Tripterygium wilfordii diagnosis and treatment prodrug, precursor compound as well as preparation method and application thereof

Abstract

The invention provides a tripterygium wilfordii diagnosis and treatment prodrug, a precursor compound as well as a preparation method and application thereof, and belongs to the technical field of biological medicines. According to the tripterygium wilfordii diagnosis and treatment prodrug, hemicyanine fluorescent dye is coupled with a neuroinflammation treatment drug, i.e., cellatrol, and diphenyl phosphinamide is introduced as a response switch, so that the tripterygium wilfordii diagnosis and treatment prodrug can detect ONOO <-> in a neuroinflammation microenvironment and realize targeted release of the cellatrol. The tripterygium wilfordii prodrug for diagnosis and treatment can effectively consume ONOO <-> and realize near-infrared fluorescence imaging in ischemic brain injury, the bioavailability of cellatrol is improved, and the toxicity of the drug is reduced, so that synchronous imaging and treatment are realized. Prodrug with structure as shown in general formula I: # imgabs0 #

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to a triptolide prodrug, a precursor compound thereof, and a preparation method and application thereof. Background Art

[0002] With the acceleration of the global population aging process, the incidence of stroke shows an upward trend. It is predicted that by 2030, the number of stroke cases will increase to 23 million, and the number of deaths is expected to reach 7.8 million. According to a report by the American Heart Association (AHA), approximately 87% of strokes are ischemic strokes (IS), which is the most common type of stroke currently. However, there is still a lack of rapid and accurate diagnostic methods clinically, resulting in many patients not being able to obtain an accurate diagnosis in a timely manner after the onset, thus missing the best treatment opportunity (usually the thrombolysis time window within 3 - 4.5 hours after the onset). Accurately identifying early ischemic lesions and evaluating the severity of the condition are important challenges faced in current clinical diagnosis and treatment.

[0003] In the treatment of ischemic stroke, although thrombolytic drugs (such as alteplase) and mechanical thrombectomy and other thrombolytic treatment methods can effectively restore blood supply to the penumbra of cerebral ischemia, the reperfusion process (CIR) may trigger secondary pathophysiological damage, including oxidative stress and inflammatory responses, thereby aggravating brain tissue damage and neurological dysfunction. Therefore, if rapid diagnosis can be achieved during the CIR process and combined with drug treatment, it will significantly improve the treatment effect of ischemic stroke.

[0004] The ischemia / reperfusion process will lead to an increase in the level of oxidative stress, generating a large amount of reactive oxygen species (ROS), especially substances such as peroxynitrite (ONOO - ) and hypochlorous acid (HOCl). These reactive oxygen species will directly damage the DNA, proteins, and lipid membranes of neurons, resulting in neuron death. It is worth noting that peroxynitrite anion (ONOO - ) as a highly toxic reactive oxygen and nitrogen species is generated in large amounts in ischemic brain injury and forms a positive feedback with the inflammatory microenvironment, further exacerbating cell damage and inflammatory responses. Therefore, how to effectively utilize and scavenge ONOO - is of great significance for promoting nerve function recovery. Developing a diagnostic and therapeutic agent based on the activation response of the stroke inflammatory microenvironment will contribute to the rapid diagnosis and precise treatment of ischemic stroke.

[0005] Natural products, due to their complex and diverse chemical structures and remarkable biological activities, have become an important source for new drug research and development. Celastrol is an important bioactive natural product extracted from Tripterygium wilfordii Hook. f., and it shows great potential and clinical value in clinics due to its strong anti-inflammatory and antioxidant properties. Research has shown that celastrol has a therapeutic effect on ischemic stroke through mechanisms such as anti-inflammation, antioxidant, and neuroprotection. However, due to its poor water solubility, low bioavailability, and the potential for adverse reactions such as liver and kidney damage caused by long-term use, its clinical application is limited. It is worth noting that the prodrug strategy can improve the physicochemical properties of drugs, achieve targeted release, thereby enhancing the therapeutic effect and reducing toxic side effects. As a typical near-infrared fluorescent dye, hemicyanine dyes have advantages such as high tissue penetration depth, low light damage, and low background autofluorescence interference, and can be used for fluorescence imaging of biological tissues in vivo. Therefore, adopting the prodrug strategy and using the endogenous ONOO in stroke - as a trigger to develop highly selective, highly efficient, low-toxic diagnostic and therapeutic prodrugs to achieve selective release of active drugs in the brain is of great significance for the efficient treatment of ischemic stroke. Summary of the Invention

[0006] In order to solve the defects existing in the prior art, the purpose of the present invention is to provide a Tripterygium wilfordii diagnostic and therapeutic prodrug, a precursor compound thereof, and a preparation method and application thereof.

[0007] In the first aspect of the present invention, a precursor compound of a Tripterygium wilfordii diagnostic and therapeutic prodrug is provided, and the precursor compound has the structure shown in the following formula:

[0008]

[0009] In the second aspect of the present invention, a preparation method of the above precursor compound is provided, and the preparation method includes the following steps:

[0010] S1. Compound 1 and 1,1,2-trimethyl-1H-benzo[e]indole are reacted under the condition of sodium acetate to obtain compound 3, and compound 3 undergoes a nucleophilic reaction with 3,5-dihydroxybenzyl alcohol to obtain compound 4; compound 4 is a hemicyanine fluorescent dye.

[0011] The synthetic route of step S1 is shown in the following formula:

[0012]

[0013] S2. p-Aminobenzyl alcohol protects the hydroxyl group through tert-butyldimethylchlorosilane to obtain compound 6, compound 6 reacts with diphenylphosphinous chloride in an organic base environment to obtain compound 7, compound 7 removes the hydroxyl protection under the condition of tetrabutylammonium fluoride to obtain compound 8, and compound 8 is brominated with phosphorus tribromide to obtain compound 9.

[0014] The synthesis route of step S2 is shown as follows:

[0015]

[0016] S3. Compound 4 and compound 9 react under alkaline conditions to form compound 10, and compound 10 is the precursor compound.

[0017] The synthesis route of step S3 is shown as follows:

[0018]

[0019] In some embodiments of the present invention, the preparation method includes the following steps:

[0020] S100. Dissolve N-[(3-(phenylaminomethylene)-2-chloro-1-cyclohexen-1-yl)methylene]aniline hydrochloride, 1,1,2-trimethyl-1H-benzo[e]indole and sodium acetate in absolute ethanol, and reflux and react under a nitrogen atmosphere to obtain compound 3;

[0021] S200. Dissolve compound 3, 3,5-dihydroxybenzyl alcohol and KHCO3 in anhydrous DMF, and react under a nitrogen atmosphere to obtain compound 4;

[0022] S300. Dissolve 4-aminobenzyl alcohol and imidazole in anhydrous dichloromethane, add tert-butyldimethylchlorosilane at 0 °C, and react to obtain compound 6;

[0023] S400. Dissolve compound 6 in anhydrous dichloromethane, add N,N-diisopropylethylamine, protect with nitrogen, and slowly add diphenylphosphinous chloride at 0 °C, and react to obtain compound 7;

[0024] S500. Dissolve compound 7 in anhydrous tetrahydrofuran, protect with nitrogen, and slowly add tetrabutylammonium fluoride at 0 °C, and react to obtain compound 8;

[0025] S600. Dissolve compound 8 in anhydrous dichloromethane, protect with nitrogen, and slowly add PBr3 at 0 °C, and react to obtain compound 9;

[0026] S700. Dissolve compound 4, compound 9, K2CO3 and KI in anhydrous DMF, protect with nitrogen, stir and react at 40 - 50 °C overnight to obtain compound 10.

[0027] In the third aspect of the present invention, there is provided the use of the above-mentioned precursor compound in the preparation of a prodrug for treating and diagnosing Tripterygium wilfordii.

[0028] In the fourth aspect of the present invention, there is provided a prodrug for treating and diagnosing Tripterygium wilfordii, and the structural formula of the prodrug for treating and diagnosing Tripterygium wilfordii is shown as follows:

[0029]

[0030] In some embodiments of the present invention, a method for preparing a prodrug of Tripterygium wilfordii for diagnosis and treatment is provided. The preparation method is as follows: Compound 10 and celastrol are subjected to a Mitsunobu reaction to obtain Compound I, and Compound I is the prodrug of Tripterygium wilfordii for diagnosis and treatment; wherein, Compound 10 is the precursor compound described in Claim 1.

[0031] In some embodiments of the present invention, the above-mentioned method for preparing the prodrug of Tripterygium wilfordii for diagnosis and treatment is specifically as follows: Triphenylphosphine and diisopropyl azodicarboxylate are dissolved in anhydrous tetrahydrofuran, protected by nitrogen, stirred at room temperature for 30 - 60 min, then Compound 10 is added, and after stirring at room temperature for 30 - 60 min, celastrol is added to obtain the prodrug of Tripterygium wilfordii for diagnosis and treatment.

[0032] In some embodiments of the present invention, in the above-mentioned method for preparing the prodrug of Tripterygium wilfordii for diagnosis and treatment, the molar ratio of triphenylphosphine, diisopropyl azodicarboxylate, Compound 10, and celastrol is 3:3:1:2.

[0033] In the fifth aspect of the present invention, an application of the above-mentioned prodrug of Tripterygium wilfordii for diagnosis and treatment in preparing a reagent for diagnosing and / or treating a disease is provided, and the disease is ischemic stroke.

[0034] In some embodiments of the present invention, the disease is neuroinflammation caused by ischemic stroke.

[0035] The present invention has the following advantages compared with the prior art:

[0036] (1) By coupling Compound 9 with a hemicyanine fluorescent dye (Compound 4) and celastrol, and introducing diphenylphosphinic amide as a response switch, the present invention realizes the response to ONOO⁻ and turns on near-infrared fluorescence imaging, thereby achieving the diagnostic effect on ischemic brain injury and the microenvironment of neuroinflammation.

[0037] (2) During the response to ONOO⁻ of the prodrug compound of Tripterygium wilfordii provided by the present invention, not only the directional release of celastrol is realized, but also the excessive ONOO⁻ is effectively consumed, thereby improving the bioavailability of the drug and reducing the treatment toxicity, achieving an efficient treatment effect on ischemic stroke and neuroinflammation.

[0038] (3) The cell viability of the prodrug of Tripterygium wilfordii provided by the present invention in PC12 cells is significantly higher than that of unmodified celastrol, which proves that its modification significantly reduces the cytotoxicity. Further in vitro experiments show that in the cell inflammation model induced by OGD / R, the prodrug of Tripterygium wilfordii of the present invention can significantly improve the cell survival rate, reflecting its excellent anti-apoptotic effect.

[0039] (4) The compounds of the present invention can effectively target mitochondria and lysosomes in PC12 cells and have good dual-targeting ability. This precise organelle localization helps to improve the drug release efficiency. Description of the Drawings

[0040] The technical solutions of the present invention will be further specifically described below through examples in combination with the drawings. The following description of the embodiments of the present invention with reference to the drawings is intended to explain the overall inventive concept of the present invention and should not be construed as a limitation to the present invention. In the drawings:

[0041] Figure 1 is the ultraviolet fluorescence spectrum diagram of the tripterygium glycosides prodrug of the present invention before and after adding ONOO- solution in a DMSO / PBS (v:v = 1:1) mixed solution and the ultraviolet fluorescence spectrum diagram of the fluorophore (Compound 4);

[0042] Figure 2 is the tripterygium glycosides prodrug of the present invention for ONOO - Fluorescence emission spectrum diagram of the response ability;

[0043] Figure 3 is the tripterygium glycosides prodrug of the present invention for ONOO - Specific response ability test result diagram, the abscissa is various substances in the physiological environment (including various anions, cations, oxidizing substances, reducing substances) and ONOO - , and the ordinate is the fluorescence intensity;

[0044] Figure 4 is the test result diagram of the neuroprotective effect of the tripterygium glycosides prodrug of the present invention at the cellular level;

[0045] Figure 5 is the test result diagram of the selective imaging of the OGD / R model of the tripterygium glycosides prodrug of the present invention;

[0046] Figure 6 is the test result diagram of the organelle targeting and localization ability of the tripterygium glycosides prodrug of the present invention;

[0047] Figure 7 is the test result diagram of the fluorescence imaging of the tripterygium glycosides prodrug of the present invention in the cerebral ischemia-reperfusion model mice;

[0048] Figure 8 is the test result diagram of the in vivo cerebral ischemia treatment effect of the tripterygium glycosides prodrug of the present invention. Detailed Embodiments

[0049] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the following will, in conjunction with the accompanying drawings in the embodiments of this application, clearly and completely describe the technical solutions in the embodiments of this application. Obviously, the described embodiments are some, rather than all, of the embodiments of this application. Based on the embodiments in this application, all other embodiments obtained by those of ordinary skill in the art without making creative efforts fall within the scope of protection of this application.

[0050] For those technical or conditions not specified in the embodiments, they shall be in accordance with the technologies or conditions described in the literature in this field or in accordance with the product specifications. For reagents or instruments whose manufacturers are not specified, they are all conventional products that can be obtained through regular channels.

[0051] For those technical or conditions not specified in the embodiments, they shall be in accordance with the technologies or conditions described in the literature in this field or in accordance with the product specifications. For reagents or instruments whose manufacturers are not specified, they are all conventional products that can be obtained through regular channels.

[0052] Unless otherwise specified, the experimental methods in the following embodiments are all conventional methods. Unless otherwise specified, the test materials used in the following embodiments are all commercially available products.

[0053] Example 1: Preparation of Compound 3

[0054] Compound 3 is: 2-((E)-2-(E)2-chloro-3-((Z)-2-(1,1,3-trimethyl-1,3-dihydro-2H-benzo[e]indol-2-ylidene)ethylidene)cyclohex-1-en-1-yl)vinyl)-1,1,3-trimethyl-1H-benzo[e]indol-3-ium.

[0055] Dissolve N-[(3-(phenylaminomethylene)-2-chloro-1-cyclohexen-1-yl)methylene]aniline hydrochloride (2 g, 5.69 mmol), 1,1,2-trimethyl-1H-benzo[e]indole (3.9 g, 11.11 mmol) and CH3COONa (1.8 g, 21.94 mmol) in anhydrous ethanol (15 mL), and reflux the reaction for 1 h under a nitrogen atmosphere. After monitoring the reaction by TLC until completion, perform suction filtration, and wash the filter cake with ethyl acetate. Vacuum dry the filter cake to obtain Compound 3 with a yield of 80%.

[0056] Example 2: Preparation of Compound 4 (hemicyanine fluorescent dye)

[0057] Compound 4 is: (E)-2-(2-(6-hydroxy-7-(hydroxymethyl)-2,3-dihydro-1H-xanthin-4-yl)vinyl)-1,1,3-trimethyl-1H-benzo[e]indol-3-ium.

[0058] Compound 3 (2.5 g, 3.52 mmol), 3,5-dihydroxybenzyl alcohol (2.46 g, 17.55 mmol) and KHCO3 (1.4 g, 13.98 mmol) were dissolved in anhydrous DMF (15 mL). The reaction was carried out at 60 °C for 2.5 h under a nitrogen atmosphere. After monitoring the reaction by TLC until completion, the reaction solution was dropped into water to precipitate a solid, which was filtered by suction, and the filter cake was dried in vacuo. Compound 4 was obtained by column chromatography purification (MeOH:DCM = 1:25, v / v) with a yield of 50%.

[0059] Example 3: Preparation of Compound 6

[0060] Compound 6 is: 4-(((tert-butyldimethylsilyl)oxy)methyl)aniline.

[0061] 4-Aminobenzyl alcohol (5 g, 40.60 mmol) and imidazole (3.04 g, 44.65 mmol) were dissolved in anhydrous dichloromethane (30 mL). tert-Butyldimethylchlorosilane (6.73 g, 44.65 mmol) was added at 0 °C. After 5 min, the temperature was restored to room temperature, and the reaction was continued with stirring for 20 min. After monitoring the reaction by TLC until completion, the reaction mixture was extracted with dichloromethane and washed with saturated brine. The organic layer was concentrated and purified by column chromatography (EA:PE = 1:5, v / v) to obtain Compound 6 with a yield of 96%.

[0062] Example 4: Preparation of Compound 7

[0063] Compound 7 is: N-(4-(((tert-butyldimethylsilyl)oxy)methyl)phenyl)-P,P-diphenylphosphonamidite.

[0064] Compound 6 (3.5 g, 14.74 mmol) was dissolved in anhydrous dichloromethane (15 mL). N,N-Diisopropylethylamine (2.1 mL, 29.48 mmol) was added, and under nitrogen protection, diphenylphosphinous chloride (4.2 mL, 22.11 mmol) was slowly added at 0 °C. After stirring for 5 min, the reaction was restored to room temperature and continued for 20 min. After monitoring the reaction by TLC until completion, the reaction mixture was extracted with dichloromethane and washed with saturated brine. The organic layer was concentrated and purified by column chromatography (MeOH:DCM = 1:25, v / v) to obtain Compound 7 with a yield of 92%.

[0065] Example 5: Preparation of Compound 8

[0066] Compound 8 is: N-(4-(hydroxymethyl)phenyl)-P,P-diphenylphosphonamidite.

[0067] Compound 7 (1 g, 2.29 mmol) was dissolved in anhydrous tetrahydrofuran (10 mL). Under nitrogen protection, tetrabutylammonium fluoride (1 M) (3.4 mL, 3.43 mmol) was slowly added at 0 °C. After stirring for 5 min, the reaction was warmed to room temperature and reacted for 1 h. After the reaction was monitored by TLC and completed, it was extracted with dichloromethane and washed with saturated brine. The organic layer was concentrated and purified by column chromatography (MeOH:DCM = 1:15, v / v) to obtain compound 8 with a yield of 98%.

[0068] The spectral data of compound 8 are as follows: 1 H NMR (500 MHz, DMSO-d6) δ 8.22 (s, 1H, NH), 7.83 (m, 4H, ArH), 7.64 - 7.56 (m, 2H, ArH), 7.53 (m, 4H, ArH), 7.12 - 7.07 (m, 4H, ArH), 5.07 (s, 1H, OH), 4.34 (s, 2H, CH2); 13 C NMR (101 MHz, DMSO) δ 143.5, 137.3, 136.4, 135.1, 134.5, 134.5, 134.4, 134.3, 131.4, 131.3, 130.0, 120.8, 120.7, 65.4; HRMS (ESI) m / z calcd for C 19 H 19 NO2P[M + H] + , 324.1153; found, 324.1140.

[0069] Example 6: Preparation of compound 10 (prodrug compound)

[0070] Compound 10 is: (E)-2-(2-(6-((4-((diphenylphosphoryl)amino)benzyl)oxy)-7-(hydroxymethyl)-2,3-dihydro-1H-xanthin-4-yl)vinyl)-1,1,3-trimethyl-1H-benzo[e]indol-3-ium.

[0071] Compound 8 (1 g, 3.09 mmol) was dissolved in anhydrous dichloromethane (10 mL). Under nitrogen protection, PBr3 (0.9 mL, 9.28 mmol) was slowly added at 0 °C and stirred for 10 min. After the reaction was monitored by TLC and completed, it was extracted with dichloromethane and washed with saturated brine. The organic layer was concentrated to obtain compound 9 with a yield of 75%;

[0072] Compound 4 (200 mg, 0.34 mmol), compound 9 (783 mg, 2.03 mmol), K2CO3 (233 mg, 1.69 mmol) and KI (561 mg, 3.38 mmol) were dissolved in anhydrous DMF (15 mL), protected by nitrogen, and stirred at 45 °C overnight. After monitoring the reaction by TLC and completion of the reaction, it was extracted with dichloromethane and washed with saturated brine. The organic layer was concentrated and purified by column chromatography (MeOH:DCM = 1:25, v / v) to obtain compound 10 with a yield of 42%.

[0073] The spectral data of compound 10 are as follows: 1 H NMR (400 MHz, DMSO-d6) δ 8.58 (d, J = 15.1 Hz, 1H, ArH), 8.42 - 8.22 (m, 2H, ArH), 8.18 - 8.07 (m, 2H, ArH), 7.92 - 7.85 (m, 1H, ArH), 7.77 - 7.65 (m, 6H, ArH), 7.52 - 7.40 (m, 8H, ArH), 7.23 (d, J = 8.0 Hz, 2H, ArH), 7.05 (d, J = 8.1 Hz, 2H, ArH), 6.98 (s, 1H, NH), 6.51 (d, J = 15.3 Hz, 1H, CH), 5.52 - 5.36 (m, 1H, CH), 5.02 (d, J = 17.4 Hz, 2H, CH2), 4.76 - 4.49 (m, 2H, CH2), 3.95 (d, J = 7.9 Hz, 3H, CH3), 2.78 - 2.51 (m, 4H, 2CH2), 1.93 (d, J = 3.1 Hz, 6H, 2CH3), 1.81 - 1.67 (m, 2H, CH2); 13 C NMR (101 MHz, DMSO-d6) δ 179.4, 161.4, 160.2, 154.5, 144.1, 142.8, 141.7, 140.4, 136.0, 133.9, 132.7, 132.6, 132.5, 132.2, 132.1, 131.1, 130.5, 130.2, 129.9, 129.3, 129.2, 128.6, 128.5, 127.5, 127.0, 126.5, 118.6, 118.6, 113.9, 113.4, 113.0, 112.4, 104.8, 100.8, 70.5, 60.4, 52.4, 33.6, 29.0, 27.4, 24.0, 23.0; HRMS (ESI) m / z calcd for C 50 H 46 N2O4P + [M + H] +,769.3190; found,769.3185.

[0074] Example 7: Preparation of the drug before Tripterygium wilfordii diagnosis and treatment (Compound I)

[0075] Compound I is: 2-((E)-2-((4-(diphenylphosphorylamino)benzyl)benzyl)benzyl)benzyl)-7-(((2R,4aS,4aS,6aS,6aS,12bR,12bR,14aS,14aS,14aS,14aS,14aS,6aS,6aS,12bR,14aS,14aS,14aS,14aS)-10-hydroxy-2,4a,6a,6a,6a-2,6a-hexamethyl-11-oxo-1,2,3,4,4,4,4a,5,6,6a,6,6a,11,12b,13,14,14,14,14a,14-octadecahydro-1H-anthracen-4-yl)vinyl)-1,1,3-trimethyl-1H-benzo[e]indol-3-ium.

[0076] Dissolve triphenylphosphine (87 mg, 0.33 mmol) and diisopropyl azodicarboxylate (0.68 mL, 0.33 mmol) in anhydrous tetrahydrofuran (8 mL), protect with nitrogen, stir at room temperature for 30 min, then add Compound 10 (100 mg, 0.11 mmol), stir at room temperature for 30 min, then add celastrol (100 mg, 0.22 mmol), and continue the reaction for 2 h. After monitoring the reaction by TLC and finishing, extract with dichloromethane, wash with saturated brine, concentrate the organic layer, and purify by column chromatography (MeOH:DCM = 1:30 - 1:15, v / v) to obtain Compound I with a yield of 45%.

[0077] The spectral data of Compound I are as follows: 11H NMR (400 MHz, DMSO-d6) δ 9.54 (s, 1H, OH), 8.65 (d, J = 14.9 Hz, 1H, ArH), 8.46 - 8.34 (m, 2H, ArH), 8.27 - 8.15 (m, 2H, ArH), 8.07 - 7.95 (m, 2H, ArH), 7.91 - 7.72 (m, 6H, ArH), 7.69 - 7.43 (m, 8H, ArH), 7.35 - 7.25 (m, 2H, ArH), 7.15 - 7.07 (m, 2H, ArH), 7.06 - 6.85 (m, 1H, ArH), 6.61 (d, J = 15.3 Hz, 1H, CH), 6.18 (d, J = 9.9 Hz, 1H, NH), 6.01 - 5.67 (m, 2H, CH2), 5.38 - 5.18 (m, 1H, CH), 5.16 - 5.04 (m, 2H, CH2), 4.86 - 4.74 (m, 2H, CH2), 4.03 (d, J = 7.2 Hz, 3H, NCH3), 3.62 - 3.42 (m, 2H, CH2), 2.89 (s, 1H, CH2), 2.73 (s, 1H, CH2), 2.72 - 2.67 (m, 2H, CH2), 2.00 (s, 6H, 2CH3), 1.93 - 1.79 (m, 4H, 2CH2), 1.72 - 1.56 (m, 2H, CH2), 1.54 - 1.43 (m, 1H, CH), 1.41 - 1.35 (m, 3H, CH3), 1.29 - 1.22 (m, 3H, CH3), 1.20 - 1.16 (m, 6H, 2CH3), 1.07 - 1.01 (m, 3H, CH3), 0.94 (d, J = 6.7 Hz, 2H, CH2), 0.88 - 0.79 (m, 4H, 2CH2), 0.78 - 0.59 (m, 3H, CH3); 1313C NMR (101 MHz, DMSO-d6) δ 179.9, 168.3, 167.8, 163.3, 144.2, 142.8, 142.8, 140.3, 134.7, 133.9, 132.9, 132.6, 132.4, 132.2, 132.1, 132.1, 131.9, 131.1, 130.5, 129.8, 129.5, 129.2, 129.2, 129.1, 127.4, 126.7, 123.1, 118.6, 115.1, 113.1, 109.9, 107.5, 55.4, 53.1, 52.6, 44.9, 44.7, 43.9, 42.5, 38.6, 34.7, 33.9, 33.0, 31.7, 31.4, 30.6, 27.3, 26.9, 26.8, 25.3, 22.6, 22.3, 22.1, 22.1, 22.0, 18.8, 14.4, 10.6; HRMS (ESI) m / z calcd for C 79 H 82 N2O7P + [M + H] + , 1201.5854; found, 1201.5845.

[0078] Example 8: Spectral Properties of the Tripterygium Precursor Drug of the Invention

[0079] The tripterygium precursor drug of the invention and the fluorophore (Compound 4) were respectively dissolved in a DMSO / PBS (v:v = 1:1) mixed solution, and the concentration of the detection solution was 10 μM. The ultraviolet spectral data of the compound of the invention before and after adding the ONOO− solution and the fluorophore (Compound 4) were measured using an ultraviolet spectrophotometer, and the data in the range of 200 - 900 nm were collected. The fluorescence spectral changes of the tripterygium precursor drug of the invention before and after adding the ONOO− solution and the fluorophore (Compound 4) were detected by fluorescence spectroscopy. The fluorescence properties were studied using 721 nm as the excitation wavelength, and the fluorescence emission spectral data in the range of 721 - 900 nm were collected by a fluorescence spectrometer, as shown in Figure 1 .

[0080] Figure 1 are the ultraviolet fluorescence spectra of the tripterygium precursor drug of the invention and the fluorophore (Compound 4) (10 μM) in a DMSO / PBS (v:v = 1:1) mixed solution. Among them, Figure 1 a is the ultraviolet spectrum of Compound I in a DMSO / PBS (v:v = 1:1) mixed solution before and after adding the ONOO− solution; Figure 1 b is the fluorescence spectrum of Compound I in a DMSO / PBS (v:v = 1:1) mixed solution before and after adding the ONOO− solution; Figure 1c is the ultraviolet fluorescence spectrum of fluorophore (4) in a DMSO / PBS (v:v = 1:1) mixed solution.

[0081] The results show ( Figure 1 ): For the prodrug of Tripterygium wilfordii Hook. f. in the present invention in a DMSO / PBS (v:v = 1:1) mixed solution, the maximum ultraviolet absorption peak is at 630 nm ( Figure 1 a), which proves that the prodrug of Tripterygium wilfordii Hook. f. in the present invention can absorb photon energy in the near-infrared light region. The maximum ultraviolet absorption of fluorophore (Compound 4) in a DMSO / PBS (v:v = 1:1) mixed solution is at 721 nm. Under excitation at 721 nm, fluorophore (Compound 4) exhibits near-infrared fluorescence at 762 nm ( Figure 1 c). After adding ONOO⁻ solution (10 μM) to the prodrug of Tripterygium wilfordii Hook. f. in the present invention in a DMSO / PBS (v:v = 1:1) mixed solution, the ultraviolet absorption peak redshifts to 731 nm, and the fluorescence at 762 nm increases instantaneously (10.14 times) ( Figure 1 b), which is consistent with the emission peak of fluorophore (Compound 4). In addition, it shows that the prodrug of Tripterygium wilfordii Hook. f. in the present invention has high sensitivity to ONOO - .

[0082] Example 9: Response ability of the prodrug of Tripterygium wilfordii Hook. f. in the present invention to ONOO - Response ability

[0083] The fluorescence spectroscopy method was used to detect the time responsiveness of the prodrug of Tripterygium wilfordii Hook. f. in the present invention to ONOO - . The specific method is to mix the compound of the present invention with ONOO - (10 μM) and measure the change in fluorescence intensity over time. Figure 2 This is the fluorescence emission spectrum diagram of the response ability of the compound of the present invention to ONOO - ; among them, Figure 2 a is the fluorescence spectrum measured over time of the prodrug of Tripterygium wilfordii Hook. f. (10 μM) in a DMSO / PBS (v:v = 1:1) mixed solution; Figure 2 b is the fluorescence spectrum measured over time of the prodrug of Tripterygium wilfordii Hook. f. (10 μM) in a DMSO / PBS (v:v = 1:1) mixed solution after adding ONOO⁻ (10 μM); the excitation wavelength is 721 nm, and the emission wavelength is 721 - 900 nm.

[0084] The results show ( Figure 2 ), that when ONOO - is added, the generated fluorescence increases with the extension of time, proving the rapid response characteristic of the prodrug of Tripterygium wilfordii Hook. f. in the present invention to ONOO - . The fluorescence activated by the prodrug of Tripterygium wilfordii Hook. f. in the present invention can remain stable for a long time within 3 hFigure 2 b), which proves that the fluorophore released in response to this compound has good fluorescence stability.

[0085] Example 10: Specific response ability test of the tripterygium pre-drug for diagnosis and treatment of the present invention to ONOO - Specific response ability test

[0086] The specific response ability of the tripterygium pre-drug for diagnosis and treatment of the present invention to ONOO was detected by fluorescence spectroscopy. - For analyzing the response selectivity of various substances (including various anions, cations, oxidizing substances, reducing substances) and ONOO in the physiological environment to the tripterygium pre-drug for diagnosis and treatment of the present invention, at room temperature, the tripterygium pre-drug for diagnosis and treatment of the present invention was respectively added to the corresponding bioanalytes (such as Na - 、K + 、K + 、Cu 2+ 、Fe 2+ 、Zn 2+ 、Mn 2+ 、Ca 2+ 、Mg 2+ 、I - 、glutathione (GSH), hydrogen peroxide (H2O2), glucose (Glu), sodium ascorbate (VcNa)) and ONOO - for incubation, and then fluorescence spectroscopy was performed.

[0087] Figure 3 The fluorescence intensity (mean ± standard deviation, n = 3) after incubation treatment for 3 h of the tripterygium pre-drug for diagnosis and treatment of the present invention (10 μM) in DMSO solution with 1 mM corresponding bioanalytes (such as Na + 、K + 、Cu 2+ 、Fe 2+ 、Zn 2+ 、Mn 2+ 、Ca 2+ 、Mg 2+ 、I - 、glutathione (GSH), hydrogen peroxide (H2O2), glucose (Glu), sodium ascorbate (VcNa)) and 10 μM ONOO - is shown.

[0088] The results show that ( Figure 3 ), when different interfering ions were respectively added to the solution of the tripterygium pre-drug for diagnosis and treatment of the present invention, the fluorescence intensity did not show obvious changes; however, when ONOO - was added, the solution showed strong fluorescence, proving that the tripterygium pre-drug for diagnosis and treatment of the present invention has good response specificity to ONOO - .

[0089] Example 11: Test of the neuroprotective effect of the lead drug of Tripterygium wilfordii Hook. f. in the present invention at the cellular level

[0090] First, the cytotoxicity of the lead drug of Tripterygium wilfordii Hook. f. in the present invention was detected by the methyl thiazolyl tetrazolium (MTT) in vitro toxicity experiment. One bottle of PC12 cells (rat adrenal medulla pheochromocytoma differentiated cell line, from the Shanghai Institute of Cell Biology, China), which were in the exponential growth phase and in good condition, was digested and made into a cell suspension with a concentration of 1×10 4 cells / mL. After plating in a 96-well plate, it was incubated at 37°C in the dark for 24 h. Then, the lead drug of Tripterygium wilfordii Hook. f. in the present invention, celastrol, and fluorophore (Compound 4) (0, 0.0078, 0.0156, 0.0313, 0.0625, 0.125, 0.25, 0.5, 1, 2 μM) were added. After continuing to culture for 24 h, MTT was added and reacted for 4 h, then the supernatant was discarded. After adding 100 μL of DMSO, the absorbance value at 490 nm was detected and the cell survival rate was calculated.

[0091] The glycemia-oxygen deprivation / reperfusion (OGD / R) model was used to induce the inflammatory response of PC12 cells. Celastrol was selected as the positive control drug to study the therapeutic effect of the lead drug of Tripterygium wilfordii Hook. f. in the present invention.

[0092] PC12 cells in logarithmic growth phase were inoculated into a 96-well plate at a density of 1×10 4 cells / well / 100 μL and incubated for 24 h. The culture medium was replaced with sugar-free DMEM complete medium and continued to be incubated in an anaerobic incubator (95% N2 + 5% CO2 mixed gas) for 6 h. After 6 h, the cells were reoxygenated, and then the culture medium was replaced with DMEM complete medium. The cells were treated with complete medium containing different concentrations (0, 0.0078, 0.0156, 0.0313, 0.0625, 0.125, 0.25, 0.5, 1, 2 μM) of the lead drug of Tripterygium wilfordii Hook. f. in the present invention, celastrol, and fluorophore (Compound 4) and incubated for 24 h to initiate and maintain reperfusion. After adding MTT and reacting for 4 h, the supernatant was discarded. After adding 100 μL of DMSO, the absorbance value at 490 nm was detected and the cell survival rate was calculated.

[0093] Cell survival rate formula:

[0094] Survival rate (%) = (OD value of the experimental group - OD value of the blank control group) / (OD value of the control group - OD value of the blank control group) × 100%

[0095] Among them, the OD value refers to the optical density value. The OD value of the experimental group is the absorbance value of the treated cells, the OD value of the control group is the absorbance value of the untreated cells, and the OD value of the blank control group is the absorbance value of the culture medium.

[0096] The oxygen-glucose deprivation / reperfusion (OGD / R) model was used to induce an inflammatory response in PC12 cells. Celastrol was selected as a positive control drug to study the anti-apoptotic effect of the tripterygium wilfordii pre-drug of the present invention. PC12 cells in logarithmic growth phase were inoculated in 6-well plates at a density of 5×10 5 cells / well / 1000 μL and incubated for 24 h. The medium was replaced with sugar-free DMEM complete medium and incubated in an anaerobic incubator (95% N2 + 5% CO2 mixed gas) for another 6 h. After 6 h, the cells were reoxygenated, and then the medium was replaced with DMEM complete medium. The cells were treated with complete medium containing different concentrations of the tripterygium wilfordii pre-drug of the present invention (0, 0.25, 0.5 μM) and celastrol (0.25 μM) and incubated for 24 h to initiate reperfusion and maintain it. Flow cytometry was used to detect apoptosis in each group.

[0097] Figure 4 This is the test result graph of the neuroprotective effect of the tripterygium wilfordii pre-drug of the present invention at the cellular level; among them, Figure 4 a shows the survival rates of PC12 cells treated with the tripterygium wilfordii pre-drug of the present invention, fluorophore (Compound 4), and celastrol at different concentrations (0, 0.0078, 0.0156, 0.0313, 0.0625, 0.125, 0.25, 0.5, 1, 2 μM). Figure 4 b shows the survival rates of PC12 cells treated with OGD / R and incubated with the tripterygium wilfordii pre-drug of the present invention, fluorophore (Compound 4), and celastrol at different concentrations (0, 0.0078, 0.0156, 0.0313, 0.0625, 0.125, 0.25, 0.5, 1, 2 μM) for 24 h. Figure 4 c shows the apoptotic flow cytometry analysis of PC12 cells treated with OGD / R and incubated with the tripterygium wilfordii pre-drug of the present invention and celastrol (0.5 μM) at different concentrations (0, 0.25, 0.5 μM) for 24 hours. Figure 4 d shows the quantitative analysis of apoptotic cells in each group. (Mean ± standard deviation, n = 3).

[0098] The results showed ( Figure 4 ) that after treatment with the tripterygium wilfordii pre-drug of the present invention at a dose of up to 1 μM, the cell viability of PC12 cells could reach 80% ( Figure 4 a). After treatment with OGD / R, the cell viability decreased significantly. After incubation with the tripterygium wilfordii pre-drug of the present invention, fluorophore (Compound 4), and celastrol, the tripterygium wilfordii pre-drug of the present invention had a better effect on improving the cell viability of PC12 cells than celastrol and fluorophore (Compound 4) at the same dose (0.125 - 0.75 μM) ( Figure 4b), showing that the pre-treatment drug of Tripterygium wilfordii Hook. f. of the present invention can effectively improve the cell viability after OGD / R injury. Further, the neuroprotective effect of the pre-treatment drug of Tripterygium wilfordii Hook. f. of the present invention on PC12 cells treated with OGD / R was verified by flow cytometry analysis. The results of Annexin V-FITC / propidium iodide (PI) double staining showed that the apoptosis rate of PC12 cells treated with OGD / R was relatively high, and the apoptosis rate was significantly reduced after treatment with the pre-treatment drug of Tripterygium wilfordii Hook. f. of the present invention, and the effect was significantly better than that of celastrol( Figure 4 c, d). It was proved that the pre-treatment drug of Tripterygium wilfordii Hook. f. of the present invention has a good protective effect on OGD / simulated ischemic stroke.

[0099] Example 12: Selective imaging test of the pre-treatment drug of Tripterygium wilfordii Hook. f. of the present invention in OGD / R model

[0100] The selective imaging ability of the pre-treatment drug of Tripterygium wilfordii Hook. f. of the present invention in the OGD / R model was evaluated by confocal fluorescence imaging. The experimental groups were divided as follows: PBS group, the compound group of the present invention, OGD / R group, OGD / R + 0.25 μM compound group of the present invention, OGD / R + 0.5 μM compound group of the present invention. The sugar oxygen deprivation model (OGD / R) was used to induce the inflammatory response of PC12 cells to study the selective imaging effect of the pre-treatment drug of Tripterygium wilfordii Hook. f. of the present invention in the OGD / R model. PC12 cells in logarithmic growth phase were inoculated in a confocal dish at a density of 5×10 4 cells / well / 1000 μL and incubated for 24 h. The culture medium was replaced with sugar-free DMEM complete medium and incubated in an anaerobic incubator (95% N2 + 5% CO2 mixed gas) for another 6 h. After 6 h, the cells were reoxygenated, and then the culture medium was replaced with DMEM complete medium. The cells were treated with complete medium containing different concentrations (0, 0.25, 0.5 μM) of the pre-treatment drug of Tripterygium wilfordii Hook. f. of the present invention and incubated for 2 h to initiate reperfusion and maintain it. The PBS group and the compound group of the present invention without OGD / R treatment were used as controls. After washing 3 times with PBS, the nuclei were stained with Hoechst 33342 and then confocal microscopy was performed. Figure 5 is the result diagram of the selective imaging test of the pre-treatment drug of Tripterygium wilfordii Hook. f. of the present invention in the OGD / R model. Among them, Figure 5 a shows the selective imaging of the pre-treatment drug of Tripterygium wilfordii Hook. f. of the present invention after OGD / R treatment of PC12 cells, scale bar = 50 μm, Figure 5 b is Figure 5 the quantitative analysis of a.

[0101] The results showed ( Figure 5 ), that the pre-treatment drug of Tripterygium wilfordii Hook. f. of the present invention itself did not produce fluorescence, but after OGD / R treatment, its fluorescence signal was significantly enhanced and showed an obvious concentration dependence ( Figure 5 a, b). It was proved that the pre-treatment drug of Tripterygium wilfordii Hook. f. of the present invention has a relatively wide cell selective imaging ability.

[0102] Example 12: Test on the organelle-targeting ability of the tripterygium pre-drug for diagnosis and treatment of the present invention

[0103] The co-localization experiment was used to evaluate the organelle-targeting ability of the tripterygium pre-drug for diagnosis and treatment of the present invention. PC12 cells were seeded into confocal dishes. After incubation for a period of time, the fresh complete medium was changed, and the cells were stimulated with LPS, and then treated with the tripterygium pre-drug for diagnosis and treatment of the present invention. The culture medium was discarded and the cells were washed with PBS. MitoTracker Green (MTG, a mitochondrial-specific dye) and LysoTracker Green (LTG, a lysosome-specific dye) were added successively for incubation, followed by washing with PBS, and then incubated with Hoechst33342 to stain the cell nuclei. Finally, confocal microscopy was used to take pictures.

[0104] Figure 6 The co-localization imaging results of the tripterygium pre-drug for diagnosis and treatment of the present invention (0.5 μM) with MitoTracker Green / LysoTracker Green in PC12 cells respectively.

[0105] The results showed ( Figure 6 Figs. a, b, c, d, e and f in

[0106] that the tripterygium pre-drug for diagnosis and treatment of the present invention had good co-localization with MTG and LTG (Person co-localization coefficient = 0.83), demonstrating the dual-targeting ability of the tripterygium pre-drug for diagnosis and treatment of the present invention in mitochondria and lysosomes.

[0107] Model establishment of middle cerebral artery occlusion (MCAO): Mice were fasted 12 h before surgery, and the anesthetic was 2.5% tribromoethanol. The anesthetized mice were placed supine on the operating table, and the four feet of the mice were fixed to ensure smooth breathing. The hair on the neck of the mice was removed with scissors, the common carotid artery, internal carotid artery and external carotid artery were separated, and a thread embolism was inserted into the middle cerebral artery and fixed. After 1 h of ischemia, the thread embolism was removed, and normal saline / the tripterygium pre-drug for diagnosis and treatment of the present invention (3.8 μmol / kg) was immediately injected into the tail vein. The mice were randomly divided into 3 groups:

[0108] 1) Sham group, i.e., the sham operation group: The mice were only separated the blood vessels but not inserted the thread embolism, and 3.8 μmol / kg of Compound I of the present invention was injected into the tail vein;

[0109] 2) Control group, the experimental mice were given an injection of 200 μL of normal saline without model establishment;

[0110] 3) MCAO + Group I of the compound I of the present invention: The mice in the model group were intravenously injected with 3.8 μmol / kg of the tripterygium wilfordii pre - drug for diagnosis and treatment of the present invention through the tail vein.

[0111] The changes in the fluorescence signal in the brains of mice were observed in real - time (5 min, 15 min, 30 min, 1 h, 2 h, 4 h) using a small animal imager. Meanwhile, the brain tissues were taken out at specific time points after drug administration for ex - vivo fluorescence imaging analysis to comprehensively evaluate the fluorescence imaging ability of the tripterygium wilfordii pre - drug for diagnosis and treatment of the present invention and its targeting to ischemic brain tissues.

[0112] Figure 7 It is the fluorescence imaging test result diagram of the mouse model of cerebral ischemia - reperfusion with the tripterygium wilfordii pre - drug for diagnosis and treatment of the present invention; among them, Figure 7 a is the fluorescence imaging of the mouse model of cerebral ischemia - reperfusion injected with the tripterygium wilfordii pre - drug for diagnosis and treatment of the present invention, the mice in the Sham group, and the mice in the normal Control group. Figure 7 b is Figure 7 the average fluorescence intensity in the brains of the mice in a (mean ± standard deviation, n = 3). Figure 7 c is the fluorescence image of the brains isolated from the mouse model of cerebral ischemia - reperfusion injected with the tripterygium wilfordii pre - drug for diagnosis and treatment of the present invention, the Sham group, and the Control group. Figure 7 d is the average fluorescence intensity of the brain tissues isolated in Figure c (mean ± standard deviation, n = 3).

[0113] The results show ( Figure 7 ) that the tripterygium wilfordii pre - drug for diagnosis and treatment of the present invention can produce clear fluorescence signals in the brain ( Figure 7 a), the signal intensity reaches the peak within a short time after injection and is significantly higher than that of the Sham group and the Control group. In addition, the fluorescence signal can last for a long time ( Figure 7 b), indicating that the tripterygium wilfordii pre - drug for diagnosis and treatment of the present invention can effectively penetrate the blood - brain barrier and be activated by ONOO - in ischemic brain tissues, releasing near - infrared fluorescence. The ex - vivo fluorescence imaging analysis of brain tissues further confirms that the signal intensity in the brains of the model group injected with the tripterygium wilfordii pre - drug for diagnosis and treatment of the present invention is significantly higher than that of the Sham group and the Control group ( Figure 7 c, d). It is proved that the tripterygium wilfordii pre - drug for diagnosis and treatment has selective fluorescence imaging ability for the cerebral ischemia disease model, providing an important basis for related diagnosis and treatment.

[0114] Example 14: In - vivo test on the therapeutic effect of the tripterygium wilfordii pre - drug for diagnosis and treatment of the present invention on cerebral ischemia

[0115] Model establishment of middle cerebral artery occlusion (MCAO): Mice were fasted for 12 h before surgery, and the anesthetic was 2.5% tribromoethanol. The anesthetized mice were placed supine on the operating table, and the four feet of the mice were fixed to ensure smooth breathing. The hair on the neck of the mice was removed with scissors, the common carotid artery, internal carotid artery and external carotid artery were separated, and a suture was inserted into the middle cerebral artery and fixed. After 1 h of ischemia, the suture was withdrawn, and normal saline / celastrol (3.8 μmol / kg) / the lead drug for treating and diagnosing Tripterygium wilfordii Hook. f. of the present invention (3.8 μmol / kg) was immediately injected into the tail vein. To evaluate the therapeutic effect of the lead drug for treating and diagnosing Tripterygium wilfordii Hook. f. of the present invention in vivo, the mice were randomly divided into four groups:

[0116] 1) sham group, i.e., the sham operation group: The blood vessels of the mice were only separated but no suture was inserted, and 200 μL of normal saline was injected into the tail vein;

[0117] 2) MCAO group, and 200 μL of normal saline was given to the experimental mice after model establishment;

[0118] 3) MCAO+celastrol group: Celastrol 3.8 μmol / kg / d was injected into the tail vein of the modeled mice, and the drug was administered twice in total for 48 h of treatment;

[0119] 4) MCAO+Compound I of the present invention group, and Compound I of the present invention 3.8 μmol / kg / d was injected into the tail vein of the modeled mice, and the drug was administered twice in total for 48 h of treatment. After two consecutive days of treatment, Longa score was performed to evaluate the neurological deficit.

[0120] Subsequently, the mice were anesthetized and the brain tissues were taken out, TTC staining was performed to measure the infarct volume, and at the same time, the pathological structure of the brain tissue and the morphological changes of neurons were observed by H&E staining to comprehensively evaluate the therapeutic effect of the lead drug for treating and diagnosing Tripterygium wilfordii Hook. f. of the present invention.

[0121] Figure 8 It is the test result diagram of the in vivo cerebral ischemia treatment effect of the lead drug for treating and diagnosing Tripterygium wilfordii Hook. f. of the present invention. Among them, Figure 8 a is the TTC staining picture of the cerebral coronal section of each group of mice. Figure 8 b is the statistical chart of the cerebral infarct volume of each group of mice (mean±standard deviation, n = 3). Figure 8 c is the neurological function score of each group of mice (mean±standard deviation, n = 6).

[0122] The results show ( Figure 8 ), compared with the sham group, the neurological function score of the mice in the MCAO group was significantly increased, and large-area infarction appeared in the brain tissue ( Figure 8a, b). The neurological function scores of the mice in the MCAO + celastrol group and the MCAO + Compound I of the present invention group were significantly reduced, and the improvement effect of the MCAO + Compound of the present invention group was more obvious ( Figure 8 c). The TTC staining results showed that after MCAO modeling, treatment with the compound of the present invention could significantly reduce the cerebral infarction volume, and the effect was better than that of the MCAO + celastrol group. Figure 8 a, b). The above results demonstrated that the present compound has good brain protection effects, can significantly reduce the cerebral infarction area, improve the pathological structure of cerebral ischemic mice, and provide a new potential strategy for the treatment of cerebral ischemic diseases.

[0123] The foregoing are only preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and changes. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A precursor compound of a medicine for treating and diagnosing Tripterygium wilfordii Hook. f., characterized in that, The precursor compound has the structure shown by the following formula:

2. A method for preparing a precursor compound as described in claim 1, characterized in that, The preparation method comprises the following steps: S1. Reacting compound 1 with 1,1,2-trimethyl-1H-benzo[e]indole under the condition of sodium acetate to obtain compound 3, and subjecting compound 3 to a nucleophilic reaction with 3,5-dihydroxybenzyl alcohol to obtain compound 4; The synthetic route of step S1 is shown by the following formula: S2. Protecting the hydroxyl group of 4-aminobenzyl alcohol with tert-butyldimethylchlorosilane to obtain compound 6, reacting compound 6 with diphenylphosphinic chloride in an organic base environment to obtain compound 7, removing the hydroxyl group protection of compound 7 under the condition of tetrabutylammonium fluoride to obtain compound 8, and brominating compound 8 with phosphorus tribromide to obtain compound 9; The synthetic route of step S2 is shown by the following formula: S3. Reacting compound 4 with compound 9 under basic conditions to generate compound 10, and compound 10 is the precursor compound; The synthetic route of step S3 is shown by the following formula:

3. The preparation method according to claim 2, characterized in that, The preparation method comprises the following steps: S100. Dissolving N-[(3-(phenylaminomethylene)-2-chloro-1-cyclohexen-1-yl)methylene]aniline hydrochloride, 1,1,2-trimethyl-1H-benzo[e]indole and sodium acetate in absolute ethanol, and refluxing and reacting under a nitrogen atmosphere to obtain compound 3; S200. Dissolving compound 3, 3,5-dihydroxybenzyl alcohol and KHCO3 in anhydrous DMF, and reacting under a nitrogen atmosphere to obtain compound 4; S300. Dissolving 4-aminobenzyl alcohol and imidazole in anhydrous dichloromethane, adding tert-butyldimethylchlorosilane at 0 °C, and reacting to obtain compound 6; S400. Dissolving compound 6 in anhydrous dichloromethane, adding N,N-diisopropylethylamine, protecting with nitrogen, and slowly adding diphenylphosphinic chloride at 0 °C, and reacting to obtain compound 7; S500. Dissolving compound 7 in anhydrous tetrahydrofuran, protecting with nitrogen, and slowly adding tetrabutylammonium fluoride at 0 °C, and reacting to obtain compound 8; S600. Dissolving compound 8 in anhydrous dichloromethane, protecting with nitrogen, and slowly adding PBr3 at 0 °C, and reacting to obtain compound 9; S700. Dissolving compound 4, compound 9, K2CO3 and KI in anhydrous DMF, protecting with nitrogen, and stirring and reacting at 40-50 °C overnight to obtain compound 10.

4. Use of the precursor compound according to claim 1 in the preparation of a triptolide prodrug for diagnosis and treatment.

5. A Tripterygium wilfordii pre-treatment medicine, characterized in that, The structural formula of the triptolide prodrug for diagnosis and treatment is shown by the following formula:

6. A preparation method of the tripterygium wilfordii pre-drug for diagnosis and treatment as described in claim 5, characterized in that, The preparation method is as follows: Performing a Mitsunobu reaction on compound 10 and triptolide to obtain compound I, and compound I is the triptolide prodrug for diagnosis and treatment; Among them, compound 10 is the precursor compound according to claim 1.

7. The preparation method according to claim 6, wherein The specific preparation method is: Dissolving triphenylphosphine and diisopropyl azodicarboxylate in anhydrous tetrahydrofuran, protecting with nitrogen, stirring and reacting at room temperature for 30-60 min, then adding compound 10, stirring and reacting at room temperature for 30-60 min, and then adding triptolide to obtain the triptolide prodrug for diagnosis and treatment.

8. The preparation method according to claim 7, characterized in that, The molar ratio of triphenylphosphine, diisopropyl azodicarboxylate, compound 10 and triptolide is 3:3:1:

2.

9. Use of the triptolide prodrug as described in claim 5 in the preparation of a reagent for diagnosing and / or treating a disease, wherein the disease is ischemic stroke.

10. The application according to claim 9, wherein The disease is neuroinflammation caused by ischemic stroke.

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