, propyl, optionally substituted heteroaryl, and optionally substituted arylalkyl; optionally substituted in connection with said substituted amine means one or more substituents selected from halogen, hydroxy, sulfonamide, carboxyl, sulfonyl, and amine; In the formula, R and R １ are the same or differ only in the isotope number of the labeling agents.

8. The symmetric tetrazine is 【Chemistry 3】 The method of claim 7, wherein the compound is selected from the group consisting of

9. the diene is an asymmetric tetrazine of formula Tz2, 【Chemistry 4】 In the formula, R ４ is —H, or (i) an isotopic labeling agent attached directly to the aromatic ring, or (ii) an isotopic labeling agent attached to said aromatic ring via a linker, said linker being —(CH ２ ) ｎ , -LO(CH ２ ) ｎ , -LNH(CH ２ ) ｎ , -LCONH(CH ２ ) ｎ , -LNHCO(CH ２ ) ｎ wherein L is selected from the group consisting of -(CH ２ ) ｍ Or - (CH ２ CH ２ O) ｍ wherein n and m are independently selected from 1 to 25; or (iii) 1,4,7,10-tetraazacyclododecane-N,N',N',N''-tetraacetic acid (DOTA), N,N'-bis(2-hydroxy-5-(carboxyethyl)benzyl)ethylenediamine-N,N'-diacetic acid (HBED-CC), 14,7-triazacyclononane-1,4,7-triacetic acid (NOTA), 2-(4,7-bis(carboxymethyl)-1,4,7-triazonan-1-yl)pentaacetate (CH2CO ... pentane diacid (NODAGA), 2-(4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl)pentanedioic acid (DOTAGA), 14,7-triazacyclononanephosphinic acid (TRAP), 14,7-triazacyclononane-1-methyl(2-carboxyethyl)phosphinic acid-4,7-bis(methyl(2-hydroxymethyl)phosphinic acid (NOPO), 3,6,9,15-tetraazabicyclopentadecane-1-yl (15-tetraazabicyclopentadecane-1-yl)pentanedioic acid ... ,11,13-triene-3,6,9-triacetic acid (PCTA), N'-(5-acetyl(hydroxy)aminopentyl-N-(5-(4-(5-aminopentyl)(hydroxy)amino-4-oxobutanoyl)amino)pentyl-N-hydroxysuccinamide (DFO), diethylenetriaminepentaacetic acid (DTPA), trans-cyclohexyl-diethylenetriaminepentaacetic acid (CHX-DTPA), 1-oxa-4,7,10-triazacyclododecane-4,7,10-triacetic acid isotopic labeling agent chelated through a chelator selected from -(CH3)-, -(CH4)-, -(CH5)-, -(CH6)-, -(CH7)-, -(CH8)-, -(CH9)-, -(CH10)-, -(CH11)-, -(CH12)-, -(CH13)-, -(CH14)-, -(CH2)-, -(CH3)-, -(CH4)-, -(CH5)-, -(CH6)-, -(CH15)-, -(CH16)-, -(CH2)-, -(CH3)-, -(CH4)-, -(CH15)-, -(CH16)-, -(CH17)-, -(CH2)-, -(CH3)-, -(CH4)-, -(CH18)-, -(CH19)-, -(CH2)-, -(CH2)-, -(CH3)-, -(CH4)-, -(CH10)-, -(CH11)-, -(CH2)-, -(CH12)-, -(CH13)-, -(CH14)-, -(CH2)-, -(CH15)-, -(CH13)-, -(CH14)-, -(CH15)-, -(CH15)-, -(CH16)-, -(CH15)-, -(CH16)-, -(CH17)-, ２ ) ｎ , -LO(CH ２ ) ｎ , -LNH(CH ２ ) ｎ , -LCONH(CH ２ ) ｎ , -LNHCO(CH ２ ) ｎ wherein L is selected from the group consisting of -(CH ２ ) ｍ Or - (CH ２ CH ２ O) ｍ where n and m are independently selected from 1 to 25; ４ is (i), (ii) or (iii), the isotopic labeling agent is １ H. ２ H. ３ H. １１ C. １２ C. １３ C. １４ C. １３ N. １４ N. １５ N. １８ F. １９ F. １２３ I, １２４ I, １２５ I, １２７ I, １３１ I, ２１１ At, １５ O. １６ O. １７ O. １８ O. ４３ Sc, ４４ Sc, ４５ Sc, ４５ Ti, ４６ Ti, ４７ Ti, ４８ Ti, ４９ Ti, ５０ Ti, ５５ Co, ５８ mCo, ５９ Co, ６０ Cu, ６１ Cu, ６３ Cu, ６４ Cu, ６５ Cu, ６７ Cu, ６７ G.A. ６８ G.A. ６９ G.A. ７１ G.A. ７６ B, ７７ B, ７９ B, ８０ mBr, ８１ B, ７２ As, ７５ 1.、 ８６ 、 ８９ 、 ９０ 、 ８９ 、 ９０ 、 ９１ 、 ９２ 、 ９４ 、 １４９ (b、) １５２ (b、) １５９ (b、) １６１ (b、) １１１ .、 １１３ .、 １１４ m.、 １１５ m.、 １７５ u、 １７７ u、 １８５ 2)、 １８６ 2)、 １８８ 2)、 ２０１ .、 ２０３ .、 ２０５ .、 ２０６ pi.、 ２０７ pi. ２０８ pi. ２１２ pi.、 ２０９ 、、 ２１２ 、、 ２１３ 、、 ３１ 0、 ３２ 0、 ３３ 0、 ３２ 3、 ３５ 3、 ４５ 3、 ４７ 3、 ８４ 32、 ８６ 32、 ８７ 32、 ８８ 32、 ８９ 32、 １６５ 、 １６６ 、 １５６ y、 １５８ y、 １６０ y、 １６１ y、 １６２ y、 １６３ y、 １６４ y、 １６５ y、 ２２７ ()、 ２３２ ()、 ５１ 、 ５２ 、 ５３ 、 ５４ 、 ７３ 3、 ７４ 3、 ７５ 3、 ７６ Se ７７ Se ７８ Se ８０ Se ８２ Se ９４ Tc ９９ｍ Tc １０３ Rh １０３ mRh １１９ Sb １２１ Sb １２３ Sb １３５ La １３８ La １３９ La １６２ Er １６４ Er １６５ Er １６６ Er １６７ Er １６８ Er １７０ Er １９３ mPt １９５ mPt １９２ Pt １９４ Pt １９５ Pt １９６ Pt １９８ Pt ２１１ At ２２３ Ra ２２５ selected from the group consisting of Ac wherein X and Y are independently selected from -CH- and -N-; In the formula, R ６ is H, or 【Chemistry 5】 where Q and Z are independently selected from -CH- and -N-, and the curved symbol indicates a bond to the tetrazine; In the formula, R ５ and R ７ is H or a hydroxy group, a sulfonamide, a carboxyl group, a sulfonyl group, an amine, a substituted amine having 1 to 5 polyethylene glycol units, -(O-CH ２ -CH ２ )-OCH ２ -COOH, n being selected from 1 to 5; or methyl, ethyl, propyl, optionally substituted heteroaryl, and optionally substituted arylalkyl; with respect to said substituted amine, optionally substituted means halogen, hydroxyl, sulfonamide, carboxyl, sulfonyl, amine, (C1-C10) alkyl, (C2-C10) alkenyl, (C2-C10) alkynyl, (C1-C10) alkylene, (C1-C10) alkoxy, (C2-C10) dialkylamino, (C1-C10) alkylthio, (C2-C10) heteroalkyl, (C2-C10) heteroalkylene, (C3-30) aryl, (C4-30) arylalkyl, (C5-30) arylalkyl, (C6-30) arylalkyl, (C7-30) arylalkyl, (C8-30) arylalkyl, (C9-30) arylalkyl, (C10) arylalkyl, (C11-30) arylalkyl, (C12-30) arylalkyl, (C13-30) arylalkyl, (C14-30) arylalkyl, (C15-30) arylalkyl, (C16-30) arylalkyl, (C17-30) arylalkyl, (C18-30) arylalkyl, (C19-30) arylalkyl, (C20-30) arylalkyl, (C21-30) arylalkyl, (C22-30) arylalkyl, (C23-30) arylalkyl, (C24-30) arylalkyl, (C25-30) arylalkyl, (C26-30) arylalkyl, (C27-30) arylalkyl, (C28-30) arylalkyl, (C29-30) arylalkyl, (C30-30) arylalkyl, (C31-30) aryl C10)cycloalkyl, (C3-C10)heterocycloalkyl, (C3-10)cycloalkylene, (C3-C10)heterocycloalkylene, (C1-C10)haloalkyl, (C1-C10)perhaloalkyl, (C2-C10)-alkenyloxy, (C3-C10)-alkynyloxy, aryloxy, arylalkyloxy, heteroaryloxy, heteroarylalkyloxy, (C1-C6)alkyloxy-(C1-C4)alkyl, optionally substituted aryl, optionally substituted heteroaryl, and optionally substituted arylalkyl; optionally substituted means one or more substituents selected from halogen, hydroxy group, sulfonamide, carboxyl group, sulfonyl group, amine, substituted amine having 1 to 5 polyethylene glycol units, -(O-CH ２ -CH ２ )-OCH ２ 10. The method of claim 1, wherein optionally substituted means one or more substituents selected from -COOH, where n is selected from 1 to 5; or H, methyl, ethyl, propyl, optionally substituted heteroaryl, and optionally substituted arylalkyl; optionally substituted in relation to said substituted amine means one or more substituents selected from halogen, hydroxyl, sulfonamide, carboxyl, sulfonyl, and amine.

10. The tetrazine is 【Chemistry 6】 The method of claim 10, wherein the asymmetric tetrazine is selected from the group consisting of:

11. The isomer-free dienophile is 【Chemistry 7】 a trans-cycloheptene (TCH), trans-cyclooctene (TCO) or trans-cyclononene (TCN) derivative selected from In the formula, X is N, NO or CR ８ and Y is N, NO or CR ８ and R ８ is -H, -F, -OH, -NH ２ , -COOH, -COOCH ３ , C.F. ３ , -Cl, -CONH ２ , CONHC ３ , -CON(CH ３ ) ２ , -CH ２ CH ２ OH, -CH ２ CH ２ N.H. ２ , -CHCH ２ N (CH ３ ) ２ is selected from the group consisting of The linker is -(CH ２ ) ｎ -(CH ２ ) ｎ NH, (CH ２ ) ｎ CO, (CH ２ ) ｎ O, (CH ２ CH ２ O) ｎ , (CH ２ CH ２ O) ｎ CH ２ CH ２ NH, (CH ２ CH ２ O) ｎ CH ２ CH ２ CO, -CO(CH) ２ -CO(CH ２ ) ｎ NH, CO(CH ２ ) ｎ CO, CO(CH ２ ) ｎ O, CO(CH ２ CH ２ O) ｎ CO(CH ２ CH ２ O) ｎ CH ２ CH ２ NH, CO(CH ２ CH ２ O) ｎ CH ２ CH ２ CO, COO(CH) ２ -COO(CH ２ ) ｎ NH, COO(CH ２ ) ｎ CO, COO(CH ２ ) ｎ O, COO(CH ２ CH2O) ｎ COO(CH ２ CH ２ O) ｎ CH ２ CH ２ NH, COO(CH ２ CH ２ O) ｎ CH ２ CH ２ CO, CONH(CH) ２ -CONH(CH ２ ) ｎ H. ２ ) ｎ P.S. ２ ) ｎ O, CONH(CH ２ CH ２ O) ｎ I'm sorry ２ CH ２ O) ｎ CH ２ CH ２ H. ２ CH ２ O) ｎ CH ２ CH ２ CO、-CONHPhCO、-COOPhCO 、-COPhCO、CONHCHMCO、(CH ２ ) ｎ NHCHMCO、(CH ２ ) ｎ OCONHCHMCO、(CH ２ ) ｎ NHCHMCO、(CH ２ ) ｎ NHCOCHMNH、(CH ２ )OCOCHMNH、(CH ２ CH ２ O) ｎ CH ２ CH ２ NHCHMCO、(CH ２ CH ２ O) ｎ CH ２ CH ２ CONHCHMCO、(CH ２ CH ２ O) ｎ CH ２ CH ２ NHCHMCO、(CH ２ CH ２ O) ｎ CH ２ CH ２ NHCOCHMNH、(CH ２ CH ２ O) ｎ COCHMNH, where n is 0 to 25, where M is H, CH ３ , C.H. ２ S.H., C.H. ２ COOH, CH ２ CH ２ COOH, CH ２ C ６ H ５ , C.H. ２ C ３ H ３ N ２ , CH(CH ３ ) CH ２ CH ３ , (CH ２ ) ４ N.H. ２ , C.H. ２ CH (CH ３ ) ２ , C.H. ２ CH ２ SCH ３ , C.H. ２ CONH ２ , (CH ２ ) ４ NHCOC ４ H ５ NCH ３ , C.H. ２ CH ２ CH ２ , C.H. ２ CH ２ CONH ２ , (CH ２ ) ３ NH-C(NH)NH ２ , C.H. ２ OH, CH(OH)CH ３ , C.H. ２ SeH, CH(CH ３ ) ２ , C.H. ２ C ８ H ６ N, C.H. ２ C ６ H ４ the side chain being selected from the group consisting of the side chains of natural amino acids which are OH; 2. The method of claim 1, wherein the targeting vector is an antibody, a nanobody, a polymer, a nanomedicine, a cell, a protein, a peptide or a small molecule.

12. The diene is a symmetrically substituted diene, and at least one of the planes of symmetry is 【Chemistry 8-1】 【Chemistry 8-2】 wherein X is CH or N; 2. The method of claim 1, wherein the nitrogen-nitrogen bond of at least one tetrazine ring is passed through the nitrogen-nitrogen bond of at least one tetrazine ring obtained from a precursor selected from:

13. The diene is 【Chemistry 9】 The method of claim 1, wherein the asymmetrically substituted diene is obtained from a precursor selected from:

14. The isomer-free dienophile targeting vector is 【Chemistry 10】 from a precursor selected from The method of claim 1 , wherein the targeting vector is an antibody, a nanobody, a polymer, a nanomedicine, a cell, a protein, a peptide or a small molecule.

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