Application of 5 '-O-triphenylmethyluridine and derivative thereof in preparation of medicine for treating cerebral infarction

Abstract

The invention discloses an application of 5 '-O-triphenylmethyluridine and derivatives thereof in preparation of drugs for treating cerebral infarction, and relates to the fields of medicines, natural products, traditional Chinese medicines and functional food, the compound has alkaloid and isoflavone structures, is novel in structure and good in druggability, contains flavone and alkaloid structures, and has a good application prospect in preparation of drugs for treating cerebral infarction. The pharmaceutical composition is composed of any excipient or carrier which is acceptable in pharmacy, nutriology and food science, has a structure shown in a formula I, and has a molecular formula of C28H26N2O6. The derivative of the compound is obtained by replacing-OCH3,-OH and = O groups in the molecular structure of the compound shown in the formula I with other acceptable groups or side chains except for H, OCH3, OH, CH3 or CF3. The compound disclosed by the invention can be used as an effective component for treating cerebral infarction diseases, inhibits ischemic brain injury, not only has a remarkable cerebral infarction treatment effect, but also has a multi-level and multi-target comprehensive pharmacological effect and multi-aspect pathological mechanism pertinence.

Description

Technical Field

[0001] The present invention relates to the fields of medicine, natural products, traditional Chinese medicine and functional foods, and particularly relates to the application of 5'-O-trityluridine and its derivatives in the preparation of drugs for treating cerebral infarction. Background Art

[0002] With the aggravation of the aging problem of the Chinese population, the incidence of ischemic stroke (IS) has also increased significantly, and China is the region with the fastest growth rate of the number of patients. According to statistics, the prevalence of stroke in the world increases year by year, the prevalence of IS is higher than that of hemorrhagic stroke, and the prevalence in men is greater than that in women. The age-standardized prevalence of IS increased from 291.2 / 100,000 in 1990 to 299.1 / 100,000 in 2013. Among them, the age-standardized prevalence of IS in men increased from 339.2 / 100,000 in 1990 to 346.08 / 100,000 in 2013, and the age-standardized prevalence of IS in women increased from 253.6 / 100,000 in 1990 to 260.4 / 100,000 in 2013.

[0003] During the 23 years, the age-standardized prevalence of global IS increased by 2.71%. Stroke is the second leading cause of death in the elderly aged 65 and above. The most significant features are high disability rate, high recurrence rate and high mortality rate. After surgical treatment, it is also an important cause of disability or poor health. Most patients are hemiplegic or even bedridden after surgery. The average annual hospitalization time of people with ischemic stroke is twice or more that of other elderly people. Compared with the caregivers of other diseases, the caregivers are more likely to have serious mental, economic and physical problems after long-term care of stroke patients; the onset of stroke not only seriously affects the quality of life of patients, but also imposes a great economic burden on their own families and society.

[0004] Through the above analysis, the problems and defects of the existing technology are as follows:

[0005] There are almost no ideal drugs in modern clinical medicine for the prevention and treatment of ischemic stroke. Although the reported research and clinically used western medicine drugs can improve the symptoms related to ischemic stroke to a certain extent, due to factors such as high price, single action target and large side effects, their clinical applications are greatly limited. At present, rapid reperfusion intravenous thrombolysis and endovascular thrombectomy are the mainstays of stroke treatment. However, due to strict inclusion criteria and many contraindications for thrombolysis, including the risk of hemorrhagic transformation, the clinical application of endovascular treatment is still limited. Therefore, it is very necessary to develop new treatment methods and drugs.

[0006] In addition, the existing patent texts retrieved mainly discuss the anti-tumor activity experiment of the drug at the cell level, and there is no pharmacological research on stroke, let alone research on drugs for cerebral infarction. SUMMARY OF THE INVENTION

[0007] An object of the present invention is to solve at least one of the technical problems existing in the prior art, and to provide the use of 5'-O-trityluridine and its derivatives in the preparation of drugs for treating cerebral infarction.

[0008] The technical solution of the present invention is as follows: 1. The use of 5'-O-trityluridine and its derivatives in the preparation of drugs for cerebral infarction.

[0009] Further, the structural formula of the 5'-O-trityluridine is as shown in Formula I:

[0010]

[0011] Further, the derivative is a compound obtained by replacing the -OCH 3 , -OH, and =O groups in the molecular structure of the compound of Formula I with other groups or side chains acceptable except H, OCH 3 , OH, CH 3 or CF 3 .

[0012] Further, the derivative has the structure of Formula II:

[0013]

[0014] Wherein, R 1 is H, OCH 3 , OH, CH 3 or CF 3 ;

[0015] R 2 is H, OCH 3 or OH;

[0016] R 3 is H, OCH 3 or OH;

[0017] R 4 is H, OCH 3 , OH, CH 3 or CF 3 .

[0018] Further, the drug includes 5'-O-trityluridine monomer and / or its derivative, and a pharmaceutically or food science acceptable excipient.

[0019] Further, the excipient is at least one of a binder, a filler, a disintegrant, wine, vinegar, and starch.

[0020] Furthermore, the synthesis method of the 5'-O-trityluridine is as follows: First, isoflavone is obtained through Suzuki coupling reaction; then, the target product is synthesized by the Mannich reaction of isoflavone and sparteine.

[0021] Furthermore, the specific steps of the synthesis method are as follows:

[0022] S1: Using 2,4,6-trihydroxyacetophenone as the raw material, adding K 2 CO 3 selectively freeing the hydroxyl groups at the 4- and 6-positions, then adding 0.5 - 3 equivalents of chloromethyl methyl ether to obtain 2-hydroxy-4,6-dimethoxymethylacetophenone, then adding N,N-dimethyl dimethylacetal to synthesize azlactone, and finally completing the synthesis of 5,7-dimethoxy-3-iodochromone under the action of elemental iodine; 5,7-dimethoxy-3-iodochromone is dissolved in dichloromethane, and the methoxymethyl group at the 5-position is removed to obtain 7-dimethoxy-3-iodochromone;

[0023] S2: Compounds 5 and 6 are synthesized into isoflavone through Suzuki coupling reaction, the methoxymethyl group at the 7-position is removed under HCl, and then the methoxy group is removed to obtain genistein;

[0024] Compound 5 is:

[0025] Compound 6 is:

[0026] S3: Sparteine, genistein and formalin are catalytically synthesized into sparteine-N-methylidene-(5,7,4'-trihydroxy)isoflavone.

[0027] The beneficial effects of the present invention are as follows:

[0028] (1) The new structure compound of the present invention can be used to treat ischemic stroke and inhibit the nerve injury caused by cerebral ischemia. The innovation of the new compound lies in that it not only has a significant anti-cerebral ischemia injury effect, but also has comprehensive pharmacological effects at multiple levels and multiple targets and is targeted at multiple pathological mechanisms, and it is an ideal monomer of anti-stroke drug in the future.

[0029] (2) The present invention provides a preparation and synthesis scheme of a compound genistein alkaloid with a novel structure, and verifies its new activity in treating stroke through relevant case implementation. The compound belongs to natural active ingredients, and at the same time has the structures of alkaloid and isoflavone, with novel structure, good drug-likeness and good clinical application prospects.

[0030] (3) The new structural compound of the present invention has significant pharmacological effects against nerve injury caused by stroke, and may become an ideal drug for treating cerebral infarction in the future. It can be used for the prevention and treatment of cerebral infarction diseases clinically, and will generate very considerable economic benefits and commercial value. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 It is a schematic diagram of the results of the new compound provided by the embodiment of the present invention in improving the oxidative stress injury of mice with cerebral ischemia model. Among them, A is the content of SOD in the serum of mice; B is the content of MDA in the serum of mice (x±SD, n = 8), ## indicates P<0.01, indicates P<0.001 compared with the Sham group; * indicates P<0.05, ** indicates P<0.01 compared with the Model group;

[0032] Figure 2 It is a schematic diagram of the results of the new compound provided by the embodiment of the present invention in improving the motor balance ability of mice with cerebral ischemia model. Among them, A is the process and steps of the rotarod test of mice; B is the pole climbing time of mice before experimental administration; C is the pole climbing time of mice before experimental administration; D is the Bederson's behavioral score of each group of mice (x±SD, n = 8), ## indicates P<0.01 compared with the Sham group; * indicates P<0.05, ** indicates P<0.01 compared with the Model group;

[0033] Figure 3 It is a schematic diagram of the results of the new compound provided by the embodiment of the present invention in improving the cerebral infarction volume of mice with cerebral ischemia model. Among them, A is the picture of the TTC staining result of mice; B is the statistical chart of the cerebral infarction volume of each group of mice (x±SD, n = 8), * indicates P<0.05, ** indicates P<0.01 compared with the Model group;

[0034] Figure 4 It is a schematic diagram of the results of the study on the effect of immunofluorescence staining method on the expression level of GFAP protein in the ischemic brain injury area provided by the embodiment of the present invention;

[0035] Figure 5It is a schematic diagram of the results of studying the effect of a new compound on the mRNA content of inflammation-related factors in the mouse brain tissue by RT-qPCR method provided by the embodiments of the present invention. Among them, A is the mRNA content of IL-6 in the mouse brain tissue; B is the mRNA content of IL-1β in the mouse brain tissue; C is the mRNA content of TNF-α in the mouse brain tissue; D is the mRNA content of TNF-β in the mouse brain tissue; E is the mRNA content of IL-4 in the mouse brain tissue; F is the mRNA content of IL-10 in the mouse brain tissue (x±SD, n = 8), # indicates P<0.05, ## indicates P<0.01, indicates P<0.001 compared with the Sham group; * indicates P<0.05, ** indicates P<0.01 compared with the Model group;

[0036] Figure 6 It is a schematic diagram of the results of the effect of genistein alkaloid on the expression levels of β-catenin and CyclinD1 proteins in the mouse brain tissue (x±SD, n = 3). Among them, A is the immunoblot result diagram of CyclinD1 and β-actin; B is the statistical chart of the β-catenin band; C is the statistical chart of the Cyclin D1 band; # indicates P<0.05, ## indicates P<0.01 compared with the Sham group; * indicates P<0.05, ** indicates P<0.01, compared with the Model group;

[0037] Figure 7 It is for the identification of neural stem cells (400×).

[0038] Figure 8 It is that GIA improves the viability of C17.2 neural stem cells. Among them, A is the survival rate of C17.2 cells after treatment with gradient concentrations of GIA; B is the quantitative analysis of cell viability; C is the growth state of C17.2 cells (200×) (x±SD, n = 3), * indicates P<0.05, ** indicates P<0.01, *** indicates P<0.001, compared with the Control group;

[0039] Figure 9 It is that GIA increases the nuclear expression of Nestin (400×);

[0040] Figure 10 It is that GIA improves the oxidative stress damage of the glucose and oxygen deprivation cell model. Among them, A is the content of SOD in C17.2 neural stem cells; B is the content of MDA in C17.2 neural stem cells; C is the content of SOD in primary neural stem cells; D is the content of MDA in primary neural stem cells (x±SD, n = 3), ## indicates P<0.01, indicates P<0.001 compared with the Control group; * indicates P<0.05, ** indicates P<0.01 compared with the OGD group;

[0041] Figure 11 To reduce neuroinflammation in the GIA-induced oxygen-glucose deprivation cell model. Among them, A, B, C, D, and C are the mRNA expression levels of IL-6, IL-1β, TNF-α, and iNOS in C17.2 neural stem cells respectively; E, F, G, and H are the mRNA expression levels of IL-6, IL-1β, TNF-α, and iNOS in primary neural stem cells respectively (x±SD, n = 3). ## indicates P < 0.01, indicates P < 0.001 compared with the Control group; * indicates P < 0.05, ** indicates P < 0.01 compared with the OGD group.

[0042] Figure 12 To increase the protein levels of β-catenin and CyclinD1 in C17.2 neural stem cells by GIA. Among them, A is the immunoblotting result graph of β-catenin, CyclinD1, and β-actin; B is the statistical graph of the β-catenin band; C is the statistical graph of the Cyclin D1 band (x±SD, n = 3); ## indicates P < 0.01 compared with the Control group; * indicates P < 0.05, ** indicates P < 0.01 compared with the OGD group;

[0043] Figure 13 To increase the protein levels of β-catenin and CyclinD1 in primary neural stem cells by GIA. Among them, A is the immunoblotting result graph of β-catenin, CyclinD1, and β-actin; B is the statistical graph of the β-catenin band; C is the statistical graph of the Cyclin D1 band (x±SD, n = 3); ## indicates P < 0.01 compared with the Control group; *P < 0.05, **P < 0.01 compared with the OGD group. Detailed implementation manners

[0044] In order to make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0045] Composed of a compound with the effect of improving cerebral infarction treatment and any pharmaceutically, nutritionally and food scientifically acceptable excipient. The compound has the structure of formula I and the molecular formula is C 28 H 26 N 2 O 6 ;

[0046]

[0047] In an embodiment of the present invention, the derivative of the compound has the structure of Formula II:

[0048]

[0049] Wherein, R1 is H, OCH 3 , OH, CH 3 or CF 3 ;

[0050] R2 is H, OCH 3 or OH;

[0051] R3 is H, OCH 3 or OH;

[0052] R4 is H, OCH 3 , OH, CH 3 or CF 3 .

[0053] In an embodiment of the present invention, structural modification of the isoflavone moiety can yield a series of derivatives, which are compounds obtained by replacing the -OCH 3 , -OH, and =O groups in the molecular structure of the compound of Formula I with other acceptable groups or side chains other than H, OCH 3 , OH, CH 3 or CF 3 . Generally, a derivative refers to a product derived from the substitution of a hydrogen atom or atomic group in a simple compound by other atoms. The derivatives of the present invention have the main structure unchanged, still being the isoflavone alkaloid structure, with the effective functional group being aromatic ketone. Isoflavone is a 3-phenyl derivative and belongs to plant secondary metabolites. Isoflavone is also an effective antioxidant that can prevent the generation of oxygen free radicals. Therefore, most compounds with an isoflavone structure have antioxidant effects and certain anti-inflammatory effects.

[0054] The present invention also provides new activities and new applications of the compound and its derivatives with the effect of improving the treatment of cerebral infarction in improving the treatment of cerebral infarction.

[0055] The present invention also provides a pharmaceutical preparation with the effect of improving the treatment of cerebral infarction, which is composed of 5'-O-trityluridine monomer and any excipient acceptable in pharmacy and food science (such as binders, fillers, disintegrants in tablets; wine and vinegar in pills; starch in powders, etc.).

[0056] Another object of the present invention is to provide a use of the compound and its derivatives with the effect of improving the treatment of cerebral infarction for preparing a drug or functional food with the effect of improving the treatment of cerebral infarction.

[0057] In an embodiment of the present invention, the preparation method of the compound includes:

[0058] Using cytisine as a raw material, the isoflavone moiety is obtained through synthesis, and the entire compound can be synthesized according to the following steps:

[0059] Step 1: A series of isoflavones are obtained through a Suzuki coupling reaction with mild reaction conditions;

[0060] Step 2: The synthesized series of isoflavones and cytisine are subjected to a Mannich reaction to synthesize the target product under the catalysis of DMAP.

[0061] In the embodiment of the present invention, the synthesis method includes the following steps:

[0062] (1) Using 2,4,6-trihydroxyacetophenone as a raw material, an appropriate amount of K 2 CO 3 is added to selectively free the hydroxyl groups at the 4- and 6-positions, then 0.5 - 3 equivalents of chloromethyl methyl ether are added to obtain 2-hydroxy-4,6-dimethylmethoxyacetophenone, and then N,N-dimethyl dimethyl acetal is added to synthesize an enamine ketone. Finally, a simple one-step cyclization reaction is carried out under the action of elemental iodine to complete the synthesis of 5,7-dimethylmethoxy-3-iodochromone. 5,7-Dimethylmethoxy-3-iodochromone (Compound 4) is dissolved in dichloromethane, and the methyl methoxy group at the 5-position is removed to obtain 3-iodochromone (Compound 5). The synthesis route is as follows:

[0063]

[0064] (2) Compound 5 and Compound 6 are synthesized into isoflavone (Compound 7) through a Suzuki coupling reaction. The methyl methoxy group at the 7-position is removed under 3M HCl, and then the methoxy group is removed to obtain genistein (Compound 9). The synthesis route is as follows:

[0065]

[0066] (3) Cytisine, genistein, and formalin are catalytically synthesized into cytisine-N-methylene-(5,7,4'-trihydroxy) isoflavone. The synthesis route is as follows:

[0067]

[0068] Example 1

[0069] (1) Using 2,4,6-trihydroxyacetophenone as a raw material, 2.1 times the mass equivalent of K 2 CO 3The hydroxyl groups at the 4- and 6-positions are selectively freed, and then 2.12 times the mass equivalent of chloromethyl methyl ether is added to obtain 2-hydroxy-4,6-dimethylmethoxyacetophenone. Then, N,N-dimethyl dimethyl acetal is added, and aminoketene is synthesized at a temperature of 74 °C with a yield of 88%. Finally, the synthesis of 5,7-dimethylmethoxy-3-iodochromone is completed in a one-step simple cyclization reaction under the action of elemental iodine with a yield of 75%. 5,7-Dimethylmethoxy-3-iodochromone (Compound 4) is dissolved in dichloromethane and placed on a stirrer and stirred at medium speed for 4 h to remove the methyl methoxy group at the 5-position to obtain 3-iodochromone (Compound 5).

[0070] (2) Using Compounds 5 and 6 as raw materials, 3.0 times the mass equivalent of K 2 CO 3 , 0.1 times the mass equivalent of Pd(PPh 3 ) 4 in dioxane and water, freeze-degassed and replaced three times, and refluxed at a temperature of 110 °C for 12 hours, isoflavone 7 can be synthesized with a yield of 75%. Subsequently, Compound 7 is de-methylated at the 7-position under 3M HCl, and then the methoxy group is removed under boron tribromide reagent to obtain genistein (Compound 9).

[0071] (3) Using sparteine and genistein as raw materials, 37 wt% formalin and 0.1 equivalent of DMAP (4-dimethylaminopyridine) are added in isopropanol, and the reaction is carried out at a temperature of 80 °C for 12 hours to synthesize sparteine-N-methylene-(5,7,4'-trihydroxy) isoflavone compound with a yield of 65%.

[0072] The following tests were carried out on the compounds obtained in Example 1 above:

[0073] 1. Establishment of a cerebral ischemia model in mice

[0074] Rose bengal is a type II photosensitizer. After photoactivation, it can directly transfer energy to oxygen molecules and generate a large amount of reactive oxygen species, which then interact with other molecules in the organism, thereby causing damage to the body. By injecting a physiological saline solution containing rose bengal through the tail vein, then exposing the skull of the mouse, and subsequently irradiating the left parietal bone with a light beam of wavelength 560 nm for 5 min to induce thrombus formation, thereby causing local infarction and achieving the ischemic process.

[0075] Grouping of mice: The experiment was divided into 6 groups, with 8 C57 mice in each group. Specifically, it was divided into a sham operation control group (normal saline), a model group (normal saline), a low-dose drug treatment group (0.3 mg / kg), a medium-dose drug treatment group (0.6 mg / kg), a high-dose drug treatment group (1.2 mg / kg), and a positive drug group (ginsenoside 20 mg / kg). The drugs were administered according to body weight for one week continuously. The drug groups used the compound monomer GIA (cytisine-N-methylidene-(5,7,4'-trihydroxy) isoflavone compound) prepared in Example 1.

[0076] 2. Determination of superoxide dismutase (SOD) and malondialdehyde (MDA)

[0077] 2.1 Determination of SOD content

[0078] According to the kit instructions, the xanthine oxidase method was used to determine the SOD content. Reagents were added according to Table 1 below, mixed well, incubated at 37 °C for 20 min, and the absorbance values of each tube were measured at a light path of 450 nm to calculate the SOD activity.

[0079] Table 1 Reagents used in the SOD test

[0080]

[0081] 2.2 Determination of MDA content

[0082] The thiobarbituric acid (TBA) method was used to detect the MDA content. Reagents were added according to Table 2 below, mixed well, and then the reagents in Table 2 were continuously added. It was placed in a metal bath at 95 °C for 80 min, taken out and cooled with running water, and centrifuged at 3500 - 4000 rpm for 10 min.

[0083] Table 2 Required reagents

[0084]

[0085] Table 3 Required reagents

[0086]

[0087] Test results Figure 1 , it can be seen that compared with the sham operation group, the SOD concentration in the model group decreased significantly, while the administration of the compound drug could significantly increase this change. Similarly, as shown in B of Figure 1 , the MDA concentration in the model group of mice increased significantly, while the effects of the drug treatment group and the positive drug could significantly reduce its concentration, and the effect was particularly obvious at medium and high doses.

[0088] 3. Behavioral experiments

[0089] 3.1 Rotarod test for mice

[0090] Before the start of the experiment and after administration of the drug, the rotarod test was performed on the mice. During the training phase of the rotarod test, each mouse was tested three times a day for three consecutive days. During training, the rotation speed was kept constant at 10 revolutions per minute. If the speed remained constant during the remaining test (90 seconds), the training was considered qualified. During the test phase (the 4th day after injury), the speed of the rod was increased from 4 revolutions per minute to 40 revolutions per minute for 300 seconds. The climbing time of the mice on the rod was recorded. Each mouse was tested three times, and the average of the three results was taken as the final time. The falling latency of the mice was scored. The test procedure referred to A in the test Figure 2 in.

[0091] 3.2 Bederson's score of mice

[0092] After administration of the drug, the animals were observed for neurobehavioral changes. Referring to the method of Zea Longa, the mouse's tail was lifted about 1 foot off the ground to observe the condition of the two front limbs. The mouse was placed on a horizontal surface, and its shoulders were pushed to observe any differences in resistance on both sides. The mouse was placed on the ground to observe its walking. A five-level scoring method (0 - 4 points) was used. The higher the score, the more severe the neurobehavioral injury of the mouse.

[0093] (1) Those with completely normal behavior were scored 0 points;

[0094] (2) When the mouse's tail was lifted off the ground, if the contralateral forelimb on the surgical side showed internal rotation and adduction, it was scored 1 point;

[0095] (3) When the mouse was placed on the ground and the resistance on both sides was checked by hand pressure, if the resistance on the contralateral side of the surgery decreased, it was scored 2 points;

[0096] (4) When the mouse was placed on the ground and its walking was observed, if it circled around the contralateral side of the surgery, it was scored 3 points;

[0097] (5) Those with extremely severe injury and unable to move independently were scored 4 points.

[0098] The test results are shown in Figure 2 B - D in. It can be seen that, as shown in Figure 2 B in, it is the climbing time of each group of mice on the rotarod before the surgical modeling experiment. It can be clearly seen from the figure that there was no significant difference in the climbing time of each group of mice before the experiment, indicating that the motor balance ability of each group of mice was similar. As shown in Figure 2 C in, after administration of the drug, compared with the sham operation group, the climbing time of the mice in the model group was significantly reduced; compared with the model group, the climbing time of the mice in the drug treatment group was significantly increased (P < 0.01), indicating that the treatment with the active compounds of Baimai powder could significantly improve the motor balance ability of the mice. As shown in Figure 2As shown in D of [reference], the Bederson's score of the mice in the sham operation group was 0. After the surgical model was established, the behavioral score of the mice increased significantly, indicating that the motor ability of the mice was significantly impaired after photochemically induced surgery. After drug treatment, the behavioral score of the mice decreased significantly, indicating that drug treatment could significantly improve the behavioral ability of the mice, and this effect was particularly obvious in the high-dose treatment group (P<0.01).

[0099] 4. Tissue Sampling

[0100] After the behavioral experiment, blood was collected by enucleating the eyeballs. Then the mice were fixed in the supine position on the operating table, the chest cavity was opened to expose the heart, and normal saline was perfused. When the liver turned white, the limbs of the mice became stiff, and the head was cut off to obtain the brain tissue.

[0101] 5. TTC Staining

[0102] After the brain tissue obtained from tissue sampling was quickly frozen in a -20°C refrigerator for 10 minutes, it was cut into 5 segments using a brain mold, and coronal brain sections (each 2 mm) were made. The sections were immersed in TTC staining solution and incubated in a 37°C incubator for 15 minutes. After staining, they were washed with water, the staining situation was observed, and photographed for record. The test results are shown in Figure 3 , it can be seen that, as Figure 3 shown in A of [reference], are the pictures of the TTC staining results of the brain tissue sections of each group of mice. Figure 3 B in [reference] is the statistical chart of the cerebral infarction volume of the mice. Compared with the Sham group, the cerebral infarction volume of the mice in the Model group increased significantly, indicating that the surgical model group was successfully established and cerebral ischemia occurred in the mice; compared with the Model group, the cerebral infarction volume of the mice in the low-dose and high-dose groups decreased significantly (P<0.05); the reduction of the cerebral infarction volume of the mice in the medium-dose group was more obvious than that in the Model group (P<0.01); the reduction of the cerebral infarction volume of the mice in the TG group was obvious compared with the Model group (P<0.05).

[0103] 6. Immunofluorescence Staining

[0104] (1) Slicing: Continuously slice the mouse brain tissue with a cryostat, with each slice being 30 μm thick, and store it at -20 °C in antifreeze; (2) Selecting slices: Select brain slices and wash them 3 times with PBS for 5 minutes each time; (3) Blocking: 1% BSA + 10% goat serum + 0.3% Triton X-100 for 60 minutes; (4) Incubating with primary antibody: Dilute Blocker (1% BSA, 0.3% Triton), incubate at room temperature for 2 hours, and overnight at 4 °C; (5) Rinsing: Wash with PBS three times for 5 minutes each time; (6) Incubating with secondary antibody: Dilute Blocker (1% BSA, 0.3% Triton) 1:400 for 120 minutes; (7) Rinsing: Wash with PBS three times for 5 minutes each time; (8) Nuclear staining: DAPI (1:1000) for 15 minutes; (9) Rinsing: Wash with PBS three times for 5 minutes each time; (10) Mounting: Mount with an anti-fluorescence quencher; (11) Photographing: Observe by photographing with a fluorescence microscope.

[0105] The test results are shown in Figure 4 , and it can be seen that the increase of GFAP in the ischemic brain tissue is also a marker signal for the central nervous system to respond to ischemic brain injury. Therefore, the expression of GFAP protein in the brain tissues of mice in each group was detected by immunofluorescence, and the results are as Figure 4 shown. After surgical modeling, the expression of GFAP protein in mice increased significantly. After treatment with medium-dose and high-dose compounds, the expression level of GFAP decreased significantly, indicating that after ischemic brain injury, the active ingredient GIA can effectively relieve the nerve injury stress response caused by cerebral ischemia and protect the neuroinflammation caused by brain injury to a certain extent.

[0106] 7. Western Blot

[0107] (1) Preparation of SDS-PAGE gel

[0108] ① Preparation of separating gel: Wash and dry the glass plates, place them in a clamp, and align them horizontally to ensure good sealing. Prepare the corresponding separating gel according to the molecular weight of the target protein. As shown in Table 4.

[0109] Table 4 Various separating gels

[0110]

[0111] Note: % is the mass concentration percentage.

[0112] Add the prepared separating gel to the glass plate and seal it with water, let it stand at room temperature for 30 minutes, discard the upper layer of water, and blot the residual liquid with filter paper;

[0113] ② Preparation of stacking gel: Prepare according to the substances in Table 5, mix the prepared stacking gel evenly and slowly add it to the glass plate.

[0114] Table 5 Stacking gel

[0115]

[0116] Note: % is the mass concentration percentage.

[0117] (2) Loading sample

[0118] Pull out the comb teeth, add 10 μL of protein sample to each well, and add 5 μL of Marker to both ends.

[0119] (3) Electrophoresis

[0120] Apply a constant voltage of 80 V first. After the sample enters the separating gel, switch to 120 V and electrophorese until the bottom.

[0121] (4) Blotting

[0122] Prepare a "sandwich" in the order of "black clamping plate - transfer filter paper - gel - PVDF membrane - transfer filter paper - white clamping plate". Place the transfer clip into the electrophoresis tank, add the transfer buffer, and transfer at 200 mA for 1.5 h. Use an ice pack to surround the electrophoresis tank to ensure the whole process is carried out at low temperature.

[0123] (5) Antibody incubation

[0124] ① Wash the PVDF membrane 3 times for 5 min each, and block it in the blocking solution at room temperature for 2 h;

[0125] ② Wash the PVDF membrane 3 times for 5 min each, dilute the primary antibody with the blocking solution, and incubate it overnight at 4 °C;

[0126] ③ Wash the PVDF membrane 3 times for 10 min each, add the secondary antibody, and incubate it at room temperature for 1.5 h; The antibody dilution is shown in Table 6:

[0127] Table 6 Antibody dilution

[0128]

[0129] (6) Gel imaging

[0130] Add the ECL chemiluminescent developing solution, react in the dark for 2 min, then develop and take pictures.

[0131] (7) Image analysis

[0132] Analyze the gray value with Image J software.

[0133] As Figure 5As shown, after modeling, the mRNA expression levels of IL-6, IL-1β, and TNF-α in mice were significantly increased, while the mRNA expression levels of anti-inflammatory factors IL-4, TNF-β, and IL-10 were significantly decreased. After drug treatment, the increase in the levels of pro-inflammatory factors could be significantly inhibited, and the mRNA expression levels of anti-inflammatory factors such as IL-4 and TNF-β were effectively increased.

[0134] As Figure 6 shown in A of [reference], they are the protein expression levels of β-catenin, CyclinD1, and β-actin in the brain tissues of mice in each experimental group.

[0135] As Figure 6 shown in B of [reference], they are the quantitative analysis results of β-catenin protein levels. Compared with the Sham group, the content of β-catenin protein in the brain tissues of mice in the Model group decreased (P < 0.001); compared with the Model group, the content of β-catenin protein in the brain tissues of mice in the high-dose and medium-dose groups increased significantly (P < 0.01). Compared with the Model group, the content of β-catenin protein in the brain tissues of mice in the TG treatment group increased (P < 0.01). As Figure 6 shown in C of [reference], they are the quantitative analysis results of Cyclin D1 protein content. Compared with the Sham group, the content of Cyclin D1 protein in the brain tissues of mice in the Model group decreased significantly (P < 0.01); compared with the Model group, the content of Cyclin D1 protein in the brain tissues of mice in the high-dose and medium-dose groups increased significantly (P < 0.01), and the effect of the low dose was particularly obvious. Compared with the Model group, the content of Cyclin D1 protein in the brain tissues of mice in the TG group also increased significantly (P < 0.01).

[0136] 8. Study on the level of cell proliferation in vitro

[0137] (1) Experimental animals: 4 pregnant SPF-grade SD rats (3000 ± 20 g), at 13 days of pregnancy, purchased from Beijing Speywood Biotechnology Co., Ltd., with the license number SCXK (Jing) 2019-0010. The embryonic brain tissues in the pregnant rats were used for the extraction experiment of primary neural stem cells.

[0138] Experimental cells: The neural stem cell line used in the experiment was murine neural progenitor cell line C17.2 cells, purchased from Beijing Hancheng Biotechnology Co., Ltd.

[0139] The test methods are as follows: Sun T, Chen L, Liu R, Liu QS, Cheng Y. Sophora alopecuroides Alleviates Neuroinflammation and Oxidative Damage of Parkinson's Disease In Vitro and In Vivo. Am J Chin Med. 2023;51(2):309-328.

[0140] Data processing: The data are expressed as X±SD, and homogeneity of variance test and normal test are performed. If the variances are homogeneous, one-way ANOVA is performed using SPSS 20.0 software; if the variances are not homogeneous, Dunnett T3 test is used; if the data are not normally distributed, rank sum test is used. P < 0.05 indicates statistical significance.

[0141] The test results are as follows:

[0142] (1) Identification of neural stem cells

[0143] As Figure 7 shown, the C17.2 cells cultured in vitro can be observed using an ordinary optical microscope, and at the same time, they are fluorescently labeled with the NSCs marker protein Nestin. The C17.2 cells cultured in vitro have good refractive index, and the C17.2 cells photographed by a laser confocal microscope can be labeled with both Nestin and DAPI at the same time, and the proportion of Nestin-positive cells reaches 100%.

[0144] (2) Determination of cell drug dosage

[0145] The CCK8 kit was used to detect the effect of GIA treatment on the viability of C17.2 cells. GIA at 0 μg / ml, 5 μg / ml, 10 μg / ml, 20 μg / ml, 40 μg / ml, 80 μg / ml, and 100 μg / ml was added respectively, and the cell viability was detected after 24 hours of culture. The results are as Figure 8 shown in A. Compared with the control group (0 μg / ml), the treatment with GIA at 5 μg / ml, 10 μg / ml, 20 μg / ml, and 40 μg / ml had no significant effect on the cell survival rate (P > 0.05), while the treatment with GIA at 80 μg / ml and 100 μg / ml significantly decreased the cell viability (P < 0.001). Therefore, the concentration of GIA should not exceed 40 μg / ml in the subsequent cell experiments.

[0146] Next, after treating the cells with GIA at concentrations of 5 μg / ml, 10 μg / ml, 20 μg / ml, and 40 μg / ml respectively, the OGD model group cells were given medium without drugs. The results are asFigure 8 As shown in Figure B, the GIA treatment at each dose concentration could significantly improve the cell survival rate, and the effect was more obvious at the two higher concentrations of 20 μg / ml and 40 μg / ml (P<0.01). This indicates that the GIA treatment within a certain concentration range can protect cells from damage caused by glucose and oxygen deprivation and increase cell viability.

[0147] In subsequent cell administration treatments, three gradient concentrations of 10 μg / ml, 20 μg / ml, and 40 μg / ml were selected to observe the cell growth status. As Figure 8 shown in Figure C, the cell growth status of the TG group and the GIA treatment group was good compared with the model group, and the number of cells increased significantly.

[0148] (3) GIA increases the nuclear expression of Nestin

[0149] After cell treatment, Nestin immunocytochemistry experiments were performed to observe the expression and localization of Nestin during cell proliferation. As Figure 9 shown, the Nestin expression in normal C17.2 cells was in the cytoplasm, and the nuclear expression of Nesitn in C17.2 cells after GIA drug treatment increased. According to the position and degree of Nestin expression in cells, Nestin was analyzed for localization.

[0150] (4) GIA alleviates oxidative stress injury of neural stem cells caused by glucose and oxygen deprivation

[0151] C17.2 neural stem cells and primary neural stem cells were respectively treated with GIA at concentrations of 10 μg / ml, 20 μg / ml, and 40 μg / ml. After 3 hours of glucose and oxygen deprivation modeling for the model group and the administration group cells, the contents of superoxide dismutase and malondialdehyde in the cells were detected. The results are as Figure 10 shown. Compared with the control group, the OGD treatment groups of both types of cells significantly reduced the content of superoxide dismutase in the cells, and the difference was statistically significant (P<0.001). After treatment with GIA at different dose concentrations, the total antioxidant content in the cells could be significantly increased (P<0.05, P<0.01). Similarly, a similar trend was also shown in the detection of malondialdehyde content. The OGD treatment groups of both types of stem cells significantly increased the malondialdehyde content in the cells, and the difference was statistically significant (P<0.01). After treatment with GIA at different dose concentrations, the GIA treatment at three dose concentrations could significantly reduce the malondialdehyde content in neural stem cells, and the difference was statistically significant (P<0.05, P<0.01). The above results indicate that after the drug protection of the active ingredient GIA, neural stem cells can alleviate the oxidative stress injury caused by glucose and oxygen deprivation and play a certain role in damage protection.

[0152] (5) GIA alleviates cellular neuroinflammation induced by glucose and oxygen deprivation

[0153] After establishing a glucose and oxygen deprivation cell ischemia model and pre - protecting C17.2 neural stem cells and primary neural stem cells with GIA at concentrations of 10 μg / ml, 20 μg / ml, and 40 μg / ml, the mRNA expression levels of pro - inflammatory factors IL - 6, IL - 1β, TNF - α, and iNOS related to inflammation were measured respectively to study the protective effect of GIA on neural inflammation of stem cells. The results are as Figure 11 shown. Compared with the blank group, the mRNA expression levels of IL - 6, IL - 1β, TNF - α, and iNOS in the OGD model group all increased significantly (P < 0.001). After treatment with different doses of GIA, the mRNA expression levels of IL - 1β, TNF - α, and iNOS could be significantly reduced. When administering GIA to C17.2 neural stem cells, low and high doses of the concentration could reduce the mRNA expression level of IL - 6, while when administering to primary neural stem cells, medium and high doses of the concentration could reduce the mRNA expression level of IL - 6 (P < 0.01). The above results all indicate that GIA can alleviate cellular neuroinflammation to a certain extent in the glucose and oxygen deprivation cell models of C17.2 neural stem cells and primary neural stem cells.

[0154] (6) GIA increases the protein levels of β - catenin and CyclinD1 in neural stem cells

[0155] As Figure 12 shown in A below, compared with the Control group, the protein contents of β - catenin and CyclinD1 in C17.2 neural stem cells decreased significantly (P < 0.01). The specific band statistical analysis is as Figure 12 shown in B and C below. Compared with the OGD model group, the protein contents of β - catenin and CyclinD1 in different concentration GIA treatment groups all increased significantly (P < 0.01). Compared with the OGD group, the CyclinD1 protein level in the low - dose GIA treatment group increased, but there was no statistical significance (P > 0.05). As Figure 13 shown in A below, compared with the Control group, the CyclinD1 protein content in primary neural stem cells decreased significantly (P < 0.01), but the β - catenin protein level decreased, but there was no statistical significance (P > 0.05). The specific band statistical analysis is as Figure 13As shown in B and C, compared with the OGD model group, the protein levels of β-catenin and CyclinD1 in the GIA treatment groups at different concentrations were significantly increased (P < 0.01). Compared with the OGD group, the Cyclin D1 protein level increased in the low-dose GIA treatment group, and the β-catenin protein level increased in the high-dose GIA treatment group, but neither had statistical significance (P > 0.05).

[0156] In summary, in vivo animal experimental studies have confirmed that GIA can promote the proliferation of endogenous NSCs and improve the motor balance ability of mice with cerebral ischemia. At the same time, it was found that GIA can promote the proliferation of neural stem cells NSCs.

[0157] In summary, the new structural compound of 5'-O-trityluridine can reduce the cerebral infarction volume of mice with photochemically induced cerebral ischemia, improve the Bederson's motor score, and significantly improve the motor ability of mice with cerebral ischemia. It can reduce oxidative stress damage in mice after ischemia, relieve neuroinflammation to a certain extent, promote the proliferation of neural stem cells, and significantly improve neurological damage after ischemic stroke.

[0158] The above-described embodiments only represent the preferred embodiments of the present invention. The description is relatively specific and detailed, but it should not be construed as a limitation on the scope of the patent of the present invention. It should be noted that for those of ordinary skill in the art, according to the technical solutions and concepts described above, various other corresponding changes and deformations can be made, and all these changes and deformations should fall within the protection scope of the claims of the present invention.

Claims

1. Application of 5'-O-trityl uridine and its derivatives in the preparation of drugs for treating cerebral infarction.

2. The use according to claim 1, characterized in that The structural formula of the 5'-O-trityl uridine is shown in Formula I:

3. The use according to claim 1, characterized in that: The derivative has the structure of formula II: Wherein, R1 is H, OCH3, OH, CH3 or CF3; R2 is H, OCH3 or OH; R3 is H, OCH3 or OH; R4 is H, OCH3, OH, CH3 or CF3.

4. The use according to claim 1, characterized in that: The drug comprises 5'-O-trityl uridine monomer and / or its derivatives and excipients acceptable in pharmaceutical or food science.

5. The use according to claim 1, characterized in that: The excipient is at least one of a binder, a filler, a disintegrant, wine, vinegar and starch.

6. The use according to claim 1, characterized in that: The synthesis method of 5'-O-trityl uridine is as follows: firstly, isoflavones are obtained by coupling reaction; and then, isoflavones and cytisine are subjected to Mannich reaction to synthesize the target product.

7. The use according to claim 6, characterized in that: The specific steps of the synthesis method are as follows: S1: 2,4,6-trihydroxyacetophenone is used as a raw material, K2CO3 is added to selectively free the hydroxyl groups at positions 4 and 6, and then 0.5-3 times the equivalent of chloromethyl methyl ether is added to obtain 2-hydroxy-4,6-dimethyl methyl ether acetophenone, and then N,N-dimethyl dimethyl acetal is added to synthesize aminoenone, and finally 5,7-dimethyl methyl ether-3-iodochromone is synthesized under the action of elemental iodine; 5,7-dimethyl methyl ether-3-iodochromone is dissolved in dichloroethane, and the methyl methyl ether at position 5 is removed to obtain 7-dimethyl methyl ether-3-iodochromone; S2: Compound 5 and compound 6 are used to synthesize isoflavones by coupling reaction, and the methyl ether at position 7 is removed under HCl, and then the methoxy group is removed to obtain broom isoflavones; The compound 5 is: The compound 6 is: S3: Cytisine, cytisine isoflavones and formalin are used to catalyze the synthesis of cytisine-N-methylene-(5,7,4 ’ -trihydroxy) isoflavones.

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