Diagnosis and treatment prodrug based on inflammatory disease activation and preparation method and application thereof

Abstract

The invention discloses a diagnosis and treatment prodrug based on inflammatory disease activation and a preparation method and application thereof, and belongs to the technical field of biological medicine. The diagnosis and treatment prodrug disclosed by the invention not only can realize accurate diagnosis on an inflammatory part through NIR fluorescence imaging, but also can selectively release ozanimod in an inflammatory tissue to play a role in efficiently treating colitis, so that the toxicity to normal tissues is reduced. Diagnosis and treatment 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 prodrug for diagnosis and treatment activated by inflammatory diseases, a preparation method thereof, and an application thereof. Background Art

[0002] Inflammatory bowel disease (IBD) is a group of immune-mediated chronic and progressive intestinal diseases, mainly including Crohn's disease and ulcerative colitis. With the acceleration of the industrialization process and the change of social lifestyle, factors such as unreasonable diet structure, overnutrition, and the acceleration of the pace of life have made the intestine in a high-load state for a long time, thereby triggering the occurrence of IBD. Due to the chronic recurrence of its course, IBD not only seriously affects the quality of life of patients, but also brings a heavy economic burden to the medical system. Although the exact cause of IBD is still unclear, early diagnosis and precise management are of great significance for improving the prognosis of patients. Therefore, it is particularly urgent to develop new diagnostic and treatment technologies to achieve the precise diagnosis and treatment of IBD.

[0003] At present, commonly used anti-inflammatory drugs in clinical practice such as 5-aminosalicylic acid (5-ASA), thiopurine drugs, and glucocorticoids only have a certain therapeutic effect on patients with mild to moderate IBD, but these drugs are often accompanied by systemic side effects, which limit their clinical application. To overcome these limitations, researchers are committed to developing drug delivery systems responsive to the inflammatory microenvironment in order to achieve precise release of drugs at the inflammatory site. However, the existing technologies still lack a diagnostic and treatment integration strategy with both therapeutic and precise visualization functions, which makes the diagnosis and treatment of inflammatory diseases still face huge challenges. The development of diagnostic and treatment integrated drugs requires more reasonable design to achieve efficient accumulation and intelligent release of drugs in inflammatory tissues, while simultaneously monitoring the circulation, distribution, and release process of drugs in the body in real time.

[0004] Developing a diagnostic and treatment integration strategy that can accurately and sensitively evaluate the severity of IBD and simultaneously achieve timely treatment will bring an important breakthrough to the clinical diagnosis and treatment of IBD. However, there is currently no report on a diagnostic and treatment integrated drug with both the treatment of inflammatory diseases and precise visualization functions. Therefore, designing a diagnostic and treatment integrated drug that can monitor the inflammatory degree of IBD in real time and provide treatment effects has important research value and application prospects. Summary of the Invention

[0005] In order to solve the defects existing in the prior art, the purpose of the present invention is to provide a prodrug for diagnosis and treatment activated by inflammatory diseases, a preparation method thereof, and an application thereof.

[0006] To achieve the above purpose, the present invention provides the following technical solutions:

[0007] In a first aspect of the present invention, there is provided a prodrug for diagnosis and treatment activated by an inflammatory disease, and the prodrug has a structure shown by general formula I:

[0008]

[0009] In a second aspect of the present invention, there is provided a method for preparing the above-mentioned prodrug for diagnosis and treatment, and the preparation method includes the following steps:

[0010] S1. Compound 1 and compound 2 react under alkaline conditions to form compound 3;

[0011] The synthesis route of step S1 is shown by the following formula:

[0012]

[0013] S2. Compound 3 undergoes an Appel reaction under the action of CBr4 and PPh3 to form the corresponding brominated product 4, and then reacts with ozanimod under the action of NaH to form compound 5, and compound 5 is a conjugate product of ozanimod and a fluorescent fragment;

[0014] The synthesis route of step S2 is shown by the following formula:

[0015]

[0016] S3. Compound 5 reacts under the action of a hydrochloric acid / methanol solution to form a compound I with a structure shown by general formula I, that is, the above-mentioned prodrug for diagnosis and treatment;

[0017] The synthesis route of step S3 is shown by the following formula:

[0018]

[0019] In a third aspect of the present invention, there is provided an application of the above-mentioned prodrug for diagnosis and treatment in the preparation of a selective imaging reagent for the diagnosis of inflammatory diseases.

[0020] In a fourth aspect of the present invention, there is provided an application of the above-mentioned prodrug for diagnosis and treatment in the preparation of a targeted therapeutic drug for inflammatory diseases.

[0021] In some embodiments of the present invention, the inflammatory disease is inflammatory bowel disease.

[0022] In some embodiments of the present invention, the inflammatory bowel disease is DSS-induced inflammatory bowel disease.

[0023] Compared with the prior art, the prodrug for diagnosis and treatment provided by the present invention has the following advantages:

[0024] (1) It can selectively monitor the activity of inflammatory levels in living cells and has high specificity.

[0025] (2) It can show a rapid response in the in - vivo inflammatory bowel disease model, and obvious NIR fluorescence signals can be observed as early as 30 minutes after injection.

[0026] (3) The fluorescence signal is mainly confined to the colon, demonstrating its ability to selectively detect and localize in in - vivo inflammatory intestinal tissues and reducing the impact on other tissues.

[0027] (4) It significantly improved colon injury in the DSS - induced mouse model, and the recovery effect of colon length was better than that of the existing drug Oza, showing its great potential in the treatment of IBD.

[0028] (5) It can not only be used for the diagnosis of IBD, but also has a therapeutic effect, demonstrating its versatility in the diagnosis and treatment of IBD. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] 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:

[0030] Figure 1 are the ultraviolet - visible and fluorescence spectral properties of the compounds of the present invention;

[0031] Figure 2 are the concentration - time responsiveness of the compounds of the present invention to LTA4H;

[0032] Figure 3 are the tests on the specific response ability of the compounds of the present invention to LTA4H;

[0033] Figure 4 are the drug release characteristics of the compounds of the present invention;

[0034] Figure 5 are the representative confocal fluorescence images of each group of RAW264.7 cells;

[0035] Figure 6 are the in - vivo imaging test result graphs of the inflammatory bowel disease (IBD) model;

[0036] Figure 7 are the evaluation result graphs of the therapeutic effect of compound I provided by the embodiments of the present invention on inflammatory bowel disease. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0037] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the following will clearly and completely describe the technical solutions in the embodiments of this application with reference to the accompanying drawings in the embodiments of this application. Obviously, the described embodiments are part of the embodiments of this application, rather than all of them. Based on the embodiments in this application, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of this application.

[0038] 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 without indicating the manufacturer, they are all conventional products that can be obtained through regular channels.

[0039] 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 without indicating the manufacturer, they are all conventional products that can be obtained through regular channels.

[0040] 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.

[0041] Example 1: Preparation of Compound 3

[0042] Compound 3 is: (R,E)-2-(2-(6-((4-(3-(benzyloxy)-2-((tert-butoxycarbonyl)amino)-3-oxopropanamido)benzyl)oxy)-7-(hydroxymethyl))-2,3-dihydro-1H-xanthen-4-yl)vinyl)-1-methylquinolin-1-ium iodide.

[0043] Dissolve (E)-4-(2-(6-hydroxy-7-(hydroxymethyl)-2,3-dihydro-1H-xanthen-4-yl)vinyl)-1-methylquinolin-1-ium iodide (591 mg, 1 mmol), (S)-4-((4-(bromomethyl)phenyl)amino)-3-((tert-butoxycarbonyl)amino)-4-oxobutanoic acid benzyl ester (476 mg, 1 mmol), KI (142.3 mg, 0.857 mmol), and potassium carbonate (42.7 mg, 0.427 mmol) in anhydrous DMF (3 ml), protect with N2, and stir at room temperature for 8 h. After the reaction is completed, dilute the reaction solution with DCM (10 mL), wash the solution with brine (3 x 10 mL), dry the organic layer over Na2SO4 and concentrate. The residue is purified by silica gel column chromatography (DCM:MeOH = 50:1, v / v) to obtain a blue solid, which is Compound 3, with a yield of 51%.

[0044] Analysis data of Compound 3: 11H NMR (CDCl3, 400 MHz) δ 8.53 (d, J = 14.6 Hz, 2H, 2 ArH), 8.14 (d, J = 8.5 Hz, 1H, ArH), 7.88 (dd, J = 10.3, 8.4 Hz, 2H, 2NH), 7.56 (t, J = 7.6 Hz, 1H, ArH), 7.48 (m, 4H, 4 ArH), 7.34 (d, J = 8.4 Hz, 1H, ArH), 7.25 (s, 4H, 4 ArH), 7.18 (d, J = 9.2 Hz, 2H, 2 ArH), 6.92 (m, 2H, 2 ArH), 6.20 (d, J = 14.9 Hz, 1H, CH=C), 5.78 (d, J = 8.6 Hz, 1H, CH=C), 5.09 (m, 3H, CH=C, CH2), 4.73 (s, 2H, CH2), 4.58 (s, 1H, OH), 3.81 (s, 2H, CH2), 2.98 (dd, J = 17.1, 4.7 Hz, 1H, CH), 2.84 (m, 2H, CH2), 2.55 (dd, J = 17.0, 6.1 Hz, 4H, 2CH2), 1.77 (t, J = 6.1 Hz, 2H, CH2), 1.39 (s, 9H, 3CH3), 1.20 (m, 3H, CH3).

[0045] Example 2: Preparation of Compound 4

[0046] Compound 4 is: (R,E)-2-(2-(6-((4-(5-(Benzyloxy)-2-((tert-butoxycarbonyl)amino)-5-oxopentanamido)benzyl)oxy)-7-(bromomethyl))-2,3-dihydro-1H-xanthen-4-yl)vinyl)-1-methylquinolin-1-ium iodide.

[0047] Compound 3 (2 g, 2.0 mmol) and CBr4 (1.35 g, 4 mmol) were dissolved in anhydrous (10 mL) THF, protected by N2, and stirred at room temperature for 1 h. Then, PPh3 (1.1 g, 4 mmol) was added, and stirring was continued for 5 h. After the reaction was completed, the reaction solution was diluted with DCM (10 mL), washed with brine (3 x 10 mL), and the organic layer was dried over Na2SO4 and concentrated. The residue was purified by silica gel column chromatography (DCM:MeOH = 100:1, v / v) to obtain a blue solid, namely Compound 4, with a yield of 76%.

[0048] Analytical data of Compound 4: 11H NMR (DMSO-d6, 400 MHz) δ 8.53 (d, J = 14.6 Hz, 2H, 2 ArH), 8.14 (d, J = 8.5 Hz, 1H, ArH), 7.88 (dd, J = 10.3, 8.4 Hz, 2H, 2NH), 7.56 (t, J = 7.6 Hz, 1H, ArH), 7.48 (m, 4H, 4 ArH), 7.34 (d, J = 8.4 Hz, 1H, ArH), 7.25 (s, 4H, 4 ArH), 7.18 (d, J = 9.2 Hz, 2H, 2 ArH), 6.92 (m, 2H, 2 ArH), 6.20 (d, J = 14.9 Hz, 1H, CH=C), 5.78 (d, J = 8.6 Hz, 1H, CH=C), 5.09 (m, 3H, CH=C, CH2), 4.73 (s, 2H, CH2), 3.81 (s, 2H, CH2), 2.98 (dd, J = 17.1, 4.7 Hz, 1H, CH), 2.84 (m, 2H, CH2), 2.55 (dd, J = 17.0, 6.1 Hz, 4H, 2CH2), 1.77 (t, J = 6.1 Hz, 2H, CH2), 1.39 (s, 9H, 3CH3), 1.20 (m, 3H, CH3). ESI-MS (m / z): calcd for C 49 H 49 N3O7Br + : 870.2348, found 870.2761.

[0049] Example 3: Preparation of Compound 5

[0050] Compound 5 is: 2-((E)-2-(6-((4-((S)-4-(Benzyloxy)-2-((tert-butoxycarbonyl)amino)-4-oxobutanamido)benzyl)oxy)-7-((2-(((R)-4-(5-(3-cyano-4-isopropoxyphenyl)-1,2,4-oxadiazol-3-yl)-2,3-dihydro-1H-inden-1-yl)amino)ethoxy)methyl)-2,3-dihydro-1H-xanthen-4-yl)vinyl)-1-methylquinolin-1-ium iodide.

[0051] The compound ozanimod (500 mg, 1.24 mmol) was dissolved in DMF (5 mL), and under an ice bath, NaH was added. The mixture was stirred at room temperature for 20 min. Then, compound 4 (1.4 g, 1.48 mmol) was added, and the reaction was carried out for 4 h. After the reaction was completed, the reaction solution was poured into ice water, and the pH was adjusted to neutral with 1 M dilute hydrochloric acid. Then, it was diluted with 50 mL of DCM, and the solution was washed with brine (3×10 mL). The organic layer was dried over Na2SO4 and concentrated. The residue was purified by silica gel column chromatography (DCM:MeOH = 50:1, v / v) to obtain a blue solid, which was compound 5 with a yield of 36%.

[0052] Analytical data of compound 5: 11H NMR (MeOD, 400 MHz) δ 8.66 (dd, J = 14.9, 6.5 Hz, 1H, ArH), 8.30 (d, J = 8.6 Hz, 1H, ArH), 8.27 (s, 1H, NH), 8.06 (dd, J = 9.0, 2.5 Hz, 1H, ArH), 8.01 (dd, J = 8.1, 2.9 Hz, 1H, ArH), 7.89 (dd, J = 7.6, 3.2 Hz, 1H, ArH), 7.81 (s, 1H, ArH), 7.74 (m, 1H, ArH), 7.66 (dd, J = 10.3, 2.9 Hz, 1H, ArH), 7.61 (d, J = 8.5 Hz, 2H, 2ArH), 7.54 (t, J = 7.7 Hz, 1H, ArH), 7.39 (dd, J = 8.4, 3.7 Hz, 2H, 2ArH), 7.32 (d, J = 6.6 Hz, 1H, ArH), 7.31 (m, 1H, ArH), 7.23 (m, 6H, 5ArH, CH=C), 7.18 (d, J = 9.2 Hz, 2H, 2ArH), 6.97 (d, J = 2.4 Hz, 1H, ArH), 6.89 (t, J = 3.1 Hz, 1H, CH=C), 6.38 (dd, J = 15.0, 3.4 Hz, 1H, CH=C), 5.14 (s, 2H, CH2), 5.04 (s, 2H, CH2), 4.83 (t, J = 6.1 Hz, 2H, CH2), 4.59 (s, 2H, CH2), 4.33 (t, J = 7.5 Hz, 1H, CH), 3.97 (m, 1H, CH), 3.60 (ddd, J = 16.0, 12.4, 7.0 Hz, 2H, CH2), 3.24 (dd, J = 15.3, 9.0 Hz, 1H, CH), 2.97 (dd, J = 14.4, 6.6 Hz, 1H, CH), 2.86 (dt, J = 16.4, 6.2 Hz, 1H, CH), 2.81 (m, 3H, CH3), 2.64 (m, 4H, 2CH2), 2.02 (dd, J = 7.1, 3.4 Hz, 7H, 2CH3, CH), 1.89 (d, J = 7.9 Hz, 2H, CH2), 1.39 (s, 9H, 3CH3), 1.27 (d, J = 2.8 Hz, 4H, 2CH2).

[0053] Example 4: Preparation of Compound I

[0054] Compound I is: 2-((E)-2-(6-((4-((S)-2-amino-4-(benzyloxy)-4-oxobutanamido)benzyl)oxy)-7-((2-(((R)-4-(5-(3-cyano-4-isopropoxyphenyl)-1,2,4-oxadiazol-3-yl)-2,3-dihydro-1H-inden-1-yl)amino)ethoxy)methyl)-2,3-dihydro-1H-xanthen-4-yl)vinyl)-1,1,3-trimethyl-1H-benzo[e]indole-3-iodide.

[0055] Compound 5 (100 mg, 0.08 mmol) was dissolved in 4 mol / L hydrochloric acid methanol solution and stirred at room temperature for 12 h. After the reaction was completed, it was concentrated under reduced pressure. Column chromatography was carried out using dichloromethane / methanol (10:1, v / v) as the eluent to obtain a blue solid, namely Compound I, with a yield of 54%.

[0056] Analytical data of Compound I: 11H NMR (MeOD, 400 MHz) δ 8.66 (dd, J = 14.9, 6.5 Hz, 1H, ArH), 8.30 (d, J = 8.6 Hz, 1H, ArH), 8.27 (m, 2H, NH2), 8.06 (dd, J = 9.0, 2.5 Hz, 1H, ArH), 8.01 (dd, J = 8.1, 2.9 Hz, 1H, ArH), 7.89 (dd, J = 7.6, 3.2 Hz, 1H, ArH), 7.81 (s, 1H, ArH), 7.74 (m, 1H, ArH), 7.66 (dd, J = 10.3, 2.9 Hz, 1H, ArH), 7.61 (d, J = 8.5 Hz, 2H, 2ArH), 7.54 (t, J = 7.7 Hz, 1H, ArH), 7.39 (dd, J = 8.4, 3.7 Hz, 2H, 2ArH), 7.32 (d, J = 6.6 Hz, 1H, ArH), 7.31 (m, 1H, ArH), 7.23 (m, 6H, 5ArH, CH=C), 6.97 (d, J = 2.4 Hz, 1H, ArH), 6.89 (t, J = 3.1 Hz, 1H, CH=C), 6.38 (dd, J = 15.0, 3.4 Hz, 1H, CH=C), 5.14 (s, 2H, CH2), 5.04 (s, 2H, CH2), 4.83 (t, J = 6.1 Hz, 2H, CH2), 4.59 (s, 2H, CH2), 4.33 (t, J = 7.5 Hz, 1H, CH), 3.97 (m, 3H, CH2, CH), 3.60 (ddd, J = 16.0, 12.4, 7.0 Hz, 2H, CH2), 3.24 (dd, J = 15.3, 9.0 Hz, 1H, CH), 2.97 (dd, J = 14.4, 6.6 Hz, 1H, CH), 2.86 (dt, J = 16.4, 6.2 Hz, 1H, CH), 2.81 (m, 3H, CH3), 2.64 (m, 4H, 2CH2), 2.02 (dd, J = 7.1, 3.4 Hz, 7H, 2CH3, CH), 1.89 (d, J = 7.9 Hz, 2H, CH2), 1.27 (d, J = 2.8 Hz, 4H, 2CH2). 1313C NMR(CDCl3, 100 MHz) δ 203.8, 179.0, 167.2, 157.4, 145.1, 136.3, 135.4, 133.4, 131.9, 131.6, 128.4, 127.7, 124.8, 123.7, 122.1, 115.7, 107.7, 102.4, 100.4, 91.0, 83.8, 83.6, 81.4, 63.7, 62.0, 49.0, 46.8, 42.0, 31.7, 29.9, 29.1, 28.8, 28.0, 25.7, 25.6, 25.4, 25.3, 22.7, 21.6, 20.9, 18.1, 17.6. ESI-MS(m / z): calcd for C 67 H 64 N7O8 + : 1094.4811, found 1094.4843.

[0057] Example 5: UV-Visible and Fluorescence Spectral Properties of Compound I of the Present Invention

[0058] Compound I of the present invention was dissolved in PBS solution containing 5% DMSO. The UV spectral data of Compound I of the present invention at different concentrations (5 μM, 10 μM, 15 μM, 20 μM) added to PBS (pH = 7.4) solution were measured using a UV spectrophotometer, and the data from 500 - 900 nm were collected. The fluorescence spectral changes of Compound I of the present invention at different concentrations (5 μM, 10 μM, 15 μM) added to PBS (pH = 7.4) solution were detected using fluorescence spectroscopy. The fluorescence properties were studied using 690 nm as the excitation wavelength, and the fluorescence emission spectral data from 700 - 900 nm were collected by a fluorescence spectrometer.

[0059] Among them, Figure 1 a is the UV absorption spectrum of Compound I of the present invention at different concentrations, Figure 1 b is the fluorescence emission spectrum of Compound I of the present invention at different concentrations.

[0060] The results showed ( Figure 1 ), Compound I of the present invention showed a strong absorption peak at 690 nm ( Figure 1 a) and fluorescence emission at 720 nm ( Figure 1 b), and with the increase of concentration, both the absorption intensity and fluorescence intensity increased, proving that the compound of the present invention can absorb the photon energy in the near-infrared light region.

[0061] Example 6: Concentration and Time Responsiveness of Compound I of the Present Invention to LTA4H

[0062] The concentration responsiveness of the compound I of the present invention to LTA4H was detected by fluorescence spectroscopy. Different concentrations of LTA4H ranging from 0 to 100 U / L were added to 4 mL of PBS solution containing 5% DMSO with the compound I of the present invention (20 μM). The excitation wavelength was set at 690 nm, and the fluorescence spectrometer collected the fluorescence emission spectrum data in the range of 700 - 900 nm.

[0063] The time responsiveness of the compound I of the present invention to LTA4H was detected by fluorescence spectroscopy. Specifically, after adding LTA4H (100 U / L), the fluorescence intensity changes with time were measured in 4 mL of PBS solution containing 5% DMSO with the compound I of the present invention (20 μM) within 120 s by absorption and fluorescence spectrometry.

[0064] Wherein Figure 2 a is the fluorescence spectrum of the compound I of the present invention after exposure to different concentrations of LTA4H in PBS buffer for 24 h. Figure 2 b is the time-dependent fluorescence emission spectrum of the compound I of the present invention in the presence of LTA4H (100 U / L).

[0065] The results showed ( Figure 2 ), as LTA4H was added in the range of 0 - 100 U / L, the fluorescence intensity increased, the peak emission center was at 710 nm, and the higher the content of LTA4H, the higher the fluorescence intensity; as the incubation time of LTA4H extended, the fluorescence intensity increased. It was proved that the compound I of the present invention had concentration-time responsiveness and high sensitivity to the detection of LTA4H.

[0066] Example 7: Test on the specific response ability of the compound I of the present invention to LTA4H

[0067] The specific response ability of the compound I of the present invention to LTA4H was detected by fluorescence spectroscopy. The selectivity of the compound I of the present invention to potential interferents such as cations, amino acids, redox agents, and other enzymes was evaluated. At room temperature, the compound I of the present invention was added to the solutions (1 μM) of corresponding bioanalytes (such as KCl, ZnCl2, LAP, L-Phe, L-Ser, NQO1, VcNa, H2O2, GST1, and LTA4H 100 U / L) respectively, with PBS buffer solution containing 5% (v / v) DMSO at 37 °C for 0.5 h. All emission spectra were excited at 690 nm and recorded at 700 - 800 nm.

[0068] Wherein, Figure 3 is the fluorescence intensity of the compound I of the present invention at 720 nm in the presence of different species.

[0069] The results showed ( Figure 3) When different interfering ions were added to the solution of Compound I of the present invention, the effects of these interferents on the fluorescence response of the compound of the present invention were negligible. Only the presence of LTA4H led to an increase in fluorescence, demonstrating the high selectivity of the compound of the present invention for LTA4H.

[0070] Example 8: Drug release characteristics of Compound I of the present invention

[0071] To further reveal the hydrolysis process of Compound I by LTA4H, we used HPLC to analyze whether the incubation of Compound I with LTA4H produced free fluorescent dye (Compound 1) and the drug ozanimod.

[0072] Among them, Figure 4 A is the HPLC chromatogram of Compound I (20 μM, in pH 7.4 PBS containing 2% DMSO) treated with LTA4H (100 U / L) for different times (0, 1, 2, 4, 6, 8, 12 hours). The signal shown by the solid arrow on the right is Compound I; the signal shown by the dotted arrow on the left is the fluorophore (Compound 1); the signal shown by the dotted arrow in the middle is ozanimod Oza. 4B is the in vitro release of the fluorescent fragment (Compound 1) and Oza after treatment with LTA4H.

[0073] The results showed that after treatment with LTA4H, Compound I gradually released the fluorescent fragment (Compound 1) (6.9 min) and the drug molecule Oza (3.7 min) ( Figure 4 A). After 12 h, about 90% release of the fluorescent fragment and Oza drug molecule was observed ( Figure 4 B). It indicates that while the diagnostic agent molecule releases the fluorescent fragment (Compound 1), it can also effectively release the drug molecule at the target site, exerting the integrated functions of treatment and diagnosis.

[0074] Example 9: Selective imaging ability of Compound I in an inflammation model

[0075] The selective imaging ability of Compound I in an inflammation model was evaluated by confocal fluorescence imaging.

[0076] The experiment was divided into four groups.

[0077] PBS control group: RAW264.7 cells were used as a control without any treatment.

[0078] Compound I group: RAW264.7 cells were incubated with Compound I for 4 h.

[0079] LPS + Compound I group: RAW264.7 cells were incubated with LPS (lipopolysaccharide, 1 μg / mL) for 24 h, and then treated with Compound I for 4 h.

[0080] LPS + Compound I + Ubenimex group: RAW264.7 cells were incubated with LPS (1 μg / mL) for 24 h, then treated with the LTA4H inhibitor Ubenimex for 24 h, subsequently treated with Compound I for 4 h, washed with PBS, and then the cell nuclei were stained with DAPI.

[0081] Images were taken using a confocal microscope. Figure 5 These are representative confocal fluorescence images of RAW264.7 cells in each group.

[0082] The results showed ( Figure 5 ), in normal and Compound I - treated RAW264.7 cells, only negligible red fluorescence was observed. In contrast, RAW264.7 cells treated with LPS + Compound I showed significant red fluorescence. The addition of the LTA4H inhibitor Ubenimex resulted in minimal fluorescence, highlighting the specific cleavage induced by LTA4H. It was demonstrated that the change in the fluorescence signal of Compound I was caused by an increase in intracellular inflammatory levels and selective cleavage by LTA4H, thus verifying the effectiveness of Compound I in monitoring inflammatory levels in live cells.

[0083] Example 10: In - vivo imaging test of Compound I in an inflammatory bowel disease (IBD) model

[0084] To evaluate the potential of Compound I for real - time detection of inflammatory bowel disease in vivo, a DSS - induced ulcerative colitis mouse model was established. Female BALB / c mice were continuously administered 3% DSS solution for 7 days, and then Compound I (1.83 mg / kg) was intravenously injected for in - vivo imaging. The change in fluorescence signal in the mice was observed in real - time using a small animal imager. Time - dependent NIR fluorescence imaging was performed at 0.5, 1, 2, 4, 8, 12, and 24 h after injection, and the results are as Figure 6 shown.

[0085] Figure 6 In, Figure A is the NIR fluorescence image of the mouse model in vivo after intravenous injection of Compound I (1.83 mg / kg) with λ ex = 690 nm. Figure 6 In, Figure B is the quantification of the fluorescence intensity in Figure A. Figure 6 In, Figure C is the tissue fluorescence imaging of the colon and major organs (i.e., heart, liver, spleen, lung, and kidney). Figure 6 In, Figure D is the quantitative determination of the average fluorescence intensity of the colon tissue and major organs.

[0086] The results showed ( Figure 6),As early as 30 min after injection, obvious NIR fluorescence appeared in the DSS-induced mice, and the peak intensity was reached approximately 8 hours later. Statistical analysis of the fluorescence signal in the colon region also showed a similar trend. In addition, in vitro imaging of the major organs (heart, liver, lung, spleen, and kidney) showed that strong fluorescence was mainly confined to the colon. These findings confirmed that Compound I could selectively and sensitively detect and localize inflamed intestinal tissues in vivo, highlighting its potential as an effective strategy for diagnosing DSS-induced IBD.

[0087] Example 11: Evaluation of the Therapeutic Effect of Compound I on Inflammatory Bowel Disease

[0088] To determine the therapeutic effect of Compound I on IBD, we conducted in vivo studies using mice with 3% DSS-induced colitis.

[0089] The mice were divided into 4 groups: PBS control group; 3% DSS-induced colitis group (3% DSS group); Oza 1.2 mg / kg + DSS as the positive control (abbreviated as Oza 1.2 mg / kg group); Compound I + DSS group (abbreviated as I 1.83 mg / kg group).

[0090] PBS control group: Only given distilled water.

[0091] 3% DSS group: Given 3% DSS in drinking water for 7 days to induce colitis.

[0092] Oza 1.2 mg / kg group: Given 3% DSS in drinking water for 7 days to induce colitis. After the model was successfully established, the mice were intravenously injected with Oza (1.2 mg / kg) on day 0.

[0093] I 1.83 mg / kg group: Given 3% DSS in drinking water for 7 days to induce colitis. After the model was successfully established, the mice were intravenously injected with Compound I (1.83 mg / kg) on day 0.

[0094] During the entire experiment, the body weight changes of all mice were monitored daily. Seven days after administration in the Oza 1.2 mg / kg group and the I 1.83 mg / kg group, all mice were euthanized, and their colons were collected for further analysis. The length of the colon was measured and photographed, and the analysis results are as Figure 7 shown.

[0095] Figure 7 In, Figure A is the body weight change graph of each group during modeling and treatment; Figure B is the quantitative graph of the colon length of each group; Figure C is the colon picture of each group of mice.

[0096] The results showed ( Figure 7), mice treated with DSS showed a trend of weight loss, while treatment with Compound I significantly increased the body weight of the mice, showing superior efficacy to Oza( Figure 7 A). In addition, compared with normal mice, treatment with 3% DSS significantly reduced the colon length of the mice by approximately 33%, suggesting the occurrence of colitis. In contrast, treatment with Compound I significantly improved this effect, with the colon length increasing to 7.78 cm, which was superior to Oza (7.72 cm)( Figure 7 B,C). This demonstrated the great potential of Compound I to repair colon damage in IBD mice.

Claims

1. A prodrug for diagnosis and treatment activated by inflammatory diseases, characterized in that, The pre-diagnosis and treatment drug has the structure shown in General Formula I:

2. A method for preparing the pre-diagnosis and treatment drug as described in claim 1, characterized in that, The preparation method includes the following steps: S1. Compound 1 and Compound 2 react under alkaline conditions to form Compound 3; The synthetic route of Step S1 is shown in the following formula: S2. Compound 3 undergoes an Appel reaction under the action of CBr4 and PPh3 to form the corresponding brominated product 4, which then reacts with ozanimod under the action of NaH to form Compound 5. Compound 5 is the conjugate product of ozanimod and the fluorescent fragment; The synthetic route of Step S2 is shown in the following formula: S3. Compound 5 reacts under the action of hydrochloric acid / methanol solution to form Compound I, that is, the pre-diagnosis and treatment drug; The synthetic route of Step S3 is shown in the following formula:

3. Use of the pre-diagnosis and treatment drug according to claim 1 in the preparation of a selective imaging reagent for the diagnosis of inflammatory diseases.

4. The application according to claim 3, characterized in that The inflammatory disease is inflammatory bowel disease.

5. Use of the pre-diagnosis and treatment drug according to claim 1 in the preparation of a targeted therapeutic drug for inflammatory diseases.

6. The application according to claim 5, characterized in that, The inflammatory disease is inflammatory bowel disease.

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