THE USE OF DIPHENOL IN PREPARATION OF MEDICINES FOR PREVENTION AND TREATMENT OF CEREBRAL ISCHEMIA

Abstract

The present invention relates to use of biphenols in the preparation of a medicament for the prevention and treatment of ischemic stroke, specifically to use of 3,3,5,5-tetraisopropyl-4,4-biphenol and salt, ester, or solvate thereof in the preparation of a medicament for the prevention and treatment of ischemic stroke injury.

Claims

1-7. (canceled)

8. A method for treating and/or preventing ischemic stroke in animal or human comprising administering to an animal or human subject an effective amount of 3,3,5,5-tetraisopropyl-4,4-biphenol and a pharmaceutically acceptable salt, ester, or solvate thereof, wherein the structure of 3,3,5,5-tetraisopropyl-4,4-biphenol is as shown in formula (I): ##STR00009##

9. The method according to claim 8, wherein the ester is a monoester or diester of 3,3,5,5-tetraisopropyl-4,4-biphenol, and preferably the ester is a monoethyl ester represented by formula (II) or a diethyl ester represented by formula (III): ##STR00010##

10. The method according to claim 8, wherein the pharmaceutically acceptable salt is a salt formed by 3,3,5,5-tetraisopropyl-4,4-biphenol with organic acid, inorganic acid or alkali metal, and wherein the pharmaceutically acceptable salts is, for example, sulfate, phosphate, hydrochloride, hydrobromide, acetate, oxalate, citrate, succinate, gluconate, tartrate, p-toluenesulfonate, benzenesulfonate, methanesulfonate, benzoate, lactate, maleate, lithium salt, sodium salt, potassium salt, or calcium salt.

11. The method according to claim 8, wherein 3,3,5,5-tetraisopropyl-4,4-biphenol and a pharmaceutically acceptable salt, ester, or solvate thereof can be formulated into a pharmaceutical composition which comprises the 3,3, 5,5-tetraisopropyl-4,4-biphenol or a pharmaceutically acceptable salt, ester, or solvate thereof, and a pharmaceutical excipient.

12. The method according to claim 8, wherein, 3,3,5,5-tetraisopropyl-4,4-biphenol and a pharmaceutically acceptable salt, ester, or solvate thereof is formulated into tablet, capsule, injection, emulsion, liposome, lyophilized powder or microsphere formulation containing it, and wherein the capsule is, for example, a soft capsule.

13. The method according to claim 8, wherein the treatment and/or prevention of ischemic stroke is achieved by improving cerebral ischemia and/or reperfusion neurological impairment; reducing cerebral ischemia and/or reperfusion cerebral infarction volume; reducing the endogenous oxygen free radical scavenger SOD consumption in brain tissues, reducing lipid peroxidation damage, while reducing serum MDA content; down-regulating cellular Fas expression in brain tissues; inhibiting brain cell apoptosis; and/or down-regulating cellular IL-1 and TNF- expression in brain tissues.

14. The method according to claim 8, wherein the ischemic stroke includes damage caused by one or more of the following conditions: cerebral thrombosis, transient ischemic attack, basal ganglia infarction, atherosclerotic thrombotic cerebral infarction, lacunar cerebral infarction, cerebral embolism, and cerebrovascular dementia.

15. A process for the preparation of 3,3,5,5-tetraisopropyl-4,4-biphenol in the method according to claim 8, which comprises the step of preparing 3,3,5,5-tetraisopropyl-4,4-biphenol from a compound represented by the formula (IV) ##STR00011##

16. The process according to claim 15, wherein 3,3,5,5-tetraisopropyl-4,4-biphenol is prepared by dissolving the compound represented by the formula (IV) in an organic solvent, preferably ethyl acetate, and then reacting it with an aqueous solution containing sodium hydrosulfite, and wherein the aqueous solution containing sodium hydrosulfite is preferably an aqueous solution formed of sodium hydrosulfite and sodium hydroxide.

17. The process according to claim 15, wherein the compound represented by the formula (IV) is obtained by dissolving propofol in an organic solvent, preferably ethyl acetate, and then adding an inorganic salt thereto, and wherein the inorganic salt is preferably silver carbonate and anhydrous magnesium sulfate.

18. The process according to claim 15 including the steps of: dissolving propofol in an organic solvent, preferably ethyl acetate, adding an inorganic salt, preferably silver carbonate and anhydrous magnesium sulfate thereto, and then stirring at room temperature; after the reaction is complete, adding water to the reaction solution, filtering and washing the solid, removing the aqueous phase, drying and filtering the ethyl acetate phase, and evaporating the filtrate to dryness, followed by optional washing, to give rosy red crystal; dissolving the rosy red crystal in ethyl acetate and mixing it with a sodium hydrosulfite solution, preferably, a sodium hydrosulfite solution of an aqueous solution of sodium hydrosulfite and NaOH, with optional stirring; then separating the ethyl acetate phase and extracting the aqueous phase with ethyl acetate, followed by drying and filtering, evaporating the filtrate to dryness to give a light yellow solid, and then washing the light yellow solid to obtain 3,3,5,5-tetraisopropyl-4,4-biphenol as a white solid.

19. (canceled)

20. (canceled)

21. The method of claim 11, wherein the ester is a monoester or diester of 3,3, 5,5-tetraisopropyl-4,4-biphenol, and preferably the ester is a monoethyl ester represented by formula (II) or a diethyl ester represented by formula (III): ##STR00012##

22. The method of claim 12, wherein the ester is a monoester or diester of 3,3, 5,5-tetraisopropyl-4,4-biphenol, and preferably the ester is a monoethyl ester represented by formula (II) or a diethyl ester represented by formula (III): ##STR00013##

23. The method of claim 13, wherein the ester is a monoester or diester of 3,3, 5,5-tetraisopropyl-4,4-biphenol, and preferably the ester is a monoethyl ester represented by formula (II) or a diethyl ester represented by formula (III): ##STR00014##

24. The method of claim 14, wherein the ester is a monoester or diester of 3,3, 5,5-tetraisopropyl-4,4-biphenol, and preferably the ester is a monoethyl ester represented by formula (II) or a diethyl ester represented by formula (III): ##STR00015##

Description

DETAILED DESCRIPTION OF THE INVENTION

[0084] The present invention will be further described with reference to the following examples. It is to be understood that these examples are merely illustrative and are not intended to limit the scope of the invention.

Example 1. Compound 1: 3,3,5,5-tetraisopropyl-4,4-biphenol

[0085] ##STR00006##

[0086] 20 g propofol was weighed and dissolved in 100 mL ethyl acetate, 24.75 g silver carbonate and 10 g anhydrous magnesium sulfate were then added thereto, stirred at room temperature for 2 h, and the reaction was checked for completion. Water was added to the reaction solution until no bubble emerged. The solid was filtered and washed with ethyl acetate, and the aqueous phase was removed. The ethyl acetate phase was dried over anhydrous sodium sulfate for 1 h and filtered. The filtrate was evaporated to dryness under reduced pressure and washed with anhydrous methanol to give 12.30 g rosy red crystal. 7 g of the above rosy red solid was dissolved in 100 mL of ethyl acetate. Then, 27.66 g sodium hydrosulfite was dissolved in 1 mol/L NaOH and added to the resultant ethyl acetate solution of the above rosy red solid, the mixture was stirred at room temperature for 1.5 h, and the reaction was checked for completion. The ethyl acetate phase was separated, the aqueous phase was extracted twice with ethyl acetate, dried over anhydrous sodium sulfate and filtered, and the filtrate was evaporated to dryness under reduced pressure to give 5 g of a light yellow solid which was washed with petroleum ether to give 4.5 g white solid. .sup.1H NMR (300 MHz, CDCl.sub.3): 7.22 (s, 4H), 4.81 (s, 2H), 3.27-3.20 (m, 4H), 1.37-1.35 (d, 24H).

Example 2. Compound 2: 4-hydroxy-3,3,5,5-tetraisopropylbiphenyl-4-acetate

[0087] ##STR00007##

[0088] 4-Benzyloxy-3,3,5,5-tetraisopropylbiphenyl-4-acetate (5 g, 10.27 mmol) was dissolved in 200 mL methanol at room temperature, 10% palladium-carbon (570 mg) was added thereto, followed by evacuation to vacuum and charging with hydrogen, which was repeated for three times, and then sealed and reacted at room temperature for 10 h. The palladium-carbon in the reaction solution was filtered, and the filtrate was evaporated under reduced pressure to obtain 4-hydroxy-3,3, 5,5-tetraisopropylbiphenyl-4-acetate (3.9 g, 95.73%) as a white solid. .sup.1H NMR (300 MHz, CDCl.sub.3) 7.19 (s, 4H), 4.86 (s, 1H), 3.37-3.32 (m, 4H), 3.16 (s, 3H), 1.20 (d, 24H).

Example 3. Compound 3: 3,3,5,5-tetraisopropylbiphenyl-4-diacetate

[0089] ##STR00008##

[0090] 4,4-dihydroxy-3,3,5,5-tetraisopropylbiphenyl (5 g, 14.10 mmol) was added to 30 mL acetic anhydride and allowed to reflux for 3 h under nitrogen. The reaction solution was cooled to room temperature, and the acetic anhydride was removed under reduced pressure. Water (200 mL) was added to the residue to give a white solid which was washed with 10% cold ethanol (100 mL) and water (200 mL) and dried to afford 3,3,5,5-tetraisopropylbiphenyl-4-diacetate (6 g, 95.06%) as a white solid.

[0091] White solid, .sup.1H NMR (300 MHz, CDCl.sub.3) 7.19 (s, 4H), 2.91-2.89 (m, 4H), 2.32 (s, 6H), 1.19 (d, 24H).

Example 4. Effects of Compound 1-3 on Neurological Impairment Score, Cerebral Infarct Volume, Fas, IL-1, and TNF- in Brain Tissues, and Cell Apoptosis in Rats with Cerebral Ischemic Reperfusion Injury

[0092] (1) Material:

[0093] An aqueous solution of compound 1-3 in PEG400 at a concentration of 10 mg/mL, in which the concentration of PEG400 in the PEG400 aqueous solution was 400 mg/ml; GL-22M low temperature centrifuge (Hubei Saite Xiangyi); BI2000 image analyzer (Chengdu Techman Software Co., Ltd.), SOD and MDA assay kits (Nanjing Jiancheng Bioengineering Research Institute); Fas and TUNEL kits (Wuhan Boside Biological Engineering Co., Ltd.), IL-1 and TNF- kits (Shanghai HengYuan Biotechnology Co., Ltd.); other reagents were made in China, analytically pure.

[0094] (2) Method:

[0095] 1. Experimental Animals and Grouping

[0096] 120 healthy male SD rats weighing 250-300 g, provided by the Experimental Animal Center of the Fourth Military Medical University, were randomly divided into 5 groups: the sham operation group, the ischemic reperfusion group, the Example 1 group (using compound 1 as the test sample), the Example 2 group (using compound 2 as the test sample) and the Example 3 group (compound 3 as the test sample).

[0097] 2. Animal Model Preparation and Treatment

[0098] Rats were intraperitoneally injected with 10% chloral hydrate 350 mg/kg after anesthesia and opened up at the center of the neck in accordance with the Zea Longa modified method. The right common carotid artery was separated, and the right external carotid artery branches was ligated. A small opening was made at the distal end of the external carotid artery, and a previously prepared thread was inserted through the common carotid artery and the external carotid artery bifurcation into the internal carotid artery, till the anterior end of the middle cerebral artery, with an immersed depth of 18 to 19 mm. The thread was then secured, and the wound was sutured layer by layer. After the operation, the rats were placed in a clean incubator to wake up. The criteria for a successfully prepared cerebral ischemia model was: after waking up, the rats showed Horner syndrome on the right side and hemiplegia on the left side. The sham operation group, the ischemic reperfusion group, and the Example groups were prepared strictly according to the requirements of the cerebral ischemic reperfusion model, while in the sham operation group the thread was put only into the external carotid artery. The animals were allowed to eat and drink freely after waking up. After 2 h, the thread was pulled out to achieve reperfusion. The sham operation group, the ischemic reperfusion group, and the Example groups were administered intravenously the aqueous solution of compound 1-3 in PEG400 at 40 mg/kg (40 mg/kg means that 40 mg of the test compound per kg of the body weight of rats) 30 min before reperfusion and 12 h after reperfusion, respectively. The sham operation group and the ischemic reperfusion group were injected with the same amount of blank PEG400 aqueous solution at the same time point.

[0099] 3. Neurological Impairment Score in Rats

[0100] The neurological impairment was scored in each group 24 h after reperfusion. The scoring was in accordance with the Longa 5-grade method: grade 0: no nerve injury symptom; grade 1: inability of stretching the contralateral forepaw; grade 2: circling to the opposite side; grade 3: tumbling to the opposite side; grade 4: no autonomous activity with loss of consciousness.

[0101] 4. Sample Collection and Preparation

[0102] 24 hours after reperfusion, 8 rats in each group were sacrificed by decapitation, the brain was removed, rinsed with PBS (pH 7.4) at 20 C. for 20 min, evenly sliced (with a thickness of 2 mm), stained in a 2% TTC solution at 37 C. in the dark for 30 min, fixed in 10% formalin for 24 h, and photographs were taken to analyze the infarct volume. A mixture of femoral arterial and venous blood was drawn from another 8 rats in each group under anesthesia, placed in a low temperature centrifuge at 4 C., centrifuged at 3500 r/min for 20 min, and the supernatant was stored in a refrigerator at 20 C. ready for SOD and MDA detection. After the blood was drawn, a perfusion needle was insert through the apex to the ascending aorta, and saline at 4 C. was rapid infused until the effluent became clear, followed by 4% paraformaldehyde phosphate buffer perfusion for fixation; the brain was removed by craniotomy, and brain tissues 2 mm in front of and behind the optic chiasma were taken and fixed, dehydrated, rendered transparent, impregnated in wax, and embedded. Brain continuous coronary tissue pathological sections were consecutively cut for later use. The remaining 8 rats in each group were sacrificed, brains were removed, the ischemic brain hemispheres were immediately taken on an ice tray, and 10% brain tissue homogenate was prepared for the detection of IL-1 and TNF-.

[0103] 5. Determination of Relative Infarct Volume

[0104] The infarct area in each brain section was analyzed and quantified by the imageJ image software, and the ratio of cerebral infarction volume in the overall brain volume was calculated.

[0105] 6. SOD and MDA Determination

[0106] SOD and MDA were determined strictly in accordance with instructions of the kit.

[0107] 7. Fas Determination

[0108] The immunohistochemical method was used for determination. Brain tissue paraffin sections were dewaxed into water, washed with 3% H.sub.2O.sub.2 to eliminate endogenous peroxidase activity, and rinsed with distilled water for 3 times. Sodium citrate buffer was used for antigen heat repairing, and blocking with calf serum was carried out at room temperature for 15 min; a rabbit anti-mouse Fas antibody was added dropwise, allowed to stand at 4 C. overnight, biotinylated goat anti-rabbit IgG was added dropwise, and heated in a water bath at 37 C. for 20 min. Washing with PBS for 5 min was continuously repeated 4 times, followed by staining with DAB and sufficient washing without counterstaining. The sections were then gradient dehydrated with alcohol, made transparent with toluene, sealed and fixed. Images were captured by a video camera under high-power optical microscope and inputted into the image analysis system for image analysis. Five non-overlapping fields were randomly selected from each section. Five areas in each field were selected for determination of gray scale. The average gray scale was calculated, and the average gray scale was inversely proportional to the rate of positive expression.

[0109] 8. Apoptosis Determination

[0110] The TUNEL method was used for determination. Brain tissue paraffin sections were dewaxed into water, washed with 3% H.sub.2O.sub.2 to eliminate endogenous peroxidase activity, and rinsed with distilled water for 2 min, repeated 3 times. A labelling solution was added for labelling at 37 C. for 2 h, and a blocking solution was added for blocking at room temperature for 30 min. A biotinylated anti-digoxin antibody was added for a reaction at 37 C. for 30 min, and SABC was added for a reaction at 37 C. for 30 min. The sections were continuously rinsed with TBS for 5 min repeatedly for 4 times, stained with DAB, sufficiently washed, mild counterstained with Hematoxylin, gradient dehydrated with alcohol, made transparent with toluene, sealed and fixed. Five non-overlapping fields in semi-dark band were randomly selected from each section and inputted into the image analysis system. The number of apoptotic cells was counted, and the average was designated the number of apoptotic cells.

[0111] 9. Determination of IL-1 and TNF-

[0112] Brain tissue homogenate was hypothermally centrifuged at 3000 rpm for 15 min, the supernatant was taken, and IL-1 and TNF- were determined in strict accordance with instructions of the kits.

[0113] (3) Results:

[0114] Compounds 1-3 can substantially improve the neurological impairment in rats with ischemic reperfusion, significantly reduce the cerebral infarction volume in rats with ischemic reperfusion, significantly reduce the consumption of the endogenous oxygen free radical scavenger SOD, reduce lipid peroxidation damage while reducing the serum MDA content, effectively down-regulate cellular Fas expression in brain tissues, effective inhibit brain cell apoptosis, and also effectively down-regulate cellular IL-1 and TNF- expression in brain tissues in rats. The results are shown in Table 1 below.

TABLE-US-00001 TABLE 1 Sham Ischemic operation reperfusion Example 1 Example 2 Example 3 group group group group group Average score 0 3.2 1.8 2.0 2.1 of neurological impairment Relative 0 35.28 5.22 16.21 3.28* 18.71 4.69* 23.22 3.12* cerebral infarction volume (%) SOD activity (U/mL) 101.54 3.45 82.14 4.37 97.16 3.85* 96.78 2.94* 94.46 2.19* MDA content 2.49 0.66 7.22 0.61 4.35 0.52* 4.74 0.67* 5.44 0.49* (mmol/mL) Fas average 175.96 5.14 134.33 6.18 165.11 5.02* 162.47 3.96* 148.36 5.33* gray scale Number of 4.62 1.54 37.26 4.10 17.32 4.49* 19.14 4.24* 24.65 3.41* apoptotic cells IL-1 content 0.39 0.08 0.92 0.14 0.60 0.08* 0.63 0.12* 0.79 0.11* (ng/mL) TNF- content 2.74 0.21 6.47 0.65 4.01 0.82* 4.11 0.72* 5.03 0.59* (ng/mL) In comparison to the ischemic reperfusion group *p < 0.05

Example 5. Effects of Compound 1-3 on Neurological Impairment Score, Cerebral Infarct Volume, Fas, IL-1, and TNF- in Brain Tissues, and Cell Apoptosis in Rats with Permanent Cerebral Ischemic Reperfusion Injury

[0115] (1) Material:

[0116] An aqueous solution of compound 1-3 in PEG400 at a concentration of 10 mg/mL, in which the concentration of PEG400 in the PEG400 aqueous solution was 400 mg/ml; GL-22M low temperature centrifuge (Hubei Saite Xiangyi); BI2000 image analyzer (Chengdu Techman Software Co., Ltd.), SOD and MDA assay kits (Nanjing Jiancheng Bioengineering Research Institute); Fas and TUNEL kits (Wuhan Boside Biological Engineering Co., Ltd.), IL-1 and TNF- kits (Shanghai HengYuan Biotechnology Co., Ltd.); other reagents were made in China, analytically pure.

[0117] (2) Method:

[0118] 1. Experimental Animals and Grouping

[0119] 120 healthy male SD rats weighing 250-300 g, provided by the Experimental Animal Center of the Fourth Military Medical University, were randomly divided into 5 groups: the sham operation group, the permanent cerebral ischemia model group, the Example 1 group (using compound 1 as the test sample), the Example 2 group (using compound 2 as the test sample) and the Example 3 group (compound 3 as the test sample).

[0120] 2. Animal Model Preparation and Treatment

[0121] Rats were intraperitoneally injected with 10% chloral hydrate 350 mg/kg after anesthesia and opened up at the center of the neck in accordance with the Zea Longa modified method. The right common carotid artery was separated, and the right external carotid artery branches was ligated. A small opening was made at the distal end of the external carotid artery, and a previously prepared thread was inserted through the common carotid artery and the external carotid artery bifurcation into the internal carotid artery, till the anterior end of the middle cerebral artery, with an immersed depth of 18 to 19 mm. The thread was then secured, and the wound was sutured layer by layer. After the operation, the rats were placed in a clean incubator to wake up. The criteria for a successfully prepared cerebral ischemia model was: after waking up, the rats showed Horner syndrome on the right side and hemiplegia on the left side. The sham operation group, the permanent cerebral ischemia model group, and the Example groups were prepared strictly according to the requirements of the cerebral ischemic reperfusion model, while in the sham operation group the thread was put only into the external carotid artery. The animals were allowed to eat and drink freely after waking up. The sham operation group, the permanent cerebral ischemia model group, and the Example groups were administered intravenously the aqueous solution of compound 1-3 in PEG400 at 40 mg/kg (40 mg/kg means that 40 mg of the test compound per kg of the body weight of rats) 30 min before insertion of thread and 12 h after embolism, respectively. The sham operation group and the permanent cerebral ischemia model group were injected with the same amount of blank PEG400 aqueous solution at the same time point.

[0122] 3. Neurological Impairment Score in Rats

[0123] The neurological impairment was scored in each group 24 h after the embolism. The scoring was in accordance with the Longa 5-grade method: grade 0: no nerve injury symptom; grade 1: inability of stretching the contralateral forepaw; grade 2: circling to the opposite side; grade 3: tumbling to the opposite side; grade 4: no autonomous activity with loss of consciousness.

[0124] 4. Sample Collection and Preparation

[0125] 24 hours after the embolism, 8 rats in each group were sacrificed by decapitation, the brain was removed, rinsed with PBS (pH 7.4) at 20 C. for 20 min, evenly sliced (with a thickness of 2 mm), stained in a 2% TTC solution at 37 C. in the dark for 30 min, fixed in 10% formalin for 24 h, and photographs were taken to analyze the infarct volume. A mixture of femoral arterial and venous blood was drawn from another 8 rats in each group under anesthesia, placed in a low temperature centrifuge at 4 C., centrifuged at 3500 r/min for 20 min, and the supernatant was stored in a refrigerator at 20 C. ready for SOD and MDA detection. After the blood was drawn, a perfusion needle was insert through the apex to the ascending aorta, and saline at 4 C. was rapid infused until the effluent became clear, followed by 4% paraformaldehyde phosphate buffer perfusion for fixation; the brain was removed by craniotomy, and brain tissues 2 mm in front of and behind the optic chiasma were taken and fixed, dehydrated, rendered transparent, impregnated in wax, and embedded. Brain continuous coronary tissue pathological sections were consecutively cut for later use. The remaining 8 rats in each group were sacrificed, brains were removed, the ischemic brain hemispheres were immediately taken on an ice tray, and 10% brain tissue homogenate was prepared for the detection of IL-1 and TNF-.

[0126] 5. Determination of Relative Infarct Volume

[0127] The infarct area in each brain section was analyzed and quantified by the imageJ image software, and the ratio of cerebral infarction volume in the overall brain volume was calculated.

[0128] 6. SOD and MDA Determination

[0129] SOD and MDA were determined strictly in accordance with instructions of the kit.

[0130] 7. Fas Determination

[0131] The immunohistochemical method was used for determination. Brain tissue paraffin sections were dewaxed into water, washed with 3% H.sub.2O.sub.2 to eliminate endogenous peroxidase activity, and rinsed with distilled water for 3 times. Sodium citrate buffer was used for antigen heat repairing, and blocking with calf serum was carried out at room temperature for 15 min; a rabbit anti-mouse Fas antibody was added dropwise, allowed to stand at 4 C. overnight, biotinylated goat anti-rabbit IgG was added dropwise, and heated in a water bath at 37 C. for 20 min. Washing with PBS for 5 min was continuously repeated 4 times, followed by staining with DAB and sufficient washing without counterstaining. The sections were then gradient dehydrated with alcohol, made transparent with toluene, sealed and fixed. Images were captured by a video camera under high-power optical microscope and inputted into the image analysis system for image analysis. Five non-overlapping fields were randomly selected from each section. Five areas in each field were selected for determination of gray scale. The average gray scale was calculated, and the average gray scale was inversely proportional to the rate of positive expression.

[0132] 8. Apoptosis Determination

[0133] The TUNEL method was used for determination. Brain tissue paraffin sections were dewaxed into water, washed with 3% H.sub.2O.sub.2 to eliminate endogenous peroxidase activity, and rinsed with distilled water for 2 min, repeated 3 times. A labelling solution was added for labelling at 37 C. for 2 h, and a blocking solution was added for blocking at room temperature for 30 min. A biotinylated anti-digoxin antibody was added for a reaction at 37 C. for 30 min, and SABC was added for a reaction at 37 C. for 30 min. The sections were continuously rinsed with TBS for 5 min repeatedly for 4 times, stained with DAB, sufficiently washed, mild counterstained with Hematoxylin, gradient dehydrated with alcohol, made transparent with toluene, sealed and fixed. Five non-overlapping fields in semi-dark band were randomly selected from each section and inputted into the image analysis system. The number of apoptotic cells was counted, and the average was designated the number of apoptotic cells.

[0134] 9. Determination of IL-1 and TNF-

[0135] Brain tissue homogenate was hypothermally centrifuged at 3000 rpm for 15 min, the supernatant was taken, and IL-1 and TNF- were determined in strict accordance with instructions of the kits.

[0136] (3) Results:

[0137] Compounds 1-3 can substantially improve the neurological impairment in permanent cerebral ischemia model rats, significantly reduce the cerebral infarction volume in permanent cerebral ischemia model rats, significantly reduce the consumption of the endogenous oxygen free radical scavenger SOD, reduce lipid peroxidation damage while reducing the serum MDA content, effectively down-regulate cellular Fas expression in brain tissues in permanent cerebral ischemia model rats, effective inhibit brain cell apoptosis, and also effectively down-regulate cellular IL-1 and TNF- expression in brain tissues in permanent cerebral ischemia model rats. The results are shown in Table 2 below.

TABLE-US-00002 TABLE 2 Permanent cerebral Sham ischemia operation model Eample 1 Example 2 Example 3 group group group group group Average 0 3.5 2.1 2.4 2.5 score of neurological impairment Relative 0 39.82 5.19 21.27 4.26* 24.71 3.76* 27.92 4.51* cerebral infarction volume (%) SOD activity 102.29 4.72 84.42 3.88 98.44 3.12* 97.36 4.51* 93.28 3.49* (U/mL) MDA 2.45 0.67 7.05 0.57 4.16 0.52* 4.49 0.59* 5.10 0.82* content (mmol/mL) Fas average 178.69 4.53 126.7 7.82 161.31 5.27* 158.28 6.66* 149.63 8.15* gray scale Number of 4.47 1.09 39.41 4.34 19.31 3.36* 20.41 5.02* 23.27 3.78* apoptotic cells IL-1 0.38 0.05 0.95 0.17 0.61 0.15* 0.64 0.09* 0.78 0.12* content (ng/mL) TNF- 2.58 0.23 6.74 0.47 4.17 0.62* 4.25 0.66* 5.15 0.88* content (ng/mL) In comparison to the permanent cerebral ischemia model group *p < 0.05

Example 6. Comparison of Efficacy of the Present Invention Over Existing Positive Drugs

[0138] The therapeutic effects of propofol and edaravone on the ischemic reperfusion model and the permanent cerebral ischemia model rats were evaluated according to the methods described above in Examples 4 and 5 (propofol 15 mg/kg; edaravone 3 mg/kg; herein, 15 mg/kg means that the rats were given 15 mg of propofol per kilogram of body weight, and 3 mg/kg means the rats were given 3 mg of edaravone per kilogram of body weight). Also, the change of behavior of the rats were observed after administration. The experimental results were shown in the following Table 3 and Table 4, respectively.

TABLE-US-00003 TABLE 3 Efficacy of positive drugs in ischemic reperfusion rat model Propofol Edaravone injection injection (30 mg/ Model group Example 1 group (100 mg/10 mL) 20 mL) Neurological 3.2 1.8 2.5 2.6 impairment score Relative cerebral 35.28 5.22 16.21 3.28 27.14 5.08* 29.31 3.95* infarct volume (%) Fas average gray 134.33 6.18 165.11 5.02 142.15 5.71* 139.65 2.05* scale SOD activity (U/mL) 82.14 4.37 97.16 3.85 88.25 2.19* 88.16 3.99* MDA content 7.22 0.61 4.35 0.52 5.58 0.48* 6.17 0.54* (mmol/mL) Number of 37.26 4.10 17.32 4.49 26.08 3.72* 28.6 4.90* apoptotic cells IL-1 content 0.92 0.14 0.60 0.08 0.73 0.09* 0.75 0.05* (ng/mL) TNF- content 6.47 0.65 4.01 0.82 5.02 0.31* 5.14 0.44* (ng/mL) Change of Sober, no Sober, no Anesthetized, Sober, no behavior of rats significant significant loss of righting significant within 30 min difference as difference as reflex difference as after the compared to compared to compared to administration pre- pre- pre- 12 h after administration administration administration reperfusion *Relative to the Example 1 group, p < 0.05.

[0139] The results in Table 3 showed that the therapeutic effect of the biphenyl derivatives on the model group was superior to that of the positive control drugs propofol and edaravone. Although most of the Examples show significant advantages over the efficacy of the positive drugs (p<0.05), Table 3 lists only the efficacy experimental results of Example 1 in comparison to the positive drugs as a reference. Further, it was found that the rats lost righting reflex after propofol administration and entered an anesthetic state while the rats in the other administration groups did not show obvious change of behavior.

TABLE-US-00004 TABLE 4 Efficacy of positive drugs in permanent cerebral ischemia rat model Propofol Edaravone injection injection (30 mg/ Model group Example 1 group (100 mg/10 mL) 20 mL) Neurological 3.5 2.1 2.7 2.8 impairment score Relative cerebral 39.82 5.19 21.27 4.26 30.11 3.58* 31.29 4.02* infarct volume (%) Fas average gray 126.7 7.82 161.31 5.27 140.51 4.84* 138.95 5.50* scale SOD activity (U/mL) 84.42 3.88 98.44 3.12 89.13 3.82* 90.62 3.81* MDA content 7.05 0.57 4.16 0.52 6.10 0.29* 5.98 0.44* (mmol/mL) Number of 39.41 4.34 19.31 3.36 31.06 3.55* 33.19 3.81* apoptotic cells IL-1 content (ng/mL) 0.95 0.17 0.61 0.15 0.78 0.07* 0.76 0.04* TNF- content 6.74 0.47 4.17 0.62 5.40 0.32* 5.53 0.54* (ng/mL) Change of Sober, no Sober, no Anesthetized, Sober, no behavior of rats significant significant loss of righting significant within 30 min difference as difference as reflex difference as after the compared to compared to compared to administration 12 h pre- pre- pre- after embolism administration administration administration *Relative to the Example 1 group, p < 0.05.

[0140] The results in Table 4 showed that the therapeutic effect of the biphenyl derivatives on the model group was superior to that of the positive control drugs propofol and edaravone. Although most of the Example groups show significant advantages over the efficacy of the positive drugs (p<0.05), Table 4 lists only the efficacy experimental results of Example 1 in comparison to the positive drugs as a reference. Further, it was found that the rats lost righting reflex after propofol administration and entered an anesthetic state while the rats in the other administration groups did not show obvious change of behavior.

Example 7. Oil-Based Preparation

[0141] The formulation of the oil-based preparation of the biphenyl derivative of the present invention can be as shown in Table 5:

TABLE-US-00005 TABLE 5 Components Amount in formulation 3,3,5,5-tetraisopropyl-4,4-biphenol 200 mg Tetrahydrofuran polyglycol ether 0.80 ml Vitamin E acetate 5 mg Benzyl alcohol 50 l Castor oil Add to 1 ml

Example 8. Tablet

[0142] The formulation of the tablet of the biphenyl derivative of the present invention can be as shown in Table 6:

TABLE-US-00006 TABLE 6 Components Amount in formulation 3,3,5,5-tetraisopropyl-4,4-biphenyl diacetate 200 mg Lactose 140 mg Microcrystalline cellulose 100 mg Starch pulp 50 mg Sodium carboxymethyl starch 10 mg Magnesium stearate 1 mg

Example 9. Capsule

[0143] The formulation of the capsule of the biphenyl derivative of the present invention can be as shown in Table 7:

TABLE-US-00007 TABLE 7 Components Amount in formulation 3,3,5,5-tetraisopropyl-4,4-biphenol 1 g Olive oil 10 g Egg yolk lecithin 1.2 g Vitamin E 0.2 g

Example 10. Emulsion

[0144] The formulation of the emulsion of the biphenyl derivative of the present invention can be as shown in Table 8:

TABLE-US-00008 TABLE 8 Components Amount in formulation 4-hydroxy-3,3,5,5-tetraisopropylbiphenyl-4- 1 g acetate Soybean oil 10 g Egg yolk lecithin 1.2 g Vitamin E 0.1 g Glycerin 2.25 g Sodium hydroxide Appropriate amount Water for injection Add to 100 ml