Biphenyl derivative and uses thereof

Abstract

The present invention relates to a biphenyl derivative and use thereof, and the biphenyl derivative has a structure represented by the formula (I) as defined in the specification. The use refers the use of the biphenyl derivative or a pharmaceutically acceptable salt or solvate thereof, or a pharmaceutical composition comprising the biphenyl derivative or a pharmaceutically acceptable salt or solvate thereof in the preparation of a medicament for the treatment and/or prevention of ischemic stroke.

Claims

1. A biphenyl derivative represented by formula I or a pharmaceutically acceptable salt or solvate thereof, ##STR00018## wherein R.sub.1R.sub.3, R.sub.4, and R.sub.6 in the biphenyl derivative are each independently n-propyl or isopropyl; R.sub.2 is selected from the group consisting of optionally substituted hydroxyl, optionally substituted carboxyl, optionally substituted acyl, optionally substituted ester, and halogen; R.sub.5 is selected from the group consisting of optionally substituted hydroxyl, optionally substituted carboxyl, optionally substituted acyl, optionally substituted ester, and halogen; with the proviso that when R.sub.2 and R.sub.5 are both hydroxyl, R.sub.1, R.sub.3, R.sub.4, and R.sub.6 are not simultaneously isopropyl; wherein the biphenyl derivative has a structure selected from a group consisting of: ##STR00019## ##STR00020##

2. The biphenyl derivative or a pharmaceutically acceptable salt or solvate thereof according to claim 1, wherein R.sub.1, R.sub.3, R.sub.4, and R.sub.6 in the biphenyl derivative are each independently isopropyl.

3. The biphenyl derivative or a pharmaceutically acceptable salt or solvate thereof according to claim 1, wherein the pharmaceutically acceptable salt includes 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.

4. A pharmaceutical composition for the treatment and/or prevention of ischemic stroke, wherein the composition comprises the biphenyl derivative or a pharmaceutically acceptable salt or solvate thereof according to claim 1 and a pharmaceutical excipient.

5. The composition according to claim 4, wherein the pharmaceutical composition is in a pharmaceutically acceptable dosage form and the dosage form is selected from a tablet, a capsule, injection, emulsion, liposome, lyophilized powder or microsphere formulation.

6. A method of treating and/or preventing ischemic stroke in an animal or human, wherein the method comprises administering to the animal or human subject an effective dose of a biphenyl derivative or a pharmaceutically acceptable salt or solvate thereof according to claim 1.

7. The method according to claim 6, 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 infarction, cerebral embolism or brain vascular dementia.

8. The method according to claim 6, wherein the treatment and/or prevention of ischemic stroke in the animal or human is achieved by improving cerebral ischemia and/or reperfusion neurological impairment; reducing cerebral ischemia and/or reperfusion cerebral infarction volume; reducing the consumption of endogenous oxygen free radical scavenger SOD, reducing lipid peroxidation damage, and lowering the serum MDA content; down-regulating the cellular expression of Fas in brain tissues; inhibiting brain cell apoptosis or; down-regulating the cellular expression of IL-1 and TNF- in brain tissues.

9. The biphenyl derivative or a pharmaceutically acceptable salt or solvate thereof according to claim 1, wherein the biphenyl derivative has a structure of: ##STR00021##

10. The pharmaceutical composition according to claim 4, wherein the biphenyl derivative has a structure of: ##STR00022##

11. The method according to claim 6, wherein the biphenyl derivative has a structure of: ##STR00023##

12. The composition according to claim 4, wherein the capsule is a soft capsule.

Description

DETAILED DESCRIPTION OF THE INVENTION

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

EXAMPLE 1

Compound 1: 4-hydroxy-3,3,5,5-tetraisopropylbiphenyl-4-acetate

(2) ##STR00006##

(3) Preparation procedure: 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 then added thereto, evacuated to vacuum and charged with hydrogen, which was repeated 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 give 4-hydroxy-3,3,5,5-tetraisopropylbiphenyl-4-acetate (3.9 g, 95.73%) as 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 2

Compound 2: 3,3,5,5-tetraisopropylbiphenyl-4-diacetate

(4) ##STR00007##

(5) Preparation procedure: 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 obtain 3,3,5,5-tetraisopropylbiphenyl-4-diacetate (6 g, 95.06%), as 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 3

Compound 3: 3,3,5,5-tetraisopropylbiphenyl-4,4-bis(oxymethylene phosphate)

(6) ##STR00008##

(7) (1) 4,4-dihydroxy-3,3,5,5-tetraisopropylbiphenyl (0.5 g, 1.4 mmol) was dissolved in dry THF (10 mL), solid NaOH (0.224 g, 5.6 mmol) and bromochloromethane (8.185 g, 84 mmol) was added thereto, and then refluxed under N.sub.2 for 2 h. The reaction solution was cooled to room temperature, filtered, and concentrated to give a yellow oil as intermediate.

(8) (2) Triethylamine (1.4 mL, 11.03 mmol) and 85% phosphoric acid (0.5 mL, 8.9 mmol) was added sequentially to 10 mL of anhydrous acetonitrile. The intermediate obtained in (1) was added to the acetonitrile solution under stirring, and then reacted at 65 C. for 2 h. The reaction solution was cooled to room temperature, the solvent was evaporated, and the residue was dissolved in 15 mL of water, adjusted to pH=1.5 with 8 M HCl, and extracted with anhydrous ether. The organic phase was combined, washed with saturated sodium chloride, dried over anhydrous sodium sulfate, and concentrated to give a yellow oil.

(9) (3) 5 mL of water was added to the above oil, adjusted to pH 9 with 20% sodium hydroxide solution, and extracted twice with toluene. The aqueous phase was concentrated to volume, and 9 mL of isopropanol was added. The mixture was heated at 70 C. until the solution became transparent, and then cooled to 0 C. White solid was precipitated, filtered, and dried under vacuum at 45 C. to give 3,3, 5,5-tetraisopropylbiphenyl-4,4-bis(oxymethylenephosphate) (50 mg, 5%). .sup.1H NMR (300 MHz, D.sub.2O) 7.29 (s, 4H), 5.20 (s, 4H), 3.36-3.12 (m, 4H), 1.12 (d, 24H).

EXAMPLE 4

Compound 4: 4-hydroxy-3,3,5,5-tetraisopropylbiphenyl-4-dimethyl carbamate, and compound 5: 3,3,5,5-tetraisopropylbiphenyl-4,4-bis(dimethylcarbamate)

(10) ##STR00009##

(11) 4,4-dihydroxy-3,3,5,5-tetraisopropylbiphenyl (1.0 g, 2.8 mmol) was dissolved in methylene chloride, solid sodium hydroxide (0.112 g, 2.8 mmol) was added thereto under stirring, and then N,N-dimethylformyl chloride (0.3 mL, 2.8 mmol) was added slowly and refluxed for 3 h. The solvent was evaporated to dryness, water was added, and the mixture was extracted with ethyl acetate, washed with saturated sodium chloride, dried over anhydrous sodium sulfate, and concentrated to give a yellow oil which was then purified with petroleum ether-ethyl acetate eluent and recrystallized from petroleum ether-ethyl acetate to give 4-hydroxy-3,3,5,5-tetraisopropylbiphenyl-4-dimethyl carbamate (0.36 g, 30.2%) as a white solid and 3,3,5,5-tetraisopropylbiphenyl-4,4-bis(dimethylcarbamate) (0.31 g, 22.3%) as a white solid.

(12) 4-hydroxy-3,3,5,5-tetraisopropylbiphenyl-4-dimethylcarbamate: white solid. .sup.1H NMR (300 MHz, CDCl.sub.3) 7.16 (s, 2H), 7.12 (s, 2H), 4.82 (s,1H), 3.11 (s,6H), 2.98-2.93 (m, 4H), 1.20 (d, 24H).

(13) 3,3,5,5-tetraisopropylbiphenyl-4,4-bis(dimethylcarbamate): white solid. .sup.1H NMR (300 MHz, CDCl.sub.3) 7.18 (s, 4H), 3.12 (s, 12H), 2.98-2.94 (m, 4H), 1.22 (d, 24H).

EXAMPLE 5

Compound 6: [4-(4-hydroxy-3,3,5,5-tetraisopropylbiphenyl)oxy]-4-carbonyl butyric acid

(14) ##STR00010##

(15) 4,4-dihydroxy-3,3,5,5-tetraisopropylbiphenyl (5.00 g, 14.10 mmol) was dissolved in DMSO (20 mL), and succinic anhydride (1.41 g, 14.09 mmol) was then added thereto and heated at 90 C. for a reaction for 5 h. The reaction solution was cooled to room temperature, into which water was added, extracted with ethyl acetate, dried over anhydrous sodium sulfate, and filtered to remove sodium sulfate. The filtrate was purified with petroleum ether-ethyl acetate eluent to give [4-(4-hydroxy-3,3,5,5-tetraisopropylbiphenyl)oxy]-4-carbonylbutyric acid (3.50 g, 54.59%) as a white solid. .sup.1H NMR (300 MHz, CDCl.sub.3) 11.10 (s, 1H), 7.65 (s, 2H), 7.51 (s, 2H), 5.35 (s, 1H), 3.07-3.04 (m, 4H), 2.71 (s, 4H), 1.20-1.18 (d, 24H).

EXAMPLE 6

Compound 7: 4,4-dihydroxy-3,3-diisopropyl-5,5-dipropylbiphenyl

(16) ##STR00011##

(17) (1) O-isopropylphenol (1.0 g, 7.3 mmol) and allyl bromide (14.6 mmol) were successively added to a 25 mL round bottom flask and dissolved in dichloromethane.

(18) (2) Benzyl tributyl ammonium bromide (0.26 g, 0.73 mmol) was added to another 50 mL flask and dissolved in a 1 M NaOH solution.

(19) (3) The solution obtained in (1) was added slowly to the solution obtained in (2) at room temperature and stirred at room temperature for 2 h. The organic phase was separated, and the aqueous phase was extracted with dichloromethane. The organic phases were combined, washed with water and saturated sodium chloride, dried over anhydrous sodium sulfate, and concentrated to give a colorless liquid. The liquid was heated under nitrogen at 250 C. for 2 h, cooled and purified with column chromatography to give a colorless liquid. The colorless liquid was dissolved in absolute ethanol and reduced by addition of Pd/C to give 2-isopropyl-6-propylphenol: .sup.1H NMR (400 MHz, CDCl.sub.3) 7.06 (dd, J=7.6, 1.6 Hz, 1H), 6.97 (dd, J=7.5, 1.6 Hz, 1H), 6.85 (t, J=7.6 Hz, 1H), 4.75 (s, 1H), 3.22-3.13 (m, 1H), 2.59-2.54 (m, 2H), 1.72-1.59 (m, 2H), 1.26 (d, J=6.9 Hz, 6H), 0.99 (t, J=7.3 Hz, 3H).

(20) (4) The obtained liquid (1.0 g, 5.6 mmol) was dissolved in 20 mL of dichloromethane, into which the catalyst Cu(OH)Cl.TMEDA (50 mg, 0.1 mmol) was added, and stirred at room temperature to give a red solid quinone which was then reduced with sodium hydrosulfite to give 4,4-dihydroxy-3,3-diisopropyl-5,5-dipropylbiphenyl (1.1 g, 55.5%).

(21) 4,4-dihydroxy-3,3-diisopropyl-5,5-dipropylbiphenyl: .sup.1H NMR (300 MHz, CDCl.sub.3) 7.29 (s, 4H), 6.52 (s, 2H), 3.13-3.08 (m, 2H), 2.43-2.40 (m, 4H), 1.51-1.43 (m, 4H), 1.03 (d, 12H), 0.84-0.81 (m, 6H).

EXAMPLE 7

Compound 8: 4,4-dihydroxy-3,3,5,5-tetrapropylbiphenyl

(22) ##STR00012##

(23) (1) O-isopropylphenol (1.0 g, 7.3 mmol) and allyl bromide (14.6 mmol) were successively added to a 25 mL round bottom flask and dissolved in dichloromethane.

(24) (2) Benzyl tributyl ammonium bromide (0.26 g, 0.73 mmol) was added to another 50 mL flask and dissolved in a 1 M NaOH solution.

(25) (3) The solution obtained in (1) was added slowly to the solution obtained in (2) at room temperature and stirred at room temperature for 2 h. The organic phase was separated, and the aqueous phase was extracted with dichloromethane. The organic phases were combined, washed with water and saturated sodium chloride, dried over anhydrous sodium sulfate, and concentrated to give a colorless liquid. The liquid was heated under nitrogen at 250 C. for 2 h, cooled and purified with column chromatography to give a colorless liquid. The colorless liquid was dissolved in absolute ethanol and reduced by addition of Pd/C to give 2,6-dipropylphenol: .sup.1H NMR (400 MHz, CDCl.sub.3) 7.06 (t, J=7.6, Hz, 1H), 6.94 (dd, J=7.6, 1.6 Hz, 2H), 5.35 (s, 1H), 2.62 (t, 4H), 1.68-1.59 (m, 4H), 0.90 (t, J=7.3 Hz, 6H).

(26) (4) The obtained liquid (1.0 g, 5.6 mmol) was dissolved in 20 mL of dichloromethane, into which the catalyst Cu(OH)Cl.TMEDA (50 mg, 0.1 mmol) was added, and stirred at room temperature to give a red solid quinone which was then reduced with sodium hydrosulfite to give 4,4-dihydroxy-3,3-diisopropyl-5,5-dipropylbiphenyl (1.1 g, 55.5%).

(27) 4,4-dihydroxy-3,3-diisopropyl-5,5-dipropylbiphenyl: .sup.1H NMR (300 MHz, CDCl.sub.3) 7.50 (s, 4H), 5.35 (s, 2H), 2.65-2.62 (t, 8H), 1.66-1.63 (m, 8H), 0.92-0.89 (t, 12H).

EXAMPLE 8

Compound 9: 4-hydroxy-3,3,5,5-tetraisopropylbiphenyl-4-carboxylic acid

(28) ##STR00013##

(29) 4,4-dihydroxy-3,3,5,5-tetraisopropylbiphenyl (10.00 g, 28.21 mmol) was dissolved in dichloromethane (50 mL), PBr.sub.3 (15.27 g, 56.41 mmol) was added dropwise thereto in an ice bath, reacted at room temperature for 5 h, neutralized with sodium bicarbonate solution, and extracted with ethyl acetate to obtain 3.23 g yellow solid. The yellow solid was dissolved in anhydrous THF, n-butyllithium (0.99 g, 15.48 mmol) was added thereto at 78 C., and then charged with nitrogen for protection. A reaction was carried out by charging carbon dioxide below the liquid interface, followed by post-treatment to give 4-hydroxy-3,35,5-tetraisopropylbiphenyl-4-carboxylic acid (1.35 g, 45.61%).

(30) 4-hydroxy-3,35,5-tetraisopropylbiphenyl-4-carboxylic acid: .sup.1H NMR (300 MHz, CDCl.sub.3) 11.0 (s,1H), 7.89 (s,2H), 7.51 (s, 2H), 5.35 (s, 1H), 3.07-3.01 (m, 2H), 2.89-2.85 (m, 2H), 1.23-1.17 (m, 24H).

EXAMPLE 9

Compound 10: 4-chloro-4-hydroxy-3,3,5,5-tetraisopropylbiphenyl, and compound 11: 4,4-dichloro-3,3,5,5-tetraisopropylbiphenyl

(31) ##STR00014##

(32) 4,4-dihydroxy-3,3,5,5-tetraisopropylbiphenyl (2 g, 5.64 mmol) was slowly added to phosphorus oxychloride (5 mL) at room temperature and refluxed for 1 h. the reaction mixture was slowly added dropwise to ice water and stirred continuously, extracted with ethyl acetate, dried over anhydrous sodium sulfate and filtered, and the filtrate was purified with petroleum ether-ethyl acetate eluent to give 4-chloro-4-hydroxy-3,3,5,5-tetraisopropylbiphenyl (500 mg, 23.76%) as a yellow solid and 4,4-dichloro-3,35,5-tetraisopropylbiphenyl (0.72 g, 32.61%) as a yellow solid.

(33) 4-chloro-4-hydroxy-3,3,5,5-tetraisopropylbiphenyl (500 mg, 23.76%) as a yellow solid; .sup.1H NMR (300 MHz, CDCl.sub.3) 7.28 (s, 2H), 7.20 (s, 2H), 4.88 (s, 1H), 3.47-3.40 (m, 2H), 3.27-3.20 (m,2H), 1.38-1.35 (d, 12H), 1.32-1.28 (t, 12H).

(34) 4,4-dichloro-3,35,5-tetraisopropylbiphenyl (0.72 g, 32.61%) as a yellow solid; .sup.1H NMR (300 MHz, CDCl.sub.3) 7.29 (s, 4H), 2.08 (s, 4H), 1.38-1.15 (m, 24H).

EXAMPLE 10

Compound 12: 4-hydroxy-3,3,5,5-tetraisopropylbiphenyl-4-carboxylate, and compound 13: 3,3,5,5-tetraisopropylbiphenyl-4,4-dicarboxylate

(35) ##STR00015##

(36) Under nitrogen protection, 3,3,5,5-tetraisopropyl-4,4-dihydroxybiphenyl (0.5 g, 2.7 mmol), 40 mL of formic acid and anhydrous aluminum chloride (3.0 g, 22.5 mmol) was added in a 100 mL round bottom flask and refluxed for 5 h. The reaction was terminated followed by addition of water, and then extracted with ethyl acetate. The organic phase was washed with saturated sodium bicarbonate and then with saturated brine, dried over anhydrous sodium sulfate and filtered, and the filtrate was purified with petroleum ether-ethyl acetate eluent to give 4-hydroxy-3,3,5,5-tetraisopropylbiphenyl-4-carboxylate (0.12 g, 20%) as a white solid; to give 3,3,5,5-tetraisopropylbiphenyl-4,4-dicarboxylate (0.3 g, 40%) as a white solid.

(37) 4-hydroxy-3,3,5,5-tetraisopropylbiphenyl-4-carboxylate: white solid; .sup.1H NMR (300 MHz, CDCl.sub.3) 9.6 (s, 1H), 7.65 (s, 2H), 7.51 (s, 2H), 3.07-3.02 (m, 4H), 1.26-1.23 (m, 24H).

(38) 3,3,5,5-tetraisopropylbiphenyl-4,4-dicarboxylate: white solid, .sup.1H NMR (300 MHz, CDCl.sub.3) 9.72 (s, 2H), 7.57 (s, 4H), 5.06-5.04 (d, 4H), 3.06-3.02 (m, 4H), 1.23-1.20 (d, 24H).

EXAMPLE 11

Compound 14: 4-hydroxy-3,3,5,5-tetraisopropylbiphenyl-2-amino-3-methylbutyrate

(39) ##STR00016##

(40) 3,3,5,5-tetraisopropyl-4,4-dihydroxybiphenyl (1 g, 2.8 mmol), Boc-valine (0.73 g, 3.36 mmol), phosphorus pentoxide (1.987 g, 14 mmol), and 30 mL dichloromethane were added in a 50 mL round bottom flask, and stirred at room temperature for 8 h. After the reaction was complete, 10 mL of water was added thereto and stirred for 1 h. An appropriate amount of ammonia was added, extracted with methylene chloride, washed with water and then with saturated sodium chloride, dried over anhydrous sodium sulfate, and purified by column chromatography to give a white solid (0.16 g, 13%).

(41) White solid, 4-hydroxy-3,3,5,5-tetraisopropylbiphenyl-2-amino-3-methylbutyrate: .sup.1H NMR (300 MHz, CDCl.sub.3) 7.57 (s, 2H), 7.51 (s, 2H), 5.35 (s, 1H), 5.12-5.10 (d, 2H),4.25-4.24 (d, 1H),3.05-3.02 (m, 4H), 2.68-2.66 (m, 1H), 1.21-1.18 (d, 24H), 0.92-0.90 (d, 6H).

EXAMPLE 12

Compound 15: Ethyl 1-(4-hydroxy-3,3,5,5-tetraisopropylbiphenyl-4-oxo)acetate

(42) ##STR00017##

(43) 3,3,5,5-tetraisopropyl-4,4-dihydroxybiphenyl (1 g, 2.8 mmol), ethyl chloroacetate (0.411 g, 3.36 mmol), sodium hydroxide (0.336 g, 8.4 mmol), and 60 mL of dichloromethane were sequentially added in a 100 mL round bottom flask, and stirred at room temperature for 8 h. After the reaction was complete, the reaction solution was filtered, extracted with dichloromethane, washed with saturated sodium chloride, dried over anhydrous sodium sulfate, and purified by column chromatography to give a white solid (0.12 g, 10%).

(44) White solid, Ethyl 1-(4-hydroxy-3,3,5,5-tetraisopropylbiphenyl-4-oxo) acetate: .sup.1H NMR (300 MHz, CDCl.sub.3) 7.57 (s, 2H), 7.48 (s,2H), 5.25 (s, 1H), 3.01-2.98 (m, 4H), 2.21 (s, 3H), 1.82-1.80 (d, 3H), 1.16-1.14 (d, 24H).

EXAMPLE 13

Effects of Compound 1-15 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

(45) (1) Material:

(46) An aqueous solution of compound 1-15 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); B12000 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.

(47) (2) Method:

(48) 1. Experimental Animals and Grouping

(49) 336 healthy male SD rats weighing 250-300 g, provided by the Experimental Animal Center of the Fourth Military Medical University, were randomly divided into 14 groups: the sham operation group, the ischemic reperfusion group, and Examples 1-12 groups (i.e., the Example groups). The biphenyl derivatives used in Examples 1 to 12 are shown in Table 1:

(50) TABLE-US-00001 TABLE 1 Example groups Biphenyl derivatives Example 1 group Compound 1 Example 2 group Compound 2 Example 3 group Compound 3 Example 4 group Compound 4 Example 5 group Compound 6 Example 6 group Compound 7 Example 7 group Compound 8 Example 8 group Compound 9 Example 9 group Compound 10 Example 10 group Compound 12 Example 11 group Compound 14 Example 12 group Compound 15

(51) 2. Animal Model Preparation and Treatment

(52) 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 the biphenyl derivatives 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.

(53) 3. Neurological Impairment Score in Rats

(54) 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.

(55) 4. Sample Collection and Preparation

(56) 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 i for 20 min, evenly sliced (with a thickness of 2 mm), stained in a 2% TTC solution at 37% 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-.

(57) 5. Determination of Relative Infarct Volume

(58) 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.

(59) 6. SOD and MDA Determination

(60) SOD and MDA were determined strictly in accordance with instructions of the kit.

(61) 7. Fas Determination

(62) 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 4t overnight, biotinylated goat anti-rabbit IgG was added dropwise, and heated in a water bath at 37a 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.

(63) 8. Apoptosis Determination

(64) 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 37t 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 37a for 30 min, and SABC was added for a reaction at 37a 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.

(65) 9. Determination of IL-1 and TNF-

(66) 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.

(67) (3) Results:

(68) 1. The biphenyl derivatives substantially improve the neurological impairment in the MCAO model rats, and the detailed scores according to the Longa scoring standard are shown in Table 2 below.

(69) TABLE-US-00002 TABLE 2 Average score of neurological Groups impairment Sham operation group 0 Ischemic reperfusion group 3.2 Example 1 group 2.0 Example 2 group 2.1 Example 3 group 2.4 Example 4 group 2.2 Example 5 group 2.2 Example 6 group 2.0 Example 7 group 2.1 Example 8 group 2.3 Example 9 group 2.4 Example 10 group 2.2 Example 11 group 2.2 Example 12 group 2.4

(70) 2. The biphenyl derivatives can significantly reduce cerebral infarction volume in the MCAO model rats: except for the sham operation group having no visible infarction focus, the other groups had different degrees of infarction occurred. With Analysis and calculation with the imageJ software, detailed data is listed in Table 3.

(71) TABLE-US-00003 TABLE 3 Relative Brain infarction Groups volume (%) Sham operation group 0 Ischemic reperfusion group 35.28 5.22 Example 1 group 18.71 4.69* Example 2 group 23.22 3.12* Example 3 group 25.41 3.59* Example 4 group 20.38 4.92* Example 5 group 23.55 5.67* Example 6 group 19.35 4.02* Example 7 group 19.29 3.54* Example 8 group 25.83 2.38* Example 9 group 24.74 6.13* Example 10 group 23.91 5.44* Example 11 group 23.59 4.11* Example 12 group 25.54 3.29* Compared with the ischemic reperfusion group *p < 0.05

(72) 3. The biphenyl derivatives can significantly reduce the consumption of endogenous oxygen free radical scavenger SOD, reduce the lipid peroxidation damage, and decrease the serum MDA content in the MCAO model rats: as compared with the sham operation group, the SOD activities of the other groups decreased, and the SOD activities of the Example groups were all higher than that of the ischemic reperfusion group; meanwhile, as compared with the sham operation group, the serum MDA contents of other groups increased, and the MDA contents of the Example group were all lower than that of the ischemic reperfusion group. Detailed data is listed in Table 4.

(73) TABLE-US-00004 TABLE 4 SOD ACTIVITY MDA CONTENT Groups (U/mL) (mmol/mL) Sham operation group 101.54 3.45 2.49 0.66 Ischemic reperfusion group 82.14 4.37 7.22 0.61 Example 1 group 96.78 2.94* 4.74 0.67* Example 2 group 94.46 2.19* 5.44 0.49* Example 3 group 93.15 3.05* 5.36 0.47* Example 4 group 95.66 3.18* 4.68 0.39* Example 5 group 95.01 3.22* 4.47 0.34* Example 6 group 96.89 3.49* 4.26 0.41* Example 7 group 94.51 2.19* 4.97 0.50* Example 8 group 96.09 3.33* 5.12 0.47* Example 9 group 92.26 2.96* 5.61 0.71* Example 10 group 95.16 3.88* 4.89 .0.36* Example 11 group 96.71 4.29* 4.92 0.52* Example 12 group 92.39 5.27* 5.37 0.55* Compared with ischemic reperfusion group *p < 0.05

(74) 4. The biphenyl derivatives can effectively down-regulate cellular Fas expression in brain tissues of the MCAO model rats: there were few Fas positive cellular expression in the sham operation group; the other groups had different degrees of expression in the cortical penumbra, and microscopic observation showed that the cell membrane and the cytoplasm in brown were Fas-positive cells. As compared with the sham operation group, the average gray scales of the other groups decreased, and the average gray scales of the Example groups were significantly higher than that of the ischemic reperfusion group. Detailed data was listed in Table 5.

(75) TABLE-US-00005 TABLE 5 Groups Fas average gray scale Sham operation group 175.96 5.14 Ischemic reperfusion group 134.33 6.18 Example 1 group 162.47 3.96* Example 2 group 148.36 5.33* Example 3 group 144.56 6.32* Example 4 group 159.68 5.06* Example 5 group 148.02 4.16* Example 6 group 155.33 3.78* Example 7 group 157.26 4.05* Example 8 group 148.82 7.62* Example 9 group 147.11 5.61* Example 10 group 145.06 4.08* Example 11 group 152.12 6.17* Example 12 group 144.26 3.47* Compared with ischemic reperfusion group *p < 0.05

(76) 5. The biphenyl derivatives can effectively inhibit brain cell apoptosis: there were few apoptotic cells in the sham operation group, while the other groups had different degrees of distribution in the cortical penumbra, and microscopic observation showed that those with brown granules in the nucleus were apoptotic cells. Compared with the sham group, the apoptotic cells were increased in the other groups, and were significantly less in the Example groups than in the ischemic reperfusion group. Detailed data was listed in Table 6.

(77) TABLE-US-00006 TABLE 6 Groups Number of apoptotic cells Sham operation group 4.62 1.54 Ischemic reperfusion group 37.26 4.10 Example 1 group 19.14 4.24* Example 2 group 24.65 3.41* Example 3 group 25.14 2.54* Example 4 group 21.49 3.18* Example 5 group 20.56 4.29* Example 6 group 19.47 5.02* Example 7 group 20.28 3.76* Example 8 group 24.81 3.35* Example 9 group 25.47 4.19* Example 10 group 22.18 5.22* Example 11 group 24.19 4.29* Example 12 group 26.27 5.37* Compared with ischemic reperfusion group *p < 0.05

(78) 6. The biphenyl derivatives can effectively down-regulate the cellular expression of IL-1 and TNF- in brain tissues of the MCAO model rats: as compared with the sham operation group, the expression of IL-1 and TNF- in the other groups significantly increased, wherein the expression of IL-1 and TNF- in the Example groups were significantly lower than that in the ischemic reperfusion group. Detailed data was listed in Table 7.

(79) TABLE-US-00007 TABLE 7 IL-1 content TNF- content Groups (ng/mL) (ng/mL) Sham operation group 0.39 0.08 2.74 0.21 Ischemic reperfusion group 0.92 0.14 6.47 0.65 Example 1 group 0.63 0.12* 4.11 0.72* Example 2 group 0.79 0.11* 5.03 0.59* Example 3 group 0.66 0.05* 4.26 0.54* Example 4 group 0.68 0.07* 4.19 0.36* Example 5 group 0.74 0.09* 5.01 0.59* Example 6 group 0.64 0.03* 4.16 0.44* Example 7 group 0.63 0.08* 4.32 0.61* Example 8 group 0.64 0.14* 4.35 0.46* Example 9 group 0.63 0.07* 5.02 0.39* Example 10 group 0.67 0.05* 4.68 0.61* Example 11 group 0.71 0.07* 5.31 0.51* Example 12 group 0.74 0.06* 4.95 0.67* Compared with ischemic reperfusion group *p < 0.05

EXAMPLE 14

Effects of Compound 1-15 on Neurological Impairment Score, Cerebral Infarct Volume, Fas, IL-1, and TNF- in Brain Tissues, and Cell Apoptosis in Rats with Permanent Cerebral Ischemia Injury

(80) (1) Material:

(81) An aqueous solution of compound 1-15 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.

(82) (2) Method:

(83) 1. Experimental Animals and Grouping

(84) 336 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, and Examples 1-12 groups. The biphenyl derivatives used in each of the Examples 1-12 groups were the same as those in Example 13.

(85) 2. Animal Model Preparation and Treatment

(86) 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 the biphenyl derivatives 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.

(87) 3. Neurological Impairment Score in Rats

(88) 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.

(89) 4. Sample Collection and Preparation

(90) 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 20i for 20 min, evenly sliced (with a thickness of 2 mm), stained in a 2% TTC solution at 37% 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-.

(91) 5. Determination of Relative Infarct Volume

(92) 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.

(93) 6. SOD and MDA Determination

(94) SOD and MDA were determined strictly in accordance with instructions of the kit.

(95) 7. Fas Determination

(96) 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 4t overnight, biotinylated goat anti-rabbit IgG was added dropwise, and heated in a water bath at 37a 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.

(97) 8. Apoptosis Determination

(98) 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 37t 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 37a for 30 min, and SABC was added for a reaction at 37a 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.

(99) 9. Determination of IL-1 and TNF-

(100) 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.

(101) (3) Results:

(102) 1. The biphenyl derivatives can substantially improve the neurological impairment in permanent cerebral ischemia model rats, and the detailed scores according to the Longa scoring standard are shown in Table 8 below.

(103) TABLE-US-00008 TABLE 8 Average score of neurological Groups impairment Sham operation group 0 Permanent cerebral 3.5 ischemia model group Example 1 group 2.4 Example 2 group 2.5 Example 3 group 2.8 Example 4 group 2.6 Example 5 group 2.4 Example 6 group 2.2 Example 7 group 2.3 Example 8 group 2.7 Example 9 group 2.8 Example 10 group 2.4 Example 11 group 2.3 Example 12 group 2.6

(104) 2. The biphenyl derivatives can significantly reduce cerebral infarction volume in the permanent cerebral ischemia model rats: except for the sham operation group having no visible infarction focus, the other groups had different degrees of infarction occurred. With Analysis and calculation with the imageJ software, detailed data is listed in Table 9.

(105) TABLE-US-00009 TABLE 9 Relative Brain infarction volume Groups (%) Sham operation group 0 Permanent cerebral 39.82 5.19 ischemia model group Example 1 group 24.71 3.76* Example 2 group 27.92 4.51* Example 3 group 29.76 2.49* Example 4 group 25.83 5.07* Example 5 group 28.37 5.02* Example 6 group 22.53 4.62* Example 7 group 21.39 4.01* Example 8 group 29.74 3.13* Example 9 group 30.11 4.01* Example 10 group 26.46 4.52* Example 11 group 28.98 5.24* Example 12 group 31.79 5.94* Compared with the permanent cerebral ischemia model group *p < 0.05

(106) 3. The biphenyl derivatives can significantly reduce the consumption of endogenous oxygen free radical scavenger SOD, reduce the lipid peroxidation damage, and decrease the serum MDA content in the permanent cerebral ischemia model rats: as compared with the sham operation group, the SOD activities of the other groups decreased, and the SOD activities of the Example groups were all higher than that of the ischemic reperfusion group; meanwhile, as compared with the sham operation group, the serum MDA contents of other groups increased, and the MDA contents of the Example group were all lower than that of the ischemic reperfusion group. Detailed data is listed in Table 10.

(107) TABLE-US-00010 TABLE 10 SOD ACTIVITY MDA CONTENT Groups (U/mL) (mmol/mL) Sham operation group 102.29 4.72 2.45 0.67 Permanent cerebral 84.42 3.88 7.05 0.57 ischemia model group Example 1 group 97.36 4.51* 4.49 0.59* Example 2 group 93.28 3.49* 5.10 0.82* Example 3 group 93.61 2.13* 5.07 0.77* Example 4 group 94.47 3.81* 4.79 0.33* Example 5 group 95.31 4.11* 4.68 0.42* Example 6 group 97.21 4.36* 4.31 0.51* Example 7 group 98.28 3.18* 4.42 0.39* Example 8 group 96.90 3.69* 5.06 0.28* Example 9 group 93.62 2.73* 5.26 0.49* Example 10 group 94.08 2.83* 4.95 .0.51* Example 11 group 95.76 3.93* 4.99. 0.45* Example 12 group 91.25 3.84* 6.03 0.36* Compared with the permanent cerebral ischemia model group *p < 0.05

(108) 4. The biphenyl derivatives can effectively down-regulate cellular Fas expression in brain tissues of the permanent cerebral ischemia model rats: there were few Fas positive cellular expression in the sham operation group; the other groups had different degrees of expression in the cortical penumbra, and microscopic observation showed that the cell membrane and the cytoplasm in brown were Fas-positive cells. As compared with the sham operation group, the average gray scales of the other groups decreased, and the average gray scales of the Example groups were significantly higher than that of the ischemic reperfusion group. Detailed data was listed in Table 11.

(109) TABLE-US-00011 TABLE 11 Groups Fas average gray scale Sham operation group 178.69 4.53 Permanent cerebral 126.7 7.82 ischemia model group Example 1 group 158.28 6.66* Example 2 group 149.63 8.15* Example 3 group 143.39 4.23* Example 4 group 156.53 4.68* Example 5 group 143.02 5.53* Example 6 group 151.63 5.58* Example 7 group 153.46 5.73* Example 8 group 145.24 5.26* Example 9 group 144.31 5.49* Example 10 group 140.62 3.85* Example 11 group 150.72 4.19* Example 12 group 139.48 5.72* Compared with the permanent cerebral ischemia model group *p < 0.05

(110) 5. The biphenyl derivatives can effectively inhibit brain cell apoptosis: there were few apoptotic cells in the sham operation group, while the other groups had different degrees of distribution in the cortical penumbra, and microscopic observation showed that those with brown granules in the nucleus were apoptotic cells. Compared with the sham group, the apoptotic cells were increased in the other groups, and were significantly less in the Example groups than in the ischemic reperfusion group. Detailed data was listed in Table 12.

(111) TABLE-US-00012 TABLE 12 Groups Number of apoptotic cells Sham operation group 4.47 1.09 Permanent cerebral 39.41 4.34 ischemia model group Example 1 group 20.41 5.02* Example 2 group 23.27 3.78* Example 3 group 29.72 3.43* Example 4 group 22.68 5.50* Example 5 group 23.48 3.62* Example 6 group 21.24 5.30* Example 7 group 20.32 4.19* Example 8 group 27.74 4.20* Example 9 group 31.44 5.19* Example 10 group 23.67 5.13* Example 11 group 26.31 3.86* Example 12 group 32.45 4.21* Compared with the permanent cerebral ischemia model group *p < 0.05

(112) 6. The biphenyl derivatives can effectively down-regulate the cellular expression of IL-1 and TNF- in brain tissues of the permanent cerebral ischemia model rats: as compared with the sham operation group, the expression of IL-1 and TNF- in the other groups significantly increased, wherein the expression of IL-1 and TNF- in the Example groups were significantly lower than that in the ischemic reperfusion group. Detailed data was listed in Table 13.

(113) TABLE-US-00013 TABLE 13 IL-1 content TNF- content Groups (ng/mL) (ng/mL) Sham operation group 0.38 0.05 2.58 0.23 Permanent cerebral 0.95 0.17 6.74 0.47 ischemia model group Example 1 group 0.64 0.09* 4.25 0.66* Example 2 group 0.78 0.12* 5.15 0.88* Example 3 group 0.67 0.08* 4.45 0.91* Example 4 group 0.71 0.06* 4.37 0.62* Example 5 group 0.72 0.11* 5.23 0.48* Example 6 group 0.55 0.05* 4.99 0.53* Example 7 group 0.57 0.08* 4.26 0.40* Example 8 group 0.65 0.08* 4.39 0.73* Example 9 group 0.62 0.12* 5.17 0.40* Example 10 group 0.66 0.06* 5.05 0.54* Example 11 group 0.76 0.04* 5.16 0.43* Example 12 group 0.75 0.04* 5.38 0.59* Compared with the permanent cerebral ischemia model group *p < 0.05.

EXAMPLE 15

Comparison of Efficacy of the Present Invention over Existing Positive Drugs

(114) The therapeutic effects of propofol and edaravone respectively on the ischemic reperfusion model and the permanent cerebral ischemia model rats were evaluated according to the methods described above in Examples 13 and 14 (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 of the two models were shown in the following Table 14 and Table 15, respectively.

(115) TABLE-US-00014 TABLE 14 Efficacy of positive drugs in ischemic reperfusion rat model Propofol injection Edaravone injection Model group Example 1 group (100 mg/10 mL) (30 mg/20 mL) Neurological impairment 3.2 2.0 2.5 2.6 score Relative brain infarction 35.28 5.22 18.71 4.69 27.14 5.08* 29.31 3.95* volume (%) Fas average gray scale 134.33 6.18 162.47 3.96 142.15 5.71* 139.65 2.05* SOD activity (U/mL) 82.14 4.37 96.78 2.94 88.25 2.19* 88.16 3.99* MDA content (mmol/mL) 7.22 0.61 4.74 0.67 5.58 0.48* 6.17 0.54* Number of apoptotic cells 37.26 4.10 19.14 4.24 26.08 3.72* 28.6 4.90* IL-1 content (ng/mL) 0.92 0.14 0.63 0.12 0.73 0.09* 0.75 0.05* TNF- content (ng/mL) 6.47 0.65 4.11 0.72 5.02 0.31* 5.14 0.44* Change of behavior of rats Sober, no Sober, no Anesthetized, loss Sober, no within 30 min after the significant significant of righting reflex significant administration 12 h after difference as difference as difference as reperfusion compared to pre- compared to pre- compared to pre- administration administration administration *Relative to Example 1, p < 0.05.

(116) The results in Table 14 showed that the therapeutic effect of the biphenyl derivatives on the models 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 14 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.

(117) TABLE-US-00015 TABLE 15 Positive drug efficacy in permanent cerebral ischemia model rats Propofol injection Edaravone injection Mode group Example 1 group (100 mg/10 mL) (30 mg/20 mL) Neurological impairment 3.5 2.4 2.7 2.8 score Brain relative infarct 39.82 5.19 24.71 3.76 30.11 3.58* 31.29 4.02* volume (%) Fas average gray value 126.7 7.82 158.28 6.66 140.51 4.84* 138.95 5.50* SOD activity (U/mL) 84.42 3.88 97.36 4.51 89.13 3.82* 90.62 3.81* MDA content (mmol/mL) 7.05 0.57 4.49 0.59 6.10 0.29* 5.98 0.44* Number of apoptotic cells 39.41 4.34 20.41 5.02 31.06 3.55* 33.19 3.81* IL-1 content (ng/mL) 0.95 0.17 0.64 0.09 0.78 0.07* 0.76 0.04* TNF- content ng/mL) 6.74 0.47 4.25 0.66 5.40 0.32* 5.53 0.54* *Relative to Example 1, p < 0.05.

(118) The results in Table 15 showed that the therapeutic effect of the biphenyl derivatives on the models 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 15 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 16

Oil-based Preparation

(119) The formulation of the Oil-based preparation of the biphenyl derivative of the present invention can be as shown in Table 16:

(120) TABLE-US-00016 TABLE 16 Amount in Amount in Components formulation Components formulation 4-hydroxy- 200 mg Benzyl Alcohol 50 l 3,3,5,5- tetraisopropyl biphenyl-4-acetate Tetrahydrofuran 0.80 ml Castor oil Add to 1 ml polyglycol ether Vitamin E acetate 5 mg

EXAMPLE 17

Tablet

(121) The formulation of the tablet of the biphenyl derivative of the present invention can be as shown in Table 17:

(122) TABLE-US-00017 TABLE 17 Amount in Amount in Components formulation Components formulation 4-hydroxy- 200 mg Starch pulp 50 mg 3,3,5,5- tetraisopropyl biphenyl-2-amino- 3-methylbutyrate lactose 140 mg Sodium 10 mg carboxymethyl starch Microcrystalline 100 mg Magnesium 1 mg cellulose stearate

EXAMPLE 18

Capsule

(123) The formulation of the capsule of the biphenyl derivative of the present invention can be as shown in Table 18:

(124) TABLE-US-00018 TABLE 18 Amount in Amound in Components formulation Components formulation 4,4-dihydroxy- 1 g Egg yolk 1.2 g 3,3-diisopropyl- lecithin 5,5-dipropylbiphenyl Olive oil 10 g Vitamin E 0.2 g

EXAMPLE 19

Emulsion

(125) The formulation of the emulsion of the biphenyl derivative of the present invention can be as shown in Table 19:

(126) TABLE-US-00019 TABLE 19 Amount in Amound in Components formulation Components formulation [4-(4-hydroxy- 1 g Glycerin 2.25 g 3,3,5,5- tetraisopropyl) biphenyl)oxy]-4- carbonylbutyric acid Soybean oil 10 g Sodium Appropriate hydroxide amount Egg yolk lecithin 1.2 g Water for Add to 100 ml Injection Vitamin E 0.1 g