CN114685537A

CN114685537A - Application of butylphthalide derivative G-3702 in preparation of medicines for preventing and treating cerebral ischemia reperfusion injury

Info

Publication number: CN114685537A
Application number: CN202011582441.9A
Authority: CN
Inventors: 汪俊松; 赵文丽
Original assignee: Nanjing University of Science and Technology
Current assignee: Nanjing University of Science and Technology
Priority date: 2020-12-28
Filing date: 2020-12-28
Publication date: 2022-07-01

Abstract

The invention discloses an application of butylphthalide derivative G-3702 in preparing a medicament for preventing and treating cerebral ischemia reperfusion injury. The structural formula of the butylphthalide derivative G-3702 is shown in the specification

The G-3702 of the invention has significant effect of resisting cerebral ischemia reperfusion injury, has better effect than butylphthalide which can significantly improve central nerve function injury of patients with ischemic stroke, and can be used for preparing medicaments for preventing and treating cerebral ischemia reperfusion injury.

Description

Application of butylphthalide derivative G-3702 in preparation of medicines for preventing and treating cerebral ischemia-reperfusion injury

Technical Field

The invention belongs to the technical field of medicines, and relates to an application of butylphthalide derivative G-3702 in preparation of medicines for preventing and treating cerebral ischemia-reperfusion injury.

Background

Cerebrovascular disease, which is a common clinical disease and frequently encountered disease, is the third most fatal disease after cardiovascular diseases and cancers, and is the first disabling factor. The incidence, the fatality rate and the disability rate of cerebrovascular diseases in China have the tendency of increasing year by year, wherein ischemic stroke accounts for most (80-88%), and particularly, the injury of reperfusion after cerebral ischemia causes the cerebrovascular diseases to have acute disease potential, high fatality rate, serious sequelae and short optimal treatment time window, so the cerebrovascular diseases become clinically difficult to treat. At present, the only drug approved by FDA for treating ischemic stroke is alteplase (Rt-PA), and the application of the Rt-PA within 4.5h of stroke has a certain promotion effect on improving nerve function, but less than 5 percent of patients benefit from the treatment due to narrow treatment window. Therefore, the search for new therapeutic drugs for ischemic stroke becomes urgent.

Butylphthalide (NBP/Enbipro) which is developed by the institute of medicine and Shiyao group of Chinese medical academy of sciences and can remarkably improve the damage of the central nervous function of a cerebral ischemic stroke patient (can rebuild the microcirculation of the cerebral ischemic area, remarkably reduce the area of cerebral infarction, protect the mitochondrial function and improve the cerebral metabolism) is widely accepted by neurologists in China, and clinical experiments of II phase are started in the United states at present. However, in China, with the clinical discovery that long-term administration of butylphthalide can cause the serum transaminase of a patient to be obviously increased, and certain liver and kidney toxicity exists; especially, the butylphthalide is accumulated in the liver and damages the liver because stroke patients are older and have poor metabolism. The structure of the artificially synthesized butylphthalide (racemization) is the same as that of the levo-apigenin extracted from the celery seeds, and a series of derivative compounds synthesized based on the artificially synthesized butylphthalide (racemization) are expected to solve the clinical adverse reaction of the butylphthalide.

Disclosure of Invention

The invention aims to provide application of butylphthalide derivative G-3702 in preparation of medicines for preventing and treating cerebral ischemia-reperfusion injury.

The purpose of the invention is realized as follows:

the butylphthalide derivative G-3702 has a structural formula as follows:

the invention also provides a preparation method of the butylphthalide derivative G-3702, which comprises the following synthetic route:

firstly, halogenated aromatic hydrocarbon and Pd (0) are subjected to oxidation addition reaction to generate Pd (II) complex Ar-Pd (II) -X,then the Pd (II) complex is reacted with Na₂CO₃The intermediate and diboron compound are subjected to metal transfer reaction to generate a complex of boron and palladium, and then reduction elimination reaction is carried out to generate corresponding aryl boron compound and Pd (0), and the method comprises the following specific steps:

o-bromobenzone, Pd (oAc)₂Mixing with THF aqueous solution until the complex formed by o-bromobenzone and Pd is mixed with Na₂CO₃After all the materials react to form organic palladium acetate compound, adding diboron compound to produce metal transfer reaction to form the intermediate pinacol o-valeryl phenylboronic acid ester, regulating pH value to 5-6 with HCl, adding NaBH₄And (3) generating a precipitate, controlling the temperature of the reaction solution to be 80-100 ℃, continuing to react for a certain time, stopping heating, standing and cooling, separating out a large amount of crystals, and performing suction filtration and washing to obtain the butylphthalide derivative G-3702.

Further, the invention provides application of the butylphthalide derivative G-3702 in preparation of medicines for preventing and treating cerebral ischemia-reperfusion injury.

The cerebral ischemia reperfusion injury comprises but is not limited to focal cerebral ischemia (MCAO) reperfusion injury, whole cerebral ischemia injury caused by four-artery ligation, acute incomplete cerebral ischemia injury and the like.

The invention carries out middle cerebral artery embolism operation (MCAO) on SD rats to cause an ischemia reperfusion model to simulate human acute ischemic stroke, and carries out intraperitoneal injection after operation to treat NBP and G-3702, and the drug effect of the SD rats is evaluated by neuroethology scoring, cerebral infarction area, death rate, HE staining section and immunofluorescence chemical staining section. The results all prove that the butylphthalide derivative G-3702 has a remarkable effect of resisting cerebral ischemia-reperfusion injury and can be used for preparing a medicine for preventing and treating the cerebral ischemia-reperfusion injury.

Drawings

FIG. 1 is a representative TTC stained section of each experimental group.

Fig. 2 is a statistical chart of animal mortality, neurobehavioral scores, and cerebral tissue infarct size for each experimental group.

Fig. 3 is a graph of HE staining for each experimental group at magnification: x 200.

FIG. 4 is a photograph of immunofluorescent stained sections of nerve cells of each experimental group.

FIG. 5 is an immunofluorescent staining section of astrocytes for each experimental group.

Figure 6 is an index plot of serum oxidative stress for each experimental group ([ p ] 0.05, [ p ] 0.01and [ p ] 0001).

Detailed Description

The invention is further described below by means of examples and figures.

Example 1

The preparation method of the butylphthalide derivative G-3702 comprises the following specific steps:

o-bromobenzone, Pd (oAc)₂Mixing with THF aqueous solution until the complex formed by o-bromobenzone and Pd is mixed with Na₂CO₃After all the reaction to form organic palladium acetate compound, adding diboron compound to produce metal transfer reaction to form intermediate pinacol o-valeryl phenylboronic acid ester, regulating pH to 5-6 with HCl, adding NaBH₄And (3) generating a precipitate, controlling the temperature of the reaction solution to be 80-100 ℃, continuing to react for a certain time, stopping heating, standing and cooling, separating out a large amount of crystals, and performing suction filtration and washing to obtain the butylphthalide derivative G-3702.

Example 2

Test of effect of butylphthalide derivative G-3702 on cerebral ischemia reperfusion injury of rats

1. Grouping and administration of drugs

After 7 days of acclimation, SD rats were randomly divided into 4 groups of 20 animals each: the Sham group (Sham), Model group (Model), NBP group (3.0mg/kg), and G-3702 group (3.0mg/kg) were administered intraperitoneally at 0, 2, and 4 hours of reperfusion (I.P.) respectively. The Sham and Model groups were given the same dose of saline as the NBP, G-3702 groups.

2. Establishment of rat cerebral ischemia reperfusion model

The Middle Cerebral Artery (MCA) is the main blood vessel supplying cerebral blood flow and blood sugar, so the method of blocking the middle cerebral artery is used to cause an experimental cerebral ischemia model, namely MCAO, to simulate human stroke. The invention adopts a wire-tying method to form a rat local cerebral ischemia reperfusion model. Rats were injected with chloral hydrate (350.00mg/kg) intraperitoneally, fixed in a supine position after anesthesia, and a longitudinal 2cm long incision was made slightly to the left in the midline of the neck, and the muscle fascia was bluntly isolated to expose the left Common Carotid Artery (CCA), External Carotid Artery (ECA), Internal Carotid Artery (ICA). Temporarily clamping the common carotid artery, dissecting the external carotid artery, making a longitudinal incision on the external carotid artery, and gently pushing the plug thread from the incision along the external carotid artery through the common carotid bifurcation into the internal carotid artery to occlude the Middle Cerebral Artery (MCA), the plug thread entering a length of about 18 to 20mm from the bifurcation. After 2h, the plug was slowly pulled out to achieve ischemia reperfusion. The MCAO model was constructed for the model group and the two administration groups, and only blood vessels were isolated in the sham operation group without inserting a plug wire. The whole operation process is carried out on a heat preservation blanket to ensure that the rectal temperature of the rat is maintained at 37 +/-1 ℃.

3. Results and analysis of the experiments

(1) Neuroethological scoring

After 24h of reperfusion, neuro-behavioral scoring was performed according to the criteria of the Berderson scoring postural reflex test (postural reflex test). Mortality and behavioral status of experimental model animals were recorded during the course of the experiment.

Bederson scores four functional grades:

0 minute: no symptoms of nerve damage

1 minute: the contralateral forepaw can not be fully extended in the tail suspension experiment

And 2, dividing: reduced resistance of forelimbs to contralateral thrust

And 3, dividing: turn round to opposite side

(2) TTC staining of brain tissue sections

After neuroethological scoring, rats were sacrificed by cervical dislocation, 6 brain tissue samples were randomly taken from each group, frozen at-20 ℃ for 30min, removed, and the two hemispheres were cut into coronal sections of about 2mm in thickness, and immersed in 15ml of 2% TTC staining solution. Incubating at 37 deg.C in dark for about 30min, and turning the upper and lower surfaces of brain slice at least once. 4% formaldehyde fixation solution overnight, and photograph. Normal brain tissue is stained dark red, and the brain infarct area is white; and the infarct size was statistically analyzed using Image J software.

(3) HE staining and immunofluorescence chemical analysis

After taking the brain, the brain was quickly dropped into the tissue fixative overnight, embedded in paraffin, sliced into 5 μm thick serial coronal sections with a microtome, and stained with HE (hematoxin-eosin) for morphological observation. And (3) performing immunofluorescence chemical analysis, testing the expression conditions of two proteins, namely NeuN and GFAP, and inspecting the states of nerve cells and astrocytes in brain tissues.

(4) Oxidative stress indicator analysis

The contents of serum superoxide dismutase (SOD), glutathione peroxidase (GSH) and Malondialdehyde (MDA) are detected according to the instruction of a kit (purchased from Nanjing to build Biotechnology Co., Ltd.) to evaluate the oxidative stress state.

(5) Analysis of Experimental results

(5.1) G-3702 improves the neurobehavioral scores of ischemia-reperfusion in rats and reduces the mortality of ischemia-reperfusion rats

The experiment records the neuroethology scoring of the rats after model building in real time, eliminates the rats with lower neuroethology scoring after reperfusion, ensures the success of the model and the consistency of the technique, and carries out the neuroethology scoring after 24h to obtain the most practical pharmacodynamic evaluation. And randomly drawing a rat brain tissue sample for TTC staining (n is 6), and calculating the infarct area. As shown in fig. 2, after NBP and G-3702 treatment, the mortality rate in 24h of rat molding was greatly reduced and the infarct size of brain tissue of rat was significantly reduced. As shown in fig. 1, a representative TTC staining picture is selected, and reduction of the infarct size can be visually seen.

(5.2) G-3702 reduction of cerebral edema in rats after ischemia reperfusion

In the experiment, when the brain tissue of the Model group is observed by naked eyes, the brain tissue can be seen to swell, the color is pale and soft, the gyrus becomes flat, the sulcus becomes light, and the weight is increased; under a microscope (figure 3), large-area infarction of right cerebral cortex can be seen, and brain tissue necrosis is liquefied to form a circular or oval hollow mesh softening range with a clear boundary; a large amount of neurons are reduced, necrotic neurons have enhanced acidophilic property, cell nucleus is deeply contracted and stained, the structure is unclear, part of necrotic neurons are dissolved and disappear, and microglial infiltration is accompanied; infarct damage invades and deeply stains the hippocampus, which is heavily shrinked by a large number of pyramidal cells in the CA3 region of the hippocampus, with a blurred nuclear and cytoplasmic boundary. In the G-3702 group, compared with the Model group, the area of the cerebral cortex necrosis liquefaction is obviously reduced, cells at the injured part are circular or oval, and the boundary is clear; the alkalophilicity of a large number of neurons and glial cells is enhanced, the cell nucleus is deeply dyed and is solidified and condensed, the cell nucleus is triangular or irregular, the nucleolus is not obvious, and the fine acidophilic property is enhanced; ipsilateral white matter nerve fibers are loose and disorganized; the cone cells in the DG region of the hippocampus are deeply contracted, strongly basophilic and compact. In the NBP group, the area of necrosed liquefaction of the cerebral cortex was significantly reduced compared to the Model group, and the degree of brain damage was similar to G-3702.

(5.3) G-3702 reduces the damage of cerebral nerve cells and astrocytes caused by ischemia and hypoxia

After treatment with NBP and G-3702, the number of surviving neural cells (NeuN + cells) was significantly increased (fig. 4); the number of astrocyte (GFAP) lesions decreased (fig. 5), and a viable healthy cell morphology was seen.

(5.4) G-3702 can reduce the oxidative stress status of rats after ischemia-reperfusion

The MCAO group with the oxidative stress marker MDA content is obviously higher than the Sham group, and the G-3702 group is obviously lower than the MCAO group (P < 0.05). Compared with the Sham group, the activities of antioxidant enzymes such as SOD, GSH and the like in the MCAO rat brain are obviously reduced (figure 6), but the contents of the antioxidant enzymes are improved after NBP and G-3702 treatment, the G-3702 reduces the accumulation of MDA (lipid peroxidation) products, improves the contents of antioxidant substances SOD and GSH, and relieves the oxidative stress of the MCAO rat.

Claims

1. The butylphthalide derivative G-3702 has the structural formula:

2. the method for preparing butylphthalide derivative G-3702 according to claim 1, comprising the following steps:

o-bromobenzone, Pd (oAc)₂Mixing with THF aqueous solution until the complex formed by o-bromobenzone and Pd is mixed with Na₂CO₃After all the reaction to form organic palladium acetate compound, adding diboron compound to produce metal transfer reaction to form intermediate pinacol o-valeryl phenylboronic acid ester, regulating pH to 5-6 with HCl, adding NaBH₄And (3) generating precipitates, controlling the temperature of the reaction solution to be 80-100 ℃, continuing to react for a certain time, stopping heating, standing and cooling, separating out a large number of crystals, and performing suction filtration and washing to obtain the butylphthalide derivative G-3702.

3. The use of butylphthalide derivative G-3702 according to claim 1 in the preparation of a medicament for the prevention and treatment of cerebral ischemia-reperfusion injury.

4. The use of claim 3, wherein the cerebral ischemic reperfusion injury is focal cerebral ischemic reperfusion injury, whole cerebral ischemic injury due to four-artery ligation, or acute incomplete cerebral ischemic injury.