CN108727447B

CN108727447B - Preparation of coumarin derivative metabolite and application of coumarin derivative metabolite in prevention and treatment of cerebral ischemia and Alzheimer's disease

Info

Publication number: CN108727447B
Application number: CN201810317196.5A
Authority: CN
Inventors: 陈乃宏; 李燕; 戴均贵; 宋修云; 盛莉; 乐田; 楚世峰; 张梓倩; 解可波; 谭圳
Original assignee: Institute of Materia Medica of CAMS
Current assignee: Institute of Materia Medica of CAMS
Priority date: 2017-04-21
Filing date: 2018-04-10
Publication date: 2021-01-12
Anticipated expiration: 2038-04-10
Also published as: CN108727447A

Abstract

The invention relates to a coumarin derivative metabolite IMM-H004-7-O-beta-glucuronic acid (IMM-H004G) with a novel structure, a preparation method thereof and application of the compound in preventing and treating cerebral ischemia and Alzheimer disease. Pharmacokinetics research shows that the half-life period of IMM-H004 in brain is less than 20min, the half-life period of metabolite IMM-H004G can reach 24H, and the exposure amount in brain is 2.3 times of that of the prototype drug. Enzymatic glucuronidation of IMM-H004 is carried out by glycosyltransferase, and a new compound IMM-H004G is obtained after separation and purification. Pharmacological tests prove that the compound can effectively inhibit the nerve cell damage induced by oxygen sugar deprivation, Abeta and okadiac acid, remarkably improve the behavioral disturbance caused by rat cerebral middle artery occlusion reperfusion, reduce the cerebral infarction area and has the anti-cerebral ischemia effect similar to that of IMM-H004. IMM-H004G is a main effective metabolite of IMM-H004 in vivo anti-cerebral ischemia, and can be used for preparing medicines for preventing and treating cerebral ischemia and Alzheimer disease.

Description

Preparation of coumarin derivative metabolite and application of coumarin derivative metabolite in prevention and treatment of cerebral ischemia and Alzheimer's disease

Technical Field

The invention belongs to the field of medicines, and particularly relates to preparation of a metabolite of coumarin derivative IMM-H004, namely a new compound IMM-H004-7-O-beta-glucuronic acid, and application of the metabolite in prevention and treatment of cerebral ischemia and Alzheimer's disease.

Background

Cerebral ischemia and Alzheimer's disease are two important senile diseases, and with the accelerated aging process in China, the two diseases further harm the life and health of the elderly in China.

At present, the clinical anti-cerebral ischemia medicaments at home and abroad mainly comprise: (1) improving cerebral blood circulation: A. thrombolytic agents, such as recombinant tissue plasminogen activator (rt-PA), Urokinase (UK); B. anticoagulants, such as heparin, heparinoids; C. antiplatelet agents such as clopidogrel, aspirin; D. defibrinating agents, such as defibrinating enzymes. (2) Neuroprotection: A. radical scavengers such as edaravone; B. neurotrophic agents, such as Naokukang, gangliosides. (3) Others, such as butylphthalide, human prourokinase phthalide. However, the cerebral blood circulation improving drugs represented by rt-PA have the disadvantages of narrow treatment time window, easy intracranial hemorrhage and unsuitability for long-term use, and the efficacy and safety of the neuroprotective agent represented by edaravone still need to be further researched and determined. Therefore, no effective medicine for treating cerebral arterial thrombosis exists.

At present, the clinical anti-Alzheimer disease drugs at home and abroad mainly comprise: (1) cholinesterase inhibitors: such as donepezil, galantamine, huperzine A. (2) NMDA receptor antagonists: such as memantine. (3) Others, such as a β antibodies, and the like. However, they can only improve the symptoms of alzheimer's disease, cannot delay its progression, and cannot fundamentally treat alzheimer's disease. Therefore, the search for new effective drugs for treating cerebral ischemia and alzheimer disease is a hot problem to be solved urgently.

The coumarin derivative IMM-H004 is a novel compound for antagonizing serious brain diseases, which is researched and developed by the inventor, and the pharmacological effects of IMM-H004 on the synthesis, cerebral ischemia resistance and Alzheimer disease resistance are disclosed in the published invention patent of 2012 with the application number of 201210176782.5. IMM-H004 has the characteristics of stable synthesis process, high bioavailability, small toxicity and good curative effect, and has practical development significance for research of candidate drugs for resisting cerebral ischemia and Alzheimer disease.

Pharmacokinetics research finds that IMM-H004 mainly generates glucuronic acid binding products IMM-H004-7-O-beta-glucuronic acid (IMM-H004G) in rat in vitro and in human liver microsomes, the exposure Amount (AUC) of IMM-H004G in blood plasma and brain is obviously higher than that of proto-drugs (7.7 times and 2.3 times of the original drugs respectively), the elimination half-life (t1/2) of IMM-H004 in brain tissues is shorter and is only 0.2H, and t1/2 of IMM-H004G is as long as 24H.

The applicant researches and discovers that a compound with a novel structure, namely IMM-H004-7-O-beta-glucuronic acid, separated from a metabolite of IMM-H004 is shown as a chemical structure in a formula I. The compound can effectively inhibit oxygen sugar deprivation (OGD), Abeta and okadaic acid (OKA) induced nerve cell damage in vitro, can remarkably improve animal behavioral disturbance caused by rat middle cerebral artery occlusion reperfusion in vivo, reduces cerebral infarction area, and has anti-cerebral ischemia effect similar to IMM-H004. At present, no research report of IMM-H004 metabolite, no research report of IMM-H004-7-O-beta-glucuronic acid for treating cerebral ischemia and Alzheimer disease, and no patent document of a preparation method of IMM-H004-7-O-beta-glucuronic acid are found.

Disclosure of Invention

The invention aims to provide a novel compound of an IMM-H004 glucuronic acid conjugate, which has a novel structure; it is still another object of the present invention to provide a method for preparing the same; the third purpose of the invention is the application of the composition in preventing and treating cerebral ischemia and Alzheimer disease.

In order to realize the purpose of the invention, the following technical scheme is adopted:

an IMM-H004 metabolite, namely IMM-H004-7-O-beta-glucuronic acid, has a novel structure, is not reported in documents and patents, and has a structure shown in formula I:

the preparation method of the IMM-H004 glucuronic acid conjugate IMM-H004-7-O-beta-glucuronic acid is characterized by comprising the following steps:

1. the compound IMM-H004 is subjected to enzymatic glucuronidation by using uridine diphosphate glucuronic acid as glycosyl donor and using glycosyltransferase UGT88D4(GenBank: BAG 31945.1).

2. The glucuronidated product of IMM-H004 was isolated and purified by ethyl acetate extraction combined with reverse semi-preparative HPLC. The product is subjected to UV, MS,¹H NMR、¹³The structure of the compound is analyzed and identified by spectroscopic means such as C NMR, HSQC and HMBC, and the compound is IMM-H004-7-O-beta-glucuronic acid, and no report of the compound and a preparation method thereof exists at present.

The invention relates to a novel compound IMM-H004-7-O-beta-glucuronic acid obtained by the method, and pharmacokinetic researches show that IMM-H004 mainly generates glucuronic acid binding products IMM-H004-7-O-beta-glucuronic acid (IMM-H004G) in rat in vitro and in human liver microsomes, the exposure (AUC) of IMM-H004G in plasma and brain is obviously higher than that of prototype drugs (7.7 times and 2.3 times of the original drugs respectively), the elimination half-life (t1/2) of IMM-H004 in brain tissues is shorter and is only 0.2H, and t1/2 of IMM-H004G is as long as 24H. In addition, the plasma protein binding rate of IMM-H004G is 17% -30% lower than that of the original drug, and the brain tissue protein binding rate is 50% lower than that of the original drug. In addition, in vitro pharmacological activity experiments show that IMM-H004G has the function of remarkably improving oxygen sugar deprivation (OGD), Abeta and okadaic acid (OKA) induced nerve cell injury in vitro, can remarkably improve animal behavioral disturbance caused by rat middle cerebral artery occlusion reperfusion in vivo, reduces cerebral infarction area, has the anti-cerebral ischemia function similar to that of IMM-H004, and has the potential of preventing and treating cerebral ischemia and Alzheimer disease.

The invention has the following technical advantages:

1. the preparation method of the novel compound IMM-H004-7-O-beta-glucuronic acid is novel and has the advantages of few reaction steps and high conversion rate, and the preparation method is not reported in documents.

2. The novel compound IMM-H004-7-O-beta-glucuronic acid has an obvious effect of preventing and treating cerebral ischemia, can remarkably inhibit the neurotoxicity effect induced by oxygen sugar deprivation, and improves the cerebral infarction area and the behavioral injury of a model rat.

3. The novel compound IMM-H004-7-O-beta-glucuronic acid has an obvious effect of preventing and treating Alzheimer's disease, and has an obvious effect of inhibiting A beta and okadiac acid-induced neurotoxicity.

4. Compared with the prototype drug IMM-H004, the novel compound IMM-H004-7-O-beta-glucuronic acid has better pharmacokinetic advantage.

5. The novel compound IMM-H004-7-O-beta-glucuronic acid has a novel structure, is not reported in documents, and has the potential of being further developed into a medicament for preventing and treating cerebral ischemia and Alzheimer disease.

Drawings

FIG. 1 IMM-H004-7-O- β -glucuronic acid;

FIG. 2 high resolution mass spectra of IMM-H004-7-O- β -glucuronic acid in positive ion mode;

FIG. 3 IMM-H004-7-O-beta-glucuronic acid¹H NMR Spectrum (DMSO-d)₆,500MHz)；

FIG. 4 is a 13C NMR spectrum (DMSO-d6,125MHz) of IMM-H004-7-O- β -glucuronic acid;

FIG. 5 HSQC spectrum of IMM-H004-7-O-beta-glucuronic acid (DMSO-d)₆,500MHz)；

FIG. 6 HMBC spectrum of IMM-H004-7-O-beta-glucuronic acid (DMSO-d)₆,500MHz)；

FIG. 7 human recombinant UGT isozyme subtypes involved in IMM-H004G production;

FIG. 8-1 IMM-H004 metabolism in human brain microvascular endothelial cells;

FIG. 8-2 IMM-H004 metabolism in human neuroblastoma cells;

figure 9 time-dependent curve of the content of IMM-H004 and its metabolite IMM-H004G in plasma after oral administration of IMM-H004 citrate (16mg/kg) in mice (n-5);

FIG. 10 is a graph of the time course of the IMM-H004 and its metabolite IMM-H004G content in plasma after intravenous injection of IMM-H004 citrate (5mg/kg) in mice (n-3);

FIG. 11 is a graph of the plasma dose time of the proto-drug versus IMM-H004G (n-3) following intravenous injection of IMM-H004 citrate (6mg/kg) in rats;

FIG. 12 plot of prototype drug versus brain tissue drug of IMM-H004G after intravenous injection of IMM-H004 citrate (6mg/kg) in rats (n-3);

FIG. 13 fecal excretion of rats intravenously injected with IMM-H004 citrate (5 mg/kg);

FIG. 14 Effect of IMM-H004G on PC12 cell injury due to oxygen sugar deprivation (means. + -. SD, n ═ 12)^###p<A 0.001vs blank control group,^**p<0.01,^***p<0.001vs model control;

FIG. 15 IMM-H004G vs. A β_1-42Influence of induced PC12 cell injury (means + -SD, n ═ 6)^###p<A 0.001vs blank control group,^*p<0.05,^**p<0.01vs model control;

FIG. 16 Effect of IMM-H004G on PC12 cell injury caused by okadiac acid (OKA) (means + -SD, n ═ 6)^###p<A 0.001vs blank control group,^*p<0.05,^**p<0.01,^***p<0.001vs model control;

FIG. 17 IMM-H004G Effect on rat behavioural behaviour due to unilateral MCAO/R-Longa's score (means + -SD, n-9-10),^*p<0.05,^**p<0.01vs MCAO/R model control group;

FIG. 18 effect of IMM-H004G on rat behavioural behaviour due to unilateral MCAO/R-screenscreen score (means. + -. SD, n ═ 9-10),^*p<0.05,^**p<0.01vs MCAO/R model control group;

FIG. 19-1 effect of IMM-H004G on the cerebral infarction of rats caused by unilateral MCAO/R-TTC staining picture;

figure 19-effect of IMM-H004G on the cerebral infarction of rats caused by unilateral MCAO/R-TTC staining to measure infarct volume (means ± SD, n ═ 9-10),^**p<0.01,^***p<0.001vs MCAO/R model control.

Detailed Description

The following examples and pharmacological activity experiments are intended to further illustrate the invention, but are not intended to limit the invention in any way.

Preparation method of IMM-H004-7-O-glucuronic acid

Example 1

Glycosyl transferase UGT88D4 catalyzing IMM-H004 for glucuronidation

The reaction system is IMM-H004304 mg (1.0mM), glycosyl donor UDP-glucuronic acid 930mg (1.5mM), glycosyl transferase (UGT88D4)80mg, and Tris-HCl buffer solution with pH 7.4 is added to make a constant volume of 80 ml. The reaction conditions were 30 ℃ water bath, shaking (60rpm) and reaction for 24 h. The conversion rate of the glycosylation reaction can reach more than 85 percent through HPLC-UV detection.

Example 2

Separation, purification and identification of IMM-H004 glucuronide

The enzymatic reaction solution was repeatedly extracted 5 times with 160ml of ethyl acetate to remove the unreacted substrate IMM-H004. Subsequently, the raffinate was reduced pressure evaporated to dryness and resuspended in 2ml of methanol. Centrifugation at 15,000g for 30min, supernatant was taken and the glycosylated product was prepared by reverse semi-preparative HPLC separation. HPLC starting mobile phase (methanol/water) ratio of 15/85, increasing mobile phase ratio gradient to 100/0 within 30 min; the flow rate is 3 ml/min; the semi-preparative column is Shiseido capcell pak C18 column (250mm × 10mm i.d., Shiseido co., ltd., Tokyo, Japan); glycosylation product t_RIt is 7.1 min. UV, MS, UV, MS,¹H NMR、¹³The compound is identified to be IMM-H004-7-O-beta-glucuronic acid by the analysis of spectral means such as C NMR, HSQC and HMBC, and the structure is shown as formula I. The compound has a novel structure and is not reported in the literature.

The spectrum information and nuclear magnetic signal attribution of the new compound are as follows:

IMM-H004-7-O- β -glucuronic acid: light yellow amorphous powder, UV λ max (MeOH) nm 327.9,255.7; HRESI-MS M/z 481.1803[ M + H ]]⁺,calcd.for C₂₂H₂₈N₂O₁₀；¹H NMR(500MHz,DMSO-d₆)δ: 6.61(1H,d,J＝2.3Hz,H-8),6.59(1H,d,J＝2.3Hz,H-6),4.99(1H,d,J＝7.1Hz,Glc-H₁),3.85 (3H,s,H-12),3.55-3.15(overlapped,Glc-H),3.26(4H,overlapped,H-14,16),3.17(4H,m,H-13, 15),2.61(3H,s,H-11),2.21(3H,s,H-17)；¹³C NMR(125MHz,DMSO-d₆)δ:172.2(Glc-6),160.2 (C-7),159.2(C-5),158..2(C-2),154.1(C-9),148.6(C-3),130.8(C-4),105.3(C-10),100.2(Glc-1), 97.3(C-6),95.1(C-8),76.6(Glc-2),74.3(Glc-5),73.1(Glc-3),72.1(Glc-4),60.7(C-13,C-15),56.8 (C-12),55.9(C-14,C-16),46.9(C-17),17.9(C-11).

Pharmacological experiments

Experimental example 1

Metabolism study of IMM-H004 citrate in rat and human liver microsomes

IMM-H004 citrate (100. mu.M) and rat and human liver microsomes (1mg/mL protein) were reduced by 99.8% after adding UDPGA (3mM) and prorocentsin (50. mu.g/mL) for 30min, and formation of 1 glucuronic acid binding product was detected. Collecting glucuronic acid binding product for nuclear magnetic analysis, wherein the product is IMM-H004-7-O-beta-glucuronic acid (IMM-H004G).

Experimental example 2

Bioconversion Studies of IMM-H004

The UGT enzyme subtypes involved in IMM-H004G production were identified using 12 human UGTs recombinases (UGT1A1, 1A3, 1A4, 1A6, 1A7, 1A8, 1A9, 1A10, 2B4, 2B7, 2B15, 2B 17). The generation site of IMM-H004G was investigated using human brain microvascular endothelial cells and human neuroblastoma cells. The results show that IMM-H004 can be metabolized to generate IMM-H004G through humanized recombinant UGT1A8, 1A3, 1A7, 1A1, 1A10, 1A9 and 2B15 (FIG. 7). Furthermore, IMM-H004 produced IMM-H004G in human brain microvascular endothelial cells, human neuroblastoma cells (FIGS. 8-1, 8-2).

Experimental example 3

Plasma pharmacokinetic study of mice following oral (16mg/kg) and intravenous (3mg/kg) IMM-H004 citrate

The content of IMM-H004G is measured by hydrolyzing plasma samples with glucuronidase at 37 deg.C for 1H. After mice take IMM-H004 citrate (16mg/kg) orally, the prototype and the metabolite IMM-H004G reach the peak 5min after the administration, the average blood drug peak concentration is 0.51 mu M and 40.67 mu M respectively, and AUC_0-t0.45 and 58.5 μ M × h, respectively (fig. 9). CL for prototype and its metabolite IMM-H004G after intravenous injection of IMM-H004 citrate (3mg/kg) in mice were 1.89 and 0.19 mg/kg/. mu.M/H, AUC_0-t1.55 and 16.0 μ M × h (fig. 10), respectively, suggesting that the proto-drug eliminated faster in mouse plasma and lower in vivo exposure.

Experimental example 4

Plasma and brain tissue pharmacokinetic studies after intravenous IMM-H004 citrate (6mg/kg) in rats

2min after intravenous injection of IMM-H004 citrate (6mg/kg) in rats, the mean blood concentration of the prototype drug is 24.29 mu M, and 0.5H after administration is lower than 1 mu M (minimum effective concentration in vitro). IMM-H004G Peak 5min post-administration, C_max16.15 μ M, and blood concentration was 1.33 μ M up to 8h after administration. AUC of proto-drug and IMM-H004G_(0-t)3.82, 29.43 μ M × h, t, respectively_1/20.64 and 3.61h, respectively, suggested faster elimination of the proto-drug in plasma and lower in vivo exposure (fig. 11).

After intravenous injection of IMM-H004 citrate (6mg/kg) in rats, the brain tissue prototype drug content is higher than 1.18nmol/g only at 2minThe concentration of the prototype drug is lower than 0.002nmol/g after 2 hours of drug administration. IMM-H004G Peak 0.25min post-administration in brain tissue, C_maxIs 0.19nmol/g, and the content concentration is lower than 0.002nmol/g in 24h after administration. AUC of proto-drug and IMM-H004G in brain tissue_(0-t)0.31, 0.72nmol/g x h, t respectively_1/2The time is 0.20 hour and 23.96 hours respectively. The prototype drug is indicated to have lower content in brain tissue and faster elimination. IMM-H004G eliminated slowly in brain tissue (FIG. 12).

Experimental example 5

Excretion studies in rats after intravenous IMM-H004 citrate (5 mg/kg).

IMM-H004G was detected in both bile and urine samples after intravenous injection of IMM-H004 citrate (5mg/kg) in rats. The dosage of the prototype drug before and after hydrolysis of the urine sample by the glucuronidase is 14.8% and 65.1% respectively. The content of the prototype drug is not obviously increased before and after hydrolysis of the feces sample by the glucuronidase, and the prototype drug accounts for 25.2% of the dosage. The total excretion of proto-drug and IMM-H004G in rat feces and urine accounted for 40.0% and 50.3% of the administered amount, respectively (FIG. 13).

Experimental example 6

IMM-H004 and IMM-H004G binding studies with rat/human plasma/brain tissue protein

The combination of the medicine and the plasma protein not only influences the in-vivo process and the medicine effect of the medicine, but also has important guiding significance for the clinical application of the medicine. Rapid equilibrium dialysis was used to study the rate of IMM-H004 and IMM-H004G binding to rat/human plasma/brain tissue protein. As shown in Table 1, the binding rate of IMM-H004(0.5-10 μ g/mL) to the plasma protein of rats is 97.39% -97.71%, and the binding rate to the plasma protein of human is 77.27% -86.62%. The binding rate of IMM-H004G (0.1-2 mug/mL) to the plasma protein of rats is 67.55% -69.09%, and the binding rate to the plasma protein of human is 64.09% -68.97%. The rat plasma protein binding rate of IMM-H004G is 30% lower than that of the original drug, and the human plasma protein binding rate of IMM-H004G is 17% -20% lower than that of the original drug. As shown in Table 2, the binding rate of IMM-H004 (0.1-1 μ g/mL) and rat brain tissue protein in 25% tissue homogenate was 89.52% -89.99%, and the binding rate of IMM-H004G (0.1-1 μ g/mL) and rat brain tissue protein was 43.00-44.22%, which was 50% lower than that of the original drug.

Table 1 IMM-H004/IMM-H004G binding rates to rat/human plasma protein (n ═ 3)

TABLE 2 binding rates of IMM-H004/IMM-H004G to rat brain tissue protein (n-3)

Pharmacological activity experiment of IMM-H004-7-O-glucuronic acid

Experimental example 7

Effect of IMM-H004-7-O-glucuronic acid on oxygen sugar deprivation injury of nerve cells in vitro

Sodium dithionite (Na)₂S₂O₄) The OGD is realized by matching with a low-sugar DMEM solution as an oxygen scavenger. Collecting full monolayer PC12 cells (simulated neuron cells), discarding original culture solution, adding DMEM complete culture solution containing 5% FBS and 5% horse serum, gently blowing with a pipette to completely disperse the cells at 5 × 10⁴The cells were inoculated at a density of 100. mu.l/ml into 96-well plates previously treated with polylysine (0.1mg/ml) and incubated for 24 hours for the experiment. The experiment is divided into a blank group, a model group and an additive group, and edaravone and nerve growth factor are used as positive controls. The blank group was given complete medium and the oxygen deprived group was added to the affected cells at a final concentration of 5mM for 24h using low-sugar DMEM solution. The drug adding group is added with 10 mu M and 1 mu M of tested compound while molding. Then 10. mu.l of 5mg/ml MTT was added, and after 4 hours the supernatant was removed, and 100. mu.l of DMSO was added to express the number of viable cells as an absorbance value at 550 nm.

The result is shown in figure 14, the survival rate of PC12 cells can be obviously reduced by oxygen sugar deprivation, and 1 and 10 mu M IMM-H004G have good improvement effect on PC12 cell damage caused by OGD, the effect is similar to that of prototype IMM-H004 and IMM-H004 citrate, and the drug effect of various forms of the IMM-H004 with equal dosage is stronger than that of the positive drug edaravone.

Experimental example 8

Influence of IMM-H004-7-O-glucuronic acid on damage of in-vitro nerve cell A beta and okadaic acid

Modeling by using main toxic component A beta of senile plaque and abnormal phosphorylation inducer okadaic acid (OKA) of Tau, and acting on the cultured PC12 cells. The experiment is divided into a blank group, a model group and an additive group, and nerve growth factor is used as positive control. Blank groups were given complete medium and model groups were added with a final concentration of 10 μ M of either A β or 30nM OKA-acting cells for 24 h. The drug adding group is added with 10 mu M and 1 mu M of tested compound while molding. Then 10. mu.l of 5mg/ml MTT was added, and after 4 hours the supernatant was removed, and 100. mu.l of DMSO was added to express the number of viable cells as an absorbance value at 550 nm.

The results are shown in fig. 15 and 16, the survival rate of PC12 cells can be obviously reduced by A beta or OKA, and the 10 mu M IMM-H004G has a good improvement effect on the PC12 cell damage caused by the two models, and the effect is similar to that of prototype IMM-H004 and IMM-H004 citrate. However, none of the low doses of IMM-H004 in its various forms has such an effect.

Experimental example 9

Effect of IMM-H004-7-O-glucuronic acid on rat focal cerebral ischemic injury

Laboratory animals and groups

Clean-grade packed male SD rat with weight of 250-280g and experimental classification into MCAO/R model group (physiological saline, 3 ml. kg)^-1I.v.), MCAO/R + IMM-H004 group (6 mg. kg)^-1I.v.), MCAO/R + IMM-H004c group (IMM-H004 citrate 10 mg/kg)^-1I.v.) and MCAO/R + IMM-H004G group (IMM-H004-7-O-glucuronic acid, 10 mg. kg^-1I.v.) 10 per group.

Establishment of cerebral ischemia reperfusion animal model

(1) Rats were fasted for 12h before surgery without water deprivation, weighed and placed in an initial anesthesia box using 3% isoflurane at 95% O₂/5％ CO₂Rats were anesthetized under guidance. After about 5min, its tail was lifted without any tension and naturally dropped with gravity indicating successful anesthesia. Fixing the rat on the operating table in a supine manner; (2) preparing skin on the neck of a rat, and disinfecting with 75% alcohol; a25 mm incision is made in the middle of the neck, and the triangle formed by the scapula-tongue bone and sternocleidomastoid muscle on the right sideExposing Common Carotid Artery (CCA), External Carotid Artery (ECA) and Internal Carotid Artery (ICA); separating ECA trunk, and bluntly separating out thyroid artery and occipital artery, and burning off; (3) ligating and burning the ECA at the position about 1cm away from the CCA bifurcation, and ligating a suture at the CCA bifurcation without tight ligation; (4) ICA was carefully isolated to the site where it originated the pterygopalatine artery (PPA) and the cranial artery. The CCA and ICA were temporarily occluded by a mini-arterial clamp, a small incision was cut at the ECA stump with an ophthalmic scissors, a nylon suture was inserted, the suture was tied, the ICA arterial clamp was released, and the suture was slowly pushed into the ICA, into the ICA into the cranial artery branch. When the plug felt resistance about 1.8cm from the ICA and ECA bifurcation, indicating that the plug had blocked the middle cerebral artery, unclamping the CCA artery and tightening the suture; cutting off redundant wire bolts; (5) a small amount of penicillin injection powder is coated on the operation wound, and the subcutaneous soft tissue and the skin are aligned and sutured. After the rat revives, observing the behavioral change of the rat to judge whether MCA is successfully blocked; (6) cutting the suture part again after 60min of blockage, carefully pulling out the nylon thread plug, tightening the suture line to prevent bleeding, and suturing the wound; (7) keeping warm after operation and supplying food water; isolating the animal after the operation, and putting the animal back into the cage after the animal is revived; (8) all the models are manufactured in a laminar flow environment, the room temperature is kept at 23-25 ℃, and the relative humidity is 60%.

Behavioral scoring

Animals were scored for neurological deficits 24h postoperatively using the Zea Longa method and the screen test. Successful cerebral ischemia reperfusion model: right Horner sign, left hemiplegia with the forelimbs as heavy. Animals without neurological deficit and coma are excluded from the experiment, and animals with neurological deficit are subjected to subsequent experiments.

The Zea Longa score raises the tail away from the ground by about one ruler, and the conditions of two forelimbs are observed; the rats were placed on the ground and the walking was observed. By using a 5-point scale (score 0-5), higher scores indicate more severe neurobehavioral impairment. 0 minute: normal, without neurological deficit; 1 minute: the left anterior paw can not be fully extended, and mild neurological deficit is caused; and 2, dividing: when walking, the rat turns to the left side (paralyzed side) and has moderate neurological deficit; and 3, dividing: when walking, the rat body was inclined to the left (paralyzed side). Severe neurological deficit; and 4, dividing: spontaneous walking and loss of consciousness; and 5, dividing: the animal died.

Screen test the screens were 60cm by 60cm mesh strips and the mesh was lcm by lcm. The left, right and upper parts of the net plate are framed by wood plates with the height of 2.5 cm. The screen is horizontally placed, a rat is placed on the screen, one end of the screen is slowly lifted, the screen is changed into a vertical position within 2s, and the screen is kept for 5s to observe whether the rat gets off the screen or the screen is gripped by a front claw, so that the gripping capability and the muscle force condition of the front claw are evaluated. The scoring criteria were divided into 4 grades. 0 minute, the front claw holds the screen for 5 seconds without falling off; 1 minute, temporarily holding the screen, sliding for a certain distance, but not falling; 2, dropping in 5 s; and 3, the rat falls down immediately when the screen rotates.

TTC staining for determining cerebral infarction volume

After the completion of the behavioral tests, the rats were anesthetized with chloral hydrate (0.35ml/100g), and the brains were decapitated and cut into 6 coronal sections 2mm thick using a sectioning groove. Then, the brain slices were quickly placed in 1.5% TTC aqueous solution, protected from light, incubated at 37 ℃ for 15min, and shaken every 5min during the incubation period. After staining, normal brain tissue was rose-red, whereas infarcted tissue was white and well-defined. After the incubation, the brain slices are put into a centrifuge tube filled with 4% paraformaldehyde for storage, each group of brain slices are arranged in order the next day, and the brain slices are shot and stored by a digital camera. The calculation formula of the cerebral infarction volume of the rat is as follows: infarct volume/(non-infarct lateral half brain volume 2)

Results of the experiment

In the experiment, a cerebral ischemia model of artery occlusion reperfusion (MCAO/R) in the middle brain of a unilateral rat is established by using a wire-embolism method, and the improvement effect of the compounds IMM-H004, IMM-H004 citrate and IMM-H004 metabolite IMM-H004-7-O-glucuronic acid (IMM-H004G) on cerebral ischemia injury is detected. Animal experiment results show that IMM-H004, IMM-H004c and IMM-H004G all have a protective effect on rat MCAO/R injury, as shown in the fact that the three medicines can obviously reduce Longa's score and screenplay score and reduce cerebral infarction volume, and the treatment effects of the three medicines are not different (figure 17-18, figure 19-1 and figure 19-2). Namely, the metabolite IMM-H004G of IMM-H004 has the effect of anti-cerebral ischemia which is consistent with the original IMM-H004 and the citrate of the original IMM-H004.

Claims

1. A coumarin derivative metabolite shown in formula I and pharmaceutically acceptable salt thereof;

2. the coumarin derivative metabolite and the pharmaceutically acceptable salts thereof according to claim 1, wherein the salts are selected from the group consisting of coumarin derivative metabolites and salts of inorganic and organic acids.

3. Preparation of a coumarin derivative metabolite according to any one of claims 1 to 2, comprising the steps of: introducing glucuronic acid into 7-position hydroxyl of IMM-H004 by using glycosyltransferase to obtain metabolite of coumarin derivative

4. A pharmaceutical composition comprising an effective amount of a coumarin derivative metabolite according to any one of claims 1 to 2 or a pharmaceutically acceptable salt thereof in combination with a pharmaceutically acceptable carrier.

5. The pharmaceutical composition of claim 4, wherein the pharmaceutical composition is selected from the group consisting of an injection, a tablet, a capsule, a pill, a granule, an oral liquid, and a suspension.

6. The use of a coumarin derivative metabolite according to any one of claims 1 to 2, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for the prevention or treatment of cerebral ischemia or alzheimer's disease.