WO2016184121A1 - Naphthalene acetamide compound - Google Patents

Abstract

A new compound (as shown in formula I) and salts thereof, which are proved to be used for treating and preventing cerebral ischemic diseases and improving sleep by experiments. When R=H, salts are respectively generated by the compound together with alkali metal sodium, magnesium, potassium or calcium and organic alkali tromethamine, diethylamine, triethylamine or the like, and have functions of treating and preventing ischemic cardiovascular and cerebrovascular diseases and improving sleep. Compared with a control group, the present invention is greater in influence on the volume of ischemic cerebral infarction and higher in plasma concentration in brain tissue; and a half-life period is obviously prolonged.

Description

Naphthylacetamide compound Technical field:

The invention belongs to the field of medicines of compounds, relates to a novel amide compound and a salt thereof, and discloses a preparation method and use thereof.

BACKGROUND OF THE INVENTION: Therapeutic drugs for ischemic stroke: The first type is vasodilators (such as dipyridamole). In the past, as long as the drug can make the cerebral blood vessels dilate, it can make the blood flow more from the blocked blood vessels. However, it has been found that dilatation of vasospasm can not do this, and it will cause blood in the lesion to flow back into healthy brain tissue (this is called brain steal syndrome), so it is no longer recommended. The second category is drugs that improve microcirculation and expand blood volume (such as low molecular weight dextran). At present, such medicinal products are more, but patients with heart disease should be used with caution, otherwise it may cause heart failure. The third category is drugs that dissolve thrombus (such as urokinase, etc.). It is most desirable to use such drugs for the purpose of dissolving emboli, but large doses are often required for systemic intravenous administration, sometimes causing bleeding. Nowadays, it is recommended to use interventional therapy for patients. It is to inject the drug directly into the infarct through the catheter to dissolve the embolus. However, before and after this treatment, a cerebral angiography is performed, which is inherently dangerous. Interventional therapy requires patients to be performed within 6 hours of getting sick, sometimes with missed opportunities. The fourth category is anticoagulant therapy (such as heparin). These drugs can prevent blood clotting, but the time and activity of prothrombin should be checked every day, and hospitals with poor conditions cannot. In addition, anticoagulant therapy also has the risk of bleeding. The fifth category is the use of calcium antagonists (such as nimodipine). These drugs can prevent calcium ions from flowing into the cells from outside the cells, slightly expanding the blood vessels of the brain, protecting brain cells, and increasing the utilization of oxygen and glucose by brain cells. The sixth category is drugs that prevent platelet aggregation (such as aspirin, etc.). The aggregation of platelets is often the beginning of cerebral thrombosis. If it can effectively block the aggregation of platelets, it may prevent further formation of blood collateral. At present, such drugs are widely used in the world, but it is more suitable as a preventive drug than a therapeutic drug, because the use of such drugs in the acute phase of stroke is not satisfactory.

Summary of the invention:

It is an object of the present invention to provide a novel amide compound for ischemic stroke, and a process for its preparation and use.

In order to achieve the above object, the present invention adopts the following technical solutions:

Animal experiments have shown that the placebo group does not reduce the volume of cerebral ischemic infarction in rats, does not improve the symptoms of cerebral ischemia, and does not have the effect of improving sleep. The compounds of the present invention have a good improvement of cerebral ischemia. The role of symptoms and can improve the sleep state of animals.

When R=H, we make a salt with an alkali metal or an organic base, which means sodium, potassium, magnesium, calcium and organic alkali tromethamine, diethylamine, triethylamine, ethanolamine, ethylenediamine. , dimethylamine, hydroxyethylethylenediamine, aminoethanolamine. R is selected from the group consisting of H, C _1-6 alkyl, C _3-6 cycloalkyl, benzyl, phenyl wherein alkylphenyl, benzyl and cycloalkyl are unsubstituted or 1-3 are independently selected from halogen Substituent substitution of a hydroxyl group, an amino group, a C1-3 alkyl group, a C1-3 alkoxy group, -CO ₂ H, CO ₂ Cl-6 and a trifluoromethyl group can be obtained by a synthesis method in the examples.

The sodium salt is preferred for the compound of formula I to form a salt with an alkali metal.

The present invention also provides a pharmaceutical composition for treating ischemic stroke characterized by comprising a therapeutically effective amount of a compound of the formula (1) or a salt thereof and a pharmaceutically acceptable carrier. It may be an oral preparation or an injection preparation.

In the treatment of improving microcirculation of the brain, it is required that the drug penetrates the blood-brain barrier to concentrate the concentration of the drug in the brain tissue, and the pharmacokinetic test proves that the compound 4 is compared with the compound D2 compound in the brain tissue. The drug concentration is significantly increased.

The experiment proves that the injection prepared by using the compound 1c in the first embodiment has been well tolerated by hemolysis and irritating experiments, and is suitable for preparing an injection for clinical use in the treatment of brain cell necrosis in stroke patients. Improve the patient's sleep.

In the effect of cerebral infarction volume in rats with focal cerebral ischemia, compound 1a was significantly more effective than compound D1 in reducing the volume of cerebral infarction. Compound 1a was significantly better than compound D1, presumably related to blood concentration in brain tissue. .

Specific embodiment:

Example 1: Preparation of each compound:

Since the compounds described herein have strong coherence, in order to describe the preparation method of each compound in detail, accurately and conveniently, it is expressed in one embodiment. The following synthesis routes indicate the number of the compound below each compound, which is more Concise explanation, replaced by serial number in the following preparation method:

(1) Synthesis of Compound 3:

Accurately weigh 93.1 g of raw material 2 and add it to 200.0 mL of thionyl chloride, and stir the reaction at 80 ° C. After the reaction was stopped, the reaction solution was cooled to room temperature. 75.7 g of triethylamine and 300 mL of tetrahydrofuran were added and stirred for 20 minutes. 1.0 g of L-valine methyl ester was dissolved in 200 mL of tetrahydrofuran, and slowly added dropwise to the above reaction solution. In the reaction, the reaction was stopped for 4 hours, and the reaction was stopped. 300 mL of 2.0 N hydrochloric acid solution and 200 mL of distilled water were added to the reaction mixture, and the mixture was stirred for 30 minutes, and the mixture was allowed to stand for separation. The organic layer was separated and the aqueous layer was extracted with ethyl acetate. 3), the organic layers were combined, washed with distilled water (200 mL × 3), and the solvent was evaporated under reduced pressure to give 121.8 g of Compound 3 in a yield of 81.7%. H NMR (400 Hz, DMSO): 7.79-7.77 (m, 1H), 7.66-7.64 (m, 1H), 7.53-7.51 (m, 1H), 7.33-7.31 (m, 1H), 7.29-7.27 (m, 1H) ), 7.20-7.18 (m, 1H), 7.12-7.10 (m, 1H), 4.29-4.27 (m, 1H), 3.88 (s, 2H), 3.68 (s, 3H), 3.52-3.50 (m, 2H) ), 2.44-2.41 (m, 2H), 2.02-1.98 (m, 2H); MS (m/z): 298.3.

(2) Compound 4 synthesis:

Accurately weigh 120.0 g of raw material 3 and 36.3 g of glycine into 200.0 mL of tetrahydrofuran, stir the reaction at 80 ° C for 6 hours, stop the reaction, and cool the reaction solution to room temperature. Add 100 mL of 1.0 N hydrochloric acid solution and 200 mL of distilled water to the reaction solution. The mixture was stirred for 30 minutes, and the mixture was partitioned. The organic layer was separated, and then evaporated, evaporated, evaporated, evaporated. Compound 4, the yield was 86.7%. H NMR (400 Hz, DMSO): 11.0 (s, 1H), 8.00 (s, 1H), 7.79-7.77 (m, 1H), 7.66-7.64 (m, 1H), 7.53-7.51 (m, 1H), 7.33 7.31(m,1H), 7.29-7.27(m,1H), 7.20-7.18(m,1H), 7.12-7.10(m,1H), 4.40-4.38(m,1H), 4.14(s,2H), 3.88 (s, 2H), 3.52-3.50 (m, 2H), 2.44-2.41 (m, 2H), 2.02-1.98 (m, 2H); MS (m/z): 298.3.

(3) Synthesis of compounds 1a, 1b, 1c:

Accurately weigh 29.7 g of Compound 4 and add it to 200.0 mL of absolute ethanol, stir at 0 ° C for 30 minutes, slowly add 23.0 g of thionyl chloride to the above reaction solution, add dropwise after about 30 minutes, and then continue the reaction. The reaction solution was naturally warmed to room temperature in an hour. The reaction was stopped, and the solvent was evaporated under reduced pressure. EtOAc (EtOAc m. The combined organic layers were evaporated to dryness crystals crystals crystals crystals crystals H NMR (400 Hz, DMSO): 8.00 (s, 1H), 7.79-7.77 (m, 1H), 7.66-7.64 (m, 1H), 7.53-7.51 (m, 1H), 7.33-7.31 (m, 1H), 7.29-7.27 (m, 1H), 7.20-7.18 (m, 1H), 7.12-7.10 (m, 1H), 4.40-4.38 (m, 1H), 4.14 (s, 2H), 4.14-4.10 (m, 2H), 3.88 (s, 2H), 3.52-3.50 (m, 2H), 2.44-2.41 (m, 2H), 2.02-1.98 (m, 2H), 1.31 (t, J = 3.6 Hz, 3H); MS (m/z): 369.5 .

The above reaction procedure was repeated to obtain 27.4 g of Compound 1b in a yield of 71.7%. H NMR (400 Hz, DMSO): 8.00 (s, 1H), 7.79-7.77 (m, 1H), 7.66-7.64 (m, 1H), 7.53-7.51 (m, 1H), 7.33-7.31 (m, 1H), 7.29-7.27 (m, 1H), 7.20-7.18 (m, 1H), 7.12-7.10 (m, 1H), 4.40-4.38 (m, 1H), 4.32-4.30 (m, 1H), 4.16 (s, 2H) ), 3.88 (s, 2H), 3.52-3.50 (m, 2H), 2.44-2.41 (m, 2H), 2.02-1.98 (m, 2H), 1.35 (d, J = 3.2 Hz, 6H); MS ( m/z): 383.4.

29.7 g of Compound 4 was accurately weighed and added to 50.0 mL of absolute ethanol, stirred at 0 ° C for 30 minutes, and 7.48 g of sodium ethoxide was added in portions to the above reaction solution, and then the reaction was continued for 1 hour, and slowly added dropwise to the reaction solution. 200.0 mL of anhydrous diethyl ether, a large amount of precipitate was precipitated in the reaction mixture, stirring was continued for 1 hour, allowed to stand for 30 minutes, suction filtration, and the filter cake was washed with anhydrous diethyl ether (50 mL×3), and dried under vacuum to obtain 22.7 g of compound 1c. The yield was 76.7%. H NMR (400 Hz, DMSO): 8.00 (s, 1H), 7.79-7.77 (m, 1H), 7.66-7.64 (m, 1H), 7.53-7.51 (m, 1H), 7.33-7.31 (m, 1H), 7.29-7.27(m,1H), 7.20-7.18(m,1H), 7.12-7.10(m,1H), 4.40-4.38(m,1H), 4.14(s,2H),3.88(s,2H), 3.52-3.50 (m, 2H), 2.44-2.41 (m, 2H), 2.02-1.98 (m, 2H); MS (m/z): 362.3.

Example 2: Effect of Compound 4 and Compound D2 in Example 1 on the volume of cerebral infarction in rats with focal cerebral ischemia

(1) Experimental materials and methods

Wistar rats weighing 250-280 g. It is kept separately before and after surgery, and it is kept at 23-25 °C at room temperature. It has its own feeding and water. The tMCAO model was prepared according to the method of Longa et al. Rats were anesthetized with 10% chloral hydrate (350 mg/kg, i.p.), body temperature was maintained at 37 ± 0.5 °C, and the supine position was fixed on the operating table. The skin was cut along the midline of the neck and the right common carotid artery (CCA), external carotid artery (ECA), and internal carotid artery (ICA) were carefully separated. Cut the ECA ligation and straighten it in line with the ICA. Cut a small opening on the ECA, and insert a 4.0 cm long, 0.26 mm diameter round head siliconized nylon rope (coated with 0.1% polylysine) to insert the ICA into the ICA about 1.85-2.00 cm, referring to the rat brain. At the beginning of the anterior artery, the blood supply to the middle cerebral artery is blocked. After 2 hours of ischemia, carefully pull out the nylon thread, ligature the ECA opening and suture the surgical incision. The contents were returned to the cage and refilled for 24 hours.

(2) Experimental grouping and administration

Thirty rats were randomly divided into 3 groups: model control group, water for injection (100 ml/kg), compound 4 in Example 1 (25, 50, 100 mg/kg), and group D2 (25, 50, 100 mg/kg). ), MCA blockade resulted in oral administration 10 minutes after ischemia.

(3) Determination of cerebral infarction volume

24 hours after reperfusion injury in rats, the brain was immediately decapitated, the olfactory bundle, cerebellum and lower brain stem were removed, and the crown was cut into 6 pieces (first to fifth pieces 2 mm/piece, sixth piece 4 mm), rapidly It was stained (37 ° C, protected from light) in 5 ml of a solution containing 1.5 ml of 4% TTC and 0.1 ml of 1 MK ₂ HPO ₄ for 20-30 minutes, during which it was flipped every 5 minutes. After TTC staining, the normal tissue was darkly stained and the infarcted tissue was white. Each group of brain slices is arranged neatly and photographed and saved. The infarct size of each piece was calculated and finally superimposed into the infarct volume. Infarct volume is expressed as a percentage of the cerebral hemisphere to eliminate the effects of cerebral edema.

Volume of cerebral infarction (%) = (surgical contralateral hemisphere volume - volume of uninfarcted part of the surgical hemisphere) / volume of the contralateral hemisphere of surgery *100%

(4) Experimental results

After 24 hours of ischemia and 2 hours of reperfusion, the volume of cerebral infarction (%) of the solvent control group was 33.8%. There were no cerebral infarctions in the sham operation group. The results of other groups of cerebral infarction volume are shown in Table 1:

Table 1: Effect of intragastric administration on cerebral infarction volume (%) in rats with ischemia

Oral administration of the compound 4 group significantly reduced the volume of cerebral infarction compared with the vehicle control group and the compound D2 group.

Example 3: Effect of Compound 1a and Compound D1 on sleep in rats:

Improve sleep function test

Animal source: Kunming mice, 18-22 grams, male. The temperature of the experimental animal breeding room was 22±2 ° C, and the relative humidity was 50-70%. In this experiment, compound 1a was administered at a dose of 25 mg/kg, and a distilled water control group was additionally provided.

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

Sample route: gavage

Experimental method: Pentobarbital sodium threshold dose hypnosis test:

Thirty male mice weighing 18-22 g were randomly divided into 3 groups, 10 rats in each group, and 30 days were continuously administered. After the sample was administered for 15 minutes on the 30th day, 50 mg/kg.bw of the animals were given to each group. Barbital sodium was intraperitoneally injected, the injection volume was 0.2ml/20g.bw, and the mouse righting reflex disappeared for more than 1 minute as the criterion for falling asleep. The sleeping time of each group of animals was observed within 60 minutes of sodium pentobarbital. sleeping time.

result:

Table 2: Effect of samples on animal body weight

As can be seen from the above table, there was no significant difference in the body weight of the animals in each dose group compared with the control group.

Table 3: Effect of supratenose dose of pentobarbital sodium on sleep time in mice

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

As can be seen from the above table, the sleep time and sleep time of the animals in the compound 1a group induced by the threshold dose of pentobarbital sodium were significantly different from those of the control group, and the time to fall asleep was shorter than that of the compound D1 and the sleep time was longer.

Pentobarbital sodium subthreshold dose hypnosis test:

Forty male mice weighing 18-22 g were randomly divided into four groups, 10 in each group, and 28 days in a row. After the sample was administered for 15 minutes on the 28th day, 30 mg/kg.bw of each group was given. Barbital sodium was injected intraperitoneally, the injection volume was 0.2ml/20g.bw, and the number of animals sleeping in each group was observed within 25 minutes after sodium pentobarbital was observed as the standard of falling into sleep. .

The results are as follows:

Table 4: Effect of subthreshold dose of pentobarbital sodium on the incidence of sleep in mice

*P<0.05 compared with the control group (cardiac test)

As can be seen from the above table, the incidence of sleeping animals and sleep in the compound 1 group and the control group induced by the subthreshold dose of pentobarbital sodium was significantly lower than that of the control group, and was higher than that of the compound D1.

Summary: Compounds 1a had a sleep-improving effect 30 days after oral administration of mouse samples.

Example 4: Injection prepared using Compound 1c of Example 1:

Accurately weigh the compound 1 prepared in Example 1 in a container, add appropriate amount of water for injection, stir until completely dissolved, adjust the pH to 8.5-8.8, add water for injection to 4000ml, add 2g of needle with activated carbon, boil for 15min The solution was decarbonized by suction filtration, and the solution was filtered through a 0.22 μm microporous membrane, and the solution was potted in a glass ampoule (94 mg per compound 1a). The preparation was autoclaved at 115 ° C for 15 min.

Example 5: Hemolysis and irritancy test of injection in Example 4

Liquid hemolytic test:

In vitro hemolysis test: adding unequal amounts to each of the liquid medicine tubes containing 2% red blood cell suspension

Example 4: Hemolysis stimulus test of compound 1c

The prepared injections were low and high (0.63 mg/mL and 1.88 mg/mL), and each tube did not produce hemolysis within 3 hours. The injection liquid prepared in Example 4 was tested to be negative for the extrahemolytic test. The specific experimental methods and experimental results are as follows:

1. Preparation of test drugs:

(1) High-dose group: Take 1 bottle of the injection (4 mL: 94 mg/vial) prepared in Example 4, aspirate 0.5 mL, and dilute to 6.25 mL with 0.9% (0.9 g/100 ml) sodium chloride injection to make A solution having a concentration of 1.88 mg/mL.

(2) Low-dose group: 2 mL of the above solution having a concentration of 1.88 mg/mL was taken, diluted to 6 mL with 0.9% (0.9 g/100 ml) of sodium chloride injection, and diluted to a solution having a concentration of 0.63 mg/mL.

2. Administration method:

A few milliliters of rabbit blood was taken and shaken for 10 minutes in a triangular flask containing glass beads to remove fibrinogen to defibrate the blood. Then, it is divided into several centrifuge tubes, each tube is added with about 10 times the amount of 0.9% sodium chloride injection, shaken, centrifuged (1500 rpm, 15 minutes), the supernatant is removed, and the precipitated red blood cells are reused. Wash 0.9% sodium chloride injection 2-3 times until the supernatant does not appear red. The obtained red blood cells were mixed with a 0.9% sodium chloride injection into a 2% suspension for testing.

Take 7 clean tubes with uniform caliber size (each tube is provided with parallel tubes). After numbering, add 2% red blood cell suspension, 0.9% sodium chloride injection, and pipette according to the ratio shown in Table 9. The water for injection and the test solution were mixed, and immediately incubated at 37 ° C in an incubator, and the observation was started every 15 minutes. After 1 hour, the observation was performed every 1 hour for a total of 3 hours.

Table 5: Preparation number of 2% red blood cell suspension

Note: 1-5 tubes are for the test tube, the sixth tube is the negative control tube, and the seventh tube is the positive control tube.

If the solution is clear red in the test, there is no cell residue at the bottom of the tube or a small amount of red blood cells remains, which means that hemolysis occurs; if the red blood cells are all sinking, the supernatant liquid is colorless and clear, indicating that no hemolysis occurs. If there is a brownish red or reddish brown flocculent precipitate in the solution, it does not disperse after shaking, indicating that red blood cell agglutination occurs. If there is a phenomenon of red blood cell condensation, it is necessary to further determine whether it is true or false. If the condensate can be evenly dispersed after shaking in the test tube, or the aggregate is placed on the slide, two drops of 0.9% sodium chloride injection are added to the edge of the cover slip, and observed under the microscope, the condensed red blood cells can be dissipated. It is a false agglomeration; if the agglomerates are not shaken or are not washed away on the slide, they are true.

3, the result is judged

When the negative control tube has no hemolysis and coagulation, and the positive control tube has hemolysis, if the test tube is in the tube The solution does not cause hemolysis and aggregation within 3 hours, and the test substance can be used for injection; if the solution in the test tube tube causes hemolysis and/or aggregation within 3 hours, the test substance is not suitable for injection.

4. Test results

Each of the drug solution tubes with a low concentration of O.63 mg/mL and a high concentration of 1.88 mg/mL injection solution did not produce hemolysis within 3 hours, and the in vitro hemolytic test was negative. See Table 6 and Table 7 for details.

Table 6: Results of hemolytic test of injection (high dose group) (visual observation)

Note: “+” means total hemolysis, “-” means no hemolysis; the sixth tube is a negative control tube, and the seventh tube is a positive control tube. Table 7: Results of hemolytic test of injection (low dose group) (visual observation)

Note: “+” means total hemolysis, “-” means no hemolysis; the sixth tube is a negative control tube, and the seventh tube is a positive control tube.

Injection vascular irritation test

Rabbit vascular irritation test: 8 healthy New Zealand rabbits were used for the test. The left and right ear veins were injected with the test drug at a dose of 5 ml/kg body weight, and the corresponding doses were administered to each drug-administered group. The injections prepared in Example 4, the low-dose group and the high-dose group were 3.15 mg/kg·bw and 9.4 mg/kg·bw, respectively. (The low and high concentrations were 0.63 mg/mL and 1.88 mg, respectively, according to the concentration. /mL, which is 0.7-1.4 times and 2-4 times the concentration of clinical intravenous infusion, and the right ear is given an equal volume of 0.9% (0.9g/100ml) sodium chloride injection as a control, once a day, for 3 consecutive times. day. Eight rabbits were given a high concentration and a low concentration of the test drug in turn, and then 0.9% sodium chloride injection was administered separately. Two rabbits each taking a low dose and a high dose were dissected 48 hours after the last administration, and the remaining 4 rabbits at low and high concentrations were examined at the end of the 2 weeks of the last administration. Results The contours of the blood vessels in the ears of 8 animals were clear. The thickness of the rabbit ears was uniform and there was no obvious change. Histopathological examination showed no toxicological changes in the blood vessels of the animals. The injection vascular irritation test prepared in Example 4 was in compliance with the regulations. The specific experimental methods and experimental results are as follows:

(1) Preparation of the test substance:

High-dose group: Take 2 bottles of the injection (4 mL: 94 mg/bottle) prepared in Example 4, aspirate 8 mL, and dilute to 100.0 mL with 0.9% (0.9 g/100 ml) sodium chloride injection to make the concentration 1.88 mg. /mL solution.

Low-dose group: 30 mL of the above solution having a concentration of 1.88 mg/mL was taken, diluted to 90.0 mL with 0.9% sodium chloride injection, and diluted to a solution having a concentration of 0.63 mg/mL.

(2) Weighing of animals: Weighed once before and after 48 hours and 14 days after the last administration.

(3) General observation and animal materials:

The reaction between the animal and the vascular injection site was observed and recorded before daily administration. At the 48th hour after the last administration, two New Zealand rabbits of high and low concentrations of the test drug were exsanguinated, and the vascular tissue reaction was visually observed and recorded. Cut the rabbit ears from the root of the ear (cut the left ear first, then cut the right ear, and mark), and then cut a section of the rabbit ear specimen and fix it in 10% neutral formaldehyde solution (the specimen is about 8cm long and about 1cm wide; The distal end cut is about 0.5 cm from the first needle, the proximal end cut is about 2 cm from the third needle, and the hanging end is near the end. 2 animals with high and low concentrations of each test drug continued to observe 14 days after the second administration, the following pathological examination was performed: the first needle was used as the boundary, and the distal end was cut; the third needle was used as the boundary, and the proximal end was cut into two segments; the blood vessel was cut at the time of tableting, and the conventional paraffin was sliced and sliced. The thickness was about 4-5 μm, HE staining, and then histopathological examination was performed.

(4), the result is judged

Comprehensive judgment was made based on the results of visual observation and pathological examination.

(5), test results

Visual observation:

Before the daily administration, the reaction of the blood-injected site of the animal was visually observed and recorded. During the administration, the high- and low-concentration parts of the test drug were visually observed, and the inner and outer sides of the vascular epidermis of the rabbit ear and the control side were red. From 0.1 cm x 0.2 cm to 0.2 cm x 1.0 cm. At 48 hours after the last administration, the contours of the bilateral rabbit ear vessels of the high concentration and low concentration of the test rabbits were clear, and the thickness of the rabbit ears was uniform and there was no significant change. See Table 12 and Table 13 for details. Four rabbits with high concentration and low concentration of the test drug were dissected 14 days after the last administration. The contours of the bilateral rabbit ear vessels were clear, and the thickness of the rabbit ears was uniform and no significant change was observed.

Pathological examination:

Four rabbits with high and low concentrations of the test drug were dissected 48 hours after the last administration, and the remaining 4 rabbits of the high and low concentrations of the test drug were examined at the end of the 2-week recovery period. No significant irritative reactions such as degeneration or necrosis of vascular tissue were observed in histopathological examination. The results are shown in Table 8 and Table 9:

Table 8: Inflammation of rabbit ear vessels by injection (high dose group) (visual observation at 48 hours after the last administration)

Table 9: Inflammation of rabbit ear vessels by injection (low dose group) (visual observation at 48 hours after the last administration)

The above results indicate that the injection prepared by using the compound 1 of Example 1 has good safety by hemolytic and vascular irritation test, and is suitable for preparation into an injection for clinical use.

Example 5: Pharmacokinetics test of compound 1c and compound D2

Instruments and reagents: Shimadzu LC.20AD HPLC system, including secondary array tube detector, CTO-10ASUP column oven, SIL-20A autosampler (Japan Shimadzu Corporation), HP-1 vacuum degasser, WD-9415 Ultrasonic Cleaner (Beijing Liuyi Machine Factory), TG16-WS Benchtop High Speed Centrifuge, XD-80A Vortex Mixer, Microsampler. Noopept reference (commercially available; purity: 99.1%; lot number: 1658-03); acetonitrile (chromatographically pure), methanol (analytical grade); distilled water.

Experimental animals: 156 clean male SD rats, both male and female, weighing (200 4-15) g.

156 rats were sequenced as 1, 2, 3, ... 78, and each experimental animal was randomly divided into 26 groups by random number table, 13 groups of compound 1c and compound D2, and 1c and compound D2 were injected into a single tail vein. After the daily dose of the experimental animals, venous blood, cerebrospinal fluid and brain tissue samples were collected simultaneously to control the dynamics of linezolid in blood, cerebrospinal fluid and brain tissue.

4. Sample collection: 1c was prepared by using physiological saline for injection to prepare 1c at a concentration of 7.5 mg/ml, and D2 was dissolved in a 1 ml/l sodium carbonate solution, and then a solution having a concentration of 7.5 mg/ml was prepared using physiological saline for injection. Rats were injected into the tail vein at a dose of 50 mg/kg. A group of rats (6 rats) were taken at 5 min, 30 min, 1 h, 2 h, 3 h, 5 h, 7 h, 9 h, 11 h, 13 h, 15 h, 17 h, 19 h after injection. Take its cerebrospinal fluid, plasma and brain tissue. The cerebrospinal fluid was extracted by cerebrospinal fluid extraction by a spinal microponic device under direct vision. After anesthesia with chloral hydrate, the skull was fixed, the back hair was cut, and a transverse incision was made at the line connecting the two ears. Cm), bluntly scrape the muscle layer of the neck and skull base, expose the occipital foramen, hold a 100ul micro-injector, take about 100ul of cerebrospinal fluid. Immediately after the rats were sacrificed, the head was decapitated, blood samples and brain tissues were collected, and the brain tissue was homogenized with 1.5 times weight of cold physiological saline, centrifuged at 5000 r/min for 15 min at low temperature, and the supernatant was taken. The venous blood samples were centrifuged. Serum was taken, and the collected cerebrospinal fluid was sealed in the dark at 20 ° C and stored in a low temperature refrigerator for 48 hours.

5. Chromatographic analysis: High performance liquid chromatography (HPLC) was used to determine the concentrations of serum, cerebrospinal fluid and brain tissue of 1C and D2. Separately absorb 100 ul of serum, cerebrospinal fluid and brain tissue supernatant, add 200 ul of methanol and mix for 5 min to precipitate protein, centrifuge at 16000 r/min for 15 min, and aspirate the supernatant to inject 90 ul. The column was an Inertsil ODS-SP column (4.6 x 250 mm, 5 m); mobile phase: water: acetonitrile (75:25, V/V); column temperature 30 ° C; flow rate 1 ml/min; detection wavelength 254 mm.

The results are shown in Table 1:

Table 1 Pharmacokinetic parameters of cerebrospinal fluid in 1c and D2

Group T _1/2 (h) C _max (μg/ml) AUC (h × μg / ml) 1c 0.41 6.80 4.99 D2 0.28 3.20 2.11

Compound 1c group T _1/2 comparative compound D2 was prolonged, and C _max and AUC were significantly improved compared with D2 group.

Claims (6)

A compound that prevents and treats cerebral vascular ischemic diseases, as shown in the following figure:

R is selected from the group consisting of H, C _1-6 alkyl, C _3-6 cycloalkyl, benzyl, phenyl wherein alkylphenyl, benzyl and cycloalkyl are unsubstituted or 1-3 are independently selected from halogen Substituted by a substituent of a hydroxyl group, an amino group, a C1-3 alkyl group, a C1-3 alkoxy group, -CO ₂ H, CO ₂ C1-6 and a trifluoromethyl group. The compound R according to claim 2 is preferably -CH ₃ , -C ₂ H ₅ , -C ₃ H ₇ , -C ₄ H ₉ or -C ₃ H ₅ . A salt formed by reacting a compound with an alkali metal according to claim R = H, from pharmaceutically acceptable sodium, potassium, magnesium, calcium and an organic base tromethamine, diethylamine, triethylamine, ethanolamine Ethylenediamine, dimethylamine, hydroxyethylethylenediamine, aminoethanolamine, etc., preferably a sodium salt. Use of a compound according to claims 1-4 for the manufacture of a medicament for the treatment of ischemic diseases. Use of a compound according to claims 1-4 for the preparation of a medicament for improving sleep.