Radioisotope tritium labeled selamectin and preparation method thereof

Abstract

According to the radioisotope tritium labeled selamectin and the preparation method thereof, doramectin is taken as a starting raw material, sodium boron tritide is taken as a tritium source, accurate tritium atom labeling is realized at a specific site of a 16-membered macrolide carbon skeleton through a'directional oxidation-tritiated reduction 'strategy, the defects of an existing labeling technology can be overcome, and the radioisotope tritium labeled selamectin is prepared. And enough high-purity and high-stability radioactive tracer can be provided for related isotope tracing research in time.

Description

Technical Field

[0001] This invention relates to the field of radiochemical synthesis, and in particular to a radioactive isotope tritium-labeled selamectin ([13-³H] selamectin) and its preparation method. Background Technology

[0002] Selamectin (CAS: 165108-07-6; English name: Selamectin), as a second-generation semi-synthetic avermectin derivative, shares structural homology with doramectin and ivermectin. After specific structural optimization, it possesses more prominent comprehensive application advantages: ① It exhibits significant broad-spectrum anthelmintic activity, effectively killing fleas, scabies mites, ear mites, hookworms, roundworms, and other internal and external parasites in dogs and cats, and can effectively prevent heartworm disease; ② Its mechanism of action is species-selective—by activating glutamate-gated chloride ion channels in invertebrates and... γ - GABA receptors cause neuromuscular paralysis in parasites, while mammals, due to blood-brain barrier protection and differences in receptor affinity, have extremely high drug safety; ③ Its unique lipid solubility allows it to be absorbed through the skin and stored in the sebaceous glands, achieving long-lasting parasite protection, making it the first drug for the prevention of heartworm disease in the form of topical drops. Based on these characteristics, selamectin has become a core product for the clinical prevention and control of parasites in small animals worldwide, laying a key foundation for the prevention and control system of parasite diseases in companion animals.

[0003] However, selamectin has unresolved safety concerns in clinical applications, necessitating systematic research. The official drug information leaflet indicates that it may cause local skin reactions (such as hair loss and erythema) and gastrointestinal symptoms (such as vomiting and diarrhea), and certain breeds, such as Border Collies carrying multidrug resistance genes, may experience toxic reactions. To accurately elucidate the mechanisms of these adverse reactions, radiolabeled selamectin must be used as a tracer to conduct pharmacokinetic studies related to safety (including tissue distribution, metabolic processes, and mass balance analysis). Radiolabeled substances offer advantages such as intuitive tracking, high sensitivity, and strong specificity, providing precise experimental data for assessing drug mechanisms of action, safety, and efficacy.

[0004] In drug pharmacokinetic studies, carbon-14 and tritium (… 3H / T is the most commonly used low-energy radionuclides, among which carbon-14 labeling is often preferred because it can stably reflect the complete metabolic pathway of the molecule. However, carbon-14 labeling technology faces significant bottlenecks for selamectin: although selamectin is a semi-synthetic derivative, it is still a complex natural product with multiple chiral centers in its molecule. Moreover, the glycosylation reaction during synthesis has low yield and poor product stability. This means that obtaining an effective amount of target label requires a large amount of high-valent carbon-14 raw material, and the long-route reaction will inevitably generate a large amount of radioactive waste, resulting in high costs for both isotope raw materials and radioactive waste disposal. Coupled with the problems of high project implementation risk and long cycle, the practical application feasibility is extremely low.

[0005] Literature reports that Ku CC et al. prepared carbon-14 labeled avermectin through microbial fermentation and fine separation purification, successfully utilizing [1- 14 [C] Sodium propionate fermentation to obtain carbon-14 labeled avermectin B 1a However, this method has insurmountable drawbacks: the strains used for fermentation are difficult to obtain, and high-efficiency fermentation processes and purification processes for fermentation mixtures have not been publicly disclosed; more importantly, the final product is a mixture of five carbon-14 labeled compounds with different labeling sites (C-3, C-7, C-11, C-13, C-23)—in biosynthesis, its precursors are randomly used for multiple propionate derivatization sites within the molecular backbone (this conclusion has been verified). 13 (As confirmed by C-NMR and mass spectrometry analysis), different metabolic fragments in metabolic studies may originate from different labeling sites, complicating the analysis of metabolic pathways and introducing uncertainties, thus failing to meet the research requirement of "precisely tracking the fate of specific structural fragments of molecules." In summary, from a technical practice perspective, the preparation of carbon-14 labeled selamectin with a sixteen-membered macrocyclic lactone carbon skeleton via microbial fermentation is objectively not feasible.

[0006] Against this backdrop, tritium labeling technology demonstrates irreplaceable advantages for the radiolabeling needs of complex natural products like selamectin. As Krauser et al. pointed out: ① High synthetic feasibility: The tritium-labeled synthetic route is significantly shorter than carbon-14 labeling, with lower technical difficulty, reducing tritium raw material consumption and significantly shortening the delivery cycle. This precisely matches the timeliness requirements of selamectin safety research, avoiding the technical risks of "30+ steps, low yield" in carbon-14 total synthesis, as well as the difficulties in obtaining strains during microbial fermentation and the lack of publicly available efficient fermentation, separation, and purification processes; ② Superior cost-effectiveness: Tritium raw materials are easier to obtain and cheaper. Combined with a shorter synthetic route and lower raw material consumption, the resulting radioactive waste volume is far less than that of carbon-14 labeling (which has extremely high radioactive waste disposal costs due to its numerous steps and byproducts). This is achieved through "less raw material +..." The dual advantages of "less waste" lower the economic threshold for technology transfer; ③ Strong application adaptability: Tritium has high specific activity and trace detection sensitivity far exceeds that of carbon-14, which can meet the needs of scenarios such as receptor binding experiments and analysis of trace metabolites. Moreover, the shorter β-ray range can improve the spatial resolution of microscopic autoradiography and help to clearly analyze tissue distribution characteristics.

[0007] In summary, the core shortcomings of existing isotope labeling technologies are as follows: the total synthesis of carbon-14 labeled selamectin suffers from high cost of isotope raw materials, high synthesis difficulty, long cycle, high risk, and large volume of radioactive waste; while the preparation of carbon-14 labeled selamectin by microbial fermentation faces difficulties in obtaining strains and complex fermentation and separation purification processes that are difficult to overcome in the short term, making it objectively unfeasible. Tritium labeling technology is not a simple alternative, but a dedicated solution that fits the structural characteristics of selamectin and the needs of downstream isotope tracing research, making it the preferred solution for its radioactive isotope labeling. However, there is no existing technology for tritium-labeled selamectin. Summary of the Invention

[0008] The purpose of this invention is to provide a radioactive isotope tritium-labeled selamectin and its preparation method. Using selamectin as the starting material and sodium borotritide as the tritium source, a "directional oxidation-tritium substitution reduction" strategy is adopted to achieve precise tritium atom labeling at specific sites on the carbon skeleton of the sixteen-membered macrocyclic lactone. This not only solves the defects of existing labeling technologies, but also provides sufficient high-purity and high-stability radioactive tracers for related isotope tracing studies in a timely manner.

[0009] To achieve the above objectives, this technical solution provides a radioactive tritium-labeled selamectin, with the following structural formula: .

[0010] This radioactive tritium-labeled selamectin is a single-site tritium label at the 13-position of the 16-membered macrocyclic lactone carbon skeleton of the selamectin molecule. Specifically, the tritium atom is directionally anchored to the 13-carbon in the core structural region of the molecule. This core structural region exhibits high chemical stability, ensuring a robust tritium label that is not easily detached during tracer experiments. Using it as a tracer allows for precise tracking of the complete metabolic trajectory of selamectin, providing accurate and complete tracer data for related research.

[0011] Correspondingly, this scheme provides a method for preparing radioactive tritium-labeled selamectin, including the following steps: S1: Doramectin was subjected to hydrolysis and desaccharification reaction to obtain doramectin aglycone.

[0012] In step S1, under inert gas protection and at room temperature, organic solvent and water are mixed to obtain a solvent solution. After cooling the solvent solution, acid is added to adjust it to acidic conditions. Doramectin is added to the solvent solution and heated to room temperature to carry out hydrolysis and desaccharification reaction. After the reaction is completed, the pH of the solvent solution is adjusted to neutral and the organic solvent is removed to obtain a concentrated solution. After washing the concentrated solution, the organic phase is extracted. After washing the organic phase, the solvent is removed under reduced pressure and the aglycone is obtained by silica gel rapid column chromatography.

[0013] In some embodiments, one or a combination of two of methanol, ethanol, isopropanol, tetrahydrofuran, and water are selected as organic solvents.

[0014] In some embodiments, the acid is selected from at least one of sulfuric acid, hydrochloric acid, and methanesulfonic acid.

[0015] In some embodiments, the hydrolysis reaction is carried out at room temperature for 24–48 h.

[0016] In some embodiments, after the reaction is completed, saturated sodium bicarbonate solution is added to adjust the pH of the reaction solution to neutral, the organic solvent is removed by vacuum concentration, the concentrate is washed with water and extracted with ethyl acetate, the organic phases are combined, washed successively with saturated sodium bicarbonate solution and saturated sodium chloride solution, the organic phase is dried with anhydrous sodium sulfate, filtered, desolventized under reduced pressure, and separated by silica gel rapid column chromatography to obtain doramectin aglycone.

[0017] The specific reaction formula is shown below: .

[0018] S2: The product protected by 5-hydroxyl silanization was prepared by reacting doramectin aglycone with a silicon protecting agent.

[0019] In some embodiments, under inert gas protection and at room temperature, a silicon protecting agent is added to a dichloromethane solution of doramectin aglycone and imidazole for reaction. After the reaction is completed, water is added to quench the reaction, the reaction solution is extracted to obtain an organic phase, the organic phase is washed and then subjected to desolvation under reduced pressure, the crude product is concentrated and separated by silica gel rapid column chromatography to obtain the product with 5-hydroxyl silanization protection.

[0020] In some embodiments, the silicon protecting group is selected from at least one of tert-butyldiphenylchlorosilane, trimethylchlorosilane, triethylchlorosilane, and tert-butyldimethylchlorosilane.

[0021] In some implementations, the organic phase is extracted with ethyl acetate, and the combined organic phases are washed with saturated sodium bicarbonate solution and saturated sodium chloride solution. The organic phase is dried with anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue is separated by silica gel rapid column chromatography to obtain the product with 5-hydroxyl silanization protection.

[0022] The specific reaction formula is shown below: .

[0023] S3: Oxidation of the 5-hydroxyl group with an oxidant to silanize the 13-hydroxyl group in the protected product yields the 13-keto-coated product.

[0024] In some embodiments, under inert gas protection and at room temperature, a mixed solution is obtained by mixing the 5-hydroxyl silylation protection product, sodium bicarbonate, and dichloromethane and cooling it to 0 °C. An oxidant is added to the mixed solution and reacted for a period of time. The temperature is then raised to room temperature to continue the reaction. After the reaction is completed, a saturated sodium bicarbonate solution is added to quench the reaction. The reaction solution is extracted to obtain an organic phase. After washing the organic phase, the solution is desoluble under reduced pressure and then separated by silica gel rapid column chromatography to obtain the 13-ketoylation product.

[0025] In some embodiments, the oxidant is selected from at least one of Desmartin oxidant, Jones reagent, Sarret reagent, Coslin reagent, PDC, and IBX oxidant.

[0026] In some embodiments, the organic phase is extracted with ethyl acetate, the organic phases are combined and washed with saturated sodium bicarbonate solution and saturated brine, the organic phase is dried with anhydrous sodium sulfate, filtered and concentrated under reduced pressure, and the residue is separated by silica gel rapid column chromatography to obtain the 13-keto-coated product.

[0027] The specific reaction formula is shown below: .

[0028] S4: At a certain temperature, the 13-keto-oxidized product is dissolved in a solvent and reduced with sodium borotrithide to obtain 5-hydroxysilyl-protected and 13-tritium-labeled doramectin aglycone.

[0029] In some embodiments, under inert gas protection and room temperature, the 13-keto-formation product is dissolved in a solvent and cooled to 0 °C. Sodium borotrithide and sodium borohydride are then added to react and the temperature is raised to room temperature. After the reaction is completed, water is added to quench the reaction. The reaction solution is extracted to obtain an organic phase. After washing the organic phase, the solution is desolvated under reduced pressure and then separated by silica gel rapid column chromatography to obtain doramectin aglycone protected by 5-hydroxysilane and labeled with 13-tritium.

[0030] In some embodiments, the temperature is 0~50 °C, and the solvent is selected from methanol, ethanol, and tetrahydrofuran.

[0031] In some embodiments, the organic phase is extracted with ethyl acetate, and the combined organic phases are washed with saturated sodium bicarbonate solution and saturated brine. The organic phase is dried with anhydrous sodium sulfate and concentrated under reduced pressure. The residue is separated by silica gel rapid column chromatography to obtain doramectin aglycone protected with 5-hydroxysilicon and labeled with 13-tritium.

[0032] The specific reaction equation is shown below: .

[0033] S5: The doramectin aglycone protected by 5-hydroxy silylation and labeled with 13-tritium is prepared by glycosylation reaction, and the reaction system of the glycosylation reaction must be strictly anhydrous.

[0034] In some embodiments, under inert gas protection and room temperature conditions, doramectin aglycone protected by 5-hydroxysilylation and labeled with 13-tritium and dried 2-{[(2 S 4 S 5 S 6 S )-5-{[dimethyl(2-methylprop-2-yl)silyl]oxy}-4-methoxy-6-methyl-3,4,5,6-tetrahydro-2 H -pyran-2-yl]oxy}-4-methoxy-6-methyl-3,4,5,6-tetrahydro-2 H [-pyran-2-yl]thio}pyridine was mixed with dichloromethane, and powdered 4Å molecular sieve was added. After reacting at room temperature, dry silver trifluoromethanesulfonate was added to continue the reaction. After the reaction was completed, the organic phase was extracted, washed, and then subjected to desolvation under reduced pressure. The resulting product was obtained by silica gel rapid column chromatography with 5,4"-hydroxysilicized protection and 13-tritium-labeled doramectin.

[0035] In some embodiments, dichloromethane and saturated sodium bicarbonate solution are added to extract and separate the organic phase; the aqueous phase is extracted with dichloromethane, and the organic phases are combined.

[0036] In some embodiments, the organic phase is washed with saturated brine and dried over anhydrous sodium sulfate, followed by desolvation under reduced pressure to obtain a residue. The residue is then separated by silica gel rapid column chromatography to obtain doramectin with 5,4"-hydroxysilylation and 13-tritium labeling and 5-hydroxysilylation and 13-tritium labeling, and doramectin aglycone. The recovered 5-hydroxysilylation and 13-tritium labeling doramectin aglycone is used to repeat the glycosylation reaction to obtain doramectin with 5,4"-hydroxysilylation and 13-tritium labeling.

[0037] The specific reaction formula is as follows: .

[0038] S6: Doramectin with 5,4"-hydroxyl group silanization protection and 13-tritium labeling is dissolved in a solvent and a desilication protection reagent is added to carry out a desilication protection reaction to obtain 13-tritium labeling doramectin.

[0039] In some embodiments, under inert gas and room temperature protection, doramectin with 5,4"-hydroxyl silanization protection and 13-tritium labeling is dissolved in a solvent, and a mixture of desilication protection reagent, pyridine and solvent is added to carry out the reaction. During the reaction, desilication protection reagent and pyridine are added. After the reaction is completed, saturated sodium bicarbonate solution is added to quench the reaction. The reaction solution is extracted to obtain an organic phase. After washing the organic phase, it is desolvated under reduced pressure and then separated by silica gel rapid column chromatography to obtain the 13-keto-coated product.

[0040] In some embodiments, the desilication protection agent is selected from at least one of hydrogen fluoride and tetra-n-butylammonium fluoride.

[0041] In some embodiments, the solvent is acetonitrile.

[0042] In some embodiments, the organic phase is extracted with ethyl acetate, the organic phases are combined and washed with water and saturated brine, the organic phase is dried with anhydrous sodium sulfate and concentrated under reduced pressure, and the residue is separated by silica gel rapid column chromatography to obtain 13-tritium-labeled doramectin.

[0043] The specific reaction formula is as follows: .

[0044] S7: Under acidic conditions, acid is added dropwise to a protic solvent to obtain an acidic solvent. 13-tritium-labeled doramectin is added to the acidic solvent to carry out a selective desaccharification reaction to obtain a 13-tritium-labeled monosaccharide doramectin product.

[0045] In some embodiments, under an inert gas and room temperature environment, acid is dropped into a protic solvent pre-cooled to 0 °C to obtain an acidic solvent. 13-tritium-labeled doramectin is added to the acidic solvent and the mixture is heated to room temperature to carry out a selective desaccharification reaction. After the reaction is completed, the pH of the reaction system is adjusted to neutral and the organic solvent is removed to obtain the residual liquid. The residual liquid is extracted and the organic phase is collected. After washing the organic phase, it is desolventized under reduced pressure and then separated by silica gel rapid column chromatography to obtain the 13-tritium-labeled monosaccharide doramectin product.

[0046] In some embodiments, the protic solvent is selected from one or a combination of two of methanol, ethanol, isopropanol, tetrahydrofuran, and water.

[0047] In some embodiments, the acid is selected from at least one of sulfuric acid, hydrochloric acid, and methanesulfonic acid.

[0048] In some embodiments, the reaction conditions are room temperature reaction for 24-48 h.

[0049] In some embodiments, the pH of the reaction system is adjusted to neutral with saturated sodium bicarbonate solution, and the organic solvent is removed under reduced pressure to obtain the residue. The residue is extracted with ethyl acetate, and the combined organic phases are washed with saturated brine. The residue is dried with anhydrous sodium sulfate and desolventized under reduced pressure to obtain the residue. The obtained residue is separated by silica gel rapid column chromatography to obtain the 13-tritium-labeled monosaccharide doramectin product.

[0050] The specific reaction equation is shown below: .

[0051] S8: Selective oxidation of the 13-position tritium-labeled monosaccharide doramectin product with an oxidant yields a ketogenic product, wherein the selectively oxidized site is the 5-position hydroxyl group.

[0052] In some embodiments, under inert gas and at room temperature, the 13-tritium-labeled monosaccharide doramectin product is dissolved in a solvent and an oxidant is added for selective oxidation. After the reaction is completed, the product is filtered through a microporous membrane and diatomaceous earth to remove impurities. The filter cake is washed and the filtrates are combined and then desolvated under reduced pressure to obtain the residue. The residue is separated by silica gel rapid column chromatography to obtain the ketylated product.

[0053] In some embodiments, the oxidant is selected from at least one of Desmartin oxidant, Jones reagent, Sarrett reagent, Coslin reagent, PDC, IBX oxidant, and manganese dioxide.

[0054] In some embodiments, after the reaction is completed, the filter cake is filtered with two layers of microporous membranes and diatomaceous earth to remove impurities, and then washed with dichloromethane.

[0055] The specific reaction formula is shown below: .

[0056] S9: An amination reagent is added to the 5-keto-ester product to prepare C. 22 -C 23 Olefinified and 13-tritium-labeled selamectin products.

[0057] In some embodiments, the 5-ketoylation product and the amination reagent are mixed and dissolved in a solvent under an inert gas and at room temperature, and then reacted. After the reaction is completed, water is added to quench the reaction, and the organic solvent is removed by vacuum concentration to obtain the residue. The organic phase is collected by extraction of the residue, washed, dried, filtered, and concentrated under vacuum to obtain the residue. The residue is then separated by silica gel rapid column chromatography to obtain C 22 -C 23 Olefinified and 13-tritium-labeled selamectin products.

[0058] In some embodiments, the amination agent is hydroxylamine hydrochloride, and the solvent is tert-butanol.

[0059] In some embodiments, after the reaction is substantially complete, water is added to quench the reaction, the solvent is removed by concentration under reduced pressure, ethyl acetate is extracted, the organic phases are combined, washed successively with saturated sodium bicarbonate solution and saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and the residue is separated by silica gel rapid column chromatography to obtain C. 22 -C 23 Olefinified and 13-tritium-labeled selamectin products.

[0060] The specific reaction formula is shown below: .

[0061] S10: Using a catalyst for C 22 -C 23 C in olefinated and 13-tritium-labeled selamectin products 22 -C 23 Selective reduction of the double bond yielded a 13-tritium-labeled selamectin product.

[0062] In some embodiments, under inert gas protection and at room temperature, C 22 -C 23 The olefinified and 13-tritium-labeled selamectin product, catalyst, and organic solvent were mixed and sealed. Hydrogen was introduced to replace the inert gas, and the reaction system was made to a pressure greater than one standard atmosphere. After reacting at the reaction temperature for a period of time, nitrogen was introduced for purging, and activated carbon was added for stirring. After stirring, the reaction solution was filtered with diatomaceous earth. The filter cake was washed, and the filtrates were combined and subjected to desolvation under reduced pressure to obtain the residue. The obtained residue was separated by rapid column chromatography to obtain the 13-tritium-labeled selamectin product.

[0063] In some embodiments, the catalyst is selected from one of rhodium reagent, palladium on carbon, and Raney nickel, the hydrogen pressure is >1 atm, and the reaction temperature is 10~50 °C.

[0064] In some embodiments, after reacting for a period of time, the mixture is cooled to 25-30 °C and the hydrogen is removed by nitrogen purging.

[0065] In some embodiments, the filter cake is washed with ethyl acetate.

[0066] The specific reaction equation is shown below: .

[0067] The overall synthetic technical route for the preparation method of this radioactive tritium-labeled selamectin is as follows: Figure 33 As shown.

[0068] Compared with existing technologies, this technical solution has the following characteristics and beneficial effects: ① The technical approach is highly targeted and adaptable to the labeling needs of complex natural products: Tritium labeling technology itself has core advantages such as short synthetic routes, low cost of isotope raw materials, low generation of radioactive waste, and low subsequent disposal costs. It is naturally adapted to the radiolabeling scenarios of natural products with complex structures and multiple chiral centers, such as selamectin, effectively avoiding the drawbacks of long routes and high risks of carbon-14 labeling, and providing an efficient and adaptable technical path for radioisotope labeling of such complex natural products.

[0069] ② The synthesis process is efficient and concise, with high feasibility: Compared to the lengthy total synthesis route requiring more than 30 steps for carbon-14 labeling, this invention adopts a stepwise strategy of "desugaring - hydroxyl protection - directed oxidation - tritium reduction - skeleton reconstruction," which has a clear logical route, controllable operation, and significantly shortens the reaction cycle. At the same time, it effectively avoids the technical bottlenecks of difficult strain acquisition and undisclosed efficient fermentation and separation / purification processes in microbial fermentation methods. The overall radiochemical yield can reach over 19%, significantly reducing the difficulty of technical implementation and project progress risks, and possessing good potential for industrialization.

[0070] ③ Cost-effective and environmentally friendly, in line with the concept of green synthesis: Using sodium borotrithide, which is readily available and costs less than 1 / 5 of the raw materials for carbon-14 labeling, as the tritium source, and combined with a short-path synthesis process, the consumption of isotopic raw materials is greatly reduced; calculated based on the same amount of product, the amount of radioactive waste generated is less than 1 / 10 of that for carbon-14 labeling, with low waste disposal costs and a small environmental burden, achieving a balance between economic efficiency and environmental protection, which is in line with the trend of green chemical development.

[0071] ④ The product exhibits excellent performance and is suitable for diverse research scenarios: The prepared tritium-labeled selamectin has a chemical purity of >98% and a radiochemical purity of >99% (meeting international standards for the use of radioactive tracers); the synthesis process utilizes site-specific reduction technology to precisely maintain the original chiral center configuration of the molecule, ensuring consistency with the biological activity of natural selamectin; combined with the high specific activity of tritium (trace detection sensitivity is 1-2 orders of magnitude higher than that of carbon-14), it can meet diverse research needs such as tissue distribution analysis, metabolite identification, mass balance studies, environmental fate tracking, and ecotoxicity assessment.

[0072] ⑤ Outstanding technological innovation and wide application value: The innovative development of the core technology of "13-position directional oxidation-tritium reduction" has overcome the technical difficulties of unclear tritium labeling sites, easy racemization of chiral configurations, and poor labeling stability in complex macrolide compounds. The tritium-labeled selamectin prepared can not only be used for non-clinical pharmacokinetic studies of selamectin, but also provide key tools for its environmental residue tracing and ecological risk assessment. At the same time, it provides a modular technical solution that can be directly referenced for the radiolabeling of similar macrolide drugs, expanding the application boundaries of directional tritium labeling technology. Attached Figure Description

[0073] Figures 1 to 3 The 1H NMR spectrum of intermediate O2: doramectin aglycone of this invention; Figure 2 for Figure 1 A magnified view of a portion of the image, corresponding to Figure 1 The chemical shift range is 6.0–3.2 ppm; Figure 3 for Figure 1 Another enlarged view of the part, corresponding to Figure 1 The chemical shift range is 2.6–0.7 ppm.

[0074] Figures 4 to 6 The 1H NMR spectrum of the intermediate O3:5-hydroxyl silanized protected product of this invention; Figure 5 for Figure 4 A magnified view of a portion of the image, corresponding to Figure 4 The chemical shift range is 6.1–3.1 ppm; Figure 6 for Figure 4 A magnified view of a portion of the image, corresponding to Figure 4 The chemical shift range is 2.5-0.1 ppm.

[0075] Figures 7 to 9 The 1H NMR spectrum of the intermediate 04:13-keto-esterified product of this invention; Figure 8 for Figure 7 A magnified view of a portion of the image, corresponding to Figure 7 The chemical shift range is 6.5-3.1 ppm; Figure 9 for Figure 7A magnified view of a portion of the image, corresponding to Figure 7 The chemical shift range is 2.7-0.1 ppm.

[0076] Figures 10 to 12 The 1H NMR spectrum of the radioactive intermediate O5: 5-hydroxyl silanized and 13-tritium-labeled doramectin aglycone of this invention; Figure 11 for Figure 10 A magnified view of a portion of the image, corresponding to Figure 10 The chemical shift range is 6.1–3.1 ppm; Figure 12 for Figure 10 A magnified view of a portion of the image, corresponding to Figure 10 The chemical shift range is 2.6-0.1 ppm.

[0077] Figures 13 to 15 The 1H NMR spectrum of latanoic acid, the radioactive intermediate of this invention, is protected by silanization of the 5,4"-hydroxyl group and labeled with tritium at the 13-position. Figure 14 for Figure 13 A magnified view of a portion of the image, corresponding to Figure 13 The chemical shift range is 6.0-3.1 ppm; Figure 15 for Figure 13 A magnified view of a portion of the image, corresponding to Figure 13 The chemical shift range is 2.6-0.0 ppm.

[0078] Figures 16 to 18 The 1H NMR spectrum of doramectin labeled with tritium at position 13, the radioactive intermediate of this invention; Figure 17 for Figure 16 A magnified view of a portion of the image, corresponding to Figure 16 The chemical shift range is 6.0-3.1 ppm; Figure 18 for Figure 16 A magnified view of a portion of the image, corresponding to Figure 16 The chemical shift range is 2.6-0.8 ppm.

[0079] Figures 19 to 21 The 1H NMR spectrum of doramectin, a radioactive intermediate of the present invention labeled with tritium at position 08:13; Figure 20 for Figure 19 A magnified view of a portion of the image, corresponding to Figure 19 The chemical shift range is 5.9–3.1 ppm; Figure 21 for Figure 21 A magnified view of a portion of the image, corresponding to Figure 19 The chemical shift range is 2.6-0.7 ppm.

[0080] Figures 22 to 24 The 1H NMR spectrum of the radioactive intermediate 09: ketylated product of this invention; Figure 23 for Figure 22 A magnified view of a portion of the image, corresponding to Figure 22 The chemical shift range is 6.6–3.1 ppm; Figure 24 for Figure 22 A magnified view of a portion of the image, corresponding to Figure 22 The chemical shift range is 2.6-0.7 ppm.

[0081] Figures 25 to 27 Radioactive intermediate 10 of the present invention: C 22 -C 23 The 1H NMR spectrum of olefinically-treated and 13-tritium-labeled selamectin; Figure 26 for Figure 25 A magnified view of a portion of the image, corresponding to Figure 25 The chemical shift range is 6.0–3.1 ppm; Figure 27 for Figure 25 A magnified view of a portion of the image, corresponding to Figure 25 The chemical shift range is 2.6-0.8 ppm.

[0082] Figures 28 to 30 The 1H NMR spectrum of selamectin (11) labeled with tritium at position 13, the target of this invention; Figure 29 for Figure 28 A magnified view of a portion of the image, corresponding to Figure 28 The chemical shift range is 6.0–3.0 ppm; Figure 30 for Figure 28 A magnified view of a portion of the image, corresponding to Figure 28 The chemical shift range is 2.6–0.7 ppm.

[0083] Figure 31 The HPLC-UV chromatogram of selamectin (11) labeled with tritium at position 13, the target of this invention, shows that the retention time of the peak is 13.185 min, the integral area is 33187040, the height is 2684307, and the integral area ratio is 100.

[0084] Figure 32 The radiometric high performance liquid chromatogram (HPLC-LSC) of selamectin (11) labeled with tritium at position 13, the target of this invention, is shown; where min is minutes and DPM is radioactivity.

[0085] Figure 33 This is the overall synthesis path diagram of this scheme. Detailed Implementation

[0086] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention are within the scope of protection of the present invention.

[0087] Example:

[0088] Under nitrogen protection and at room temperature, tetrahydrofuran (30 mL) was mixed with water (3 mL), cooled in an ice bath, and concentrated sulfuric acid (3 mL) was added dropwise while stirring until homogeneous. Compound 01 (5000 mg, 5.50 mmol) was then added, and the mixture was heated to room temperature and stirred for 24 h. TLC analysis: R f (02) = 0.50, R f (01) = 0.10, V EA : V PE =1:1. After the reaction, the pH of the reaction solution was adjusted to neutral with saturated sodium bicarbonate solution, and the organic solvent was removed by concentration under reduced pressure. The concentrate was washed with water and extracted with ethyl acetate (200 mL × 3). The organic phases were combined and washed successively with saturated sodium bicarbonate solution (200 mL × 2) and saturated sodium chloride solution (200 mL × 2). The organic phase was dried over anhydrous sodium sulfate, filtered, and then dissolved under reduced pressure. The residue was subjected to silica gel rapid column chromatography (…). V EA : V PE Separation was performed using a ratio of 1:2 to 1:1 to obtain a white solid O2 (1.3 g, yield 38%). 1 H NMR (500 MHz, CDCl3) δ 5.85(dt, J = 8.1, 2.4 Hz, 1H), 5.80 – 5.70 (m, 3H), 5.54 (dd, J = 9.9, 2.6 Hz, 1H), 5.41 (q, J = 2.8 Hz, 1H), 5.40 – 5.30 (m, 2H), 4.73 – 4.63 (m, 2H), 4.30 (d, J = 4.8 Hz, 1H), 4.01 (s, 2H), 3.96 (d, J= 6.2 Hz, 1H), 3.92 – 3.84 (m, 1H), 3.34 – 3.25 (m, 2H), 2.53 (tt, J = 6.9, 2.4 Hz, 1H), 2.30 (d, J = 10.5 Hz, 1H), 2.27 (dt, J = 7.1, 2.5 Hz, 1H), 2.00 (ddd, J = 12.0, 4.8, 1.9 Hz, 1H), 1.87 (t, J = 2.0 Hz, 4H), 1.82 – 1.76 (m, 2H), 1.69 (d, J = 9.4 Hz, 1H), 1.62 (d, J =10.6 Hz, 5H), 1.52 (s, 5H), 1.44 (d, J = 11.7 Hz, 1H), 1.27 – 1.24 (m, 2H), 1.18 (d, J = 7.0 Hz, 3H), 1.13 (t, J = 6.6 Hz, 1H), 0.92 (d, J = 7.2 Hz, 3H), 0.85 (d, J = 12.0 Hz, 1H). ESI-MS(+) m / z 633[M+Na] + .

[0089]

[0090] Under nitrogen protection and at room temperature, TBSCl (690 mg, 4.30 mmol) was added to a solution of compound O2 (920 mg, 1.50 mmol) and imidazole (520 mg, 7.60 mmol) in dichloromethane (8 mL), and the mixture was stirred at room temperature for 5 h. TLC analysis: R f (02) = 0.20, R f (03) = 0.8, V EA : V PE=1:5; Compound 02 reacted essentially to completion. The reaction was quenched with water (20 mL), and the mixture was extracted with ethyl acetate (20 mL × 3). The combined organic phases were washed successively with saturated sodium bicarbonate solution (20 mL) and saturated sodium chloride solution (20 mL × 2). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was subjected to silica gel rapid column chromatography (…). V EA : V PE =1:10) Separation yielded a white solid O3 (900 mg, yield 82%): 1 H NMR (500 MHz, CDCl3) δ 5.82 (d, J = 10.1 Hz, 1H), 5.79 – 5.67 (m, 3H), 5.53 (dd, J = 9.9, 2.6Hz, 1H), 5.35 (d, J = 4.6 Hz, 1H), 5.32 (q, J = 1.8 Hz, 1H), 4.73 – 4.51 (m,2H), 4.43 (d, J = 5.5 Hz, 1H), 4.01 (s, 1H), 3.91 – 3.83 (m, 1H), 3.82 (d, J =5.5 Hz, 1H), 3.37 (q, J = 2.5 Hz, 1H), 3.29 (d, J = 9.9 Hz, 1H), 2.56 – 2.46(m, 1H), 2.29 (q, J = 7.7 Hz, 3H), 2.02 (ddd, J = 12.0, 4.9, 2.0 Hz, 1H), 1.85(s, 1H), 1.79 (p, J = 1.2 Hz, 3H), 1.70 (s, 1H), 1.59 (d, J = 21.8 Hz, 6H),1.53 (s, 3H), 1.45 (t, J = 11.8 Hz, 1H), 1.31 – 1.24 (m, 8H), 1.17 (d, J = 7.0Hz, 3H), 0.93 (s, 9H), 0.92 – 0.91 (m, 3H), 0.88 (s, 1H), 0.13 (s, 6H). ESI-MS(+)m / z 747[M+Na] + .

[0091]

[0092] Under nitrogen protection and at room temperature, compound O3 (880 mg, 1.20 mmol), sodium bicarbonate (505 mg, 6.00 mmol), and dichloromethane (8 mL) were mixed and cooled to 0 °C in an ice bath; Dysmartin oxidant (764 mg, 1.80 mmol) was added, and the mixture was stirred for 10 min; the mixture was then brought to room temperature and stirred for another 1 h. TLC analysis: R f (03) = 0.40, R f (04) = 0.50, V EA : V PE =1:5, UV color development; compound 03 reacted essentially completely. The reaction was quenched with saturated sodium bicarbonate solution (20 mL), extracted with ethyl acetate (20 mL × 3), the organic phases were combined, washed with sodium bicarbonate solution (20 mL) and saturated brine (20 mL × 2), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and the residue was subjected to silica gel rapid column chromatography (…). V EA : V PE =1:10) Separation yielded a white solid O4 (570 mg, 65%): 1 H NMR (500 MHz, CDCl3) δ 6.23 (ddd, J = 9.2, 7.6, 1.6Hz, 1H), 6.04 (dd, J = 14.8, 11.2 Hz, 1H), 5.86 (dt, J = 11.2, 2.5 Hz, 1H), 5.75 (dd, J = 9.9, 1.8 Hz, 1H), 5.53 (dd, J = 9.9, 2.6 Hz, 1H), 5.45 (dd, J =14.9, 10.1 Hz, 1H), 5.39 – 5.31 (m, 2H), 4.73 (dd, J = 14.7, 2.5 Hz, 1H), 4.62(dd, J= 14.7, 2.4 Hz, 1H), 4.44 (ddd, J = 5.5, 2.7, 1.4 Hz, 1H), 3.83 (dd, J =11.3, 5.6 Hz, 2H), 3.69 – 3.62 (m, 1H), 3.43 (d, J = 2.4 Hz, 1H), 3.28 (dd, J =10.1, 1.8 Hz, 1H), 2.57 (ddd, J = 12.3, 7.6, 4.7 Hz, 1H), 2.33 – 2.18 (m, 2H), 2.02 (ddd, J = 12.2, 5.0, 1.8 Hz, 1H), 1.81 (t, J = 2.4 Hz, 3H), 1.79 (dt, J =2.6, 1.2 Hz, 3H), 1.74 – 1.66 (m, 2H), 1.60 (s, 3H), 1.53 – 1.47 (m, 3H), 1.30 – 1.21 (m, 5H), 1.17 (d, J = 6.7 Hz, 3H), 1.06 (q, J = 11.8 Hz, 1H), 0.94– 0.91 (m, 13H), 0.14 (s, 6H). ESI-MS(+) m / z 723[M+H] + .

[0093]

[0094] Under nitrogen protection and at room temperature, a solution of compound O4 (325 mg, 0.45 mmol) in anhydrous ethanol (3.0 mL) was cooled to 0 °C using an ice bath; sodium borotrithide (13 mg, 5.92E+9 Bq, 1.85E+10 Bq / mmol) and sodium borohydride (4 mg, 0.11 mmol) were added, and the mixture was stirred for 10 min; the mixture was then brought to room temperature and stirred for another 1 h. TLC analysis: R f (04) = 0.50, R f (05) = 0.60, V EA : V PE=1:5, compound 04 reacted essentially to completion. The reaction was quenched with water (20 mL), extracted with ethyl acetate (20 mL × 3), the organic phases were combined, washed with saturated sodium bicarbonate solution (20 mL) and saturated brine (20 mL × 2), dried over anhydrous sodium sulfate, dissolved under reduced pressure, and the residue was subjected to silica gel rapid column chromatography (…). V EA : V PE =1:10) Separation yielded a white solid 05 (5.55E+9 Bq, radiochemical yield 94%): 1 H NMR (500 MHz, CDCl3) δ 5.85 – 5.79 (m, 1H), 5.79– 5.66 (m, 3H), 5.53 (dd, J = 9.9, 2.6 Hz, 1H), 5.41 – 5.24 (m, 3H), 4.63(ddd, J = 49.5, 14.5, 2.3 Hz, 2H), 4.43 (ddt, J = 5.8, 2.8, 1.3 Hz, 1H), 3.91 –3.84 (m, 1H), 3.81 (d, J = 5.5 Hz, 1H), 3.36 (q, J = 2.5 Hz, 1H), 3.29 (d, J =9.9 Hz, 1H), 2.51 (dq, J = 8.8, 6.9 Hz, 1H), 2.36 – 2.21 (m, 3H), 2.02 (ddd, J = 12.1, 4.9, 1.9 Hz, 1H), 1.90 – 1.80 (m, 2H), 1.78 (dt, J = 2.6, 1.2 Hz, 3H), 1.76 (dt, J = 4.2, 1.9 Hz, 1H), 1.69 (d, J = 6.6 Hz, 1H), 1.65 – 1.57 (m, 4H), 1.53 (t, J = 1.3 Hz, 4H), 1.45 (t, J = 11.8 Hz, 1H), 1.32 – 1.23 (m, 4H), 1.17(d, J= 7.0 Hz, 3H), 0.95 – 0.85 (m, 13H), 0.13 (s, 6H). ESI-MS(+) m / z 747[M+Na] + .

[0095]

[0096] Under nitrogen protection and at room temperature, compound 05 (5.55E+9 Bq) and dry 2-{[(2 S 4 S 5 S 6 S )-5-{[dimethyl(2-methylprop-2-yl)silyl]oxy}-4-methoxy-6-methyl-3,4,5,6-tetrahydro-2 H -pyran-2-yl]oxy}-4-methoxy-6-methyl-3,4,5,6-tetrahydro-2 H [-pyran-2-yl]thio}pyridine (540 mg, 1.05 mmol) was mixed with dichloromethane (4 mL), and powdered 4 Å molecular sieve was added. The mixture was stirred at room temperature for 30 min. Dry silver trifluoromethanesulfonate (325 mg, 1.26 mmol) was then added, and stirring continued for 1 h. TLC analysis: R f (05) = 0.50, R f (06) = 0.30, V EA : V PE =1:1. Add dichloromethane (20 mL) and saturated sodium bicarbonate solution (20 mL) to extract and separate the organic phase; extract the aqueous phase with dichloromethane (20 mL × 3), combine the organic phases, wash with saturated brine (100 mL × 2), dry to anhydrous sodium sulfate, and remove solvent under reduced pressure. The resulting residue is subjected to silica gel rapid column chromatography (…). V EA : V PE =1:10) to separate white solid 06 and compound 05; the recovered compound 05 was fed three times, and the results were combined to obtain compound 06 (4.44E+9 Bq, radiochemical yield 80%). 1 H NMR (500MHz, CDCl3) δ 5.88 – 5.81 (m, 1H), 5.80 – 5.69 (m, 3H), 5.54 (dd, J = 9.9, 2.6Hz, 1H), 5.39 (tt, J= 11.6, 4.8 Hz, 1H), 5.34 – 5.29 (m, 2H), 5.01 (dd, J =11.0, 3.5 Hz, 1H), 4.80 – 4.75 (m, 1H), 4.63 (ddd, J = 49.0, 14.5, 2.4 Hz,2H), 4.46 – 4.40 (m, 1H), 4.12 (s, 1H), 3.94 (s, 1H), 3.90 – 3.85 (m, 1H),3.82 (q, J = 5.2 Hz, 2H), 3.69 (dq, J = 9.1, 6.3 Hz, 1H), 3.61 (ddd, J = 11.2,8.5, 4.7 Hz, 1H), 3.43 (s, 3H), 3.39 (q, J = 2.5 Hz, 1H), 3.37 (d, J = 4.8 Hz,1H), 3.34 (s, 3H), 3.32 – 3.29 (m, 1H), 3.21 (t, J = 9.0 Hz, 1H), 3.14 (t, J =8.8 Hz, 1H), 2.52 (ddd, J = 9.3, 7.0, 2.5 Hz, 1H), 2.34 – 2.20 (m, 5H), 2.01(ddd, J = 12.1, 5.0, 1.9 Hz, 1H), 1.79 (dt, J = 2.5, 1.2 Hz, 5H), 1.70 – 1.53(m, 8H), 1.50 (s, 3H), 1.46 (d, J = 11.7 Hz, 1H), 1.27 – 1.24 (m, 5H), 1.21(d, J = 6.2 Hz, 4H), 1.16 (d, J = 6.9 Hz, 3H), 0.93 (s, 9H), 0.92 – 0.90 (m,4H), 0.89 (s, 9H), 0.13 (s, 6H), 0.08 (d, J = 9.5 Hz, 6H). ESI-MS(+) m / z: 1151[M+Na] + .

[0097]

[0098] Under nitrogen protection and at room temperature, a 1 mL solution of acetonitrile in compound 06 (4.44E+9 Bq) was added dropwise to a mixture of 1 drop of 48% hydrofluoric acid aqueous solution, 0.2 mL of pyridine, and 10 mL of acetonitrile. The mixture was stirred at room temperature for 72 h. Pyridine and 48% hydrofluoric acid could be added during the reaction until complete. TLC analysis: R f (06) = 0.70, R f (07) = 0.20, V EA : V PE =1:2. After the reaction was completed, the reaction was quenched with saturated sodium bicarbonate solution (50 mL), extracted with ethyl acetate (50 mL × 3), the organic phases were combined, washed with water (50 mL) and saturated brine (50 mL), dried over anhydrous sodium sulfate, dissolved under reduced pressure, and the residue was subjected to silica gel rapid column chromatography (…). V EA : V PE =1:5~1:2) Separation yielded a white solid 07 (4.00E+9 Bq, radiochemical yield 90%): 1 H NMR (500 MHz, CDCl3) δ 5.92 – 5.85 (m, 1H), 5.82 – 5.68 (m, 3H), 5.54 (dd, J =9.9, 2.5 Hz, 1H), 5.47 – 5.40 (m, 2H), 5.39 (d, J = 4.0 Hz, 1H), 5.05 – 4.97(m, 1H), 4.79 (d, J = 3.7 Hz, 1H), 4.74 – 4.64 (m, 2H), 4.35 – 4.26 (m, 1H), 3.97 (d, J = 6.3 Hz, 1H), 3.94 (s, 1H), 3.88 (td, J = 10.8, 4.1 Hz, 1H), 3.85 –3.80 (m, 1H), 3.79 – 3.73 (m, 1H), 3.62 (ddd, J= 11.2, 8.5, 4.7 Hz, 1H), 3.51– 3.45 (m, 1H), 3.42 (d, J = 5.2 Hz, 5H), 3.34 – 3.28 (m, 2H), 3.24 (t, J = 9.0Hz, 1H), 3.17 (t, J = 9.1 Hz, 1H), 2.58 – 2.47 (m, 1H), 2.38 – 2.18 (m, 6H), 2.00 (ddd, J = 12.0, 4.8, 1.8 Hz, 1H), 1.88 (t, J = 2.1 Hz, 3H), 1.83 – 1.75(m, 4H), 1.66 (t, J = 14.1 Hz, 3H), 1.60 – 1.51 (m, 5H), 1.49 (d, J = 7.4 Hz,5H), 1.32 – 1.22 (m, 10H), 1.17 (d, J = 6.9 Hz, 3H), 0.93 (d, J = 7.2 Hz, 3H), 0.85 (q, J = 12.0 Hz, 1H). ESI-MS(+) m / z 923[M+Na] + .

[0099]

[0100] Under nitrogen protection and at room temperature, concentrated sulfuric acid (0.05 mL) was added dropwise to isopropanol (5 mL) pre-cooled to 0 °C, and the mixture was stirred until homogeneous. Then, compound 07 (4.00E+9 Bq) was added, the mixture was brought to room temperature, and stirred for 16 h. TLC analysis: R f (07) = 0.20, R f (08) = 0.30, V EA : V PE =1:1, the raw materials reacted almost completely. The pH of the reaction solution was adjusted to neutral with saturated sodium bicarbonate solution, the organic solvent was removed under reduced pressure, and the residue was extracted with ethyl acetate (80 mL × 3). The organic phases were combined, washed with saturated brine (50 mL), dried over anhydrous sodium sulfate and dissolved under reduced pressure. The residue was subjected to silica gel rapid column chromatography (…).V EA : V PE Separation at a ratio of 1:2 to 1:1 yielded a white solid 08 (3.11E+9 Bq, radiochemical yield 78%). 1 H NMR (500 MHz, CDCl3) δ 5.89(dt, J = 10.3, 2.4 Hz, 1H), 5.79 – 5.69 (m, 3H), 5.54 (dd, J = 9.9, 2.6 Hz,1H), 5.46 – 5.38 (m, 2H), 5.01 – 4.96 (m, 1H), 4.83 (d, J = 3.8 Hz, 1H), 4.73– 4.63 (m, 2H), 4.31 – 4.26 (m, 1H), 3.98 – 3.95 (m, 2H), 3.90 – 3.83 (m,2H), 3.58 – 3.53 (m, 1H), 3.48 (s, 3H), 3.32 – 3.28 (m, 2H), 3.16 (t, J = 9.1Hz, 1H), 2.52 (ddd, J = 9.3, 6.9, 2.6 Hz, 1H), 2.33 – 2.24 (m, 4H), 2.02 (d, J = 1.9 Hz, 1H), 1.87 (t, J = 2.1 Hz, 3H), 1.83 – 1.73 (m, 4H), 1.71 – 1.61 (m,3H), 1.58 – 1.52 (m, 4H), 1.50 (d, J = 1.7 Hz, 3H), 1.46 (d, J = 11.8 Hz, 1H), 1.27 (d, J = 6.3 Hz, 5H), 1.15 (d, J = 6.9 Hz, 3H), 1.14 – 1.11 (m, 1H), 0.92(d, J = 7.2 Hz, 3H), 0.85 (d, J = 12.2 Hz, 1H). ESI-MS (+) m / z 757[M+H] + .

[0101] Under nitrogen protection and at room temperature, activated manganese dioxide (500 mg, 5.73 mmol) was added to a 3 mL solution of compound 08 (3.11E+9 Bq) in dichloromethane and stirred for 16 h; activated manganese dioxide (500 mg, 5.73 mmol) was then added, and stirring continued for another 8 h. TLC analysis: R f (08) = 0.10, R f (09) = 0.2, V EA : V PE The reaction mixture had a ratio of 1:2, with a small amount of reactants remaining. The reaction solution was filtered through two microporous membranes and diatomaceous earth to remove impurities. The filter cake was washed with dichloromethane, and the combined filtrates were subjected to desolvation under reduced pressure. The resulting residue was then subjected to silica gel rapid column chromatography (…). V EA : V PE =1:10) Separation yielded a white solid 09 (3.11E+9 Bq, radiochemical yield 57%): 1 H NMR (500 MHz, CDCl3) δ 6.61 – 6.54 (m, 1H), 5.96 (dt, J = 10.9, 2.5Hz, 1H), 5.86 – 5.67 (m, 3H), 5.55 (dd, J = 9.9, 2.6 Hz, 1H), 5.48 (tt, J =11.4, 4.8 Hz, 1H), 5.01 (d, J = 10.9 Hz, 1H), 4.83 (d, J = 3.7 Hz, 1H), 4.80 –4.69 (m, 2H), 3.97 (s, 1H), 3.94 – 3.86 (m, 2H), 3.86 (s, 1H), 3.61 – 3.58(m, 1H), 3.58 – 3.52 (m, 1H), 3.48 (s, 3H), 3.36 – 3.27 (m, 1H), 3.17 (t, J =9.1 Hz, 1H), 2.54 (ddd, J = 9.8, 6.9, 2.8 Hz, 1H), 2.34 – 2.22 (m, 4H), 2.03(ddd, J= 11.9, 4.8, 1.8 Hz, 1H), 1.89 (dd, J = 2.6, 1.5 Hz, 3H), 1.84 – 1.77(m, 3H), 1.66 (d, J = 15.1 Hz, 3H), 1.62 – 1.53 (m, 6H), 1.50 (d, J = 2.4 Hz, 3H), 1.27 (d, J = 6.2 Hz, 5H), 1.16 (d, J = 7.0 Hz, 3H), 0.93 (d, J = 7.2 Hz, 3H), 0.91 – 0.87 (m, 1H). ESI-MS (+) m / z 777[M+Na] + .

[0102]

[0103] Under nitrogen protection and at room temperature, compound 09 (1.78E+9 Bq) and hydroxylamine hydrochloride (55 mg, 0.77 mmol) were added sequentially to a 10 mL round-bottom flask, dissolved in 2 mL of tert-butanol, and stirred at room temperature for 2 days. TLC analysis was then performed. R f (09) = 0.30, R f (10) = 0.20, V EA : V PE The ratio was 1:2, and the reaction was almost complete. The reaction was quenched with water (50 mL), the solvent was removed by concentration under reduced pressure, and the mixture was extracted with ethyl acetate (10 mL × 3). The organic phases were combined and washed successively with saturated sodium bicarbonate solution (20 mL × 1) and saturated sodium chloride solution (10 mL × 2). The mixture was dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and the residue was subjected to silica gel rapid column chromatography (…). V EA : V PE =1:5~1:2) Separation yielded a white solid 10 (1.15E+9 Bq, radiochemical yield 65%): 1 H NMR (500MHz, ) δ 5.96 (dt, J = 10.4, 2.6 Hz, 1H), 5.83 (dd, J= 2.5, 1.4 Hz, 1H), 5.82– 5.69 (m, 3H), 5.55 (dd, J = 9.9, 2.6 Hz, 1H), 5.46 (td, J = 11.3, 5.6 Hz,1H), 4.99 (d, J = 11.0 Hz, 1H), 4.83 (d, J = 3.8 Hz, 1H), 4.80 – 4.68 (m, 2H),4.68 (s, 1H), 3.96 (s, 1H), 3.87 (dd, J = 9.3, 6.2 Hz, 2H), 3.57 (ddd, J =11.4, 8.8, 4.7 Hz, 1H), 3.49 (s, 3H), 3.43 – 3.39 (m, 1H), 3.32 (d, J = 9.9Hz, 1H), 3.17 (t, J = 9.1 Hz, 1H), 2.52 (d, J = 7.0 Hz, 1H), 2.34 – 2.24 (m,4H), 2.03 – 1.99 (m, 1H), 1.94 (dd, J = 2.5, 1.4 Hz, 3H), 1.79 (t, J = 11.0 Hz,4H), 1.67 (t, J = 13.4 Hz, 3H), 1.58 – 1.52 (m, 4H), 1.51 – 1.49 (m, 3H), 1.27(d, J = 6.3 Hz, 4H), 1.25 (t, J = 3.6 Hz, 2H), 1.16 (d, J = 7.0 Hz, 3H), 0.93(d, J = 7.1 Hz, 3H), 0.87 (d, J = 11.5 Hz, 1H). ESI-MS(+) m / z :770[M+H] + 。

[0104]

[0105] Compound 10 (1.15E+9 Bq), triphenylphosphine rhodium chloride catalyst (2.8 mg, 0.003 mmol), and acetone (5 mL) were mixed under nitrogen protection and at room temperature. The mixture was sealed and purged with nitrogen five times. Hydrogen was then introduced to 3.5 bar, and the mixture was heated to 50 °C and stirred for 6 h. The mixture was cooled to 25–30 °C, and hydrogen was removed by nitrogen purging. Activated carbon (80 mg, 5% w / w) was added, and the mixture was stirred at 25–30 °C for 1 h. The reaction solution was filtered through diatomaceous earth, and the filter cake was washed with ethyl acetate (50 mL). The filtrates were combined and dissolved under reduced pressure. The residue was subjected to rapid column chromatography (…). V EA : V PE =1:5~1:2) to separate white solid 11 (1.14E+9 Bq, radiochemical yield 99%): 1 H NMR (500 MHz, CDCl3) δ 5.95 (dt, J = 10.3, 2.5 Hz, 1H), 5.83 (dd, J =2.5, 1.5 Hz, 1H), 5.81 – 5.69 (m, 2H), 5.43 (tt, J = 11.1, 4.9 Hz, 1H), 4.97(dd, J = 10.3, 2.7 Hz, 1H), 4.83 (d, J = 3.7 Hz, 1H), 4.80 – 4.68 (m, 2H), 4.67(s, 1H), 3.96 (s, 1H), 3.87 (dq, J = 9.4, 6.2 Hz, 1H), 3.70 – 3.61 (m, 1H), 3.58 (ddd, J = 11.5, 8.9, 4.7 Hz, 1H), 3.48 (s, 3H), 3.40 (t, J = 2.4 Hz, 1H), 3.17 (t, J = 9.1 Hz, 1H), 3.07 (d, J = 7.7 Hz, 1H), 2.52 (ddd, J = 9.3, 7.0, 2.5Hz, 1H), 2.39 – 2.20 (m, 4H), 1.99 – 1.95 (m, 1H), 1.94 (dd, J = 2.5, 1.4 Hz, 3H), 1.79 (d, J= 12.7 Hz, 3H), 1.69 – 1.55 (m, 5H), 1.55 – 1.51 (m, 3H), 1.50 (s, 4H), 1.44 (t, J = 11.2 Hz, 2H), 1.37 (t, J = 11.7 Hz, 1H), 1.27 (d, J = 6.2Hz, 4H), 1.22 (dd, J = 22.3, 10.2 Hz, 3H), 1.16 (d, J = 6.9 Hz, 3H), 0.87 –0.81 (m, 1H), 0.79 (d, J = 5.1 Hz, 3H). ESI-MS(+) m / z :772[M+H] + .

[0106] HPLC conditions: HPLC retention time of white solid (11) t The time was 13.185 min. HPLC chromatographic conditions: Diamonsil C18 column (5 µm, 4.6 mm × 250 mm, Dima Corporation, USA); flow rate 1.00 mL / min; wavelength 254 nm; column temperature 30 ℃; injection volume 10 µL; gradient elution (min / %B) control: 0 / 90, 1 / 90, 15 / 100, 20 / 100; A was an aqueous solution containing 0.05% formic acid, and B was acetonitrile.

[0107] The conventional analytical methods for quality indicators of isotope-labeled compounds were used (Yu Zhiyang, Yang Zhengmin, Li Mengxue, et al. Radioisotopes). 14 Synthesis of C-labeled remdesivir [J]. Nuclear Chemistry and Radiochemistry, 2025, 47(1): 93-102), and the quality indicators of the target compound (11) were detected. The detection results showed that the quality indicators of the white solid (11) were as follows: total activity was 1.14E+9 Bq; specific activity was 1.11E+10 Bq / mmol; chemical purity and radiochemical purity were both greater than 98%.

[0108] Based on the above and in conjunction with a comparison with the literature, the white solid (11) was confirmed to be the target compound [13-³H]seramycin.

[0109] Those skilled in the art should understand that the technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments have been described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0110] The above embodiments are merely illustrative of several implementation methods of this application, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of this application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the protection scope of this application. Therefore, the protection scope of this application should be determined by the appended claims.

Claims

1. A radioactive tritium-labeled selamectin, characterized in that, The structural formula is as follows: 。 2. A method for preparing radioactive tritium-labeled selamectin, characterized in that, include: S1: Doramectin is subjected to hydrolysis and desaccharification reaction to obtain doramectin aglycone; S2: The product protected by 5-hydroxyl silanization was prepared by reacting doramectin aglycone with a silicon protecting agent. S3: Oxidation of the 5-hydroxyl group at the silanization protected product with an oxidizing agent to obtain the 13-keto-coated product; S4: At a certain temperature, the 13-keto-coated product is dissolved in a solvent and reduced with sodium borotrithide to obtain a 5-hydroxysilyl-protected and 13-tritium-labeled doramectin aglycone. S5: The doramectin aglycone protected by 5-hydroxy silylation and labeled with 13-tritium is prepared by glycosylation reaction. The reaction system for the glycosylation reaction must be strictly anhydrous. S6: Doramectin with 5,4"-hydroxyl group silanization protection and 13-tritium labeling is dissolved in a solvent and a desilication protection reagent is added to carry out a desilication protection reaction to obtain 13-tritium labeling doramectin; S7: Under acidic conditions, acid is added dropwise to a protic solvent to obtain an acidic solvent. 13-tritium-labeled doramectin is added to the acidic solvent to carry out a selective desaccharification reaction to obtain a 13-tritium-labeled monosaccharide doramectin product. S8: Selective oxidation of the 13-position tritium-labeled monosaccharide doramectin product with an oxidant yields a ketogenic product, wherein the selective oxidation site is the 5-position hydroxyl group; S9: An amination reagent is added to the 5-keto-ester product to prepare C. 22 -C 23 Olefinified and 13-tritium-labeled selamectin product; S10: Using a catalyst for C 22 -C 23 C in olefinated and 13-tritium-labeled selamectin products 22 -C 23 Selective reduction of the double bond yielded a 13-tritium-labeled selamectin product.

3. The method for preparing radioactive tritium-labeled selamectin according to claim 2, characterized in that, In step S1, under inert gas protection and at room temperature, an organic solvent and water are mixed to obtain a solvent solution. After cooling the solvent solution, acid is added to adjust it to acidic conditions. Doramectin is then added to the solvent solution and the temperature is raised to room temperature to carry out a hydrolysis reaction.

4. The method for preparing radioactive tritium-labeled selamectin according to claim 2, characterized in that, In step S2, under inert gas protection and at room temperature, a silicon protective reagent is added to a dichloromethane solution containing doramectin aglycone and imidazole to carry out the reaction.

5. The method for preparing radioactive tritium-labeled selamectin according to claim 2, characterized in that, In step S3, under inert gas protection and at room temperature, the 5-hydroxyl silylation protection product, sodium bicarbonate and dichloromethane are mixed to obtain a mixed solution and cooled to 0 °C. An oxidant is added to the mixed solution and reacted for a period of time before the temperature is raised to room temperature to continue the reaction.

6. The method for preparing radioactive tritium-labeled selamectin according to claim 2, characterized in that, In step S5, under inert gas protection and room temperature, doramectin aglycone protected by 5-hydroxyl silylation and labeled with 13-tritium and dried 2-{[(2 S 4 S 5 S 6 S )-5-{[dimethyl(2-methylprop-2-yl)silyl]oxy}-4-methoxy-6-methyl-3,4,5,6-tetrahydro-2 H -pyran-2-yl]oxy}-4-methoxy-6-methyl-3,4,5,6-tetrahydro-2 H [-pyran-2-yl]thio}pyridine was dissolved in dichloromethane, and powdered 4Å molecular sieve was added. After reacting at room temperature, dry silver trifluoromethanesulfonate was added to continue the reaction.

7. The method for preparing radioactive tritium-labeled selamectin according to claim 2, characterized in that, In step S6, under inert gas and room temperature protection, doramectin with 5,4"-hydroxyl silanization protection and 13-tritium labeling is dissolved in a solvent, and a mixture of desilication protection reagent, pyridine and solvent is added to carry out the reaction, and desilication protection reagent and pyridine are added during the reaction.

8. The method for preparing radioactive tritium-labeled selamectin according to claim 2, characterized in that, In step S7, under an inert gas and room temperature environment, acid is dropped into a protic solvent pre-cooled to 0 °C to obtain an acidic solvent. 13-tritium-labeled doramectin is added to the acidic solvent and the temperature is raised to room temperature to carry out a selective desaccharification reaction.

9. The method for preparing radioactive tritium-labeled selamectin according to claim 2, characterized in that, In step S9, the 5-keto-oxidized product and the amination reagent are mixed and dissolved in a solvent under an inert gas and at room temperature, and then the reaction is carried out.

10. The method for preparing radioactive tritium-labeled selamectin according to claim 2, characterized in that, In step S10, under inert gas protection and at room temperature, C is... 22 -C 23 The olefin-modified and 13-tritium-labeled selamectin product, catalyst, and organic solvent were mixed and sealed. Hydrogen was introduced to replace the inert gas, and the pressure of the reaction system was increased to more than one standard atmosphere. After reacting at the reaction temperature for a period of time, nitrogen was introduced for purging, and activated carbon was added for post-treatment.

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