Methods for providing labeled single isomeric chemical entity targeting vectors based on use of isomer-free dienophiles

Abstract

The present invention relates to a method for providing a single isomer chemical entity targeting vector suitable for providing a label of the targeting vector. The method applies a specific combination between dienes and dienophiles with complementary reverse electron demand Diels-Alder cycloaddition reactivity that will form a single isomer form of the compound after ligation and then oxidation. The labeled single isomer chemical entity targeting vectors are useful in therapy, radiotherapy, therapeutic diagnosis, diagnosis, and imaging. The methods employ click chemistry in which one chemical entity conjugated to the label is clicked together with a second chemical entity having complementary anti-electron demand Diels-Alder cycloaddition reactivity that is conjugated to a targeting carrier and then rapidly oxidized to form a single isomeride compound.

Description

Technical Field

[0001] The present invention relates to a method for providing a labeled single isomer chemical entity targeting vector, the obtained targeting vector and the use of the targeting vector. The labeled single isomer chemical entity targeting vector can be used for treatment, such as radiotherapy, diagnosis, imaging and other photochemical methods. Background Art

[0002] Labeled targeting vectors based on click chemistry between dienes and dienophiles can be used for both imaging purposes, such as diagnostic and other photochemical imaging methods, and therapeutic purposes. For example, such targeting vectors have been labeled with a radioactive label and can be applied for diagnostic and / or therapeutic purposes. The specific use depends on the identity of the radioactive label used, as different radionuclides are used for different purposes. In addition, the specific use depends on the specific target to which the vector is directed. Several combinations of radioactive labels and vectors are currently used for diagnostic, therapeutic, diagnostic and imaging purposes. There are different chemical entities that connect the radioactive labeled entity to the targeting entity, and the present invention is based on click chemistry, in which the diene and the corresponding dienophile are connected, thereby bridging the radioactive label and the targeting entity.

[0003] The term "click chemistry" refers to a class of reactions that are rapid, simple to use, versatile, chemoselective, and high product yielding. These reactions have been widely used in various research fields, including materials science, polymer chemistry, and pharmaceutical science. Radiochemistry is one of the fields that shows the real potential of click chemistry, for example, as disclosed by Zeng et al. in Journal of Nuclear Medicine, 54, 829-832, 2013. Basically, the selectivity, ease of use, rapidity, and modularity of click ligation make it almost perfect for the construction of radiotracers, a process that usually involves working with biomolecules under aqueous conditions with fast-decaying radioisotopes. Among the different click chemistries, one of the most suitable and used 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 usually trans-cyclooctene (TCO). The reaction is initiated by a reverse electron demand Diels-Alder reaction followed by N 2 This is in contrast to other known click reactions, such as the Staudinger ligation (0.003 M -1 s -1 ) and strain-promoted azide-alkyne cycloaddition (SPAAC) (0.1 M -1 s -1). Therefore, due to its specificity, Tz ligation is very suitable for synthon-based labeling. A major disadvantage of conventional ligation of Tz to TCO is that the ligation occurs without any regiospecificity. In addition, conventional ligation of Tz to TCO produces several tautomeric forms. This means that conventional ligation of Tz to TCO typically produces a complex reaction mixture of at least sixteen unique isomeric products (Scheme 1). This mixture of isomeric products cannot be used directly for pharmaceutical purposes because the specific isomeric form of the compound affects the pharmacokinetics of the therapeutic agent and any potential toxicological effects of a single isomeric form cannot be distinguished. Although the various dihydropyridazines obtained from conventional ligation can be slowly auto-oxidized to the corresponding pyridazines over the course of hours to days, thereby slowly reducing the number of tautomeric forms of the product (WO2012 / 121746), this slow oxidative conversion does not reduce the number of regioisomers and stereoisomers. Methods for accelerating the conversion of dihydropyridines to pyridazines have been described (Karaki-Fumika et al., Tetrahedron, 97, 132411, 2021; et al., ACS Medicinal Chemistry Letters, 7, 62-66, 2015), however, in no case has the oxidation been reported to be complete within two hours, which is required if the pyridazines are to be used as radiopharmaceuticals. WO2017 / 059397, WO2020 / 242948, et al., ACS Chemical Neuroscience, 11, 4460-4468, 2020 and WO2012 / 121746 disclose connections between tetrazines and TCOs, which will inevitably provide several isomeric chemical entities.

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

[0006] Radiopharmaceuticals are increasingly used in diagnostics, particularly in oncology, for diagnostic imaging and targeted radionuclide therapy. Positron emission tomography (PET) is the gold standard for nuclear imaging, with better resolution and quantification than other modalities. In 2019, 2,200,800 clinical PET scans were performed in the United States alone. Targeted radionuclide therapy is more effective in treating cancer than many state-of-the-art chemotherapies. It also has advantages over external beam radiation therapy (e.g., “gamma knife”) because it provides a means of limiting the delivered dose to the tumor and its surrounding area, which is particularly interesting in the radiotherapy of micrometastatic disease. The combination of diagnostic imaging and targeted radiotherapy can be used in “theranostics,” a concept with powerful applications in personalized medicine involving patient selection, dose finding, and monitoring of treatment response. A theranostic pair is two radionuclides that can be substituted for each other without changing the pharmacokinetics of the radiopharmaceutical, but shifting its application between diagnostic imaging and radionuclide therapy.

[0007] The two most widely used diagnostic imaging methods are nuclear-based PET and SPECT. Both methods rely on the combination of a radionuclide with a carrier that specifically targets cancer cells. In imaging, this radiolabeled carrier is called a "radiotracer." The radiotracer accumulates in tumor lesions, and its location can then be visualized by detecting the emitted radiation. Globally, the ratio of SPECT cameras used in hospitals to PET cameras 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. One or more gamma cameras acquire a series of 2D projection images of the distribution of the radiotracer in the body from multiple angles. These projection images are then combined to produce a 3D image.

[0009] Positron emission tomography (PET) is currently considered the most advanced form of nuclear imaging. The key uses of PET in oncology are diagnosis and therapy monitoring, especially of metastatic cancer. Compared to previous modalities, especially SPECT, PET offers improved resolution and sensitivity and generally higher quality images. These properties are particularly relevant in the detection and subsequent treatment of very small metastases. New innovations, especially whole-body PET, which significantly improves sensitivity and detects more metastases, are expected to further strengthen the dominant position of PET in modern clinics. PET relies on the use of radionuclides that emit positrons when they decay. These positrons travel a limited distance and then annihilate with electrons in the surrounding medium. This produces two annihilation photons, each with an energy of 511 keV, which are emitted in opposite directions. These photons can be detected by the PET scanner.

[0010] The best radionuclide for PET is fluorine-18 ( 18 F). 18 F has a decay half-life of 110 minutes and emits 97% positrons per decay, making it close to ideal for clinical PET applications. This is particularly true for small molecules and peptide-based radiopharmaceuticals, which represent the vast majority of relevant PET tracers. Equally important, 18 F can actually be produced in large quantities (>300 doses per run) on standard biomedical cyclotrons, which are readily available in most parts of the world, with more than 200 in Europe alone. 18 F is not concerned with its closest competitor (the generator produces radioactive gallium-68 ( 68Compared with the most widely used diagnostic radionuclide, the SPECT radionuclide technetium-99m, 18 F provides the highest quality images through its status as a PET radionuclide. 18 F is expected to become a key diagnostic radionuclide in the future.

[0011] Radioactive iodine is widely used in SPECT imaging. Traditionally, iodine-131 ( 131 I), but now, clinical SPECT scanning usually uses iodine-123 ( 123 I) is carried out. This is due to 123 I has a shorter half-life (T1 / 2 = 13.2 hours), and lacks β-negative emission. 131 I has a more favorable radiotoxicity. Its gamma photon energy of 159 keV is very suitable for clinical SPECT imaging. Due to the intrinsic accumulation of iodine in the thyroid gland, the free form 123 I is widely used for imaging of thyroid diseases. As a component of SPECT radiotracers, 123 I is used, for example, in the imaging agents MIBG (oncology) and ioflupane (CNS). 123 I and clinically used beta-negative emitting therapeutic radionuclides 131 I and investigational Auger electron radiotherapy agents 125 I forms a theranostic pair. Iodine-123 is produced in a cyclotron by irradiating xenon in a capsule with protons and is commercially available.

[0012] Iodine-124( 124 I) can be used for PET imaging. It is usually produced by bombarding enriched tellurium-124 in a cyclotron. However, 124 The imaging properties of I are not ideal. It has a complex decay scheme with lots of high-energy gamma radiation. Only 23% of decays result in positron emission.

[0013] Astatine-211( 211 At) is primarily a therapeutic nuclide that emits alpha particles when it decays. Alpha particles are absorbed by only 100μm of tissue and cannot be detected by external scanners. However, 211 One of the decay branches of At also produces X-rays in the 70-90 keV range, which can be imaged with a gamma camera or a SPECT scanner. 211 Distribution of At-labeled radiopharmaceuticals in patients.

[0014] However, there are challenges in introducing the above radionuclides into molecules, which limits the practical application of radionuclide-based therapy, diagnosis and imaging.

[0015] Most of the PET, SPECT and therapeutic radionuclides mentioned above are radiometals. The traditional method of introducing radiometals into carrier molecules is to use chelator groups that form coordination bonds with the radiometal atoms. The radiolabeling procedure generally involves mixing the radiolabeling precursor (carrier with chelator groups) with the radiometal ion and heating the mixture to allow the chelation reaction to proceed. Although chelation of radiometals is conceptually simple, it has a number of disadvantages, namely:

[0016] - Radiolabeled products are usually not separable from unlabeled precursors because the differences in physicochemical properties are not significant;

[0017] - Chelation reactions are sensitive to trace metal impurities in the solutions used for radiolabeling, which makes scale-up problematic;

[0018] -Heating is necessary to overcome the activation barrier of the chelation reaction and may degrade temperature-sensitive carriers.

[0019] Compared with its radioactive metal alternatives, 18 F is a halogen and requires covalent bonding to the targeting vector. This is in stark contrast to chelator-based labeling techniques used for radioactive metals. Covalent bonds are currently usually formed by direct nucleophilic displacement of leaving groups such as triflates. This chemistry is harsh, lengthy, and poorly scalable, and is therefore incompatible with many vectors, especially peptides, which are becoming increasingly important.

[0020] Small molecule radiopharmaceuticals containing radioiodine are typically prepared using either electrophilic demethylamination or iodine-iodine exchange radiochemistry. The former is a mild, versatile, and practical reaction in which the radioiodide is oxidized to a positively charged iodine species, which then displaces a leaving group, typically a tin group, in an aromatic substitution reaction. The reaction occurs at room temperature and yields are typically quantitative. Iodine-iodine isotope exchange is used when high molar activity is not a problem and the substrate can withstand harsh conditions. The exchange is performed at elevated temperatures with acids and copper as catalysts.

[0021] Like fluorine and iodine, astaxanthin-211 is a halogen and can be covalently linked to the targeting carrier. Aliphatic astatine carbon bonds do not provide sufficient in vivo stability, so 211 At is usually introduced into the aromatic ring to form an astatine moiety. Unlike iodine, statin cannot be stably coupled to the tyrosine residues of proteins: 211 At was found to form a weak bond with the sulfhydryl group of cysteine, but not with tyrosine. 211 At labeling requires the synthesis of specialized precursors with a suitable leaving group, such as a trialkyltin group attached to an aromatic ring. 211At labeling protocols use oxidizing agents such as chloramine-T or N-chlorosuccinimide. Such agents may degrade the biomolecules used as targeting carriers.

[0022] 18 F has long been the radionuclide of choice for diagnostic PET imaging, and 211 At is the most promising therapeutic radionuclide in alpha therapy. Iodine radioisotopes 123 I. 124 I. 125 I and 131 I can be used for SPECT imaging, PET imaging, Auger therapy, and beta therapy, respectively. In addition, all three elements can be introduced into aromatic rings to form fluorine / iodine / steroidal aromatic moieties with maximum structural similarity, which is crucial for therapeutic pairs. However, there is no information on 18 F and 211 At or radioactive iodine 123 I. 124 I. 125 I. 131 I combined with reports of therapeutic diagnostic applications.

[0023] As mentioned above, development 18 F / 211 At and 18 F / 123 I, 124 I, 125 I, 131 One obstacle to theranostic pairing is the demanding labeling chemistry, which prevents direct regioselective labeling of biomolecular targeting vectors with these radionuclides. Another obstacle is that not all targeting vectors contain an aromatic moiety, which must therefore be introduced as a prosthetic group, also known as a synthon.

[0024] Because of these challenges, synthon-based approaches have been investigated for the preparation of radiopharmaceuticals. These approaches involve the direct labeling of a single intermediate compound (the “synthon”) which is subsequently coupled to a carrier under mild conditions. The carrier is therefore not subject to the harsh conditions of direct radiolabeling, although it does need to be modified with a chemical label that is complementary to the radiolabeled synthon. Crucial, then, is the chemistry used to conjugate the synthon to the carrier. This chemistry must be specific, compatible with pharmaceutically relevant aqueous media, and produced in high yield to avoid radioactivity losses and minimize the need for subsequent purification. Crucially, it must also be extremely fast, as the procedure for preparing radiopharmaceuticals is performed at nano- to micromolar concentrations and must be completed or nearly completed within minutes due to the decay of the radionuclide. In light of this, click chemistry has emerged as a strategic approach for radiolabeling a range of targeted carriers such as monoclonal antibodies, nanomedicines, peptides, or small molecules.

[0025] Targeted radionuclide therapy (TRT) can be based on beta emitters, Auger electron emitters, and alpha emitters.

[0026] Beta-particle emitting radionuclides (such as 90 Y. 177 Lu, 131 I) decay by emitting high-energy electrons (beta particles), which can travel up to about 12 mm in the tissue. The decay energy is deposited in the largest tissue volume of the three treatment types mentioned here. Beta-emitters are therefore suitable for treating medium-sized tumors, where most of the dose will be absorbed by the cancer cells. However, for micrometastases or heterogeneous tumors, even with perfect radionuclide concentrations in and around the tumor, a large part of the irradiation dose will be absorbed by the surrounding healthy cells. Therefore, beta emitters are not the best choice for treating micrometastases or heterogeneous tumors. This is an important disadvantage of beta emitters, as micrometastases are one of the main causes of cancer recurrence and cancer mortality.

[0027] Alpha emitters (such as 225 Ac, 211 At and 212 Pb) decays with the emission of alpha particles. Alpha particles are much heavier than beta particles, and their trajectories are straight and short, on the order of 30-100 μm, comparable to the diameter of a few mammalian cells. Therefore, all the energy from the decay is delivered to only a few neighboring cells. Alpha rays are more cytotoxic than beta rays and can be delivered to micrometastases in a highly focused manner.

[0028] In a clear example of the advantages of alpha therapy, a novel candidate for the flagship beta-tumor therapeutic 177 Lu-PSMA (prostate-specific membrane antigen)-resistant metastatic prostate cancer patients undergoing α-tumor therapy 225 Complete remission was achieved after three cycles of Ac-PSMA.

[0029] Auger electron radiotherapy (AeRT) uses radionuclides that emit extremely short range showers of electrons when they decay via electron capture (EC) or internal conversion (IC). Through advanced drug delivery techniques, these special radionuclides can be delivered to the nucleus of cancer cells. Here, the emitted Auger electrons damage DNA and kill the cancer cells. Notably, the short range of Auger electrons ensures that their energy is primarily deposited within the target cell, resulting in extremely localized treatment. 123 I and 125 I have high Auger electron yield and are suitable for AeRT.

[0030] Iodine-131( 131I) is a beta particle emitter widely used in clinical radionuclide therapy. It has a decay half-life of 8.0 days and mainly emits beta particles with a maximum energy of 606keV at 90% abundance. The maximum range of these beta particles in tissues is about 2mm, making 131 I is able to treat small and medium-sized tumor lesions. Due to its inherent accumulation in thyroid tissue, it is widely used for thyroid ablation. In addition, a therapeutic variant of MIBG is available, using 131 I was radiolabeled, 131 I is used in radioimmunotherapy. 123 I (SPECT) and 124 I(PET) forms a theranostic pair. Both iodine-123 and iodine-125 have large Auger electron emissions, about 10 and 20 electrons, respectively. This makes them suitable for use in Auger electron radiotherapy, a form of radionuclide therapy currently under investigation.

[0031] At-211 is an alpha 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 provides a number of translational advantages. First, there are limited toxic side effects of the radioactive daughter nuclides that are released from the targeting carrier as a result of the initial decay. Second, radiation dosimetry calculations are simplified. In addition, due to 211 The half-life of At (therapeutic nuclide) is relatively short, which can enhance the control of radiation dose to patients.

[0032] against 211 Phase I / II clinical studies of At-labeled tumor-targeted radiopharmaceuticals have shown low acute toxicity and no radiation side effects. Preclinical studies have shown that in animal models, 211 At can effectively eradicate tumors. 211 At is a promising therapeutic radionuclide.

[0033] However, in order for click chemistries such as Tz-TCO ligations to provide end products that are viable in regulatory and commercial settings, it is critical that only one ligation product is produced. This is not possible using existing methods because ligation typically produces a large number of isomeric products that cannot be separated, making them impractical for clinical translation due to toxicity issues and unpredictable pharmacokinetics. The present invention provides a method whereby certain combinations of chemical entities with complementary counter-electrons that require Diels-Alder cycloaddition reactivity, after ligation, are rapidly oxidized to form a single compound. This means that only one isomeric product is obtained, and therefore separation of the isomeric products is not required. The method advantageously enables the production of a single compound with unparalleled efficiency and practicality, for example with 18 F. 123 I.124 I. 125 I or 131 I and 211 At any tracer is radiolabeled. Summary of the invention

[0034] The present invention provides a method for providing a labeled single isomeric chemical entity targeting vector. The method uses click chemistry, in which a chemical entity conjugated with a label is clicked with a second chemical entity having a complementary anti-electron demand Diels-Alder cycloaddition reactivity, which is conjugated with a targeting vector and then rapidly oxidized to form a single isomeric compound. The advantage of this method is that a single isomeric final product is provided in the shortest possible time, thereby reducing clinical transformation and production costs.

[0035] The method of providing a labeled single isomeric chemical entity targeting vector comprises the following steps:

[0036] a) labeling a first chemical entity having anti-electron demanding Diels-Alder cycloaddition reactivity and conjugated to a pharmaceutical, imaging or therapeutic agent with a labeling agent; wherein the first chemical entity is selected from: a symmetrically substituted diene, an asymmetrically substituted diene or an isomer-free dienophile, wherein at least one plane of symmetry passes through the nitrogen-nitrogen bond of at least one tetrazine ring; and

[0037] b) linking the labeled first chemical entity obtained in step a) to a second chemical entity having a complementary anti-electron demand Diels-Alder cycloaddition reactivity and conjugated to the targeting carrier; wherein the second chemical entity is selected from: a symmetrically substituted diene, an asymmetrically substituted diene or an isomer-free dienophile wherein at least one plane of symmetry passes through the nitrogen-nitrogen bond of at least one tetrazine ring;

[0038] The inverse electron demand Diels-Alder cycloaddition reaction kinetics between the first and second chemical entities was carried out in phosphate buffered saline (PBS) at 25°C with 500 M -1 s -1 The minimum second-order rate constant,

[0039] Provided that when the labeling agent in step a) is 94 Tc, 99 mTc, 211 At 223 Ra or 225 When Ac, the labeling agent is conjugated to the asymmetrically substituted diene; and

[0040] c) by adding 1 to 100 equivalents of chloran, fluoroaniline, DDQ (2,3-dichloro-5,6-dicyano-1,4-benzoquinone) or NaNO 2, oxidizing the ligated tagged targeting vector obtained in step b) at a temperature of 15°C to 50°C for up to 60 minutes.

[0041] Such labeling agents can be any agent useful as a label, imaging agent, therapeutic agent or theranostic agent, including radionuclides and fluorescent entities.

[0042] The targeting vector can be any suitable vector for a specific target, including antibodies, nanobodies, polymers, nanomedicines, cells, proteins, peptides and small molecules.

[0043] Several examples of symmetrically substituted dienes, asymmetrically substituted dienes and isomer-free dienophiles suitable for use in the process are disclosed. Suitable dienes include, for example, tetrazines. Suitable dienophiles include, for example, trans-cycloheptenes (TCH's), trans-cyclooctenes (TCO's) and trans-cyclononenes (TCN's).

[0044] The method of the present invention also includes embodiments in which the first chemical entity and / or the second chemical entity are obtained from specific precursors. These precursors include precursors for obtaining symmetrically substituted dienes, for obtaining asymmetrically substituted dienes, and for obtaining isomer-free dienophiles, respectively.

[0045] Furthermore, the present invention provides the use of the labeled single isomeric chemical entity targeting carrier obtained by this method in theranostics, treatment, radiotherapy, diagnosis and imaging. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 : Scheme showing the synthesis of symmetrical tetrazines.

[0047] Figure 2 : Showing an alternative synthetic scheme for symmetrical tetrazines.

[0048] Figure 3 :[ 18 Radio-HPLC of [F]I at the end of deprotection.

[0049] Figure 4 :show[ 18 F] The ultraviolet trajectory of XX.

[0050] Figure 5 :show[ 18 Radioactive traces of F]XX.

[0051] Figure 6 :show[ 18 F]XX analysis of UV and radioactive traces by HPLC.

[0052] Figure 7 :[ 18 UV trace of F]X-semipreparative HPLC.

[0053] Figure 8 :[ 18 Radioactive traces of F]X-semipreparative HPLC.

[0054] Fig. 9 :[ 18 F]X analysis of HPLC for UV and radioactive traces.

[0055] Fig.10 :[ 18 UV trace-semipreparative HPLC of F]XI.

[0056] Fig.11 : Radioactive tracer-[ 18 Semi-preparative HPLC of [F]XI.

[0057] Fig.12 :[ 18 UV and radiolabeling-analytical HPLC of [F]XI.

[0058] Fig.13 :thick[ 125 I] Radioactive high performance liquid chromatography of XVII.

[0059] Fig.14 : UV and radioactive traces - [ 211 Semi-preparative HPLC of At]XIV.

[0060] Fig.15 :Purified [ 211 Radioactive traces of At]XIV - analytical HPLC.

[0061] Fig.16 : Table showing the results of the radiolabeled tetrazine click experiment in Example 11.

[0062] Fig.17 : Protocol for click reactions performed using 27 and data table for click reactions performed using 27.

[0063] Fig.18 : Click reaction schemes involving a Tz and two different TCOs, and data tables for these click reactions. .

[0064] Fig.19 : HPLC analysis of the tetrazine TCO tested in Example 12 after pyridazine oxidation.

[0065] Fig. 20 :[ 18 F] Protocols for click reactions performed with Tz and four different TCOs and a data table for these click reactions.

[0066] Fig.21: The carrier structure tested in Example 13.

[0067] Fig. 22 : Shows the carrier oxidation results table of Example 13. DETAILED DESCRIPTION

[0068] The present invention provides a method for providing a labeled single isomeric chemical entity targeting carrier in a first aspect. The method uses a specific combination of a diene and a dienophile, which have complementary anti-electron demand Diels-Alder cycloaddition reactivity, and after ligation, a single isomeric form of the compound will be formed after oxidation.

[0069] 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 is conjugated to a targeting vector of interest, respectively.

[0070] The connection between the diene and the dienophile is based on the anti-electron demand Diels-Alder cycloaddition reactivity, and therefore, the diene and dienophile to be connected must have complementary anti-electron demand Diels-Alder cycloaddition activities. In addition, the connection between the diene and the dienophile should have a reaction kinetics with a minimum second-order rate constant of 500 M in PBS at 25 °C as determined by stopped-flow spectrophotometry. -1 s -1 , in order to be relevant to the present method. In phosphate buffered saline (PBS) at 25°C, the second-order rate constant is less than 500 M -1 s -1 The attachment of the radiolabeled target will take a long time to provide a labeled targeting vector, as the time available for imaging and / or therapy after the attachment step is usually limited.

[0071] The second-order rate constant can be measured by different methods, but is usually measured by stopped-flow spectrophotometry, for example as described in (Chance, Rev. Sci. Instrum. 1951, 22, 619-627). Herein, the method described by Battisti et al. in J. Med. Chem. 2021, 64, 20, 15297–15312 has been applied.

[0072] In order to provide a labeled chemical entity or targeting vector that will only result in a single isomeric form when the diene and dienophile are linked with complementary Diels-Alder cycloaddition reactivity, a combination of two requirements is necessary. The first requirement involves the selection of the structures of the diene and dienophile to be linked. It has been found herein that two linking options can be used to combine the diene and dienophile to provide a linking (i.e., click) product of a targeting vector that only provides one isomeric form upon oxidation: linking option i) asymmetrically substituted dienes and isomer-free dienophiles; linking option ii) a symmetrically substituted diene and isomer-free dienophiles.

[0073] The second requirement concerns the oxidation step. This paper shows that not all oxidants are suitable for this approach, either because the oxidation is not efficient enough when working in the required time frame or because the oxidation affects the structure of the targeting vector, which may prevent 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 a single isomeric form after the oxidation step. Oxidation conditions that provide less than 90% of the product in a single isomeric form are not pure enough for therapeutic / imaging / diagnostic use and require additional toxicology studies.

[0074] It is shown herein that suitable oxidation is carried out at a temperature range of 15°C to 50°C for up to 60 minutes by adding 1 to 100 equivalents of an oxidant selected from chloran, fluoroaniline, DDQ and NaNO 2 .

[0075] Thus, a method of providing a labeled single isomeric chemical entity targeting vector comprises:

[0076] a) labeling a first chemical entity having anti-electron demand Diels-Alder cycloaddition reactivity and conjugated to a pharmaceutical, imaging or therapeutic agent with a labeling agent; wherein the first chemical entity is selected from: a symmetrically substituted diene, an asymmetrically substituted diene or an isomer-free dienophile, wherein at least one plane of symmetry passes through the nitrogen-nitrogen bond of at least one tetrazine ring; and

[0077] b) linking the labeled first chemical entity obtained in step a) to a second chemical entity having a complementary anti-electron demand Diels-Alder cycloaddition reactivity and conjugated to the targeting carrier; wherein the second chemical entity is selected from: a symmetrically substituted diene, an asymmetrically substituted diene or an isomer-free dienophile wherein at least one plane of symmetry passes through the nitrogen-nitrogen bond of at least one tetrazine ring,

[0078] The minimum rate constant for the reverse electron demand Diels-Alder cycloaddition reaction between the first and second chemical entities in PBS at 25°C was 500 M, as determined by spectrophotometry.-1 s -1 ;

[0079] Provided that when the labeling agent in step a) is 94 Tc, 99m Tc, 211 At 223 Ra or 225 When Ac, the labeling agent is conjugated to the asymmetrically substituted diene; and

[0080] c) by adding 1 to 100 equivalents of a quinone oxidant, such as chlorobenzoquinone, tetrafluorobenzoquinone, DDQ or NaNO at a temperature ranging from 15°C to 50°C. 2 , the ligated tagged targeting vector obtained in step b) was oxidized for up to 60 minutes.

[0081] In some embodiments, the pharmaceutical agent, imaging agent or therapeutic agent that is conjugated to the first chemical entity is the same as the labeling agent. For example, this may occur when the labeling agent is an agent that can be used as both a label and a therapeutic agent or imaging agent.

[0082] In some embodiments, the labeling agent is a radionuclide. Some radionuclides can be used for imaging, diagnosis and / or treatment, and, in these embodiments, the same radionuclide has been used as a labeling agent and an imaging or therapeutic agent. Labeling a diene or dienophile with a radionuclide will generally not provide 100% radionuclide labeling efficiency, and some of the labeled products will inevitably be labeled with stable isotopes of the corresponding radionuclide element.

[0083] If a symmetrically substituted diene is used as a starting point as the entity to be radiolabeled, it is only possible to provide a radiolabeled single isomeric chemical entity targeting vector if the radiolabel is present in both radioactive and stable forms, since the symmetrically substituted diene will contain two targets for the radionuclide / stable isotope.

[0084] However, some radionuclides of interest in therapy and imaging are not available in a stable form. Therefore, there is no corresponding element that can label the symmetric position of the labeling target, which will inevitably lead to multiple isomeric forms of the final product. Therefore, if the labeling agent is 211 At 223 Ra or 225 Ac, symmetrically substituted dienes are not available, and therefore, when these radionuclides are used as labels and / or as diagnostic / therapeutic agents, the linking combination option ii) should be applied, in which the asymmetrically substituted diene is linked to an isomer-free dienophile. Therefore, the method is used under the condition that when the labeling agent in step a) is 94 Tc, 99m Tc, 211 At223 Ra or 225 When Ac, the labeling agent is conjugated to the asymmetrically substituted diene; and

[0085] This method enables labeling of any targeting vector with unparalleled efficiency and practicality, e.g. 18 F. Radioactive iodine ( 123 I. 124 I. 125 I or 131 I) and 211 The breakthrough nature of this method is the potential to form a single end product within 60 minutes, often in less than 60 minutes, such as within 1-20 minutes, thereby simplifying clinical translation. In contrast, traditional tetrazine ligation produces multiple products, thus requiring large and unmanageable toxicology packages or tedious and time-consuming separations.

[0086] definition

[0087] Symmetric tetrazine: In the context of the present invention, a symmetrical tetrazine refers to any tetrazine that, as a "cold" reference or after radiolabeling / deprotection, exhibits one or more planes of symmetry in the chemical structure. One of the planes of symmetry passes through the nitrogen-nitrogen bonds of the tetrazine ring.

[0088] Cold Reference: In the context of the present invention, a cold reference refers to a compound labeled with a non-radioactive isotope of an atom, where a radioactive isotope of the same atom is required to provide a radiolabeled form of the same compound. In addition, in the context of the present invention, the term cold reference also includes compounds containing one or more protecting groups that will be replaced by the labeling agent upon labeling.

[0089] Asymmetric tetrazine: In the context of the present invention, an asymmetric tetrazine refers to one that has no nitrogen-nitrogen bonds through the tetrazine ring as a "cold" reference or after radiolabeling / deprotection.

[0090] Isomer-free dienophile or isomer-free TCH / TCO / TCN: In the context of the present invention, isomer-free dienophile or isomer-free TCH / TCO / TCN refers to any dienophile or any TCH, TCO and TCN, respectively, which forms only one isomer / enantiomer after reaction and oxidation with the corresponding tetrazine.

[0091] TCH: In the context of the present invention, TCH refers to any 7-membered ring with at least one double bond in trans configuration, capable of reacting as a dienophile in a reverse electron demand Diels-Alder cycloaddition reaction.

[0092] TCO: In the context of the present invention, TCO refers to any 8-membered ring having at least one double bond in the trans configuration, capable of reacting as a dienophile in a reverse electron demand Diels-Alder cycloaddition reaction.

[0093] TCN: In the context of the present invention, TCN refers to any 9-membered ring with at least one double bond in trans configuration, capable of reacting as a dienophile in a reverse electron demand Diels-Alder cycloaddition reaction.

[0094] As is well known, in click chemistry, dienes, such as tetrazines, are clicked with dienophiles, such as trans-cycloheptenes (TCH's), trans-cyclooctenes (TCO's) or trans-cyclononenes (TCN's), with complementary anti-electron demanding Diels-Alder cycloaddition reactivity. In a preferred embodiment, the method of providing a labeled single isomeric chemical entity targeting carrier wherein the diene is a tetrazine and the dienophile is a trans-cycloheptene (TCH), a trans-cyclooctene (TCO) or a trans-cyclononene (TCN).

[0095] The labeled monoisomeric chemical entity targeting carrier obtainable according to the method of the present invention can be used for various purposes, depending on the characteristics of the reagent used as the label. Labeling agents suitable for the method include radioactive labels and fluorescent labels.

[0096] In a preferred embodiment, the labeling agent used in step a) of the method for providing a labeled single isomeric chemical entity targeting carrier is a radionuclide or a stable isotope of the corresponding element. The properties and corresponding uses of the commonly used different radionuclides are well known in the art.

[0097] In the method of providing a labeled single isomeric chemical entity targeting carrier, radionuclide labeling agents and stable isotopes of corresponding elements suitable for use as labeling agents in step a) include: 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 You, 46 You, 47 You, 48 You, 49 You, 50 You, 55 Co, 58 mCo, 59 Co, 60 Cu, 61 Cu, 63 Cu, 64 Cu, 65 Cu, 67 Cu, 67 Language, 68 Language, 69 Language, 71 Language, 76 Br, 77 Br, 79 Br, 80 mBr, 81 Br, 72 And, 75 And, 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 You, 113 You, 114 doesn't, 115 doesn't, 175 In, 177 In, 185 Re, 186 Re, 188 Re, 201 Tl, 203 Tl, 205 Tl, 206 Pb, 207 Pb, 208 Pb, 212 Pb, 209 Wind, 212 Wind, 2 13 Wind, 31 P, 32 P, 33 P,32 S, 35 S 45 Sc, 47 Sc, 84 Wed, 86 Wed, 87 Wed, 88 Wed, 89 Wed, 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 If, 74 If, 75 If, 76 If, 77 If, 78 If, 80 If, 82 If, 94 Tc, 99m Tc, 103 Rh, 103 mRh, 119 Sb, 121 Sb, 123 Sb, 135 Here, 138 Here, 139 Here, 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 Ac。

[0098] In a preferred embodiment, the radionuclide labeling agent is selected from: 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 stable isotopes of the corresponding element selected from: 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 corresponding stable isotopes are particularly useful in positron emission tomography (PET).

[0099] In another preferred embodiment, the radionuclide labeling agent is selected from: 64 Cu, 67 Cu, 67 Ga, 111 In, 131 I, 177 Lu, 186 Re, 201 Tl, 212Pb, 213 Bi; the corresponding stable isotope of the element is selected from: 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 corresponding stable isotopes are particularly useful in single photon emission computed tomography (SPECT).

[0100] In another preferred embodiment, the radionuclide labeling agent is selected from: 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; the stable isotopes of the corresponding elements are selected from: 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, 162 Dy, 163 Dy, 164 Dy, 175 Lu, 185 Re. These radionuclides are beta particle emitters and these radionuclides and their corresponding stable isotopes are used therapeutically, for example in connection with the treatment of various neoplastic diseases.

[0101] In another preferred embodiment, the radionuclide labeling agent is selected from: 149Tb, 212 Pb, 212 Bi, 213 Bi, 227 Th; the corresponding stable isotope of the element is selected from: 159 Tb, 206 Pb, 207 Pb, 208 Pb, 209 Bi, 232 Th. These radionuclides are alpha particle emitters and these radionuclides and their corresponding stable isotopes are used therapeutically, for example in connection with the treatment of various neoplastic diseases.

[0102] In another preferred embodiment, the radionuclide labeling agent is selected from: 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; the stable isotopes of the corresponding elements are selected from: 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 emit electrons via the Auger effect with low kinetic energy. These radionuclides and their stable isotopes are used in Auger therapy, for example in connection with the highly targeted treatment of various neoplastic diseases.

[0103] In another preferred embodiment, the radionuclide labeling agent is selected from 3 H, 14 C and 35 S, the stable isotope of the corresponding element is selected from 1 H, 2 H, 12 C, 13 C, 32 S. These radionuclides are used for in vitro studies such as displacement and tritium determination.

[0104] In another preferred embodiment, the radionuclide labeling agent is selected from: 11 C, 13 N, 18 F, 123 I, 125 I, 131 I, or 211 At; and the corresponding stable isotope of the element (when the stable isotope of the element is available) is selected from: 12 C, 14 N, 19 F, 127 I. These radionuclides and their corresponding stable isotopes (where available) are among the most commonly used radionuclides in therapy and imaging today.

[0105] The targeting carrier coupled to the diene or dienophile mentioned in step b) to provide a labeled single isomeric chemical entity targeting carrier can be any type of targeting carrier suitable for treatment, imaging or diagnosis. Such common targeting carriers suitable for the present method include antibodies, nanobodies, polymers, nanomedicines, cells, proteins, peptides and small molecules.

[0106] Common targeting carriers suitable for the present method include: peptides, such as octreotide, octreotide acid, AE105; small molecules, such as FAPI derivatives and PSMA derivatives.

[0107] In a preferred embodiment, the targeting vector used in the method for providing a labeled single isomeric chemical entity targeting vector is selected from the group consisting of: octreotide, octreotate, AE105, FAPI derivatives, and PSMA derivatives.

[0108] The oxidation step c) in the method of providing a labeled single isomer chemical entity targeting carrier is carried out at a certain temperature and time by adding a specific oxidant to the linked compound obtained in step b). These conditions ensure that the efficiency of the oxidation step is ≥ 90%, thereby meeting the speed required for the treatment, diagnosis or imaging use of the labeled single isomer chemical entity targeting carrier.

[0109] The time required to obtain an oxidation efficiency of ≥ 90% depends on the specific compound being oxidized, the temperature, the oxidant equivalent and the oxidant. Under the conditions used in the present method, an oxidation efficiency of ≥ 90% will be 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, an oxidation efficiency of ≥ 90% is obtained within 0-20 minutes.

[0110] The temperature of the oxidation step is 15°C-50°C, such as 15°C-45°C, 15°C-40°C, 15°C-35°C, 20°C-30°C or about 20°C-25°C. The preferred temperature is room temperature, such as 20°C-25°C.

[0111] The oxidant should be a quinone oxidant selected from tetrachlorobenzoquinone, tetrafluorobenzoquinone, DDQ or NaNO 2 . It is surprisingly found herein that the use of other types of oxidants will not provide the desired single isomeric form of the labeled chemical entity targeting vector, or will negatively affect the structure of the targeting vector. In the method for providing a labeled single isomeric chemical entity targeting vector, an oxidant is added to the connected labeled compound obtained from step b), and the labeled single isomeric chemical entity targeting vector is 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 oxidant is added to the labeled compound obtained from step b).

[0112] In one embodiment, the oxidant is supported by a solid. Any conventional solid support is suitable, such as an oxidant supported by alumina, silica gel, polymer, montmorillonite, zeolite or nanomaterial. Advantages of using solid supported oxidants generally include ease of removal from the reaction by filtration, excess reagent can be used to drive the reaction to completion without introducing purification difficulties, easy handling, and recovery of the recovered reagent is economical and effective.

[0113] The conventional connection between tetrazines and TCOs will generate many different tautomers, regioisomers, and enantiomers as shown in Scheme 1.

[0114]

[0115] Scheme 1. Overview of all isomeric forms after conventional tetrazine ligation.

[0116] Including tautomers, enantiomers and regioisomers

[0117] Scheme 2 below is an example of the most advanced tetrazine ligation. A reaction that produces many isomeric products:

[0118]

[0119] Scheme 2. Detailed example of the current state-of-the-art tetrazine ligation

[0120] (All isomeric forms are omitted, an overview of the isomeric forms is shown in Scheme 1)

[0121] In this reaction a variety of isomers (at least 12 isomers) can be formed, all of which have very similar polarity and are therefore difficult to separate, for example, by HPLC.

[0122] Here, we describe a method that allows the use of a labeled first chemical entity, such as a radiolabeled tetrazine-based synthon, for radiolabeling isomer-free dienophiles, such as isomer-free trans-cycloheptene (TCH), isomer-free trans-cyclooctene (TCO), and isomer-free trans-cyclononene (TCN) functionalized supports, and vice versa, with subsequent chemical oxidation yielding a single final compound in a short time, such as within 0-60 min ( Fig.17 ). The method comprises two steps: a ligation step followed by an oxidation step.

[0123] In a preferred embodiment, the method of providing a labeled single isomeric chemical entity targeting vector comprises:

[0124] a) labeling a first chemical entity having inverse electron demand Diels-Alder cycloaddition reactivity and conjugated to a pharmaceutical agent, imaging agent or therapeutic agent with a labeling agent; wherein the first chemical entity is selected from: a symmetrical tetrazine wherein at least one plane of symmetry passes through a nitrogen-nitrogen bond of at least one tetrazine ring, an asymmetrical tetrazine, isomer-free trans-cycloheptene (TCH), isomer-free trans-cyclooctene (TCO) and isomer-free trans-cyclononene (TCN),

[0125] b) linking the labeled first chemical entity obtained in step a) to a second chemical entity having a complementary inverse electron demand Diels-Alder cycloaddition reactivity and conjugated to the targeting carrier; wherein the second chemical entity is selected from: symmetrical tetrazines, asymmetrical tetrazines, isomer-free trans-cycloheptene (TCH), isomer-free trans-cyclooctene (TCO), isomer-free trans-cyclononene (TCN) and pre-meso-trans-cycloheptene (TCH), wherein at least one plane of symmetry passes through the nitrogen-nitrogen bond of at least one tetrazine ring,

[0126] The inverse electron demand Diels-Alder cycloaddition reaction kinetics between the first and second chemical entities was carried out in PBS at 25 °C with 500 M -1 s -1 The minimum rate constant, determined by stopped-flow spectrophotometry,

[0127] However, the prerequisite is that when the labeling agent in step a) is 94 Tc, 99m Tc, 211 At, 223 Ra or 225 When Ac, the labeling agent is conjugated to the asymmetrically substituted diene; and

[0128] c) by adding 1 to 100 equivalents of chloranil, tetrafluorobenzoquinone, DDQ or NaNO 2 , oxidizing the ligated tagged targeting vector obtained from step b) at a temperature of 20°C to 30°C for up to 20 minutes.

[0129] The starting entities to be attached are an isomer-free dienophile, such as isomer-free TCH, TCO or TCN, and a diene, such as a tetrazine. The diene can be an asymmetrically substituted diene, such as an asymmetrically substituted tetrazine, or a symmetrically substituted tetrazine in which at least one plane of symmetry passes through the nitrogen-nitrogen bond of at least one tetrazine ring, such as a symmetrical tetrazine. The use of an isomer-free dienophile, such as isomer-free TCH, TCO or TCN, can reduce the number of click products formed by eliminating all enantiomeric and regioisomeric products. The tautomers formed, such as dihydropyridazines, will subsequently be oxidized to the corresponding single isomers, such as pyridazine, to produce a single product. The "R" substituent on the diene used in this method, such as tetrazine, will typically be replaced with a 18 F. 123 I. 124 I. 125 I. 131 I or 211 At substituted aryl functionalization.

[0130] The following schemes 2 and 3 are illustrations of connection examples according to the method of the present invention, where the use of asymmetric tetrazine and TCO conjugated with a targeting carrier is used as an example:

[0131]

[0132] Solution 3.

[0133]

[0134] Solution 4.

[0135] The targeting vector is an antibody, nanobody, polymer, nanodrug, cell, protein, peptide or small molecule.

[0136] When the labeling agent in step a) is 211 At, 223 Ra or 225 In the case of Ac, the labeling agent must be conjugated to the asymmetrically substituted diene.

[0137] The following dienes are examples of symmetrical tetrazines of formula Tz1 suitable for attachment in step b) of the method for providing a labeled single isomeric chemical entity targeting carrier:

[0138] Where R and R 1 yes The curl symbol indicates the link to the tetrazine; R 2 is -H or (i) an isotope-labeled agent directly attached to an aromatic ring; or (ii) an isotope-labeled agent attached to an aromatic ring via a linker selected from (CH 2 ) n ,-LO(CH 2 ) n ,-LNH(CH 2 ) n ,-LCONH(CH 2 ) n ,-LNHCO(CH 2 ) n , where L is -(CH 2 ) m or -O(CH 2 CH 2 O) m, wherein n and m are independently selected from 1-25; or (iii) an isotope labeling agent chelated by a chelating agent selected from: 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( 1,4,7-triazacyclononanephosphonic acid (TRAP), 1,4,7-triazacyclononane-1-methyl (2-carboxyethyl)phosphonic acid-4,7-bis(methyl (2-hydroxymethyl)phosphonic acid (NOPO), 3,6,9,15-tetraazacyclododecane-1-yl) 1,4,7-triazacyclononanephosphonic acid (TRAP), 1,4,7-triazacyclononanephosphonic acid-4,7-bis(methyl (2-hydroxymethyl)phosphonic acid (NOPO), 1,4,7-triazacyclononanephosphonic acid (TRAP), 1,4,7-triazacyclononanephosphonic acid-1,4,7-triazacyclododecane-1-yl) 1,4,7-triazacyclononanephosphonic acid (NOPO), 1,4,7-triazacyclononanephosphonic acid (TRAP), 1,4,7-triazacyclononanephosphonic acid- ... Azoheterocycles 9.3.1.pentadeca-1(15), 11,13-triene-3,6,9-triacetic acid N'-(5-acetyl(hydroxy)aminopentyl-N-(5-(5-aminopentyl)(hydroxy)amino-4-oxobutanoyl)amino)pentyl-N-hydroxysuccinamide (DFO), diethylenetriaminepentaacetic acid (DTPA), trans-cyclohexyldiethylenetriaminepentaacetic acid (CHX-DTPA), 1-oxo-4,7,10- Triazacyclododecane-4,7,10-triacetic 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) are attached to the aromatic ring via a linker selected from (CH 2 )n,-LO(CH 2 )n,-LNH(CH 2 )n,-LCONH(CH 2 )n,-LNHCO(CH 2 )n, where L is -(CH 2 )m or -O(CH 2 CH 2 O) m, n and m are independently selected from 1-25;

[0139] Among them, when R 2 In case of (i) or (ii), the isotope labeling agent is selected from:

[0140] 1 H, 2 H, 3 H, 11 C, 12 C,13 C, 13 N, 14 N, 15 N 18 F, 19 F, 123 OF, 124 OF, 125 OF, 127 OF, 131 OF, 211 And, 15 O, 16 O, 17 O, 18 O, 32 S, 35 S, 43 Sc, 44 Sc, 45 Sc, 45 Water, 46 Water, 47 Water, 48 Water, 49 Water, 50 Water, 55 Co, 58 mCo, 59 Co, 60 The, 61 The, 63 The, 64 The, 65 The, 67 The, 67 Come on, 68 Come on, 69 Come on, 71 Come on, 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 Through, 113 Through, 114 name, 115 name, 175Ridiculous, 177 Ridiculous, 185 Too, 186 Too, 188 Too, 201 Tl, 203 Tl, 205 Tl, 206 Pb, 207 Pb, 208 Pb, 212 Pb, 209 Purpose, 212 Purpose, 2 13 Purpose, 31 P, 32 P, 33 P, 45 Sc, 47 Sc, 84 Wed, 86 Wed, 87 Wed, 88 Wed, 89 Wed, 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 If, 74 If, 75 If, 76 If, 77 If, 78 If, 80 If, 82 If, 94 Tc, 99m Tc, 103 Rh, 103 mRh, 119 Sb, 121 Sb, 123 Sb, 135 Here, 138 Here, 139 Here, 162 Er, 164 Er, 165 Er, 166Er, 167 Er, 168 Er, 170 Er, 193 mPt, 195 mPt, 192 Pt, 194 Pt, 195 Pt, 196 Pt, 198 Pt,

[0141] wherein X and Y are independently selected from: -CH- and -N-;

[0142] Where R 3 independently selected from H or selected from hydroxyl, sulfonamide, carboxyl, sulfonyl, amine, substituted amine having 1-5 polyethylene glycol units, -(O-CH 2 -CH 2 ) 1-5 -OCH 2 -COOH, methyl, ethyl, propyl, optionally substituted heteroaryl and optionally substituted arylalkyl; wherein, the optional substitution relative to the substituted amine refers to a 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) heteroalkyl, (C3-30 (C1-C10)cycloalkyl, (C3-C10)heterocycloalkyl, (C3-10)cycloalkyl, (C3-C10)heterocycloalkyl, (C1-C10)haloalkyl, (C1-C10)perhaloalkyl, (C2-C10)-alkenyloxy, (C3-C10)-alkynyloxy, aryloxy, aralkyloxy, heteroaryloxy, heteroaralkyloxy, (C1-C6)alkoxy-(C1-C4)alkyl, optionally substituted aryl, optionally substituted heteroaryl and optionally substituted aralkyl; wherein optionally substituted refers to halogen, hydroxy, sulfonamide, carboxyl, sulfonyl, amine, substituted amine with 1-5 polyethylene glycol units, -(O-CH 2 -CH 2 ) 1-5 -OCH 2 -COOH, H, methyl, ethyl, propyl, one or more substituents of optionally substituted heteroaryl and optionally substituted arylalkyl; wherein the optional substitution relative to the substituted amine refers to one or more substituents selected from halogen, hydroxyl, sulfonamide, carboxyl, sulfonyl and amine;

[0143] Where R and R 1 The same or differ only in the isotope number of the labeling agent.

[0144] The following tetrazines are preferred tetrazines of formula Tz1 suitable for the attachment of step b) in the method of providing a labeled single isomeric chemical entity targeting carrier:

[0145]

[0146] In addition, the unsymmetrical tetrazine of formula Tz2 is a preferred diene embodiment suitable for attachment in step b) of the method for providing a labeled single isomeric chemical entity targeting carrier:

[0147]

[0148] Where R 4 is -H or (i) an isotope-labeled agent directly attached to an aromatic ring, or (ii) an isotope-labeled agent attached to an aromatic ring via a linker selected from (CH 2 ) n , -LO(CH 2 ) n , -LNH(CH 2 ) n , -LCONH(CH 2 ) n , -LNHCO(CH 2 ) n , where L is -(CH2) m or -O(CH 2 CH 2 O) m, wherein n and m are independently selected from 1-25, or (iii) the isotope labeling agent chelated by a chelating agent is selected from: 1,4,7,10-tetraazacyclododecane NE-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-Triazacyclononane-1-yl)pentanedioic acid (NODAGA), 2-(4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane-1-yl)pentanedione iodic acid (DOTAGA), 14,7-triazacyclononanephosphonic acid (TRAP), 14,7-triazacyclononane-1-methyl(2-carboxyethyl)phosphonic acid-4,7-bis(methyl(2-hydroxymethyl)phosphonic acid (NOPO), 3,6,9,15-tetraazabicyclo9 .3.1.pentadeca-1(15), 11,13-triene-3,6,9-triacetic acid (PCTA), N'-(5-acetyl(hydroxy)aminopentyl-N-(5-(aminopentyl)(hydroxy)amino-4-oxobutanoyl)amino)pentyl-N-hydroxysuccinamide (DFO), diethylenetriaminepentaacetic acid (DTPA), trans-cyclohexyldiethylenetriaminepentaacetic acid (CHX-DTPA), 1-oxa-4,7,10-triazine heterocyclododecane-4,7,10-triacetic 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), and connected to the aromatic ring via a linker selected from (CH 2 ) n ,-LO(CH 2 ) n ,-LNH(CH 2 ) n ,-LCONH(CH 2 ) n ,-LNHCO(CH 2 ) n , where L is -(CH 2 ) m or -O(CH 2 CH 2 O) m, , where L is -(CH 2 ) m or -O(CH 2 CH 2 O) m, n and m are independently selected from 1-25; wherein, when R 4 In the case of (i), (ii) or (iii), the isotope labeling agent is selected from: 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, 91 Zr, 92 Zr, 94 Zr,149 Tb, 152 Tb, 159 Tb, 161 Tb, 111 You, 113 You, 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 Purpose, 212 Purpose, 2 13 Purpose, 31 P, 32 P, 33 P, 45 Sc, 47 Sc, 84 Wed, 86 Wed, 87 Wed, 88 Wed, 89 Wed, 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 If, 74 If, 75 If, 76 If, 77 If, 78 If, 80 If, 82 If, 94 Tc, 99m Tc, 103 Rh, 103 mRh, 119Sb, 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,

[0149] wherein X and Y are independently selected from: -CH- and -N-;

[0150] Where R 6 is H or wherein Q and Z are independently selected from: -CH- and -N- and wherein the curl symbol indicates attachment to a tetrazine;

[0151] Where R 5 and R 7 independently selected from H or selected from hydroxyl, sulfonamide, carboxyl, sulfonyl, amine, substituted amine having 1-5 polyethylene glycol units, -(O-CH 2 -CH 2 ) 1-5 -OCH 2-COOH, methyl, ethyl, propyl, optionally substituted heteroaryl and optionally substituted arylalkyl; wherein, the optional substitution relative to the substituted amine refers to a 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) heteroalkyl, (C3-30C10) cycloalkyl, (C3-C10) heterocyclic alkyl, (C3-10) cycloalkyl, (C3-C10) heterocycloalkylene, (C1-C10) haloalkyl, (C1-C10) perhaloalkyl, (C2-C10) alkenyloxy, (C3-C10) alkynyloxy, aryloxy, arylalkoxy, heteroaryloxy, heteroarylalkoxy, (C1-C6) alkoxy-(C1-C4) alkyl, optionally substituted aryl, optionally substituted heteroaryl and optionally substituted arylalkyl; wherein optionally substituted refers to halogen, hydroxy, sulfonamide, carboxyl, sulfonyl, amine, substituted amine with 1-5 polyethylene glycol units, -(O-CH 2 -CH 2 ) 1-5 -OCH 2 -COOH, H, methyl, ethyl, propyl, optionally substituted heteroaryl and optionally substituted arylalkyl, wherein the optional substitution relative to the substituted amine refers to one or more substituents selected from halogen, hydroxyl, sulfonamide, carboxyl, sulfonyl and amine.

[0152] The following tetrazines are preferred tetrazines of formula Tz2 for attachment in step b) of the method for providing a labeled single isomeric chemical entity targeting carrier:

[0153]

[0154] The following trans-cycloheptenes (TCH's), trans-cyclooctenes (TCO's) and trans-cyclononenes (TCNs) are preferred isomer-free dienophiles for attachment in step b) of the method of providing a labeled single isomer chemical entity targeting vector:

[0155]

[0156] Where X is N, NO or CR 8 ; Y is N, NO or CR 8 ; R 8 Selected from: -H, -F, -OH, -NH 2 ,-COOH,-COOCH 3 ,CF 3 ,-Cl,-CONH 2,CONHCH 3 ,-CON(CH 3 ) 2 ,-CH 2 CH 2 OH,-CH 2 CH 2 NH 2 ,-CHCH 2 N(CH 3 ) 2 , and

[0157] wherein the linker is selected from: -(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(CH2 ) 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 NH,CO(CH) 2 ) n CO,CO(CH) 2 ) n O,CONH(CH 2 CH 2 O) n ,CONH(CH) 2 CH 2 O) n CH 2 CH 2 NH,CO(CH) 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, wherein n is 0-25, wherein M is a side chain selected from the group consisting of natural amino acid side chains: H, CH 3 ,CH 2 SH,CH 2 COOH,CH 2 CH 2 COOH,CH 2 C 6 H 5 ,CH 2 C 3 H 3 N 2 ,CH(CH 3 )CH 2 CH 3 ,(CH 2 ) 4 NH 2 ,CH 2 CH(CH 3 ) 2 ,CH 2 CH 2 SCH 3 ,CH 2 CONH 2 ,(CH 2 ) 4 NHCOC 4 H 5 NCH 3 ,CH 2 CH 2 CH 2 ,CH 2 CH 2 CONH 2 ,(CH 2 ) 3 NH-C(NH)NH 2 ,CH 2 OH,CH(OH)CH 3 ,CH 2 SeH,CH(CH 3 ) 2 ,CH 2 C 8 H 6N,CH 2 C 6 H 4 OH;

[0158] The targeting vector is an antibody, nanobody, polymer, nanodrug, cell, protein, peptide or small molecule.

[0159] Examples of asymmetric TZs suitable for ligation in step b) of the method of providing a labeled single isomeric chemical entity targeting vector are:

[0160]

[0161] Examples of monocyclic isomer-free TCH and TCN that are suitable for use in step b) of the method for providing a labeled single isomeric chemical entity targeting vector:

[0162]

[0163] Wherein the linker is selected from: -(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 NH,CO(CH) 2 ) n CO,CO(CH) 2 ) n O,CONH(CH 2 CH 2 O) n ,CONH(CH) 2 CH 2 O) n CH 2 CH 2 NH,CO(CH) 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 CH2 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 0-25, where M is a side chain selected from the side chains of natural amino acids: H, CH 3 ,CH 2 SH,CH 2 COOH,CH 2 CH 2 COOH,CH 2 C 6 H 5 ,CH 2 C 3 H 3 N 2 ,CH(CH 3 )CH 2 CH 3 ,(CH 2 ) 4 NH 2 ,CH 2 CH(CH 3 ) 2 ,CH 2 CH 2 SCH 3 ,CH 2 CONH 2 ,(CH 2 ) 4 NHCOC 4 H 5 NCH 3 ,CH 2 CH 2 CH 2 ,CH 2 CH 2 CONH 2 ,(CH 2 ) 3NH-C(NH)NH 2 ,CH 2 OH,CH(OH)CH 3 ,CH 2 SeH,CH(CH 3 ) 2 ,CH 2 C 8 H 6 N,CH 2 C 6 H 4 OH; wherein the targeting vector is an antibody, a nanobody, a polymer, a nanomedicine, a cell, a protein, a peptide or a small molecule.

[0164] Examples of bicyclic isomer-free TCOs suitable for attachment in step b) of the method for providing a labeled single isomeric chemical entity targeting vector:

[0165]

[0166] Where R 8 Yes: H, F, OH, NH 2 ,CH 3 ,COOH,COOCH 3 ,CF 3 ,Cl,CONH 2 ,CONHCH 3 ,CON(CH 3 ) 2 ,CH 2 CH 2 OH,CH 2 CH 2 NH 2 ,CHCH 2 N(CH 3 ) 2 , and the linker is selected from: -(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 CH2 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 NH,CO(CH) 2 ) n CO,CO(CH) 2 ) n O,CONH(CH 2 CH 2 O) n ,CONH(CH) 2 CH 2 O) n CH 2 CH 2 NH,CO(CH) 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, wherein n is 0-25, wherein M is a side chain selected from the group consisting of natural amino acid side chains: H, CH 3 ,CH 2 SH,CH 2 COOH,CH 2 CH 2 COOH,CH 2 C 6 H 5 ,CH 2 C 3 H 3 N 2 ,CH(CH 3 )CH 2 CH 3 ,(CH 2 ) 4 NH 2 ,CH 2 CH(CH3 ) 2 ,CH 2 CH 2 SCH 3 ,CH 2 CONH 2 ,(CH 2 ) 4 NHCOC 4 H 5 NCH 3 ,CH 2 CH 2 CH 2 ,CH 2 CH 2 CONH 2 ,(CH 2 ) 3 NH-C(NH)NH 2 ,CH 2 OH,CH(OH)CH 3 ,CH 2 SeH,CH(CH 3 ) 2 ,CH 2 C 8 H 6 N,CH 2 C 6 H 4 OH, wherein the targeting vector is an antibody, a nanobody, a polymer, a nanomedicine, a cell, a protein, a peptide or a small molecule.

[0167] Examples of tricyclic isomer-free TCOs suitable for attachment in step b) of the method for providing a labeled single isomeric chemical entity targeting vector:

[0168]

[0169] Where X is N, NO or CR 8 , Y is N, NO or CR 8 ; R 8 Selected from: -H, -F, -OH, -NH 2 ,-COOH,-COOCH 3 ,CF 3 ,-Cl,-CONH 2 ,CONHCH 3 ,-CON(CH 3 ) 2 ,-CH 2 CH 2 OH,-CH 2 CH 2 NH 2 ,-CHCH 2N(CH 3 ) 2 and

[0170] wherein the linker is selected from: -(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(CH2 CH 2 O) n CH 2 CH 2 CO,CONH(CH) 2 -CONH(CH) 2 ) n NH,CO(CH) 2 ) n CO,CO(CH) 2 ) n O,CONH(CH 2 CH 2 O) n ,CONH(CH) 2 CH 2 O) n CH 2 CH 2 NH,CO(CH) 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 CH2 O) n COCHMNH, wherein n is 0-25, wherein M is a side chain selected from the group consisting of natural amino acid side chains: H, CH 3 ,CH 2 SH,CH 2 COOH,CH 2 CH 2 COOH,CH 2 C 6 H 5 ,CH 2 C 3 H 3 N 2 ,CH(CH 3 )CH 2 CH 3 ,(CH 2 ) 4 NH 2 ,CH 2 CH(CH 3 ) 2 ,CH 2 CH 2 SCH 3 ,CH 2 CONH 2 ,(CH 2 ) 4 NHCOC 4 H 5 NCH 3 ,CH 2 CH 2 CH 2 ,CH 2 CH 2 CONH 2 ,(CH 2 ) 3 NH-C(NH)NH 2 ,CH 2 OH,CH(OH)CH 3 ,CH 2 SeH,CH(CH 3 ) 2 ,CH 2 C 8 H 6 N,CH 2 C 6 H 4 OH;

[0171] The targeting vector is an antibody, nanobody, polymer, nanodrug, cell, protein, peptide or small molecule.

[0172] Examples of tetracyclic isomer-free TCOs that are suitable for attachment in step b) of the method for providing a labeled single isomeric chemical entity targeting vector are as follows:

[0173]

[0174] Where X is N, NO or CR 8 ; Y is N, NO or CR 8 ; R 8 Selected from: -H, -F, -OH, -NH 2 ,-COOH,-COOCH 3 ,CF 3 ,-Cl,-CONH 2 ,CONHCH 3 ,-CON(CH 3 ) 2 ,-CH 2 CH 2 OH,-CH 2 CH 2 NH 2 ,-CHCH 2 N(CH 3 ) 2 And the linker is selected from: -(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 CH2 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 NH,CO(CH) 2 ) n CO,CO(CH) 2 ) n O,CONH(CH 2 CH 2 O) n ,CONH(CH) 2 CH 2 O) n CH 2 CH 2 NH,CO(CH) 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 ) nNHCOCHMNH,(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, wherein n is 0-25, wherein M is a side chain selected from the group consisting of natural amino acid side chains: H, CH 3 ,CH 2 SH,CH 2 COOH,CH 2 CH 2 COOH,CH 2 C 6 H 5 ,CH 2 C 3 H 3 N 2 ,CH(CH 3 )CH 2 CH 3 ,(CH 2 ) 4 NH 2 ,CH 2 CH(CH 3 ) 2 ,CH 2 CH 2 SCH 3 ,CH 2 CONH 2 ,(CH 2 ) 4 NHCOC 4 H 5 NCH 3 ,CH 2 CH 2 CH 2,CH 2 CH 2 CONH 2 ,(CH 2 ) 3 NH-C(NH)NH 2 ,CH 2 OH,CH(OH)CH 3 ,CH 2 SeH,CH(CH 3 ) 2 ,CH 2 C 8 H 6 N,CH 2 C 6 H 4 OH;

[0175] The targeting vector is an antibody, nanobody, polymer, nanodrug, cell, protein, peptide or small molecule.

[0176] Step c) is an oxidation step in the method of providing a labeled single isomer chemical entity targeting carrier. Although the auto-oxidation of the connection entity targeting carrier (e.g., pyridazine) obtained in step b) of the method occurs spontaneously, the process is very slow and can last for hours to days. Step c) in the method provides a rapid method for oxidizing pyridazine compounds, wherein only a single isomeric form is obtained at least within 60 minutes (e.g., within 1-20 minutes). To facilitate the process, dihydropyridazine is oxidized by a standard oxidant or a solid-supported oxidant, preferably a solid-supported oxidant. The oxidation step can be carried out at a temperature of 15 to 50°C, for example, at 20-30°C, preferably at room temperature, for about 10 to 60 minutes, preferably less than 20 minutes. To promote oxidation, 1 to 100 equivalents (preferably 1) of an oxidant are added to the connection compound obtained from the connection step. The oxidant needs to be selective for the oxidation of dihydropyrazine to pyridazine (with an efficiency of 95%). The targeting carrier must not be chemically modified by the oxidant. The oxidant is a quinone oxidant selected from the group consisting of chloranil, tetrafluorobenzoquinone, DDQ, NaNO 2 .

[0177] In a preferred embodiment, the diene is a symmetrically substituted diene in which at least one plane of symmetry passes through the nitrogen-nitrogen bond of at least one tetrazine ring, obtained from a precursor selected from:

[0178]

[0179]

[0180] wherein X is CH or N.

[0181] In another preferred embodiment, the diene is an asymmetrically substituted diene obtained from a precursor selected from:

[0182]

[0183] In another preferred embodiment, the isomer-free dienophile targeting vector is obtained from a precursor selected from the group consisting of:

[0184]

[0185] The targeting carrier is an antibody, nanobody, polymer, nanodrug, cell, protein, peptide or small molecule.

[0186] The labeled single isomer chemical entity targeting vector provided by the method of providing a labeled single isomer chemical entity targeting vector can be used for therapy, radiotherapy, theranostics, diagnosis or imaging, depending on the labeling agent or agent, or imaging agent or therapeutic agent and the targeting vector.

[0187] 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 group on the targeting vector.

[0188] In a preferred embodiment, the labeled single isomeric chemical entity targeting vector provided by the method of providing a labeled single isomeric chemical entity targeting vector is used for therapy.

[0189] In another preferred embodiment, the labeled single isomeric chemical entity targeting vector provided by the method of providing a labeled single isomeric chemical entity targeting vector is used for radiation therapy.

[0190] In another preferred embodiment, the labeled single isomeric chemical entity targeting vector provided by the method of providing a labeled single isomeric chemical entity targeting vector is used for theranostic use.

[0191] In another preferred embodiment, the labeled single isomeric chemical entity targeting vector provided by the method of providing a labeled single isomeric chemical entity targeting vector is used for diagnosis.

[0192] In another preferred embodiment, the labeled single isomeric chemical entity targeting vector provided by the method of providing a labeled single isomeric chemical entity targeting vector is used for imaging.

[0193] The following examples describe (1) the synthesis of tetrazines and TCOs representatively used in steps a) and b) of the present method for providing labeled single isomeric chemical entity targeting vectors, and (2) the click reaction and oxidation between these compounds to produce single isomeric pyridazines.

[0194] Example

[0195] Overview

[0196] All reagents and solvents were dried according to standard methods before use. Commercial reagents were used without further purification. TLC (thin layer chromatography) analysis was performed using silica gel 60F254 (Merck), and the samples were analyzed by UV absorption and / or immersion in 7% ethanolic sulfuric acid solution or KMnO4 solution (prepared with 1.5 g KMnO 4 , 10g K 2 CO 3 and 1.25 mL 10% NaOH dissolved in 200 mL water) and then observed the carbonization for detection. The results were analyzed by silica gel column chromatography (40-60 μm, ) or CombiFlashNextGen 300+ (Teledyne ISCO) for compound purification. Chloroform-d, methanol-d 4 or DMSO-d 6 As deuterated solvent, and residual solvent as internal standard, the HPLC was recorded on Brucker (400 and 600 MHz instruments). 1 H and 13 C NMR spectra. In all NMR experiments, the deuterated solvent signal was used as an internal standard. Chemical shifts are expressed in parts per million (ppm). Coupling constants (J values) are in Hertz (Hz). The multiplicity of 1H NMR signals is reported as follows: s, singlet; d, doublet; dd, doublet in a doublet; ddd, a pair of doublets in a triplet; dt, doublet in a triplet; t, triplet; q, quartet; m, multiplet; br, broad signal. NMR spectra of all compounds were reprocessed from the original FID files using MestReNova software (version 12.0.22023). Mass spectrometry was performed using MS-Acquity-A: Waters Acquity UPLC and QDa detector. Purification was performed by preparative HPLC on an Agilent 1260infinity system using a SymmetryPrep-C18 column, 17 mL / min H 2 O-MeCN gradient 50-100% in 15 minutes with 0.1% trifluoroacetic acid. All final compounds were >95% pure as determined by analytical HPLC. Analytical HPLC method: (Thermo UltiMate 3000) using a C-18 column ( 5u C18(2) 150x 4.6mm), eluent: A:H 2O contains 0.1% TFA, B: MeCN contains 0.1% TFA. Gradient from 100% A to 100% B in 15 minutes, back to 100% A in 4 minutes, flow rate 1.5 mL / min. Detection was by UV absorption at λ=254 nm on a UVD 170U detector.

[0197] Example 1

[0198] Synthesis of Symmetric Tetrazines and Their Precursors

[0199] Compounds I and XXXIV

[0200] Figure 1 . The reaction scheme for the synthesis of symmetrical tetrazines is shown. The reagents and conditions are as follows: i) NH 2 (CH 2 ) 2 R, MeCN, 12 hours, room temperature; ii) Boc 2 O, Et 3 N, DCM, 12 hours, room temperature; iii) Zn(OTf) 2 , NH 2 NH 2 , H 2 O, EtOH, 65°C, 24 hours; iv) HCl, dioxane, room temperature, 4 hours; v) tert-butyl bromoacetate, Et 3 N, DMF, 50°C, 12 hours; vi) TFA, DCM, room temperature, 2 hours; vii) MsCl, Et 3 N, DMAP, DCM, room temperature, 12 hours.

[0201] Figure 2 :Reagents and conditions: i) NH 2 (CH 2 ) 2 OH, MeCN, 12 hours, room temperature; ii) Boc 2 O, Et 3 N, DCM, 12 hours, room temperature; iii) Zn(OTf) 2 , NH 2 NH 2 ·H 2 O, EtOH, 65°C, 24 hours; iv) HCl, dioxane, room temperature, 4 hours; v) tert-butyl bromoacetate, Et 3 N, DMF, 50°C, 12 hours; vi) DAST, DCM, -78°C to room temperature, 4 hours; vii) TFA, DCM, room temperature, 2 hours.

[0202] Synthesis of 4-(((2-hydroxyethyl)amino)methyl)benzonitrile (3)

[0203] To a solution of ethanolamine in DCM (60 mL) was added dropwise a solution of 4-(bromomethyl)benzonitrile (4 g, 20.20 mmol) in DCM (20 mL). The reaction was stirred at room temperature for 1 hour. The organic phase was then washed with water (3 x 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); 1 H NMR (400 MHz, CDCl 3 )δ8.01–7.52(m,2H),7.47–7.30(m,2),4.05–3.76(m,2H),3.74–3.43(m,2H),2.73(dtd,J=10.7,6.0,1.7Hz,2H),2.47(s,1H); 13 C NMR (101 MHz, CDCl 3 )δ145.76,132.22,128.67,118.88,110.71,60.91,53.07,50.78.

[0204] Synthesis of tert-butyl (4-cyanobenzyl)(2-hydroxyethyl)carbamate (4)

[0205] 4-(((2-hydroxyethyl)amino)methyl)benzonitrile (1.62 g, 9.19 mmol) and Et 3 To a solution of N (2.56 mL, 18.39 mmol) in DCM (30 mL) was added Boc 2 O (2.10 gr, 9.65 mmol). The reaction was stirred at room temperature for 12 hours. Then water (50 mL) and K 2 CO 3 The solution was washed with saturated solution (50 mL) and then with anhydrous Na 2 SO 4 Drying, filtration and concentration under reduced pressure gave 2.36 g (93%) of the desired crude product (mixture of rotamers). Rf. = 0.4 (heptane / EtOAc 50 / 50); 1 H NMR (600 MHz, CDCl 3 )δ7.63(d,J=7.9Hz,2H),4.55–4.52(m,2H),3.74(s,2H),3.51–2.94(m,2H),2.69(s,1H),1.76–0.53(m,9H); 13 C NMR (151 MHz, CDCl 3)δ156.91,144.11,132.43,128.02,127.50,118.72,111.19,81.02,62.17,61.46,52.10,51.17,50.37,49.51,28.32.

[0206] Synthesis of 4-((2-fluoroethyl)amino)methyl)benzonitrile (5)

[0207] To 4-(bromomethyl)benzonitrile (0.78 g, 4.00 mmol) was added CH 3 KCN (40 mL) was added to the solution 2 CO 3 (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 and MgSO 4 Dry, filter and concentrate 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). 1 H NMR (400 MHz, CDCl 3 )δ7.55(d,J=8.2Hz,2H),7.40(d,J=8.0Hz,2H),4.63–4.48(m,1H),4.47–4. 37(m,1H),3.84(s,2H),2.93–2.84(m,1H),2.84–2.72(m,1H),1.65(s,1H). 13 C NMR (101 MHz, CDCl 3 ) δ145.6, 132.3, 128.6, 118.9, 110.9, 83.5 (d, J = 165.5Hz), 53.1, 49.1 (d, J = 19.7Hz).

[0208] Synthesis of tert-butyl 4-cyanobenzyl(2-fluoroethyl)carbamate (6)

[0209] To 4-((2-fluoroethyl)amino)methyl)benzonitrile (540 mg, 3.03 mmol) and Et 3 N (1.27 mL, 9.09 mmol) CH 2 Cl 2 Add Boc (40 mL) to the solution 2O (790 mg, 3.63 mmol), and the mixture was stirred at room temperature for 12 hours. 2 CO 3 Wash the solution with Na 2 SO 4 Dry, filter and concentrate 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). 1 H NMR (400 MHz, CDCl 3 )δ7.55(d,J=7.8Hz,2H),7.27(d,J=7.8Hz,2H),4.79–4.10(m,4H),3.62–3.28(m,2H),1.96–1.05(m,9H). 13 C NMR (101 MHz, CDCl 3 )δ155.4,144.2,143.8,132.4,128.1,127.5,118.7,111.1,83.2(d,J=168.2Hz),82.7(d,J=170.5Hz),52.1,51.2,47.7,28.3.

[0210] 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-(4-(tert-butoxycarbonyl)(2-fluoroethyl)amino)methyl)phenyl)-1,2,4,5-tetrazine-3-yl)benzyl)(2-hydroxyethyl)carbamate (8)

[0211] To tert-butyl 4-cyanobenzyl(2-fluoroethyl)carbamate (1.1 g, 3.95 mmol), tert-butyl (4-cyanobenzyl)(2-hydroxyethyl)carbamate (0.27 g, 0.99 mmol) and Zn(Otf) were added. 2 To a suspension of 4-nitropropene (0.72 g, 1.98 mmol) in ethanol (30 mL) was added hydrazine monohydrate (3.83 mL, 79 mmol). The mixture was stirred at 70 °C for 22 hours and after completion of the reaction, cooled at room temperature. The volatiles were removed under reduced pressure and the residue was dissolved in EtOH (40 mL). To the crude reaction was added NaNO 2(5.52 g, 80.00 mmol) in water (20 mL) was then added dropwise with HCl (2 M) until gas evolution ceased and the pH reached 2-3, resulting in a red mixture. The crude reaction was extracted with DCM (3 × 40 mL) and washed with brine (3 × 20 mL). The organic phase was collected and washed with MgSO 4 Dry, filter and concentrate under reduced pressure. Purify by flash chromatography (DCM / MeOH 98 / 2) to give 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); 1 H NMR (600 MHz, CDCl 3 )δ8.63(d,J=8.0Hz,4H),7.50(d,J=7.0Hz,4H),5.11–4.38(m,8H),3.97–3.26(m,4H),1.95–0.57(m,18H); 13 C NMR (151 MHz, CDCl 3 )δ163.77,155.61,155.57,143.70,130.81,128.51,128.21,127.87,83.18(d,J=167.9Hz),82.6 1(d,J=169.3Hz),72.44,65.78,52.09,51.09,47.67(d,J=19.9Hz), 47.19(d,J=21.0Hz),28.38.

[0212] 0.08 g (7%) of the second polar component, corresponding to tert-butyl (4-(6-((4-(tert-butoxycarbonyl)(2-fluoroethyl)amino)methyl)phenyl)-1,2,4,5-tetrazin-3-yl)benzyl)(2-hydroxyethyl)carbamate, was isolated as a red solid (mixture of rotamers). Rf=0.22 (DCM / MeOH 98 / 2); 1 H NMR (400 MHz, CDCl 3 )δ8.54(d,J=7.9Hz,4H),7.40(d,J=8.1Hz,4H),4.79–4.34(m,8H),3.52–3.25(m,5H),1.38(d,J=8.8Hz,18H).

[0213] Synthesis of N,N'-(((1,2,4,5-tetrazine-3,6-diyl)bis(4,1-phenylene))bis(methylene))bis(2-fluoroethane-1-amine)(9)

[0214] Di-tert-butyl(((1,2,4,5-tetrazine-3,6-diyl)bis(4,1-phenylene))bis(methylene))bis((2-fluoroethyl)carbamate) (0.15 g, 0.25 mmol) was treated with HCl (4M) in dioxane (1 mL). A precipitate formed. Filtration gave 0.1 g (83%) of the desired product as the hydrochloride salt. 1 H NMR(600MHz,DMSO)δ9.70(s,4H),8.61(d,J=8.0Hz,4H),7.89(d,J=8.0Hz,4H) ,4.86(t,J=4.6Hz,2H),4.78(t,J=4.6Hz,2H),4.37(s,4H),3.40-3.25(m,4H); 13 C NMR (151MHz, DMSO) δ 163.63, 136.83, 132.81, 131.66, 128.28, 80.09 (d, J = 165.1Hz), 50.24, 47.21 (d, J = 19.8Hz).

[0215] Synthesis of 2,2'-((((1,2,4,5-tetrazine-3,6-diyl)bis(4,1-phenylene))bis(methylene))bis((2-fluoroethyl)azadiacyl))diacetate (10)

[0216] To a suspension of N,N'-(1,2,4,5-tetrazine-3,6-diyl)bis(4,1-phenylene)bis(methylene)bis(2-fluoroethane-1-amine) (0.09 g, 0.20 mmol) in anhydrous DMF (3 mL) hydrochloride was added Et 3 N (0.13 mL, 1.00 mmol). . 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 × 15 mL) and brine (2 × 15 mL). The organic phase was collected and washed with MgSO 4 Drying, filtration and concentration under reduced pressure gave 0.11 g (91%) of the desired compound as a red oil. Rf = 0.48 (Heptane / EtOAc 70 / 30); 1 H NMR (400 MHz, CDCl 3)δ8.62(d,J=8.4Hz,4H),7.65(d,J=8.2Hz,4H),4.64(t,J=5.0Hz,2H),4.53(t,J=5.0Hz,2 H),4.04(s,4H),3.42(s,4H),3.14(t,J=5.0Hz,2H),3.07(t,J=5.0Hz,2H),1.51(s,18H); 13 C NMR (101 MHz, CDCl 3 )δ170.58,163.82,144.39,130.80,129.63,128.02,83.08(d,J=167.6Hz),81.19,58.48,55.69,53.70(d,J=20.0Hz),28.22.

[0217] Synthesis of 2,2'-((((1,2,4,5-tetrazine-3,6-diyl)bis(4,1-phenylene))bis(methylene))bis((2-fluoroethyl)azadiacyl))diacetic acid (I)

[0218] Di-tert-butyl 2,2'-(((((1,2,4,5-tetrazine-3,6-diyl)bis(4,1-phenylene))bis(methylene))bis((2-fluoroethyl)azepine))diacetate (0.12 g, 0.19 mmol) was added to 5 mL of CH 2 Cl 2 2 mL of TFA was added to the solution in 4% paraformaldehyde. The mixture was stirred at room temperature for 4 hours. The solvent was then removed under reduced pressure. Purification by preparative HPLC gave 0.70 g (50%) of the desired compound (TFA salt) as a red solid. 1 H NMR (600MHz, DMSO) δ8.51(d,J=8.2Hz,4H),7.67(d,J=7.9Hz,4H),4.60(t,J=5.0H z,2H),4.53(t,J=5.0Hz,2H),4.02(s,4H),3.46(s,4H),3.06(s,2H),3.02(s,2H); 13 C NMR (151MHz, DMSO) δ 172.57, 163.68, 144.56, 131.23, 130.11, 128.01, 82.92 (d, J = 165.3Hz), 58.08, 54.66, 53.64 (d, J = 19.8Hz).

[0219] Synthesis of 2-((4-(6-(4-((2-fluoroethyl)amino)methyl)phenyl)-1,2,4,5-tetrazine-3-yl)benzyl)amino)ethan-1-ol (11)

[0220] (4-(6-(4-((tert-Butyloxycarbonyl)(2-fluoroethyl)amino)methyl)phenyl)-1,2,4,5-tetrazin-3-yl)benzyl)(2-hydroxyethyl)carbamate (0.06 g, 0.1 mmol) was treated with HCl (4M) in dioxane (1 mL). A precipitate formed. Filtration gave 0.035 g (75%) of the desired product as the hydrochloride salt. 1 H NMR(600MHz,DMSO)δ9.64(s,4H),8.61(d,J=8.3Hz,4H),8.02–7.53(m,4H),5.28(s,1H),4.85(t,J=4.6Hz,1H),4.77 (t,J=4.6Hz,1H),4.37(s,2H),4.34(s,2H),3.73(s,2H),3.40(t,J=4.8Hz,1H),3.35(t,J=4.8Hz,1H),3.04(s,2H); 13 C NMR(151MHz,DMSO)δ163.63,137.00,136.86,132.81,132.74,131.64,128.29,1 28.27, 80.12 (d, J = 165.3Hz), 56.84, 50.27, 50.01, 49.12, 47.24 (d, J = 19.9Hz).

[0221] Synthesis of 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)glycine tert-butyl ester (12)

[0222] To a suspension of 2-((4-((6-((2-fluoroethyl)amino)methyl)phenyl)-1,2,4,5-tetrazin-3-yl)benzyl)amino)ethan-1-ol (0.03 g, 0.077 mmol) in anhydrous DMF (3 mL) hydrochloride was added Et 3 N (0.05 mL, 0.38 mmol). Tert-butyl bromoacetate (0.04 mL, 0.3 mmol) was added and the reaction was stirred at 50 °C for 6 hours. The mixture was cooled to room temperature and diluted with EtOAc (30 mL). The organic phase was washed with water (2 × 15 mL) and brine (2 × 15 mL). The organic phase was collected and washed with MgSO 4 Drying, filtration and concentration under reduced pressure gave 0.03 g (64%) of the desired compound as a red oil. Rf = 0.25 heptane / EtOAc 70 / 30); 1 H NMR (400 MHz, CDCl 3)δ9.44–8.40(m,2H),7.61(dd,J=9.6,8.2Hz,2H),4.62(t,J=5.0Hz,1H),4.50(t,J=5.0Hz,1H),4.02(s,2H),3.96(s,2H),3.63(s,2H) ,3.39(s,2H),3.29(s,2H),3.23(s,1H),3.11(t,J=5.0Hz,1H),3.04(t,J=5.0Hz,1H),2.91(t,J=5.1Hz,2H),1.48(s,9H),1.46(s,9H); 13 C NMR (151 MHz, CDCl 3 )δ170.99,170.60,163.87,163.75,144.47,143.63,131.04,130.76,129.75,129.64,128.15,128.05,83.09 (d,J=167.8Hz),81.71,81.20,59.12,58.56,58.48,56.99,55.71,55.52,53.70(d,J=20.1Hz),28.22,28.12.

[0223] Synthesis of 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)glycine tert-butyl ester (XXXIV)

[0224] To the 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 CH 2 Cl 2 To the solution in (10 mL) was added formyl chloride (0.01 g, 0.019 mmol) and DMAP (0.001 g, 0.01 mmol). The reaction was stirred at room temperature for 12 hours. The solvent was removed under reduced pressure. Purification by flash chromatography (80 / 20 heptane / ethyl acetate) gave 0.020 (48%) of the desired product as a red solid. Rf = 0.55 (Heptane / EtOAc 70 / 30); 1 H NMR (400 MHz, CDCl 3)δ8.60(d,J=8.1Hz,4H),7.62(dd,J=8.4,3.0Hz,4H),4.62(t,J=5.0Hz,1H),4.50(t,J=5.0Hz,1H),4.11–3.89( m,4H),3.57(t,J=6.8Hz,2H),3.39(s,2H),3.36(s,2H),3.15-3.10(m,3H),3.04(t,J=5.0Hz,1H),1.48(s,18H); 13 CNMR (101MHz, CDCl 3 )δ170.51,163.84,163.81,130.87,130.79,129.64,129.56,128.05,128.03,83.08(d,J= 167.8Hz), 81.35, 81.19, 58.48, 58.12, 55.96, 55.70, 55.50, 53.70 (d, J = 20.0Hz), 28.23.

[0225] Synthesis of 2,2'-((((1,2,4,5-tetrazine-3,6-diyl)bis(4,1-phenylene))bis(methylene))bis((2-hydroxyethyl)azadiacyl))diacetate (13)

[0226] To tert-butyl (4-cyanobenzyl)(2-hydroxyethyl)carbamate (4 g, 14.47 mmol) and Zn(OTf) 2 To a suspension of 40 mL of 4-nitropropene (2.63 g, 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 hours and after completion of the reaction, it was cooled at room temperature. The volatiles were removed under reduced pressure and the residue was dissolved in EtOH (80 mL). To the crude reaction was added NaNO 2 (19.97 g, 289.00 mmol) in water (50 mL), then HCl (2 M) was added dropwise until gas evolution stopped and the pH reached 2-3, resulting in a red mixture. The crude reaction was extracted with DCM (3 x 60 mL) and washed with brine (3 x 50 mL). The organic phase was collected and precipitated with MgSO 4 Dry, filter and concentrate under reduced pressure. Purify by flash chromatography (DCM / MeOH 95 / 5) to give 1.2 g (28%) of the desired product as a red solid (mixture of rotamers). Rf=0.21 (DCM / MeOH 95 / 5); 1 H NMR (600 MHz, CDCl 3)δ8.63(d,J=7.9Hz,4H),7.49(d,J=7.9Hz,4H),4.62(s,4H),3.79(s,4H),3.60–3.37(m,4H),3.01(s,2H),1.48(s,18H); 13 C NMR (151 MHz, CDCl 3 )δ163.74,157.21,156.07,143.55,130.84,128.25,127.88,80.91,62.19,61.44,52.12,51.13,50.28,49.41,28.39.

[0227] Synthesis of 2,2'-((((1,2,4,5-tetrazine-3,6-diyl)bis(4,1-phenylene))bis(methylene))bis((2-hydroxyethyl)azadiacyl))diacetate (14)

[0228] (4-(6-(4-((tert-Butyloxycarbonyl)(2-fluoroethyl)amino)methyl)phenyl)-1,2,4,5-tetrazin-3-yl)benzyl)(2-hydroxyethyl)carbamate (0.9 g, 1.55 mmol) was treated with a solution of HCl (4M) in dioxane (15 mL). A precipitate formed. Filtration gave 0.68 g (97%) of the desired product as the hydrochloride salt. 1 H NMR (400MHz, DMSO) δ9.41 (s, 4H), 8.59 (d, J = 8.4Hz, 4H), 7.89 (d, J = 8.4Hz, 4H), 5.29 (s, 2H), 4.33 (s, 4H), 3.74 (s, 4H), 3.04 (s, 4H); 13 C NMR (101MHz, DMSO) δ163.62,137.03,132.71,131.65,128.23,56.84,49.98,49.14.

[0229] Synthesis of 2,2'-((((1,2,4,5-tetrazine-3,6-diyl)bis(4,1-phenylene))bis(methylene))bis((2-hydroxyethyl)azadiacyl))diacetate (15)

[0230] To a suspension of the hydrochloride salt of 2,2'-((((1,2,4,5-tetrazine-3,6-diyl)bis(4,1-phenylene))bis(methylene))bis(2-hydroxyethyl)azadienyl)diacetate (0.25 g, 0.55 mmol) in anhydrous DMF (10 mL) was added Et3N (0.38 mL, 2.75 mmol). 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 (2×15 mL) and brine (2×15 mL). The organic phase was collected and precipitated with MgSO 4 Drying, filtration and concentration under reduced pressure gave 0.31 g (91%) of the desired compound as a red oil. Rf = 0.23 (heptane / EtOAc 60 / 40); 1 H NMR (400 MHz, CDCl 3 )δ8.28(d,J=8.0Hz,4H),7.27(d,J=8.0Hz,4H),3.62(s,4H),3.31(t,J=5.2Hz,4H),2.96(s,4H),2.58(t,J=5.1Hz,4H),1.14(s,18H); 13 C NMR (101 MHz, CDCl 3 )δ171.14,163.75,143.96,130.90,129.66,128.11,81.56,59.21,58.57,56.95,55.67.

[0231] Synthesis of 2,2'-(((((1,2,4,5-tetrazine-3,6-diyl)bis(4,1-phenylene))bis(methylene))bis((2-fluoroethyl)azadienyl))diacetate (10) and 2,2'-((((1,2,4,5-tetrazine-3,6-diyl)bis(4,1-phenylene))bis(methylene))bis((2-hydroxyethyl)azadienyl))diacetate (12)

[0232] 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)azepine))diacetate (0.24 g, 0.39 mmol) in anhydrous THF (15 mL) was added DAST (0.055 mL, 0.39 mmol). The resulting mixture was stirred at −78° C. for 1 hour and at room temperature for another 3 hours. Subsequently, NaHCO 3The saturated solution (10 mL) was quenched and stirred for 30 minutes. The reaction mixture was extracted with DCM (3×30 mL) and washed with brine (3×30 mL). The organic phase was collected and washed with MgSO 4 The mixture was dried, filtered and concentrated under reduced pressure. Purification by flash chromatography (DCM / MeOH 95 / 5) gave 0.12 g (50%) of 2,2'-((((1,2,4,5-tetrazine-3,6-diyl)bis(4,1-phenylene))bis(methylene))bis((2-fluoroethyl)azepine))diacetate (see Example x for NMR). At the same time, 0.04 g (14%) of a more polar component was isolated, corresponding to 2,2'-di-tert-butyl((((1,2,4,5-tetrazine-3,6-diyl)bis(4,1-phenylene))bis(methylene))bis((2-hydroxyethyl)azepine))diacetate.

[0233] Example 2

[0234] Synthesis of Asymmetric Tetrazines and Their Precursors

[0235] Compounds XII, XIII and their precursors XLVIII, L were obtained as disclosed in the following documents: García-Vázquez, R.; Battisti, UM; JT; Shalgunov, V.; Hvass, L.; Stares, DL; Petersen, IN; Crestey, FC; A.; Svatunek, D.; et al. Direct Cu-mediated aromatic 18F-labeling of highly reactive tetrazines for pretargeted bioorthogonal PETimaging. Chem. Sci. 2021, 12, 11668–11675 and Battisti, UM; Bratteby, K.; JT; Hvass, L.; Shalgunov, V.; Mikula, H.; A.; Herth,MMDevelopment of theFirst Aliphatic 18F-Labeled Tetrazine Suitable for Pretargeted PET Imaging—Expanding the Bioorthogonal Tool Box.J.Med.Chem.2021,64,15297–15312

[0236] Example 2.1

[0237] Compound XLVIII

[0238]

[0239] Scheme 5. Shows the synthesis of precursor XLVIII. Reagents and conditions: i) NBS, AIBN, CH 3 CN, reflux, 24 hours, 49%; ii) a) glycine tert-butyl ester hydrochloride, K 2 CO 3 ,CH 3 CN,25℃,24h,b)Boc 2 O,CH 2 Cl 2 ,rt,12h,91%;iii)a)CH 2 Cl 2 ,S 8 ,NH 2 NH 2 H 2 O, EtOH, 50℃, 24h, b) NaNO 2 ,AcOH,rt,30min,27%;iv)(Me 3 Sn) 2 ,Pd(PPh 3 ) 4 ,THF,65℃,MW,3h,47%.

[0240] 3-(Bromomethyl)-5-iodobenzonitrile(23)

[0241] To 3-iodo-5-methylbenzonitrile (2.50 g, 10.28 mmol) and N-bromosuccinimide (2.28 g, 12.86 mmol) was added CH 3 AIBN (0.67 g, 4.11 mmol) was added to the CN (40 mL) solution. The reaction was refluxed for 24 h. The solvent was removed under vacuum and the crude product was purified by flash chromatography (95 / 5 n-heptane / ethyl acetate) to give 1.61 g (49%) of the desired product as a white solid. Rf = 0.28 (95 / 5 n-heptane / EtOAc); 1 H NMR (400 MHz, CDCl 3 )δ7.96(d,J=1.6Hz,1H),7.89(d,J=1.6Hz,1H),7.64(t,J=1.6Hz,1H),4.38(s,2H) 13 C NMR (101 MHz, CDCl 3)δ142.20,140.90,140.12,131.67,116.55,114.56,94.05,29.90.

[0242] Tert-butyl 2-((tert-butoxycarbonyl)(3-cyano-5-iodophenyl)amino)acetate (24)

[0243] To 3-(bromomethyl)-5-iodobenzonitrile (2.00 g, 6.21 mmol) was added CH 3 KCN (40 mL) was added to the solution 2 CO 3 (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), and MgSO 4 Dry, filter and concentrate. Purify by flash chromatography (80 / 20 n-heptane / ethyl acetate); 1.91 g (83%) of the desired product was obtained as a colorless oil. Rf = 0.23 (80 / 20 n-heptane / EtOAC); 1H NMR (400 MHz, CDCl 3 )δ7.89(s,1H),7.79(s,1H),7.56(s,1H),3.72(s,2H),3.21(s,2H),1.91(s,1H),1.41(s,9H); 13C NMR(101MHz, CDCl 3 )δ171.24,143.30,141.56,139.06,130.89,117.13,114.11,93.95,81.68,51.74,50.76,28.13.

[0244] To the amine (1.9 g, 5.10 mmol) and Et 3 N (1.71 mL, 12.25 mmol) CH 2 Cl 2 Add Boc (40 mL) to the solution 2 O (1.41 g, 6.12 mmol). The reaction was stirred at room temperature for 12 hours. Then water (50 mL) and K 2 CO 3 The solution was washed with saturated solution (50 mL) and then with anhydrous Na 2 SO 4Drying, filtration and concentration under reduced pressure gave 2.5 g of crude product. Purification by flash chromatography (80 / 20 n-heptane / ethyl acetate) gave 2.2 g (91%) of the desired product as a colorless oil (60 / 40 unspecified rotational isomer mixture). Rf = 0.38 (80 / 20 n-heptane / EtOAc); 1 H NMR (400 MHz, CDCl 3 )δ7.99–7.80(m,2H),7.71–7.41(m,1H),4.48(s,1.2H),4.42(s,0.8H),3.86(s,0.8H),3.70(s,1.2H),1.51–1.42(m,18H); 13 C NMR (101 MHz, CDCl 3 )δ168.43,155.62,142.14,141.49,141.13,140.79,139.30,131.33,130.50,130.04,117. 00,114.29,94.03,82.03,81.93,81.30,81.13,50.85,50.56,49.78,49.53,28.22,28.05.

[0245] tert-Butyl 2-((tert-Butyloxycarbonyl)(3-iodo-5-(1,2,4,5-tetrazin-3-yl)benzyl)amino)acetate (25)

[0246] CH 2 Cl 2 (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) and tert-butyl 2-((tert-butyloxycarbonyl)(3-cyano-5-iodobenzyl)amino)acetate (2.10 g, 4.44 mmol) were added to a Biotage microwave vial (10-20 mL) equipped with a stir bar. The vessel was sealed and the reaction mixture was heated to 50° C. for 24 hours, then cooled to room temperature and unsealed. Then 3 ml of CH 2 Cl 2 and NaNO 2 (1.84 g, 26.73 mmol) in water (30 ml) was added to the now yellow mixture, and then acetic acid (10 mL) was added dropwise to give a red mixture. The reaction mixture was washed with CH 2 Cl 2 Extract, wash with brine, and precipitate with MgSO 4Dry, filter and concentrate in vacuo. The crude product was purified by flash chromatography (95 / 5 n-heptane / EtOAc) to give 0.64 g (27%) of the desired product as a red solid (60 / 40 unspecified rotamer mixture). Rf = 0.31 (80 / 20 n-heptane / EtOAc); 1 H NMR (400 MHz, CDCl 3 )δ10.18(s,1H),8.84–8.71(m,1H),8.45–8.36(m,1H),7.87–7.74(m,1H),4.54(s,1 .2H),4.47(s,0.8H),3.85(s,0.8H),3.69(s,1.2H),1.44–1.36(m,9H),1.38(s,9H); 13 C NMR (101 MHz, CDCl 3 )δ168.63,165.14,158.04,155.71,155.39,141.70,141.46,141.26,140.93,136.06,135.93,133.58,133 .52,126.70,126.48,95.21,81.83,81.74,81.07,80.85,51.11,50.71,49.47,49.33,28.31,28.26,28.07.

[0247] N-(3-(1,2,4,5-tetrazine-3-yl)-5-(trimethyltinyl)benzyl)-N-(tert-butyloxycarbonyl)glycine tert-butyl ester (XLVIII)

[0248] Pd(PPh 3 ) 4 (19.4 mg, 10%) and hexamethylditin (87 μL, 0.42 mmol) were added sequentially into a microwave vial equipped with a stirring bar, which was then sealed and heated with N 2 Purge. A solution of tert-butyl 2-((tert-butyloxycarbonyl)(3-iodo-5-(1,2,4,5-tetrazine-3-yl)benzyl)amino)acetate (0.08 g, 0.17 mmol) in anhydrous THF (2.5 mL) was added via syringe and the reaction was stirred in a microwave oven at 65 °C for 3 hours. The reaction was cooled to room temperature and unsealed, then quenched with saturated aqueous KF solution (1 mL). CH 2 Cl 2 The solution was extracted with (3 x 10 mL), washed with brine (10 mL), and precipitated with MgSO 4Dry, filter and concentrate under reduced pressure. The crude product was purified by flash chromatography (95 / 5 n-heptane / EtOAc) to give 0.055 g (66%) of the desired product as a purple oil (60 / 40 unspecified rotational isomer mixture). Rf = 0.38 (80 / 20 n-heptane / ethylene oxide); 1 H NMR (400 MHz, CDCl 3 )δ10.21(s,1H),8.74–8.60(m,1H),8.45–8.37(m,1H),7.70–7.61(m,2H),4.65(s,1.2H ),4.58(s,0.8H),3.90(s,0.8H),3.75(s,1.2H),1.49(s,9H),1.44(s,9H),0.36(s,9H); 13 C NMR(101MHz,Chloroform-d)δ168.85,166.70,157.76,155.81,155.62,144.69,140.25,139.75,138.58,138.33 ,134.55,131.09,127.41,81.62,81.51,80.69,80.51,51.60,51.17,49.25,48.95,28.37,28.29,28.07,-9.34.

[0249] Example 3

[0250] Synthetic isomer-free TCO

[0251]

[0252] Scheme 6. Synthesis of isomer-free TCO. i) mCPBA, THF, H 2 O, 0℃→RT, 17 hours, quantitative; ii) HClO 4 , THF, H 2 O, room temperature, 24 hours (48%); iii) silver nitrate, AgNO 3 , hV, room temperature, 8 hours, 60%; iv) oxalyl chloride, DMSO, Et 3 N, DCM, -78°C, 3 hours (62%); v) 2-aminoaniline, MeOH, room temperature, 0.2 hours, quantitative.

[0253] (Z)-9-Oxacyclo[6.1.0]non-4-ene (26). Cyclooctadiene (1.00 g, 9.24 mmol) was dissolved in dry DCM (20 mL), cooled to 0°C, and mCBPA (2.07 g, 9.24 mmol) was added portionwise. After addition, the reaction was stirred at 0°C for 30 minutes, and then the reaction was warmed to room temperature. The reaction was stirred for 17 hours. 2M K 2 CO 3 (20 mL) to quench the reaction and extract the crude product with 3×DCM (15 mL). The combined organic phases were washed with K 2 CO 3 (25 mL) and brine (25 ml). The solvent was evaporated and the crude product was used in the next reaction without purification.

[0254] (Z)-cyclooct-5-ene-1,2-diol (27). 25 (1.15 g, 9.24 mmol) was dissolved in HClO 4 (250 μL, 2.90 mmol) in a mixture of THF / water (3:2 v / v, 50 mL). The reaction was stirred at room temperature for 24 hours. Water (10 mL) was added to the reaction mixture and 3×Et 2 O (20 mL) to extract the product. The combined ether fractions were washed with 2 × 2 M NaHCO 3 (25 mL) and washed. The crude product was purified by CombiFlash to give a clear viscous oil (624 mg, 4.40 mmol, 48%). 1 H NMR (600 MHz, deuterated chloroform) δ 5.64–5.56 (m, 2H), 3.72–3.65 (m, 2H), 2.41–2.33 (m, 2H), 2.17–2.09 (m, 4H), 1.63–1.55 (m, 2H); 13 C NMR (151 MHz, deuterated chloroform) δ 129.3, 74.1, 33.6, 22.9.

[0255] (E)-Cyclooct-5-ene-1,2-diol (28)

[0256] The compound was obtained as described in the following literature, 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. The bottom of the flash cylinder (220 g, screw cap, Luer lock interface, product number FCSTLL-220-6) was filled with 8 cm of silica (particle size 15-40 microns) and continued to be filled with silver nitrate impregnated silica until the top. The chromatographic column was rinsed with 9:1 ether / n-heptane (500 mL) and protected from light with aluminum foil. The cooling fence and UV lamp were turned on, and no silver leakage was detected after 10 minutes. Methyl benzoate (1 mL), 27 (1 g) and another 50 mL of 9:1 ether / n-heptane solution were added to a round-bottom flask. The mixture was then added to a quartz flask. The pump was turned on (flow rate = 100 mL / min) and the photoreactor was started to carry out the photoisomerization reaction for 8 hours. After 8 hours, the photoreactor was turned off and the chromatographic column was dried with air flow. 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 minutes, filtered and the organic layer was collected. The organic layer was washed with brine and MgSO 4 Drying, filtration and concentration gave 0.60 g (60%) of the product as a pale yellow oil (a mixture of axial and equatorial isomers); 1 H NMR (CDCl 3 ,400MHz)δ5.45-5.36(m,2H),3.58(bs,2H),3.47(m,2H),2.35-2.21(m,4H),2.02-1.98(m,2H),1.76-1.65(m,2H). 13 C NMR (CDCl3, 100MHz) δ 132.6, 76.0, 40.7, 32.6.

[0257] (E)-Cyclooct-5-ene-1,2-dione (29)

[0258] Under argon atmosphere, at -78 ° C, a solution of DMSO (1.05 mL, 14.77 mmol) in anhydrous DCM (30 mL) was added dropwise to a solution of (COCl) 2 (736 μL, 8.44 mmol) in anhydrous DCM (15 mL). After addition, the reaction mixture was stirred at -78 ° C for 30 minutes. A solution of 28 (500 mg, 3.52 mmol) in anhydrous DCM (10 mL) was added dropwise, and the reaction mixture was stirred for another 30 minutes at -78 ° C. Et3N (4.90 mL, 35.16 mmol) was added dropwise, and the reaction mixture was kept at -78 ° C for another 60 minutes. The reaction mixture was allowed to warm to room temperature and stirred for another hour. The reaction mixture was washed with water (50 mL), 2×0.5 M HCl (aqueous solution) (50 mL) and brine (30 mL). The organic phase was washed with MgSO 4 Drying and evaporation gave the desired compound as a clear liquid (302 mg, 2.19 mmol, 62%).

[0259] (E)-6,7,10,11-Tetrahydrocycloocta[b]quinoxaline (30)

[0260] The compound was obtained as described 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). The corresponding 2-aminoaniline (110 mg, 1 mmol) was added to the solution and the mixture was stirred at room temperature. After 30 minutes, the mixture was heated with CH 2 Cl 2 The crude mixture was extracted with 10 mL (3 × 10 mL). The combined organic layers were dried (MgSO 4 ), after filtration and removal of the solvent, 0.2 g (97%) of the desired product was obtained. 1 H NMR (400 MHz, CDCl 3 )δ8.23–7.93(m,2H),7.77–7.55(m,2H),6.19–5.93(m,1H),5.24–5.09(m,1H),3.40–3.23(m,2H),3.22–3.12(m ,1H),3.02(td,J=12.8,2.8Hz,1H),2.80–2.69(m,1H),2.69–2.54(m,1H),2.50–2.38(m,1H),2.36–2.25(m,1H).

[0261] Example 3.1

[0262]

[0263] Scheme 7. Synthesis of isomer-free TCO. i) sulfuric acid, nitric acid, 80 ° C, 0.5 hours (56%); ii) urea, 150 ° C, 6 hours (87%); iii) tin (II) chloride, ethanol, reflux, 15 hours (quantitative); iv) Cs 2 CO 3 , tert-butyl bromoacetate or methyl bromoacetate, DMF, 120°C, 4 hours (33-49%); v) Method A: tert-butyl 2-(5,6-diamino-1,3-dioxoisoindolin-2-yl) acetate, TFA, DCM, room temperature, 4 hours (99%); Method B: methyl 2-(5,6-diamino-1,3-diketoisoindolin-2-yl) acetate, concentrated hydrochloric acid (aqueous solution), dioxane, 70°C, 24 hours (99%); vi) acetic acid,

[0264] (E)-cyclooct-5-ene-1,2-dione, DMF, room temperature, 6 hours (27%); vii) LiOH, THF, H 2 O, rt, 3 h (89%); viii) N-hydroxysuccinimide, DCC, DMF, rt, 18 h (72%).

[0265] 5-Chloro-6-nitrosoisoindoline-1,3-dione (31)

[0266] 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 hours. After cooling to room temperature, the dark red solution was poured into ice. The resulting yellow precipitate was collected by filtration, washed with water, and dried in vacuo to give a light yellow solid, which was recrystallized from EtOH to give 3.5 grams (56% yield) of the desired product. Rf = 0.45 (n-Heptane / EtOAc 60 / 40); 1 H NMR (400MHz, DMSO) δ11.87(s,1H),8.51(s,1H),8.27(s,1H); 13 CNMR(101MHz,DMSO)δ167.21,167.17,151.86,136.54,133.00,130.89,126.60,120.20.

[0267] 5-Amino-6-nitrosoisoindoline-1,3-dione (32)

[0268] The compound was synthesized according to the literature. 1 A mixture of 5-chloro-6-nitrosoisoindoline-1,3-dione (2.4 g, 10.59) and urea (6.36 g, 105.92 mmol) was stirred and heated to 150°C. Place under argon for 6 hours. After cooling to room temperature, the solid was suspended in hot water (80°C), filtered, and washed with hot water (3x50 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); 1 H NMR (600MHz, DMSO) δ11.43(s,1H),8.56–8.05(m,3H),7.40(s,1H); 13 C NMR (151MHz, DMSO) δ168.01, 150.64, 138.24, 132.50, 122.38, 117.74, 114.65.

[0269] 5,6-Diaminoindoline-1,3-dione (33)

[0270] The compound was synthesized according to the literature. 1 A suspension of 4-amino-5-nitrophthalimide (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 hours. The resulting bright red suspension was cooled to room temperature, and the red solid was collected by filtration, washed with ethanol and dried in vacuo to obtain a reddish-orange solid. Rf = 0.15 (95 / 5 DCM / MeOH); 1 H NMR (400MHz, DMSO) δ10.25(s,1H),6.81(s,2H),5.50(s,4H); 13 C NMR (101MHz, DMSO) δ170.72, 140.30, 122.83, 107.03.

[0271] tert-Butyl 2-(5,6-diamino-1,3-dioxoisoindolin-2-yl)acetate (34)

[0272] To Cs 2 CO 3 To a suspension of 5,6-diaminoindoline-1,3-dione (1.93 g, 5.92 mmol) and 5,6-diaminoindoline-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 reactants were heated at 120 ° C for 4 hours under argon. The mixture was cooled to room temperature and water (20 mL) was added. The reaction mixture was extracted with DCM (3×50 mL). The organic phase was washed with anhydrous MgSO 4Dry, filter and concentrate under reduced pressure. The resulting residue was purified by 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); 1 H NMR (600MHz, DMSO) δ6.89(s,2H),5.61(s,4H),4.11(s,2H),1.40(s,9H); 13 C NMR (151MHz, DMSO) δ168.63, 167.62, 140.46, 121.55, 107.34, 82.08, 40.55, 28.08.

[0273] Methyl 2-(5,6-diamino-1,3-dioxoisoindolin-2-yl)acetate (35)

[0274] To Cs 2 CO 3 To a suspension of 5,6-diaminoindoline-1,3-dione (1.93 g, 5.92 mmol) and 5,6-diaminoindoline-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 reactants were heated at 120 °C under argon for 4 hours. The mixture was cooled to room temperature and water (20 mL) was added. The reaction mixture was extracted with DCM (3 × 50 mL). The organic phase was washed with anhydrous MgSO 4 Dry, filter and concentrate under reduced pressure. The resulting residue was purified by 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); 1 H NMR (400MHz, DMSO) δ6.90(s,2H),5.63(s,4H),4.25(s,2H),3.67(s,3H); 13 C NMR (101MHz, DMSO) δ169.13, 168.50, 140.52, 121.47, 107.37, 52.72, 38.69.

[0275] 2-(5,6-Diamino-1,3-dioxoisoindolin-2-yl)acetic acid (36)

[0276] Method A

[0277] 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 hours 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); 1 H NMR (400MHz, DMSO) δ6.90 (s, 2H), 4.13 (s, 2H); 13 C NMR (101MHz, DMSO) δ169.95,168.60,140.16,121.80,107.71,38.86.

[0278] Method B

[0279] 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 hydrochloric acid (1 mL). The reaction was stirred at 70 °C for 24 hours 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); 1 H NMR (400MHz, DMSO) δ6.90 (s, 2H), 4.13 (s, 2H); 13C NMR (101MHz, DMSO) δ 169.95, 168.60, 140.16, 121.80, 107.71, 38.86.

[0280] (E)-tert-Butyl 2-(1,3-dioxo-1,3,6,7,10,11-hexahydro-2H-cycloocta[b]pyrrolo[3,4-g]quinoxalin-2-yl)acetate (37)

[0281] 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 hours. The solution was diluted with 1 mL of water and directly purified by preparative HPLC to give 0.09 g (17%) of the desired compound as a yellow solid. Rf = 0.36 (heptane / EtOAc 75 / 25); 1H NMR (400MHz, CDCl3) δ8.61–8.37(m,2H),6.10(ddd,J=15.8,10.7,3.9Hz,1H),5.15(ddd,J=16.3,9.5,5.8Hz,1H),4.42(s,2H),3.40–3.28(m ,2H),3.24(dd,J=10.9,5.1Hz,1H),3.08(td,J=12.8,2.8Hz,1H),2.85–2.66(m,2H),2.45(q,J=11.9Hz,1H),2.39–2.24(m,1H),1.48(s,9H).

[0282] (E)-2-(1,3-dioxo-1,3,6,7,10,11-hexahydro-2H-cycloocta[b]pyrrolo[3,4-g]quinoxalin-2-yl)acetate (38)

[0283] To (E)-cyclooct-5-ene-1,2-dione (0.21 g, 1.54 mmol) was added 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). The reaction was stirred at room temperature in the dark for 6 hours. The solution was diluted with 10 mL of water and directly purified by preparative HPLC to give 0.15 g (26%) of the desired compound as a yellow solid. Rf = 0.31 (heptane / EtOAc 75 / 25); 1 H NMR(600MHz, CDCl3)δ8.53–8.46(m,2H),6.10(ddd,J=15.7,10.8,3.9Hz,1H), 5.14(ddd,J=16.2,9.5,5.8Hz,1H),4.53(s,2H),3.79(s,3H),3.34(ddt,J=14 .9,11.1,5.4Hz,2H),3.24(dd,J=11.2,5.1Hz,1H),3.08(td,J=12.8,2.8Hz,1 H), 2.84–2.65 (m, 2H), 2.50–2.39 (m, 1H), 2.33 (ddd, J = 11.1, 9.3, 5.5Hz, 1H).

[0284] (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)

[0285] To (E)-cyclooct-5-ene-1,2-dione (0.21 g, 1.54 mmol) was added 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). The reaction was stirred at room temperature in the dark for 6 hours. The solution was diluted with 10 mL of water and directly purified 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); 1 H NMR (600MHz, DMSO) δ13.33(s,1H),8.47(d,J=9.4Hz,2H),6.18(ddd,J=17.2,10.7,3.8Hz,1H),5.17–5.08(m,1H),4.42(s,2H),3.52–3.43(m,1H),3 .23(dd,J=9.2,3.1Hz,2H),3.12(dd,J=11.2,5.1Hz,1H),2.71(tt,J=17. 0,6.0Hz,2H),2.32(qd,J=11.0,7.6Hz,1H),2.17(tt,J=11.3,5.4Hz,1H); 13 C NMR (151MHz, DMSO) δ169.10,166.50,161.93,161.59,143.21,143.07,137 .16,132.73,130.81,130.63,125.34,125.12,46.31,41.35,34.30,28.33.

[0286] 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)

[0287] To a solution of (E)-2-(1,3-dioxo-1,3,6,7,10,11-hexahydro-2H-cycloocta[b]pyrrolo[3,4-g]quinoxaline-2-yl)acetic acid (0.06 g, 0.18 mmol) in anhydrous DMF (3 mL) was added DCC (0.04 g, 0.19 mmol) followed by N-hydroxysuccinimide (0.04 g, 0.35 mmol). The reactants were stirred at room temperature overnight under argon. EtOAc (10 mL) was added and the organic layer was washed with water (2 x 10 mL) and brine (2 x 10 mL). The organic phase was washed with anhydrous MgSO 4Dry, filter and concentrate 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 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); 1 H NMR (400MHz, CDCl3) δ8.56–8.47(m,2H),6.10(td,J=12.1,5.9Hz,1H),5.25–5.07(m,1H),4.87(s,2H),3.40–3.30(m ,2H),3.30–3.21(m,1H),3.15–3.04(m,1H),2.92–2.68(m,6H),2.45(q,J=11.9Hz,1H),2.33(dt,J=12.1,6.1Hz,1H); 13 C NMR (101MHz, CDCl3) δ168.16,165.58,163.15,161.67,161.31,143.40,143.24,136.5 4,132.67,130.16,130.00,126.26,126.07,46.74,41.81,37.05,34.19,28.26,25.55.

[0288] Example 3.2

[0289]

[0290] Scheme 8. Synthesis of isomer-free TCO. i) AcOH, glycine methyl ester HCl, reflux, 16 hours (58%); ii) NaN 3 , MeOH, room temperature, 17 hours (89%); iii) Pd / C (10%), H 2 , EtOH, room temperature, 24 hours (95%); iv) AcOH, (E)-cyclooct-5-ene-1,2-dione, DMF, room temperature, 6 hours (24%).

[0291] 2-(3,4-Dichloro-2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetic acid methyl ester (41)

[0292] To a solution of dichloromaleic anhydride (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 at reflux for 16 hours. The solvent was removed under reduced pressure. Water (100 mL) was added and the resulting suspension was stirred for 1 hour. The solid was filtered to give 4.15 g (58%) of the desired product as a beige solid. 1H NMR (400MHz, DMSO) δ4.41 (s, 2H), 3.70 (s, 3H); 13 C NMR (101MHz, DMSO) δ167.96, 162.82, 133.39, 53.09, 40.13.

[0293] 2-(3,4-diazide-2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetic acid methyl ester (42)

[0294] 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 in vacuo, and 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 in vacuo to give the compound (3.89 g, 89%) as a yellow solid. 1 H NMR (400MHz, DMSO) δ4.34 (s, 2H), 3.70 (s, 3H); 13 C NMR (101MHz, DMSO) δ168.14, 164.29, 120.16, 53.03, 39.25.

[0295] 2-(3,4-diazide-2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetic acid methyl ester (43)

[0296] 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 H 2 The catalyst was then filtered on a pad of celite and the solvent removed to give 1.2 g (95%) of the desired compound as a red oil. NMR was normal. 1 H NMR (400MHz, DMSO) δ4.94 (s, 4H), 4.09 (s, 2H), 3.65 (s, 4H); 13 C NMR (101MHz, DMSO) δ169.38, 169.16, 118.89, 52.63, 38.46.

[0297] (E)-2-(1,3-Dioxo-1,3,5,6,9,10-hexahydro-2H-cycloocta[b]pyrrolo[3,4-E]pyrazin-2-yl)acetate (45)

[0298] A solution of methyl 2-(3,4-diazide-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 hours. The solution was diluted with 10 mL of water and directly purified by preparative HPLC to give 0.1 g (24%) of the desired compound as a yellow solid. Rf = 0.28 (Heptane / EtOAc 75 / 25); 1 H NMR (600MHz, CDCl3) δ6.11(ddd,J=15.7,10.8,3.9Hz,1H),5.13(ddd,J=16.2,9.5,5.8Hz,1H),4.53(s,2H),3.77(s,3H),3.35(ddt,J=14.9,11.1, 5.4Hz,2H),3.26(dd,J=11.2,5.1Hz,1H),3.08(td,J=12.8,2.8Hz,1H),2 .85–2.63(m,2H),2.50–2.39(m,1H),2.32(ddd,J=11.1,9.3,5.5Hz,1H): 13 C NMR (151MHz, CDCl3) δ167.20,164.20,163.25,143.86,128.71,52.87,38.83,35.36,27.06.

[0299] Example 3.3

[0300]

[0301] Scheme 9. Synthesis of isomer-free TCO. i) NH 4 CO 3 , methyl 4-oxobutanoate, MeOH, room temperature, 16 hours (63%), ii)

[0302] LiOH,H 2 O, THF, rt, 3 h (76%).

[0303] (E)-3-(4,5,8,9-tetrahydro-1H-cycloocta[d]imidazol-2-yl)propanoic acid methyl ester (46)

[0304] In a 15 mL screw neck vial, ammonium bicarbonate (384 mg, 4.86 mmol) and methyl 4-oxobutanoate (85%, 331 mg, 2.43 mmol) were stirred in 2 mL MeOH at room temperature for 15 min. (E)-Cyclooct-5-ene-1,2-dione (305 mg, 2.21 mmol) was added in 3 mL MeOH and stirred for 16 h. The reaction mixture was diluted with 0.1% TFA (aq.) and submitted to preparative HPLC to give methyl (E)-3-(4,5,8,9-tetrahydro-1H-cyclooct[d]imidazol-2-yl)propanoate as a beige viscous liquid (325 mg, 1.39 mmol, 63%). 1 H NMR (599.65MHz, MeOD) δ = 5.64–5.56 (m, 2H), 3.70 (s, 3H), 3.20–3.15 (m, 2H), 2.95 (dt, J = 13.8, 6. 5Hz,2H),2.88(t,J=7.0Hz,2H),2.76–2.70(m,2H),2.35(d,J=6.1Hz,2H),2.28(h,J=6.7Hz,2H). 13 C NMR (150.78MHz, MeOD) δ = 173.2, 136.0, 129.9, 56.6, 52.5, 31.5, 30.81, 29.0, 22.1.

[0305] (E)-3-(4,5,8,9-Tetrahydro-1H-cycloocta[d]imidazol-2-yl)propanoic acid (47)

[0306] In a 5mL screw neck vial, 1M LiOH solution (861μL, 861μmol) was added to a 2.5mL THF / water (3:2v / v) solution of (E)-3-(4,5,8,9-tetrahydro-1H-cyclooctaalkylimidazol-2-yl)methyl propionate (100mg, 287μmol). The suspension was stirred at room temperature for 3 hours. The reaction was quenched with 1M TFA (aqueous solution) and preparative HPLC was performed to obtain (E)-3-(4,5,8,9-tetrahydro-1H-cycloocta[d]imidazol-2-yl)propionic acid as a light yellow amorphous solid. (73mg, 218μmol, 76%). 1H NMR (599.65MHz, DMSO) δ = 12.52 (s, 1H), 5.57–5.48 (m, 2H), 3.03 (t, J = 7.2Hz, 2H), 2.88 (dt, J = 14.2, 6.4Hz, 2H), 2.77 (t, J =7.2Hz,2H),2.65(ddd,J=14.4,6.7,4.9Hz,2H),2.24(ddd,J=11.5,9.4,4.5Hz,2H),2.16(ddd,J=12.1,7.4,4.3Hz,2H). 13 CNMR (150.78MHz, DMSO) δ = 172.6, 142.67, 134.9, 127.6, 30.5, 29.2, 27.8, 20.8.

[0307]

[0308] Scheme 10. Synthesis of E,E-cyclooctadiene. v) AcOOH, Na 2 CO 3 ,DCM,0℃→room temperature,48h(65%);vi)nBuLi,PH(Ph) 2 ,AcOH,H 2 O 2 ,THF,-78℃ to room temperature, 12 hours, 52%; vii)NaH,THF,-78℃ to room temperature, 12 hours, 25%.

[0309] cis,cis-5,10-dioxacyclo[7.1.0.04,6]decane (48)

[0310] The synthesis method of this compound is described in the following literature: 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. Sodium carbonate (20.5 g, 50.84 mmol, 1.1 eq) was added to a stirred solution of (Z,Z)-1,5-cyclooctadiene (5 g, 46.2 mmol, 1.0 eq) in DCM (25 mL). The suspension was cooled to 0 °C and peracetic acid (36%, 17.9 mL, 97.1 mmol, 2.1 eq) was added. The mixture was warmed to room temperature and stirred until the reaction was complete after 48 h. The reaction mixture was heated ... 2 CO3 The reaction was quenched with 1% ethyl acetate and then extracted with EtOAc (4 × 25 mL). The combined organic layers were washed with MgSO 4 Drying, concentration and distillation gave cis,cis-5,10-dioxacyclo[7.1.0.04,6]decane (4.19 g, 29.89 mmol, 65%). 1 H NMR (400 MHz, CDCl 3 )δ2.97–2.88(m,4H),2.00–1.89(m,4H),1.89–1.77(m,4H); 13 C NMR (101 MHz, CDCl 3 )δ56.02,22.01.

[0311] ((trans,trans)-2,6-dihydroxycyclooctane-1,5-diyl)bis(diphenylphosphine oxide)(49)

[0312] The synthesis method of this compound is described in the following literature: H.; Neves, AA; Day, HA; Stairs, S.; Brindle, KM; Leeper, FJ (E,E)-1,5-Cyclooctadiene: a small and fastclick-chemistry multitalent. Chemical Communications 2011, 47, 7203-7205.

[0313] To a solution of diphenylphosphine (10.43 mL, 11.16 g, 59.92 mmol, 2.1 eq) and cis, cis-5,10-dioxacyclo-[7.1.0.04,6]decane (4.0 g, 28.5 mmol, 1.0 eq) in THF (120 mL) was added BuLi (2 M in hexane, 29.96 mL, 59.92 mmol, 2.1 eq). The mixture was stirred for 1 hour, warmed to room temperature and stirred for 12 hours. The brown solution was cooled to 0 °C and diluted with THF (86 mL) and the reaction mixture was stirred by adding AcOH (4.9 mL, 5.14 g, 85.60 mmol, 3 eq) and H 2 O 2(30%, 8.7 mL, 85.60 mmol, 3 equiv.) was quenched. The mixture became clear and a white precipitate was subsequently formed. The mixture was removed from the ice bath and vigorously stirred at 25 °C for 2 hours, then the suspension was transferred to a separatory funnel, EtOAc (150 mL) and H2O (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 washed with MgSO 4 Drying, filtration and concentration afforded a white solid. The remaining AcOH was removed azeotropically with toluene to afford an isomeric mixture of phosphine oxides (8.14 g, 14.9 mmol, 52%). The spectral data were consistent with previously published data.

[0314] (E,E)-1,5-cyclooctadiene(50)

[0315] The synthesis method of the compound is described in the following literature: 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.

[0316] The phosphine oxide mixture (6.00 g, 11.0 mmol, 1.0 eq.) was dissolved in DMF (30 mL) under stirring under an Ar atmosphere. The clear solution became cloudy and NaH (60% mineral oil, 1.44 g, 34.2 mmol, 3.1 eq.) was added in portions at room temperature over 5 minutes under a positive argon stream. The reaction mixture was stirred at room temperature for 12 hours, cooled to 0°C, diluted with pentane (50 mL), and washed with half-saturated NH 4 The reaction mixture was quenched with Cl (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 afford (E,E)-1,5-cyclooctadiene (25% yield). The spectral data were consistent with previously published data.

[0317]

[0318] Scheme 11. Synthesis of isomer-free TCO X. i) CHCl 3 , reflux, 2 days 63%) ii) 33, CH 2 Cl 2 , room temperature, 18 hours, (50%).

[0319] 6-Benzyl-5,7-dioxo-6,7-dihydro-5H-pyrrolo[3,4-d]pyridazine-1,4-dicarboxylic acid dimethyl ester (51) 1,2,4,5-tetrazine-3,6-dicarboxylic acid dimethyl ester (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 under reflux for two days. After the reaction, the dihydropyridazine spontaneously oxidized to the corresponding pyridazine. After complete consumption of 1,2,4,5-tetrazine-3,6-dicarboxylic acid dimethyl ester, the reaction was cooled to room temperature. The product was purified by CombiFlash to give a light solid (54 mg, 0.16 mmol, 63%). 1 H NMR (600 MHz, CDCl 3 )δ7.54(dd,J=8.3,6.8Hz,2H),7.50–7.47(m,1H),7.42–7.38(m,2H),4.17(s,6H). 13 C NMR (151 MHz, CDCl 3 )δ162.5,162.2,159.2,149.5,138.2,130.3,129.7,126.5,54.3.

[0320] (E)-1,3-Dioxo-2-phenyl-2,3,5,6,9,10-hexahydro-1H-cycloocta[f]isoindole-4,11-dicarboxylic acid dimethyl ester (52)

[0321] To a CH 2 Cl 2 To the solution, compound 33 dissolved in pentane (0.06 mmol) was added. The solution was stirred at room temperature for 18 hours. The volatiles were then removed under reduced pressure. Purification by flash chromatography gave 12 mg (50%) of the desired product as a white solid. 1 H NMR (400 MHz, CDCl 3 )δ7.45(dd,J=8.4,6.9Hz,2H),7.37(t,J=7.4Hz,1H),7.35–7.30(m,2H),5.77(t,J=5 .5Hz,2H),3.89(s,6H),2.61–2.36(m,2H),2.36–2.21(m,2H),1.65(d,J=8.1Hz,4H).

[0322] Example 4

[0323] Connecting asymmetric tetrazines to isomer-free TCOs

[0324]

[0325] Solution 12.

[0326] 1-(4-Fluorophenyl)-5,6,13,14-tetrahydropyridazine[4',5':5,6]cycloocta[1,2-b]quinoxaline(53)

[0327] 3-(4-fluorophenyl)-1,2,4,5-tetrazine was obtained as described in the following literature, A.; Petersen Ida, N.; Herth Matthias, M.; Kristensen Jesper, L.

[0328] Compound 30 (12 mg, 0.057 mmol) was dissolved in MeCN / H 2 O (4 / 1 v / v, 2.5 mL), 3-(4-fluorophenyl)-1,2,4,5-tetrazine (10 mg, 0.057 mmol) was added thereto. The reaction was stirred at room temperature for 30 minutes. p-Chlorobenzoquinone (17 mg, 0.068 mmol) was added and the reaction was stirred for 10 minutes. Then, the reaction was treated with H 2 O (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. 1 H NMR (600 MHz, CDCl 3 )δ8.71(s,1H),8.04–7.72(m,2H),7.70–7.56(m,2H),7.42(ddd,J=8.5,5.3,1.0Hz,2H),3 .56(t,J=7.4Hz,2H), 3.45(t,J=7.4Hz,2H), 3.32(t,J=7.4Hz,2H), 3.16(t,J=7.5Hz,2H); 13 C NMR (151 MHz, CDCl 3)δ163.25(d,J=248.9Hz),161.19,154.70(d,J=3.2Hz),151.67,141.20,141.01,138.61,136.66,133.02(d,J=3.5 Hz), 130.93 (d, J = 8.3Hz), 129.66, 128.54 (d, J = 7.8Hz), 122.46, 115.59 (d, J = 21.6Hz), 35.62, 34.52, 30.11, 27.39.

[0329]

[0330] Solution 13.

[0331] 2-(1-(4-(2-fluoroethoxy)phenyl)-9,11-dioxo-5,6,9,11,14,15-hexahydro-10H-pyridazin[4',5':5,6]cycloocta[1,2-b]pyrrolo[3,4-g]quinoxalin-10-yl)acetic acid (54)

[0332] 3-(4-(2-Fluoroethoxy)phenyl)-1,2,4,5-tetrazine was obtained as described in the following literature: García Vázquez 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 methyl (E)-2-(1,3-dioxo-1,3,6,7,10,11-hexahydro-2H-cycloocta[b]pyrrolo[3,4-g]quinoxalin-2-yl)acetic acid (5 mg, 0.0148 mmol) was added in H 2 O (2 mL) was added to the stirred solution. p-Chlorobenzoquinone (18 mg, 0.074 mmol) was added and the reaction was stirred for 15 minutes. Then, the reactants were treated with H 2 The solution was concentrated under reduced pressure and purified by preparative HPLC, and all fractions containing pure product were lyophilized to give the product as a white solid (5 mg, 0.0095 mmol, 64%). 1 H NMR (600 MHz, DMSO-d 6)δ13.31(br s,1H),8.92(d,J=1.9Hz,1H),8.42(d,J=8.5Hz,2H),7.43–7.35(m,2H),7.22–7.14(m,2H),4.87–4.84(m,1H),4.80–4.76(m,1H), 4.41–4.36(m,3H),4.36–4.33(m,1H),3.68(t,J=7.2Hz,2H),3.57(t,J=7.2Hz,2H),3.44(t,J=7.3Hz,2H),3.27(d,J=14.6Hz,2H); 13 C NMR (151MHz, DMSO) δ169.03,166.40,166.38,161.23,159.76,159.61,159.07,151.52,143.51,143.39,139.90,137.24,130. 95,130.93,130.84,129.80,125.13,125.08,114.91,82.64(d,J=166.5Hz),67.68(d,J=18.9Hz),35.03,34.82,28.80,27.08.

[0333]

[0334] Solution 14.

[0335] 3-(6-(4-(2-fluoroethoxy)phenyl)-4,5,10,11-tetrahydro-1H-imidazo[4',5':5,6]cycloocta[1,2-d]pyridazin-2-yl)propanoic acid methyl ester 2,2,2-trifluoroacetate (55)

[0336] 3-(4-(2-Fluoroethoxy)phenyl)-1,2,4,5-tetrazine was obtained as described in García Vázquez 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 (E)-3-(4,5,8,9-tetrahydro-1H-cyclooctaimidazol-2-yl)propanoic acid methyl ester (15 mg, 0.043 mmol) in MeCN / H2O (50 v / v%, 1 mL). p-Chlorobenzoquinone (53 mg, 0.215 mmol) was added and the reaction was stirred for 25 minutes. The reactants were then treated with H 2O (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 give the product as a yellow solid (5 mg, 0.012 mmol, 27%). 1 H NMR (600MHz, MeOD) δ8.99(s,1H),7.41(d,J=8.7Hz,2H),7.15(d,J=8.6Hz,2H),4.83–4.72(m,2H),4.37–4.28(m,2H),3.62(s,3H),3. 37(t,J=7.0Hz,2H), 3.26(t,J=7.0Hz,2H), 3.21(t,J=7.0Hz,2H), 3.05(t,J=6.9Hz,2H), 2.98(t,J=7.0Hz,2H), 2.79(t,J=6.9Hz,2H).

[0337]

[0338] Solution 15.

[0339] 3-(6-(4-iodophenyl)-4,5,10,11-tetrahydro-1H-imidazo[4',5':5,6]cycloocta[1,2-d]pyridazin-2-yl)propanoic acid methyl ester 2,2,2-trifluoroacetate (56)

[0340] 3-(4-Iodophenyl)-1,2,4,5-tetrazine was obtained according to the following literature, García Vázquez 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 (E)-3-(4,5,8,9-tetrahydro-1H-cyclooctaimidazol-2-yl)propanoic acid methyl ester (20 mg, 0.057 mmol) was added in MeCN / H 2 O (50 v / v%, 1 mL) was added to a stirred solution. p-Chloroaniline (71 mg, 0.287 mmol) was added and the reaction was stirred for 15 minutes. 2 The reaction was diluted with 0.2% O (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 give the product as a yellow solid (21 mg, 0.034 mmol, 61%). 1H NMR (600MHz, MeOD) δ9.03 (s, 1H), 7.94 (d, J = 8.3Hz, 2H), 7.24 (d, J = 8.3Hz, 2H), 3.62 (s, 3H), 3.37 (t, J = 7. 0Hz, 2H), 3.21 (td, J = 7.0, 4.0Hz, 4H), 3.05 (t, J = 6.9Hz, 2H), 2.97 (t, J = 7.0Hz, 2H), 2.79 (t, J = 7.0Hz, 2H). 13 C NMR(151MHz,MeOD)δ173.11,162.90,153.09,146.19,142.67,139.88,139.08,137.3 2,131.95,128.12,127.91,96.19,52.53,31.21,28.48,26.91,26.11,24.82,22.02.

[0341] Example 5

[0342] Connection between symmetric tetrazines and isomer-free TCOs

[0343]

[0344] Solution 16.

[0345] 1,4-Bis(pyridin-2-yl)-5,6,13,14-tetrahydropyridazine[4',5':5,6]cycloocta[1,2-b]quinoxaline. (57)

[0346] 30 (50 mg, 0.24 mmol) was dissolved in MeCN / H 2 O (4 / 1 v / v, 5 mL), 3,6-di(pyridin-2-yl)-1,2,4,5-tetrazine (56 mg, 0.24 mmol) was added thereto. The reaction was stirred at room temperature for 30 minutes. p-Chlorobenzoquinone (117 mg, 0.47 mmol) was added and the reaction was stirred for 10 minutes. Then, the reaction was treated with H 2 O (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. 1 H NMR (400 MHz, CDCl 3 )δ8.79(d,J=4.8Hz,2H),8.05–7.93(m,4H),7.91(t,J=7.7Hz,2H),7.68(dt,J=6.4,3 .7Hz,2H),7.42(dd,J=7.5,4.9Hz,2H),3.62(t,J=6.4Hz,4H),3.51(t,J=6.6Hz,4H);13 C NMR (101 MHz, CDCl 3 )δ158.39,156.56,156.12,148.64,140.73,140.42,137.11,129.42,128.43,125.09,123.61,36.57,27.69.

[0347] Radiolabeling

[0348] General approach:

[0349] Unless otherwise stated, all reagents and solvents were purchased from ABX, Sigma-Aldrich, Fluorochem, and VWR 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 performed under an inert atmosphere (nitrogen) and using oven-dried glassware (152°C). Thin layer chromatography (TLC) was performed on silica-coated aluminum plates (Silica Gel 60F254) from Merck, and spots were visualized by UV at 254nm, anisaldehyde, and / or potassium permanganate staining.

[0350] Example 6

[0351] Symmetric tetrazine 18 F radiolabeling

[0352]

[0353] Scheme 17. Radiolabeling of symmetrical tetrazines. I) [ 18 F]Bu 4 NF / Bu 4 NOMs PO 4 3- ,t-BuOH / DMSO,100℃,5min; ii)TFA,CH 3 CN,80℃,10min.

[0354] Symmetrical 18 F-labeled tetrazine [ 18 The preparation method of F]I is as follows:

[0355] Received from the cyclotron 18 F] The fluoride aqueous solution was passed through a pretreated anion exchange resin (Sep-PakLight QMA column (cartridge)). 3 PO 4 The QMA was then rinsed with 10 mL of H2 O washing to pre-treat QMA. 18 F]F- was eluted from QMA into a 4 mL v-shaped vial with 1 mL Bu 4 NOMs were dissolved in MeOH. The eluate was dried at 100 °C for 5 min under a nitrogen stream. Precursor XXXIV (9.3 μmol, 6 mg) was dissolved in 167 μL DMSO and then diluted with 833 μL tBuOH. The solution was added to dry [ 18 F] fluoride solution and allowed to react at 100 °C for 5 minutes. After adding 3 mL of H 2 The reaction was cooled to 50°C with air before evaporation. The radiochemical conversion (RCC) after the first step was 54% as determined by radioHPLC. The crude mixture was applied to a Sep-pak plus C18 solid phase extraction (SPE) column which was purified by eluting with 10 mL of EtOH and 10 mL of H 2 Pre-treat the SPE with another 5 mL of HO. 2 O and dried with N2. The product was eluted from the SPE with 2 mL MeCN into a 7 mL v-shaped vial containing 600 μL TFA. The mixture was reacted at 80°C for 10 minutes. The RCC of [18F]I determined by radio-HPLC was 95% ( Figure 3 ).

[0356] Radio-HPLC was performed on a Luna 5 μm C18(2) column (150 × 4.6 mm) using acetonitrile (CH 3 CN) in water containing 0.1% TFA. Gradient conditions: 0 min–0% CH 3 CN, 0-10min–CH 3 CN content increases linearly to 100%, 10-12min–100% CH 3 CN, 12-13min–CH 3 CN content decreases linearly to 0%, 13-15min – 0% CH 3 CN, elution rate 2mL / min.

[0357] Figure 3 The end of deprotection is shown. 18 Radio-HPLC of [F]I.

[0358] Example 7

[0359] Asymmetric tetrazine 18 F radiolabeling

[0360] Asymmetry 18 F-labeled tetrazine [ 18F] XIII is prepared from the Nosyl precursor (p-nitrobenzenesulfonyl precursor) XXXI by nucleophilic substitution as described by Battisti, UM; Bratteby, K.; JT; Hvass, L.; Shalgunov, V.; Mikula, H.; A. Herth, MM Development of the First Aliphatic18F-Labeled Tetrazine Suitable for Pretargeted PET Imaging—Expanding the Bioorthogonal Tool Box. J. Med. Chem. 2021, 64, 15297–15312.

[0361] Example 8

[0362] Asymmetric tetrazine 18 F radiolabeling

[0363] Prepared using the general method described in the following literature 18 F-labeled tetrazines, with slight modifications, were prepared by García-Vázquez et al. (doi:10.3390 / ph15020245).

[0364]

[0365] Solution 18.

[0366] The [18F] fluoride aqueous solution received from the cyclotron was diluted with 10 mL of 0.5 M K 3 PO 4 Pre-treated Sep-Pak Light QMA column. Use Bu4NOMs solution (20mM MeOH solution, 1mL) to 18 F]F- was eluted from the QMA 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 evaporation of the methanol, acetonitrile (0.5 mL) was added to the same vial and evaporated under the same conditions to remove traces of water. Two acetonitrile additions were performed. Nosyl precursor LIX (1.5 mg) was dissolved in anhydrous DMSO (0.2 mL), diluted with tBuOH (0.8 mL), and dried [ 18 After 5 min at 100 °C, the reaction was cooled to 80 °C with ambient air flow, diluted with water (2 mL), and purified by semi-preparative HPLC: Luna 5μM C18 (2) 250mm×10mm column, isocratic elution with 66% acetonitrile in 20mm citrate buffer pH 6.1, elution rate 6mL / min. The product peak (retention time 10.2min) was collected, diluted with water (50-100mL), and passed through a Sep-pak Plus C18 short solid phase extraction column (Waters, USA), which was pre-treated by flushing with EtOH / water mixture (1 / 1v / min, 10mL). The Sep-pak column was then flushed with additional water (5mL), purged with nitrogen, eluted with 1-2mL of organic solvent (MeCN or EtOH), and diluted with water to achieve the desired organic solvent concentration for click / oxidation experiments.

[0367] Obtained 18 The radiochemical yield of [F]XX was 39±13% (n=4) and the radiochemical purity was >95%.

[0368] Figure 4 :show[ 18 F] The ultraviolet trajectory of XX.

[0369] Figure 5 :show[ 18 Radioactive traces of F]XX.

[0370] Figure 6 :show[ 18 F]XX analysis of UV and radioactive traces by HPLC.

[0371] HPLC conditions: Luna 5 μm C18(2) column (150×4.6 mm), acetonitrile (CH 3 CN) in water containing 0.1% TFA. Gradient conditions: 0-1 min–25% CH 3 CN, 1-8min–CH 3 CN content increases linearly to 95%, 8-9 min – 95% CH 3 CN, 9-9.5min–CH 3 CN content decreases linearly to 25%, 9.5-10min–25% CH 3 CN, elution rate 1.5mL / min.

[0372]

[0373] Solution 19.

[0374] Received from the cyclotron 18 F] Fluoride aqueous solution was prepared by using 10 mL of 0.5 M K 3 PO 4Pre-treated Sep-Pak Light QMA columns. 4 NOTf solution (20 mM MeOH solution, 1 mL) was added 18 F]F- was eluted from the QMA column 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 evaporation of the methanol, acetonitrile (0.5 mL) was added to the same vial and evaporated under the same conditions to remove traces of water. Nosyl precursor XLVI (1.5 mg) was dissolved in anhydrous acetonitrile (0.2 mL), diluted with tBuOH (0.8 mL), and dried [ 18 After 5 min at 100 °C, the reaction was cooled to 80 °C with ambient air flow, diluted with water (2 mL), and purified by semi-preparative HPLC: Luna 5μM C18 (2) 250mm×10mm column, isocratically eluted with 55% acetonitrile in 20mm citrate buffer pH 6.1 at 5mL / min. The product peak (retention time 11.5min) was collected, diluted with water (50-100mL), and passed through a Sep-pak Plus C18 short solid phase extraction column (Waters, USA), which was pre-treated by flushing with an ethanol / water mixture (1 / 1v / min, 10mL). The Sep-pak column was then flushed with additional water (5mL), purged with nitrogen, eluted with 1-2mL of organic solvent (MeCN or EtOH), and diluted with water to achieve the desired organic solvent concentration for click / oxidation experiments.

[0375] Obtained 18 The radiochemical yield of [F]X was 11±5% (n=3) and the radiochemical purity was >95%.

[0376] Figure 7 :[ 18 UV trace of F]X-semipreparative HPLC.

[0377] Figure 8 :[ 18 Radioactive traces of F]X-semipreparative HPLC.

[0378] Fig. 9 :[ 18 F]X analysis of HPLC for UV and radioactive traces.

[0379] HPLC conditions: Luna 5 μm C18(2) column (150×4.6 mm), acetonitrile (CH 3 CN) in water containing 0.1% TFA. Gradient conditions: 0-1 min–15% CH 3CN, 1-8min–CH 3 CN content increased linearly to 85%, 8-9 min–85% CH 3 CN, 9-9.5min–CH 3 CN content decreased linearly to 15%, 9.5-10min–15% CH 3 CN, elution rate 1.5mL / min.

[0380]

[0381] Plan 20.

[0382] Received from the cyclotron 18 F] The fluoride aqueous solution was prepared by using 5 mL of 0.5 M K 3 PO 4 Pretreated 45 mg PS anion exchange column (Synthra). 4 NOTf solution (20 mM in MeOH, 0.6 mL) was added 18 F]F- was eluted from the PS column 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 evaporation of the methanol, acetonitrile (0.5 mL) was added to the same vial and evaporated under the same conditions to remove traces of water. Two acetonitrile additions were performed. Nosyl precursor XLVII (1.5 mg) was dissolved in anhydrous acetonitrile (0.3 mL), diluted with tBuOH (0.7 mL), and dried [ 18 F] fluoride residue. After 5 minutes of reaction at 100°C, 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 5μm, 250mm×10mm column, isocratic elution with 50% acetonitrile in 20mm citrate buffer pH 6.1, elution rate 5mL / min. The product peak (retention time 9.0min) was collected, diluted with water (50-100mL), and passed through a Sep-pak Plus C18 short solid phase extraction column (Waters, USA), which was pre-treated by flushing with an EtOH / water mixture (1 / 1v, 10mL). The Sep-pak column was then flushed with additional water (5 mL), purged with nitrogen, eluted with 1-2 mL of organic solvent (MeCN or EtOH), and diluted with water to achieve the desired organic solvent concentration for click / oxidation experiments.

[0383] Obtained 18 The radiochemical yield of [F]XI was 16±10% (n=4) and the radiochemical purity was >95%.

[0384] Fig.10UV trace-semi-preparative HPLC of [18F]XI is shown

[0385] Fig.11 Showing traces of radioactivity of [18F]XI - Semi-preparative HPLC

[0386] Fig.12 UV and radioactivity traces of [18F]XI are shown - Analytical HPLC

[0387] HPLC conditions: Luna 5 μm C18(2) column (150×4.6 mm), acetonitrile (CH 3 CN) in water containing 0.1% TFA. Gradient conditions: 0-1 min–25% CH 3 CN, 1-8min–CH 3 CN content increases linearly to 60%, 8-9 min–60% CH 3 CN, 9-9.5min–CH 3 CN content decreases linearly to 25%, 9.5-10min–25% CH 3 CN, elution rate 1.5mL / min.

[0388] Example 9

[0389] Asymmetric tetrazine 125 I radiolabeling

[0390] Asymmetry 125 I-labeled tetrazine [ 125 I] XVII was prepared from the tin-based precursor LVI as described by BattistiUmberto, M.; Herth Matthias, M.; A.;Garcia,R.NUCLIDE LABELLED H-TETRAZINESAND USE THEREOF FOR PET AND SPECT PRETARGETED IMAGING AND RADIONUCLIDETHERAPY.WO 2021 / 228992A1,2021 / 05 / 12,2021.

[0391] Fig.13 Shows rough 125 I] Radio-HPLC of XVII

[0392] Example 10

[0393] 211 Radioactive Labeling of Asymmetric Tetrazine with At

[0394] Asymmetry 211At-labeled tetrazine [ 211 At] XIV was prepared from the tin-based precursor LIV as described by Battisti Umberto, M.; Herth Matthias, M.; A.;Garcia,R.NUCLIDE LABELLED H-TETRAZINES AND USE THEREOF FOR PET AND SPECT PRETARGETED IMAGING ANDRADIONUCLIDE THERAPY.WO 2021 / 228992 A1,2021 / 05 / 12,2021.

[0395] Fig.14 Shows [ 211 UV and radioactive traces of At]XIV-semi-preparative HPLC

[0396] Fig.15 The purified [ 211 Radioactivity trace of At]XIV - analytical HPLC

[0397] Embodiment 11

[0398] Click assay with radiolabeled tetrazines

[0399] In the click experiment, excess TCO reacts with non-carrier added radiolabeled 18 F-Tz reaction. The half-life of a bimolecular reaction is determined by the concentration of the excess reagent. Therefore, in a click experiment, the observed conversion time is determined by the TCO concentration and all 18 F Tz.

[0400] In combination with 18 TCO stock solution (20-2000 μM) was prepared in a solvent mixture matching the F-Tz solution. 18 The F-Tz solution was mixed rapidly at a 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 allowed to stand at room temperature for a certain period of time, and then a sample was injected on a radio-HPLC to evaluate the click conversion rate. The results are shown in Fig.16 shown.

[0401] Example 12

[0402] Oxidant screening for the oxidation of dihydropyridazines to pyridazines yields a single end product

[0403] Fig.17 and Fig.18The top shows the reaction between tetrazine and TCO dissolved in 1:1H 2 O / EtOH (% v / v). The cycloaddition was completed within 5 minutes to obtain several isomers. The oxidant was then added to obtain the final single isomer product. Each oxidant (5 equivalents) was added to the mixture and the reaction was analyzed by HPLC-MS after 60 minutes. These screening experiments surprisingly showed that not all oxidants can be used to provide a single isomeric form of tetrazine-TCO-pyridazine. For all quinone-based oxidants, the corresponding pyridine is formed. Inorganic-based embodiments cannot convert the click product (dihydropyridazine) into the corresponding pyridine within 60 minutes. Fig.19 HPLC analysis after oxidation of the tested tetrazine-TCO-pyridazines is shown.

[0404] Oxidation of Radiolabeled Compounds

[0405] Fig. 20 The top shows the solution in 1:1H 2 O / EtOH (% v / v) 18 Reaction between F-tetrazine and TCO. As described in the click section, by mixing 18 F-Tz and TCO solutions 18 Crude solutions of F-click products (50-100 μL) were used for oxidation experiments. The oxidation protocol included the introduction of an oxidant. The oxidation mixture was placed in a closed vial at room temperature for the desired period of time, after which a sample was removed and analyzed by HPLC.

[0406] Example 13

[0407] Compatibility of targeting vectors with oxidative conditions:

[0408] To test whether conditions that lead to oxidation of click products do not result in degradation of typical targeting vectors, we subjected a series of vectors relevant to therapeutic radiopharmaceutical development to oxidative conditions that have previously been shown to efficiently convert dihydropyridazines to mono-product pyridazines.

[0409] The structure of the test vector is as follows Fig.21 shown.

[0410] Vector Oxidation Test Procedure: Solutions of targeted vectors (70 μM) and oxidants (350 μM, 5 equiv.) in EtOH / water mixtures (89-94% EtOH v / v) were 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 degradation, meaning that they did not react with the oxidants. Results are shown in Table 1. Fig. 22 As shown, these supports are compatible with the oxidants tested.

[0411] Embodiment 14

[0412] Measurement of second-order rate constants

[0413] According to the method described in the following literature, Battisti et al. J. Med. Chem. 2021, 64, 20, 15297–15312 (see page 15310 for experimental details and influencing factors), the secondary rate constants of all click reactions performed in the previous examples were measured by stopped flow spectroscopy in phosphate buffered saline (PBS) at 25 ° C. In brief, stopped flow measurements were performed using an SX20-LED stopped flow spectrophotometer (Applied Photophysics) equipped with a 535nm LED (optical path 10mm, full width half maximum 34nm) to monitor the characteristic tetrazine visible light absorbance (520-540nm). The reagent syringe was filled with an axial TCO-PEG4 solution, and the instrument was pretreated. Subsequent data for each tetrazine were collected in triplicate. The reaction was carried out in PBS at 25 ° C and automatically recorded during acquisition. The 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.

[0414] Only in phosphate buffered saline at 25°C did it show a minimum second-order rate constant of 500 M -1 s -1 Reactions with lower kinetics are considered suitable to provide adequate rate kinetics and, therefore, reactions with lower kinetics are ignored for the purposes of the present method.

Claims

1. A method for providing a labeled single isomer chemical entity targeting vector, the method comprising: a) labeling a first chemical entity having anti-electron demanding Diels-Alder cycloaddition reactivity and conjugated to a pharmaceutical, imaging or therapeutic agent with a labeling agent; wherein the first chemical entity is selected from: a symmetrically substituted diene, an asymmetrically substituted diene or an isomer-free dienophile, wherein at least one plane of symmetry passes through the nitrogen-nitrogen bond of at least one tetrazine ring; and b) linking the labeled first chemical entity obtained in step a) to a second chemical entity having a complementary anti-electron demand Diels-Alder cycloaddition reactivity and conjugated to the targeting carrier; wherein the second chemical entity is selected from: a symmetrically substituted diene, an asymmetrically substituted diene or an isomer-free dienophile wherein at least one plane of symmetry passes through the nitrogen-nitrogen bond of at least one tetrazine ring, The inverse electron demand Diels-Alder cycloaddition reaction kinetics between the first and second chemical entities was carried out in phosphate buffered saline (PBS) at 25°C with 500 M -1 s -1 The minimum rate constant, determined by stopped-flow spectrophotometry, The premise is that when the labeling agent in step a) is 94 Tc, 99m Tc, 211 At 223 Ra or 225 When Ac, the labeling agent is conjugated to the asymmetrically substituted diene; and c) oxidizing the ligated tagged targeting vector obtained from step b) by adding 1 to 100 equivalents of chlorobenzoquinone, tetrafluorobenzoquinone, DDQ or NaNO2 at a temperature of 15°C to 50°C for up to 60 minutes.

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

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 method according to claim 3, wherein the labeling agent is selected from: 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 You, 113 You, 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 Purpose, 212 Purpose, 213 Purpose, 31 P, 32 P, 33 P, 32 S, 35 S, 45 Sc, 47 Sc, 84 Wed, 86 Wed, 87 Wed, 88 Wed, 89 Wed, 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 If, 74 If, 75 If, 76 If, 77 If, 78 If, 80 If, 82 If, 94 Tc, 99m Tc, 103 Rh, 103 mRh, 119 Sb, 121 Sb, 123 Sb, 135 Let, 138 Let, 139 Let, 162 Is, 164 Is, 165 Is, 166 Is, 167 Is, 168 Is, 170 Is, 193 mPt, 195 mPt, 192 Currently, 194 Currently, 195 Currently, 196 Currently, 198 Currently, 211 To, 223 Raw, 225 Ac.

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

6. A method according to any one of the preceding claims, wherein the oxidant is supported by a solid phase.

7. The process of claim 1, wherein the diene is a symmetrical tetrazine having the formula Tz1: Where R and R1 are wherein the curl symbol indicates a link to a tetrazine; wherein R2 is -H or (i) an isotope labeling agent directly linked to an aromatic ring; or (ii) an isotope labeling agent linked to an aromatic ring via a linker selected from (CH2) n ,-LO(CH2) n ,-LNH(CH2) n ,-LCONH(CH2) n ,-LNHCO(CH2) n , where L is -(CH2) m or -(CH2CH2O) m , wherein n and m are independently selected from 1-25; or (iii) an isotope labeling agent chelated by a chelating agent selected from: 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-triazacyclo-1-yl) Glutaric acid (NODAGA), 2-(4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane-1-yl)glutaraldehyde (DOTAGA), 14,7-triazacyclononanephosphonic acid (TRAP), 14,7-triazacyclononane-1-methyl(2-carboxyethyl)phosphonic acid-4,7-bis(methyl(2-hydroxymethyl)phosphonic acid (NOPO), 3,6,9,15-tetraazacyclopentadecan-1(15), 11,13-triene-3,6,9-triacetic acid N'-(5 -Acetyl(hydroxy)aminopentyl-N-(5-(5-aminopentyl)(hydroxy)amino-4-oxobutanoyl)amino)pentyl-N-hydroxysuccinamide (DFO), diethylenetriaminepentaacetic acid (DTPA), trans-cyclohexyldiethylenetriaminepentaacetic acid (CHX-DTPA), 1-oxo-4,7,10-triazacyclododecane-4,7,10-triacetic 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) are connected to the aromatic ring via a linker selected from (CH2)n, -LO(CH2)n, -LNH(CH2)n, -LCONH(CH2)n, -LNHCO(CH2)n, wherein L is -(CH2)m or -O(CH2CH2O)m, and n and m are independently selected from 1-25; in, When R2 is (i) or (ii), the isotope labeling agent is selected from: 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 OF, 124 OF, 125 OF, 127 OF, 131 OF, 211 And, 15 O, 16 O, 17 O, 18 Oh,, 43 Sc, 44 Sc, 45 Sc, 45 Water, 46 Water, 47 Water, 48 Water, 49 Water, 50 Water, 55 Co, 58 mCo, 59 Co, 60 The, 61 The, 63 The, 64 The, 65 The, 67 The, 67 Come on, 68 Come on, 69 Come on, 71 Come on, 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 Through, 113 You, 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 Purpose, 212 Purpose, 213 Purpose, 31 P, 32 P, 33 P, 32 S, 35 S 45 Sc, 47 Sc, 84 Wed, 86 Wed, 87 Wed, 88 Wed, 89 Wed, 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 If, 74 If, 75 If, 76 If, 77 If, 78 If, 80 If, 82 If, 94 Tc, 99m Tc, 103 Rh, 103 mRh, 119 Sb, 121 Sb, 123 Sb, 135 Here, 138 Let, 139 Let, 162 Is, 164 Is, 165 Is, 166 Is, 167 Is, 168 Is, 170 Is, 193 mPt, 195 mPt, 192 Currently, 194 Currently, 195 Currently, 196 Currently, 198 Currently, wherein X and Y are independently selected from: -CH- and -N-; wherein R3 is independently selected from H or selected from hydroxyl, sulfonamide, carboxyl, sulfonyl, amine, substituted amine having 1-5 polyethylene glycol units, -(O-CH2-CH2) n -OCH2-COOH, n is selected from 1-5, or methyl, ethyl, propyl, optionally substituted heteroaryl and optionally substituted arylalkyl; wherein, the optional substitution relative to the substituted amine refers to 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) heteroalkyl, (C3-30C10) cycloalkyl, (C3- ... 10) heterocycloalkyl, (C3-10) cycloalkyl, (C3-C10) (0) heterocycloalkyl, (C1-C10) haloalkyl, (C1-C10) perhaloalkyl, (C2-C10) -alkenyloxy, (C3-C10) -alkynyloxy, aryloxy, aralkyloxy, heteroaryloxy, heteroaralkyloxy, (C1-C6) alkoxy-(C1-C4) alkyl, optionally substituted aryl, optionally substituted heteroaryl and optionally substituted aralkyl; wherein optionally substituted means selected from halogen, hydroxy, sulfonamide, carboxyl, sulfonyl, amine, substituted amine with 1 to 5 polyethylene glycol units, -(O-CH2-CH2) n -OCH2-COOH, wherein n is selected from 1-5, or one or more substituents of H, methyl, ethyl, propyl, optionally substituted heteroaryl and optionally substituted arylalkyl; wherein the optional substitution relative to the substituted amine refers to one or more substituents selected from halogen, hydroxyl, sulfonamide, carboxyl, sulfonyl and amine; Wherein R and R1 are the same or differ only in the isotope number of the labeling agent.

8. The method according to claim 7, wherein the symmetrical tetrazine is selected from:

9. The process of claim 1, wherein the diene is an asymmetric tetrazine having the formula Tz2: wherein R4 is -H or (i) an isotope labeling agent directly attached to the aromatic ring, or (ii) an isotope labeling agent attached to the aromatic ring via a linker selected from (CH2) n , -LO(CH2) n , -LNH(CH2) n , -LCONH(CH2) n , -LNHCO(CH2) n , where L is -(CH2) m or -(CH2CH2O) m , wherein n and m are independently selected from 1-25, or (iii) the isotope labeling agent chelated by a chelating agent is selected from: 1,4,7,10-tetraazacyclododecane NE-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-triazacyclononane-1-yl)pentanedioic acid (NODAGA), 2-(4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane-1-yl)pentanedione iodic acid (DOTAGA), 14,7-triazacyclononanephosphonic acid (TRAP), 14,7-triazacyclononane-1-methyl(2-carboxyethyl)phosphonic acid-4,7-bis(methyl(2-hydroxymethyl)phosphonic acid (NOPO), 3,6,9,15-tetraazacyclononanephosphonic acid (TAP), 1,4,7-tri ... Bicyclo 9.3.1.pentadeca-1(15),11,13-triene-3,6,9-triacetic acid (PCTA), N'-(5-acetyl(hydroxy)aminopentyl-N-(5-(aminopentyl)(hydroxy)amino-4-oxobutanoyl)amino)pentyl-N-hydroxysuccinamide (DFO), diethylenetriaminepentaacetic acid (DTPA), trans-cyclohexyldiethylenetriaminepentaacetic acid (CHX-DTPA), 1-oxa-4,7,10-triazacyclodecane dioxane-4,7,10-triacetic 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), and connected to the aromatic ring via a linker selected from (CH2) n ,-LO(CH2) n ,-LNH(CH2) n ,-LCONH(CH2) n ,-LNHCO(CH2) n , where L is -(CH2) m or -O(CH2CH2O) m , n and m are independently selected from 1-25; wherein, When R4 is (i), (ii) or (iii), the isotope labeling agent is selected from: 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 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 You, 113 You, 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 Purpose, 212 Purpose, 213 Purpose, 31 P, 32 P, 33 P, 32 S, 35 S, 45 Sc, 47 Sc, 84 Wed, 86 Wed, 87 Wed, 88 Wed, 89 Wed, 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 If, 74 If, 75 If, 76 If, 77 If, 78 If, 80 If, 82 If, 94 Tc, 99m Tc, 103 Rh, 103 mRh, 119 Sb, 121 Sb, 123 Sb, 135 Let, 138 Let, 139 Let, 162 Is, 164 Is, 165 Is, 166 Is, 167 Is, 168 Is, 170 Is, 193 mPt, 195 mPt, 192 Currently, 194 Currently, 195 Currently, 196 Currently, 198 Currently, 211 To, 223 Raw, 225 Ac, wherein X and Y are independently selected from: -CH- and -N-; Where R6 is H or wherein Q and Z are independently selected from: -CH- and -N- and wherein the curl symbol indicates attachment to a tetrazine; wherein R5 and R7 are independently selected from H or selected from hydroxyl, sulfonamide, carboxyl, sulfonyl, amine, a substituted amine having 1-5 polyethylene glycol units, -(O-CH2-CH2)-OCH2-COOH, wherein n is selected from 1-5, or methyl, ethyl, propyl, optionally substituted heteroaryl and optionally substituted arylalkyl; wherein, optionally substituted with respect to the substituted amine refers to 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)heteroalkyl, (C3-30 C10)cycloalkyl, (C3-C10)heterocycloalkyl, (C3-10)cycloalkyl, (C3-C10)heterocycloalkylene, (C1-C10)haloalkyl, (C1-C10)perhaloalkyl, (C2-C10)-alkenyloxy, (C3-C10)-alkynyloxy, aryloxy, arylalkoxy, heteroaryloxy, heteroarylalkoxy, (C1-C6)alkoxy-(C1-C4)alkyl, optionally substituted aryl, optionally substituted heteroaryl and optionally substituted arylalkoxy wherein optionally substituted refers to one or more substituents selected from halogen, hydroxyl, sulfonamide, carboxyl, sulfonyl, amine, a substituted amine having 1-5 polyethylene glycol units, -(O-CH2-CH2)-OCH2-COOH, wherein n is selected from 1-5, or H, methyl, ethyl, propyl, optionally substituted heteroaryl and optionally substituted arylalkyl; wherein optionally substituted relative to the substituted amine refers to one or more substituents selected from halogen, hydroxyl, sulfonamide, carboxyl, sulfonyl and amine.

10. The method according to claim 10, wherein the tetrazine is an asymmetric tetrazine selected from the group consisting of:

11. The method of claim 1, wherein the isomer-free dienophile is a trans-cycloheptene (TCH), trans-cyclooctene (TCO) or trans-cyclononene (TCN) derivative selected from: wherein X is N, NO or CR8; Y is N, NO or CR8; R8 is selected from: -H, -F, -OH, -NH2, -COOH, -COOCH3, CF3, -Cl, -CONH2, CONHCH3, -CON(CH3)2, -CH2CH2OH, -CH2CH2NH2, -CHCH2N(CH3)2, and wherein the linker is selected from: -(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) n NHCHMCO,(CH2) n NHCOCHMNH,(CH2)OCOCHMNH,(CH2CH2O) n CH2CH2NHCHMCO,(CH2CH2O) n CH2CH2CONHCHMCO,(CH2CH2O) n CH2CH2NHCHMCO,(CH2CH2O) n CH2CH2NHCOCHMNH,(CH2CH2O) n COCHMNH, wherein n is 0-25, wherein M is a side chain selected from the group consisting of natural amino acid side chains: 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; And wherein the targeting carrier is an antibody, a nanobody, a polymer, a nanomedicine, a cell, a protein, a peptide or a small molecule.

12. The process according to claim 1, wherein when the diene is a symmetrically substituted diene in which at least one plane of symmetry passes through the nitrogen-nitrogen bond of at least one tetrazine ring, it is obtained from a precursor selected from: wherein X is CH or N.

13. The method according to claim 1, wherein: When the diene is an asymmetrically substituted diene, it is obtained from a precursor selected from:

14. The method of claim 1, wherein the isomer-free dienophile targeting vector is obtained from a precursor selected from the group consisting of: in, The targeting carrier is an antibody, a nanobody, a polymer, a nanodrug, a cell, a protein, a peptide or a small molecule.

Images