WO2014176826A1

WO2014176826A1 - Acylated tanshinol derivative, composition comprising same and use thereof

Info

Publication number: WO2014176826A1
Application number: PCT/CN2013/079153
Authority: WO
Inventors: 熊利泽; 文爱东; 孙晓莉; 奚苗苗; 贾艳艳; 郭超; 殷英; 朱艳荣; 翁琰; 关月; 权伟
Original assignee: 中国人民解放军第四军医大学
Priority date: 2013-04-28
Filing date: 2013-07-10
Publication date: 2014-11-06

Abstract

The invention relates to an acylated tanshinol derivative of the following general formula (I), a pharmaceutical composition comprising same, and use thereof in the preparation of drugs for preventing or treating ischemia/reperfusion injury.

Description

Acylated Danshensu Derivatives, Compositions Thereof, and Applications Cross-Reference to Related Applications

This application claims Chinese patent application (Application No.: 201310152264.4) and "April 28, 2013", entitled "Acylated Danshensu Derivatives and Their Applications in Prevention and Treatment of Cerebral Vascular and Liver and Kidney Diseases", submitted on April 28, 2013 The priority of the Chinese Patent Application (Application No.: 201310152187.2), entitled "Application of the acylated Danshensu Derivative in the Prevention and Treatment of Cardiovascular Diseases and Complications", is hereby incorporated by reference. Technical field

The present invention relates to the field of medicinal chemistry, and in particular to an acylated danshensu derivative, a pharmaceutical composition comprising the same, and use thereof for the preparation of a medicament for preventing or treating ischemia/reperfusion injury. Background technique

China has long used salvia miltiorrhiza to treat diseases such as angina pectoris and myocardial infarction, and has developed a number of drugs based on salvia miltiorrhiza, such as Danhong injection and compound Danshen dripping pills. Danshensu is the main active ingredient of Salvia miltiorrhiza Bunge. It has a good preventive and therapeutic effect on cardiovascular-related ischemia/reperfusion injury. However, the efficacy of Danshensu in the treatment of ischemic diseases and ischemia/reperfusion injury associated with cerebrovascular disease is not clear. In addition, the phenolic hydroxyl group in the structure of Danshensu is easily oxidized and has poor stability.

Therefore, there is a need to develop a Danshensu drug that can prevent and treat ischemia/reperfusion injury.

Summary of the invention

In one aspect, the invention relates to a compound of the formula ω or a pharmaceutically acceptable salt thereof:

among them

Ri> and are independently selected from H or -COR, wherein R is selected from CM alkyl with, phenyl or alkyl with d_ ₅ substituted phenyl;

, and the same or different, but not H at the same time;

M is selected from the group consisting of pharmaceutically acceptable cations: H ⁺ , NH ₄ ⁺ , Li ⁺ , Na ⁺ , K ⁺ , Mg ²⁺ or

2+

Ca

In another aspect, the invention relates to a pharmaceutical composition comprising a compound of the invention and/or a pharmaceutically acceptable salt or adjuvant thereof.

In a further aspect, the invention relates to the use of a compound of the invention and/or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the prevention or treatment of ischemia/reperfusion injury.

In a further aspect, the invention relates to a method of preventing or treating ischemia/reperfusion injury comprising administering a compound of the invention and/or a pharmaceutically acceptable salt thereof to a subject having ischemia/reperfusion injury. DRAWINGS

Figure 1: Myocardial infarct size in each group after different treatments ±s, n=8)

Sham: sham operation group; MI/R: myocardial ischemia/reperfusion group; A, B, C: different acylated danshensu derivative groups; DHI: Danhong injection group.

P<0.05 compared with Sham group; <0.05ο compared with MI/R group

Figure 2: Determination of in vitro viability of cardiomyocytes after different treatments (±s, n=5)

Control: control group; SI/R: simulated ischemia/reperfusion group; A, B, C: different acylated Danshensu derivative group; DHI: Danhong injection group.

P < 0.01 compared with the Control group; *P < 0.05, < 0.01 compared with the SI/R group. Figure 3: Determination of LDH in cell supernatant after different treatments of cardiomyocytes ^±s, n=5)

P<0.01 compared with the Control group; *P < 0.05, < 0.01 compared with the SI/R group. Figure 4: Neurological scores of rats in different groups after treatment (n=8)

Sham: sham operation group; CI/R: cerebral ischemia/reperfusion group; A, B, C: different acylated danshen derivatives treatment group; Eda: edaravone injection group.

P<0.05 compared with the Sham group; compared with the CI/R group, <

Figure 5: Comparison of cerebral infarct size in rats after different treatments ±s, n=8)

P<0.05 compared with the Sham group; V compared to the CI/R group

Figure 6: Neuron-specific enolase activity in serum of rats after different treatments «=8) Sham: sham operation group; CI/R: cerebral ischemia/reperfusion group; A, B, C: different acyl groups Danshensu derivative treatment group; Eda: edaravone injection group.

Compared with the Sham group, P<0.05 compared with the CI/R group, <0.05, "Ρ Figure 7: Determination of in vitro survival rate after different treatments of neuronal cells ^±s, n=5)

Control: control group; SI/R: simulated ischemia/reperfusion group; A, B, C: different acylated Danshensu derivative treatment group; Eda: edaravone injection group.

Compared with the Control group, P is *P < 0.05, < compared with the SI/R group. Figure 8: Determination of LDH in cell supernatants after different treatments of neuronal cells (±s, n=5)

Control: control group; SI/R: simulated ischemia/reperfusion group; A, B, C: different acylated Danshensu derivative groups; Eda: edaravone injection group.

Compared with the Control group, ##Ρ is compared with the SI/R group, < 0.05, "P <

In one aspect, the invention relates to a compound of the formula ω or a pharmaceutically acceptable compound thereof

among them

And independently selected from hydrazine or -COR, wherein R is selected from phenyl fluorenyl, phenyl or d- ₅ fluorenyl substituted phenyl; , the same or different, but not H at the same time;

M is a pharmaceutically acceptable cation: H ⁺ , NH ₄ ⁺ , Li ⁺ , Na ⁺ , K ⁺ , Mg ²⁺ or Ca ²⁺ . Unless otherwise indicated, the following terms will have the following meaning throughout this specification. The term "CM thiol" as used herein, refers to a straight or branched, saturated monovalent hydrocarbon radical having 1, 2, 3, 4 or 5 carbon atoms. Examples of the d- ₅ fluorenyl group include methyl, ethyl, propyl, butyl, pentyl, isopropyl, isobutyl, sec-butyl, tert-butyl, isopentyl, 2-methylbutyl, 1 -methylbutyl, 1-ethylpropyl, 1,2-dimethylpropyl, neopentyl and 1,1-dimethylpropyl. Preferably, the d- ₅ thiol group is methyl, ethyl, propyl or isopropyl.

Similarly, the term "mercapto" as used herein, refers to a straight or branched, saturated monovalent hydrocarbon radical having 1, 2 or 3 carbon atoms. Examples of sulfhydryl groups are methyl, ethyl, propyl and isopropyl.

The phenyl group in the compound of the formula 1 of the present invention is optionally substituted by a CM thiol group, such as methylphenyl, ethylphenyl, propylphenyl, isopropylphenyl, butylphenyl, isobutyl Phenyl. Preferably, the above phenyl substituted by CM thiol is methylphenyl and ethylphenyl.

The term "pharmaceutically acceptable cation" as used herein includes, but is not limited to, the following cations: H ⁺ , Li ₄ ⁺ , Li ⁺ , Na ⁺ , K ⁺ , Mg ²⁺ , Ca ²⁺ .

The term "pharmaceutically acceptable salt" as used herein refers to a relatively non-toxic, inorganic or organic acid addition salt of a compound of the invention. See, for example, S. M. Berge et al., "Pharmaceutical Salts," J. Pharm. Sci. 1977, 66, 1-19. Pharmaceutically acceptable salts of the compounds of the invention include, but are not limited to, salts selected from the group consisting of hydrochlorides, nitrates, sulfates, hydrogen sulfates, diacid salts, acetates, lactates, citric acids Salt, tartrate, maleate, fumarate, methanesulfonate or gluconate. Preferably, the pharmaceutically acceptable salt of the compound of the invention is the hydrochloride salt.

In one embodiment, R is selected from phenyl, phenyl or d- ₃ fluorenyl substituted phenyl, preferably methyl, ethyl, propyl, phenyl or methylphenyl.

In one embodiment, M is H ⁺ , NH ₄ ⁺ , Li ⁺ , Na ⁺ , K ⁺ , Mg ²⁺ or Ca ²⁺ . Preferably, M is H+, Na ⁺ or K ⁺ .

In one embodiment, the compound of the formula of the invention, or a pharmaceutically acceptable salt thereof, is selected from the group consisting of: , -'Ο

Oh,

ο , '

, ο

A c and the above compounds are pharmaceutically acceptable,

The compounds of the invention, or pharmaceutically acceptable salts thereof, may act systemically and/or locally. The compound of the present invention or a pharmaceutically acceptable salt thereof can be administered by a suitable method, including, but not limited to, oral, injection, parenteral administration, topical administration, rectal administration, transdermal administration, and the like, as needed.

The compound of the present invention or a pharmaceutically acceptable salt thereof may be formulated into a desired administration form according to different routes of administration, including but not limited to injections, solutions, suspensions, emulsions, aerosols, tablets, powders. , capsules, granules, pills, pills, suppositories, etc. This can be done by prior art methods. For example, it can be accomplished by mixing a compound of the present invention or a pharmaceutically acceptable salt thereof with a pharmaceutically suitable adjuvant. Examples of pharmaceutically suitable excipients that can be used in the present invention include, but are not limited to, solvents, emulsifiers, dispersants, wetting agents, binders, stabilizers, colorants, and odor and/or taste masking agents.

In another aspect, the present invention is also a pharmaceutical composition comprising a compound of the present invention and/or a pharmaceutically acceptable salt thereof, and a pharmaceutically suitable excipient.

The dose of the compound of the present invention or a pharmaceutically acceptable salt thereof contained in the pharmaceutical composition of the present invention depends on the type and severity of the disease or condition, and the characteristics of the subject, such as general health, age, sex, body weight and drug Tolerance. One skilled in the art will be able to determine the appropriate dosage of the active compound of the present invention based on these or other factors.

In still another aspect, the present invention provides the use of a compound of the present invention and/or a pharmaceutically acceptable salt thereof for the preparation of a medicament for preventing or treating ischemia/reperfusion injury.

The term "treating" as used herein includes overcoming, alleviating, alleviating, relieving or ameliorating an injury, disease or condition. The term "prevention" refers to the prevention of potentially expected damage, disease or condition to some extent. In some cases, the term "treatment" also includes "prevention."

Studies have shown that reperfusion after a period of ischemia leads to inflammatory reactions involving the components of the innate immune system, including the complement and coagulation cascades. Both parenchymal and bone marrow cells produce free radicals, nitric oxide, and pro- and anti-inflammatory cytokines. These substances will attack this part and regain blood. The cells within the tissue are supplied, thereby causing cell damage. Thus, the term "ischemia/reperfusion injury" as used herein refers to damage that occurs after the blood circulation is restarted in an organism tissue that is suffering from ischemia. Ischemia/reperfusion injury also occurs when the blood circulation is restarted after stopping due to organ transplantation. Ischemia/reperfusion injury can occur in many tissues, such as kidney, liver, lung, pancreas, skeletal muscle, smooth muscle soft tissue, skin and intestine, as well as important tissues and organs such as the heart and brain. Exemplary ischemia/reperfusion injuries include, but are not limited to, cerebral ischemia/reperfusion injury, myocardial ischemia/reperfusion injury, hepatic ischemia/reperfusion injury, renal ischemia/reperfusion injury.

A further aspect of the invention provides a method of preventing or treating ischemia/reperfusion injury comprising administering a compound of the invention and/or a pharmaceutically acceptable salt thereof to a subject having ischemia/reperfusion injury.

The term "subject" as used herein includes mammals, preferably humans.

The Danshensu derivatives of the present invention have improved fat solubility and stability, and are excellent for preventing and treating ischemia/reperfusion injury. Example

The following examples are for illustrative purposes only and are not to be considered as limiting. Example 1: Preparation of Compound A

Add 5g of sodium danshensu to a 100ml three-necked flask, add 50ml of acetic anhydride, stir for 10 minutes, add lm perchloric acid dropwise at room temperature, raise the temperature to 40 ° C, stir at 40 ° C for one hour, stop Stir. The reaction solution was cooled to room temperature, and the reaction mixture was poured into 50 ml of ice water. After the ice was completely melted, it was extracted once with 50 ml of ethyl acetate, and the mixture was separated, and the aqueous phase was extracted three times with 20 ml of ethyl acetate.

The organic phase is distilled under reduced pressure into an oil, and an appropriate amount of ethyl acetate is added thereto, and an appropriate amount of silica gel is added thereto, and the mixture is sampled, sampled, and subjected to 100-200 mesh silica gel column chromatography, and the mobile phase is petroleum ether: ethyl acetate = 10:1 At 1:1, the product fraction was collected, and the solvent was evaporated under reduced pressure to give a white solid product with a yield of 45% and purity 98.9% (HPLC). The product was confirmed to be Compound A by NMR spectrum.

M.p.: 57-58 °C;

3⁄4 NMR (500 MHz, CDC1 ₃ ): δ 2.10 (s, 3H), 2.28 (s, 6H), 3.14 (m, 2H), 5.22 (q, 1H), 7.10-7.14 (t, 3H) ₀ Example 2: Preparation of Compound B

Add 5g of sodium danshensu to a 100ml three-necked flask, add 50ml of propionic anhydride, stir for 10 minutes, add lm perchloric acid dropwise at room temperature, raise the temperature to 40 ° C, stir at 40 ° C for one hour, stop Stir. The reaction solution was cooled to room temperature, and the reaction mixture was poured into 50 ml of ice water. After the ice was completely melted, it was extracted once with 50 ml of ethyl acetate, and the mixture was separated, and the aqueous phase was extracted three times with 20 ml of ethyl acetate.

The organic phase is distilled under reduced pressure into an oil, and an appropriate amount of ethyl acetate is added thereto, and an appropriate amount of silica gel is added thereto, and the mixture is sampled, sampled, and subjected to 100-200 mesh silica gel column chromatography, and the mobile phase is petroleum ether: ethyl acetate = 10:1 The product fraction was collected to 1:1, and the solvent was evaporated under reduced pressure to yield white solid product (yield: 41%, purity 98.1% (HPLC). The product was confirmed to be Compound 8 by NMR spectrum.

M.p.: 58-59 °C;

3⁄4 NMR (500 MHz, CDC1 ₃ ): 51.14 (t, 3H), 1.28 (m, 6H), 2.40 (q, 2H), 2.60 (q, 4H), 3.20 (t, 2H), 5.26 (q, 1H) ), 7.11-7.16 (t, 3H). Example 3: Preparation of Compound C

5 g of sodium danshensu was added to a 100 ml three-necked flask, 5 ml of benzoyl chloride, 10 ml of pyridine, 20 ml of toluene were added, and the mixture was refluxed for four hours. The reaction liquid was cooled to room temperature, water was added, the organic phase was separated, and the organic phase was distilled under reduced pressure to oil. Add the appropriate amount of ethyl acetate, add appropriate amount of silica gel, mix the sample, prepare the sample, and use 100-200 mesh silica gel column chromatography, the mobile phase is petroleum ether: ethyl acetate = 10:1 to 1:1, collect the product part, The solvent was evaporated under reduced pressure to give a white solid product (yield: 37%, purity 98.5% (HPLC). The product was confirmed to be Compound C by NMR spectrum.

M.p. : 55-56 ° C ;

3⁄4 NMR (500 MHz, CDC1 ₃ ): δ 3.42 (m, 2H), 5.56 (q, 1H), 7.35-7.56 (m, 12H), 8.04-8.10 (m, 6H). Test case

Experimental reagents / kits:

Danhong Injection (DHI) (Specification 10ml, Heze Buchang Pharmaceutical Co., Ltd., Lot No.: 12051067); Eda (Eda) (Specification 10mg/5ml, Nanjing Xiansheng Pharmaceutical Co., Ltd., batch number: H20031342); Acid kinase isoenzyme (CK-MB) quantification kit (Shanghai Xitang Biotechnology Co., Ltd. Company); lactate dehydrogenase (LDH) quantification kit, alanine aminotransferase (ALT) quantification kit, aspartate aminotransferase (AST) quantification kit, superoxide dismutase (SOD) quantification Kit, malondialdehyde (MDA) quantification kit, urine protein (PU) quantification test kit; Coomassie brilliant blue protein quantification kit (Nanjing Institute of Bioengineering); 2,3,5-triphenyl chloride Azolium blue (TTC) stain (China Pharmaceutical Group Shanghai Chemical Reagent Company); Evans blue (EB), tetramethylazozolium blue (MTT), 5-bromodeoxyuridine (BrdU), trypsin and small Bovine serum (Sigma-Aldrich, USA); DMEM medium (Gibco, USA); Neuron specific enolase (NSE) kit (Shanghai Jianglai Biotechnology Co., Ltd.); Serum muscle liver (Cre) kit and Urea nitrogen (BUN) kit (Beijing Beihua Kangtai Clinical Reagent Co., Ltd.). laboratory apparatus:

HX-100E animal ventilator, BL-420S multi-channel physiological recorder (Chengdu Taimeng Technology Co., Ltd.); 5417 low-temperature high-speed centrifuge (Eppendorf); COOLPIX S1 camera (Nikon, Japan); C680 type enzyme label Instrument, Model 450 Automatic Enzyme Labeling Tester (BIO-RAD); Ultra-clean Workbench (Suzhou Purification Equipment Company); Inverted Microscope (Olympus, Japan); Cell Culture Incubator (Hem, USA); Model 721 Spectrophotometer (Shanghai Precision Scientific Instrument Co., Ltd.) Test Example 1: Protective effects of Compounds A, B and C on myocardial ischemia/reperfusion (MI/R) injury in rats

(1) Experimental animals

Male SD rats weighing 240 ± 20 g were purchased from the Animal Center of the Fourth Military Medical University.

(2) Experimental plan and results

1 Establishment of a rat model of myocardial ischemia/reperfusion injury

Rats were randomly divided into 6 groups: sham operation group (Sham, 2% sodium citrate), model group (MI/R, 2% sodium citrate), administration group (Compound A group, Compound B) Group, Compound C group, dissolved in 2% sodium citrate), positive control group (8 ml/kg Danhong injection, DHI). The administration time of each group of rats was at the beginning of reperfusion.

Establishment of myocardial ischemia/reperfusion injury model: After anesthesia in rats, connect ECG monitoring, intubate the trachea, cut the sternum, expose the heart, ligation of the left anterior descending coronary artery to cause ischemia, and restore blood flow after 30 min ischemia. Perfusion (see: Mochizuki et aL, 2012, Resuscitation). Where Sham The rats in the group were not ligated to the left anterior descending coronary artery, and the rest were the same as the model group.

2 myocardial infarct size determination

Three hours after reperfusion, the heart was stained with Evans blue and counterstained with TTC. After staining, the white area represents the area of necrosis (AN), the red area represents the ischemic but not infarcted area, the blue area represents the normal area, and the area at risk (AAR) is the area of the red area and the white area. with. Analysis was performed using Image-Pro Plus 6.0 image analysis software. The degree of infarction is expressed as the AN/AAR ratio.

The results showed that compared with the sham group, the myocardial infarct size of the model group was significantly increased (P < 0.05), indicating that the MI/R model was successfully established. Compared with the model group, rats in the compound group A had significantly reduced myocardial infarct size (P<0.05), and were equivalent to the therapeutic effect of the positive control drug (P>0.05). See Figure 1 for the specific results.

3 Determination of serum biochemical indicators

Three hours after reperfusion, blood was taken from the abdominal aorta and centrifuged to obtain serum. The activity of CK-MB and LDH was measured using a kit.

The results showed that serum CK-MB and LDH levels were significantly increased in the model group compared with the Sham group (P<0.05). The levels of serum CK-MB and LDH in the compound group A were significantly lower than those in the model group (P<0.05). See Table 1 for the specific results.

1. Serum CK-MB and LDH levels in each group of rats 0 ± n = 8)

Note: Compared with the sham group, ^# Ρ < 0.05 _; compared with the model group, < 0.05. Test Example 2: Protective effects of compounds A, B and C on simulated ischemia/reperfusion (SI/R) injury in rat primary cardiomyocytes

(1) Experimental animals

Neonatal SD rats (1-2 days), the same source as in Example 1.

(2) Experimental plan and results

1 primary culture of cardiomyocytes

The heart of the newborn SD rat was removed and the myocardial tissue was broken. 0.125% trypsin was added, incubated at 37 ° C for 5 min, the supernatant was discarded, 0.125% trypsin was added again, and the reaction was repeated 5 times. DMEM medium containing calf serum was added, and the supernatant was discarded after centrifugation. The cells were again resuspended in DMEM medium containing 10% calf serum, and after mixing, the cells were seeded into the culture plate. The cardiomyocytes were purified by differential adherence method, and the appropriate amount of BrdU (final concentration O.Ol mM) was added and cultured in a 37 ° C C0 ₂ incubator. The medium was changed after 24 h.

2 Modeling of myocardial cell simulation ischemia/reperfusion injury model

The medium was changed to serum-free DMEM medium saturated with 95% N ₂ 1 5% CO ₂ mixture, and rapidly transferred to a device filled with a mixture of 95% N ₂ / 5% CO ₂ . After 2 h, the medium was transferred to a conventional incubator and incubated for 24 h to establish a model of myocardial hypoxia/reoxygenation injury.

The experiment was divided into 6 groups: blank control group (Control), simulated ischemia/reperfusion injury group (SI/R), administration group (Compound A group, Compound B group and Compound C group, each 10 μΜ) and positive control. Group (2% Danhong injection, DHI).

3 Determination of myocardial cell survival rate

After the reperfusion of primary cardiomyocytes, 20×L 5 mg/ml MTT was added to each group, and the cells were incubated for 4 h, and the culture solution was discarded. Each group was further added with 150 L of dimethyl sulfoxide (DMSO), which was then detected at 490 nm using a microplate reader.

As shown in Figure 2, myocardial cell survival was significantly reduced after simulated ischemia/reperfusion injury (P < 0.01). In the drug-administered group, Compound A significantly enhanced the survival rate of cardiomyocytes (P < 0.05 or 0.01), and the effect of Compound A was equivalent to the positive control drug DHI > 0.05).

4 Myocardial cell supernatant Determination of LDH release

After the reperfusion of primary cardiomyocytes, the supernatant cells were taken from each group, and the LDH activity of the cell supernatant was measured using a kit.

As shown in Figure 3, myocardial LDH release was significantly increased after ischemia/reperfusion injury (P < 0.01), while Compounds A and B significantly reduced the LDH content of the cell supernatant < 0.05 or ³ < 0.01), and the effect of Compound A was equivalent to the positive control drug DHI > 0.05). Test Example 3: Protective effects of compounds A, B and C on cerebral ischemia/reperfusion (CI/R) injury in rats

(1) Experimental animals

The animal species and source were the same as test case 1, and the body weight was 260 ± 20 g.

(2) Experimental plan and results

1 Establishment of a rat model of cerebral ischemia/reperfusion injury

Rats were randomly divided into 6 groups: sham operation group (Sham, 2% sodium citrate), model group (CI/R,

2% sodium citrate), administration group (Compound A group, Compound C group, Compound E group, 30 mg/kg each, dissolved in 2% sodium citrate), positive control group (Eda, 3 mg/ Kg edaravone injection). Each group of rats was administered or solvent time and at the beginning of reperfusion.

Model establishment method: After anesthesia, the median incision in the neck, the right common artery of the neck (CCA), the external carotid artery (ECA) and the pterygopalatine artery, and a small vertical incision under the bifurcation of the common carotid artery. Place the nylon thread (3-0, USA) in the internal carotid artery for about 17~18 mm until there is a slight resistance. After blocking the blood flow for 2 h, carefully pull out the plug to form reperfusion (see: Koizumi). Et al., 1986, stroke) The Sham group was not inserted with a nylon thread, and the remaining steps were the same as the surgery group.

2 neurological function score

After 24 h of cerebral ischemia/reperfusion, the neurological deficit scores of each group were evaluated and recorded according to the Longa score: 0 points, no dysfunction; 1 point, unable to extend the left forelimb; 2 points, rotated to the left; 3 Points, dumped to the left; 4 points, no autonomic activities with disturbance of consciousness. The higher the score, the greater the neurological damage of the animal.

As shown in Figure 4, both Compounds A and B significantly improved neurological function in CI/R rats compared to the model group (PO.05, n=8), and the effects of Compounds A and B were compared with the positive control reagent Eda. Quite (P Xi.OS, n=8).

3 cerebral infarct size determination

Rats with neurological function scores were decapitated and cut into 2 mm thick sections, then stained with 2% TTC and fixed with paraformaldehyde solution 24 and the area of cerebral infarction was calculated. The red area was normal tissue and the white area was infarct area. . As shown in Figure 5, the infarct size of the rats was significantly reduced after treatment with Compounds A, B, and C (P < 0.05), and the efficacy of Compound A was comparable to that of the positive control reagent Eda.

4 Determination of serum specific enolase (NSE) in serum

After 24 hours of reperfusion, blood was taken from the abdominal aorta of the rats, and the supernatant was centrifuged. The kit was used to detect the activity of NSE in the serum.

As shown in Figure 6, the NSE activity in the serum of the CI/R group was significantly higher than that of the Sham group (Z ³ < 0.05). Compared with the CI/R group, compounds A, B and C all significantly reduced serum NSE activity (P < 0.01 or 0.05), and the effects of compounds A and B were comparable to the positive control reagent Eda. Test Example 4: Protective effects of compounds A, B and C on ischemia/reperfusion (SI/R) injury in rat primary neuronal cells

(1) Experimental animals

Female SD rats (14-15 days of pregnancy), the source is the same as test case 1.

(2) Experimental plan and results

1 Primary culture of neurons

The fetus was removed from the pregnant rat and the cerebral cortex was removed from the fetal rat brain tissue. The cerebral cortex was transferred to a pre-cooled eppendorf tube, and 500 μM of trypsin was added and digested at 37 ° C for 10 min. Digestion was carried out by adding 200 l of FBS and resuspended. Add 3 ml of 10% DEME medium, resuspend, filter, and discard the supernatant. Add 2 1^ 10% 0 ^^ medium and: 827 (50 times) 40 ^, resuspend. The suspension was added to a poly-lysine pre-coated 24-well plate at a density of 2.5 χ ^{10 5} neurons/well and cultured at 37 ° C in a 5% (0 ₂ incubator).

2 Neuron SI/R model establishment

The procedure for establishing the myocardial cell SI/R model in Example 2 was the same.

The experiment was divided into 6 groups: blank control group (Control), simulated ischemia/reperfusion injury group (SI/R), administration group (compound group A, compound B group and compound C group, each 10 μΜ) and positive. Control group (5% edaravone, Eda).

3 Neuronal cell survival rate determination:

The procedure for measuring the survival rate of cardiomyocytes in Example 2 was the same.

As shown in Figure 7, neuronal cell survival was significantly reduced after simulated ischemia/reperfusion injury. 0.01). Compounds A and B significantly enhanced the survival rate of neuronal cells (P O.05 or 0.01), and the effect of Compound A was comparable to the positive control drug Eda > 0.05).

4 Neuronal cell supernatant Determination of LDH release

After the primary neuronal cell reperfusion, the supernatant cells were taken from each group, and the LDH activity of the cell supernatant was measured using a kit.

As shown in Figure 8, the release of LDH from neuronal cells was significantly increased by < 0.01 after ischemia/reperfusion injury. Compounds A and B significantly reduced LDH levels in cell supernatants < 0.05 or 0.01), and compound A was equivalent to the positive control drug Eda > 0.05). Test Example 5: Protection of Compounds A, B and C against Hepatic Ischemia/Reperfusion (HI/R) Injury in Rats

(1) Experimental animals

The animal species and source were the same as Test Example 1, and the body weight was 250 ± 30 g.

(2) Experimental plan and results

1 Rat HI/R injury model preparation

Rats were randomly divided into 5 groups: sham operation group (Sham, 2% sodium citrate), model group (HI/R, 2% sodium citrate), and administration group (compound group A, compound B). Group, Compound C, each 20 mg/kg, dissolved in 2% sodium citrate). Each group was given a drug or solvent (tail vein injection) at the beginning of reperfusion, and the dose was determined based on the pre-experimental pharmacodynamic curve.

Establishment of a rat model of hepatic ischemia/reperfusion: After anesthesia in animals, the hepatic hilum was exposed after laparotomy, and the hepatic artery, portal vein and bile duct were clamped together with non-invasive vascular clamps, and the blood flow was blocked when the hepatic hilum was blocked for 30 minutes. Infusion for 1 h, an animal model of liver ischemia/reperfusion lung injury (see: Oguz et al., 2013, Eur Rev Med Pharmacol Sci). Sham only used anesthesia, laparotomy, and did not block blood flow.

2 biochemical indicators analysis

After lh reperfusion, the abdominal aorta of the rats was bled, and the liver tissue was taken to make a 10% tissue homogenate, centrifuged and serum and tissue supernatant were collected. Serum ALT, AST and changes in SOD and MDA in liver tissue were measured according to the kit instructions.

The results of the experiment are shown in Table 1-2: Compared with the model group, the serum AST and ALT activities of the compound A, B, and C treatment groups were significantly lower than <0.05); the MDA level in the liver tissue was significantly reduced and the SOD activity was significantly increased ( P < 0.05), and the best in group B. Table 2. Serum ALT and AST levels in each group of rats 0 ± n = 8)

Note: Sham: sham operation group; HI/R: liver ischemia/reperfusion group; A, B and C: different acylated danshensu derivatives; compared with Sham group, #Ρ<0.01; and HI/R group In comparison, *P < 0.05.

Table 3. Activity of MDA and SOD in liver tissues of rats in each group 0± s, n=8)

Note: Sham: sham operation group; HI/R: liver ischemia/reperfusion group; A, B and C: different acylated danshensu derivatives; compared with Sham group, #Ρ<0.01; and HI/R group In comparison, *P < 0.05. Test Example 6: Protection of Compounds A, B and C against Renal Ischemia/Reperfusion (RI/R) Injury in Rats

(1) Experimental animals

The animal species and source were the same as test case 1, and the body weight was 260±20g.

(2) Experimental plan and results 1 Preparation of rat RI/R injury model

Rats were randomly divided into 5 groups: sham operation group (Sham, 2% sodium citrate), model group (RI/R, 2% sodium citrate), administration group (compound group A, compound B) Group, Compound C, 20 mg/kg each, dissolved in 2% sodium citrate). Each group of administration or solvent time was at the beginning of reperfusion.

Model establishment method: After anesthesia in rats, the mid-abdominal incision exposes the bilateral kidneys, the bilateral renal arteries are separated, and the bilateral renal arteries are clamped with arterial clips. After the renal clamp is closed for 1 h, the arterial clip is released to restore perfusion, and then the wound is sutured (see : Bi et al" 2009, European Journal of Pharmacology. The Sham group only exposed bilateral renal pedicles (no ligation), and the remaining steps were the same as the model group.

2 Determination of serum biochemical indicators

24 hours after reperfusion of the rat kidney, blood was taken from the abdominal aorta, centrifuged, and the supernatant was taken. Serum levels of MDA, PU, Cre, and BU were measured according to the kit instructions.

Compared with the sham group, the levels of MDA, PU, Cre and BUN in the serum of the model group were significantly increased (P<0.05), while the treatment groups of the eight, B and C groups significantly reduced the elevated levels of these indicators in the serum of rats. (P<0.05), and the effect of compound C was the best, and the results are shown in Table 4. Table 4. Effects of serum MDA, PU, Cre, BUN in each group of rats (± n=8)

Note: Sham: sham operation group; RI/R: renal ischemia/reperfusion group; A, B and C: different acylated Danshensu derivatives; compared with Sham group, #Ρ < 0.05; and RI/R group In comparison, 0.05.

Claims

Claim

Compound of the formula

And independently selected from H or -COR, wherein R is selected from phenyl fluorenyl, phenyl or d- ₅ fluorenyl substituted phenyl;

, and the same or different, but not H at the same time;

M is a pharmaceutically acceptable cation: H ⁺ , NH ₄ ⁺ , Li ⁺ , Na ⁺ , K ⁺ , Mg ²⁺ or Ca ²⁺ .

2. A compound of the formula or a pharmaceutically acceptable salt thereof according to claim 1 wherein R is selected from the group consisting of fluorenyl, phenyl or d- ₃ fluorenyl substituted phenyl.

3. A compound of the formula or a pharmaceutically acceptable salt thereof according to claim 2, wherein R is selected from the group consisting of methyl, ethyl, propyl, phenyl, methylphenyl.

4. A compound of formula 1 or a pharmaceutically acceptable salt thereof according to claim 3 selected from the group consisting of:

A Β C

And pharmaceutically acceptable salts of the above compounds.

A pharmaceutical composition comprising the compound according to any one of claims 1 to 4 and/or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable adjuvant.

6. The pharmaceutical composition according to claim 5, which is a dosage form selected from the group consisting of injections, solutions, suspensions, emulsions, aerosols, tablets, powders, capsules, granules, pills Agents, pills and suppositories.

The use of the compound according to any one of claims 1 to 4 and/or a pharmaceutically acceptable salt thereof for the preparation of a medicament for preventing or treating ischemia/reperfusion injury.

8. The use according to claim 7, wherein the ischemia/reperfusion injury is selected from the group consisting of: cerebral ischemia/reperfusion injury, myocardial ischemia/reperfusion injury, hepatic ischemia/reperfusion injury, renal deficiency Blood/reperfusion injury.