Process for the rapid oxidation of dihydropyridazines to pyridazines

Abstract

The invention relates to a method for providing pyridazines by fast oxidation of dihydropyridazines. The pyridazines can be used in treatments such as radiotherapy, diagnostics, imaging and other photochemical methods. The method is suitable for dihydropyridazine mixtures generated by a click reaction between a first chemical entity which is a 1,2,4,5-tetrazine or a strained alkene with retro Diels-Alder cycloaddition reactivity and a second chemical entity which is a 1,2,4,5-tetrazine or a strained alkene with complementary retro Diels-Alder cycloaddition reactivity. The linkage between the first chemical entity and the second chemical entity is followed by fast acid catalyzed oxidation.

Description

Technical Field

[0001] This invention relates to a method for providing pyridazine via the rapid oxidation of dihydropyridazine. This pyridazine can be used in treatments such as radiotherapy, diagnostics, imaging, and other photochemical methods. Background Technology

[0002] Tetraazine-linked labeled targeting carriers can be used for imaging purposes (e.g., diagnostics and other photochemical imaging methods) and for therapeutic purposes. For example, such targeting carriers are labeled with radiolabels that can be used for diagnostics and / or therapy. Specific uses depend on the characteristics of the radiolabel used, as different radionuclides are provided for different purposes. Furthermore, specific uses also depend on the specific target to which the carrier is targeted. Currently, several combinations of radiolabels and carriers are used in diagnostics, therapy, therapeutic diagnostics, and imaging. Different chemical entities exist that link the radiolabeled entity to the target entity. This invention is based on tetraazine ligation, in which tetraazine (Tz) and trans-cyclooctene (TCO) are linked, followed by the rapid oxidation of the corresponding click product (i.e., dihydropyridazine) to the corresponding pyridazine, thereby reducing the complexity of the reaction products bridging the radiolabel and the target entity.

[0003] The term "tetraazine linkage" refers to a class of rapid, simple, versatile, chemoselective click reactions that offer high product yields. These reactions have been widely applied in numerous research fields, including materials science, polymer chemistry, and pharmaceutical science. Radiochemistry is one area that truly showcases the potential of click chemistry, as illustrated by Zeng et al. in the *Journal of Nuclear Medicine*, 54, 829-832, 2013. Essentially, the selectivity, ease of use, speed, and modularity of click linkages make them almost perfectly suited for the construction of radiotracers, a process often involving working with biomolecules under aqueous conditions with rapidly decaying radioisotopes.

[0004] Tetraazine linkages are characterized by the formation of a covalent bond between 1,2,4,5-tetraazine (Tz) and a typical trans-cyclooctene (TCO). The reaction is initiated by a reverse electron-demanding Diels-Alder reaction, followed by a reverse Diels-Alder reaction driven by N2 expulsion. Tetraazine linkages are among the fastest known chemical linkages, with a second-order rate constant as high as 10⁻⁶ in MeCN at 25 °C. 6 M -1 s -1 This is contrary to other known click responses, such as the Staudinger connection (0.003M). -1s -1 ) and strain-promoted azide-alkyne cycloaddition (SPAAC) (0.1M -1 s -1 For this reason, and due to its specificity, Tz linkage is well-suited for syntheticon-based labeling. A major drawback of conventional Tz-TCO linkage is that it lacks any regiospecificity when it occurs. Furthermore, conventional Tz-TCO linkage also produces several tautomeric forms. This means that conventional Tz-TCO linkage typically yields a complex reaction mixture of at least sixteen distinct isomers (Scheme 1). Such mixtures of isomers cannot be used directly for pharmaceutical purposes because the specific isomers of the compound affect the pharmacokinetics of the therapeutic agent, and it is impossible to distinguish any potential toxicological effects of individual isomers. While the various dihydropyridazines obtained from conventional linkage can be slowly and spontaneously oxidized to the corresponding pyridazine over hours to days, thus slowly reducing the number of tautomeric forms of the product (WO2012 / 121746), this slow oxidative transformation does not reduce the number of regio and stereoisomers. Methods to accelerate the conversion of dihydropyridine to pyridazine have been described (Karaki Fumika et al., Tetrahedron, 97, 132411, 2021; Keinänen et al., ACS Medicinal Chemistry Letters, 7, 62-66, 2015). However, in all reported cases, the oxidation was not completed within two hours, which is required if the pyridazine is to be used as a radiopharmaceutical. WO2017 / 059397, WO2020 / 242948, Syvänen et al., ACS Chemical Neuroscience, 11, 4460-4468, 2020, and WO2012 / 121746 disclose the connection between tetrazine and TCO, which will inevitably provide several isomeric chemical entities.

[0005] Alternatively, pyridazines can be prepared by linking Tz with strained cyclic alkynes, but the second-order rate constant of this reaction is slow.

[0006] Radiopharmaceuticals are increasingly used in therapeutic diagnostics, particularly in oncology, for both diagnostic imaging and targeted radionuclide therapy. Positron emission tomography (PET) is the gold standard for nuclear imaging, offering superior resolution and quantification compared to other modalities. In 2019 alone, 2,200,800 clinical PET scans were performed in the United States. Targeted radionuclide therapy treats cancer more effectively than many of the most advanced chemotherapy therapies. It also offers advantages over external beam radiotherapy (such as Gamma Knife) because it provides a method for confining the delivered dose to the tumor and its surrounding area, which is particularly significant in the radiotherapy of micrometastatic disease. The combination of diagnostic imaging and targeted radiotherapy can be used for "therapeutic diagnostics," a concept with powerful applications in personalized medicine involving patient selection, dose discovery, and monitoring of treatment response. Therapeutic diagnostic pairings involve two radionuclides that can be substituted for each other without altering the pharmacokinetics of the radiopharmaceutical, but whose application is shifted 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 binding of a radionuclide to a carrier that targets cancer cells. In imaging, this radiolabeled carrier is called a "radiotracer." The radiotracer accumulates in the tumor lesion, and its location can then be observed by detecting the emitted radiation. PET is strongly favored in oncology, while SPECT dominates in cardiology and is used for bone scans and certain other specific organ scans. Globally, the ratio of SPECT cameras to PET cameras used in hospitals is approximately 5:1.

[0008] Single-photon emission computed tomography (SPECT) is the earlier of the two methods. SPECT imaging uses gamma photons emitted by a radionuclide, typically in the range of 100-200 keV. A series of two-dimensional projection images of the distribution of the radiotracer in the body are acquired from multiple angles using one or more gamma cameras. These projection images are then combined to generate a 3D image.

[0009] Positron emission tomography (PET) is currently considered the most advanced form of nuclear imaging. A key application of PET in oncology is in diagnostic and therapeutic monitoring, particularly for metastatic cancer. Compared to previous modalities, especially SPECT, PET offers improved resolution and sensitivity, and generally provides higher quality images. These properties are particularly relevant in the detection of very small metastases and subsequent treatment. New innovations, especially whole-body PET, have significantly improved sensitivity and detected more metastases, promising to further solidify PET's dominant position in modern clinical practice. PET relies on the use of radionuclides that emit positrons upon decay. These positrons travel a limited distance and then annihilate with electrons in the surrounding medium. This produces two annihilation photons, each at 511 keV, emitted in opposite directions. These photons can be detected by a PET scanner.

[0010] The ideal radionuclide for PET is fluorine-18 (F18). 18 F). 18 The decay half-life of F is 110 minutes, and each decay emits 97% positrons. 18 F is close to being an ideal choice for clinical PET applications. This is especially true for small molecule and peptide-based radiopharmaceuticals, which represent the vast majority of relevant PET tracers. Equally important, 18 F can actually be mass-produced (>300 doses per batch) on standard biomedical cyclotrons, which are readily available in most parts of the world, with over 200 existing in Europe alone. Therefore, 18 F is unconcerned about its closest competitor (the generator produces radioactive gallium-68). 68 Ample supply of Ga) related to Ga) 18 The lower the positron energy of F, the higher the resolution of the image. Compared to the most widely used diagnostic radionuclide (SPECT radionuclide technetium-99m), 18 F provides the highest quality images through its state as a PET radionuclide. Therefore, 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 (I-131) is used. 131 I), but nowadays, clinical SPECT scans typically use iodine-123 (I), 123 I) proceed. This is because... 123 I has a shorter half-life (t½ = 13.2 hours) and lacks β-emission, compared to 131Iodine exhibits more favorable radiotoxicity. Its 159 keV gamma photon energy is ideal 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 thyroid diseases. It is also a component of the SPECT radiotracer. 123 I is used, for example, in imaging agents MIBG (oncology) and ioflupane (CNS). 123 I and clinically used β-emission therapeutic radionuclides 131 I and research Auger electron beam radiotherapy agents 125 Iodine-123 is used for diagnosis and treatment matching. It is produced in a cyclotron by proton irradiation of xenon gas in a capsule and is commercially available.

[0012] Iodine-124 ( 124 I) It can be used for PET imaging. It is typically produced in a cyclotron by bombarding tellurium-124 enriched with it. However, 124 Imaging characteristics of I are not ideal. It has a complex decay scheme with many high-energy gamma rays. Only 23% of the decays result in positron emission.

[0013] Astatine-211 ( 211 Alpha (α) is primarily a therapeutic radionuclide that emits alpha particles upon decay. Alpha particles are absorbed by tissues up to 100 µm in diameter and cannot be detected by external scanners. However, 211 One decay branch of At also produces X-rays in the 70-90 keV range, which can be imaged using a gamma camera or SPECT scanner. Therefore, SPECT imaging can be used to... 211 Clinical assessment of the distribution of At-labeled radiopharmaceuticals in patients.

[0014] However, introducing these radionuclides into molecules remains a challenge, which limits the practical application of radionuclides in therapy, diagnosis, and imaging.

[0015] Most of the aforementioned PET, SPECT, and therapeutic radionuclides are radioactive metals. The traditional method for introducing radioactive metals into carrier molecules involves using chelating agent groups that form coordinate bonds with the radioactive metal atoms. Radiolabeling procedures typically involve mixing a radiolabeled precursor (the carrier with the chelating agent group) with radioactive metal ions and heating the mixture to allow the chelation reaction to proceed. While the chelation of radioactive metals is conceptually simple, it has several drawbacks:

[0016] - Due to the insignificant differences in physicochemical properties, radiolabeled products are usually impossible to separate from unlabeled precursors;

[0017] - The chelation reaction is sensitive to trace metal impurities in the solution used for radiolabeling, which makes scale-up problematic;

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

[0019] Compared to its radioactive metal alternatives, 18 F is a halogen and needs to be covalently bonded to the targeting carrier. This contrasts sharply with chelator-based labeling techniques used for radiometals. Covalent bonds are currently typically formed by direct nucleophilic substitution of the leaving group (e.g., trifluoromethanesulfonates). This chemical reaction is demanding, time-consuming, and poorly scalable, making it incompatible with many carriers, especially the increasingly important peptides.

[0020] Small-molecule radiopharmaceuticals containing radioactive iodine are typically prepared using electrophilic detannylation or iodine-iodine exchange radiochemistry. The former is a mild, versatile, and practical reaction in which the radioactive iodide is oxidized to a positively charged iodine species, which is then substituted with a leaving group (usually a tin group) in an aromatic substitution reaction. This reaction occurs at room temperature and the yield is usually quantitative. Iodine-iodine isotope exchange is used when high molar activity is not an issue and the substrate can withstand harsh conditions. The exchange is carried out at high temperatures using acid and copper as catalysts.

[0021] Similar to fluorine and iodine, astatine-211 is a halogen that can be covalently linked to a target carrier. Aliphatic astatine-carbon bonds do not provide sufficient in vivo stability, therefore they are typically... 211 At is introduced onto the aromatic ring, forming an astatine aryl moiety. Unlike iodine, astatine cannot stably couple to tyrosine residues in proteins. 211 At was found to form a weak bond with the sulfhydryl group of cysteine, rather than with tyrosine. Therefore, 211 At-labeling requires the synthesis of a specific precursor with a suitable leaving group (e.g., a trialkyltin group attached to an aromatic ring). Standard 211 At-labeling schemes use oxidizing agents, such as chloramine-T or N-chlorosuccinimide. These agents may degrade biomolecules used as targeting carriers.

[0022] 18 F has long been the preferred radionuclide for diagnostic PET imaging, and 211 At is the most promising therapeutic radionuclide for alpha therapy. Iodine radioisotopes 123 I, 124 I, 125 I and 131I can be used for SPECT imaging, PET imaging, Auger therapy, and β-therapy, respectively. Furthermore, all three elements can be introduced into an aromatic ring to form a fluorine / iodine / astatine aryl moiety with maximum structural similarity, which is crucial for therapeutic diagnostic pairing. However, there is no information regarding... 18 F and 211 At or radioactive iodine 123 I, 124 I, 125 I, 131 Reports of I being used in combination for therapeutic and diagnostic applications.

[0023] As mentioned above, development 18 F / 211 At and 18 F / 123 I, 124 I, 125 I, 131 One obstacle to I-1 therapy-diagnostic pairing is the demanding labeling chemistry, which prevents the direct regioselective labeling of biomolecular targeting vectors with these radionuclides. Another obstacle is that not all targeting vectors contain an aryl moiety, so it must be introduced as a prosthetic group (also known as a synthon).

[0024] Due to these challenges, synthon-based methods have been investigated for the preparation of radiopharmaceuticals. These methods involve the direct labeling of a single intermediate compound (“synthesis”) and then conjugating it to a carrier under mild conditions. Therefore, the carrier is unaffected by the harsh conditions of direct radiolabeling, although it does require modification with a chemical label complementary to the radiolabeled synthon. Crucially, the chemistry used to conjugate the synthon to the carrier is essential. This chemistry must be specific, compatible with pharmaceutically relevant aqueous media, and yield high enough to avoid radioactive loss and minimize the need for subsequent purification. Crucially, it must also be very rapid, as the preparation of radiopharmaceuticals is carried out at nanomolar 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 β-emitters, Auger electron emitters, and α-emitters.

[0026] Radioactive nuclides that emit beta particles (e.g.) 90 Y、 177 Lu、 131I) By emitting high-energy electrons (β particles) that decay, these electrons can travel a distance of approximately 12 mm within tissue. The decay energy is deposited within the largest tissue volume of the three treatment types mentioned here. Therefore, β emitters are suitable for treating medium-sized tumors, where most of the dose is absorbed by cancer cells. However, for micrometastases or heterogeneous tumors, even with perfect concentrations of radionuclides within and around the tumor, most of the radiation dose is still absorbed by surrounding healthy cells. Therefore, β emitters are not the optimal choice for treating micrometastases or heterogeneous tumors. This is a significant drawback of β emitters, as micrometastases are one of the leading causes of cancer recurrence and cancer death.

[0027] α-emitters (e.g.) 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—approximately 30–100 μm, comparable to the diameter of a few mammalian cells. Therefore, all the energy produced by decay is transferred to only a few neighboring cells. Compared to beta radiation, alpha radiation is more cytotoxic and can be delivered to microtransfers in a highly focused manner.

[0028] A clear example of the advantages of alpha therapy: a flagship beta-therapeutic agent 177 Lu-PSMA (prostate-specific membrane antigen) resistant metastatic prostate cancer patients in alpha-therapy 225 Complete remission was achieved after three cycles of Ac-PSMA.

[0029] Auger electron beam radiotherapy (AeRT) utilizes radionuclides that emit short-range electron showers upon decaying via electron trapping (EC) or internal conversion (IC). Using advanced drug delivery technologies, these specialized radionuclides can be delivered directly to the nucleus of cancer cells. Here, the emitted Auger electrons damage DNA and kill the cancer cells. Notably, the short range of the Auger electrons ensures that their energy is primarily deposited within the target cells, resulting in highly localized therapy. 123 I and 125 I has a high Auger electron production rate and is suitable for AeRT.

[0030] Iodine-131 ( 131 I) is a beta particle emitter widely used in clinical radionuclide therapy. Its decay half-life is 8.0 days, primarily emitting beta particles with a maximum energy of 606 keV at 90% abundance. These beta particles have a maximum tissue size of approximately 2 mm, making... 131I can treat small to medium-sized tumors. Due to its inherent accumulation in thyroid tissue, 131 I is widely used in thyroid ablation. In addition, it is used... 131 Therapeutic variants of radiolabeled MIBG are available. 131 I is used in radioimmunotherapy. It is related to... 123 I (SPECT) and 124 I (PET) formation therapy diagnostic pairing. Both iodine-123 and iodine-125 exhibit high Auger electron emission, approximately 10 and 20 electrons respectively. This makes them suitable for Auger electron radiotherapy, a currently investigated form of radionuclide therapy.

[0031] Astatine-211 is an alpha-emitting radionuclide with a half-life of 7.2 hours. Unlike most other alpha-emitting nuclides used in targeted alpha therapy, 211 At produces an alpha particle on each decay chain, which offers several advantages for conversion. First, the toxic side effects of the radionuclides are limited, as these nuclides are released from the target carrier due to initial decay. Second, radiation dosimetry calculations are simplified. Furthermore, because... 211 At has a relatively short half-life (for therapeutic nuclides), which can enhance the control of radiation dose delivered to patients.

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

[0033] However, to enable click chemistry (e.g., Tz-TCO linking) to deliver viable end products in both regulatory and commercial settings, it is necessary to reduce the number of linking products prepared. Currently available methods produce a large number of inseparable isomers, hindering clinical translation due to toxicity issues and unpredictable pharmacokinetics. Furthermore, a limitation to the practical application of radiolabeled products is that the radiolabeled compounds must be administered to patients for imaging, treatment, or diagnostic purposes within a very limited timeframe after production. Therefore, any method that reduces the linking steps would be highly beneficial, especially when radioactive reagents are used as labelers, diagnostic agents, therapeutic agents, therapeutic diagnostic agents, or in combination with other reagents.

[0034] This invention provides a rapid method for providing labeled pyridazine products, wherein the ligation step takes less than 180 minutes, and wherein a form or only a few forms of pyridazine are provided. This means that the complexity of the reverse electron-demanding Diels-Alder reaction is reduced, which simplifies the clinical translation and production of these compounds. This method advantageously enables the radiolabeling of any tracer with unparalleled efficiency and practicality, for example using... 18 F, 123 I, 124 I, 125 I or 131 I and 211 At. Summary of the Invention

[0035] This invention provides a method for the rapid oxidation of dihydropyridazines to pyridazines. The method is applicable to mixtures of dihydropyridazines generated by a click reaction between a first chemical entity and a second chemical entity, wherein the first chemical entity is a 1,2,4,5-tetraazine or a strained olefin with a reverse electron-demanding Diels-Alder cycloaddition reactivity, and the second chemical entity is a 1,2,4,5-tetraazine or a strained olefin with a complementary reverse electron-demanding Diels-Alder cycloaddition reactivity. One of the first or second chemical entities is coupled to a label, and the other is coupled to a targeting support. The connection between the first and second chemical entities is subsequently followed by rapid acid-catalyzed oxidation. The advantages of this method are that it oxidizes dihydropyridazines to pyridazines in a minimal time and significantly reduces the complexity of the reaction mixture, thereby simplifying clinical translation and reducing production costs.

[0036] A method for providing the rapid oxidation of dihydropyridazine to pyridazine includes the following steps:

[0037] a) A first chemical entity is labeled with a labeling agent, the first chemical entity having a reverse electron-demanding Diels-Alder cycloaddition reactivity and being coupled to a pharmaceutical, diagnostic, or therapeutic agent; wherein the first chemical entity is a 1,2,4,5-tetraazine or a dienophile selected from: a cyclic unsaturated hydrocarbon having at least one trans-configured double bond; an unsaturated heterocycle having at least one trans-configured double bond; and

[0038] b) Connect the labeled first chemical entity obtained in step a) to a second chemical entity, the second chemical entity having complementary anti-electron demand Diels-Alder cycloaddition reactivity and being coupled to a target support; wherein the second chemical entity is a 1,2,4,5-tetraazine or a dienophile selected from: a cyclic unsaturated hydrocarbon having at least one trans configuration double bond; an unsaturated heterocycle having at least one trans configuration double bond;

[0039] c) The resulting mixture of the dihydropyridazine-labeled targeting support obtained in step b) is oxidized in a solvent at a temperature of 15°C to 50°C for up to 180 minutes by adding an acid catalyst and an oxidant, wherein the acid catalyst has a pKa of less than 5 in H₂O at 25°C, is added to the mixture obtained in step b) at a concentration of 0.1 M to 5 M, and the oxidant is selected from quinone oxidants, Bobbit salt oxidants, peroxide oxidants, peroxyacid oxidants, or oxidants with a minimum UV irradiance of 2 mW / cm². 2 Ultraviolet light.

[0040] The labeling agent can be any reagent that can be used as a marker, imaging agent, therapeutic agent, or therapeutic diagnostic agent, including radionuclides and fluorescent entities.

[0041] The targeting vector can be any suitable vector for a specific target, including antibodies, nanobodies, polymers, nanomedicines, cells, proteins, peptides, and small molecules.

[0042] Furthermore, the present invention also provides the application of the labeled pyridazine-conjugated targeting vector obtained by this method in therapeutic diagnosis, treatment, radiotherapy, diagnosis and imaging. Attached Figure Description

[0043] Figure 1 The reaction schemes for oxidation testing of linkage products using different chemical oxidants and TFA, along with tables listing the specific compounds and the results.

[0044] Figure 2 The reaction schemes for oxidation testing of the linkage products using ultraviolet light or air as oxidant under different conditions (conditions 1-5), and a table listing the specific compounds and the time (minutes) used to obtain ≥90% oxidation efficiency.

[0045] Figure 3 The reaction schemes for oxidation testing of the linkage products using ultraviolet light or air as oxidants under different conditions (conditions 6-11), and a table listing the specific compounds and the time (minutes) used to obtain ≥90% oxidation efficiency.

[0046] Figure 4 : Reaction schemes for specific linkage products tested by oxidation with chemical oxidants or ultraviolet light under different conditions (conditions 3-13), and tables showing the time (minutes) to obtain ≥90% oxidation efficiency.

[0047] Figure 5The reaction schemes for specific ligation products tested by UV light under different conditions (conditions 14-21), and a table showing the time (minutes) required to obtain ≥90% oxidation efficiency.

[0048] Figure 6 The reaction schemes for oxidation testing of the linkage products under different conditions (conditions 21-22) using ultraviolet light are listed, along with tables showing the specific compounds and the time (in minutes) required to obtain ≥90% oxidation efficiency.

[0049] Figure 7 The reaction schemes for the ligation products tested under different conditions (conditions 6-11), and a table listing the specific compounds and the time (minutes) required to obtain ≥90% oxidation efficiency.

[0050] Figure 8 The graph above shows the time (in minutes) for the click reaction to the F label to achieve ≥90% oxidation efficiency under different conditions.

[0051] Figure 9 The graph above shows the time (in minutes) for the click reaction targeting the I label to achieve ≥90% oxidation efficiency under different conditions.

[0052] Figure 10 The reaction schemes for the ligation products tested under different conditions (conditions 1-12) are listed, along with tables showing the specific compounds and the time (in minutes) required to achieve ≥90% oxidation efficiency.

[0053] Figure 11 The reaction schemes for the linkage products tested under different conditions (conditions 1-4) are listed, along with tables showing the specific compounds and the time (in minutes) required to achieve ≥90% oxidation efficiency.

[0054] Figure 12 : Shows the synthesis scheme of compound 55.

[0055] Figure 13 : Shows the scheme for oxidizing radiolabeled dihydropyridazine to radiolabeled pyridazine containing a PSMA-targeting carrier, and a table showing the HPLC yield (%) of oxidized radiolabeled pyridazine containing a PSMA-targeting carrier.

[0056] definition

[0057] A container is an entity in which a chemical reaction (such as an oxidation step) takes place. In this context, a container is a laboratory glassware, such as a bottle. Bottles can be made of borosilicate, quartz, or other suitable materials.

[0058] In this document, a labeler refers to an imaging agent that is attached to a chemical entity that exhibits a reverse electron-demanding Diels-Alder cycloaddition reactivity and is coupled to a pharmaceutical, diagnostic, or therapeutic agent. In some embodiments, the pharmaceutical, diagnostic, or therapeutic agent coupled to the chemical entity is the same as the labeler. For example, when the labeler is something that can be used as both a labeler and a therapeutic or diagnostic agent (e.g., a diagnostic imaging agent). 18 This is the case when using reagents of F). Labeling agents include radionuclides. However, since labeling dienes or dienophiles with radionuclides does not typically provide 100% labeling efficiency (using radionuclides), some labeled products will inevitably be labeled with stable isotopes of the corresponding radionuclide elements.

[0059] In this article, "connection" refers to a click reaction between a first chemical entity with a reverse electron-demanding Diels-Alder cycloaddition reactivity and a second chemical entity with a complementary reverse electron-demanding Diels-Alder cycloaddition reactivity.

[0060] "First chemical entity" and "second chemical entity" refer to a pair of compounds with complementary reverse electron-demanding Diels-Alder cycloaddition reactivity. This pair is a 1,2,4,5-tetraazine and a diephile, the diephile being a cyclic unsaturated hydrocarbon having at least one trans-configured double bond or an unsaturated heterocycle having at least one trans-configured double bond. One of the 1,2,4,5-tetraazine or the diephile is labeled with a labeling agent and coupled to a pharmaceutical, diagnostic, or therapeutic agent, while the other is coupled to a targeting carrier. Thus, in some embodiments, the "first chemical entity" is a 1,2,4,5-tetraazine, and the "second chemical entity" is a diephile being a cyclic unsaturated hydrocarbon having at least one trans-configured double bond or an unsaturated heterocycle having at least one trans-configured double bond. In other embodiments, the "first chemical entity" is a dienophile, which is a cyclic unsaturated hydrocarbon having at least one trans configuration double bond or an unsaturated heterocycle having at least one trans configuration double bond, and the "second chemical entity" is 1,2,4,5-tetraazine.

[0061] In this article, the solvent is a mixture of water and water-miscible substances.

[0062] Ultraviolet irradiance in mW / cm 2 The unit is ultraviolet irradiance reaching the surface of the reaction vessel, and it is the unit specified on commercially available ultraviolet lamps. An ultraviolet irradiance of at least 2 mW / cm² is required. 2 The lamp is sufficient to provide at least 90% oxidation efficiency of pyridazine in glass and quartz cuvettes with an optical path of 2 mm (700 μl) to 30 mm (10 ml). Detailed Implementation

[0063] In a first aspect, the present invention provides a method for the rapid oxidation of dihydropyridazines to pyridazines. This method is applicable to the oxidation of various dihydropyridazines generated by the linkage of a 1,2,4,5-tetraazine with a cyclic unsaturated hydrocarbon having at least one trans-configured double bond or an unsaturated heterocycle having at least one trans-configured double bond. Rapid oxidation is achieved by employing a combination of acid and various oxidants (including chemical oxidants) and ultraviolet photocatalytic oxidation.

[0064] Tetraazine or strained olefins are coupled to the reagent of interest (e.g., pharmaceuticals, diagnostic agents, imaging agents, or therapeutic agents) and labeled with a labeling agent. Compatible dienes or dienophiles are then coupled to the target carrier of interest.

[0065] The linkage between tetrazine and the strained olefin is based on the reverse electron-demanding Diels-Alder cycloaddition reactivity; therefore, the tetrazine and the strained olefin must possess complementary reverse electron-demanding Diels-Alder cycloaddition reactivity. The linkage between the tetrazine and the strained olefin should exhibit reaction kinetics as specified, with a minimum second-order rate constant of 250 M, determined by stop-flow spectrophotometry in PBS at 25 °C. -1 s -1 The connection between the tetrazine and the strained olefin specified herein ensures that the second-order rate constant in phosphate-buffered saline (PBS) at 25°C is avoided to be below 250 M. -1 s -1 The connection was such that, at 25°C in phosphate-buffered saline (PBS), the second-order rate constant was less than 250 M. -1 s -1 The binding process will take a long time to provide the labeled target carrier because the time available for the radiolabeled agent to be used for imaging and / or treatment after the binding step is usually limited.

[0066] The second-order rate constant can be determined by various methods, but is typically determined by stop-flow spectrophotometry, as described in (Chance, Rev. Sci. Instrum. 1951, 22, 619-627). In this paper, the method described in Battisi et al., J. Med. Chem. 2021, 64, 20, 15297-15312 is used.

[0067] In order to rapidly provide labeled chemical entities or targeting carriers with improved homogeneity (which will carry pyridazines derived from the linkage between tetrazine and strained olefins), it is necessary to oxidize the resulting composite mixtures of various dihydropyridazines.

[0068] This paper demonstrates that, under specific conditions, oxidation performed by chemical or photochemical methods can be rapidly accelerated by adding acid. It also shows that not all oxidants are suitable for this method, either because oxidation is not efficient enough to operate within the required timeframe or produces byproducts, or because oxidation affects the structure of the target carrier, potentially preventing the labeled target carrier from binding to its target. The oxidation efficiency of step c) in this method is at least 90%, meaning that at least 90% of the labeled and clicked target carrier is in pyridazine form after the oxidation step. Oxidation conditions providing less than 90% product will not have sufficient purity for therapeutic / imaging / diagnostic use, requiring additional toxicology studies and / or purification procedures.

[0069] This paper demonstrates that by adding a combination of acid catalyst and oxidant, suitable oxidation can be achieved in a solvent at temperatures ranging from 15°C to 50°C for up to 180 minutes. The acid catalyst has a pKa value below 5 in H₂O at 25°C and is added at a concentration of 0.1 M to 5 M. The oxidant is a quinone oxidant, a Bobbit salt oxidant, a peroxide oxidant, a peroxyacid oxidant, or ultraviolet light (minimum ultraviolet irradiance of 2 mW / cm²). 2 When using ultraviolet light as the oxidant, a photosensitizer may optionally be added during the oxidation step to further accelerate the reaction. The photosensitizer depends on the wavelength of the applied ultraviolet light; therefore, the photosensitizer should be selected according to the wavelength of the ultraviolet light applied in the oxidation step. It is well known which photosensitizer is suitable at a given wavelength, and it is anticipated that oxidation step c) in this method can be carried out at an ultraviolet light wavelength of 200-400 nm, and the photosensitizer can be selected accordingly. In a preferred embodiment, the oxidation in step c) is carried out at 200-300 nm (e.g., 200-350 nm, 250-350 nm, 250-300 nm) or, for example, at 254 nm. When applied to ultraviolet light at wavelengths of 200-350 nm (e.g., at 254 nm), the preferred photosensitizer is a fluorescein or porphyrin photosensitizer, such as fluorescein or substituted 4,4',4''-(20-phenylporphyrin-5,10,15-triyl)tribenzenesulfonic acid or substituted 4,4',4''-(20-phenylporphyrin-5,10,15-triyl)tris(1-methylpyridin-1-onium).

[0070] The term "acid" used in step c) refers to any molecule having a pKa value of at least 5 or less for protons as measured in H₂O at 25°C. Suitable acids have pKa values ​​from -10 to 5, such as -8 to 3, -8 to 1, -6 to 1, -4 to 1, -2 to 1, or, for example, -10 to 2, -9 to 1, -8 to 0, -10 to -2, -10 to -4, -10 to -6, or, for example, -8 to -10. The lower the pKa, the better the reaction rate. Therefore, in a preferred embodiment, the acid has a pKa from -10 to 0, such as -10 to -8, -10 to -6, -10 to -4, -10 to -2, and -8 to 0.

[0071] Furthermore, the concentration of acid in the solvent must be between 0.1M and 5M, for example, 0.5M-5M, 1M-5M, 1.5M-5M, 2M-5M, 2.5M-5M, 3M-5M, 3.5M-5M, 4M-5M, or 4.5M-5M. In a preferred embodiment, the concentration of acid in the solvent is between 0.5M and 3M, for example, 0.5M-2M, or for example, 1M.

[0072] Therefore, methods for the rapid oxidation of dihydropyridazine to pyridazine include:

[0073] a) A first chemical entity is labeled with a labeling agent, the first chemical entity having a reverse electron-demanding Diels-Alder cycloaddition reactivity and being coupled to a pharmaceutical, diagnostic, or therapeutic agent; wherein the first chemical entity is a 1,2,4,5-tetraazine or a dienophile selected from: a cyclic unsaturated hydrocarbon having at least one trans-configured double bond; an unsaturated heterocycle having at least one trans-configured double bond; and

[0074] b) Connect the labeled first chemical entity obtained in step a) to a second chemical entity, the second chemical entity having complementary anti-electron demand Diels-Alder cycloaddition reactivity and being coupled to a target support; wherein the second chemical entity is a 1,2,4,5-tetraazine or a dienophile selected from: a cyclic unsaturated hydrocarbon having at least one trans-configured double bond; an unsaturated heterocycle having at least one trans-configured double bond; and

[0075] c) The resulting mixture of the dihydropyridazine-labeled targeting support obtained in step b) is oxidized in a solvent at a temperature of 15°C to 50°C for up to 180 minutes by adding an acid catalyst and an oxidant, wherein the acid catalyst has a pKa of less than 5 in H₂O at 25°C, is added to the mixture obtained in step b) at a concentration of 0.1 M to 5 M, and the oxidant is selected from quinone oxidants, Bobbit salt oxidants, peroxide oxidants, peroxyacid oxidants, or oxidants with a minimum UV irradiance of 2 mW / cm². 2Ultraviolet light.

[0076] In some implementations, the first chemical entity is the same as the coupled pharmaceutical, diagnostic, or therapeutic agent and the labeling agent. This is the case, for example, when the labeling agent is a reagent that can be used as both a labeling agent and a therapeutic or diagnostic agent.

[0077] In some implementations, the labeling agent is a radionuclide. Certain radionuclides can be used for imaging, diagnosis, and / or treatment; in this example, the same radionuclide can be used as both a labeling agent and an imaging or therapeutic agent. Labeling dienes or dienophiles with radionuclides typically does not provide 100% labeling efficiency (with radionuclides), and some labeled products will inevitably be labeled with stable isotopes of the corresponding radionuclide elements.

[0078] This method can label any target vector with unparalleled efficiency and practicality, for example, using... 18 F. Radioactive iodine ( 123 I, 124 I, 125 I or 131 I) and 211 At is used for radiolabeling. The breakthrough of this method lies in its ability to form a final product with improved homogeneity within 180 minutes (typically within 60 minutes, e.g., within 1–20 minutes), thus simplifying clinical translation. In contrast, conventional tetrazine linkages produce multiple products, thus requiring large and difficult-to-manage toxicological packaging or cumbersome and time-consuming separation.

[0079] The pyridazine-containing targeting carriers obtained by the method of this invention can be applied to various purposes depending on the properties of the reagents used as markers. Suitable markers for this method include radiolabelers and fluorescent markers.

[0080] In a preferred embodiment, the labeling agent used in step a) of the method for providing a targeting carrier containing labeled pyridazine is a radionuclide or a stable isotope of the corresponding element. The properties and corresponding uses of the various radionuclides commonly used are well known in the art.

[0081] In step a) of the method for providing a targeting carrier containing labeled pyridazine, suitable radionuclide labeling agents and stable isotopes of the corresponding elements 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 OF, 124 OF, 125 OF, 127 OF, 131 OF, 15 O、 16 O、 17 O、 18 O、 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 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 Through, 113 Through, 114 name, 115 name, 175 Head, 177 Head, 185 Re、 186 Re、 188 Re、 201 Tl、 203 Tl、205 Tl、 206 Pb、 207 Pb、 208 Pb、 212 Pb、 209 Bi、 212 Bi、 213 Bi、 31 P、 32 P、 33 P、 32 S、 35 S、 45 Sc、 47 Sc、 84 Sr、 86 Sr、 87 Sr、 88 Sr、 89 Sr、 165 Your, 166 Your, 156 Dy、 158 Dy、 160 Dy、 161 Dy、 162 Dy、 163 Dy、 164 Dy、 165 Dy、 227 Th、 232 Th、 51 Cr、 52 Cr、 53 Cr、 54 Cr、 73 The、 74 The、 75 The、 76 The、 77 The、 78 The、 80 The、 82 The、 94 Tc、 99m Tc、 103 Rh、 103 mRh、 119 Sb、 121 Sb、 123 Sb、 135 The、 138 The、 139 The、 162 Er、 164 Er、 165 Er、 166 Er、 167 Er、 168 Er、 170 Er、 193 mPt、 195mPt, 192 Pt, 194 Pt, 195 Pt, 196 Pt, 198 Pt, 211 At、 223 Ra、 225 Ac.

[0082] 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; the stable isotopes of the corresponding elements are 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).

[0083] 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、 212 Pb, 213 Bi; the stable isotopes of the corresponding elements are 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).

[0084] 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 in therapy, for example, in the treatment of various cancerous diseases.

[0085] In another preferred embodiment, the radionuclide labeling agent is selected from: 149 Tb, 212 Pb, 212 Bi、 213 Bi、 227 Th; the stable isotopes of the corresponding elements are selected from: 159 Tb, 206 Pb, 207 Pb, 208 Pb, 209 Bi、 232 These radionuclides are alpha particle emitters, and these radionuclides and their corresponding stable isotopes are used in therapy, for example, in relation to the treatment of various cancers.

[0086] 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, 99 mTc, 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、 78Se、 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 corresponding stable isotopes are used in Auger therapy, for example, in highly targeted treatment of various cancers.

[0087] In another preferred embodiment, the radionuclide labeling agent is selected from: 3 H, 14 C and 35 S, the stable isotopes of the corresponding element are selected from: 1 H, 2 H, 12 C 13 C and 32 S. These radionuclides are used in in vitro studies, such as displacement and tritium ionization experiments.

[0088] 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; the stable isotopes of the corresponding elements (when stable isotopes of such elements are available) are selected from: 12 C 14 N、 19 F and 127 I. These radionuclides and their corresponding stable isotopes (if available) are among the most commonly used radionuclides in current therapeutics and imaging.

[0089] The targeting carrier mentioned in step b) of the method for providing a targeting carrier containing a labeled pyridazine (the targeting carrier coupled with a tetrazine or with a cyclic unsaturated hydrocarbon (having at least one trans-configured double bond) or an unsaturated heterocycle (having at least one trans-configured double bond) can be any kind of targeting carrier suitable for therapeutic, imaging, or diagnostic purposes, such as a targeting carrier with [missing information - likely a specific type of target carrier]. Commonly used targeting carriers of this type suitable for this method include antibodies, nanobody, polymer, nanomedicine, cells, proteins, peptides, and small molecules.

[0090] Commonly used targeting vectors suitable for this method include: peptides, such as octreotide, octreotate, and AE105; and small molecules, such as FAPI derivatives and PSMA derivatives.

[0091] In a preferred embodiment, the target carrier used in the method for providing a target carrier containing labeled pyridazine is selected from: octreotide derivatives, octreotide acid derivatives, AE105 derivatives, FAPI derivatives, and PSMA derivatives.

[0092] The linkage of the compounds involved in steps a) and b) of the method will be determined by a minimum second-order rate constant of 250 M by stop-flow spectrophotometry in phosphate-buffered saline (PBS) at 25°C. -1 s -1 It occurs under certain conditions (due to the structure of these compounds).

[0093] In the method for providing a pyridazine-containing targeting vector, the oxidation step c) is carried out at a specific temperature and time by adding a specific oxidant to the mixture of linking compounds obtained in step b). These conditions ensure that the oxidation step is ≥90% efficient, thereby meeting the speed required for therapeutic, diagnostic, or imaging applications of the pyridazine-containing targeting vector.

[0094] The time required to achieve an oxidation efficiency of ≥90% depends on the specific compound being oxidized, the temperature, the oxidant equivalent, and the oxidant itself. Under the conditions employed in this method, an oxidation efficiency of ≥90% will be achieved within 180 minutes (e.g., 0-160 minutes, 0-120 minutes, 0-90 minutes, 0-60 minutes, 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 achieved within 0-20 minutes.

[0095] The oxidation step is performed at a temperature of 15°C-50°C, for example, 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, for example, between 20°C and 25°C.

[0096] All oxidations should be catalyzed by the addition of an acid. In one embodiment, the oxidant should be a chemical oxidant, preferably a quinone oxidant selected from quinone oxidants, Bobbit salt oxidants, peroxide oxidants, or peroxyacid oxidants. Surprisingly, it has been found here that using other types of oxidants will either fail to provide the desired pyridazine-containing target carrier or will negatively affect the structure of the target carrier. The acid and chemical oxidant are added to a complex mixture of various dihydropyridazines obtained by linking tetrazines and strained olefins (this mixture is obtained in step b) of the method for providing a target carrier containing labeled pyridazines), wherein the product obtained in step b) is in amounts from 1 to 100 equivalents, for example, 10 to 90 equivalents, 20 to 80 equivalents, 30 to 70 equivalents, 40 to 60 equivalents, or 50 equivalents. Preferably, 1 to 10 equivalents (e.g., 1 equivalent) of the oxidant are added to the labeled compound obtained in step b).

[0097] In another embodiment, oxidation shall be carried out in the presence of an acid by ultraviolet light (minimum ultraviolet irradiance of 2 mW / cm²). 2 Photocatalysis is performed. An acid is added to a composite mixture of various dihydropyridazines obtained by linking tetrazines and strained olefins obtained in step b), and the mixture is exposed to ultraviolet light, for example, 200 to 400 nm, 200 to 280 nm (UVC), 280 to 315 nm (UVB), and 315 to 400 nm (UVA). Preferably, 254 nm.

[0098] In another embodiment, oxidation should be carried out in the presence of acid by ultraviolet light (minimum ultraviolet irradiance of 2 mW / cm²). 2 ), or by ultraviolet light (minimum ultraviolet irradiance of 2mW / cm). 2 (A photosensitizer (e.g., a fluorescein or porphyrin photosensitizer) is added) for photochemical catalysis. An acid is added to a complex mixture of various dihydropyridazines obtained by linking a tetrazine with a strained olefin obtained in step b), and the mixture is exposed to ultraviolet light, for example, 200 to 400 nm, 200 to 280 nm (UVC), 280 to 315 nm (UVB), or 315 to 400 nm (UVA). Preferably, the exposure is at 254 nm.

[0099] In a preferred embodiment, the oxidation in step c) is performed by ultraviolet light (minimum ultraviolet irradiance of 2 mW / cm²). 2 ) or by ultraviolet light (minimum ultraviolet irradiance of 2mW / cm²) 2 (where a photosensitizer is added) to perform photochemical catalysis, wherein the 1,2,4,5-tetraazine in step a) or step b) is bispyridyltetraazine.

[0100] In another preferred embodiment, the oxidation in step c) is photocatalyzed by ultraviolet light, wherein the ultraviolet irradiance is, for example, 2 to 50 mW / cm². 2 2 to 40 mW / cm 2 2 to 20 mW / cm 2 2 to 15 mW / cm 2 5 to 15 mW / cm 2 10 to 15 mW / cm 2 12mW / cm is preferred 2 .

[0101] In a preferred embodiment, the method for oxidizing dihydropyridazine to pyridazine includes:

[0102] a) A first chemical entity is labeled with a labeling agent, the first chemical entity having a reverse electron-demanding Diels-Alder cycloaddition reactivity and being coupled to a pharmaceutical, diagnostic, or therapeutic agent; wherein the first chemical entity is a 1,2,4,5-tetraazine or a dienophile selected from: a cyclic unsaturated hydrocarbon having at least one trans-configured double bond; an unsaturated heterocycle having at least one trans-configured double bond; and

[0103] b) Connect the labeled first chemical entity obtained in step a) to a second chemical entity, the second chemical entity having complementary anti-electron demand Diels-Alder cycloaddition reactivity and being coupled to a target support, wherein the second chemical entity is a 1,2,4,5-tetraazine or a dienophile selected from: a cyclic unsaturated hydrocarbon having at least one trans-configured double bond; an unsaturated heterocycle having at least one trans-configured double bond; and

[0104] c) The resulting mixture of the dihydropyridazine-labeled targeting support obtained in step b) is oxidized in a solvent at a temperature of 15°C to 50°C for up to 180 minutes by adding an acid catalyst and an oxidant, wherein the acid catalyst has a pKa of less than 5 in H₂O at 25°C, is added to the mixture obtained in step b) at a concentration of 0.1 M to 5 M, and the oxidant is selected from quinone oxidants, Bobbit salt oxidants, peroxide oxidants, peroxyacid oxidants, or oxidants with a minimum UV irradiance of 2 mW / cm². 2 The oxidation must be carried out in a quartz container if the oxidant is ultraviolet light and the tetrazine in step a) or b) is bispyridyltetrazine.

[0105] When the oxidation in step c) is passed through ultraviolet light (minimum ultraviolet irradiance of 2mW / cm²), 2 ) or by ultraviolet light (minimum ultraviolet irradiance of 2mW / cm²) 2When photocatalysis is performed by adding a photosensitizer, it has been found that the solvent may affect the oxidation rate, thereby affecting the time required to complete oxidation to at least 90%. The more water present in the solvent, the faster the oxidation rate. Therefore, in a preferred embodiment, when the oxidant in step c) is a minimum UV irradiance of 2 mW / cm², 2 When ultraviolet light (optionally with added photosensitizer) is applied, the solvent in step c) contains 1-99% water, such as 5-95% water, such as 20-95%, 30-95%, 40-95%, 50-95%, 60-95%, 70-95%, 80-95%, or 90-95% water.

[0106] It was also found that when oxidation in step c) is carried out by ultraviolet light (minimum ultraviolet irradiance of 2 mW / cm²), 2 Alternatively, through ultraviolet light (minimum ultraviolet irradiance of 2 mW / cm²). 2 When photocatalysis is performed by adding a photosensitizer, the material of the container (e.g., a bottle) in which oxidation takes place can affect the reaction rate of the oxidation step, and consequently the time required to complete oxidation to at least 90%. As shown in the examples, the reaction time for UV photocatalytic oxidation is significantly reduced when the oxidation step is performed in a quartz bottle compared to a standard borosilicate glass bottle. Therefore, in a preferred embodiment, when the oxidant in step c) is UV light (minimum UV irradiance of 2 mW / cm²), the reaction time is significantly reduced. 2 When optionally adding a photosensitizer, step c) is carried out in a quartz container.

[0107] Furthermore, it was found that when oxidation in step c) is carried out by ultraviolet light (minimum ultraviolet irradiance of 2 mW / cm²), 2 Alternatively, through ultraviolet light (minimum ultraviolet irradiance of 2 mW / cm²). 2 When photocatalysis is performed by adding a photosensitizer, using an open container (e.g., an uncapped vial) for the oxidation step may affect the reaction rate of the oxidation step, thus affecting the time required to complete oxidation to at least 90%. As shown in the examples, the reaction time for UV photocatalytic oxidation is reduced when the oxidation step is performed in an open bottle compared to a closed bottle. Therefore, in a preferred embodiment, when the oxidant in step c) is UV light (optionally with the addition of a photosensitizer), step c) is performed in an open container.

[0108] This study found that when oxidation in step c) is catalyzed by ultraviolet light or by photochemical catalysis with the addition of a photosensitizer, the intensity of the ultraviolet light source may affect the reaction rate of the oxidation step. Therefore, oxidation should be performed using ultraviolet light (with a minimum ultraviolet irradiance of 2 mW / cm²). 2 The reaction is carried out to allow for rapid oxidation (i.e., within 180 minutes).

[0109] In one embodiment, the oxidant is solid-supported. Any commonly used solid support is suitable, such as oxidants supported on alumina, silica gel, polymers, montmorillonite, zeolite, or nanomaterials. The advantages of using solid-supported oxidants generally include: ease of removal from the reaction by filtration; the ability to use excess reagent to advance the reaction without introducing purification difficulties; ease of operation; and economical and efficient recycling of the recovered reagent.

[0110] Conventional linkage between tetrazine and TCO will produce complex mixtures of different tautomers, regioisomers and enantiomers, as schematically shown in Scheme 1.

[0111]

[0112] Option 1: An overview of all isomers after conventional tetrazine linkage, including tautomers, enantiomers, and regiomers.

[0113] Scheme 2 below is an example of the most advanced tetrazine linkage to date. This reaction produces an excessive number of isomers:

[0114]

[0115] Option 2. A specific example of the current state-of-the-art tetrazine linkage. (Not all isomers are shown here; for an overview of possible isomers, see Option 1).

[0116] This reaction can form a variety of isomers (at least 12 isomers), all of which have very similar polarities and are therefore difficult to separate, for example by HPLC.

[0117] In this paper, we describe a method that reduces complexity by improving the uniformity of application of a labeled first chemical entity (e.g., a radiolabeled tetrazine synthon) in the radiolabeling of cyclic unsaturated hydrocarbons (having at least one trans-configured double bond); and unsaturated heterocyclic (having at least one trans-configured double bond) functionalized supports (and vice versa), followed by oxidation to generate a pyridazine-containing targeting support within a short time (e.g., within 0–180 minutes). The method comprises three steps: a labeling step, a linking step, and an oxidation step.

[0118] The starting entity to be joined is a cyclic unsaturated hydrocarbon (with at least one trans configuration double bond) or an unsaturated heterocycle (with at least one trans configuration double bond) and 1,2,4,5-tetraazine.

[0119] The following schemes 2 and 3 are illustrations of examples of connections performed according to the method of the present invention, exemplified here by asymmetric tetrazine and TCO coupled to a target carrier:

[0120]

[0121] Option 3. Connection of symmetrical Tz with bicyclic unsaturated hydrocarbons (with at least one trans configuration double bond).

[0122]

[0123] Option 4: Connection of asymmetric Tz with a bicyclic unsaturated hydrocarbon (with at least one trans configuration double bond).

[0124]

[0125] Option 5: Connection of asymmetric Tz with a cyclic unsaturated hydrocarbon (with at least one trans configuration double bond).

[0126]

[0127] Option 6: Linkage of symmetrical tetrazine with a cyclic unsaturated hydrocarbon (having at least one trans configuration double bond)

[0128] The targeting carrier can be an antibody, nanobody, polymer, nanomedicine, cell, protein, peptide or small molecule.

[0129] The following tetrazine of formula Tz1 is suitable for the linkage in step b) of the method for providing a targeting vector containing a labeled pyridazine:

[0130]

[0131] Where R and R1 are independently selected from -H, -Me, or Wherein, the wavy line symbol indicates a connection to tetrazine; wherein, R2 is -H or (i) an isotopic labeling agent directly connected to the aromatic ring; or (ii) an isotopic labeling agent 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 stands for -(CH2). m Or -(CH2CH2O) mWherein, n and m are independently selected from 1-25; or (iii) an isotopic labeling agent chelated by a linker and linked to an aromatic ring, said chelating agent being 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-triazacyclononane-1-yl)glutaric acid (NODAGA), 2-(4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane-1-yl)glutaric 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-pentadecano-1(15),11,13-triene-3,6,9-triacetic acid (PCTA), N'-(5-acetyl(hydroxy)aminopentyl-N-(5-(4-(5-aminopentyl)(hydroxy)amino-4-oxobutyryl)amino)pentyl-N-hydroxysuccinamide (DFO), diethylenetriaminepentaacetic acid (DTPA), trans-cyclohexyl-diethylenetriaminepentaacetic acid (CHX-DTPA) 1-Oxa-4,7,10-triazacyclododecane-4,7,10-triacetic acid (OXO-Do3A), benzyl-DTPA (SCN-BZ-DTPA), 1-(benzyl-4-isothiocyanate)-3-methyl-DTPA (1B3M), 2-(benzyl-4-isothiocyanate)-4-methyl-DTPA (1M3B), and 1-(2)-methyl-4-isocyanate benzyl-DTPA (MX-DTPA), wherein the linker is selected from: -(CH2) n -LO(CH2) n -LNH(CH2) n -LCONH(CH2) n -LNHCO(CH2) n Where L stands for -(CH2). m Or -(CH2CH2O) m n and m are independently selected from 1-25;

[0132] When R2 is (i) or (ii), the isotope labeling agent is selected from:

[0133] 1 H, 2 H, 3 H, 11 C 12 C 13C、 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 O、 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 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 Through, 113 Through, 114 name, 115 name, 175 Head, 177 Head,185 Too, 186 Too, 188 Too, 201 Tl、 203 Tl、 205 Tl、 206 Pb、 207 Pb、 208 Pb、 212 Pb、 209 Bi、 212 Bi、 213 Bi、 31 P、 32 P、 33 P、 32 S、 35 S、 45 Sc、 47 Sc、 84 Sr、 86 Sr、 87 Sr、 88 Sr、 89 Sr、 165 Your, 166 Your, 156 Dy、 158 Dy、 160 Dy、 161 Dy、 162 Dy、 163 Dy、 164 Dy、 165 Dy、 227 Th、 232 Th、 51 Cr、 52 Cr、 53 Cr、 54 Cr、 73 The、 74 The、 75 The、 76 The、 77 The、 78 The、 80 The、 82 The、 94 Tc、 99m Tc、 103 Rh、 103 mRh、 119 Sb、 121 Sb、 123 Sb、 135 The、 138 The、 139 The、 162 Er、 164 Er、 165 Er、 166Er、 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,

[0134] Where X and Y are independently selected from: -CH and -N-;

[0135] R3 is independently selected from H or from hydroxyl, sulfonamide, guanidinyl, carboxyl, sulfonyl, amine, substituted amine having 1-25 polyethylene glycol units, and -(O-CH2-CH2). n The part of -OCH2-COOH, n is selected from 1-25; or methyl, ethyl, propyl, optionally substituted heteroaryl and optionally substituted arylalkyl; wherein, relative to the substituted amine, optional substitution means one or more substituents selected from: halogen, hydroxyl, sulfonamide, carboxyl, sulfonyl, amine, (C1-C10)alkyl, (C2-C10)alkenyl, (C2-C10)alkynyl, (C1-C10)alkylene, (C1-C10)alkoxy, (C2-C10)dialkylamino, (C1-C10)alkylthio, (C2-C10)heteroalkyl, (C2-C10)heteroalkylene, (C3-C10)cycloalkyl, (C3-C10)... Heterocyclic alkyl, (C3-C10)cycloalkylene, (C3-C10) heterocyclic alkylene, (C1-C10) haloalkyl, (C1-C10) perhaloalkyl, (C2-C10)-olefin, (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 means selected from one or more of the following substituents: halogen, hydroxyl, sulfonamide, carboxyl, sulfonyl, amine, substituted amine having 1-25 polyethylene glycol units, -(O-CH2-CH2). n -OCH2-COOH (n selected from 1-25); or H, methyl, ethyl, propyl, optionally substituted heteroaryl and optionally substituted arylalkyl; wherein, relative to the substituted amine, optional substitution refers to one or more substituents selected from halogen, hydroxyl, sulfonamide, carboxyl, sulfonyl and amine;

[0136] R and R1 are the same or differ only in the number of isotopes of the labeling agent.

[0137] The following tetrazine is a preferred tetrazine of formula Tz1, used for linking in step b) of the method for providing a targeting vector containing a labeled pyridazine:

[0138]

[0139] The following structures are preferred cyclic unsaturated hydrocarbons (having at least one trans-configured double bond) or unsaturated heterocycles (having at least one trans-configured double bond) for use in step b) of the method for providing a targeting support containing labeled pyridazine:

[0140]

[0141] Where X is NH, O, S, CH2, OCONH, OCSNH, NHCO; Y is N, NO, or CR8; Z is N, NO, or CR8; R8 is selected from: -H, -F, -OH, -NH2, -COOH, -COOCH3, CF3, -Cl, -CONH2, CONHCH3, -CON(CH3)2, -CH2OH, -CH2NH2, -CH2CH2OH, -CH2CH2NH2, -CHCH2N(CH3)2, and

[0142] The connector 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) nCH2CH2CO, 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, where n is 0-25, and M is a side chain selected from the group consisting of side chains of natural amino acids: 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;

[0143] Among them, the targeting carrier is an antibody, nanobody, polymer, nanomedicine, cell, protein, peptide or small molecule.

[0144] Examples of monocyclic unsaturated hydrocarbons (having at least one trans-configured double bond) or monocyclic unsaturated heterocycles (having at least one trans-configured double bond) (for connection in step b) of the method for providing a targeting vector containing labeled pyridazine):

[0145]

[0146] Among them, 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) nCH2CH2NHCOCHMNH, (CH2CH2O) n COCHMNH, where n is 0-25, and M is a side chain selected from the group consisting of side chains of natural amino acids: 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;

[0147] Among them, the targeting carrier is an antibody, nanobody, polymer, nanomedicine, cell, protein, peptide or small molecule.

[0148] Examples of bicyclic unsaturated hydrocarbons (having at least one trans-configured double bond) or bicyclic unsaturated heterocycles (having at least one trans-configured double bond) (for connection in step b) of the method for providing a targeting vector containing labeled pyridazine):

[0149]

[0150] 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) nCH2CH2NH, 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, where n is 0-25, and M is a side chain selected from the group consisting of side chains of natural amino acids: 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;

[0151] Among them, the targeting carrier is an antibody, nanobody, polymer, nanomedicine, cell, protein, peptide or small molecule.

[0152] Examples of tricyclic unsaturated hydrocarbons (having at least one trans-configured double bond) or tricyclic unsaturated heterocycles (having at least one trans-configured double bond) (for connection in step b) of the method for providing a targeting vector containing labeled pyridazine):

[0153]

[0154] Where 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

[0155] 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) nNHCHMCO, (CH2) n NHCOCHMNH, (CH2)OCOCHMNH, (CH2CH2O) n CH2CH2NHCHMCO, (CH2CH2O) n CH2CH2CONHCHMCO, (CH2CH2O) n CH2CH2NHCHMCO, (CH2CH2O) n CH2CH2NHCOCHMNH, (CH2CH2O) n COCHMNH, where n is 0-25, and M is a side chain selected from the group consisting of side chains of natural amino acids: 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;

[0156] Among them, the targeting carrier is an antibody, nanobody, polymer, nanomedicine, cell, protein, peptide or small molecule.

[0157] Examples of tetracyclic unsaturated hydrocarbons (having at least one trans-configured double bond) or tetracyclic unsaturated heterocycles (having at least one trans-configured double bond) (for connection in step b) of the method for providing a targeting vector containing labeled pyridazine):

[0158]

[0159] 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 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 NH₄CO(CH₂) n CO, CO(CH2) n O, CO(CH2CH2O) n CO(CH2CH2O) n CH2CH2NH, CO(CH2CH2O) n CH2CH2CO, COO(CH)2-COO(CH2) n NH₃COO(CH₂) n CO, COO(CH2) n O, COO(CH2CH2O) n COO(CH2CH2O) n CH2CH2NH, COO(CH2CH2O) n CH2CH2CO, CONH(CH)2-CONH(CH2) n NH, CONH(CH2) n CO, CONH(CH2) n O, CONH(CH2CH2O) n CONH(CH2CH2O) n CH2CH2NH, CONH(CH2CH2O) n CH2CH2CO、-CONHPhCO、-COOPhCO、-COPhCO、CONHCHMCO、(CH2) n NHCHMCO, (CH2) n OCONHCHMCO, (CH2) n NHCHMCO, (CH2) n NHCOCHMNH、(CH2)OCOCHMNH、(CH2CH2O) n CH2CH2NHCHMCO, (CH2CH2O) n CH2CH2CONHCHMCO, (CH2CH2O) n CH2CH2NHCHMCO, (CH2CH2O) n CH2CH2NHCOCHMNH, (CH2CH2O) nCOCHMNH, where n is 0-25, and M is a side chain selected from the group consisting of side chains of natural amino acids: 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;

[0160] Among them, the targeting carrier is an antibody, nanobody, polymer, nanomedicine, cell, protein, peptide or small molecule.

[0161] Step c) in the method for providing a targeting carrier containing pyridazine is an oxidation step. Although the self-oxidation of the linked entity targeting carrier (e.g., pyridazine) obtained in step b) of the method occurs spontaneously, the process is extremely slow and can last for hours or even days. Step c) of the method provides a method for rapidly oxidizing a mixture of dihydropyridazines to obtain the pyridazine compound via a catalytic acid, wherein the pyridazine-containing targeting carrier is obtained within at least 180 minutes (e.g., within 1-20 minutes). These conditions ensure that the efficiency of the oxidation step is ≥90%, thereby meeting the speed required for therapeutic, diagnostic, or imaging applications of the targeting carrier containing labeled pyridazine. To facilitate this process, the dihydropyridazine is oxidized by a standard or solid-loaded oxidant (preferably solid-loaded). The oxidation step can be carried out at a temperature of 15 to 50°C, for example at 20 to 30°C, preferably at room temperature, for about 10 to 180 minutes, preferably less than 20 minutes. To facilitate oxidation, 1 to 100 equivalents (preferably 1 equivalent) of a chemical oxidant and an acid are added to the linked compound obtained in the linking step. The oxidant must be selective, used to oxidize dihydropyridazine to pyridazine. The targeting carrier must not be chemically modified by the oxidant. The oxidant can be a quinone oxidant, a Bobbit salt oxidant, a peroxide oxidant, a peroxyacid oxidant, or ultraviolet light (minimum ultraviolet irradiance of 2 mW / cm²). 2 ), optionally add photosensitizer.

[0162] The acid is added to the complex mixture of various dihydropyridazines obtained in step b) by linking tetrazines and strained olefins, and exposed to ultraviolet light, for example, 200 to 400 nm, 200 to 280 nm (UVC), 280 to 315 nm (UVB), or 315 to 400 nm (UVA). Preferably, 254 nm.

[0163] In a preferred embodiment, oxidation is carried out in the presence of acid by ultraviolet light (minimum ultraviolet irradiance of 2 mW / cm²). 2 Alternatively, through ultraviolet light (minimum ultraviolet irradiance of 2 mW / cm²). 2 Photocatalysis was achieved by adding photosensitizers (e.g., fluorescein or porphyrin photosensitizers). An acid was added to a complex mixture of various dihydropyridazines obtained in step b), which consisted of tetrazines and strained olefins, and then exposed to ultraviolet light (minimum UV irradiance of 2 mW / cm²). 2 The wavelengths are ultraviolet light, such as 200 to 400 nm, 200 to 280 nm (UVC), 280 to 315 nm (UVB), or 315 to 400 nm (UVA). Preferably, the wavelength is UVC, for example, 254 nm.

[0164] Depending on the labeling agent, pharmaceutical agent, imaging agent, or therapeutic agent, and depending on the targeting carrier, the targeting carrier containing labeled pyridazine provided by the method can be used for treatment, radiotherapy, therapeutic diagnosis, diagnosis, or imaging.

[0165] Preferably, the targeting support is coupled to the linker via a nitrogen atom on the targeting support. Alternatively, preferably, the targeting support is coupled to the linker via a carbonyl group on the targeting support.

[0166] In a preferred embodiment, a targeting vector containing labeled pyridazine is used for treatment.

[0167] In another preferred embodiment, a targeting carrier containing labeled pyridazine is used for radiotherapy.

[0168] In another preferred embodiment, the targeting vector containing labeled pyridazine is used for therapeutic diagnosis.

[0169] In another preferred embodiment, a targeting vector containing labeled pyridazine is used for diagnosis.

[0170] In another preferred embodiment, a targeting vector containing labeled pyridazine is used for imaging.

[0171] The following examples describe: (1) the synthesis of tetrazine and TCO used in steps a) and b) of the method of this application for providing a targeting vector containing a labeled pyridazine; and (2) the click reaction and oxidation between such compounds to generate pyridazine. Example

[0172] Overview

[0173] All reagents and solvents were dried according to standard methods before use. Commercially available reagents were used, and no further purification was required. Analytical thin-layer chromatography (TLC) was performed using silica gel 60F254 (Merck), with detection performed by carbonization following UV absorption and / or immersion in 7% sulfuric acid EtOH solution or KMnO4 solution (1.5 g KMnO4, 10 g K2CO3, and 1.25 mL 10% NaOH in 200 mL water). Compound purification was performed by silica gel column chromatography (40–60 μm, 60 Å) or using a CombiFlash NextGen 300+ (Teledyne ISCO). Recordings were performed on Bruker instruments (400 and 600 MHz). 1 H and 13 C NMR spectroscopy, using chloroform- d MeOH- d 4 or DMSO- d 6 As a deuterated solvent, the residual solvent was used as an internal standard. The deuterated solvent signal was used as an internal standard for all NMR experiments. Chemical shifts are expressed in parts per million (ppm). Coupling constants (J values) are expressed in Hertz (Hz). 1The multiplexing of the 1H NMR signals is reported as follows: s, singlet; d, doublet; dd, doublet; ddd, doublet of doublets of doublets; dt, doublet triplet; t, triplet; q, quartet; m, multiplet; br, broad signal. The NMR spectra of all compounds were reprocessed based on the original FID files using MestReNova software (version 12.0.22023). Mass spectrometry analysis was performed using MS-Acquity-A: Waters Acquity UPLC with a QDa detector. Purification was performed by preparative HPLC on an Agilent 1260 Infinity system with a SymmetryPrep-C18 column, 17 mL / min H2O-MeCN gradient 50-100%, 15 min, using 0.1% trifluoroacetic acid. The purity of all final compounds was >95% as determined by analytical HPLC. Analytical HPLC method: (Thermo Fisher® UltiMate 3000), C-18 column (Luna® 5u C18(2) 100Å, 150×4.6mm), eluent: A: H2O containing 0.1% TFA, B: MeCN containing 0.1% TFA. Gradient elution: from 100% A to 100% B in 15 minutes, and back to 100% A in 4 minutes, at a flow rate of 1.5 mL / min. Detection was performed on a UVD 170U detector by UV absorption at λ = 254 nm.

[0174] Example 1

[0175] Synthesis of tetrazine and its precursors

[0176] Example 1.1

[0177]

[0178] Scheme 7: Shows the synthesis reaction scheme of tetrazine Tz-I. Reagents and conditions: i) NH2(CH2)2R, MeCN, 12 h, room temperature; ii) Boc2O, Et3N, DCM, 12 h, room temperature; iii) Zn(OTf)2, NH2NH2·H2O, EtOH, 65℃, 24 h; iv) HCl, dioxane, room temperature, 4 h.

[0179] Synthesis of 4-(((2-fluoroethyl)amino)methyl)benzonitrile (2)

[0180] To a solution of 4-(bromomethyl)benzonitrile (0.78 g, 4.00 mmol) in CH3CN (40 mL), K2CO3 (0.33 g, 24.0 mmol) and 2-fluoroethylamine hydrochloride (0.16 g, 16.0 mmol) were added. The mixture was stirred overnight at room temperature. The solvent was removed under reduced pressure, and the residue was diluted with water (20 mL) and extracted with EtOAc. The combined organic layers were washed with brine, dried over MgSO4, filtered, and concentrated under reduced pressure. The crude product was purified by rapid column chromatography in heptane using EtOAc (heptane / EtOAc 50 / 50) to give 0.54 g (76%) of the desired product as a colorless oil. f = 0.24 (heptane / EtOAc 40 / 60). 1 H NMR (400 MHz, CDCl3) δ 7.55 (d, J = 8.2 Hz, 2H), 7.40 (d, J = 8.0 Hz, 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, CDCl3) δ 145.6, 132.3,128.6, 118.9, 110.9, 83.5 (d, J = 165.5 Hz), 53.1, 49.1 (d, J = 19.7 Hz).

[0181] Synthesis of 4-cyanobenzyl(2-fluoroethyl)carbamate tert-butyl ester (3)

[0182] Boc2O (790 mg, 3.63 mmol) was added to a solution of 4-(((2-fluoroethyl)amino)methyl)benzonitrile (540 mg, 3.03 mmol) and Et3N (1.27 mL, 9.09 mmol) in CH2Cl2 (40 mL), and the mixture was stirred at room temperature for 12 h. The solution was washed with water and saturated K2CO3 solution, dried over Na2SO4, filtered, and concentrated under reduced pressure. The crude product was purified by rapid column chromatography (heptane / EtOAc 70 / 30) to give 0.710 g (84%) of the desired product as a colorless oil (a mixture of rotational isomers). f = 0.42 (heptane / EtOAc 80 / 20). 1H NMR (400 MHz, CDCl3) δ7.55 (d, J = 7.8 Hz, 2H), 7.27 (d, J = 7.8 Hz, 2H), 4.79 - 4.10 (m, 4H), 3.62- 3.28 (m, 2H), 1.96 - 1.05 (m, 9H). 13 C NMR (101 MHz, CDCl3) δ 155.4, 144.2,143.8, 132.4, 128.1, 127.5, 118.7, 111.1, 83.2 (d, J = 168.2 Hz), 82.7 (d, J = 170.5 Hz), 52.1, 51.2, 47.7, 28.3.

[0183] Synthesis of di-tert-butyl(((1,2,4,5-tetraazine-3,6-diyl)bis(4,1-phenylene))bis(methylene))bis((2-fluoroethyl)carbamate) (4)

[0184] To a suspension of tert-butyl 4-cyanobenzyl(2-fluoroethyl)carbamate (1.1 g, 3.95 mmol) and Zn(OTf)₂ (0.72 g, 1.98 mmol) in EtOH (30 mL), hydrazine monohydrate (3.83 mL, 79 mmol) was added. The mixture was stirred at 70 °C for 22 hours. After the reaction was complete, it was cooled to room temperature. Volatile substances were removed under reduced pressure, and the residue was dissolved in EtOH (40 mL). A solution of NaNO₂ (5.52 g, 80.00 mmol) in water (20 mL) was added to the crude reaction mixture, followed by dropwise addition of HCl (2 M) until gas evolution stopped, the pH reached 2-3, and a red mixture was obtained. The crude reaction mixture was extracted with DCM (3 × 40 mL) and washed with brine (3 × 20 mL). The organic phase was collected, dried over MgSO₄, filtered, and concentrated under reduced pressure. Purification by rapid chromatography (DCM / MeOH 98 / 2) yielded 0.300 g (26%) of red solid. f = 0.45 (DCM / MeOH 98 / 2). 1 H NMR (600MHz, CDCl3) δ 8.63 (d, J = 8.0 Hz, 4H), 7.50 (d, J= 7.0 Hz, 4H), 5.11 - 4.38 (m, 8H), 3.97 - 3.26 (m, 4H), 1.95 - 0.57 (m, 18H); 13 C NMR (151 MHz, CDCl3) δ163.77, 155.61, 155.57, 143.70, 130.81, 128.51, 128.21, 127.87, 83.18 (d, J =167.9 Hz), 82.61 (d, J = 169.3 Hz), 72.44, 65.78, 52.09, 51.09, 47.67 (d, J =19.9 Hz), 47.19 (d, J = 21.0 Hz), 28.38.

[0185] Synthesis of N,N'-(((1,2,4,5-tetraazine-3,6-diyl)bis(4,1-phenylene))bis(methylene))bis(2-fluoroethane-1-amine) (Tz-I)

[0186] Di-tert-butyl(((1,2,4,5-tetraazine-3,6-diyl)bis(4,1-phenylene))bis(methylene))bis((2-fluoroethyl)carbamate) (0.15 g, 0.25 mmol) was treated with a solution of HCl (4 M) in dioxane (1 mL). A precipitate formed. Filtration gave 0.1 g (83%) of the desired product as hydrochloride. 1 H NMR (600 MHz, DMSO) δ 9.70 (s, 4H), 8.61 (d, J = 8.0 Hz, 4H), 7.89 (d, J = 8.0 Hz, 4H), 4.86 (t, J = 4.6 Hz, 2H), 4.78 (t, J = 4.6 Hz, 2H), 4.37 (s, 4H), 3.40 - 3.25 (m, 4H); 13 C NMR (151 MHz, DMSO) δ 163.63, 136.83, 132.81, 131.66, 128.28, 80.09 (d, J = 165.1 Hz), 50.24, 47.21 (d, J = 19.8 Hz).

[0187] Example 1.2

[0188]

[0189] Scheme 8: Shows the synthesis reaction scheme for tetrazine Tz-II. Reagents and conditions: i) K2CO3, MeCN, 2 h, reflux; ii) S8, NH2NH2·H2O, EtOH, 65 °C, 24 h.

[0190] Synthesis of 5-(2-fluoroethoxy)pyridinecarboxynitrile (6)

[0191] To a solution of 5-hydroxypyridine carboxynitrile (1 g, 8.35 mmol) and CH3CN (30 mL), K2CO3 (2.30 g, 16.65 mmol) and 1-fluoroiodoethane (813 µL, 10.00 mmol) were added. The reaction was refluxed until all the starting material was consumed. Water was added, and the solution was extracted with EtOAc. The solvent was removed under reduced pressure to give 1.32 g of a dark solid (95%). 1 H NMR (400 MHz, CDCl3) δ 8.42 (d, J = 2.9 Hz, 1H), 7.66 (d, J = 8.6 Hz, 1H), 7.29 (dd, J =8.6, 2.9 Hz, 2H), 4.90 - 4.83 (m, 1H), 4.77 - 4.69 (m, 1H), 4.40 - 4.35 (m, 1H), 4.32 - 4.25 (m, 1H).

[0192] Synthesis of 3,6-bis(5-(2-fluoroethoxy)pyridin-2-yl)-1,2,4,5-tetraazine (Tz-II)

[0193] Hydrazine monohydrate (0.89 mL, 18.27 mmol) was added to a suspension of 5-(2-fluoroethoxy)pyridinium carboxylate (810 mg, 4.87 mmol) and S8 (0.72 g, 1.98 mmol) in EtOH (1.8 mL). The mixture was stirred at 90 °C for 4 hours, and after the reaction was complete, it was cooled to room temperature. Volatile substances were removed under reduced pressure, and the residue was dissolved in EtOH (40 mL). A solution of NaNO2 (5.52 g, 80.00 mmol) in water (20 mL) was added to the crude reaction mixture, followed by dropwise addition of HCl (2 M) until gas evolution stopped, the pH reached 2-3, and a red mixture was obtained. The crude reaction mixture was extracted with DCM (3 × 40 mL) and washed with brine (3 × 20 mL). The organic phase was collected, dried over MgSO4, filtered, and concentrated under reduced pressure. Purification by rapid chromatography (DCM / MeOH 98 / 2) yielded 125 mg (28%) of red solid. f =0.45 (DCM / MeOH9 8 / 2).

[0194] Synthesis of 3-(5-iodopyridin-2-yl)-6-(pyridin-2-yl)-1,2,4,5-tetraazine (Tz-VII)

[0195] This compound was described by Albu et al., 125 The procedure was obtained as reported in "I-Tetraazine and Reverse Electron Demand Diels-Alder Chemistry: A Convenient Radioiodination Strategy for Biomolecular Labeling, Screening and Biodistribution Studies", Bioconjug Chem. 2016;27,207-216.

[0196] Synthesis of 2,2'-((2-(2-fluoroethoxy)-4-(1,2,4,5-tetraazine-3-yl)benzyl)azadiyl)diacetic acid (Tz-XII)

[0197] The compound was obtained according to the procedure reported by Battisi et al., “Development of the first aliphatic 18F-labeled tetrazine suitable for pre-targeted PET imaging – an extension of the bioorthogonal toolbox,” J. Med. Chem. 2021, 64, 20, 15297-15312.

[0198] Example 1.3

[0199]

[0200] Scheme 9: Shows the synthesis reaction scheme for tetrazine Tz-XX. Reagents and conditions: i) K2CO3, MeCN, 2 h, reflux; ii) S8, NH2NH2·H2O, EtOH, 65 °C, 24 h.

[0201] Synthesis of 3-(bromomethyl)-5-iodobenzonitrile (8)

[0202] In a 100 mL round-bottom flask equipped with a stir bar, 3-iodo-5-methylbenzonitrile (2 g, 8.23 ​​mmol), N-bromosuccinimide (2.20 g, 12.34 mmol), and AIBN (540 mg, 3.29 mmol) were dissolved in 25 mL of MeOH. The reaction was refluxed at 80–85 °C for 6 hours. Water and DCM were added. The organic layer was washed twice with water, and the aqueous phase was extracted twice with DCM each time. The combined organic phases were washed in brine, dried over MgSO4, filtered, and concentrated under vacuum. The residue was dissolved in DCM and adsorbed onto diatomaceous earth. The crude product was then purified by rapid chromatography (heptane / DCM 85:15 -> 70:30) to give 2 g (75.6%) of NB35 as a white solid. f = 0.11 (heptane:DCM 8:2). 1 H-NMR (CDCl3, 600MHz): 4.38 (s, 2H), 7.65 (t, 1H, J = 1.6), 7.91 (t, 1H, J = 1.55), 7.97 (t, 1H, J = 1.65). 13 C-NMR (CDCl3, 600 MHz): 30.0, 94.2, 114.7, 116.7, 131.8, 140.29, 141.1, 142.4.

[0203] Synthesis of 3-(hydroxymethyl)-5-iodobenzonitrile (9)

[0204] 3-(bromomethyl)-5-iodobenzonitrile (1 g, 3.11 mmol) and barium carbonate (1.23 g, 6.21 mmol) were refluxed in a 1:1 MeCN / deionized water (24 mL) at 75–80 °C. The reaction was stirred overnight. LCMS showed the reaction progress with more MeCN. After 28 hours, the reaction was still proceeding slowly, so more MeCN, water, and barium carbonate (1 equivalent) were added. The reaction was refluxed and stirred overnight. After 45 hours, the reaction was observed to be complete by LCMS. The mixture was cooled to room temperature. The solids were removed by filtration. The filtrate was extracted three times with DCM. The combined organic phases were dried over MgSO4, filtered, and concentrated. The residue was redissolved in DCM and adsorbed onto diatomaceous earth. The compound was purified by rapid chromatography (5–20% EtOAc, in heptane) to a white color. Yield: 582 mg (69%). f = 0.36 (7:3 heptane: EtOAc).1 H-NMR (CDCl3, 600MHz): 1.88 (t, 1H, J = 6.76), 4.72 (d, 2H, J = 5.7), 7.62 - 7.64 (m, 1H), 7.88 - 7.90 (m, 1H), 7-95 - 7.97(m, 1H). 13 C-NMR (CDCl3, 600 MHz): 63.3, 94.1, 114.4, 117.2, 129.4, 139.47,140.1, 144.2.

[0205] Synthesis of (3-iodo-5-(1,2,4,5-tetraazine-3-yl)phenyl)methanol (10)

[0206] 3-(hydroxymethyl)-5-iodobenzonitrile (548 mg, 2.12 mmol), DCM (135 μL, 2.12 mmol), S8 (136 mg, 0.53 mmol), and ethanol (3 mL) were mixed together in a 20 mL microwave-safe reaction tube. Then, hydrazine monohydrate (825 μL, 16.9 mmol) was slowly added with stirring. The container was sealed, and the reaction mixture was heated to 50 °C for 22 hours. The reaction darkened, and a yellow precipitate appeared. Next, 5 mL of DCM and sodium nitrite (1.46 g, 21.15 mmol) in 20 mL of H₂O were added to the mixture. Then, excess acetic acid (6 mL) was slowly added, during which time the solution turned bright red. The reaction mixture was stirred for approximately 10 minutes. The aqueous phase was then extracted four times with DCM. The organic phase was dried over anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure. The residue (769 mg) was purified by rapid chromatography (heptane / EtOAc) to give 262 mg (39%) of product as a red / pink solid. f = 0.24 (heptane / EtOAc 70 / 30). 1 H-NMR (CDCl3, 600MHz): 1.88 (t, 1H, J = 5.9), 4.82(d, 2H, J = 4.8Hz), 8.03 - 8.05 (m, 1H), 8.59 - 8.60 (m, 1H), 8.90 - 8.93 (m,1H), 10.26 (s, 1H). 13C-NMR (CDCl3, 600 MHz): 64.1, 95.2, 125.8, 133.7, 136.3, 140.3, 144.4, 158.2, 165.4.

[0207] Synthesis of di-tert-butylformamidinyl (3-iodo-5-(1,2,4,5-tetraazine-3-yl)benzyl)carbamate (11)

[0208] (3-Iodo-5-(1,2,4,5-tetraazine-3-yl)phenyl)methanol (184 mg, 0.59 mmol) in 8 mL of anhydrous THF was stirred under argon atmosphere with 1,3-bis(tert-butoxycarboxyl)guanidine (167 mg, 0.64 mmol) and triphenylphosphine (310 mg, 1.17 mmol). DIAD (230 μL, 1.17 mmol) was added dropwise. Initially, the mixture darkened, but after 15 minutes, due to Tz, the mixture turned red / pink. After 3 hours, LCMS showed complete conversion. The solvent was evaporated, and the resulting crude product was reconstituted in EtOAc, washed with 2× water and 2× brine, dried over MgSO4, filtered, and concentrated. The compound was dissolved in EtOAc and adsorbed onto diatomaceous earth. The compound was purified by rapid chromatography (95:5 heptane:EtOAc). Yield: 186 mg (45%). f = 0.36 (8:2 heptane: EtOAc). 1 H-NMR (CDCl3, 400MHz): 1.45 (s, 9H), 1.52 (s, 9H), 5.23 (s, 2H), 8.05 - 8.08 (m, 1H), 8.60 - 8.63 (m, 1H), 8.89 (s, 1H), 9.25 (s, 1H), 9.43(s, 1H), 10.24(s, 1H). 13 C-NMR (CDCl3, 400 MHz): 28.1, 28.5, 46.9, 79.3,85.0, 94.8, 127.2, 133.4, 135.9, 142.0, 142.2, 154.7, 158.2, 160.5, 163.7,165.4.

[0209] Synthesis of 1-(3-iodo-5-(1,2,4,5-tetraazine-3-yl)benzyl)guanidine (Tz-XX)

[0210] Di-tert-butylformamidinyl (3-iodo-5-(1,2,4,5-tetraazine-3-yl)benzyl)carbamate (30 mg, 0.054 mmol) was deprotected with a solution of 4 M HCl in dioxane (4 mL) for 41 hours, and the residue was then concentrated under vacuum. The crude product was dissolved in 5 mL of H₂O containing 0.1% TFA and transferred to prep-HPLC. The fractions containing the purified product were combined and lyophilized. The yield was 17 mg (1×TFA: 69%, 2×TFA: 56%). 1 H-NMR(MeOD, 600MHz): 4.55 (s, 2H), 8.02 -8.03 (m, 1H), 8.56 - 8.57 (m, 1H), 8.88 - 8.90 (m, 1H), 10.40 (s, 1H). 13 C-NMR (MeOD, 600 MHz): 44.9, 95.8, 126.8, 136.0, 137.3, 141.3, 141.5, 158.9, 159.7,166.4.

[0211] Example 2

[0212] Synthesis of cyclic unsaturated hydrocarbons with at least one trans configuration double bond and their precursors

[0213] Example 2.1

[0214]

[0215] Scheme 10: Synthesis of TCO without isomers. i) mCPBA, THF, H2O, 0℃ → room temperature, 17 hours, 47%; ii) LiAlH4, THF, 0℃ → room temperature, 12 hours, 99%; iii) a) Et3N, DMAP, CH2Cl2, 0℃ → room temperature, 12 hours, 51%; iv) Crystallization from pentane, 41%; v) NaOH, THF, H2O, reflux, 2 hours, 52%; vi) AgNO3, hV, room temperature, 8 hours, 44%.

[0216] (Z)-9-oxabicyclo[6.1.0]non-4-ene (13)

[0217] Cis,cis-1,5-cyclooctadiene (22.0 g, 203.36 mmol, 1.00 equivalent) and dried CH2Cl2 (300 mL) were added to a 500 mL round-bottom flask. The mixture was cooled to 0 °C in an ice bath, and mCPBA (45.57 g, 203.36 mmol, 1.00 equivalent) was added in portions to obtain a white suspension. The mixture was brought to room temperature and stirred overnight. The mixture was filtered and washed with saturated NaHCO3 solution (3 × 100 mL) and saturated NaCl solution (1 × 100 mL). The organic layer was collected, dried over MgSO4, filtered, and concentrated under reduced pressure. Purification by rapid chromatography (n-heptane / EtOAc, 90:10) yielded (Z)-9-oxabicyclo[6.1.0]non-4-ene (11.82 g, 95.16 mmol, 47%) as a colorless oil. 1 H NMR (600 MHz, CDCl3)δ 5.69 - 5.48 (m, 2H), 3.15 - 2.91 (m, 2H), 2.55 - 2.35 (m, 2H), 2.21 - 2.08(m, 2H), 2.08 - 1.86 (m, 4H); 13 C NMR (151 MHz, CDCl3) δ 129.00, 56.87, 28.25, 23.82.

[0218] (Z)-Cyclooct-4-enol (14)

[0219] Lithium aluminum hydride flakes (3.26 g, 85.93 mmol, 3.00 equivalent) were added to a dried 500 mL three-necked round-bottom flask. The flask was sealed and purged with argon. The flask was cooled to 0 °C using an ice bath, and dry THF (120 mL) was slowly added while stirring vigorously to obtain a gray suspension. 1,2-epoxy-5-cyclooctene (3.56 g, 28.64 mmol, 1.00 equivalent) was added dropwise to dry THF (10 mL), and the mixture was brought to room temperature and stirred overnight. The mixture was cooled to 0 °C in an ice bath and quenched with EtOAc (120 mL). A saturated solution of Rochelle salt (100 mL) was added, and the mixture was stirred vigorously for 10 minutes. The mixture was transferred to a separatory funnel, and the organic layer was collected. The aqueous layer was extracted with DCM (3 × 150 mL). The combined organic layers were washed with water (200 mL), dried with MgSO4, filtered, and concentrated under reduced pressure to give (Z)-cyclooctyl-4-enol (3.49 g, 28.45 mmol, 99%). 1H NMR (600 MHz, CDCl3) δ 5.75 - 5.63 (m, 1H), 5.61 - 5.52 (m,1H), 3.86 - 3.75 (m, 1H), 2.36 - 2.24 (m, 1H), 2.20 - 2.04 (m, 3H), 1.97 (s,1H), 1.93 - 1.88 (m, 1H), 1.86 - 1.81 (m, 1H), 1.75 - 1.68 (m, 1H), 1.67 -1.59 (m, 1H), 1.56 - 1.46 (m, 2H); 13 C NMR (151 MHz, CDCl3) δ 130.23, 129.63, 72.85, 37.75, 36.36, 25.75, 24.97, 22.88.

[0220] (Z)-Cyclooct-4-en-1-yl(1R,4S)-4,7,7-trimethyl-3-oxo-2-oxabicyclo[2.2.1]heptane-1-carboxylic acid ester (a mixture of epimers) (16)

[0221] (±)-(Z)-cyclooctyl-4-enol (4.5 g, 35.65 mmol) was dissolved in dry CH2Cl2 (100 mL), and DMAP (0.87 g, 7.13 mmol) and Et3N (14.9 mL, 106.97 mmol) were added to the mixture. The solution was cooled to 0 °C, and (1S)-(-)-camphoryl chloride (8.5 g, 38.22 mmol) was added to the mixture in portions. The resulting solution was stirred at room temperature for 17 hours. The mixture was washed with saturated NaHCO3 solution (3 × 100 mL) and saturated NaCl solution (1 × 100 mL). The organic layer was collected, dried over MgSO4, filtered, and concentrated under reduced pressure to give a mixture of 5.63 g (51%) of the desired epimers. Recrystallization from pentane yielded 2.31 g (41%) of (Z)-cyclooct-4-en-1-yl(1R,4S)-4,7,7-trimethyl-3-oxo-2-oxabicyclo[2.2.1]heptane-1-carboxylic acid ester, in crystal form (needle-like). 1H NMR (400 MHz, CDCl3) δ 5.75 - 5.58 (m, 2H), 5.05- 4.94 (m, 1H), 2.46 - 2.29 (m, 2H), 2.28 - 2.08 (m, 3H), 2.06 - 1.84 (m,4H), 1.84 - 1.74 (m, 1H), 1.74 - 1.33 (m, 4H), 1.10 (s, 3H), 1.04 (s, 3H,).0.95 (s, 3H); 13 C NMR (101 MHz, CDCl3) δ 178.46, 166.97, 130.05, 129.99,129.59, 129.56, 91.23, 54.96, 54.21, 33.87, 33.85, 33.81, 30.69, 29.12,29.10, 25.67, 25.65, 24.92, 24.88, 22.37, 22.35, 17.01, 16.95, 16.88, 9.84.

[0222] (S,Z)-Cyclooct-4-en-1-ol (19)

[0223] (S,Z)-cyclooctyl-4-en-1-yl(1R,4S)-4,7,7-trimethyl-3-oxo-2-oxabicyclo[2.2.1]heptane-1-carboxylic acid ester (0.26 g, 0.85 mmol) was dissolved in THF (10 mL), and a solution of NaOH (0.2 g, 4.24 mmol) in water (1 mL) was added. The reaction was stirred vigorously under reflux for 2 hours. The mixture was quenched with H2O (10 mL), and the aqueous layer was extracted with DCM (3 × 30 mL). The combined organic layers were washed with H2O (20 mL), dried over MgSO4, filtered, and concentrated under reduced pressure to give (S,Z)-cyclooctyl-4-enol (0.056 g, 52%) as a colorless oil. 1H NMR (600 MHz, CDCl3) δ 5.75 - 5.63(m, 1H), 5.61 - 5.52 (m, 1H), 3.86 - 3.75 (m, 1H), 2.36 - 2.24 (m, 1H), 2.20- 2.04 (m, 3H), 1.97 (s, 1H), 1.93 - 1.88 (m, 1H), 1.86 - 1.81 (m, 1H), 1.75 - 1.68 (m, 1H), 1.67 - 1.59 (m, 1H), 1.56 - 1.46 (m, 2H); 13 C NMR (151 MHz, CDCl3) δ 130.23, 129.63, 72.85, 37.75, 36.36, 25.75, 24.97, 22.88.

[0224] (S,E)-Cyclooct-4-en-1-ol (20)

[0225] The bottom of a flash evaporator (220g, screw cap, Luer lock connector, catalog number: FCSTLL-220-6) was filled with 8cm of silica (15-40µm), and then filled with silver nitrate-impregnated silica until the top. The column was flushed with 500mL of 9:1 diethyl ether / n-heptane and protected from light with aluminum foil. The cooling grid and UV lamp were turned on, and no silver leakage was detected after 10 minutes. Methyl benzoate (1mL), (S,Z)-cyclooctyl-4-en-1-ol (1g), and another 50mL of 9:1 diethyl ether / n-heptane solution were added to a round-bottom flask. The mixture was then transferred to a quartz flask. The pump was turned on (flow rate = 100mL / min), and the photoreactor was started for photoisomerization for 8 hours. After 8 hours, the photoreactor was turned off, and the column was dried with a gas stream. Silica was removed from the column, and the column was washed with 400mL of ammonia and 400mL of DCM. The mixture was stirred for 30 minutes, filtered, and the organic layer was collected. The organic layer was washed with brine, dried with MgSO4, filtered, and concentrated to give 0.44 g (44%) of product, a yellow oily substance (a mixture of axial and equatorial isomers). Main equatorial... 1 H NMR (400 MHz, CDCl3) δ 5.57 (ddd, J = 15.3, 10.6, 4.1Hz, 1H), 5.38 (ddd, J = 15.7, 10.9, 3.6 Hz, 1H), 3.50 - 3.40 (m, 1H), 2.32(tq, J= 17.3, 5.1 Hz, 3H), 2.01 - 1.87 (m, 4H), 1.74 - 1.50 (m, 3H). Minoraxial 1 H NMR (400 MHz, CDCl3) δ 6.02 - 5.19 (m, 2H), 4.48 - 3.85 (m, 1H), 2.37(dddd, J = 15.2, 10.5, 7.0, 4.8 Hz, 1H), 2.30 - 1.96 (m, 4H), 2.00 - 1.75 (m,3H), 1.65 (dddd, J = 14.4, 12.8, 5.0, 1.7 Hz, 1H), 1.26 (dddd, J = 13.6,10.9, 3.5, 0.9 Hz, 1H).

[0226] Example 2.2

[0227]

[0228] Scheme 11: Synthesis of cis-cyclooctyl-5-en-1,2-diol. i) NMO, OsO4, THF, H2O, acetone, 0°C to room temperature, 12 hours, 93%; ii) AgNO3, hV, room temperature, 8 hours, 0°C to room temperature, 12 hours, 51%;

[0229] cis-Z-cyclooct-5-ene-1,2-diol (21)

[0230] Osmium tetroxide (12.7 mg, 0.05 mmol) was added to a stirred mixture of 1,5-cyclooctadiene (1.22 mL, 12.9 mmol), 4-methylmorpholine N-oxide (1.87 g, 13.75 mmol), and THF:H₂O:acetone (1:1:1) (90 mL) at 0 °C. After reacting the reaction mixture at 25 °C for 12 hours, the reaction mixture was poured into a saturated aqueous solution of NaHSO₃ (60 mL), extracted with EtOAc (3 × 150 mL), and washed with water (2 × 50 mL) and brine (50 mL). The mixture was dried (MgSO₄), concentrated, and then subjected to rapid chromatography (silica, 30% EtOAc in heptane) to give 1.70 g (93%) of the desired compound. 1HNMR (400 MHz, CDCl3) δ 5.72 - 5.61 (m, 2H), 4.03 - 3.96 (m, 2H), 2.56 - 2.44(m, 2H), 2.10 - 1.96 (m, 4H), 1.86 - 1.74 (m, 2H); 13 C NMR (101 MHz, CDCl3) δ130.09, 75.18, 32.08, 23.11.

[0231] cis-E-cyclooct-5-ene-1,2-diol (22)

[0232] The bottom of a flash evaporator (220g, screw cap, Luer lock connector, catalog number: FCSTLL-220-6) was filled with 8cm of silica (15-40µm), and then filled with silver nitrate-impregnated silica until the top. The column was flushed with 500mL of 9:1 diethyl ether / n-heptane and protected from light with aluminum foil. The cooling grid and UV lamp were turned on, and no silver leakage was detected after 10 minutes. Methyl benzoate (1mL), cis-Z-cyclooctyl-5-ene-1,2-diol (1g), and another 50mL of 9:1 diethyl ether / n-heptane solution were added to a round-bottom flask. The mixture was then transferred to a quartz flask. The pump was turned on (flow rate = 100mL / min), and the photoreactor was started for photoisomerization for 8 hours. After 8 hours, the photoreactor was turned off, and the column was dried with a gas stream. Silica was removed from the column, and the column was washed with 400mL of ammonia and 400mL of DCM. The mixture was stirred for 30 minutes, filtered, and the organic layer was collected. The organic layer was washed with brine, dried with MgSO4, filtered, and concentrated to give 0.51 g (51%) of product, which was a colorless oil.

[0233] Example 3

[0234] Synthesis of unsaturated heterocycles with at least one trans configuration double bond and their precursors

[0235] Example 3.1

[0236]

[0237] Scheme 12: Synthesis of TCO without isomers. i) mCPBA, THF, H2O, 0℃ → room temperature, 17 hours, quantitative; ii) HClO4, THF, H2O, room temperature, 24 hours (48%); iii) AgNO3, hV, room temperature, 8 hours, 60%; iv) oxalyl chloride, DMSO, Et3N, DCM, -78℃, 3 hours (62%); v) 2-aminoaniline, MeOH, room temperature, 0.2 hours, quantitative.

[0238] (Z)-9-oxabicyclo[6.1.0]non-4-ene (23)

[0239] Cyclooctadiene (1.00 g, 9.24 mmol) was dissolved in dry DCM (20 mL) and cooled to 0 °C. mCBPA (2.07 g, 9.24 mmol) was added in portions. After addition, the reaction mixture was stirred at 0 °C for 30 min, then heated to room temperature. The reaction was stirred for 17 h. The reaction was quenched with 2 M K₂CO₃ (20 mL), and the crude product was extracted with 3× DCM (15 mL). The combined organic phases were washed with K₂CO₃ (25 mL) and brine (25 mL). The solvent was evaporated, and the crude product was used directly in the next reaction without purification.

[0240] (Z)-Cyclooct-5-ene-1,2-diol (24)

[0241] Compound 25 (1.15 g, 9.24 mmol) was dissolved in a THF / water mixture (3:2 v / v, 50 mL) containing HClO4 (250 μL, 2.90 mmol). The reaction mixture was stirred at room temperature for 24 hours. Water (10 mL) was added to the reaction mixture, and the product was extracted with 3×Et2O (20 mL). The combined ether fractions were washed with 2×2M NaHCO3 (25 mL). The crude product was purified by CombiFlash to give a clear, viscous oil (624 mg, 4.40 mmol, 48%). 1 H NMR (600 MHz, Chloroform-d) 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, Chloroform-d) δ 129.3, 74.1, 33.6,22.9.

[0242] (E)-Cyclooct-5-ene-1,2-diol (25)

[0243] This compound was obtained as described in the following literature: Royzen, M.; Yap, GPA; Fox, JM. A Photochemical Synthesis of Functionalized trans-Cyclooctenes Driven by MetalComplexation. Journal of the American Chemical Society 2008, 130, 3760-3761.

[0244] The bottom of a flash evaporator (220g, screw cap, Luer lock connector, catalog number FCSTLL-220-6) was filled with 8cm of silica (15-40µm), and then filled with silver nitrate-impregnated silica until the top. The column was flushed with 500mL of 9:1 diethyl ether / n-heptane and protected from light with aluminum foil. The cooling grid and UV lamp were turned on, and no silver leakage was detected after 10 minutes. Methyl benzoate (1mL), compound 27 (1g), and another 50mL of 9:1 diethyl ether / n-heptane solution were added to a round-bottom flask. The mixture was then transferred to a quartz flask. The pump (flow rate = 100mL / min) was started, and the photoreactor was started for photoisomerization for 8 hours. After 8 hours, the photoreactor was turned off, and the column was dried with a gas stream. Silica was removed from the column, and the column was washed with 400mL of ammonia and 400mL of DCM. The mixture was stirred for 30 minutes, filtered, and the organic layer was collected. The organic layer was washed with brine, dried with MgSO4, filtered, and concentrated to give 0.60 g (60%) of product, which was a yellow oily substance (a mixture of axial and equatorial isomers). 1 H NMR (CDCl3, 400 MHz) δ 5.45-5.36 (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, 100 MHz) δ 132.6, 76.0, 40.7, 32.6.

[0245] (E)-Cyclooct-5-ene-1,2-dione (26)

[0246] Under an argon atmosphere and at -78°C, a solution of DMSO (1.05 mL, 14.77 mmol) in dry DCM (30 mL) was added dropwise to a solution of (COCl)₂ (736 μL, 8.44 mmol) in dry DCM (15 mL). After addition, the reaction mixture was stirred at -78°C for 30 minutes. A solution of compound 28 (500 mg, 3.52 mmol) in dry DCM (10 mL) was added dropwise, and the reaction mixture was stirred at -78°C for another 30 minutes. Et₃N (4.90 mL, 35.16 mmol) was added dropwise, and the reaction mixture was maintained at -78°C for another 60 minutes. The reaction mixture was then warmed to room temperature and stirred for another hour. The reaction mixture was then dissolved in water (50 mL) and 2 × 0.5 M HCl. （aq） Wash with 50 mL of saline solution and 30 mL of brine. Dry the organic phase with MgSO4 and evaporate to give the desired compound as a clear liquid. (302 mg, 2.19 mmol, 62%).

[0247] (E)-6,7,10,11-tetrahydrocyclooctyl[b]quinoxaline (27)

[0248] The compound was obtained according to the procedure described in the following literature: Delpivo, C.; Micheletti, G.; Boga, C. A Green Synthesis of Quinoxalines and 2,3-Dihydropyrazines. Synthesis 2013, 45, 1546-1552. A 50 mL round-bottom flask was fed with a solution of the appropriate compound 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 crude mixture was extracted with CH2Cl2 (3 × 10 mL). The combined organic layers were dried (MgSO4), filtered, and the solvent was removed to give 0.2 g (97%) of the desired product. 1 H NMR (400 MHz, CDCl3) δ 8.23 ​​- 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, 2.8 Hz, 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).

[0249] Example 3.2

[0250]

[0251] Scheme 13: Synthesis of TCO without isomers. i) Sulfuric acid, nitric acid, 80°C, 0.5 h (56%); ii) Urea, 150°C, 6 h (87%); iii) Stannous chloride (II), EtOH, reflux, 15 h (quantitative); iv) Cs₂CO₃, tert-butyl bromoacetate or methyl bromoacetate, DMF, 120°C, 4 h (33-49%); v) Method A: tert-butyl 2-(5,6-diamino-1,3-dioxoisoindoline-2-yl)acetate, TFA, DCM, room temperature, 4 h (99%); Method B: methyl 2-(5,6-diamino-1,3-dioxoisoindoline-2-yl)acetate, concentrated HCl （溶液） dioxane, 70°C, 24 hours (99%); vi) AcOH, (E)-cyclooctyl-5-ene-1,2-dione, DMF, room temperature, 6 hours (27%); vii) LiOH, THF, H2O, room temperature, 3 hours (89%); viiii) N-hydroxysuccinimide, DCC, DMF, room temperature, 18 hours (72%).

[0252] 5-Chloro-6-nitroisoindoline-1,3-dione (29)

[0253] The synthesis of this compound was based on literature. A mixture of concentrated sulfuric acid (33.1 mL) and fuming nitric acid (2.26 mL) was added dropwise to 4-chlorophthalimide (5.00 g, 27.53 mmol). The reaction mixture was heated at 80 °C for 0.5 h. After cooling to room temperature, the deep red solution was poured onto ice. The resulting yellow precipitate was collected by filtration, washed with water, and dried under vacuum to give a pale yellow solid, which was recrystallized from EtOH to give 3.5 g (56% yield) of the desired product. f = 0.45 (n-heptane / EtOAc 60 / 40). 1 H NMR (400 MHz, DMSO) δ 11.87 (s, 1H), 8.51 (s, 1H), 8.27 (s, 1H); 13C NMR (101 MHz, DMSO) δ 167.21, 167.17, 151.86, 136.54, 133.00, 130.89, 126.60, 120.20.

[0254] 5-Amino-6-nitroisoindoline-1,3-dione (30)

[0255] This compound was synthesized according to literature. A mixture of 5-chloro-6-nitroisoindoline-1,3-dione (2.4 g, 10.5 g) and urea (6.36 g, 105.92 mmol) was stirred under argon and heated to 150 °C for 6 hours. After cooling to room temperature, the solid was suspended in hot water (80 °C), filtered, and washed with hot water (3 × 50 mL). The solid was recrystallized from EtOH to give 1.92 g (87%) of a yellow solid. f = 0.28 (n-heptane / EtOAc 60 / 40). ¹H NMR (600 MHz, DMSO- d 6 ) δ 11.43 (s, 1H), 8.56 - 8.05 (m, 3H), 7.40 (s, 1H); 13 C NMR (151 MHz, DMSO-d6) δ 168.01, 150.64, 138.24, 132.50, 122.38, 117.74, 114.65.

[0256] 5,6-Diaminoisoindoline-1,3-dione (31)

[0257] This compound was synthesized according to literature. 1. A suspension of 4-amino-5-nitrophthalimide (1.0 g, 4.27 mmol) and stannous(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, filtered to collect the red solid, washed with ethanol, and dried under vacuum to give a red-orange solid. R f = 0.15 (95 / 5DCM / MeOH); 1 H NMR (400 MHz, DMSO) δ 10.25 (s, 1H), 6.81 (s, 2H), 5.50 (s, 4H); 13 C NMR (101 MHz, DMSO) δ 170.72, 140.30, 122.83, 107.03.

[0258] 2-(5,6-diamino-1,3-dioxoisoindoline-2-yl)tert-butyl acetate (32)

[0259] To a suspension of Cs₂CO₃ (1.93 g, 5.92 mmol) and 5,6-diaminoisoindoline-1,3-dione (1.0 g, 5.64 mmol) in anhydrous DMF (5 mL), tert-butyl bromide acetate (0.87 mL, 5.92 mmol) was added. The reaction mixture was heated at 120 °C for 4 h under argon atmosphere. 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 dried over anhydrous MgSO₄, filtered, and concentrated under reduced pressure. The residue was purified by rapid chromatography (98 / 2 DCM / MeOH) to give 0.8 g (49%) of the desired compound as a yellow solid. R f = 0.42 (DCM / MeOH 93 / 7); 1 H NMR (600MHz, DMSO) δ 6.89 (s, 2H), 5.61 (s, 4H), 4.11 (s, 2H), 1.40 (s, 9H); 13 C NMR (151 MHz, DMSO) δ 168.63, 167.62, 140.46, 121.55, 107.34, 82.08, 40.55, 28.08.

[0260] 2-(5,6-diamino-1,3-dioxoisoindoline-2-yl)methyl acetate (33)

[0261] Methyl bromoacetate (0.63 mL, 5.92 mmol) was added to a suspension of Cs₂CO₃ (1.93 g, 5.92 mmol) and 5,6-diaminoisoindoline-1,3-dione (1.0 g, 5.64 mmol) in anhydrous DMF (5 mL). The reaction mixture was heated at 120 °C for 4 h under argon atmosphere. 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 dried over anhydrous MgSO₄, filtered, and concentrated under reduced pressure. The residue was purified by rapid chromatography (98 / 2 DCM / MeOH) to give 0.45 g (33%) of the desired compound as a yellow solid. R f = 0.40 (DCM / MeOH 93 / 7); 1 H NMR (400 MHz, DMSO) δ 6.90 (s, 2H), 5.63 (s, 4H), 4.25 (s, 2H), 3.67 (s, 3H); 13C NMR (101MHz, DMSO) δ 169.13, 168.50, 140.52, 121.47, 107.37, 52.72, 38.69.

[0262] 2-(5,6-diamino-1,3-dioxoisoindoline-2-yl)acetic acid (34)

[0263] Method A

[0264] TFA (2 mL) was added to a solution of 0.4 g (1.37 mmol) of tert-butyl 2-(5,6-diamino-1,3-dioxoisoindoline-2-yl)acetate in DCM (6 mL). The reaction was stirred at room temperature for 4 hours, then concentrated under reduced pressure to give 0.32 g (99%) of the desired compound as a yellow solid. f = 0.29 (DCM / MeOH 99 / 1+0.1% AcOH). 1 H NMR (400 MHz, DMSO) δ 6.90 (s, 2H), 4.13 (s, 2H); 13 C NMR (101 MHz, DMSO) δ 169.95, 168.60, 140.16, 121.80, 107.71, 38.86.

[0265] Method B

[0266] Concentrated hydrochloric acid (1 mL) was added to a solution of methyl 2-(5,6-diamino-1,3-dioxoisoindoline-2-yl)acetate (0.25 g, 1.01 mmol) in dioxane (6 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. f = 0.29 (DCM / MeOH 99 / 1+0.1% AcOH); 1 H NMR (400 MHz, DMSO) δ 6.90 (s, 2H), 4.13 (s, 2H); 13 C NMR (101 MHz, DMSO) δ 169.95, 168.60, 140.16, 121.80, 107.71, 38.86.

[0267] (E)-2-(1,3-dioxo-1,3,6,7,10,11-hexahydro-2H-cyclooctyl[b]pyrrolo[3,4-g]quinoxalin-2-yl)tert-butyl acetate (35)

[0268] A solution of tert-butyl 2-(5,6-diamino-1,3-dioxoisoindoline-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 10 mL of water and purified directly by preparative HPLC to give 0.09 g (17%) of the desired compound as a yellow solid. f = 0.36 (heptane / EtOAc 75 / 25); 1 H NMR (400 MHz, CDCl3) δ 8.61 - 8.37 (m, 2H), 6.10 (ddd, J = 15.8, 10.7, 3.9Hz, 1H), 5.15 (ddd, J = 16.3, 9.5, 5.8 Hz, 1H), 4.42 (s, 2H), 3.40 - 3.28 (m,2H), 3.24 (dd, J = 10.9, 5.1 Hz, 1H), 3.08 (td, J = 12.8, 2.8 Hz, 1H), 2.85 -2.66 (m, 2H), 2.45 (q, J = 11.9 Hz, 1H), 2.39 - 2.24 (m, 1H), 1.48 (s, 9H).

[0269] (E)-2-(1,3-dioxo-1,3,6,7,10,11-hexahydro-2H-cyclooctyl[b]pyrrolo[3,4-g]quinoxalo-2-yl)methyl acetate (36)

[0270] A solution of methyl 2-(5,6-diamino-1,3-dioxoisoindoline-2-yl)acetate (0.38 g, 1.40 mmol) and acetic acid (0.8 mL, 14.03 mmol) in DMF (5 mL) and water (1 mL) was added to (E)-cyclooct-5-ene-1,2-dione (0.21 g, 1.54 mmol). The reaction was stirred at room temperature in the dark for 6 hours. The solution was diluted with 10 mL of water and purified directly by preparative HPLC to give 0.15 g (26%) of the desired compound as a yellow solid. f = 0.31 (heptane / EtOAc 75 / 25); 1H NMR (600 MHz, CDCl3) δ 8.53 - 8.46 (m, 2H), 6.10 (ddd, J = 15.7, 10.8, 3.9Hz, 1H), 5.14 (ddd, J = 16.2, 9.5, 5.8 Hz, 1H), 4.53 (s, 2H), 3.79 (s, 3H), 3.34 (ddt, J = 14.9, 11.1, 5.4 Hz, 2H), 3.24 (dd, J = 11.2, 5.1 Hz, 1H), 3.08(td, J = 12.8, 2.8 Hz, 1H), 2.84 - 2.65 (m, 2H), 2.50 - 2.39 (m, 1H), 2.33(ddd, J = 11.1, 9.3, 5.5 Hz, 1H).

[0271] (E)-2-(1,3-dioxo-1,3,6,7,10,11-hexahydro-2H-cyclooctyl[b]pyrrolo[3,4-g]quinoxalo-2-yl)acetic acid (37)

[0272] A solution of 2-(5,6-diamino-1,3-dioxoisoindololin-2-yl)acetic acid (0.33 g, 1.40 mmol) and acetic acid (0.8 mL, 14.03 mmol) in DMF (5 mL) and water (1 mL) was added to (E)-cyclooct-5-en-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 purified directly by preparative HPLC to give 0.13 g (27%) of the desired compound as a yellow solid. R f = 0.45 (DCM / MeOH 99 / 1+0.1%AcOH); 1 H NMR (600 MHz, DMSO) δ 13.33 (s, 1H), 8.47 (d, J = 9.4 Hz, 2H), 6.18(ddd, J = 17.2, 10.7, 3.8 Hz, 1H), 5.17 - 5.08 (m, 1H), 4.42 (s, 2H), 3.52 -3.43 (m, 1H), 3.23 (dd, J = 9.2, 3.1 Hz, 2H), 3.12 (dd,J = 11.2, 5.1 Hz, 1H), 2.71 (tt, J = 17.0, 6.0 Hz, 2H), 2.32 (qd, J = 11.0, 7.6 Hz, 1H), 2.17(tt, J = 11.3, 5.4 Hz, 1H); 13 C NMR (151 MHz, DMSO) δ 169.10, 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.

[0273] 2,5-Dioxopyrrolidine-1-yl(E)-2-(1,3-dioxo-1,3,6,7,10,11-hexahydro-2H-cyclooctyl[b]pyrrolo[3,4-g]quinoxaloline-2-yl)acetate (38)

[0274] Under argon atmosphere, DCC (0.04 g, 0.19 mmol) was added to a solution of (E)-2-(1,3-dioxo-1,3,6,7,10,11-hexahydro-2H-cyclooctyl[b]pyrrolo[3,4-g]quinoxaloline-2-yl)acetic acid (0.06 g, 0.18 mmol) in anhydrous DMF (3 mL), followed by N-hydroxysuccinimide (0.04 g, 0.35 mmol). The reaction was stirred overnight under argon atmosphere at room temperature. EtOAc (10 mL) was added, and the organic layer was washed with water (2 × 10 mL) and brine (2 × 10 mL). The organic phase was dried over anhydrous MgSO4, filtered, and concentrated under reduced pressure. The residue was dissolved in dry, cold MECN (4 mL), filtered, and concentrated to give a yellow solid. The solid was ground in diethyl ether, filtered, and given 0.056 g (72%) of the desired compound as a yellow solid. f = 0.41 (n-heptane / EtOAc 50 / 50); 1 H NMR (400 MHz, CDCl3) δ 8.56 - 8.47 (m, 2H), 6.10 (td, J=12.1, 5.9 Hz, 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 =11.9 Hz, 1H), 2.33 (dt, J = 12.1, 6.1 Hz, 1H); 13 C NMR (101 MHz, CDCl3) δ168.16, 165.58, 163.15, 161.67, 161.31, 143.40, 143.24, 136.54, 132.67,130.16, 130.00, 126.26, 126.07, 46.74, 41.81, 37.05, 34.19, 28.26, 25.55.

[0275] Example 3.3

[0276]

[0277] Scheme 14: Synthesis of TCO without isomers. i) AcOH, glycine methyl ester hydrochloride, reflux, 16 h (58%); ii) NaN3, MeOH, room temperature, 17 h (89%); iii) Pd / C (10%), H2, EtOH, room temperature, 24 h (95%); iv) AcOH, (E)-cyclooct-5-ene-1,2-dione, DMF, room temperature, 6 h (24%).

[0278] 2-(3,4-dichloro-2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)methyl acetate (40)

[0279] To a solution of dichloromaleic anhydride (5.0 g, 29.95 mmol) in acetic acid (100 mL), glycine methyl ester hydrochloride (4.51 g, 35.94 mmol) was added. The mixture was refluxed and heated 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. 1 H NMR (400 MHz, DMSO) δ 4.41 (s, 2H), 3.70 (s, 3H); 13C NMR (101 MHz, DMSO) δ 167.96, 162.82, 133.39, 53.09, 40.13.

[0280] 2-(3,4-diazido-2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)methyl acetate (41)

[0281] A solution of 2-(3,4-dichloro-2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)acetate (4.15 g, 17.43 mmol) and sodium azide (2.72 g, 41.84 mmol) in methanol (70 mL) was stirred overnight. The solvent was removed under reduced pressure, 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 under vacuum to give the compound (3.89 g, 89%) as a yellow solid. 1 H NMR (400 MHz, DMSO) δ4.34 (s, 2H), 3.70 (s, 3H); 13 C NMR (101 MHz, DMSO) δ 168.14, 164.29, 120.16, 53.03, 39.25.

[0282] 2-(3,4-diazido-2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)methyl acetate (42)

[0283] To a solution of 1.6 g (6.37 mmol) of 2-(3,4-diazido-2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)acetate in EtOH (40 mL), 10% Pd / C (0.34 g, 0.31 mmol) was added. The mixture was stirred at room temperature and allowed to stand for 24 hours under a H2 atmosphere. The catalyst was then filtered through a diatomaceous earth mat to remove the solvent, yielding 1.2 g (95%) of the desired compound as a red oil. The NMR spectrum was normal. The NMR was good. 1 H NMR (400 MHz, DMSO) δ4.94 (s, 4H), 4.09 (s, 2H), 3.65 (s, 4H); 13 C NMR (101 MHz, DMSO) δ 169.38,169.16, 118.89, 52.63, 38.46.

[0284] (E)-2-(1,3-dioxo-1,3,5,6,9,10-hexahydro-2H-cyclooctyl[b]pyrrolo[3,4-e]pyrazin-2-yl)methyl acetate (43)

[0285] A solution of methyl 2-(3,4-diazido-2,5-dioxo-2,5-dihydro-1H-pyrrolo-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-en-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 purified directly by preparative HPLC to give 0.1 g (24%) of the desired compound as a yellow solid. Rf = 0.28 (heptane / EtOAc 75 / 25); 1 H NMR (600 MHz, CDCl3) δ6.11 (ddd, J = 15.7, 10.8, 3.9 Hz, 1H),5.13 (ddd, J = 16.2, 9.5, 5.8 Hz, 1H), 4.53 (s, 2H), 3.77 (s, 3H), 3.35 (ddt, J = 14.9, 11.1, 5.4 Hz, 2H), 3.26 (dd, J = 11.2, 5.1 Hz, 1H), 3.08 (td, J =12.8, 2.8 Hz, 1H), 2.85 - 2.63 (m, 2H), 2.50 - 2.39 (m, 1H), 2.32 (ddd, J =11.1, 9.3, 5.5 Hz, 1H): 13 C NMR (151 MHz, CDCl3) δ 167.20, 164.20, 163.25, 143.86, 128.71, 52.87, 38.83, 35.36, 27.06.

[0286] Example 3.4

[0287]

[0288] Scheme 15: Synthesis of TCO without isomers. i) NH4CO3, methyl 4-oxobutyrate, MeOH, room temperature, 16 hours (63%); ii) LiOH, H2O, THF, room temperature, 3 hours (76%).

[0289] (E)-3-(4,5,8,9-tetrahydro-1H-cyclooct[d]imidazol-2-yl)propionate (44)

[0290] In a 15 mL screw-neck vial, ammonium bicarbonate (384 mg, 4.86 mmol) and methyl 4-oxobutyrate (85%, 331 mg, 2.43 mmol) were stirred in 2 mL of MeOH at room temperature for 15 minutes. (E)-cyclooctyl-5-en-1,2-dione (305 mg, 2.21 mmol) in 3 mL of MeOH was added to the suspension, and the mixture was stirred for 16 hours. The reaction mixture was then diluted with 0.1% TFA. (溶液) The solution was diluted and transferred to a preparative HPLC to give methyl (E)-3-(4,5,8,9-tetrahydro-1H-cyclooct[d]imidazole-2-yl)propionate as a beige viscous liquid (325 mg, 1.39 mmol, 63%). 1 H NMR (599.65 MHz, MeOD) δ = 5.64 - 5.56(m, 2H), 3.70 (s, 3H), 3.20 - 3.15 (m, 2H), 2.95 (dt, J = 13.8, 6.5 Hz, 2H), 2.88 (t, J = 7.0 Hz, 2H), 2.76 - 2.70 (m, 2H), 2.35 (d, J = 6.1 Hz, 2H), 2.28(h, J = 6.7 Hz, 2H). 13 C NMR (150.78 MHz, MeOD) δ = 173.2, 136.0, 129.9, 56.6, 52.5, 31.5, 30.81, 29.0, 22.1.

[0291] (E)-3-(4,5,8,9-tetrahydro-1H-cyclooct[d]imidazol-2-yl)propionic acid (45)

[0292] In a 5 mL screw-neck vial, add 861 μL (1 M LiOH solution, 861 μmol) to 100 mg (287 μmol) of methyl (E)-3-(4,5,8,9-tetrahydro-1H-cyclooct[d]imidazole-2-yl)propionate in 2.5 mL THF / water (3:2 v / v). Stir the suspension at room temperature for 3 hours. Then add 1 M TFA. （溶液） The reaction was quenched and transferred to preparative HPLC to give (E)-3-(4,5,8,9-tetrahydro-1H-cyclooct[d]imidazole-2-yl)propionic acid as a pale yellow amorphous solid (73 mg, 218 μmol, 76%). 1H NMR (599.65 MHz, DMSO) δ = 12.52 (s, 1H), 5.57 - 5.48 (m, 2H), 3.03 (t, J = 7.2 Hz, 2H), 2.88 (dt, J = 14.2, 6.4 Hz, 2H), 2.77 (t, J = 7.2 Hz, 2H), 2.65 (ddd, J = 14.4, 6.7, 4.9 Hz, 2H), 2.24 (ddd, J = 11.5, 9.4, 4.5 Hz, 2H),2.16 (ddd, J = 12.1, 7.4, 4.3 Hz, 2H). 13 C NMR (150.78 MHz, DMSO) δ = 172.6, 142.67, 134.9, 127.6, 30.5, 29.2, 27.8, 20.8.

[0293] Example 3.4

[0294]

[0295] Scheme 16: Synthesis of E,E-cyclooctadiene. v) AcOOH, Na2CO3, DCM, 0℃ → room temperature, 48 hours (65%); vi) nBuLi, PH(Ph)2, AcOH, H2O2, THF, -78℃ to room temperature, 12 hours (52%); vii) NaH, THF, -78℃ to room temperature, 12 hours, 25%; iv) CHCl3, reflux, 2 days (63%); v) Compound 33, CH2Cl2, room temperature, 18 hours (50%).

[0296] cis,cis-5,10-dioxatricyclo[7.1.0.04,6]decane (45)

[0297] This compound was synthesized as described in the following literature: Stöckmann, 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.

[0298] Sodium carbonate (20.5 g, 50.84 mmol, 1.1 equivalents) was added to a stirred solution of (Z,Z)-1,5-cyclooctadiene (5 g, 46.2 mmol, 1.0 equivalents) in DCM (25 mL). The suspension was cooled to 0 °C and peracetic acid (36%, 17.9 mL, 97.1 mmol, 2.1 equivalents) was added. The mixture was heated to room temperature and stirred until the reaction was complete after 48 hours. The reaction was quenched with saturated Na₂CO₃ and then extracted with EtOAc (4 × 25 mL). The combined organic layers were dried over MgSO₄, concentrated, and distilled to give cis,cis-5,10-dioxatricyclo[7.1.0.04,6]decane (4.19 g, 29.89 mmol, 65%). 1 HNMR (400 MHz, CDCl3) δ 2.97 - 2.88 (m, 4H), 2.00 - 1.89 (m, 4H), 1.89 - 1.77(m, 4H); 13 C NMR (101 MHz, CDCl3) δ 56.02, 22.01.

[0299] ((trans,trans)-2,6-dihydroxycyclooctane-1,5-diyl)bis(diphenylphosphine oxide) (46)

[0300] This compound was synthesized as described in the following literature: Stöckmann, 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.

[0301] To a solution of diphenylphosphine (10.43 mL, 11.16 g, 59.92 mmol, 2.1 equivalents) and cis,cis-5,10-dioxatricyclo[7.1.0.04,6]decane (4.0 g, 28.5 mmol, 1.0 equivalents) in THF (120 mL), BuLi (2 M hexane solution, 29.96 mL, 59.92 mmol, 2.1 equivalents) was added dropwise at -78 °C. The mixture was stirred for 1 hour, then heated to room temperature and stirred for 12 hours. The brown solution was cooled to 0 °C, diluted with THF (86 mL), and quenched by adding AcOH (4.9 mL, 5.14 g, 85.60 mmol, 3 equivalents) and H2O2 (30%, 8.7 mL, 85.60 mmol, 3 equivalents). The mixture became clear, followed by the formation of a white precipitate. The mixture was removed from the ice bath and vigorously stirred at 25°C for 2 hours. The suspension was then transferred to a separatory funnel, and 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 removed by filtration to give ((trans,trans)-5,8-dihydroxycyclooctane-1,4-diyl)bis(diphenylphosphine oxide). The combined organic layers were dried over MgSO₄, filtered, and concentrated to give a white solid. The remaining AcOH was removed by azeotropic reaction with toluene to give an isomeric mixture of phosphine oxides (8.14 g, 14.9 mmol, 52%). The spectroscopic data were consistent with previously published data.

[0302] (E,E)-1,5-Cyclooctadiene (47)

[0303] This compound was synthesized as described in the following literature: Stöckmann, 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.

[0304] A mixture of phosphine oxides (6.00 g, 11.0 mmol, 1.0 equivalent) was dissolved in DMF (30 mL) and stirred under an Ar atmosphere. The clear solution became turbid, and NaH (60% in mineral oil, 1.44 g, 34.2 mmol, 3.1 equivalent) was added in portions over a positive Ar atmosphere at room temperature for 5 minutes. The reaction mixture was stirred at room temperature for 12 hours, cooled to 0 °C, diluted with pentane (50 mL), and quenched with semi-saturated NH4Cl (22 mL). The aqueous layer was extracted with pentane (50 mL), and the combined organic layers were washed with water (5 × 22 mL) and brine (1 × 22 mL) to give (E,E)-1,5-cyclooctadiene (25% yield). The spectroscopic data were consistent with previously published data.

[0305] 6-Benzyl-5,7-dioxo-6,7-dihydro-5H-pyrrolo[3,4-d]pyridazine-1,4-dicarboxylic acid dimethyl ester (50)

[0306] Dimethyl 1,2,4,5-tetraazine-3,6-dicarboxylate (50 mg, 0.25 mmol) was dissolved in CHCl3 (1 mL), and 1-phenyl-1H-pyrrole-2,5-dione (53 mg, 0.30 mmol) was added. The reaction was refluxed and stirred for two days. After the reaction, the dihydropyridazine spontaneously oxidized to the corresponding pyridazine. After the dimethyl 1,2,4,5-tetraazine-3,6-dicarboxylate was completely consumed, the reaction was cooled to room temperature. The product was purified by CombiFlash to give a grayish-white solid (54 mg, 0.16 mmol, 63%). 1 H NMR (600MHz, CDCl3) δ 7.54 (dd, J = 8.3, 6.8 Hz, 2H), 7.50 - 7.47 (m, 1H), 7.42 -7.38 (m, 2H), 4.17 (s, 6H). 13 C NMR (151 MHz, CDCl3) δ 162.5, 162.2, 159.2, 149.5, 138.2, 130.3, 129.7, 126.5, 54.3.

[0307] (E)-1,3-dioxo-2-phenyl-2,3,5,6,9,10-hexahydro-1H-cyclooctyl[f]isoindole-4,11-dicarboxylic acid dimethyl ester (51)

[0308] Compound 33 (0.06 mmol) in pentane was added to a solution of compound 35 (20 mg, 0.06 mmol) in CH₂Cl₂. The solution was stirred at room temperature for 18 hours. Then, volatile substances were removed under reduced pressure. Purification by rapid chromatography yielded 12 mg (50%) of the desired product as a white solid. 1 H NMR (400 MHz, CDCl3) δ 7.45 (dd, J =8.4, 6.9 Hz, 2H), 7.37 (t, J = 7.4 Hz, 1H), 7.35 - 7.30 (m, 2H), 5.77 (t, J =5.5 Hz, 2H), 3.89 (s, 6H), 2.61 - 2.36 (m, 2H), 2.36 - 2.21 (m, 2H), 1.65 (d, J = 8.1 Hz, 4H).

[0309] Example 4

[0310] Screening of oxidants for the oxidation of dihydropyridazine to pyridazine

[0311] Figure 1-4 The reaction between tetrazine and TCO is shown, dissolved in a 1:1 H₂O / EtOH (%v / v). Figure 2 , 3 Figures 4 and 5 show the results obtained under different conditions during the oxidation step. Cycloaddition was completed within 5 minutes, yielding several isomeric dihydropyridazines. Then, an acid and an oxidant were added to give the final pyridazine product. Each oxidant (5 equivalents) was added to the mixture, and the reaction was analyzed by HPLC-MS after 60 minutes. The time required for the oxidation reaction is also shown in the figures. These screening experiments unexpectedly showed that not all combinations of oxidants and acids are suitable for providing pyridazines. In most cases, the addition of an acid (TFA) catalyzed the process. For all quinone oxidants, the corresponding pyridazine was formed, with quinones exhibiting higher electron deficiency exhibiting better performance, i.e., faster oxidation. The examples of the listed inorganic oxidants failed to convert the click product (dihydropyridazine) to the corresponding pyridazine within 60 minutes. In the case of Bobbitt salts, the addition of an acid (TFA) catalyzed the process. In the case of the listed examples of organic oxidants, mixed results were obtained. For peroxides and peracids, the addition of an acid (TFA) catalyzed the process.

[0312] Figure 4-7 The results of photocatalytic oxidation are shown. Photocatalytic oxidation was performed using a 16×254nm 35W lamp (RPR-2537A, UV irradiance 12mW / cm²).2 The results were obtained in a Rayonet® reactor, with the addition of acid and / or photosensitizer. The combination of these parameters showed a significant non-additive effect. Figure 8 and Figure 9 This shows data on the use of different oxidants to oxidize two different dihydropyridazine compounds.

[0313] for Figure 2 The test shown is conducted under the following conditions (con.):

[0314] Tz (1 equivalent, 0.0018 mmol), TCO(OH)2 (2 equivalents, 0.0035 mmol), 1:1 H2O / EtOH (v / v%), in borosilicate glass. Time in minutes until >90% conversion.

[0315] Condition 1: TFA (1% v / v), fluorescein (0.4 equivalent), UV （254nm） 40℃

[0316] Condition 2: TFA (1% v / v), fluorescein (0.4 equivalent), UV （254nm） room temperature

[0317] Condition 3: Fluorescein (0.4 equivalents), UV （254nm） 40℃

[0318] Condition 4: TFA (1% v / v), 5-(4-carboxyphenyl)-10,15,20-(tri-N-methyl-4-pyridyl)porphyrin trichloride (0.4 equivalents), UV （254nm） room temperature

[0319] Condition 5: TFA (1% v / v), 5-(4-carboxypropylcarbamoylphenyl)-10,15,20-(tris-4-sulfonylphenyl)porphyrin triammonium (0.4 equivalents), UV （254nm） , room temperature.

[0320] for Figure 3 The test shown is conducted under the following conditions (con.):

[0321] Tz (1 equivalent, 0.0018 mmol), TCO(OH)2 (2 equivalents, 0.0035 mmol), 1:1 H2O / EtOH (v / v%), in borosilicate glass. Time in minutes until >90% conversion.

[0322] Condition 1: TFA (1% v / v), UV （254nm） 40℃

[0323] Condition 2: TFA (1% v / v), UV（254nm） room temperature

[0324] Condition 3: TFA (1% v / v), sunlight, room temperature (control)

[0325] Condition 4: UV （365nm） Room temperature (control)

[0326] Condition 5: TFA (1% v / v), darkness, room temperature (control)

[0327] Condition 6: Sunlight, room temperature (control)

[0328] for Figure 4 The test shown is conducted under the following conditions (con.):

[0329] Tz (1 equivalent, 0.0018 mmol), TCO (1 equivalent, 0.0018 mmol), 1:1 H2O / EtOH (v / v%). Time is in minutes, until >90% conversion.

[0330] Condition 1: TFA (1% v / v), mCPBA, room temperature

[0331] Condition 2: mCPBA, room temperature (control)

[0332] Condition 3: TFA (1% v / v), Bobbitt salt, room temperature

[0333] Condition 4: Bobbitt salt, room temperature (control)

[0334] Condition 5: TFA (1% v / v), D-α-tocopherol quinone, room temperature

[0335] Condition 6: D-α-tocopherol quinone, room temperature (control)

[0336] Condition 7: TFA (1% v / v), fluorescein (0.4 equivalent), UV （254nm） Room temperature (in borosilicate glass)

[0337] Condition 8: TFA (1% v / v), UV （254nm） Room temperature (in borosilicate glass)

[0338] Condition 9: TFA (1% v / v), 5-(4-carboxyphenyl)-10,15,20-(tri-N-methyl-4-pyridyl)porphyrin trichloride (0.4 equivalents), UV （254nm） Room temperature (in borosilicate glass)

[0339] Condition 10: TFA (1% v / v), 5-(4-carboxypropylcarbamoylphenyl)-10,15,20-(tris-4-sulfonylphenyl)porphyrin triammonium (0.4 equivalents), UV （254nm） Room temperature (in borosilicate glass)

[0340] Condition 11: TFA (1% v / v), sunlight (in borosilicate glass) (control).

[0341] for Figure 5 The test shown is conducted under the following conditions (con.):

[0342] Tz (1 equivalent, 0.0018 mmol), TCO(OH)2 (2 equivalents, 0.0035 mmol), 1:1 H2O / EtOH (v / v%), in borosilicate glass. Time in minutes until >90% conversion.

[0343] Condition 1: TFA (0.1M), UV (254nm) Room temperature

[0344] Condition 2: HCl (0.1M), UV (254nm) Room temperature

[0345] Condition 3: H3PO4 (0.5M), UV (254nm) Room temperature

[0346] Condition 4: TFA (0.5M), UV (254nm) Room temperature

[0347] Condition 5: AcOH (1M), UV (254nm) Room temperature

[0348] Condition 6: TFA (1M), UV (254nm) Room temperature

[0349] Condition 7: H2SO4 (1M), UV (254nm) Room temperature

[0350] Condition 8: HCl (1M), UV (254nm) Room temperature.

[0351] against Figure 6 The test shown is conducted under the following conditions (con.):

[0352] Tz (1 equivalent, 0.0018 mmol), TCO(OH)2 (2 equivalents, 0.0035 mmol), 1:1 H2O / EtOH (v / v%). Time is in minutes until >90% conversion.

[0353] Condition 1: HCl (1M), UV（254nm） Room temperature, borosilicate bottle (open).

[0354] Condition 2: HCl (1M), UV （254nm） At room temperature, in an open quartz flask.

[0355] against Figure 7 The test shown is conducted under the following conditions (con.):

[0356] Tz (1 equivalent, 0.0018 mmol), TCO(OH)2 (2 equivalents, 0.0035 mmol), 1:1 H2O / EtOH (v / v%). Time is in minutes until >90% conversion.

[0357] Condition 1: TFA (1% v / v), UV （254nm） 40℃

[0358] Condition 2: TFA (1% v / v), UV （254nm） room temperature

[0359] Condition 3: TFA (1% v / v), sunlight, room temperature (control)

[0360] Condition 4: UV （254nm） Room temperature (control)

[0361] Condition 5: TFA (1% v / v), darkness, room temperature (control)

[0362] Condition 6: Sunlight, room temperature (control).

[0363] Figure 8 and Figure 9 The non-additive effect of the combination of UV light and acid is shown when the resulting dihydropyridazine (obtained by the linkage between Tz and a cyclic unsaturated hydrocarbon (having at least one trans configuration double bond, as shown above each figure) is oxidized to provide the corresponding pyridazine.

[0364] Example 5

[0365] Screening of oxidants for oxidizing radiolabeled dihydropyridazine to radiolabeled pyridazine.

[0366] Oxidation of radiolabeled compounds

[0367] Will be through mixing 18 Obtained from F-Tz and TCO solution 18 A crude solution (50-100 µL) of the F-click product (as described in the click section (Example 4)) was used for the oxidation experiment. The oxidation protocol consisted of two steps: acidification and introduction of an oxidant.

[0368] Acidification: Mix the solution of the click product with an acid solution at a ratio of 19:1 or 9:1 v / v. The acid solution is pure acid or an aqueous solution of the desired acid.

[0369] Oxidation using a solid oxidant: In the same organic co-solvent used in the click reaction (i.e., EtOH for ethanol-water mixtures and acetonitrile for acetonitrile-water mixtures), a solution of the acidified click product is further mixed with a solution of 10 mg / mL of the desired solid oxidant at a ratio of 9:1 v / v. If the oxidant cannot be dissolved to 10 mg / mL in the selected co-solvent, a saturated solution is used instead. The oxidation mixture is placed in a sealed vial and left at room temperature for the desired time. The sample is then removed and analyzed by HPLC.

[0370] Oxidation using air oxygen: Place a solution (60-100 µL) of the acidified click product in an open vial and leave it at room temperature for the required time. Then, remove the sample and analyze it by HPLC.

[0371] Oxidation using medical oxygen: At room temperature, medical oxygen from a rubber balloon is bubbled through an acidified solution of the click product (60-100 µL). When the total volume of the solution decreases by more than two-fold due to evaporation, reconstruction is performed using the same water-organic mixture used in the click reaction. The sample is then removed and analyzed by HPLC.

[0372] UV-assisted oxidation: A solution of the acidified click product is mixed with a concentrated solution of the desired photosensitizer in water or a suitable organic co-solvent at a ratio of 9:1 v / v (for fluorescent dyes). The resulting mixture is irradiated under a CAMAG UV lamp equipped with 2 × 254 nm 8W lamps (UV irradiance of 2 mW / cm²). 2 The required time period is determined, and then the sample is removed and analyzed by HPLC.

[0373] Radioactive oxidation using chemical oxidants:

[0374] Here and thereafter, the time required to achieve >90% oxidation is as follows: Figure 10 and Figure 11 As shown.

[0375] for Figure 10 The test shown is conducted under the following conditions (con.):

[0376] Condition 1: TCO (30 μM), 0.1 M HCl in a 9:1 MeCN / H2O (v / v%) solution, in an open vial at room temperature (control).

[0377] Condition 2: TCO (30 μM), 0.1 M HCl in a 9:1 MeCN / H2O (v / v%) solution, 1 mg / mL tetrafluorobenzoquinone (Fluoranil), sealed vial, room temperature.

[0378] Condition 3: TCO (30 μM), 0.1 M HCl in a 9:1 MeCN / H2O (v / v%) solution, O2 gas bubbling, open bottle, room temperature (control).

[0379] Condition 4: TCO (30 μM), 1% TFA in 1:1 EtOH / H2O (v / v%), in an open vial, at room temperature (control group).

[0380] Condition 5: TCO (30 μM), 1% TFA in 1:1 EtOH / H2O (v / v%), 1 mM duroquinone, sealed vial, room temperature.

[0381] Condition 6: TCO (30 μM), 1% TFA in 1:1 EtOH / H2O (v / v%), 1 mM Bobbitt salt, sealed vial, room temperature.

[0382] Condition 7: TCO (30 μM), 1% TFA in 1:1 EtOH / H2O (v / v%), 1 mM mCPBA, sealed vial, room temperature.

[0383] Condition 8: TCO (30 μM), 1% TFA in 1:1 EtOH / H2O (v / v%), 3% H2O2, sealed vial, room temperature.

[0384] Condition 9: TCO (30 μM), 1% TFA in 1:1 EtOH / H2O (v / v%), 1 mM PIDA, sealed vial, room temperature.

[0385] Condition 10: TCO (30 μM), 1% TFA in 1:1 EtOH / H2O (v / v%), 1 mM TEMPO, sealed vial, room temperature.

[0386] Condition 11: TCO (30 μM), 1% TFA in 1:1 EtOH / H2O (v / v%), 1 mM D-α-tocopherol quinone, sealed vial, room temperature.

[0387] Condition 12: TCO (10 μM), 0.1 M HCl in 9:1 MeCN / H2O (v / v%), 1 mg / mL tetrachlorobenzoquinone (chloranil), sealed vial, room temperature.

[0388] against Figure 11 The test shown is conducted under the following conditions (con.):

[0389] Condition 1: TCO (30 μM), 0.1 M HCl in a 9:1 MeCN / H2O (v / v%) environment, UV （254nm） Small, open bottle, room temperature

[0390] Condition 2: TCO (5 μM), 0.1 M HCl in a 9:1 MeCN / H2O (v / v%) environment, UV （254nm） Small, open bottle, room temperature

[0391] Condition 3: TCO (5 μM), 0.1 M HCl in a 9:1 MeCN / H2O (v / v%) solution, O2 saturated solution, UV （254nm） Small, open bottle, room temperature

[0392] Condition 4: TCO (5 μM), 0.1 M HCl in a 9:1 MeCN / H2O (v / v%) solution, 50 μg / mL fluorescein, UV （254nm） Small, open bottle, room temperature

[0393] Example 6

[0394] Measurement of second-order rate constant

[0395] The second-order rate constants of all click reactions carried out in the previous examples were measured by stopped-flow spectroscopy in phosphate-buffered saline (PBS) at 25 °C, 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)). Briefly, stopped-flow measurements were performed using an SX20-LED stopped-flow spectrophotometer (Applied Photophysics) equipped with a 535 nm LED (10 mm path length, 34 nm full width at half maximum) to monitor the visible absorbance (520-540 nm) of the characteristic tetrazines. A reagent syringe filled with a solution of axial-TCO-PEG4 was used to pre-treat the instrument. Subsequent data for each tetrazine were collected in triplicate. Reactions were carried out in PBS at 25 °C and recorded automatically at acquisition. The dataset was analyzed by fitting exponential decay using Prism 6 (GraphPad) to calculate the observed pseudo-first-order rate constant, which was converted to a second-order rate constant by dividing by the concentration of excess TCO compound.

[0396] Only at 25°C in phosphate-buffered saline does it exhibit a minimum second-order rate constant of 250 M. -1 s -1Only reactions that meet these criteria are considered suitable for providing sufficient rate kinetics. These measurements confirm that click reactions between 1,2,4,5-tetraazine and dienophiles (cyclic unsaturated hydrocarbons with at least one trans-configured double bond) and click reactions between 1,2,4,5-tetraazine and dienophiles (unsaturated heterocycles with at least one trans-configured double bond) both exhibit a 250 M [response value missing]. -1 s -1 The minimum second-order rate constant.

[0397] Example 7

[0398] Radiolabeled dihydropyridazine was oxidized to radiolabeled pyridazine containing a PSMA targeting vector.

[0399] Synthesis of Compound 52 Figure 12 ).

[0400] The resin-bound and tert-butyl-protected binding motif was synthesized as described above. 5 4 equivalents of Fmoc-L-2-Nal-OH were activated in DMF with 3.92 equivalents of HATU (1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate) and 4 equivalents of DIPEA relative to the resin (0.22 mmol). After 2 min, the solution was added to resin-fixed glutamic acid-urea-lysine and shaken for 1 h. The Fmoc protecting group was cleaved with a mixture of DMF and piperidine (1:1). The product (12) was reacted with 4 equivalents of trans-4-(Fmoc-aminomethyl)cyclohexanecarboxylic acid (N-Fmoc-tranexamic acid), which was activated with 3.92 equivalents of HATU and 4 equivalents of DIPEA. Deprotection of the Fmoc group was carried out with a mixture of DMF and piperidine (1:1) to give product 13. The product was cleaved from the resin and deprotected with trifluoroacetic acid (TFA), triisopropylsilane (TIPS), and water (95:2.5:2.5). Preparative HPLC yielded 0.110 g of the desired compound 52. 1 H NMR (600 MHz, DMSO) δ 12.74 - 12.36 (m, 2H), 12.14 (s, 1H), 7.98 - 7.93 (m, 2H), 7.86 (dd, J = 7.7, 1.6 Hz, 1H), 7.82 - 7.77 (m, 2H), 7.69 (d, J = 1.7 Hz, 1H), 7.60 (s, 3H), 7.49 - 7.43 (m, 2H), 7.40 (dd, J=8.4, 1.7 Hz, 1H), 6.34 - 6.25 (m, 2H), 4.55 (td, J = 9.0, 5.1 Hz, 1H), 4.11(td, J = 8.3, 5.2 Hz, 1H), 4.03 (td, J = 8.2, 5.2 Hz, 1H), 3.12 (dd, J =13.7, 5.0 Hz, 1H), 3.06 (dq, J = 13.0, 6.6 Hz, 1H), 2.99 (dq, J = 13.0, 6.7Hz, 1H), 2.93 (dd, J = 13.7, 9.5 Hz, 1H), 2.66 - 2.59 (m, 3H), 2.31 - 2.18(m, 2H), 2.09 (ddt, J = 12.0, 7.5, 3.6 Hz, 1H), 1.93 (dddd, J = 14.1, 9.3,6.7, 5.2 Hz, 1H), 1.77 - 1.56 (m, 5H), 1.55 - 1.19 (m, 9H), 1.05 (qd, J =13.0, 3.5 Hz, 1H), 0.93 - 0.81 (m, 2H); ESI-MS [M+H] + = 656.3.

[0401] Synthesis of Compound 53 ( Figure 12 )

[0402] Relative to resin and compound 52 (0.22 mmol), 4 equivalents of Fmoc-D-Glu-OH were activated in DMF with 3.92 equivalents of HATU (1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate) and 4 equivalents of DIPEA. After 2 minutes, the solution was added to resin-fixed glutamic acid-urea-lysine and shaken for 1 hour. The Fmoc protecting group was cleaved with a mixture of DMF and piperidine (1:1). The product was reacted with 4 equivalents of Fmoc-D-Arg(Pbf)-OH, which was activated with 3.92 equivalents of HATU and 4 equivalents of DIPEA. Deprotection of the Fmoc group was carried out with a mixture of DMF and piperidine (1:1) to give product 53. The product was cleaved from the resin and deprotected with trifluoroacetic acid (TFA), triisopropylsilane (TIPS), and water (95:2.5:2.5). Preparative HPLC yielded 0.07 g of the desired compound 23. ESI-MS [M+H] + =1226.8.

[0403] Synthesis of Compound 54 Figure 12 )

[0404] Under argon atmosphere, 4-methylmorpholine (0.016 mL, 0.15 mmol) was added to a solution of compound 53 (0.026 g, 0.016 mmol) and compound 5 (0.007 g, 0.016 mmol) in dry DMF (3 mL). The reaction was stirred at room temperature for 6 hours. The mixture was then diluted with 10 mL of mobile phase A (1% TFA, in H2O) and purified by preparative HPLC. The collected fraction was lyophilized to give 0.015 g (51%) of the desired compound as a white solid. ESI-MS [M+H] + =1546.3.

[0405] Synthesis of Compound 55 Figure 12 )

[0406] Compound 1 (0.0005 g, 0.0023 mmol) was added to a solution of compound 54 (0.00 g, 0.0023 mmol) in ACN (2 mL) and 1% TFA in H₂O (2 mL). The reaction was stirred at room temperature for 5 hours. The mixture was purified directly by preparative HPLC. The collected fraction was freeze-dried to give 0.002 g (45%) of the desired compound as a white solid. ESI-MS [M+H] + =1736.6.

[0407] Radiolabeled dihydropyridazine was oxidized to radiolabeled pyridazine containing a PSMA targeting vector. Figure 13 )

[0408] Figure 13 Showing PSMA TCO-compound 54 and 18 The reaction protocol of F-Tz, and a table listing the solvents, acids, and acid concentrations (M) used in the click and oxidation steps, as well as the HPCL yields (%) of the resulting radiolabeled pyridazine PSMA-targeted vectors.

[0409] Will be through mixing 18 Obtained from F-Tz and TCO solution 18 A crude solution (50-100 μL) of the F-click product (as described in the click section (Example 4)) was used for oxidation experiments with TCO-compound 54. The oxidation protocol consisted of two steps: acidification and introduction of an oxidant.

[0410] Acidification: The solution of the click product is mixed with an acid solution of TFA, HCl, or H3PO4 at a ratio of 19:1 or 9:1 v / v. The acid solution is pure acid or an aqueous solution of the desired acid.

[0411] UV-assisted oxidation: The solution of the acidified click product was irradiated under a CAMAG UV lamp equipped with 2×366nm 8W lamps (UV irradiance of 2mW / cm²). 2 The required time period is determined, and then the sample is removed and analyzed by HPLC.

[0412] In all experiments using PSMA, UV irradiation was consistently set at 5 minutes. Figure 13 As shown, the HPLC yield (conversion) of the oxidation product is 53-95%.

[0413] against Figure 13 The test shown is conducted under the following conditions (con.):

[0414] Compound 54 (50 μM), acid in solvent:water 9:1 v / v, UV （365nm） Irradiate for 5 minutes in an open quartz vial at room temperature.

Claims

1. A method for oxidizing dihydropyridazine to pyridazine: a) A first chemical entity is labeled with a labeling agent, the first chemical entity having a reverse electron-demanding Diels-Alder cycloaddition reactivity and being coupled to a pharmaceutical, diagnostic, or therapeutic agent; wherein... The first chemical entity is a 1,2,4,5-tetraazine or a dienophile selected from: a cyclic unsaturated hydrocarbon having at least one trans-configured double bond; an unsaturated heterocycle having at least one trans-configured double bond; and b) Connect the labeled first chemical entity obtained in step a) to a second chemical entity, the second chemical entity having complementary anti-electron demand Diels-Alder cycloaddition reactivity and being coupled to a target support; wherein the second chemical entity is a 1,2,4,5-tetraazine or a dienophile selected from: a cyclic unsaturated hydrocarbon having at least one trans-configured double bond; an unsaturated heterocycle having at least one trans-configured double bond; and c) The resulting mixture of the dihydropyridazine-labeled targeting support obtained in step b) is oxidized in a solvent at a temperature of 15°C to 50°C for up to 180 minutes by adding an acid catalyst and an oxidant, wherein the acid catalyst has a pKa of less than 5 in H₂O at 25°C, is added to the mixture obtained in step b) at a concentration of 0.1 M to 5 M, and the oxidant is selected from quinone oxidants, Bobbit salt oxidants, peroxide oxidants, peroxyacid oxidants, and has a minimum UV irradiance of 2 mW / cm². 2 Ultraviolet light.

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

3. The method according to claim 2, wherein, The marker in step a) 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 In、 113 In、 114 mIn、 115 mIn、 175 Ridiculous, 177 Ridiculous, 185 Too, 186 Too, 188 Too, 201 Tl、 203 Tl、 205 Tl、 206 Pb、 207 Pb、 208 Pb、 212 Pb、 209 Bi、 212 Bi、 213 Bi、 31 P、 32 P、 33 P、 32 S、 35 S、 45 Sc、 47 Sc、 84 Sr、 86 Sr、 87 Sr、 88 Sr、 89 Sr、 165 Your, 166 Your, 156 Dy、 158 Dy、 160 Dy、 161 Dy、 162 Dy、 163 Dy、 164 Dy、 165 Dy、 227 Th、 232 Th、 51 Cr、 52 Cr、 53 Cr、 54 Cr、 73 The、 74 The、 75 The、 76 The、 77 The、 78 The、 80 The、 82 The、 94 Tc、 99m Tc、 103 Rh、 103 mRh、 119 Sb、 121 Sb、 123 Sb、 135 The、 138 Let's go. 139 Let's go. 162 Is, 164 Is, 165 Is, 166 Is, 167 Is, 168 Is, 170 Is, 193 mPt, 195 mPt, 192 At this time, 194 At this time, 195 At this time, 196 At this time, 198 At this time, 211 To, 223 Raw, 225 Ac.

4. The method according to any one of the preceding claims, wherein, The targeting carrier in step b) is an antibody, nanobody, polymer, nanomedicine, cell, protein, peptide or small molecule.

5. The method according to any one of the preceding claims, wherein, The solvent in step c) contains 1% to 99% water, and the oxidant in step c) has a minimum UV irradiance of 2 mW / cm². 2 Ultraviolet light.

6. The method according to any one of the preceding claims, wherein, The solvent in step c) contains 5% to 95% water, and the oxidant in step c) has a minimum UV irradiance of 2 mW / cm². 2 Ultraviolet light.

7. The method according to any one of the preceding claims, wherein, Step c) is carried out in a quartz container.

8. The method according to any one of the preceding claims, wherein, Step c) is carried out in an open container.

9. The method according to any one of the preceding claims, wherein, The oxidant in step c) is solid-supported.

10. The method according to any one of the preceding claims, wherein, The method further includes: using an oxidant with a minimum ultraviolet irradiance of 2 mW / cm². 2 When exposed to ultraviolet light, a photosensitizer is added in step c).

11. The method according to claim 10, wherein, The photosensitizer is a fluorescein-based photosensitizer or a porphyrin-based photosensitizer.

12. The method according to any one of the preceding claims, wherein, The tetrazine is of formula Tz1: ， Where R and R1 are independently selected from -H, -Me, or Wherein, the wavy line symbol indicates a connection to tetrazine; wherein, R2 is -H or (i) an isotopic labeling agent directly connected to the aromatic ring; or (ii) an isotopic labeling agent 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 stands for -(CH2). m Or -(CH2CH2O) m Wherein, n and m are independently selected from 1-25; or (iii) an isotopic labeling agent chelated by a linker and linked to an aromatic ring, said chelating agent being 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-triazacyclononane-1-yl)glutaric acid (NODAGA), 2-(4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane-1-yl)glutaric 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-(4-(5-aminopentyl)(hydroxy)amino-4-oxobutyryl)amino)pentyl-N-hydroxysuccinamide (DFO), diethylenetriaminepentaacetic acid (DTPA), trans-cyclohexyl-diethylenetriaminepentaacetic acid (CHX-DTPA), 1 -oxa-4,7,10-triazacyclododecane-4,7,10-triacetic acid (OXO-Do3A), benzyl-DTPA p-isothiocyanate (SCN-BZ-DTPA), 1-(benzyl-3-isothiocyanate)-3-methyl-DTPA (1B3M), 2-(benzyl-4-isothiocyanate)-4-methyl-DTPA (1M3B), and 1-(2)-methyl-4-isocyanate benzyl-DTPA (MX-DTPA), wherein the linker is selected from: -(CH2) n -LO(CH2) n -LNH(CH2) n -LCONH(CH2) n -LNHCO(CH2) n Where L stands for -(CH2). m Or -(CH2CH2O) m n and m are independently selected from 1-25; 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 O、 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 Cu、 61 Cu、 63 Cu、 64 Cu、 65 Cu、 67 Cu、 67 Ga、 68 Ga、 69 Ga、 71 Ga、 76 Br、 77 Br、 79 Br、 80 mBr、 81 Br、 72 As、 75 As、 86 Y、 89 Y、 90 Y、 89 Zr、 90 Zr、 91 Zr、 92 Zr、 94 Zr、 149 Tb、 152 Tb、 159 Tb、 161 Tb、 111 In、 113 In、 114 mIn、 115 mIn、 175 Ridiculous, 177 Ridiculous, 185 Too, 186 Too, 188 Too, 201 Tl、 203 Tl、 205 Tl、 206 Pb、 207 Pb、 208 Pb、 212 Pb、 209 Bi、 212 Bi、 213 Bi、 31 P、 32 P、 33 P、 32 S、 35 S、 45 Sc、 47 Sc、 84 Sr、 86 Sr、 87 Sr、 88 Sr、 89 Sr、 165 Your, 166 Your, 156 Dy、 158 Dy、 160 Dy、 161 Dy、 162 Dy、 163 Dy、 164 Dy、 165 Dy、 227 Th、 232 Th、 51 Cr、 52 Cr、 53 Cr、 54 Cr、 73 The、 74 The、 75 The、 76 The、 77 The、 78 The、 80 The、 82 The、 94 Tc、 99m Tc、 103 Rh、 103 mRh、 119 Sb、 121 Sb、 123 Sb、 135 The、 138 Let's go. 139 Let's go. 162 Is, 164 Is, 165 Is, 166 Is, 167 Is, 168 Is, 170 Is, 193 mPt, 195 mPt, 192 At this time, 194 At this time, 195 At this time, 196 At this time, 198 At this time, 211 To, 223 Raw, 225 Ac, Where X and Y are independently selected from: -CH and -N-; R3 is independently selected from H or from hydroxyl, sulfonamide, guanidinyl, carboxyl, sulfonyl, amine, substituted amine having 1-25 polyethylene glycol units, and -(O-CH2-CH2). n The part of -OCH2-COOH, n is selected from 1-25; or methyl, ethyl, propyl, optionally substituted heteroaryl and optionally substituted arylalkyl; wherein, relative to the substituted amine, optional substitution means one or more substituents selected from the following: halogen, hydroxyl, sulfonamide, carboxyl, sulfonyl, amine, (C1-C10)alkyl, (C2-C10)alkenyl, (C2-C10)alkynyl, (C1-C10)alkylene, (C1-C10)alkoxy, (C2-C10)dialkylamino, (C1-C10)alkylthio, (C2-C10)heteroalkyl, (C2-C10)heteroalkylene, (C3-C10)cycloalkyl, (C3-C10)... Heterocyclic alkyl, (C3-C10)cycloalkylene, (C3-C10)heterocyclic alkylene, (C1-C10)haloalkyl, (C1-C10)perhaloalkyl, (C2-C10)-olefin, (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 means selected from one or more of the following substituents: halogen, hydroxyl, sulfonamide, carboxyl, sulfonyl, amine, substituted amine having 1-25 polyethylene glycol units, -(O-CH2-CH2). n -OCH2-COOH, n is selected from 1-25; or H, methyl, ethyl, propyl, optionally substituted heteroaryl and optionally substituted arylalkyl; wherein, relative to the substituted amine, optional substitution means one or more substituents selected from halogen, hydroxyl, sulfonamide, carboxyl, sulfonyl and amine; R and R1 are the same or differ only in the number of isotopes of the labeling agent.

13. The method according to claim 12, wherein, The tetrazine is selected from: 。 14. The method according to any one of the preceding claims, wherein, The second chemical entity is selected from: ， Where X is NH, O, S, CH2, OCONH, OCSNH, NHCO; Y is N, NO, or CR8; Z is N, NO, or CR8; R8 is selected from: -H, -F, -OH, -NH2, -COOH, -COOCH3, CF3, -Cl, -CONH2, CONHCH3, -CON(CH3)2, -CH2OH, -CH2NH2, -CH2CH2OH, -CH2CH2NH2, -CHCH2N(CH3)2, and Among them, 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, where n is 0-25, and M is a side chain selected from the group consisting of side chains of natural amino acids: 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; Among them, the targeting carriers are antibodies, nanobodies, polymers, nanomedicines, cells, proteins, peptides, or small molecules.

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