HK1170725B - A butylphthalide derivative and the preparing method and use thereof - Google Patents

Description

Butylphthalide derivative and preparation method and application thereof

the technical field is as follows:

the invention belongs to the field of medicines of compounds, and relates to (-) - (S) -3- (3' -hydroxy) -butyl phthalide and ester generated by the same and acid, and discloses a preparation method and application thereof.

Background art:

the butylphthalide has the effects of improving the damage of the central nervous function of a patient with acute ischemic stroke, promoting the functional recovery of the patient, and mainly converting into two metabolites, namely 3-3- (3' -hydroxy) butylphthalide and 3-hydroxy-3-butylphthalide:

3-3- (3' -hydroxy) butylphthalide (metabolite I)

3-hydroxy-3-butylphthalide (metabolite II)

Succinate, glycinate and phosphate of metabolite I are synthesized by Guangdong Zhongke medicine research company and Shandong green leaf natural medicine research company Limited in 2008, and application of the succinate, the glycinate and the phosphate in preventing cerebral ischemia diseases is described. Patent application 200410036628.3 discloses a new use of butylphthalide analogs such as 3- (3' -hydroxy) butylphthalide and 3-hydroxy-3-butylphthalide. Animal experiments prove that the two have the following effects:

1) the nerve symptoms of the rats caused by cerebral ischemia due to cerebral trauma are obviously improved;

2) improving the dysmnesia of rats caused by cerebral ischemia;

3) relieving cerebral edema caused by cerebral ischemia of rats;

4) reducing cerebral apoplexy of rats caused by cerebral ischemia;

5) improving the energy metabolism of rats caused by cerebral ischemia;

6) increasing cerebral blood flow in the cerebral blood region;

7) reducing cerebral infarction area of rats with local cerebral ischemia and relieving nerve function deficiency symptoms;

8) platelet aggregation resistance and antithrombotic resistance;

9) preventing and treating dementia.

Researchers of our company found that metabolite I has two different optical isomers, and prepared the S configuration and R configuration of compound 3-3- (3' -hydroxy) butylphthalide (metabolite I) by an asymmetric synthesis method, as shown in the following figure:

s configuration R configuration

Animal experiments show that R configuration does not affect cerebral infarction volume of cerebral ischemic rats and can not improve cerebral ischemia symptoms, S configuration has the effects of improving cerebral ischemia symptoms and reducing cerebral infarction volume of cerebral ischemic rats, and metabolite I (the compound of claim 1) of S configuration has the effect of improving sleep.

Since the metabolite I (compound of claim 1) with S configuration is oily liquid and insoluble in water, in order to prepare injection formulation, we further study to make it form ester with acid, and then salt, to prepare compound which can be dissolved in water, to suit the needs of preparation.

The invention content is as follows:

the invention aims to provide a derivative of 3- (3' -hydroxyl) butylphthalide for cerebral arterial thrombosis, a preparation method and application thereof.

In order to achieve the purpose, the invention adopts the following technical scheme:

by asymmetric synthesis, compounds of formula I shown below are prepared:

formula I (S configuration)

Simultaneously preparing a compound with an R configuration, as shown in the following figure:

r configuration

Animal experiments prove that the R-configuration compound has no effect of reducing the cerebral ischemic infarction volume of rats, can not improve the symptoms of cerebral ischemia and can not improve the sleep, and the R-configuration compound has good effect of improving the symptoms of cerebral ischemia and can improve the sleep state of animals.

Since the compound of formula I is an oily water-insoluble substance, we form an ester with an acid, where the acid refers to a pharmaceutically acceptable inorganic acid or an organic acid, where the inorganic acid refers to nitric acid, sulfuric acid or phosphoric acid, and the organic acid contains at least one group selected from amino, hydroxyl or carboxyl in addition to an acid radical. The salt after ester formation can be dissolved in water, and can be prepared into injection or lyophilized powder for injection.

The organic acid may be amino acids, specifically glycine, alanine, lysine, arginine, serine, phenylalanine, proline, tyrosine, aspartic acid, glutamic acid, histidine, leucine, methionine, threonine, pyroglutamic acid, tryptophan or valine.

Among them, preferred is an ester with glycine as shown in the following figure:

the organic acid may also be dicarboxylic acid, specifically camphoric acid, malic acid, citric acid, maleic acid, succinic acid, oxalic acid, glutaric acid, oxalic acid, or malonic acid.

Among them, preferred is an ester with succinic acid as shown in the following figure:

the organic acid may also be pamoic acid, hydroxynaphthoic acid, gentisic acid, salicylic acid, glycolic acid, mandelic acid, lactic acid, 4-acetamidobenzoic acid or nicotinic acid.

The compounds of formula I form esters with inorganic acids, of which phosphoric acid is preferred, as shown in the following figure:

the ester is further salified, so that a compound which is soluble in water can be prepared, the problem of water solubility can be solved, and the compound can be used for preparing injection preparations.

An ester of a compound of formula I with glycine, preferably the hydrochloride salt, as shown in the following figure:

the invention also provides a salt of the dibasic acid ester of the compound shown in the formula I, which is a salt formed by potassium, sodium, magnesium or organic amine, wherein the organic amine radical can be tromethamine, diethanol amine, triethanol amine, glycine, lysine or arginine.

Among them, sodium salts are preferred, and the formula shown in the following figure:

the ester formed by the compound shown in the formula I and phosphoric acid can also be continuously salified with physiologically acceptable alkali to solve the problem of water solubility, and the salt is sodium, potassium, magnesium or organic amine salt, and the organic amine comprises lysine, glycine, arginine, tromethamine, diethanolamine or triethanolamine.

The ester of the compound of formula I with phosphoric acid is preferably the disodium salt, and the formula is shown in the following figure:

the invention also provides a pharmaceutical composition for treating ischemic stroke, which is characterized by comprising a therapeutically effective amount of the compound of the general formula (1) or a salt thereof and a pharmaceutically acceptable carrier. Can be made into oral preparation or injection preparation.

Muscle and blood vessel irritation experiments show that the ester generated by the compound shown in the formula I and acid is further salified and then is injected for administration, the muscle and blood vessel irritation is not caused, and the compound can be used for injection preparations. The specific embodiment is as follows:

example 1: preparation of each compound:

because of the strong consistency of the compounds described herein, the following synthetic schemes, in which the numbers of the compounds are indicated below, are substituted for the numbers in the following preparations, in order to describe the preparation of each compound in detail, accurately and conveniently, are expressed in 1 example:

different compounds 1a, 1b and 1c are obtained when R in the scheme above is a different group.

(1) And synthesis of compound 3:

diisopropylamine (1.3mol) was placed in a 2000mL round-bottom flask, 1000mL of anhydrous dichloromethane was added, triethylamine (2.0mol) was added after dissolution, Compound 2(1.0mol) was added dropwise in an ice bath, and after completion of the addition, the temperature was gradually raised to room temperature, and the mixture was stirred overnight. After diluting with 500mL of dichloromethane, the mixture was washed with 5% dilute hydrochloric acid (500 mL. times.1), water (500 mL. times.1), and saturated brine (500 mL. times.1), dried over anhydrous sodium sulfate, and concentrated to obtain 202.4 g of Compound 3, crude yield 99%, MS (m/z): 206.1.

(2) and synthesis of compound 4;

dissolving the compound 3(0.8mol) in 500mL of dry tetrahydrofuran, gradually adding tert-butyl lithium (1.0mol) dropwise at-78 ℃, adding tetramethylethylenediamine (1.2mol) after the dropwise addition is finished, and stirring at-78 ℃ for 30 min. Lactone (1.0mol) is added into the mixture dropwise, the mixture is gradually warmed to room temperature, the reaction is continued for 5 hours, saturated ammonium chloride solution is added to terminate the reaction, the organic solvent is removed by distillation under reduced pressure, the mixture is extracted by ethyl acetate, the concentration under reduced pressure is carried out, and the recrystallization is carried out, so that 222.4 g of compound 4 is obtained, and the yield is 91%. HNMR (400Hz, CDCl)₃)：8.08-8.06(m，1H)，8.04-8.02(m，1H)，7.63-7.61(m，1H)，7.60-7.58(m，1H)，3.95-3.93(m，2H)，3.40-3.38(m，1H)，2.55(t，J＝1.2Hz，2H)，1.64-1.62(m，2H)，1.25(d，J＝1.5Hz，12H)，1.21(d，J＝1.6Hz，3H)；MS(m/z)：306.2。

(3) Synthesis of compound 5:

dissolving 220 g of compound 4(0.7mol) in 500mL of methanol, adding sodium borohydride (1.40mol) in small portions under ice bath, stirring overnight at room temperature under the protection of nitrogen, and adding about 20mL of concentrated hydrochloric acid dropwise to decompose excessive sodium borohydride after the reaction is finished. The methanol was distilled off under reduced pressure, extracted with dichloromethane, washed with water, brine, dried over anhydrous sodium sulfate, concentrated, and recrystallized to give 204.8 g of compound 5 in 95% yield, MS (m/z): 308.2.

(4) synthesis of compound 6:

dissolving compound 5(0.60mol) in 500mL of toluene, adding catalytic amount of p-toluenesulfonic acid (about 1%), slightly boiling under nitrogen protection and refluxing for 8 days, distilling under reduced pressure to remove toluene, adding dichloromethane for dilution, washing with water, washing with brine, drying over anhydrous magnesium sulfate, and passing through a column to obtain 106.4 g of compound 6 with 86% yield, MS (m/z): 207.2.

(5) synthesis of compound 7:

dissolving compound 6(0.2mol) in 200mL of methanol, adding 16 g/40 mL of sodium hydroxide aqueous solution under stirring, adding the solution completely within 15 minutes, heating the solution until the system becomes a uniform solution, continuing to react for 2 hours at room temperature, stopping the reaction, distilling the methanol under reduced pressure, adding a proper amount of distilled water into the residual solution for dilution, cooling the system to below-5 ℃, adjusting the pH to be 3-4 by using a 5% hydrochloric acid solution under stirring, extracting the solution with diethyl ether (100 × 3), combining diethyl ether extracts, cooling the solution to below-5 ℃, slowly dropwise adding 0.2mol of (+) - (R) - α -phenylethylamine, keeping the temperature of the system below-5 ℃, standing for 3 hours, precipitating a large amount of crystals, filtering and collecting the crystals, recrystallizing the crystals twice by using acetone or ethyl ester, wherein the crystallization concentration is 15 g of crystals per 100mL of solvent, obtaining 20.3 g of crystals, dissolving the crystals with 10 times of distilled water, adding the sodium hydroxide to adjust the pH to be 13, extracting and recovering the diethyl ether(+) - (R) - α -phenylethylamine, adjusting pH of water phase to 2 with hydrochloric acid, extracting with diethyl ether, drying, concentrating to obtain crude (+) - (R) -3- (3' -hydroxy) butylbenzene peptide, recrystallizing the crude product with ethanol to obtain 8.7 g of compound 7 with 21% yield [ α ]]_D＝+66.80(c＝1.02，CH₃OH)。HNMR(400Hz，CDCl₃)：7.91-7.89(m，1H)，7.41-7.39(m，1H)，7.33-7.31(m，1H)，7.30-7.28(m，1H)，5.24(t，J＝1.2Hz，1H)，3.40-3.38(m，1H)，2.55(t，J＝1.2Hz，2H)，2.05-2.03(m，2H)，1.45-1.43(m，2H)，1.21(d，J＝1.6Hz，3H)；MS(m/z)：207.2。

(6) Synthesis of compound 8:

dissolving compound 6(0.2mol) in 200mL of methanol, adding 16 g/40 mL of sodium hydroxide aqueous solution under stirring, adding the solution completely within 15 minutes, heating the solution until the system becomes a uniform solution, continuing to react for 2 hours at room temperature, stopping the reaction, distilling the methanol under reduced pressure, adding an appropriate amount of distilled water to the residual solution for dilution, cooling the system to below-5 ℃, adjusting the pH of the system to be 3-4 by using 5% hydrochloric acid solution under stirring, extracting the solution by using diethyl ether (100 ×), combining the diethyl ether extract, cooling the solution to below-5 ℃, slowly dropwise adding 0.2mol of (S) - α -phenylethylamine, keeping the temperature of the system below-5 ℃, standing for 3 hours, precipitating a large amount of crystals, filtering and collecting the crystals, recrystallizing the crystals twice by using acetone or ethyl ester, wherein the concentration of the crystals is 15 g/100 mL of the solvent, obtaining 20.3 g of the crystals, dissolving the crystals by using 10-fold volume of distilled water, adding sodium hydroxide to adjust the solution pH to be 13, extracting the diethyl ether for recovering the (S) - α -phenylethylamine, adjusting the aqueous phase by using hydrochloric acid, extracting the solution to be 2g of the crude product, extracting the product of (S3), and drying the crude product of the crude product, and obtaining the crude product of (367), and concentrating the crude product]_D＝-66.80(c＝1.02，CH₃OH)。HNMR(400Hz，CDCl₃)：7.91-7.89(m，1H)，7.41-7.39(m，1H)，7.33-7.31(m，1H)，7.30-7.28(m，1H)，5.24(t，J＝1.2Hz，1H)，3.40-3.38(m，1H)，2.55(t，J＝1.2Hz，2H)，2.05-2.03(m，2H)，1.45-1.43(m，2H)，1.21(d，J＝1.6Hz，3H)；MS(m/z)：207.2。

(7) Synthesis of compound 1 a:

dissolving 10.0mmol of compound 8 and 10.0mmol of succinic anhydride and 10mmol of DMAP in 50mL of DMF, heating to 85 ℃, reacting for 6 hours, stopping the reaction, pouring the reaction solution into 200mL of ice water, adjusting the pH value to 2-3 by 0.1N hydrochloric acid, extracting with ethyl acetate (100mL of × 3), combining organic phases, washing with saturated saline water for three times, drying with anhydrous sodium sulfate, distilling under reduced pressure to obtain(s) -3- (3' -succinate) butyl phenyl peptide, recrystallizing to obtain 2.8 g of white solid powder, wherein the yield is 91 percent₃)：7.8-7.78(m，1H)，7.65-7.63(m，1H)，7.49-7.47(m，1H)，7.44-7.42(m，1H)，5.50-5.46(m，1H)，3.82-3.74(m，1H)，2.72-2.66(m，2H)，2.62-2.56(m，2H)，2.05-2.03(m，2H)，1.45-1.43(m，2H)，1.21(d，J＝1.6Hz，3H)；MS(m/z)：307.3。

Dissolving the(s) -3- (3 '-succinate) butylphenol peptide in 4mL of 50mL of methanol and 10% sodium hydroxide solution, heating and refluxing for 2 hours, and concentrating to obtain(s) -3- (3' -succinate) butylphenol peptide sodium salt (compound 1a), MS (m/z): 305.3.

(8) synthesis of compound 1 b:

dissolving 10.0mmol of compound 8 in 50mL of ethyl acetate, adding 1.0 mL of pyridine and 10mmol of DMAP, cooling to 0-5 ℃, adding 10.0mmol of glycine under stirring, keeping the temperature at 0-5 ℃ for 6 hours, stopping the reaction, pouring the reaction solution into 100mL of ice water, adjusting the pH to 7 with 0.1N hydrochloric acid, standing to separate an organic layer, extracting an aqueous phase with ethyl acetate (50mL × 3), combining the organic phases, washing with saturated saline solution for three times, drying with anhydrous sodium sulfate, distilling under reduced pressure to obtain(s) -3- (3' -glycinate) butyl phenyl peptide, and recombining ethanolAfter crystallization 2.3 g of a white solid powder are obtained in 87% yield. HNMR (400Hz, CDCl)₃)：7.8-7.78(m，1H)，7.65-7.63(m，1H)，7.49-7.47(m，1H)，7.44-7.42(m，1H)，5.50-5.46(m，1H)，3.82-3.74(m，1H)，3.62(s，2H)，2.05-2.03(m，2H)，1.45-1.43(m，2H)，1.21(d，J＝1.6Hz，3H)；MS(m/z)：264.3。

The(s) -3- (3 '-glycinate) butylphenyl peptide was dissolved in 50mL of acetone and 10mL of diethyl ether, a solution of hydrogen chloride in diethyl ether was added dropwise to adjust the pH to 2, and a large amount of white solid was precipitated, which was then filtered and dried to obtain 2.4 g of(s) -3- (3' -glycinate) butylphenyl peptide hydrochloride (compound 1b), yield 80%, MS (m/z): 264.3.

(9) synthesis of compound 1 c:

adding 20.0 mL of pyridine to 100mL of DMF, cooling to-10 ℃, adding 25.0mL of phosphorus oxychloride with stirring, stirring for 30min, adding 10.0mmol of compound 8 in portions, stirring for 3 h, stopping the reaction, pouring the reaction solution into 100mL of ice water, adjusting the pH to 2 with 0.1N hydrochloric acid, extracting with ethyl acetate (50mL of × 3), combining the organic phases, washing three times with saturated saline solution, drying with anhydrous sodium sulfate, distilling under reduced pressure to remove the solvent to obtain (S) -3- (3 '-phosphate) butylphenyl peptide, dissolving with 20mL of ethanol, adding 10.6 sodium carbonate, keeping the temperature at 30 ℃ for 2 h, adding 100mL of acetone after stopping the reaction, standing at 5 ℃ for crystallization, filtering, and drying under vacuum to obtain 2.8 g of (S) -3- (3' -phosphate disodium salt (compound 1c) which is white solid powder with a yield of 85%. HNMR (400Hz, CDCl₃)：7.91-7.89(m，1H)，7.41-7.39(m，1H)，7.33-7.31(m，1H)，7.30-7.28(m，1H)，5.24(t，J＝1.2Hz，1H)，3.40-3.38(m，1H)，2.05-2.03(m，2H)，1.65-1.63(m，2H)，1.21(d，J＝1.6Hz，3H)；MS(m/z)：331.2。

Example 2: preparation of (-) - (S) -3- (3' -disodium phosphate) butylphthalide for injection (Compound 1c in example 1) lyophilized powder

Taking 10g of prepared (S) -3- (3' -phosphate) butylphthalide sodium salt (compound 1c), adding 1000ml of water for injection, dissolving, adding 60g of mannitol, fully dissolving, adding the water for injection to 1200ml, filtering through a microporous filter membrane after active carbon decarburization, subpackaging in 7ml of penicillin bottles with 3ml of each bottle, plugging, freeze-drying and capping to obtain the compound. Specification: 20 mg/bottle.

Example 3: preparation of (S) -3- (3' -disodium phosphate) butylphthalide (Compound 1C) capsules

Accurately weighing (S) -3- (3' -phosphate) butyl phthalide sodium salt with constant weight according to the prescription amount, sieving with a 100-mesh sieve, adding lactose which is dried at 80 ℃ and sieved with a 80-mesh sieve according to the prescription amount, uniformly mixing, detecting the content, and filling into No. 1 capsules after the content is qualified.

Implementation 4: preparation of (S) -3- (3' -disodium phosphate) butylphthalide (1c compound) injection

Accurately weighing (S) -3- (3' -phosphate) butylphthalide sodium salt (compound 1c) according to the prescription amount, placing in a container, adding appropriate amount of water for injection, adjusting pH to 6.5-7.2, adding water for injection to 4000ml, adding 2g of needle activated carbon, boiling for 15min, suction filtering for decarbonization, filtering the solution through a 0.22 mu m microporous membrane, filling the solution in a glass ampoule, and pressure sterilizing the preparation for 30min at 115 ℃.

Example 5: influence on cerebral infarction volume of rats with local cerebral ischemia

(1) Test materials and methods

Wistar rat, weight 250-280 g. Feeding separately before and after operation, maintaining room temperature at 23-25 deg.C, and feeding food and water. The tMCAO model was prepared according to longa et al. Rats were anesthetized with 10% chloral hydrate (350mg/kg, i.p.), body temperature was maintained at 37 ± 0.5 ℃, and were mounted on an operating table in the supine position. The skin was dissected along the median cervical line and the right Common Carotid Artery (CCA), External Carotid Artery (ECA), Internal Carotid Artery (ICA) were carefully isolated. The ECA ligations were cut and straightened out in line with the ICA. A small opening was cut in ECA, and a round-head siliconized nylon string (coated with 0.1% polylysine) of 4.0cm in length and 0.26mm in diameter was inserted through the opening into ICA at about 1.85-2.00cm, which is the beginning of the anterior cerebral artery in rats, to block the blood supply to the middle cerebral artery. After 2 hours of ischemia, the nylon thread was carefully withdrawn, the ECA opening was ligated and the surgical incision was closed, and the animals were returned to their cages for another 24 hours.

(2) Experimental grouping and administration

Rats were randomized into 12 groups: model control group, water for injection (100ml/kg), compound 7 administration group (25, 50, 100mg/kg) in example 1, compound 8 administration group (25, 50, 100mg/kg) in example, and DL-3- (3' -hydroxy) -butylphthalide (abbreviated as DL) administration group (25, 50, 100mg/kg) in total were 12 groups. The MCA blockade was administered orally 10 minutes after the ischemia.

(3) Determination of cerebral infarct volume

After 24 hours of reperfusion injury of rats, brains were immediately decapitated and harvested, the olfactory tracts, cerebellum and lower brainstem were removed, and the coronaries were cut into 6 pieces (2 mm/piece for the first to fifth pieces, 4mm for the sixth piece), and rapidly placed in 5ml containing 1.5ml 4% TTC and 0.1ml1MK₂HPO₄The solution was stained (37 ℃ C., protected from light) for 20-30 minutes while turning over every 5 minutes. After TTC staining, normal tissue was deep-stained red and infarcted tissue was white. The brain slices of each group are arranged in order, and are photographed and stored. Calculating the infarct size of each tablet, and finally adding and converting into the infarct volume. Infarct volume is expressed as a percentage of the cerebral hemisphere,to eliminate the effects of cerebral edema.

Cerebral infarct volume (%) (volume of contralateral hemisphere-volume of non-infarcted portion of contralateral hemisphere) volume of contralateral hemisphere/volume of contralateral hemisphere 100%

(4) Results of the experiment

The solvent control had a cerebral infarct volume of 33.8% after 2 hours of ischemia and 24 hours of reperfusion. The sham-operated group did not have any cerebral infarction. The volume results of the other groups of cerebral infarctions are shown in table 1:

table 1: influence of intragastric administration on cerebral infarction volume of ischemic rat

Compared with the solvent control group, the oral administration of the compound 8 and the DL-3- (3 ' -hydroxy) -butylphthalide group can obviously reduce the volume of cerebral infarction, the compound 7 group does not effectively reduce the volume of cerebral infarction, and the S-configuration (-) - (S) -3- (3 ' -hydroxy) butylphthalide, namely the compound 8 group is superior to the DL-3- (3 ' -hydroxy) -butylphthalide, which indicates that the compound 8 in the S configuration is an effective active ingredient, while the compound 7 in the R configuration is not found to have the effect of reducing the volume of cerebral infarction caused by cerebral ischemia.

Example 12: effect of injection administration of 1a, 1b, 1c on cerebral infarct volume

(1) Experimental grouping and administration

The rats were subjected to ischemia block model of example 11. The rats were randomly divided into 12 groups: the sham-operated group, the model control group, (water for injection, 10mg/kg), the 1a group (2.5mg/kg, 5.0mg/kg, 10mg/kg), the 1b group (2.5mg/kg, 5.0mg/kg, 10mg/kg), and the 1c group (2.5mg/kg, 5.0mg/kg, 10 mg/kg). MCA blockade was administered intravenously 10 minutes after the ischemia.

(2) Results and discussion

The volume of cerebral infarction was measured in the same manner as in example 4. The cerebral infarct volume in the solvent control group was 33.6% after reperfusion for 24 hours after 2 hours of ischemia. The sham operation group did not have any cerebral infarction, and compared with the solvent control group, each group could significantly reduce the volume of cerebral infarction compared with the solvent control group, and all sample groups could significantly reduce the volume of cerebral infarction, as shown in table 2.

Table 2: effect of intravenous administration of salt of MI ester on cerebral infarct volume of ischemic rats

Example 13: 1a, 1b and 1c vascular irritation test

1) And (3) experimental design:

dissolving 1a, 1b and 1c in water for injection, and respectively preparing two concentration groups: 4.2mg/ml, 1.4mg/ml in the low concentration group, 5ml in the rabbit marginal vein injection.

2) The administration method comprises the following steps:

8 healthy New Zealand rabbits were selected and injected with high and low concentrations of the test drug into the left ear margin vein of the rabbit, and with an equal volume of sodium chloride injection into the right ear margin vein of the rabbit as a control. After the 8 rabbits are sequentially given the high concentration and the low concentration of the test drug, 0.9% sodium chloride injection is respectively given. Once daily for 3 consecutive days. Before dosing and 48 hours and 14 days after the last dose were weighed once each.

3) General observations and animal draw:

the reaction of animals and the injection site of blood vessels was observed and recorded before administration every day, 2 new zealand rabbits with high concentration and low concentration of the tested drug were killed by bleeding respectively 48 hours after the last administration, after the reaction of the vascular tissues was observed and recorded by naked eyes, double rabbit ears were cut off from the root of the ear (first, the left ear was cut off, then, the right ear was cut off, and labeled), then, a section of rabbit ear specimen was cut off and fixed in 10% neutral formaldehyde solution respectively (the specimen was about 8cm long and about 1cm wide; the incision at the distal end was about 0.5cm from the first eye, the incision at the proximal end was about 2cm from the third eye, and the string end was the proximal end). Each of 2 animals, which left a high and low concentration of the test drug, was observed continuously until 14 days after the last administration, and subjected to pathological examination.

Cutting a section at the far end by taking the first needle eye as a boundary; cutting two sections at the proximal end by taking the third needle eye as a boundary; the blood vessels were transected, sectioned with normal paraffin, approximately 4-5 μm thick, stained with H-E, and then examined histopathologically.

5) Determination of results

And carrying out comprehensive judgment according to the results of visual observation and pathological examination. The reaction of the injection part of the animal blood vessel is observed and recorded visually before the administration every day, the administration side of part of the animal with high concentration and low concentration of the tested medicament and the inner and outer sides of the blood vessel epidermis of the needle insertion part of the rabbit ear on the control side are red visually during the administration, and the area is from 0.1cm multiplied by 0.2cm to 0.2cm multiplied by 1.0 cm. At 48 hours after the last administration, the blood vessels of the bilateral rabbit ears of the 4 rabbits with high concentration and low concentration of the tested medicament are relatively clear in outline, and the thickness of the rabbit ears is uniform and is not obviously changed. 4 rabbits with high concentration and low concentration of the tested drug are necropsied 14 days after the last administration, the blood vessels of the ears of the rabbits on both sides are clear, and the ears of the rabbits are uniform in thickness and have no obvious change.

4 rabbits with high and low concentrations of the test drug were necropsied 48 hours after the last dose and 4 rabbits with high and low concentrations of the remaining test drug were necropsied at the end of the 2-week recovery period. No remarkable irritation reaction such as degeneration or necrosis of vascular tissues is seen in the pathological histological examination.

Example 13: effect of compound 7, compound 8 and DL-3- (3' -hydroxy) -butylphthalide on rat sleep:

test for improving sleep

Sample properties: in the capsule preparation of the compounds 7 and 8, the content is brown yellow particles.

Animal sources: kunming white mouse, 18-22g, male, clean grade animal supplied by Guangdong laboratory animal center. The temperature of the laboratory animal feeding room is 22 +/-2 ℃, the relative humidity is 50-70%, and the animal feed is provided by the Guangdong laboratory animal center.

In the experiment, 25mg/kg of compound 7, compound 8 and DL-3- (3' -hydroxy) -butylphthalide (DL for short) are respectively set, and a distilled water control group is additionally set.

Sample treatment: 25mg of each sample was added with distilled water to 20ml to obtain a uniform suspension for testing.

The sample administration route comprises the following steps: gavage stomach

The experimental method comprises the following steps:

pentobarbital sodium suprathreshold dose hypnotic test:

40 male mice with the weight of 18-22g are selected and randomly divided into four groups, 10 mice are taken for continuous sample feeding for 30 days, after the sample is perfused for 15 minutes on the 30 th day, 50mg/kg.b.w of sodium pentobarbital is injected into the abdominal cavity of each group of animals, the injection amount is 0.2ml/20g.b.w, the disappearance of the righting reflex of the mice reaches more than 1 minute is taken as a sleep-onset judgment standard, and the sleep-onset time and the sleep time of each group of animals within 60 minutes of the sodium pentobarbital are observed.

As a result:

effect of samples on animal body weight

As can be seen from the above table, the animal body weight of each dose group of the sample has no significant difference compared with the control group.

Effect of suprathreshold dose of sodium pentobarbital on inducing sleep time in mice

^*P < 0.05 compared with the control group (by analysis of variance)

As can be seen from the above table, the time to sleep and the sleep time of the sample animals in the compound 8((-) - (S) -3- (3' -hydroxy) -butylphthalide) group induced by pentobarbital sodium in suprathreshold dose were significantly different from those in the control group.

Pentobarbital sodium subthreshold dose hypnosis test

40 male mice with the weight of 18-22g are selected and randomly divided into four groups, 10 male mice are taken for 28 days, samples are continuously taken for 28 days, 30mg/kg.b.w of sodium pentobarbital are injected into the abdominal cavity of each group of animals after the samples are perfused for 15 minutes on the 28 th day, the injection amount is 0.2ml/20g.b.w, and the number of animals which sleep in 25 minutes after the animals sleep in each group are fed with the sodium pentobarbital is observed by taking the disappearance of the righting reflex of the mice for more than 1 minute as a sleep-entering judgment standard.

As a result:

effect of subthreshold dose of sodium pentobarbital on the incidence of sleep in mice

^*P < 0.05 compared with the control group (tested by chi fang)

As can be seen from the above table, both the compound 8 group and the DL group had significantly lower incidence of sleeping animals and sleep induced by the sub-threshold dose of sodium pentobarbital than the control group.

To summarize: after 30 days of oral administration of the mouse sample, the compound 8((-) - (S) -3- (3 '-hydroxy) -butylphthalide) and DL group (3- (3' -hydroxy) -butylphthalide) have sleep improving effect,

the S configuration has larger effect than racemic DL configuration, and the DL configuration is larger than R configuration.

Claims (6)

1. The application of the compound of the formula I in preparing the sleep improvement medicine,

2. the use as claimed in claim 1, wherein the compound of formula i is reacted with an acid to form an ester, the acid being selected from pharmaceutically acceptable inorganic or organic acids.

3. The use according to claim 2, wherein the inorganic acid is selected from nitric acid, sulfuric acid or phosphoric acid; the organic acid is selected from amino acids: glycine, alanine, lysine, arginine, serine, phenylalanine, proline, tyrosine, aspartic acid, glutamic acid, histidine, leucine, methionine, threonine, pyroglutamic acid, tryptophan or valine; dibasic acid: camphoric acid, malic acid, maleic acid, succinic acid, oxalic acid, glutaric acid, oxalic acid, or malonic acid; tribasic acid: citric acid; pamoic acid, hydroxynaphthoic acid, gentisic acid, salicylic acid, glycolic acid, mandelic acid, lactic acid, 4-acetamidobenzoic acid or nicotinic acid.

4. The use of claim 2, wherein the acid is glycine, succinic acid or phosphoric acid and has the formula ii, iii, iv:

5. use according to claim 3 or 4, wherein the ester obtained by reacting the compound of formula I with an amino acid is reacted with sulphuric acid, phosphoric acid, sulphonic acid or hydrochloric acid to obtain a salt; reacting the ester obtained by reacting the compound shown in the formula I with dibasic acid with sodium, potassium, magnesium, tromethamine, diethanol amine, triethanol amine, glycine, lysine or arginine to obtain salt; the ester obtained by reacting the compound of formula I with phosphoric acid is reacted with sodium, potassium, magnesium, lysine, glycine, arginine, tromethamine, diethanolamine or triethanolamine to obtain the salt.

6. The use as claimed in claim 5 wherein the salt has the formula V, VI or VII respectively: