CN113620932A - Preparation method of specific medicine for treating eye angiogenesis - Google Patents
Preparation method of specific medicine for treating eye angiogenesis Download PDFInfo
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- CN113620932A CN113620932A CN202110737699.XA CN202110737699A CN113620932A CN 113620932 A CN113620932 A CN 113620932A CN 202110737699 A CN202110737699 A CN 202110737699A CN 113620932 A CN113620932 A CN 113620932A
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Abstract
The invention provides a preparation method of a specific medicine for treating ocular angiogenesis, which comprises the following steps: (1) adding 1eq of sunitinib and 1eq of rhein into a reaction bottle, and adding 100ml of dichloromethane for dissolving; (2) after the reaction system is cooled to normal temperature, 1.1eq of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and 1.1eq of 1-hydroxybenzotriazole are sequentially added, and after the addition is finished, the reaction system is stirred for 10 hours at normal temperature; (3) adding water into the reaction system to quench the reaction, extracting with 50ml of ethyl acetate, washing the organic phase with 100ml of saturated NaCl, and passing through anhydrous NaSO4Drying, filtering and removing the solvent to obtain the product. The invention has the beneficial effects that:the modified drug can specifically target eyes and inhibit the formation of new blood vessels of the eyes, thereby achieving the effect of preventing or treating related diseases of angiogenesis of the eyes.
Description
Technical Field
The invention relates to the technical field of biomedicine, in particular to a preparation method of a specific medicine for treating ocular angiogenesis.
Background
There are many pathogenesis of eye diseases, and excessive angiogenesis caused by various reasons is one of the main pathological factors of eye diseases, such as retinal neovascular eye disease, corneal neovascular eye disease, iris neovascular eye disease and the like. Inhibition of angiogenesis is critical in the treatment of such diseases.
The traditional treatment method mainly adopts traditional Chinese medicine therapy, laser and other physical therapies, but the curative effect is not ideal. Blocking the growth of new blood vessels may become a new method for treating such diseases, so that angiogenesis inhibitors are a class of drugs which are of great importance for the current treatment of diseases with abnormal new blood vessels. Although these vascular inhibitors currently on the market present great promise, their drawbacks are also quite evident: the traditional medicines for inhibiting angiogenesis, such as sunitinib, apatinib and the like, have non-specific action targets, low specificity and selectivity on tissue-specific blood vessels and limited effect, so the dosage is large and large side effect is easy to cause. Thus, a good anti-angiogenic drug should be selective for tissue-specific blood vessels, so that only low doses of the drug are required to achieve a highly effective angiogenesis-inhibiting effect. Currently, there is a high need to develop new drugs for specifically inhibiting ocular angiogenesis, and the drugs of the present invention specifically for treating ocular angiogenesis would be new options for treating such ocular diseases.
Sunitinib is an oral small molecule multi-target Receptor Tyrosine Kinase (RTK), can inhibit cell signaling by targeting multiple receptor tyrosine kinases including all receptors of Vascular Endothelial Growth Factor Receptor (VEGFR) and platelet-derived growth factor (PDGF-R), and can play a role in tumor angiogenesis and cell proliferation. The tissue-specific sunitinib or sunitinib analogue administration mode is provided, so that the sunitinib or sunitinib analogue administration mode can better exert the drug effect and reduce side effects. However, sunitinib drugs currently on the market have significant drawbacks in their specific selection for recipient tissues.
Fluorescence is one of the inherent properties of a variety of bio-organic molecules, including proteins. Small molecule drugs such as doxorubicin (doxorubicin), daunorubicin (daunorubicin), menadione (menadione), ellipticine (ellipticine), and hamalol (harmalol), which are currently found to carry fluorophores, allow visualization at the subcellular level in cell culture, indicating sites of function. In addition, some strongly fluorescent compounds have a blocking specificity profile in vivo, which makes possible the synthesis of tissue-specific engineered drugs.
Disclosure of Invention
The invention aims to provide a preparation method of a medicine specifically used for treating ocular angiogenesis, and aims to solve the problems of non-specific action target and large dosage of the existing sunitinib medicine.
In order to achieve the purpose, the invention adopts the following technical scheme: a method for preparing a medicament specifically used for treating ocular angiogenesis, which comprises the following steps:
(1) adding 1eq of sunitinib and 1eq of rhein into a reaction bottle, and adding 100ml of dichloromethane for dissolving;
(2) after the reaction system is cooled to normal temperature, 1.1eq of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and 1.1eq of 1-hydroxybenzotriazole are sequentially added, and after the addition is finished, the reaction system is stirred for 10 hours at normal temperature;
(3) water was added to the reaction system to quench the reaction, followed by extraction with ethyl acetate (50 ml), washing of the organic phase with saturated NaCl100ml, and passage over anhydrous NaSO4Drying, filtering and removing the solvent to obtain the product.
As a further optimized scheme of the synthetic method of the medicine for specifically inhibiting the intraocular angiogenesis, the step (2) is completed by adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and 1-hydroxybenzotriazole as condensing agents.
As a further optimization scheme of the synthesis method of the medicine for specifically inhibiting the intraocular angiogenesis, the dosage of sunitinib in the step (1) is 5.05g,9.717 mmol.
As a further optimization scheme of the synthesis method of the medicine for specifically inhibiting the intraocular angiogenesis, the dosage of the rhein in the step (1) is 2.784g,9.717 mmol.
As a further optimized scheme of the synthetic method of the drug for specifically inhibiting the intraocular angiogenesis, the dosage of the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride in the step (2) is 2.06g and 10.69 mmol.
As a further optimized scheme of the synthetic method of the medicine for specifically inhibiting the intraocular angiogenesis, the dosage of the 1-hydroxybenzotriazole in the step (2) is 1.46g and 10.69 mmol.
As a further optimization scheme of the synthesis method of the medicine specifically used for inhibiting the intraocular angiogenesis, the number of times of extraction with ethyl acetate in the step (3) is five.
Compared with the prior art, the invention has the beneficial effects that:
(1) the medicine is generated by condensation reaction of sunitinib and rhein, wherein the sunitinib can inhibit cell signal conduction by targeting multiple receptor tyrosine kinases, effectively inhibit angiogenesis, and thus achieve the function of resisting angiogenesis-related diseases; rhein is a small molecular compound with red fluorescence characteristic, has specific distribution in eyes, and can be used for assisting the synthesis of tissue-specific targeted drugs.
(2) The modified drug can specifically target eyes and inhibit the formation of new blood vessels of the eyes, thereby achieving the effect of preventing or treating related diseases of angiogenesis of the eyes.
(3) The medicine can specifically target eyes, experiments prove that the medicine is diluted into working solution with the concentration of 15 mu M, the zebra fish juvenile fish of three days is soaked, and observation is carried out through confocal observation on the sixth day, so that the medicine can specifically affinity the zebra fish eyes.
(4) In the aspect of inhibiting the formation of blood vessels of eyes, the medicine can obviously inhibit the formation of the blood vessels of the retina at the concentration of 15 mu M by in vivo imaging and confocal observation, and the inhibition rate is more than 75 percent.
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FIG. 1 is a scheme of the synthesis process of the drug of the present invention.
FIG. 2 is a schematic diagram showing the tissue-specific affinity of the drug of the present invention in zebrafish.
FIG. 3 is a phenotypic picture of the inhibition of retinal angiogenesis by the drug of the present invention on zebra fish.
FIG. 4 is a table of the treatment of excessive neovascularization of retinal vessels in zebra fish by the drug of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. Of course, the specific embodiments described herein are merely illustrative of the invention and are not intended to be limiting.
Example 1
Referring to FIGS. 1 to 4, the present invention provides the technical scheme that the medicine of the present invention has specific targeting effect on eyes
The compatibility of the medicine in different tissues of the juvenile zebra fish is detected by adopting a zebra fish living body imaging technology and confocal microscopic observation.
Collecting wild zebra fish embryos, collecting embryos from at least three reproduction groups, mixing the embryos, adding E3 culture solution, placing into a constant-temperature incubator at 28.5 ℃ for culture, and removing dead embryos and replacing the culture solution in time.
The zebra fish has small embryo volume, fast development, large embryo yield and transparent embryo, can realize a high-throughput experiment by combining with a 96-well plate for processing, and can optimize parameters such as different processing time points, processing time periods, drug concentrations and the like in parallel. The specific method comprises the following steps: 5 zebra fish embryos (2dpf,3dpf and 4dpf) at a specific development period were placed in each well of a 96-well plate, and after completely discarding the E3 culture solution in the wells, 200. mu.L of the working solution of the present invention (5. mu.M, 10. mu.M, 15. mu.M, 20. mu.M and 30. mu.M) at different concentrations were added and placed in an incubator at 28 ℃ for culture.
And observing the affinity condition of the drug tissues in the juvenile fish by using a body type fluorescence microscope and a laser confocal microscope seven days before the processed embryo develops.
The specific operation is as follows: the zebra fish embryo fixing solution is prepared according to the following table (2), and the tissue specificity affinity condition of the drug in the zebra fish juvenile fish is observed under a fluorescence microscope after the embryo is fixed by the embryo fixing solution. The experimental result is shown in figure 2, and the medicine can specifically target the eye parts of the zebra fish embryos.
(Table 2)
Example 2
Influence of the medicine of the invention on the angiogenesis of the retina of the zebra fish
The influence of the medicine on the angiogenesis of the retina of the zebra fish is detected by adopting a zebra fish living body imaging technology and confocal microscopic observation.
Transgenic zebrafish Tg (fli1ep: EGFP-CAAX) embryos were collected, embryos were collected from at least three breeding groups, mixed and added with E3 medium and placed in a 28.5 ℃ incubator for culture until use, during which time dead embryos were removed and the medium was replaced.
Based on the different treatment times, treatment periods and treatment concentration experiments in the above examples, we finally selected the following treatment protocol: culturing to the third day, placing 5 pieces of zebra fish juvenile fish in each small hole of a 96-well plate, completely sucking out the residual E3 culture solution in the hole, adding 200 μ L of 15 μ M of the inventive drug working solution, and soaking in an incubator at 28 deg.C for three days.
Observing the retinal vessel development phenotype of the juvenile fish by a body-mounted fluorescence microscope and a laser confocal microscope on the sixth day of zebra fish embryonic development.
And performing data statistics on the retinal vascular development phenotype of the zebra fish juvenile fish. Differential analysis was performed on GraphPad Prism8 software, with P <0.05 representing significant differences. The experimental results are shown in fig. 3, and the number of new blood vessels is significantly reduced under the drug treatment of the present invention compared with the normal control group.
Example 3
The medicine has the treatment effect on the excessive neogenesis of the retinal vessels of the zebra fish
The influence of the medicine on the excessive regeneration of the retinal vessels of the zebra fish is detected by adopting a zebra fish living body imaging technology and confocal microscopic observation.
Transgenic zebrafish Tg (fli1ep: EGFP-CAAX) embryos were collected, embryos were collected from at least three breeding groups, mixed and added with E3 medium and placed in a 28.5 ℃ incubator for culture until use, during which time dead embryos were removed and the medium was replaced.
2dpf of zebrafish embryos were collected and treated with embryo culture medium (E3) containing 300mM glucose to induce a zebrafish embryo angiogenesis model.
Culturing to 3dpf, placing 5 pieces of the above high-sugar treated zebra fish juvenile fish in each small hole of a 96-well plate, completely sucking out the residual E3 culture solution in the hole, adding 200 μ L of 15 μ M medicinal working solution, and placing in an incubator at 28 deg.C for soaking for three days.
Observing the retinal vessel development phenotype of the juvenile fish by a body-mounted fluorescence microscope and a laser confocal microscope on the sixth day of zebra fish embryonic development.
And performing data statistics on the retinal vascular development phenotype of the zebra fish juvenile fish. Differential analysis was performed on GraphPad Prism8 software, with P <0.05 representing significant differences. The experimental result is shown in figure 4, and compared with a pathological model, the excessive neogenesis phenotype of the zebra fish embryo blood vessels under the treatment of the medicine is obviously saved.
The above description is only a preferred embodiment of the present invention, and should not be taken as limiting the invention, and any local variations in the formulation and process thereof should be considered within the scope of the present invention.
Claims (7)
1. A method for preparing a drug specifically used for treating ocular angiogenesis, which is characterized by comprising the following steps:
(1) adding 1eq of sunitinib and 1eq of rhein into a reaction bottle, and adding 100ml of dichloromethane for dissolving;
(2) after the reaction system is cooled to normal temperature, 1.1eq of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and 1.1eq of 1-hydroxybenzotriazole are sequentially added, and after the addition is finished, the reaction system is stirred for 10 hours at normal temperature;
(3) adding water into the reaction system to quench the reaction, extracting with 50ml of ethyl acetate, washing the organic phase with 100ml of saturated NaCl, and passing through anhydrous NaSO4Drying, filtering and removing the solvent to obtain the product.
2. The method for preparing a drug specifically for the treatment of ocular angiogenesis according to claim 1, wherein said step (2) is performed by adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and 1-hydroxybenzotriazole as condensing agents.
3. The process for the preparation of a medicament specifically for the treatment of ocular angiogenesis according to claim 1 or 2, wherein the amount of sunitinib used in step (1) is 5.05g,9.717 mmol.
4. The method for preparing the medicine for treating ocular angiogenesis according to claim 1, wherein the rhein in the step (1) is used in an amount of 2.784g,9.717 mmol.
5. The method for preparing the drug specifically used for treating ocular angiogenesis according to claim 1, wherein the amount of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride used in the step (2) is 2.06g and 10.69 mmol.
6. The method for preparing the drug specifically used for treating ocular angiogenesis according to claim 1, wherein the amount of 1-hydroxybenzotriazole used in step (2) is 1.46g and 10.69 mmol.
7. The process for the preparation of a drug specifically for the treatment of ocular angiogenesis according to claim 1, wherein the number of extractions with ethyl acetate in step (3) is five.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101163473A (en) * | 2005-05-12 | 2008-04-16 | 辉瑞大药厂 | Anticancer combination therapy using sunitinib malate |
| CN109232358A (en) * | 2017-07-10 | 2019-01-18 | 复旦大学 | Indole derivatives or its salt and its preparation method and application |
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- 2021-06-30 CN CN202110737699.XA patent/CN113620932B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101163473A (en) * | 2005-05-12 | 2008-04-16 | 辉瑞大药厂 | Anticancer combination therapy using sunitinib malate |
| CN109232358A (en) * | 2017-07-10 | 2019-01-18 | 复旦大学 | Indole derivatives or its salt and its preparation method and application |
Non-Patent Citations (2)
| Title |
|---|
| 许军等: "《药物化学（第2版）》", 31 August 2018, 中国医药科技出版社 * |
| 陈临溪等: "《血管内皮细胞药理与临床》", 31 December 2012, 人民军医出版社 * |
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