Application of compound based on 3-phenyl-1, 2, 4-oxadiazole-5-formamide skeleton in preparation of medicine for treating pathological retinal neovascular diseases

Abstract

The invention provides an application of a compound based on a 3-phenyl-1, 2, 4-oxadiazole-5-formamide skeleton in preparation of a medicine for treating pathological retinal neovascular diseases, and belongs to the technical field of biological medicines. The compound based on the 3-phenyl-1, 2, 4-oxadiazole-5-formamide skeleton developed by the invention can be used as an inhibitor of PPT1, and the palmitoylation modification level of a substrate is improved by inhibiting the enzyme activity of the PPT1. According to the compound, the palmitoylation modification of Gpx1 is improved by inhibiting PPT1, so that the generation of pathological retinal neovascularization is inhibited.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to the use of a compound based on a 3-phenyl-1,2,4-oxadiazole-5-carboxamide skeleton in the preparation of a drug for treating pathological retinal neovascular diseases. Background Art

[0002] Pathological angiogenesis refers to the formation of new blood vessels under abnormal physiological conditions such as infection, inflammation and oxidative stress. The formation of these abnormal blood vessels can lead to a variety of health problems, including cancer, chronic inflammation and retinopathy. Angiogenesis is a complex process involving the fine regulation of multiple cytokines and signaling pathways. In this process, the expression of pro-angiogenic factors plays a vital role, especially vascular endothelial growth factor (VEGF), which plays a core role in angiogenesis and is the main driving force of angiogenesis. Oxidative stress is one of the key mechanisms. In a hypoxic environment, hypoxia-inducible factor 1α (HIF-1α) is stabilized and accumulated, and then transferred to the cell nucleus to bind to HIF-1β to form an active HIF-1α / HIF-1β dimer. This dimer can activate the transcription of the VEGF gene, thereby initiating the angiogenesis program. This process is particularly evident in tumor growth, wound healing and retinopathy under certain pathological conditions.

[0003] In the treatment at home and abroad, a series of anti-VEGF drugs represented by bevacizumab have shown significant effects in the treatment of cancer and retinal pathological vascular diseases such as age-related macular degeneration (AMD) and diabetic macular edema (DME). However, expanding its application scope often promotes the consequences of drug-induced resistance. In addition, the use of such drugs also leads to serious adverse reactions such as hypertension, proteinuria, photoreceptor damage and even blindness. Therefore, it is particularly important to find new targets or pathways to limit the expression of vascular endothelial growth factor. Summary of the invention

[0004] The purpose of the present invention is to provide an application of a compound based on a 3-phenyl-1,2,4-oxadiazole-5-carboxamide skeleton in the preparation of a drug for treating pathological retinal neovascular diseases, so as to solve the above technical problems.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides an application of a compound based on a 3-phenyl-1,2,4-oxadiazole-5-carboxamide skeleton in the preparation of a drug for treating pathological retinal neovascular diseases. The compound based on a 3-phenyl-1,2,4-oxadiazole-5-carboxamide skeleton has the following general structural formula:

[0007]

[0008] Among them, R 1 Contains a nitrogen heterocyclic hydrocarbon group or a tertiary amine group; R 2 Contains straight-chain alkyl, cycloalkyl, aryl or ether groups.

[0009] Furthermore, the R 1 Selected from the following structures:

[0010]

[0011] The R 2 Selected from the following structures:

[0012]

[0013] Furthermore, the compound based on the 3-phenyl-1,2,4-oxadiazole-5-carboxamide skeleton comprises the following structural formula:

[0014]

[0015]

[0016] Furthermore, the compound based on the 3-phenyl-1,2,4-oxadiazole-5-carboxamide skeleton increases the palmitoylation modification level of the substrate by inhibiting the enzyme activity of PPT1, thereby inhibiting the formation of pathological retinal neovascularization.

[0017] Furthermore, the concentration of the compound based on the 3-phenyl-1,2,4-oxadiazole-5-carboxamide skeleton is 5 to 20 μM.

[0018] The present invention also provides a pharmaceutical composition for treating pathological retinal neovascular diseases, comprising an active ingredient and a pharmaceutically or immunologically acceptable excipient, carrier or diluent;

[0019] The active component comprises a compound based on a 3-phenyl-1,2,4-oxadiazole-5-carboxamide skeleton.

[0020] Furthermore, the pharmaceutical composition is in the form of an injection, eye drops, liposomes or aerosols.

[0021] Beneficial effects of the present invention:

[0022] The compound based on the 3-phenyl-1,2,4-oxadiazole-5-carboxamide skeleton developed by the present invention can be used as an inhibitor of PPT1, and can increase the palmitoylation modification level of its substrate by inhibiting the activity of PPT1 enzyme. The compound can inhibit the formation of retinal pathological neovascularization by inhibiting PPT1 to increase the palmitoylation modification of Gpx1.

[0023] Gpx1 is a palmitoylated protein, and PPT1 can dynamically regulate the palmitoylation of Gpx1. The increase in the palmitoylation level of Gpx1 can inhibit the formation of retinal pathological neovascularization. Compared with wild-type OIR mice, the retinal pathological neovascularization of PPT1-KO OIR mice is significantly reduced. Based on this, the present invention has developed an intervention method that effectively inhibits PPT1 to reduce retinal neovascularization. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The results of the effect of Drug21 compounds on HUVECs cell proliferation, cell formation and migration ability; A: HUVECs cells were incubated with 7.5 μmol of Drug21 compounds at different time points, and the palmitoylation modification of Gpx1 in HUVECs cells was detected by ABE and Western Blot experiments; B: Quantitative analysis of Figure A; C: CCK8 was used to detect the effect of different concentrations of Drug21 compounds on HUVECs cell proliferation. D: The effect of different concentrations of Drug21 compounds on the lumen formation ability of HUVECs cells was detected by lumen formation experiment. E: Quantitative analysis of Figure D. F: The effect of different concentrations of Drug21 compounds on the migration ability of HUVECs cells was detected by wound healing experiment. G: Quantitative analysis of Figure F, **** indicates p<0.0001.

[0025] Figure 2 The figure shows the inhibitory effect of Drug21 compound on the formation of retinal pathological neovascularization; A: OIR mice were randomly divided into two groups on the 14th day. The DMSO group was injected with 1μl 0.1% DMSO in the vitreous cavity, and the Drug21 compound group was injected with 1μl 25μm Drug21 compound in the vitreous cavity. Retinal tissues were taken on the 17th day, and the palmitoylation modification of Gpx1 in the retinal tissue was detected by ABE and Western Blot; B: Quantitative analysis of Figure A. C: OIR mice were randomly divided into two groups on the 14th day. The DMSO group was injected with 1μl 0.1% DMSO in the vitreous cavity, and the Drug21 compound group was injected with 1μl 25μm Drug21 compound in the vitreous cavity. Retinal tissues were taken on the 17th day, and retinal flat mounts were performed to observe the pathological neovascularization of the mouse retina (the retinal neovascularization in the Drug21 compound group was reduced). D: Quantitative analysis of Figure C. DETAILED DESCRIPTION

[0026] The present invention provides an application of a compound based on a 3-phenyl-1,2,4-oxadiazole-5-carboxamide skeleton in the preparation of a drug for treating pathological retinal neovascular diseases. The compound based on a 3-phenyl-1,2,4-oxadiazole-5-carboxamide skeleton has the following general structural formula:

[0027]

[0028] Among them, R 1 Contains a nitrogen heterocyclic hydrocarbon group or a tertiary amine group; R 2 Contains straight-chain alkyl, cycloalkyl, aryl or ether groups.

[0029] In the present invention, the R 1 Preferably selected from the following structures:

[0030]

[0031] The R 2 Preferably selected from the following structures:

[0032]

[0033]

[0034] In the present invention, the compound based on the 3-phenyl-1,2,4-oxadiazole-5-carboxamide skeleton is preferably of the following structural formula:

[0035] Table 1

[0036]

[0037]

[0038] In the present invention, the preparation method of the compound of formula I comprises the following steps:

[0039]

[0040] (1) Commercially available (E)-4-bromo-N'-hydroxybenzamidine (5.00 g, 23.25 mmol) and triethylamine (5.06 ml, 46.50 mmol) were dissolved in 50 mL of acetonitrile, and ethyl oxalyl chloride (6.32 g, 46.50 mmol) was added dropwise under ice bath, and the mixture was stirred for 0.5 h at the same temperature, and then the mixture was heated to 72° C. and refluxed for 7 h. After the reaction was completed, the solid in the reaction system was removed by suction filtration, and the solvent was removed from the filtrate under reduced pressure. The concentrate was diluted with ethyl acetate, extracted with ethyl acetate, washed with water and saturated brine, and the organic phase was dried over anhydrous sodium sulfate and purified by column chromatography to obtain intermediate 1.

[0041]

[0042] (2) Dissolve the intermediate 1 in 50 ml of ethanol, add sodium hydroxide solution at room temperature to adjust the pH to alkaline, hydrolyze at room temperature for 3 hours, remove the solvent under reduced pressure, add water to dissolve, slowly add dilute HCl in an ice bath to adjust the pH to acidic until a large amount of solid is generated, filter, wash the filter cake with ice water or petroleum ether, and finally dry at 45°C to obtain a white solid 2;

[0043]

[0044] (3) Dissolve intermediate 2 (1.5 equiv.), substituted aromatic amine or aliphatic amine (1 equiv.), TCFH (1.1 equiv.) and NMI (2.1 equiv.) in 50 ml of tetrahydrofuran and stir at room temperature for 5-8 hours. After the reaction is completed, remove the solvent under reduced pressure, dilute the concentrate with ethyl acetate, extract with ethyl acetate, wash with water and saturated brine, dry the organic phase with anhydrous sodium sulfate, and purify by column chromatography to obtain intermediate 3;

[0045]

[0046] (4) Dissolve the intermediate 3 (1 equiv.), substituted aromatic amine or aliphatic amine (1.5 equiv.), Pd2(dba)3 (0.1 equiv.), BINAP (0.5 equiv.) and K2CO3 (2.0 equiv.) in 50 ml of toluene and stir at 85°C for 5-8 h under nitrogen protection. After the reaction is completed, cool to room temperature, remove the solvent under reduced pressure, dilute the concentrate with ethyl acetate, extract with ethyl acetate, wash with water and saturated brine, dry the organic phase with anhydrous sodium sulfate, and purify by column chromatography to obtain the final product.

[0047] In the present invention, the compound based on the 3-phenyl-1,2,4-oxadiazole-5-carboxamide skeleton increases the palmitoylation modification level of the substrate by inhibiting the enzyme activity of PPT1, thereby inhibiting the formation of retinal pathological neovascularization.

[0048] In the present invention, the concentration of the compound based on the 3-phenyl-1,2,4-oxadiazole-5-carboxamide skeleton is 5 to 20 μM, preferably 7.5 to 15 μM.

[0049] The present invention also provides a pharmaceutical composition for treating pathological retinal neovascular diseases, comprising an active ingredient and a pharmaceutically or immunologically acceptable excipient, carrier or diluent;

[0050] The active component comprises a compound based on a 3-phenyl-1,2,4-oxadiazole-5-carboxamide skeleton.

[0051] In the present invention, the pharmaceutical composition is in the form of injection, eye drops, liposome or aerosol.

[0052] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0053] Example

[0054] 1.ABE (acyl-biotinyl exchange)

[0055] (1) Take 2 mg of protein in a 15 mL EP tube, adjust the protein volume to 2 mg / mL (total volume is 1 mL) with LB (=1 mL - protein volume), and then add 10 μL PI (protease inhibitor);

[0056] (2) CM protein precipitation: add 4 mL methanol, 1.5 mL chloroform, and 3 mL sterile water, mix well, and centrifuge for 10 min (4000 rpm). Aspirate about 7 mL of supernatant, leaving about 3 mL. Add 3 mL of methanol to the wall, mix well, and centrifuge again for 10 min at 4000 rpm. Pour off the supernatant, air dry, and wipe dry;

[0057] (3) NEM blocking: add 200 μL SB to dissolve the protein, pipette and beat evenly, place in a 50°C water bath for 10 min, then add 800 μL NEM liquid, place in a 4°C cold storage and incubate overnight with rapid shaking; (NEM blocking solution ratio = 0.00625 g NEM + 800 μL LB + 10 μL PI)

[0058] (4) The next day, thaw 4 ml of MHPDP-biotin and PI in advance. After taking out the protein from the 4°C cold storage, separate it.

[0059] (5) CM protein precipitation: add 4 mL methanol, 1.5 mL chloroform, and 3 mL sterile water, mix well, and centrifuge for 10 min at 4000 rpm. Aspirate 7 mL of supernatant, leaving about 2 mL. Add 3 mL methanol to the cell wall, mix well, and centrifuge again for 10 min at 4000 rpm. Pour off the supernatant.

[0060] (6) Wash 2-3 times with 3 ml of 70% methanol, 5 min each time, 4000 rpm, discard the supernatant after the last wash, and air dry;

[0061] (7) Add 460 μL SB to the protein sample until the liquid becomes clear and centrifuge for 1 min.

[0062] (8) Take 1.5 ml EP tubes and label them +HA, -HA, and Input respectively;

[0063] +HA tube, add 200 μL protein and 800 μL solution A;

[0064] -HA tube, add 200 μL protein and 800 μL solution B;

[0065] HA reagent preparation:

[0066] HA (about 1 ml): 0.069 HA + 0.791 ml PBS + 0.194 ml 5 M NaOH; total pH adjusted to 7.4;

[0067] Table 2

[0068] Solution A(+HA) Solution(-HA) HA(light-proof) 700ul - 4mMHPDP-biotin 250ul 250ul 10% TritionX-100 (protect from light) 20ul 20ul PI 10ul 10ul 1mM Tris / HCl, pH 7.4 - 50ul PBS 20ul 670ul Total 1ml 1ml

[0069] After each tube is mixed, incubate it in a shaker in the dark room for 2-3 hours at 350 rpm, and shake the tube quickly in the dark.

[0070] (9) Add 60 μL protein to the input tube and 15 μL loading tube.

[0071] Cook the sample at 100℃ for 10 min, then centrifuge again and store in a -20℃ refrigerator;

[0072] (10) After incubation for 2-3 hours in step (8), take a 15 ml centrifuge tube and precipitate CM again with the same steps as above (4 ml methanol, 1.5 ml chloroform, 3 ml water, 4000 rpm; centrifuge for 10 min), then wash 2-3 times with 3 ml 70% methanol, 5 min each time, 4000 rpm. Aspirate the remaining liquid, then air-dry the protein and wipe the bottle dry with paper.

[0073] (11) After washing and air-drying the protein, add 80-100 μL TB and blow evenly (heat in a 50°C water bath for 5-10 min), then add 950 μL LB + 10 μL PI and cook in a water bath for 5-30 min to fully dissolve the protein; if the sample is clear when taken out of the water bath, centrifugation is not required. If there is precipitation, high-speed centrifugation can be performed for 2 min (7000-8000 rpm).

[0074] (12) Prepare a new 1.5 ml EP tube, add 30 μL of STREPTAVIDIN AGAROSE and leave it for a few minutes. Observe whether the amount of precipitated beads is consistent. Then add 1 ml of the supernatant from the centrifugation in step (11), shake well, and incubate at room temperature in the dark with rapid shaking for 3 h or at 4 °C overnight.

[0075] (13) Wash the beads five times with cold Wash Buffer, centrifuge for 1 min (2500 rpm), discard 0.9 μL of the supernatant, aspirate to the 0.1 mark, add 1 ml of cold Wash Buffer (prepared in advance and placed at 4°C), mix, centrifuge for 1 min (2500 rpm), and then discard 1 ml of the supernatant. Repeat 5 times;

[0076] (14) For the last time (after the fifth centrifugation), discard the supernatant and add 60-80 μL LB elution buffer (LB elution buffer: LB 960 μL + 5*loading 240 μL + β-ME 12 μL);

[0077] (15) After adding the eluent, cook the sample at 100°C for 10 min (750 rpm); then centrifuge for 2 min (high speed) and store at -20°C.

[0078] (16) Detect target protein by Western Blot.

[0079] 2. Cell Tube Formation Experiment

[0080] (1) Preparation of materials: One day in advance, take out the matrix gel from the -80°C refrigerator and place it at 4°C to thaw. At the same time, place the 96-well plate and 200 μL of the buffer in a 4°C refrigerator to pre-cool.

[0081] (2) On ice, add 50 μL of matrix gel to a 96-well plate, making sure there are no bubbles. After shaking to make the plate evenly distributed, place the plate in a 37°C incubator to solidify for 30-60 minutes.

[0082] (3) Prepare the treated cells in advance and count the number of cells using a cell counting plate.

[0083] (4) In a 96-well plate, 2 × 10 4 cells.

[0084] (5) After 4 hours, observe the tube formation of cells under a 10x microscope and take photos and count them in time.

[0085] 3. Cell Proliferation Assay

[0086] (1) After treating the cells according to the experimental purpose, you can start the CCK8 proliferation assay.

[0087] (2) Collect the treated cells and count them using a cell counting plate.

[0088] (3) Four time points were set for each group of samples: 0, 24, 48, and 72 hours. The number of cells in each group was adjusted to 3,000 / 100 μL / well and seeded into 96-well plates. Each sample was replicated four times.

[0089] (4) After the cells have attached for 4-6 hours, add 10 μL of CCK8 to each well and continue incubating in the incubator for 2 hours.

[0090] (5) Turn on the microplate reader 20 minutes in advance, measure the absorbance of the cells at 450 nm, and record the statistics.

[0091] 4. Cell scratch assay

[0092] (1) After treating the cells according to the experimental purpose, you can start the cell scratch experiment.

[0093] (2) Collect the treated cells and count them using a cell counting plate.

[0094] (3) Calculate the volume of cells to be inoculated in the insert based on the number of cells, so that each chamber is inoculated with 1×10 5 cells, and then fill up with 100 μL of DMEM complete medium, and then return to the incubator at 37°C and 5% CO2 to continue culturing until the cells are fully grown in the chamber, which usually takes 12 hours.

[0095] (4) After observing that the cells have grown completely in the chamber and are in good condition, carefully remove the plug with sterilized forceps, then wash the cells once with PBS and add DMEM containing 0.375% FBS to continue culturing.

[0096] (5) After replacing the DMEM medium with 0.375% FBS, the cells were photographed using a 10x fluorescence microscope. Three fields of view (upper, middle, and lower) were taken for each group and recorded as the 0-hour image.

[0097] (6) After 8 hours, the wound healing was observed, the floating cells were washed away with PBS, and then photographed under a 10x fluorescence microscope with the same parameters; each group of experiments was repeated 3 times.

[0098] 5. Retinal Staining

[0099] (1) Fixing tissue: Experimental mice were killed by CO2 method, the eyeballs were clamped with forceps and placed in a 24-well plate, and 500 μL 4% PFA was added to fix the eyes for 0.5-1 h (slow shaking table). Afterwards, a hole was made at the pupil with a syringe and fixed for another 10 min.

[0100] (2) Wash the fixed eyeballs three times with PBS (fast shaking), each time for 5 minutes.

[0101] (3) Carefully remove the retinal tissue under a microscope and wash it three times in a PBS shaker for 5 minutes each time. Discard the PBS after the last wash.

[0102] (5) Retinal tissue staining: Add 500μL lectin solution to each well and incubate overnight at room temperature in the dark with slow shaking. Lectin solution configuration: 500μg Isolectin B4-594 (I21413) powder + 500μL 1mM CaCl2 + 49.5ml 1mM CaCl2 (packed separately and stored at -20℃ for use)

[0103] (6) Wash the retina with PBS three times (fast shaking), 5 min each time.

[0104] (7) Slide: Carefully transfer the stained retinal tissue to a slide, remove impurities in the cup again under a microscope, then cut it 1 mm away from the optic nerve root, then transfer it to the opposite side and cut it 1 mm away from the optic nerve root, then carefully unfold the retina and cut it evenly into a four-leaf clover shape. Evenly drop a drop of sealing agent on the cover glass, be careful to avoid bubbles, and then carefully cover the retina. Keep the whole process away from light, and try to complete each retina within 2 minutes.

[0105] (8) Drying: Dry in a light-proof oven at 37°C or air-dry at room temperature, then store the slides at 4°C in a light-proof manner and take photos and observe them promptly.

[0106] 6. Establishment of OIR Mouse Model

[0107] (1) C57BL / 6J newborn mice were raised with their mothers under normoxia for 7 days (the first day after birth was designated as P0). On P7, the mice and their mothers were transferred to an oxygen chamber with an oxygen concentration of 75±2% and continued to be raised for 5 days. On P12, the mice were taken out and continued to be raised under normoxia.

[0108] (2) The retinas of mice were taken at P17 and retinal flat mounts were made to observe the blood vessel conditions. The retinal flat mounts of mice in the normal group showed uniform and neat distribution, smooth and non-tortuous blood vessels, and no pathological new blood vessels; the retinal flat mounts of mice in the OIR group showed a large area of ​​non-irrigated area in the center, disordered and tortuous peripheral blood vessels, and a large number of new blood vessel clusters at the junction, indicating that the modeling was successful.

[0109] 7. Intravitreal Injection in Mice

[0110] (1) Preparation and grouping of experimental animals: Before the experiment begins, mice are randomly assigned to a control group and an experimental group, with at least 3 mice in each group, to ensure the statistical significance of the experimental results. Ensure that the two groups of mice are comparable in gender, age, and weight.

[0111] (2) Preparation of injection solution: Under sterile conditions, accurately weigh the Drug21 compound and dissolve it in physiological saline containing 0.1% DMSO to ensure that the final concentration is 25 μM. Before the experiment, the prepared injection solution is stabilized at room temperature for a period of time to ensure that the drug is completely dissolved.

[0112] (3) Intravitreal injection therapy was performed at P14 in the OIR model:

[0113] ①: According to the weight of the mouse, intraperitoneal injection of sodium pentobarbital (0.1 ml for every 10 g of body weight)

[0114] ②: After the mouse is anesthetized, use lidocaine eye drops for local anesthesia and mydriasis to keep the mouse's eyeball at the level of the corneal margin.

[0115] ③: Squeeze out the eyeball, fully expose the cornea and scleral limbus under sterile conditions, pierce a hole 1mm behind the corneal limbus with a 1ml syringe, be careful to avoid blood vessels, and then slowly inject 1μL of the mixed solution into the vitreous cavity with a microsyringe, stay in the vitreous cavity for 30s, and then quickly remove the needle.

[0116] ④: Gently press the cornea to make the eyeball enter the eye socket and reposition the eyeball; then observe the mouse to wake up and continue to keep it in the cage according to the experimental design.

[0117] 8. Statistical analysis of data

[0118] SPSS13.0 and GraphPadPrism8.0 statistical software were used for data statistical analysis, and one-way analysis of variance and independent sample t-test were used for the control group and experimental group. Data were expressed as mean ± standard deviation, and P < 0.05 was considered statistically significant.

[0119] Experimental Example 1

[0120] HUVECs cells were incubated with 7.5 μmol of 20# at different time points, and the palmitoylation modification of Gpx1 in HUVECs cells by Drug21 was detected by ABE and Western Blot technology. The results showed that Drug21 compound could effectively upregulate the level of palm-Gpx1 in a time-dependent manner ( Figure 1A, 1B). Tube formation is a key step in the angiogenesis process, while cell migration and proliferation are the basis for the expansion of the vascular network. Next, HUVECs were treated with different concentrations of Drug21 compounds, and CCK8 was used to detect the effects of different concentrations of Drug21 compounds on HUVECs cell proliferation, and the tube formation experiment was used to detect the effects of different concentrations of Drug21 compounds on the tube formation ability of HUVECs cells, and the wound healing experiment was used to detect the effects of different concentrations of Drug21 compounds on the migration ability of HUVECs cells. The evaluation of the results of tube formation, scratch healing and CCK8 proliferation experiments showed that Drug21 compounds inhibited the tube formation of HUVECs in a dose-dependent manner ( Figure 1 D, 1E), migration ( Figure 1 F, 1G) and proliferation ( Figure 1 C).

[0121] Experimental Example 2

[0122] To further examine the effects of Drug21 compounds in vivo, we established an OIR mouse model and randomly divided the mice into two groups. The control group (DMSO) was injected with 1 μL of 0.1% DMSO into the vitreous cavity, and the experimental group (Drug21 compounds) was injected with 1 μL of 25 μm Drug21 compounds into the vitreous cavity. Both groups were treated by intravitreal injection on P14, and retinal tissue samples of mice were collected on P17. The palmitoylation modification of Gpx1 in retinal tissue was detected by ABE and Western Blot, and it was observed that the palmitoylation level of Gpx1 in the retinal tissue of mice in the Drug21 compound group was significantly enhanced ( Figure 2 A, 2B). This result is consistent with the in vitro experimental results, indicating that the Drug21 compound can also effectively inhibit PPT1 activity in vivo and increase the palmitoylation modification of Gpx1. Figure 2 C is the retinal flat mount of P17 mice in the control group and the experimental group. It was observed that the retinal neovascularization in the experimental group was significantly reduced ( Figure 2 D). In addition, the observation results of retinal flat mounts also showed that the Drug21 compound significantly reduced the relative area of ​​new blood vessels in the mouse retina, which is direct evidence of reduced angiogenesis. These experimental results together prove that the Drug21 compound can effectively inhibit angiogenesis in the OIR disease model by targeting PPT1 and enhancing Gpx1 palmitoylation.

[0123] Similarly, Drug1 to Drug20 compounds were also verified, and this series of compounds had similar properties to Drug21 in inhibiting PPT1.

[0124] As can be seen from the above examples, the present invention provides a use of a compound based on a 3-phenyl-1,2,4-oxadiazole-5-carboxamide skeleton in the preparation of a drug for treating pathological retinal neovascular diseases. The compound based on a 3-phenyl-1,2,4-oxadiazole-5-carboxamide skeleton developed by the present invention can be used as an inhibitor of PPT1, and the palmitoylation modification level of its substrate is increased by inhibiting the activity of PPT1 enzyme. The compound increases the palmitoylation modification of Gpx1 by inhibiting PPT1 to inhibit the formation of pathological retinal neovascularization.

[0125] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Use of a compound based on a 3-phenyl-1,2,4-oxadiazole-5-carboxamide skeleton in the preparation of a drug for treating pathological retinal neovascular diseases, characterized in that: The compound based on the 3-phenyl-1,2,4-oxadiazole-5-carboxamide skeleton has the following general structural formula: Among them, R 1 Contains a nitrogen heterocyclic hydrocarbon group or a tertiary amine group; R 2 Contains straight-chain alkyl, cycloalkyl, aryl or ether groups.

2. The use according to claim 1, characterized in that: The R 1 Selected from the following structures: The R 2 Selected from the following structures:

3. The use according to claim 1, characterized in that: The compound based on the 3-phenyl-1,2,4-oxadiazole-5-carboxamide skeleton comprises the following structural formula:

4. The use according to any one of claims 1 to 3, characterized in that: The compound based on the 3-phenyl-1,2,4-oxadiazole-5-carboxamide skeleton increases the palmitoylation modification level of the substrate by inhibiting the enzyme activity of PPT 1, thereby inhibiting the formation of retinal pathological neovascularization.

5. The use according to claim 4, characterized in that: The concentration of the compound based on the 3-phenyl-1,2,4-oxadiazole-5-carboxamide skeleton is 5 to 20 μM.

6. A pharmaceutical composition for treating pathological retinal neovascular diseases, characterized in that: Containing an active ingredient and a pharmaceutically or immunologically acceptable excipient, carrier or diluent; The active component comprises a compound based on a 3-phenyl-1,2,4-oxadiazole-5-carboxamide skeleton.

7. The pharmaceutical composition for treating pathological retinal neovascular diseases according to claim 6, characterized in that: The pharmaceutical composition may be prepared in the form of injection, eye drops, liposome or aerosol.

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