CN113577066A - Use of arylguanidine compounds or pharmaceutically acceptable salts thereof - Google Patents

Use of arylguanidine compounds or pharmaceutically acceptable salts thereof Download PDF

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CN113577066A
CN113577066A CN202010362195.XA CN202010362195A CN113577066A CN 113577066 A CN113577066 A CN 113577066A CN 202010362195 A CN202010362195 A CN 202010362195A CN 113577066 A CN113577066 A CN 113577066A
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substituted
heteroaryl
unsubstituted
dcz3301
alkyl
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CN113577066B (en
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李波
赵晨
许凯
徐志建
颜标
张勇
蔡婷婷
朱维良
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Shanghai Institute of Materia Medica of CAS
Eye and ENT Hospital of Fudan University
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Eye and ENT Hospital of Fudan University
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    • A61K31/00—Medicinal preparations containing organic active ingredients
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    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
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    • A—HUMAN NECESSITIES
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    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
    • A61K31/444—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a six-membered ring with nitrogen as a ring heteroatom, e.g. amrinone
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    • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D213/60—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
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Abstract

The invention relates to an application of an arylguanidine compound shown in a formula 1 or a pharmaceutically acceptable salt thereof, in particular to an application of an arylguanidine compound shown in a formula 1 or a pharmaceutically acceptable salt thereof in preparing a medicament for preventing or treating angiogenesis-related ophthalmic diseases.

Description

Use of arylguanidine compounds or pharmaceutically acceptable salts thereof
Technical Field
The invention relates to the field of medicinal chemistry, in particular to application of an arylguanidine compound or a pharmaceutically acceptable salt thereof in preparing a medicament for treating or preventing angiogenesis-related ophthalmic diseases.
Background
Pathological neovascularization occurs in a variety of ocular tissues, including the retina, choroid, and cornea. It is a major cause of vision impairment due to numerous ocular diseases, such as Diabetic Retinopathy (DR), age-related macular degeneration (AMD), and keratitis. Corneal neovascularization (CoNV) typically occurs in inflammatory or infectious ocular surface diseases, and CoNV can cause scarring, edema, and inflammation of the cornea, ultimately resulting in and worsening of vision. Therefore, inhibition of corneal neovascularization is an important strategy for preventing or treating related ocular surface diseases.
Disclosure of Invention
The invention aims to provide application of aryl guanidine compounds or pharmaceutically acceptable salts thereof in preparing medicines for preventing or treating angiogenesis-related ophthalmic diseases.
The invention provides an application of an arylguanidine compound shown as a formula 1 or a pharmaceutically acceptable salt thereof in preparing a medicament for treating or preventing angiogenesis-related ophthalmic diseases:
Figure BDA0002475387400000011
wherein:
q is
Figure BDA0002475387400000012
Wherein the content of the first and second substances,
Figure BDA0002475387400000013
representing a joint;
R1、R2each independently selected from unsubstituted or substituted C6-C20 aryl or C3-C14 heteroaryl, wherein the heteroaryl has one or more heteroatoms selected from: o, N or S;
l is unsubstituted or substituted- (CH)2)n-, or unsubstituted or substituted
Figure BDA0002475387400000014
Wherein m is 0, 1, 2, 3 or 4, n is 0, 1, 2, 3 or 4, 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;
R1、R2each said substitution in L and Z is independently substituted with 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, haloC 1-C6 alkyl, carbonyl, carboxy, amido, cyano, hydroxymethyl, haloC 1-C6 alkoxy, mercapto, or sulfamoyl, phenyl, phenoxy.
In an embodiment, R1、R2Each independently selected from unsubstituted or substituted C6 aryl or C5-C6 heteroaryl, wherein said heteroaryl has one or more heteroatoms selected from: o, N or S; the substitution is substituted by halogen or amido;
l is
Figure BDA0002475387400000022
Or- (CH)2) n-, wherein m is 0, n is 0, Y is O, Ν oh, 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 is substituted by one or more substituents selected from the group consisting of: halogen, C1-C6 alkyl, C1-C6 alkoxy, halogenated C1-C6 alkyl.
In an embodiment, R1、R2Each independently selected from unsubstituted or substituted C6 aryl or C5-C6 heteroaryl, wherein said heteroaryl has one or more heteroatoms selected from: o, N or S; the substitution is substituted by halogen or amido;
l is
Figure BDA0002475387400000023
Or- (CH)2) n-, wherein m is 0, n is 0, and Y is O;
z is 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 is substituted by one or more substituents selected from the group consisting of: halogen, C1-C6 alkoxy, halogenated C1-C6 alkyl.
In an embodiment, the arylguanidine compound of formula 1 is selected from any one of the compounds represented by the following structural formulae:
Figure BDA0002475387400000021
Figure BDA0002475387400000031
the synthesis of specific compounds can be found in CN 201610107774.3. Compound 1, numbered 1 above, is also referred to below as DCZ 3301.
In an embodiment, the arylguanidine compound of formula 1 or a pharmaceutically acceptable salt thereof treats or prevents a neovascularization-associated ophthalmic disease by inhibiting corneal neovascularization.
Advantageous effects
The aryl guanidine compound of the invention is found to have anti-angiogenesis activity through activity test on an ophthalmic disease model, which indicates that the compound has potential application in preventing or treating angiogenesis-related ophthalmic diseases.
Drawings
FIGS. 1A-1F show that DCZ3301 reduces Human Umbilical Vein Endothelial Cell (HUVEC) cell activity and induces spontaneous apoptosis of HUVEC. Chemical structure of dcz3301 (compound 1); ATP content of HUVEC 24h and 48h after dcz3301 treatment; expression levels of cleaved PARP and cleaved Caspase-3 proteins in HUVECs 24h after DCZ3301 treatment; 1D.z-VAD can reduce the decrease of HUVEC ATP content; 1e. clipping PARP protein expression levels in HUVEC after addition of z-VAD; 1F.z-VAD inhibited the decrease in HUVEC ATP levels caused by DCZ3301, while Hela and HT29 cells did not inhibit the decrease in HUVEC ATP levels caused by DCZ 3301.
FIGS. 2A-2D show that DCZ3301 can inhibit the proliferation, migration, and tube-forming behavior of HUVEC. BrdU/PI double staining flow cytometry analysis; scratch test, DCZ3301 inhibited the healing process of HUVEC (n ═ 3), scale bar: 250 μm; transwell assay, DCZ3301 reduced the number of transmembrane cells (n-3), scale bar: 100 μm, P < 0.0001; dcz3301 inhibited HUVEC luminal formation, decreasing branch length (n-3), scale bar: 100 μm, P < 0.01.
FIGS. 3A-3B show that DCZ3301 inhibited the PI3K/AKT and ERK1/2 signaling pathways. p-PI3K, p-AKT and p-ERK1/2 expression levels in HUVEC 24h after DCZ3301 treatment; 3B.p-PI3K, p-AKT and p-ERK1/2 <0.05, <0.01, <0.001, < 0.0001.
FIGS. 4A-4B show that DCZ3301 inhibited choroidal microvascular sprouting and corneal neovascular area following alkali burn. Day 6, two images of choroidal microvascular sprouting (n ═ 6), scale bar: the thickness of the film is 250 mu m,**p<0.01; anterior segment photography of mouse cornea (n-6 per group) and corneal tile images after FITC-dextran apex perfusion (n-6), scale bar: 250 μm; in corneal applanation, the ratio of corneal neovascularization area to total corneal area (n-6) p<0.01、****p<0.0001。
Detailed Description
The invention will now be further illustrated, but is not limited, by the following specific examples.
Example (b):
material
Laboratory animal
Inbred BALB/c mice, male, 6-8 weeks old, and 20-30g in weight were purchased from Nanjing university of medical laboratory animal center (Nanjing, China). The mice are all raised in animal rooms of eye-ear-nose-throat hospitals affiliated to the university of Compound Dan, the light and shade cycles are carried out for 12 hours, the raising environment temperature is maintained at 22 +/-2 ℃, the relative humidity is 40-70%, and sufficient mouse food and drinking water are provided. Animals were anesthetized by intraperitoneal injection at doses of ketamine (80mg/kg) and xylazine (10mg/kg), and mice were sacrificed by cervical dislocation after completion of the experiment. The procedures of animal experiments in the study were all reviewed by the animal ethics committee of the eye-nose-throat hospital affiliated, university of redden, and followed the declaration of the Association for Research in Vision and Ophthalmology, ARVO, on animal experiments.
The main reagents are listed in table 1 below.
TABLE 1
Figure BDA0002475387400000041
Figure BDA0002475387400000051
Figure BDA0002475387400000061
The main instrumentation is listed in table 2 below.
TABLE 2
Figure BDA0002475387400000062
Example 1 Effect of DCZ3301 on the cellular Activity of HUVEC and spontaneous apoptosis of HUVEC
1.1 cell culture
Human Umbilical Vein Endothelial Cells (HUVEC) were purchased from a double denier IBS cell bank, and a Human cervical cancer cell line HeLa and a Human colorectal adenocarcinoma cell line HT29 were purchased from a China academy of sciences type culture Collection cell bank. HUVEC were cultured in M199 culture medium containing 10% fetal bovine serum, HeLa and HT29 were cultured in DMEM culture medium containing 10% fetal bovine serum, and the incubator was maintained at 37 ℃ and 5% CO2The environment of (2). Digestion was performed with 0.25% trypsin and cells were passaged in either 1:3 or 1:4 fashion.
1.2 cell Activity assays
CellTiter-
Figure BDA0002475387400000063
The method for measuring the activity of the luminous cells is a method for measuring the number of cells with metabolic activity based on the content of ATP in the cells. The specific operation is as follows:
1) taking HUVEC cells in logarithmic growth phase, spreading the cell suspension in a 96-well plate, and setting 6 wells in each group;
2) when cell growth was confluent to 90%, different concentrations of DCZ3301 or 0.1% DMSO were added, and according to
Adding z-VAD to the working concentration of 20 mu M, and continuing to culture for 24 or 48 hours;
3) add 25. mu.l of the prepared CellTiter-Glo reagent into each well;
4) shaking on shaking table for 12min at room temperature, mixing well and lysing cells;
5) each well sucks 100 mul of liquid and transfers to a light-tight 96-well plate;
6) the luminescence intensity was read in a microplate reader (unit: light intensity).
7) The luminosity was recorded, the relative percentage of each group was calculated against the 0.1% DMSO group and a histogram was plotted.
1.3 protein extraction and concentration determination
1.3.1 extraction of Total cellular protein:
the cells were scraped off, centrifuged, rinsed with PBS, and centrifuged. Adding RIPA lysate containing protease inhibitor, phosphatase inhibitor and PMSF, placing on ice, and cracking for 20min, and blowing once every 5 min. The supernatant was then centrifuged at 12000rpm 10min at 4 ℃ and transferred to a new EP tube.
1.3.2 BCA method protein quantification:
1) mixing the solution A and the solution B in the BCA kit according to the required hole number according to the ratio of 50: 1;
2) the standard protein BSA is diluted to 0.5 mg/ml;
setting 8 holes in a 96-well plate for standard curve preparation, setting 3 holes as blank control, and adding a sample;
3) incubating at 37 deg.C for 30 min;
4) detecting with enzyme-labeling instrument at wavelength of 562nm, and reading absorbance;
5) drawing a standard curve according to the measured numerical values and obtaining a calculation formula, and then calculating the concentration of each sample;
6) diluting each sample to 2mg/ml with loading buffer solution, and filling the part with insufficient volume with PBS;
7) boiling at 100 deg.C for 10min, and storing at-80 deg.C.
1.4 Western blot assay (Western blot)
1.4.1 preparation of glue
1) Selecting a glass plate with the specification of 1.5mm, washing the glass plate with double distilled water, drying and fixing the glass plate on a sponge base;
2) the gel was prepared using the Bio-rad FastCast premix acrylamide solution kit, adding for each gel: 4ml of the solution A, 4ml of the solution B, 40 mul of 10% ammonium persulfate and 4 mul of TEMED, mixing uniformly, adding into a glass plate, and flattening the liquid surface by double distilled water;
3) standing at room temperature, pouring out double distilled water when an obvious boundary line between the separation gel and water appears, and sucking the double distilled water by using filter paper;
4) preparing concentrated glue, adding each concentrated glue: 1.5ml of concentrated solution A +1.5ml of concentrated solution B + 15. mu.l of 10% ammonium persulfate + 3. mu.l of TEMED; and (3) uniformly mixing, adding a glass plate, inserting a comb with the specification of 1.5mm, and standing at room temperature until the mixture is completely solidified.
1.4.2 electrophoresis
1) Fixing the rubber plate in an electrophoresis tank, pouring electrophoresis liquid, pulling out a comb, and washing out residual rubber in the lane;
2) adding a proper marker according to different target bands, loading the sample by 20 or 30ug, loading the sample with equal concentration and equal volume, and filling the rest lanes with 1 multiplied sample loading buffer solution;
3) the electrophoresis tank is connected with an electrophoresis apparatus, the constant voltage is 80V, and the constant voltage is 120V after the strips run to the separation gel.
1.4.3 transfer film
1) Preparing a membrane transferring solution (1L containing 200ml of methanol), and burying in ice to be cooled;
2) cutting the PVDF membrane to a proper size, and soaking in methanol for 30 min;
3) pouring the membrane transferring liquid into a tray, taking out the separation gel from the electrophoresis tank, cutting, and installing a membrane transferring sandwich: the blackboard, the sponge, the 3 layers of filter paper, the separation glue, the PVDF membrane, the 3 layers of filter paper, the sponge and the transparent plate are soaked in the film transfer liquid in the whole process, bubbles are removed, and the blackboard, the sponge, the PVDF membrane, the 3 layers of filter paper, the sponge and the transparent plate are placed in a film transfer groove after being clamped;
4) constant current of 250mA, 80 min-100 min.
1.4.4 immunoassay and ECL chemiluminescence
1) And (3) sealing: preparing confining liquid (2.5g of skimmed milk powder dissolved in 50ml of 1 XTSST), taking out a PVDF membrane, cutting to a proper size, putting into a dressing box, and standing on a shaking table at room temperature for 1 h;
2) primary anti-reaction: washing the membrane with TBST, diluting with 5% BSA according to the antibody titer, and reacting at 4 ℃ overnight;
3) secondary antibody reaction: washing the membrane with TBST, diluting the secondary antibody marked with HRP with a confining liquid, standing the secondary antibody on a shaking table at room temperature for 1h, and washing the membrane with TBST after the reaction is finished;
4) ECL chemiluminescence detection: preparing a developer, and keeping out of the sun after mixing the solution A and the solution B of the ECL luminescence kit in a ratio of 1: 1; spreading the PVDF film on a flat plate of a chemiluminescence imaging instrument, absorbing liquid on the film by absorbent paper, dripping a developer, and performing ECL chemiluminescence imaging. Quantitative analysis was performed by analyzing the band gray values using Image J (version 1.8.0) software.
The experimental results are as follows:
the relative molecular mass of DCZ3301 was 464.0 (fig. 1A). Using CellTiter-
Figure BDA0002475387400000081
The kit for detecting the activity of the luminous cells evaluates the activity of the cells by detecting the content of ATP in the cells, and calculates the percentage of each group by taking the luminosity of a 0.1% DMSO group as a reference. The minimum effective concentration of DCZ3301 was 1. mu.M, with increasing concentration, the lower the degree of cell activity; and over time, 48h of cells were less active compared to 24h data, indicating that DCZ3301 inhibited the activity of the cells in a time and dose dependent manner (fig. 1B).
The Caspase (Caspase) family is a key regulatory molecule of apoptosis, acting in a cascade. Wherein Caspase-3 is located at downstream, and is an important executive molecule, and when Caspase is cleaved and activated, it can cleave and activate target protein. Poly (ADP-ribose) polymerase (PARP) is the most important substrate for Caspase-3, and plays an important role in DNA repair and cell stability. After apoptosis is started, the PARP of 116kD is cut into two fragments of 89kD and 24kD to lose enzyme activity, accelerate the instability of cells and finally cause apoptosis. Caspase-3 and PARP hydrolysis are considered to be important markers of apoptosis, and therefore, here, the expression levels of these two proteins in HUVEC cells were examined. In the Western blot results, the expression of cleaved PARP and cleaved Caspase-3 was increased with increasing DCZ3301 concentration (FIG. 1C), which is consistent with the data from the cell activity assay in a dose-dependent manner, and the surface DCZ3301 induced apoptosis of HUVEC.
In addition, Caspase inhibitors z-VAD-FMK (hereinafter referred to as z-VAD) were further used for identification. The z-VAD is a broad-spectrum Caspase inhibitor, can effectively prevent Caspase activation in the process of apoptosis, and is widely applied to the research of apoptosis. The decrease in intracellular ATP levels caused by DCZ3301 was found to reverse this process to varying degrees after the addition of z-VAD (20 μ M), and the magnitude of the decrease in cellular ATP levels was greatly attenuated (FIG. 1D), indicating that DCZ3301 inhibits HUVEC cellular activity because DCZ3301 promotes HUVEC apoptosis, as further confirmed in the Western blot assay. As shown in FIG. 1E, in the non-z-VAD group, a sheared PARP band was observed, indicating that apoptosis had occurred; in the group with the addition of z-VAD, PARP was not hydrolyzed, indicating that apoptosis was inhibited. It can be seen that DCZ3301 promotes spontaneous apoptosis in HUVEC cells and is a Caspase pathway dependent approach.
In addition, as shown in FIG. 1F, after treatment with the same concentration of DCZ3301, ATP levels were significantly reduced in the HUVEC group, while ATP levels were increased after the addition of z-VAD; in the Hela group, although the ATP content is obviously reduced, the addition of z-VAD can not reverse the process; in the HT29 group, DCZ3301 had no effect on HT 29. These data demonstrate that DCZ3301 has no apoptotic promoting effect on both epithelial cells, which also means that DCZ3301 has selective specificity.
Taken together, DCZ3301 induced spontaneous apoptosis of HUVEC in a Caspase-dependent manner.
Example 2 Effect of DCZ3301 on the proliferation, migration and tubulogenesis behavior of HUVECs
The experimental method comprises the following steps:
2.1 BrdU/PI double staining flow cytometry
5-bromo-2' -deoxyuracil (BrdU) is a thymidine analog that can be incorporated into newly synthesized DNA during DNA synthesis, and DNA replication and cell proliferation can be detected using FITC-conjugated anti-BrdU antibodies. Propidium Iodide (PI) is an ethidium bromide analog and can be inserted into double-stranded DNA, and the content of the DNA can be detected by red fluorescence of the PI. Through BrdU/PI double staining flow cytometry analysis, the content of cell DNA can be quantified simultaneously, and the cell proliferation state can be effectively reflected. The specific experimental steps are as follows:
1) mixing HUVEC (2X 10)5One/dish) is inoculated into a 60mm culture dish and cultured for 24 hours;
2) adding DCZ3301 to a new M199 culture solution to a final concentration of 5. mu.M, adding an equal volume of DMSO to the control group, and continuing to culture for 24 h;
3) after PBS washing, adding BrdU to a new culture solution until the working concentration is 10 mu M, and incubating for 2h at 37 ℃;
4) digesting the fine particles with trypsinCells, centrifugation 1000rpm x 5min, then supernatant (same below) discarded, 5ml of pre-cooled PBS was added to wash the cells, centrifugation was performed, and the cells were resuspended in pre-cooled PBS to a cell density of 1 x 106Per ml;
5) adding 5ml of precooled ethanol to fix the cells, and standing overnight at 4 ℃;
6) centrifuging to completely remove ethanol;
7) resuspend cells with 1ml 2N HCl/0.5% Triton-100 and incubate at room temperature for 30min to denature double stranded DNA into single stranded DNA;
8) the HCl was removed by centrifugation and 1ml of 0.1M Na was added2B4O7Resuspend cells at pH 8.5 to neutralize acidity;
9) after centrifugation, cells were washed with 2ml of 1% BSA in PBS;
10) after centrifugation, 0.5% Tween-20, PBS containing 1% BSA, and FITC-conjugated anti-BrdU antibody were mixed and added to the cells, which were slowly shaken on a shaker and incubated in the dark at room temperature for 30 minutes;
11) washed with 2ml of 0.5% Tween-20/1% BSA/PBS and resuspended in 0.6ml PBS containing 5. mu.l of 2mg/ml RNase and 5. mu.l of PI (1mg/ml), incubated for 20 min;
12) the cells were flow cytometrically analyzed for BrdU content (FITC) and DNA content (PI) using a facsrot flow cytometer, with cells not labeled with BrdU as negative controls.
2.2 cell migration assay
2.2.1 scratch test
1) Placing the Ibidi scratch experiment culture insert into a 6-hole plate by using sterile forceps;
2) HUVEC at 1X 105The individual/ml density was inoculated into the insert, 70. mu.l each on either side;
3) after 24h incubation, the inserts were nipped with sterile forceps and washed with PBS to remove floating cells;
4) adding DCZ3301 into 2ml M199 culture solution to the final concentration of 5 μ M, adding DMSO with the same volume into the control group culture solution, mixing well, and adding into 6-well plate;
5) placing into a living cell workstation, randomly selecting a shooting coordinate for each well under a matched inverted microscope to be used as a 0h time point, then shooting at a fixed point every 2h, and continuously culturing and observing for 24 h.
6) Image analysis is performed through ImageJ software, and the scratch area of every 2 hours is calculated to obtain the healing rate, wherein the calculation formula is as follows: the healing rate (time point) — [ scratch area (0h) -scratch area (time point) ]/scratch area (0h) × 100%, and a healing curve was plotted.
2.2.2 Transwell test
1) Experiments were performed using Transwell plates with 8 μ M pore size, DCZ3301(5 μ M) or DMSO (0.1%) was added to M199 medium containing 10% FBS (concentration calculated as 800 μ l final volume per well) and 600 μ l was actually added to the lower chamber;
2) selecting HUVEC in logarithmic growth phase, subjecting to trypsinization, blowing into cell suspension with FBS-free M199 culture medium, and diluting to cell density of 1.5 × 105Adding 200 mul of cell suspension into a Transwell upper chamber, and culturing for 24 h;
3) discarding the upper chamber liquid, moving the small chamber to a new 24-pore plate, fixing with 4% PFA for 10min, rinsing with PBS, and then placing the small chamber on absorbent paper for natural air drying;
4) preparing 0.1% crystal violet staining solution (used as prepared) and storing in dark after filtering;
5) after the small chamber is completely dried, placing the small chamber in a 24-pore plate containing crystal violet, and dyeing for 20min in a dark place;
6) rinsing the chamber with double distilled water to wash out crystal violet, gently wiping off cells which are not migrated at the bottom of the upper chamber with a cotton swab, and rinsing with double distilled water;
7) photographed under an inverted microscope, five fields of view were randomly selected for each membrane;
8) the number of migrated cells was counted using ImageJ software.
2.3 tube formation test
1) Removing growth factor matrigel, melting overnight at 4 ℃, and precooling a 96-well plate and a gun head in advance;
2) in a clean bench, a 96-hole plate is flatly placed on an ice box, 60 mu l of liquid matrigel is slowly added into each hole, and bubbles are prevented from being generated during adding;
3) incubating the 96-well plate in an incubator for 30min to solidify the matrigel;
4) HUVEC in logarithmic growth phase were selected, and after trypsinization, single cell suspensions were prepared in M199 medium containing DCZ3301 (5. mu.M) or DMSO (0.1%) and adjusted to a cell density of 2X 105Per ml;
5) uniformly inoculating 100 mu l/hole in a 96-hole plate, and putting the 96-hole plate into a cell culture box for incubation;
6) after 4h, observing the tube cavity sample structure under an inverted phase contrast microscope, and shooting the central visual field of each hole;
analysis was performed using ImageJ software and the total branch length in each graph was counted.
The experimental results are as follows:
BrdU/PI double stain flow cytometry was used to investigate whether DCZ3301 affected the proliferation of HUVEC. HUVEC without DCZ3301 treatment, DNA replication was not affected, in the form of an inverted U-shaped structure; whereas, in HUVECs treated with DCZ3301, DNA replication and cell cycle were significantly inhibited, exhibiting an irregular morphology (fig. 2A), indicating that DCZ3301 inhibited HUVEC proliferation.
The migration of endothelial cells is critical for the formation of new blood vessels, and the extent of the effect of DCZ3301 on the migration behavior of HUVECs was investigated by performing a scratch test and a Transwell test. After DCZ 33015. mu.M treatment, the healing area of 12h HUVEC cells was significantly less than the control group. The curve of the healing rate shows that the DCZ3301 group was lower than the control group and that time was linear with the area of healing (fig. 2B). In the Transwell assay, it was found that the number of cells migrated to the membrane of the Transwell chamber was significantly less in the DCZ3301 group than in the control group (FIG. 2C). These two experiments indicate that DCZ3301 inhibits the migration behavior of HUVEC.
In addition, the effect of DCZ3301 on the sprouting behavior of blood vessels was investigated by tube-forming experiments. After 4 hours of incubation, luminal-like structures were significantly less in the DCZ3301 group than in the control group; the total branch length was then measured for both groups and was significantly reduced after treatment with DCZ3301 (fig. 2D), indicating that DCZ3301 inhibited the tube-forming behavior of HUVEC.
The above experiments demonstrate that DCZ3301 inhibits the proliferation, migration and tube-forming behavior of HUVEC, presenting anti-angiogenic characteristics.
Example 3: effect of DCZ3301 on PI3K/AKT and ERK1/2 Signal pathways
The experimental method comprises the following steps:
3.1 the protein extraction method and Western blot operation method were the same as those described in Experimental example 1 above.
The experimental results are as follows:
during the process of DCZ3301 inducing HUVEC apoptosis, the expression of several key proteins was tested. The PI3K/AKT pathway is a very classical signaling pathway that is closely related to cell transformation, tumorigenesis and metastasis and has been shown to play an important role in regulating neovascularization. Total protein of HUVEC cells was extracted 24 hours after treatment with different concentrations of DCZ3301, and the expression level of this pathway was examined by Western blot assay. As a result, the expression of phosphorylated PI3K and phosphorylated AKT decreased gradually with increasing concentration of DCZ3301, showing a dose-dependent relationship (fig. 3A), indicating that DCZ3301 inhibited the PI3K/AKT signaling pathway of HUVEC.
The ERK1/2 signaling pathway regulates endothelial cell proliferation, migration, arterial differentiation, and vascular homeostasis, and thus further examined the expression of p-ERK1/2, and DCZ3301 inhibited ERK1/2 phosphorylation and ERK1/2 signaling pathway in a dose-dependent manner, as compared to the control group (FIG. 3B).
DCZ3301 therefore induced apoptosis of HUVEC by inhibiting PI3K/AKT and ERK1/2 signaling pathways.
Example 4 Effect of DCZ3301 on mouse choroidal microvascular sprouting and corneal neovascularization
The experimental method comprises the following steps:
4.1. constructing mouse choroid microvascular sprouting in vitro model:
1) taking male BALB/c mice of 6-8 weeks, killing the mice by dislocation of cervical vertebrae after anesthesia, taking out eyeballs, and immediately storing the eyeballs in precooled DME/F-121: 1 culture solution;
2) microscopically, and operating on ice, cutting out fascia and extraocular muscles on the surface of an eyeball, removing anterior segment tissues such as cornea, crystal, iris, ciliary body and the like, separating retinal nerve epithelial layer, and leaving retinal pigment epithelial layer (RPE) -choroid-sclera complex, wherein the choroid complex of the experiment is taken from the equatorial part and the RPE-choroid-sclera complex is cut into a size of 1mm × 1mm because choroid blood vessels near the near-sighted part grow slowly and choroid blood vessels at the peripheral part grow too fast;
3) adding 40 mu L of growth factor-removed matrigel into a precooled 24-pore plate, and placing the RPE-choroid-sclera transplant in the center of the matrigel;
4) after the rest of the grafts are placed, putting the 24-hole plate into a cell culture box, and placing for 10 minutes at 37 ℃ to polymerize the liquid matrigel into a solid state;
5) 500. mu.L of DME/F-121: 1 medium containing 10% FBS and 1% penicillin/streptomycin was added to each well, and the mixture was placed in a cell culture chamber (day 0) and changed every 24 hours;
6) on day 3, 0.1% DMSO or DCZ3301(5 μ M), n ═ 6; observing on day 6, and shooting under an inverted phase contrast microscope;
7) area was measured using ImageJ software, and budding microvascular area-total area-area of central graft.
4.2. Construction and medication method of cornea neogenesis blood vessel model (Alkali burn-induced cornea neovasilization model) induced by Alkali burn
1) Immersing a circular piece of filter paper with a diameter of about 2mm in a 1M NaOH solution;
2) anesthetizing the mice, and selecting the right eye of each mouse for alkali treatment;
3) dripping 0.4% oxybuprocaine hydrochloride eye drops on the surface of the cornea to relieve pain locally;
4) operating under an ophthalmic operating microscope, attaching a piece of filter paper soaked by NaOH to the center of a cornea for 30 seconds by using sterile forceps;
5) immediately washing the eyeball with 20ml of PBS after taking off the filter paper to wash away residual NaOH solution;
6) applying ofloxacin eye ointment to prevent infection;
7) DCZ3301 was diluted in 0.9% saline to make eye drops at concentrations of 50 μ M and 200 μ M, the control group was dissolved in saline with DMSO (n ═ 6 per group), and 6 mice without alkali burn treatment were treated with saline alone; the medicine is applied from the day of molding, and each mouse is dropped with 5 mul of eye 3 times a day for continuous treatment for 7 days.
4.3 anterior segment photography and corneal Patch preparation
4.3.1 anterior Ocular photographing
To record the morphology of the neovasculature of the mouse cornea, on day 7 post-molding, the mouse was anesthetized and placed in front of a slit lamp, and the mouse cornea was photographed with an associated digital camera under white light.
4.3.2 corneal Panels
FITC-dextran (FITC-dextran, 2000kDa) was dissolved in PBS at a concentration of 50mg/ml and stored in the dark. The four limbs of the mouse were fixed to the console under anesthesia. After opening the chest, a 1ml syringe was inserted into the apex of the mouse heart and FITC-dextran 0.5ml was slowly infused until 3 min. The eyeball was removed, fixed in 4% PFA at room temperature in the dark for 1h, and transferred to PBS. The eye was dissected under an ophthalmic microscope, an incision was made 2mm behind the limbus to retain the blood vessels at the limbus, the anterior segment tissue was dissected away, and the adherent iris and exudate on the inner surface of the cornea were removed. Cutting the cornea into four pieces, cutting into the shape of herba Tetrastigmatis Hypoglauci, transferring to glass slide, sucking water with absorbent paper, dripping the fluorescence quenching blocking tablet, and covering with cover glass. After mounting, the morphology of the corneal neovascularization was observed and photographed by a fluorescence microscope. And (4) performing quantitative analysis by using ImageJ software, measuring the area of the neovascular cornea and the area of the whole cornea, and calculating the proportion of the area of the neovascular cornea.
4.4 statistical analysis
The SPSS 20.0 statistical software is adopted, data results are expressed by mean +/-standard deviation, and data quantified by each experimental group are tested to be in accordance with normal distribution and uniform in variance. The two-group comparison adopts a pairing t test, the one-way ANOVA is used for comparison in the multi-group comparison, and the difference is considered to have statistical significance when the P is less than 0.05.
The experimental results are as follows:
in this experiment, a choroidal microvascular sprouting in vitro model and an alkali burn induced corneal neovascularization (hereinafter abbreviated as CNV) model were constructed using BALB/c mice. The choroidal microvascular sprouting assay is an effective and reproducible ex vivo angiogenesis assay that can effectively assess the effects of DCZ3301 on microangiogenesis. On day 3, 5 μ M DCZ3301 was added and the results were recorded on day 6. As shown in FIG. 4A, DCZ3301 significantly inhibited the area of choroidal microvascular sprouting compared to the control group. In the corneal neovascular model, CNV formation was observed on day 2 after molding and gradually grew from the limbus to the corneal center, and different sets of anterior segment photographs were taken on day 7. Corneal clouding and neovascularization were observed in all the models compared to the non-models, and corneal epithelial defects, corneal ulceration, corneal perforation and infection were not observed in these mice. Since alkali burns also simultaneously induce the formation of iris neovascularization, an atrial chamber was observed in a few mice. The day 7 images show a significant reduction in neovascularisation in DCZ3301(50 μ M and 200 μ M) compared to the DMSO group (fig. 4B).
The effect of DCZ3301 on corneal neovascularization was further quantified by perfusing the apex of the heart with FITC-dextran, dissecting the eyeball of the mouse and making a corneal tile, and collecting the area of CNV on the corneal tile (fig. 4B). The CNV area and the area of the whole cornea were measured by ImageJ software, and the proportion of CNV was calculated. Both the CNV areas were significantly smaller in the DCZ3301 (50. mu.M and 200. mu.M) groups compared to the DMSO group (P <0.01, P <0.0001, respectively). Comparing the data of the 50 μ M group and the 200 μ M group, the post-operative CNV area difference between the two groups was not statistically significant (P ═ 0.0577), indicating that 50 μ M DCZ3301 could effectively inhibit the formation of CNV in mice.

Claims (5)

1. Use of an arylguanidine compound represented by the following formula 1 or a pharmaceutically acceptable salt thereof for the preparation of a medicament for treating or preventing a angiogenesis-related ophthalmic disease:
Figure FDA0002475387390000011
wherein:
q is
Figure FDA0002475387390000012
Wherein the content of the first and second substances,
Figure FDA0002475387390000013
representing a joint;
R1、R2each independently selected from unsubstituted or substituted C6-C20 aryl or C3-C14 heteroaryl, wherein the heteroaryl has one or more heteroatoms selected from: o, N or S;
l is unsubstituted or substituted- (CH)2)n-, or unsubstituted or substituted
Figure FDA0002475387390000014
Wherein m is 0, 1, 2, 3 or 4, n is 0, 1, 2, 3 or 4, 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;
R1、R2each said substitution in L and Z is independently substituted with 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, haloC 1-C6 alkyl, carbonyl, carboxy, amido, cyano, hydroxymethyl, haloC 1-C6 alkoxy, mercapto, or sulfamoyl, phenyl, phenoxy.
2. Use according to claim 1, wherein R1、R2Each independently selected from unsubstituted or substituted C6 aryl or C5-C6 heteroaryl, wherein said heteroaryl has one or more heteroatoms selected from: o, N or S; the substitution is substituted by halogen or amido;
l is
Figure FDA0002475387390000015
Or- (CH)2) n-, wherein m is 0, n is 0, Y is O, Ν oh, 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 is substituted by one or more substituents selected from the group consisting of: halogen, C1-C6 alkyl, C1-C6 alkoxy, halogenated C1-C6 alkyl.
3. Use according to claim 1, wherein R1、R2Each independently selected from unsubstituted or substituted C6 aryl or C5-C6 heteroaryl, wherein said heteroaryl has one or more heteroatoms selected from: o, N or S; the substitution is substituted by halogen or amido;
l is
Figure FDA0002475387390000021
Or- (CH)2) n-, wherein m is 0, n is 0, and Y is O;
z is 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 is substituted by one or more substituents selected from the group consisting of: halogen, C1-C6 alkoxy, halogenated C1-C6 alkyl.
4. The use according to claim 1, wherein the arylguanidine compound represented by formula 1 is selected from any one of compounds represented by the following structural formulae:
Figure FDA0002475387390000022
Figure FDA0002475387390000031
5. the use according to claim 1, wherein the arylguanidine compound represented by formula 1 or a pharmaceutically acceptable salt thereof treats or prevents a neovascularization-associated ophthalmic disease by inhibiting corneal neovascularization.
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