WO2021218884A1

WO2021218884A1 - Use of aryl guanidine compound or pharmaceutically acceptable salt thereof

Info

Publication number: WO2021218884A1
Application number: PCT/CN2021/089785
Authority: WO
Inventors: 李波; 赵晨; 章淑杰; 徐志建; 许凯; 颜标; 张勇; 蔡婷婷; 朱维良
Original assignee: 中国科学院上海药物研究所; 复旦大学附属眼耳鼻喉科医院
Priority date: 2020-04-30
Filing date: 2021-04-26
Publication date: 2021-11-04
Also published as: CN113577066B; CN113577066A

Abstract

The use of an aryl guanidine compound as represented by formula 1 or a pharmaceutically acceptable salt thereof in the preparation of a drug for preventing or treating angiogenesis-related ophthalmic disorders.

Description

Use of arylguanidine compound or pharmaceutically acceptable salt thereof

Technical field

The present invention relates to the field of medicinal chemistry, in particular to the use of an arylguanidine compound or a pharmaceutically acceptable salt thereof in the preparation of a medicine for the treatment or prevention of ophthalmic diseases related to neovascularization.

Background technique

Pathological neovascularization occurs in multiple ocular tissues, including the retina, choroid, and cornea. It is the main cause of visual impairment caused by many eye diseases, such as diabetic retinopathy (DR), age-related macular degeneration (AMD) and keratitis. Corneal neovascularization (CoNV) usually occurs in inflammatory or infectious ocular surface diseases. CoNV can cause corneal scarring, edema and inflammation, and ultimately lead to vision deterioration. Therefore, inhibiting corneal neovascularization is an important strategy to prevent or treat related ocular surface diseases.

Summary of the invention

The purpose of the present invention is to provide the use of a class of arylguanidine compounds or pharmaceutically acceptable salts thereof in the preparation of drugs for the prevention or treatment of ophthalmic diseases related to neovascularization.

The present invention provides a use of an arylguanidine compound represented by formula 1 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for the treatment or prevention of ophthalmic diseases related to neovascularization:

in:

Q is

in,

Indicates the connection point;

R ₁ and R ₂ are each independently selected from an unsubstituted or substituted C6-C20 aryl group or a C3-C14 heteroaryl group, wherein the heteroaryl group has one or more heteroatoms selected from the following group: O, N Or S;

L is unsubstituted or substituted -(CH ₂ ) _n -, or unsubstituted or substituted

Wherein m is 0, 1, 2, 3 or 4, n is 0, 1, 2, 3 or 4, and Y is O, NH or S;

Z is hydrogen, alkyl, unsubstituted or substituted C6-C20 aryl or C3-C14 heteroaryl, wherein the heteroaryl has one or more heteroatoms selected from the group consisting of O, N or S;

Each of the substitutions in R ₁ , R ₂ , L and Z refers to being independently substituted by one or more substituents selected from the group consisting of halogen, hydroxy, C1-C6 alkyl, C3-C8 cycloalkyl, C1-C6 alkoxy, amino, -NH (C1-C6 alkyl), -N (C1-C6 alkyl) (C1-C6 alkyl), nitro, C2-C6 alkenyl, halogenated C1-C6 Alkyl, carbonyl, carboxy, amido, cyano, hydroxymethyl, halogenated C1-C6 alkoxy, mercapto, or sulfamoyl, phenyl, phenoxy.

In an embodiment, R ₁ and R ₂ are each independently selected from an unsubstituted or substituted C6 aryl group or a C5-C6 heteroaryl group, wherein the heteroaryl group has one or more heteroatoms selected from the following group: O, N or S; the substitution refers to substitution by halogen or amide;

L is

Or -(CH ₂ )n-, where m is 0, n is 0, and Y is O, ΝΗ or S;

Z is tert-butyl, unsubstituted or substituted C6 aryl or unsubstituted or substituted C5-C6 heteroaryl, wherein the heteroaryl has one or more heteroatoms selected from the group consisting of O, N or S; The substitution refers to substitution by one or more substituents selected from the group consisting of halogen, C1-C6 alkyl, C1-C6 alkoxy, and halogenated C1-C6 alkyl.

L is

Or -(CH ₂ )n-, where m is 0, n is 0, and Y is O;

Z is an unsubstituted or substituted C6 aryl group or an unsubstituted or substituted C5-C6 heteroaryl group, wherein the heteroaryl group has one or more heteroatoms selected from the group consisting of O, N or S; Substitution refers to substitution by one or more substituents selected from the group consisting of halogen, C1-C6 alkoxy, and halogenated C1-C6 alkyl.

In an embodiment, the arylguanidine compound represented by Formula 1 is selected from any one of the compounds represented by the following structural formulas:

For the synthesis of specific compounds, please refer to CN 201610107774.3. The compound 1 numbered above is also referred to as DCZ3301 below.

In an embodiment, the arylguanidine compound represented by Formula 1 or a pharmaceutically acceptable salt thereof can treat or prevent ophthalmic diseases related to neovascularization by inhibiting corneal neovascularization.

Beneficial effect

Through the activity test on the ophthalmic disease model, it is found that the arylguanidine compound of the present invention has anti-angiogenic activity, indicating that the compound has the potential to prevent or treat neovascularization-related ophthalmic diseases.

Description of the drawings

Figures 1A-1F show that DCZ3301 reduces human umbilical vein endothelial cells (Human umbilical vein endothelial cells, HUVEC) cell viability and induces spontaneous apoptosis of HUVEC. 1A. The chemical structure of DCZ3301 (Compound 1); 1B. The ATP content of HUVEC after 24h and 48h treatment with DCZ3301; 1C. The expression levels of cleavage PARP and cleavage Caspase-3 protein in HUVEC after 24h treatment with DCZ3301; 1D. z-VAD can reduce the decrease of HUVEC ATP content; 1E. After adding z-VAD, the expression level of PARP protein in HUVEC is cleaved; 1F.z-VAD can inhibit the decrease of HUVEC ATP content caused by DCZ3301, while Hela and HT29 cells Can not inhibit the reduction of HUVEC ATP content caused by DCZ3301.

Figures 2A-2D show that DCZ3301 can inhibit the proliferation, migration and tube formation of HUVEC. 2A. BrdU/PI double staining flow cytometry; 2B. Scratch test, DCZ3301 inhibited the healing process of HUVEC (n=3), scale bar: 250μm; 2C. Transwell test, DCZ3301 reduced the number of transmembrane cells (n =3), scale bar: 100μm, ****P<0.0001; 2D.DCZ3301 inhibits the formation of HUVEC lumen and reduces the branch length (n=3), scale bar: 100μm, **P<0.01.

Figures 3A-3B show that DCZ3301 can inhibit PI3K/AKT and ERK1/2 signaling pathways. 3A. After 24 hours of treatment with DCZ3301, the expression levels of p-PI3K, p-AKT and p-ERK1/2 in HUVEC; 3B. Quantitative analysis of p-PI3K, p-AKT and p-ERK1/2*p<0.05, ** p<0.01, ***p<0.001, ****p<0.0001.

Figures 4A-4B show that DCZ3301 can inhibit the sprouting of choroidal microvessels and the area of corneal neovascularization after alkali burns. 4A. On the 6th day, two groups of choroidal microvessel sprouting images (n=6), scale bar: 250μm, ^** p<0.01; 4B. Photograph of anterior segment of mouse cornea (n=6 per group) and FITC-dextran Corneal tile image after apical perfusion (n=6), scale bar: 250μm; in corneal tile, the ratio of corneal neovascularization area to the entire corneal area (n=6)**p<0.01, **** p<0.0001.

Detailed ways

The present invention will be further described below in conjunction with specific embodiments, but the present invention is not limited.

Examples:

Material

Experimental animal

Inbred BALB/c mice, males, 6-8 weeks old, 20-30 g in weight, were purchased from the Medical Experimental Animal Center of Nanjing Medical University (Nanjing, China). The mice were all raised in the animal room of the Eye, Ear, Nose, and Throat Hospital Affiliated to Fudan University, with a 12-hour light-dark cycle. The breeding environment was maintained at 22±2°C and a relative humidity of 40-70%. Sufficient rat food and drinking water were provided. The animals were anesthetized by intraperitoneal injection at the doses of ketamine (80mg/kg) and xylazine (10mg/kg). After the experiment, the mice were sacrificed by cervical dislocation. The operating procedures of animal experiments in the study have been reviewed by the Animal Ethics Committee of the Eye, Ear, Nose and Throat Hospital Affiliated to Fudan University, and follow the Association for Research in Vision and Ophthalmology (ARVO) declarations on animal experiments.

The main reagents are listed in Table 1 below.

Table 1

The main instruments and equipment are listed in Table 2 below.

Table 2

Example 1 The effect of DCZ3301 on the cell viability of HUVEC and the spontaneous apoptosis of HUVEC

1.1 Cell culture

Human umbilical vein endothelial cells (HUVEC) were purchased from Fudan IBS cell bank, human cervical cancer cell line HeLa and human colorectal adenocarcinoma cell line HT29 were purchased from cell bank of the Chinese Academy of Sciences Type Culture Collection Committee . HUVEC was cultured in M199 medium containing 10% fetal bovine serum, HeLa and HT29 were cultured in DMEM medium containing 10% fetal bovine serum, and the incubator was maintained at 37°C and 5% CO ₂ . Use 0.25% trypsin for digestion, and pass the cells in a 1:3 or 1:4 manner.

1.2 Cell viability detection

The luminescent cell viability assay is a method for detecting the number of metabolically active cells based on the content of ATP in the cell. The specific operations are as follows:

1) Take HUVEC cells in logarithmic growth phase and spread the cell suspension in a 96-well plate, with 6 wells in each group;

2) When the cells grow and converge to 90%, add different concentrations of DCZ3301 or 0.1% DMSO, and add z-VAD to a working concentration of 20 μM as needed, and continue culturing for 24 or 48 hours;

3) Add 25μl of configured CellTiter-Glo reagent to each well;

4) At room temperature, shake on a shaker for 12 minutes, mix well and lyse the cells;

5) Aspirate 100μl of liquid from each well and transfer to an opaque 96-well plate;

6) Put it into the microplate reader to read the luminous intensity (unit: light intensity).

7) Record the luminosity, take the 0.1% DMSO group as a control, calculate the relative percentage of each group, and draw a histogram.

1.3 Protein extraction and concentration determination

1.3.1 Extraction of total cell protein:

The cells were scraped off by the cell shovel and centrifuged, rinsed with PBS and centrifuged. Add RIPA lysis buffer containing protease inhibitor, phosphatase inhibitor and PMSF, place it on ice and lyse for 20 minutes, pipetting once every 5 minutes. Then centrifuge at 4°C, 12000rpm×10min, and transfer the supernatant to a new EP tube.

1.3.2 BCA method protein quantification:

1) According to the required number of holes, mix A and B in the BCA kit at a ratio of 50:1;

2) The standard protein BSA is diluted to 0.5mg/ml;

Set 8 holes in a 96-well plate for standard curve preparation, set 3 holes as blank control, and add samples;

3) Incubate in a 37°C incubator for 30 minutes;

4) Put it into the microplate reader for detection, the wavelength is 562nm, and read the absorbance;

5) Draw a standard curve based on the measured values and obtain a calculation formula, and then calculate the concentration of each sample;

6) Dilute each sample to 2mg/ml with loading buffer, and fill up the insufficient volume with PBS;

7) Boil at 100°C for 10 minutes, and store at -80°C.

1.4 Western blot test (Western blot)

1.4.1 Glue

1) Choose a 1.5mm glass plate, wash the glass plate with double distilled water, dry it, and fix it on the sponge base;

2) Use Bio-rad's FastCast pre-mixed acrylamide solution kit to prepare the glue, add each piece of separation glue: 4ml separation A solution + 4ml separation solution B + 40μl 10% ammonium persulfate + 4μl TEMED, mix well and add to the glass plate , And flatten the liquid surface with double distilled water;

3) Let it stand at room temperature. When there is a clear dividing line between the separating glue and water, pour out the double distilled water and blot it dry with filter paper;

4) Configure concentrated glue, add each concentrated glue: 1.5ml concentrated A solution + 1.5ml concentrated solution B + 15μl 10% ammonium persulfate + 3μl TEMED; after mixing, add to the glass plate, insert a 1.5mm comb, and let stand at room temperature Until completely solidified.

1.4.2 Electrophoresis

1) Fix the gel plate in the electrophoresis tank, pour the electrophoresis solution, pull out the comb, and wash out the residual gel in the swimming lane;

2) According to different purpose bands, add appropriate markers, load 20 or 30ug of sample, load the sample with equal concentration and equal volume, and fill up the remaining lanes with 1× loading buffer;

3) Connect the electrophoresis tank to the electrophoresis instrument, and the constant voltage is 80V, and after the strip runs to the separation gel, it is converted to a constant voltage of 120V.

1.4.3 Transfer film

1) Configure transfer fluid (1L contains 200ml methanol), bury it in ice and get cold;

2) The PVDF membrane is cut to an appropriate size and soaked in methanol for 30 minutes;

3) Pour the transfer membrane solution into the tray, take the separation gel out of the electrophoresis tank and cut it, install the transfer membrane sandwich: blackboard + sponge + 3 layers of filter paper + separation glue + PVDF membrane + 3 layers of filter paper + sponge + transparent board, soak the whole process In the film transfer liquid, drive out the air bubbles, clamp and put it into the film transfer tank;

4) Constant current 250mA, 80min～100min.

1.4.4 Immunoassay and ECL chemiluminescence

1) Sealing: Prepare a sealing solution (2.5g skimmed milk powder dissolved in 50ml 1×TBST), take out the PVDF membrane, cut it to a suitable size, put it in an incubator, and place it on a shaker at room temperature for 1 hour;

2) Primary antibody reaction: After washing the membrane with TBST, dilute with 5% BSA according to the antibody titer, and react overnight at 4°C;

3) Secondary antibody reaction: After washing the membrane with TBST, dilute the HRP-labeled secondary antibody with blocking solution, place it on a shaker at room temperature for 1 hour, and wash the membrane with TBST after the reaction;

4) ECL chemiluminescence detection: configure the developer, mix A and B of the ECL luminescence kit 1:1 and then avoid light; spread the PVDF film on the flat plate of the chemiluminescence imaging instrument, and absorb the water on the film with absorbent paper. Liquid, drop developer, ECL chemiluminescence imaging. Use Image J (version 1.8.0) software to analyze the band gray value for quantitative analysis.

Experimental results:

The relative molecular mass of DCZ3301 is 464.0 (Figure 1A). used

The luminescent cell activity detection kit evaluates the activity of cells by detecting the content of ATP in the cells. The luminosity of the 0.1% DMSO group is used as a reference to calculate the percentage of each group. The lowest effective concentration of DCZ3301 is 1μM. As the concentration increases, the degree of cell activity decreases; and as time goes by, compared with the data of 24h, the cell activity at 48h is lower, indicating that DCZ3301 is in a time- and dose-dependent manner Inhibit cell activity (Figure 1B).

The Caspase family is a key regulator of cell apoptosis, acting in a cascade manner. Caspase-3 is located downstream and is an important executive molecule. When Caspase is cleaved and activated, it can cleave and activate the target protein. Poly(ADP-ribose) polymerase (PARP) is the most important substrate of Caspase-3, which plays a vital role in DNA repair and cell stability. After cell apoptosis is initiated, 116kD PARP is cut into 89kD and 24kD fragments and loses enzyme activity, accelerating cell instability, and finally causing cell apoptosis. Caspase-3 and PARP hydrolysis are considered to be important signs of cell apoptosis. Therefore, here, the expression levels of these two proteins in HUVEC cells were detected. In the Western blot results, as the concentration of DCZ3301 increased, the expression of cleaved PARP and cleaved Caspase-3 increased (Figure 1C). This dose-dependent manner is consistent with the data of cell viability detection. The surface DCZ3301 can induce HUVEC apoptosis.

In addition, the Caspase inhibitor z-VAD-FMK (hereinafter referred to as z-VAD) was further used for identification. z-VAD is a broad-spectrum inhibitor of caspase, which can effectively prevent the activation of caspase during the process of apoptosis and has been widely used in the study of cell apoptosis. The results showed that the decrease in intracellular ATP content caused by DCZ3301, after adding z-VAD (20μM), reversed this process to varying degrees, and the decrease in cellular ATP content was greatly weakened (Figure 1D), indicating that DCZ3301 inhibited HUVEC The cell viability is due to DCZ3301 promoting HUVEC cell apoptosis, which was further verified in the Western blot test. As shown in Figure 1E, in the group without z-VAD, sheared PARP bands can be seen, indicating that apoptosis has occurred; while in the group with z-VAD, PARP is not hydrolyzed, indicating that apoptosis is inhibited NS. It can be seen that DCZ3301 can promote the spontaneous apoptosis of HUVEC cells and is dependent on the Caspase pathway.

In addition, as shown in Figure 1F, after the same concentration of DCZ3301 treatment, the HUVEC group, the ATP content was significantly reduced, but after adding z-VAD, the ATP content was increased; in the Hela group, although the ATP content was significantly reduced, but adding z- VAD cannot reverse this process; in the HT29 group, DCZ3301 has no effect on HT29. These sets of data indicate that DCZ3301 has no effect on promoting apoptosis of these two types of epithelial cells, which also means that DCZ3301 has selective specificity.

Based on the above results, DCZ3301 induces spontaneous apoptosis of HUVEC in a way that depends on the Caspase pathway.

Example 2 The effect of DCZ3301 on the proliferation, migration and tube formation behavior of HUVEC

experimental method:

2.1 BrdU/PI double staining flow cytometry analysis

5-bromo-2'-deoxyuridine (BrdU) is a thymidine analogue, which can be incorporated into newly synthesized DNA during DNA synthesis. The FITC-conjugated anti-BrdU antibody can be used DNA replication and cell proliferation are detected. Propidium Iodide (PI) is an analogue of ethidium bromide, which can be inserted into double-stranded DNA. The red fluorescence of PI can detect the content of DNA. Through BrdU/PI double-staining flow cytometry analysis, the content of cell DNA can be quantified at the same time, effectively reflecting the state of cell proliferation. The specific experimental steps are as follows:

1) Inoculate HUVEC (2×10 ⁵ pcs/dish) into a 60mm petri dish and culture for 24h;

2) Add DCZ3301 to the new M199 culture medium to a final concentration of 5μM, add an equal volume of DMSO to the control group, and continue to culture for 24h;

3) After washing with PBS, add BrdU to a new culture medium to a working concentration of 10μM, and incubate at 37°C for 2h;

4) Digest the cells with trypsin, centrifuge at 1000rpm×5min, discard the supernatant (the same below), add 5ml of pre-cooled PBS to wash the cells, centrifuge, and then resuspend the cells in pre-cooled PBS to make the cell density 1×10 ⁶ pcs/ml;

5) Add 5ml of pre-cooled ethanol to fix the cells, overnight at 4°C;

6) Centrifuge to completely remove ethanol;

7) Resuspend the cells with 1ml 2N HCl/0.5% Triton-100, and incubate at room temperature for 30 minutes to denature double-stranded DNA to form single-stranded DNA;

8) Centrifuge to remove HCl, add 1ml 0.1M Na ₂ B ₄ O ₇ PH 8.5 to resuspend the cells to neutralize acidity;

9) After centrifugation, wash the cells with 2ml of PBS solution containing 1% BSA;

10) After centrifugation, mix 0.5% Tween-20, 1% BSA-containing PBS, and FITC-conjugated anti-BrdU antibody, add the cells, place on a shaker, shake slowly, and incubate for 30 minutes at room temperature in the dark;

11) Wash with 2ml 0.5% Tween-20/1% BSA/PBS, and resuspend in 0.6ml PBS containing 5μl 2mg/ml RNase and 5μl PI (1mg/ml), and incubate for 20min;

12) Using a FACSORT flow cytometer, perform flow cytometric analysis of BrdU content (FITC) and DNA content (PI) on the cells, and cells not labeled with BrdU are used as a negative control.

2.2 Cell migration test

2.2.1 Scratch test

1) Use sterile forceps to put the Ibidi scratch experiment culture insert into the 6-well plate;

2) Inoculate HUVEC into the plug-in unit at a density of 1×10 ^{5 cells/ml, 70 μl on each side;}

3) After culturing for 24 hours, use sterile tweezers to pick up the insert and wash with PBS to remove floating cells;

4) Add DCZ3301 to 2ml M199 culture medium to a final concentration of 5μM, add an equal volume of DMSO to the control culture medium, mix well and add to a 6-well plate;

5) Put it into the living cell workstation, under the matched inverted microscope, randomly select a shooting coordinate for each hole as the 0h time point, then shoot at fixed points every 2h, and continuously cultivate and observe for 24h.

6) Perform image analysis with ImageJ software to calculate the scratch area every 2 hours to get the healing rate. The calculation formula is: healing rate (time point) = [scratch area (0h)-scratch area (time point)] /Scratch area (0h)×100%, and draw a healing curve.

2.2.2 Transwell test

1) Use a Transwell plate with 8μm pore size for experiment, add DCZ3301 (5μM) or DMSO (0.1%) to M199 medium containing 10% FBS (calculate the concentration with a final volume of 800μl per well), actually take 600μl and add it to the lower chamber ；

2) Select HUVEC in the logarithmic growth phase, digest with trypsin, pipette into cell suspension with M199 medium without FBS, and dilute to a cell density of 1.5×10 ⁵ cells/ml, and add 200μl of cell suspension to it Go to the upper room of Transwell and incubate for 24 hours;

3) Discard the upper chamber liquid and move the chamber to a new 24-well plate, fix it with 4% PFA for 10 minutes, rinse with PBS, and place the chamber upside down on absorbent paper to air dry;

4) Prepare 0.1% crystal violet staining solution (prepared for current use), filtered and stored away from light;

5) After the chamber is completely dry, place it in a 24-well plate containing crystal violet and dye for 20 minutes in the dark;

6) Rinse the cell with double-distilled water to wash away the crystal violet, gently wipe off the unmigrated cells at the bottom of the upper chamber with a cotton swab, and then rinse with double-distilled water;

7) Shooting under an inverted microscope, five fields of view are randomly selected for each film;

8) Count the number of migrating cells with ImageJ software.

2.3 Tube test

1) De-growth factor Matrigel is melted overnight at 4°C, and the 96-well plate and pipette tips are pre-cooled in advance;

2) In the ultra-clean workbench, place the 96-well plate flat on the ice box, and slowly add 60μl of liquid Matrigel to each well to avoid bubbles when adding;

3) Place the 96-well plate in the incubator and incubate for 30 minutes to solidify the Matrigel;

4) Select HUVEC in the logarithmic growth phase, after trypsinization, prepare a single cell suspension with M199 medium containing DCZ3301 (5μM) or DMSO (0.1%), and adjust the cell density to 2×10 ⁵ cells/ml ；

5) 100μl/well is evenly seeded in a 96-well plate and placed in a cell incubator for incubation;

6) 4h later, observe the luminal structure under an inverted phase contrast microscope, and photograph the central field of view of each hole;

Use ImageJ software to analyze and count the total branch length in each figure.

Experimental results:

BrdU/PI double staining flow cytometry was used to study whether DCZ3301 affects the proliferation of HUVEC. HUVEC without DCZ3301 treatment, DNA replication is not affected, showing an inverted U-shaped structure; while HUVEC treated with DCZ3301, DNA replication and cell cycle are significantly inhibited, showing an irregular shape (Figure 2A), It shows that DCZ3301 inhibits the proliferation of HUVEC.

The migration of endothelial cells is very important for the formation of new blood vessels. The scratch test and Transwell test were carried out to explore the influence of DCZ3301 on the migration behavior of HUVEC. After DCZ3301 5μM treatment, the healing area of HUVEC cells at 12h was significantly less than that of the control group. The curve of the healing rate showed that the DCZ3301 group was lower than the control group, and there was a linear relationship between time and healing area (Figure 2B). In the Transwell test, it was found that the number of cells in the DCZ3301 group that migrated to the Transwell compartment membrane was significantly less than that of the control group (Figure 2C). These two experiments show that DCZ3301 inhibits the migration behavior of HUVEC.

In addition, the effect of DCZ3301 on the sprouting behavior of blood vessels was studied through a tube test. After 4 hours of incubation, the lumen-like structure in the DCZ3301 group was significantly less than that in the control group; then the total branch length of the two groups was measured, and the total branch length was significantly reduced after DCZ3301 treatment (Figure 2D), indicating that DCZ3301 inhibited HUVEC的成管行为。

The above experiments confirmed that DCZ3301 inhibited the proliferation, migration and tube formation of HUVEC, showing anti-angiogenesis characteristics.

Example 3: The influence of DCZ3301 on PI3K/AKT and ERK1/2 signal pathways

experimental method:

3.1 The protein extraction method and the Western blot operation method are the same as in the above experimental example 1.

Experimental results:

In the process of DCZ3301 inducing HUVEC cell apoptosis, the expression of several key proteins was detected. The PI3K/AKT pathway is a very classic signaling pathway, which is closely related to cell transformation, tumor occurrence and metastasis, and has been proven to play an important role in regulating the formation of new blood vessels. After 24 hours of treatment with different concentrations of DCZ3301, the total protein of HUVEC cells was extracted, and the expression level of this pathway was detected by Western blot test. The results showed that as the concentration of DCZ3301 increased, the expression of phosphorylated PI3K and phosphorylated AKT gradually decreased, showing a dose-dependent relationship (Figure 3A), indicating that DCZ3301 inhibited the PI3K/AKT signaling pathway of HUVEC.

The ERK1/2 signaling pathway regulates the proliferation, migration, arterial differentiation and vascular homeostasis of endothelial cells. Therefore, the expression of p-ERK1/2 was further tested. Compared with the control group, DCZ3301 inhibited ERK1/2 in a dose-dependent manner. The phosphorylation of ERK1/2 inhibits the ERK1/2 signaling pathway (Figure 3B).

Therefore, DCZ3301 induced HUVEC cell apoptosis by inhibiting PI3K/AKT and ERK1/2 signaling pathways.

Example 4 The effect of DCZ3301 on mouse choroidal microvessel sprouting and corneal neovascularization

experimental method:

4.1. Construction of an in vitro model of mouse choroidal microvascular sprouting:

1) Take 6-8 weeks old male BALB/c mice, sacrifice them with cervical dislocation after anesthesia, take out the eyeballs, and immediately store them in pre-chilled DME/F-12 1:1 culture medium;

2) Under a microscope and operating on ice, cut off the fascia and extraocular muscles on the surface of the eyeball, remove the cornea, lens, iris, ciliary body and other anterior segment tissues, separate the retinal neuroepithelial layer, and leave the retinal pigment epithelium layer (retinal pigment epithelium, RPE)-choroid-sclera complex. Since the choroidal blood vessels near the optic disc grow slowly, and the peripheral choroidal blood vessels grow too fast, the choroidal complexes in this experiment are all taken from the equator. The RPE-choroid-sclera complex is cut into a size of 1mm×1mm;

3) Take 40 μL of de-growth factor matrigel and add it to a pre-cooled 24-well plate, and place the RPE-choroid-sclera graft in the center of the matrigel;

4) After the rest of the graft is placed, put the 24-well plate in the cell culture box and place it at 37°C for 10 minutes to polymerize the liquid matrigel into a solid state;

5) Add 500μL of DME/F-12 1:1 culture medium containing 10% FBS and 1% penicillin/streptomycin to each well, put it in the cell culture box (day 0), and change the medium every 24h;

6) Add 0.1% DMSO or DCZ3301 (5μM) on the 3rd day, n=6; observe on the 6th day, take pictures under an inverted phase contrast microscope;

7) Measure the area using ImageJ software, the sprouting microvessel area = total area-central graft area.

4.2. Construction and medication method of Alkali burn-induced corneal neovascularization model

1) Immerse a circular filter paper sheet with a diameter of about 2mm into 1M NaOH solution;

2) Anesthetize the mice, select the right eye of each mouse for alkali treatment;

3) Instill 0.4% Obucaine Hydrochloride Eye Drops on the surface of the cornea for local analgesia;

4) Operate under an ophthalmic operating microscope, use sterile forceps to attach the filter paper soaked in NaOH to the center of the cornea for 30 seconds;

5) After removing the filter paper, immediately rinse the eyeball with 20ml PBS to wash away the remaining NaOH solution;

6) Apply ofloxacin ointment to prevent infection;

7) Dilute DCZ3301 in 0.9% normal saline to prepare eye drops of 50μM and 200μM. The control group was dissolved in normal saline with DMSO (n=6 per group), and the other 6 mice without alkali burn treatment were used alone Physiological saline treatment; medication was administered from the day of modeling, and each mouse was given eye drops 3 times a day, 5 μl each time, for 7 consecutive days.

4.3 Photographs of the anterior segment and production of corneal tiles

4.3.1 Photograph of anterior segment

In order to record the morphology of corneal neovascularization in mice, on the 7th day after modeling, the mice were anesthetized and placed in front of a slit lamp, and the corneas of the mice were photographed with a connected digital camera under white light.

4.3.2 Corneal tiles

FITC-dextran (FITC-dextran, 2000kDa) was dissolved in PBS at a concentration of 50mg/ml and stored in the dark. Under anesthesia, fix the limbs on the operating table. After opening the chest cavity, a 1ml syringe was inserted into the apex of the mouse's heart, and 0.5ml of FITC-dextran was slowly perfused for 3 minutes. The eyeballs were removed, put in 4% PFA and fixed at room temperature and protected from light for 1 hour, and then transferred to PBS. Then the eyeball was dissected under an ophthalmological microscope, an incision was made 2mm behind the limbus cornea to preserve the blood vessels of the limbus cornea, the anterior segment of the eye was dissected and separated, and the iris and exudate membrane adhered to the inner surface of the cornea were removed. The cornea is cut into four petals, cut into a "four-leaf clover" shape, and then transferred to a glass slide, the water on the glass slide is absorbed by absorbent paper, an anti-fluorescence quenching mount is dropped, and a cover glass is placed. After mounting the slide, observe and photograph the morphology of corneal neovascularization with a fluorescence microscope. Use ImageJ software for quantitative analysis, measure the area of corneal neovascularization and the area of the entire cornea, and calculate the proportion of neovascularization area.

4.4 Statistical analysis

Using SPSS 20.0 statistical software, the data results are expressed as mean±standard deviation, and the quantified data of each experimental group are in accordance with the normal distribution and showed uniform variance after inspection. Paired t test was used for comparison between two groups, and one-way ANOVA was used for comparison between multiple groups. P<0.05 was considered as statistically significant.

Experimental results:

In this experiment, BALB/c mice were used to construct an in vitro model of choroidal microvascular sprouting and an alkaline burn-induced corneal neovascularization (CNV) model. The choroidal microvessel sprouting test is an effective and reproducible in vitro angiogenesis test, which can effectively evaluate the effect of DCZ3301 on microangiogenesis. 5μM DCZ3301 was added on the 3rd day, and the results were recorded on the 6th day. As shown in Figure 4A, compared with the control group, DCZ3301 significantly inhibited the sprouting area of choroidal microvessels. In the corneal neovascularization model, the formation of CNV can be observed on the second day after modeling, and it gradually grows from the limbus to the center of the cornea. On the seventh day, different groups of anterior segments were taken. Compared with the unmade models, corneal clouding and neovascularization were observed in all models. There were no corneal epithelial defects, corneal ulcers, corneal perforations and infections in these mice. Since alkali burns also induced the formation of iris neovascularization, hemorrhage in the anterior chamber was observed in a small number of mice. The image on day 7 showed that compared with the DMSO group, DCZ3301 (50 μM group and 200 μM) had a significant reduction in new blood vessels (Figure 4B).

The apex of mice was perfused with FITC-dextran, the mouse’s eyeballs were dissected and corneal tiles were made, and the area of CNV on the corneal tiles was collected to further quantify the effect of DCZ3301 on corneal neovascularization (Figure 4B). Through ImageJ software, the area of CNV and the area of the entire cornea were measured, and the proportion of CNV was calculated. Compared with the DMSO group, the area of CNV in the DCZ3301 (50μM and 200μM) group was significantly smaller (P<0.01, P<0.0001, respectively). Comparing the data of the 50μM group and the 200μM group, it was found that the postoperative CNV area difference between the two groups was not statistically significant (P=0.0577), indicating that 50μM DCZ3301 can effectively inhibit the formation of CNV in mice.

Claims

Use of the arylguanidine compound represented by the following formula 1 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for the treatment or prevention of ophthalmic diseases related to neovascularization:

in:

Q is
in,
Indicates the connection point;

R 1 and R 2 are each independently selected from an unsubstituted or substituted C6-C20 aryl group or a C3-C14 heteroaryl group, wherein the heteroaryl group has one or more heteroatoms selected from the following group: O, N Or S;

L is unsubstituted or substituted -(CH 2 ) n -, or unsubstituted or substituted
Wherein m is 0, 1, 2, 3 or 4, n is 0, 1, 2, 3 or 4, and Y is O, NH or S;

Z is hydrogen, alkyl, unsubstituted or substituted C6-C20 aryl or C3-C14 heteroaryl, wherein the heteroaryl has one or more heteroatoms selected from the group consisting of O, N or S;

Each of the substitutions in R 1 , R 2 , L and Z refers to being independently substituted by one or more substituents selected from the group consisting of halogen, hydroxy, C1-C6 alkyl, C3-C8 cycloalkyl, C1-C6 alkoxy, amino, -NH (C1-C6 alkyl), -N (C1-C6 alkyl) (C1-C6 alkyl), nitro, C2-C6 alkenyl, halogenated C1-C6 Alkyl, carbonyl, carboxy, amido, cyano, hydroxymethyl, halogenated C1-C6 alkoxy, mercapto, or sulfamoyl, phenyl, phenoxy.
The use according to claim 1, wherein R 1 and R 2 are each independently selected from unsubstituted or substituted C6 aryl or C5-C6 heteroaryl, wherein the heteroaryl has one or more selected from Heteroatoms of the following group: O, N or S; the substitution refers to substitution by halogen or amide;

L is
Or -(CH 2 )n-, where m is 0, n is 0, and Y is O, ΝΗ or S;

Z is tert-butyl, unsubstituted or substituted C6 aryl or unsubstituted or substituted C5-C6 heteroaryl, wherein the heteroaryl has one or more heteroatoms selected from the group consisting of O, N or S; The substitution refers to substitution by one or more substituents selected from the group consisting of halogen, C1-C6 alkyl, C1-C6 alkoxy, and halogenated C1-C6 alkyl.
The use according to claim 1, wherein R 1 and R 2 are each independently selected from unsubstituted or substituted C6 aryl or C5-C6 heteroaryl, wherein the heteroaryl has one or more selected from Heteroatoms of the following group: O, N or S; the substitution refers to substitution by halogen or amide;

L is
Or -(CH 2 )n-, where m is 0, n is 0, and Y is O;

Z is an unsubstituted or substituted C6 aryl group or an unsubstituted or substituted C5-C6 heteroaryl group, wherein the heteroaryl group has one or more heteroatoms selected from the group consisting of O, N or S; Substitution refers to substitution by one or more substituents selected from the group consisting of halogen, C1-C6 alkoxy, and halogenated C1-C6 alkyl.
The use according to claim 1, wherein the arylguanidine compound represented by formula 1 is selected from any one of the compounds represented by the following structural formulas:
The use according to claim 1, wherein the arylguanidine compound represented by formula 1 or a pharmaceutically acceptable salt thereof treats or prevents ophthalmic diseases related to neovascularization by inhibiting corneal neovascularization.