CN110256390B

CN110256390B - Coumarin compound, preparation method and application of coumarin compound in preparation of medicine for treating cerebral apoplexy

Info

Publication number: CN110256390B
Application number: CN201910540845.2A
Authority: CN
Inventors: 李侠; 王钧; 李明凯; 张文彤; 马波; 樊朝阳
Original assignee: Fourth Military Medical University FMMU
Current assignee: Fourth Military Medical University FMMU
Priority date: 2019-06-21
Filing date: 2019-06-21
Publication date: 2020-11-24
Anticipated expiration: 2039-06-21
Also published as: CN110256390A

Abstract

The invention provides a coumarin compound, a preparation method and application of preparing a medicine for treating cerebral apoplexy, and mainly aims to provide an ideal IS brain protection medicine which meets part or all standards: has multifaceted effects (such as antioxidant effect), and can non-selectively block acute IS cascade caused by OS; the volume of the cerebral infarction can be obviously reduced: the vascular tension is not influenced, and no bleeding event occurs in the research process; promoting the metabolism of toxic substances by brain tissues, and having slight side effect of the medicine; before use, differential diagnosis is not needed, so that early treatment is efficient and feasible.

Description

Coumarin compound, preparation method and application of coumarin compound in preparation of medicine for treating cerebral apoplexy

Technical Field

The invention belongs to the field of chemical drugs, and particularly relates to a coumarin compound, a preparation method and application of the coumarin compound in preparation of drugs for treating cerebral apoplexy.

Background

In recent years, the incidence rate of stroke ranks first in the world in China, and the third national cause of death investigation result proves that the stroke becomes the first cause of death in China, the annual growth rate exceeds 8.7%, and the life health of the people in China is seriously influenced. The stroke IS mainly divided into Ischemic Stroke (IS) and Hemorrhagic Stroke (HS), wherein the IS type patients account for three quarters of the total disease patients and are the main type of stroke disease. The key of IS treatment IS 'early diagnosis and early intervention', various current treatments try to save ischemic brain tissues by recanalizing blood flow of early infarcted brain tissues, but the short 'treatment time window' hinders the popularization of the treatment mode. For many patients, particularly in poor and basic areas with relatively backward medical conditions and relatively weak stroke treatment systems, the patients often exceed a treatment time window when hospitalizing, and the existing thrombolytic therapy is far from achieving the purpose of effective treatment. Therefore, an efficient and convenient-to-popularize IS treatment method IS urgently sought.

Disclosure of Invention

The invention aims to provide a coumarin compound, which has the structure of the formula (I):

Compound 1：R＝H；

Compound 2：R＝3-CH₃；

Compound 3：R＝3-OCH₃；

Compound 4：R＝3-Oph；

Compound 5：R＝3-Br；

Compound6：R＝3-F。

optionally, the preparation of the coumarin compound comprises: 20mmol of 4-hydroxycoumarin and 10mmol of aromatic aldehyde synthetic reagent

Heating and refluxing in organic solvent, R is H, R is 3-CH₃、R＝3-OCH₃And R-3-Oph, R-3-Br or R-3-F, precipitating white solid particles, and recrystallizing the white solid particles to obtain the target product.

Optionally, the organic solvent is ethanol, and the recrystallization is performed in ethanol with a volume percentage concentration of 95%.

A process for the preparation of a coumarin compound as claimed in claim 1, which comprises: 20mmol of 4-hydroxycoumarin and 10mmol of aromatic aldehyde synthetic reagent

A coumarin compound having the structure of formula (ii):

optionally, the preparation of the coumarin compound comprises: 20mmol of 4-hydroxycoumarin and 10mmol

Heating and refluxing the mixture in an organic solvent until white solid particles are separated out, and recrystallizing the white solid particles to obtain the target product.

Optionally, the organic solvent is ethanol, and the recrystallization is carried out in ethanol with a volume percentage concentration of 95%.

The coumarin compound is applied to preparing drugs for treating cerebral apoplexy.

The coumarin compound prepared by the preparation method of the coumarin compound is applied to preparing drugs for treating cerebral apoplexy.

The present study IS directed to the invention of an ideal IS brain protection drug that meets some or all of the following criteria: has multifaceted effects (such as antioxidant effect), and can non-selectively block acute IS cascade caused by OS; the volume of the cerebral infarction can be obviously reduced: the vascular tension is not influenced, and no bleeding event occurs in the research process; promoting the metabolism of toxic substances by brain tissues, and having slight side effect of the medicine; before use, differential diagnosis is not needed, so that early treatment is efficient and feasible.

Drawings

The accompanying drawings, which are included to provide a further understanding of the disclosure and are incorporated in and constitute a part of this specification, illustrate embodiments of the disclosure and together with the description serve to explain the disclosure without limiting the disclosure. In the drawings:

FIGS. 1-6 are nuclear magnetic spectra of compounds 1-6, respectively;

fig. 7 is a graph of the results of cell stability of COM 3 in primary neurons, and the effect on cell survival in OGD-injured neurons;

FIG. 8 is an observation of normal, OGD neurons, and under-lens morphological changes of COM 32.5, 5, 10 μ g/ml neurons after OGD injury;

FIG. 9 shows the results of PI/Hoechst double staining. A is a randomly selected fluorescent field; b is COM 3 pair OGD neuron PI⁺Statistical analysis of the effect of cell number;

FIG. 10 is a graph of the effect of COM 3 on the amount of LDH release from OGD neurons;

FIG. 11 is a staining of post MCAO cerebral infarct volume; a is a representative picture of staining; b IS the statistical analysis of COM 3 to IS mouse cerebral infarction volume calculation; c IS analysis of the effect of COM 3 on IS mouse neurobehavioral;

FIG. 12 IS a graph showing the effect of COM 3 on the amount of LDH release from brain tissue of mice after IS;

FIG. 13 IS a graph showing the effect of COM 3 on brain edema in IS mice.

Detailed Description

Early brain protection treatment of IS as important as thrombolytic therapy. The thrombolytic reflux IS the precondition of successful treatment in the acute stage of IS, and the cerebral protection treatment IS the decisive factor influencing the prognosis of patients, and the thrombolytic reflux and the cerebral protection treatment complement each other and are not indispensable for successfully rescuing the brain tissue. The blockade of the ischemic cascade chain mediated by Oxidative Stress (OS) by brain protection therapy IS the key to effective treatment of IS. Brain protection IS the blocking of the cascade of IS ischemia at one or more targets (calcium overload, oxidative free radical activation, neuronal death), blocking or reversing the pathological process of ischemic penumbra neurons. post-IS neuronal OS damage IS a key step in initiating a series of pathological cellular processes including inflammation, apoptosis, autophagy, and the like. If the injury can be blocked, the oxidation resistance of brain tissue IS improved, the ischemia tolerance time of nerve cells after IS can be prolonged, and the time IS strived for effectively opening blood vessels; it can also prevent blood flow Reperfusion-induced delayed nerve cell death, i.e., Ischemia Reperfusion Injury (CIRI). At present, under the guidance of domestic and foreign research, the research aims to invent an ideal IS brain protection medicament which meets the following partial or all standards: has multifaceted effects (such as antioxidant effect), and can non-selectively block acute IS cascade caused by OS; the volume of the cerebral infarction can be obviously reduced: the vascular tension is not influenced, and no bleeding event occurs in the research process; promoting the metabolism of toxic substances by brain tissues, and having slight side effect of the medicine; before use, differential diagnosis is not needed, so that early treatment is efficient and feasible.

The compound of the invention has the structural formula as R ═ H and 3-CH₃、3-OCH₃3-Oph, 3-Br or 3-F, indicates the name of the substituent bearing at the corresponding site on the parent nucleus structure, the designation "3" indicates the corresponding substituent at the third site, if not so noted, e.g. R ═ H, indicates R ═ H at all of

sites

1, 2 and 3.

In the same way, the labeling principle of the series aromatic aldehyde synthetic reagents used in the preparation process is the same.

In the present invention, Compound represents Compound, and the corresponding following numbers represent Compound numbers, and in the following detailed experimental section, Compound is also represented by COM, and the numbers following COM represent Compound numbers.

The following are specific examples provided by the inventors to further explain the technical solution of the present invention: (the% appearing in the following experiment is the volume% concentration)

Firstly, the compound of the invention has a general synthesis formula and synthesis steps:

Compound 1：R＝H；Compound 2：R＝3-CH₃；Compound 3：R＝3-OCH₃；Compound 4：R＝3-Oph；Compound 5：R＝3-Br；Compound6：R＝3-F。

the synthesis steps are as follows: synthesizing a novel coumarin compound: at 250mL of threeAdding 4-hydroxycoumarin (20mmol) and simultaneously adding 100mL of absolute ethyl alcohol into a flask, heating until the absolute ethyl alcohol is completely dissolved, and then adding a series of aromatic aldehyde synthetic reagents:

(Compound 1：R＝H；Compound 2：R＝3-CH₃；Compound 3：R＝3-OCH₃(ii) a Compound 4: r is 3-Oph; compound 5: r is 3-Br; compound 6: r is 3-F; 10mmol) and heated and refluxed for 4 hours until white solid particles are separated out, the heating is continued for about 1 hour, after the reaction is finished, the mixture is naturally cooled and filtered, and ethanol (95 percent) is recrystallized to finally obtain the target pure white granular crystal.

II, compound structure identification:

compound1-6 was characterized for molecular weight, structure, and purity by Nuclear Magnetic Resonance Spectroscopy (NMR). The identification results are shown below:

Compound 1：

3, 3' -benzylidene-bis-4-hydroxycoumarin;

3,3′-Benzylidene-bis-(4-hydroxycoumarin)；

1H NMR (CDCl3, ppm):11.528(s,1H),11.299(s,1H),7.994-8.080(q,2H),7.606-7.649(m,2H),7.215-7.421(m,9H),6.104(s, 1H). The specific nuclear magnetic spectrum is shown in figure 1.

Copmound 2：

3, 3' - (3-methylbenzylidene) -bis-4-hydroxycoumarin;

3,3′-(3-Methylbenzylidene)-bis-(4-hydroxycoumarin)；

1H NMR (CDCl3, ppm):11.528(s,1H),11.285(s,1H),8.009-8.088(q,2H),7.623-7.654(t,2H),7.415-7.432(d,4H),7.206-7.236(t,1H),7.082-7.097(d,1H),7.012-7.042(t,2H),6.080(s,1H),2.312(s, 3H). The specific NMR spectrum is shown in FIG. 2.

Compound 3：

3, 3' - (3-methoxybenzylidene) -bis-4-hydroxycoumarin;

3,3′-(3-Methoxybenzylidene)-bis-(4-hydroxycoumarin)；

1H NMR (DMSO-d6, ppm) 7.891-7.910(d,2H),7.565-7.604(t,2H),7.301-7.371(m,4H),7.146-7.175(t,2H),6.840-6.906(q,2H),6.244(s,1H),3.573(s, 3H). The specific NMR spectrum is shown in FIG. 3.

Compound 4：

3, 3' - (3-phenoxybenzylidene) -bis-4-hydroxycoumarin;

3,3′-(3-Phenoxylbenzylidene)-bis-(4-hydroxycoumarin)；

1H NMR (CDCl3, ppm):11.655(s,1H),11.319(s,1H),8.009-8.091(q,2H),7.626-7.668(m,2H),7.412-7.433(d,4H),7.274-7.326(m,3H),6.999-7.055(m,4H),6.900-6.950(m,2H),6.107(s, 1H). The specific NMR spectrum is shown in FIG. 4.

Compound 5：

3, 3' - (3-bromobenzylidene) -bis-4-hydroxycoumarin

3,3′-(3-Bromobenzylidene)-bis-(4-hydroxycoumarin)

1H NMR (CDCl3, ppm):11.593(s,1H),11.316(s,1H),8.028-8.109(q,2H),7.649-7.691(m,2H),7.360-7.458(m,6H),7.179-7.245(m,2H),6.086(s, 1H). The specific NMR spectrum is shown in FIG. 5.

Compound 6：

3, 3' - (3-fluorobenzylidene) -bis-4-hydroxycoumarin

3,3′-(3-Fluorobenzylidene)-bis-(4-hydroxycoumarin)

1H NMR (CDCl3, ppm):11.616(s,1H),11.327(s,1H),8.023-8.109(q,2H),7.645-7.688(m,2H),7.435-7.456(d,4H),7.301-7.340(m,1H),6.941-7.047(m,3H),6.095(s, 1H). The specific NMR spectrum is shown in FIG. 6.

Description of cells, experimental animals, reagents, etc.: the primary culture neurons are taken from cerebral cortex of a C57BL/6 fetal mouse, and C57BL/6 pregnant mice and male mice are purchased from the experimental animal center of the university of air military medical science and are raised in the animal laboratory of the pharmacological department of the university of air military medical science, and the treated animals strictly comply with the ethical regulations of animal experiments.

DMEM, Neurobasal, B27, FBS, 0.25% trypsin was purchased from Gibco. SOD and GSH-Px detection kits are purchased from Biyuntian biology, Inc. The PI/Hoechst detection kit is purchased from Nanjing Biotechnology Limited. TTC was purchased from Sigma, USA. CCK-8 is available from Dojindo, Japan.

And thirdly, screening compounds based on the influence on the antioxidant capacity of the neurons:

(1) compound solution preparation, 0.1% DMSO for promoting dissolution, and filtration sterilization. Dilutions were designed to appropriate concentrations according to the experimental design.

(2) Determining the influence of computer 1-6 on the oxidation resistance of oxidative damage neurons, selecting two kinds of antioxidases, namely Superoxide Dismutase (SOD) and Glutathione Peroxidase (GSH-Px), as detection indexes, and determining by using a kit microplate method. The primary neurons are taken from 14-16-day-old fetal mice, meninges and blood vessels are removed through micromanipulation, then the fetal neurons are digested by 0.2% trypsin, the fetal neurons are inoculated in a culture medium of DMED + 10% FBS, the Neurobasal + 2% B27 culture medium is completely changed on the next day, and then liquid is changed in half every three days. Inoculating neuron in 96-well plate at density of 5 × 10⁴A hole. Neurons were grown for 7 days with 500. mu. mol H₂O₂The neuron is damaged for 24 hours, then the influence of Compound1-6

concentration

1, 5, 10, 25, 50 mug/ml on the neuron antioxidant enzyme SOD and GSH-Px is determined, and 4 Compound holes are arranged in each group.

(3) The results show that

Experimental results show that Compound 3(COM 3) in Compound1-6 has a significant up-regulation of the activity of the oxidative damage primary neuronal antioxidant enzymes SOD and GSH-Px at concentrations of 5. mu.g/ml, 10. mu.g/ml, 25. mu.g/ml (tables 1 and 2). The results suggest that COM 3 has significant antioxidant effects in neurons and IS likely to have neuroprotective effects against IS.

TABLE 1 Effect of Compound1-6 on neuronal SOD and GSH-Px antioxidant enzymes

TABLE 2 Effect of Compound1-6 on neuronal GSH-Px antioxidant enzymes

P <0.05, p <0.01 compared to saline group.

Protective effect of compound on Oxygen Deprivation (OGD) neuron

(1) CCK-8 was used to test the effect of COM 3 on neuronal cell stability: CCK-8 measures the cell stability of COM 3 in neurons; culturing primary neurons by the method to the 7 th day, co-culturing the neurons with COM 3 concentrations of 1, 2.5, 5, 10, 25 and 50 μ g/ml for 24h, and adding Neurobasal medium containing 10% CCK-8 to incubate for 2h at 37 ℃. The absorbance was measured at a wavelength of 450nm, and 4 duplicate wells were set for each group. Examining the effect of COM 3 on cell survival of OGD neurons: using OGD (Total replacement of sugar-free EBSS medium, 95% N)₂，5％CO₂And (3) damaging primary neurons at 37 ℃ for 6h and reoxygenation for 18h, and detecting the protective effect of COM 3 on the neurons by using CCK-8 by the same cell stability detection method. Detecting the absorbance of the solution at a wavelength of 450nm, and setting 4 copies in each groupAnd (4) a hole.

(2) The effect of COM 3 on the number of OGD neuron deaths is detected by PI/Hoechst double staining and LDH release amount. The neuron dose is 1.2 multiplied by 10⁶The density of each well was inoculated in a six-well plate, and cultured as described above until the seventh day to perform OGD damage on neurons. The experiment is divided into five groups of Control, OGD + COM 32.5 μ g/ml, OGD + COM 35 μ g/ml and OGD + COM 310 μ g/ml, and each group is provided with 4 compound holes. After each group is correspondingly treated, washing with PBS for 3 times, adding 10 mul of Hoechst dye into each hole, incubating for 20min, washing with PBS for three times, adding 5 mul of PI dye into each hole, incubating for 15min, observing Hoechst and PI fluorescence at excitation wavelengths of 352 and 488nm, and randomly taking three visual fields in each group. The amount of released LDH in the cell supernatant was measured, treated according to standard procedures, and then the absorbance was measured at 450nm, with four duplicate wells per group.

(3) The results show that

The results of CCK-8 measurements for neuronal cell stability showed no statistical difference in cell stability from the Control (CTRL) group at COM 3 concentrations of 1, 2.5, 5, 10 and 20 μ g/ml, suggesting no significant cytotoxicity for primary neurons at COM 3 concentrations less than 20 μ g/ml (fig. 7A). The results of cell survival of IS in vitro neuron injury simulation with OGD showed that the neuron survival after OGD injury was significantly reduced, whereas the neuron cell survival was significantly increased under the COM 3 treatment group concentrations of 2.5, 5, 10 and 20 μ g/ml, wherein the increase in cell survival was most significant at the concentration of 10 μ g/ml (FIG. 7B), suggesting that COM 3 has significant protective effect on OGD neurons. The primary neurons were damaged by OGD under the light mirror and the pattern after COM 3 treatment is shown in FIG. 8. PI/Hoechst staining results suggest that neurons PI after OGD injury are Positive (PI)⁺) The number of cells is obviously increased compared with the Control group, and PI can be obviously reduced compared with the OGD group after the COM 3 is treated⁺The number of positive cells suggests that COM 3 can significantly reduce the number of neuronal deaths after OGD injury, and significantly promote neuronal survival (fig. 9A, B). The LDH release amount in the supernatant of the neuron cell is further measured to verify the results, and the LDH release amount measurement results show that COM can remarkably reduce the LDH release amount of the neuron after OGD, thereby prompting that COM 3 can remarkably reduce the damage of OGD to the neuron (figure)10)。

Fifth, protective effect of compounds on MCAO mice:

(1) an MCAO model IS established to simulate IS injury at the animal level and neuro-behavioral scoring IS performed. Selecting a C57BL/6 male mouse with the weight of 20-22 g, anaesthetizing, fixing in a supine position, making a lateral incision on the neck, separating the common carotid artery, the external carotid artery and the internal carotid artery, blocking the blood flow in the corresponding blood vessel, making an incision on the external carotid artery, sending a thrombus wire, pulling the external carotid artery outwards and downwards to enable the thrombus wire to run into the internal carotid artery, pushing the male mouse for a proper distance, placing the male mouse at the root of the middle cerebral artery to form thrombus, and blocking the blood supply of lateral branches. The control group is not treated, the MCAO group is not administrated after only operation, COM 3(2.5, 5 and 10 mu g/ml) is injected into the abdominal cavity 30min after the administration group is finished, the thrombus line is pulled out after 1h to recover blood flow perfusion, and modeling is finished after 24h of infusion. Neuro-behavioral scoring was performed by Longa penny method, i.e., mice after molding were scored, and the scoring details are shown in table 3.

TABLE 3 Fine rules of neuro-behavioral Scoring

(2)2, 3, 5-triphenyltetrazolium chloride (2, 3, 5-triphenylyte-tetrazolium chloride, TTC) staining was used to detect the effect of COM 3 on the brain infarct volume of IS mice. Each group of mice after modeling is deeply anesthetized and killed, brain tissues are completely taken out on ice, the mice are placed at the temperature of minus 20 ℃ for 30min, the slices with the coronal plane and the coronal plane are fixed in shape, the thickness is 2mm, and 5 slices are continuously cut at the midpoint of the intersection of the frontal pole and the visual plane by a first knife. Immediately using 2% TTC solution to incubate for 30min in the dark at 37 ℃, taking out and then fixing 4% paraformaldehyde overnight, and taking pictures for observation the next day. Cerebral infarct volume was computationally analyzed with Image Pro Plus 6.0 software. Each group guaranteed 6 mice. The method IS characterized in that the influence of COM 3 on the brain tissue damage degree of IS mice IS detected by using the release amount of the brain tissue LDH, the brain tissue homogenate of the corresponding position of each group of mice IS prepared into 10% suspension, and the other detection methods are the same as the step of detecting LDH by using cells.

(3) And (3) detecting the brain edema of the mice after the IS, and researching the influence of COM 3 on the brain edema of the mice after the IS. After the treatment of each group of mice, taking out brain tissue, immediately weighing the wet weight of the brain tissue, then placing the brain tissue into a constant-temperature electric heating box at 70 ℃ for 48h, and then weighing the dry weight of the brain tissue, wherein the brain moisture content is (wet weight-dry weight)/wet weight multiplied by 100%. Brain edema volume after data acquisition using Image Pro Plus 6.0 software, the calculated formula is edema volume ═ contralateral volume/contralateral volume x 100%.

(4) The results show that

MCAO modelling was performed to reduce the course of mouse IS injury. TTC staining results suggested that the volume of infarcted (white) zone of the brain tissue in the MCAO group mice was statistically significantly different from the control group. After the COM 3 was intraperitoneally injected, the cerebral infarction volume was significantly reduced at an intraperitoneal COM 35 and 10mg/kg concentration, suggesting that COM 3 could significantly reduce the cerebral infarction volume of IS mice (fig. 11A, B). The neuro-behavioral scoring results suggest that COM 3 also improved neuro-behavioral performance of the mice while reducing the volume of brain tissue infarct in IS mice, resulting in a recovery of IS hindbrain function (fig. 11C). The detection result of the LDH release amount of IS brain tissue suggests that compared with the MCAO group, COM 3 intraperitoneal injection can significantly reduce the LDH release amount of the brain tissue, namely, the brain tissue damage degree IS reduced, and the protective effect of COM 3 on IS brain tissue IS further verified (fig. 12). The brain edema detection result indicates that the COM 3 reduces the brain edema degree while relieving the brain tissue injury, and the COM 3 has statistical difference on the relieving of the brain edema under the concentration of 10mg/kg, which proves that the COM 3 also has a certain relieving effect on the IS hindbrain edema (fig. 13A and B).

The preferred embodiments of the present disclosure are described in detail with reference to the accompanying drawings, however, the present disclosure is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solution of the present disclosure within the technical idea of the present disclosure, and these simple modifications all belong to the protection scope of the present disclosure.

It should be noted that, in the foregoing embodiments, various features described in the above embodiments may be combined in any suitable manner, and in order to avoid unnecessary repetition, various combinations that are possible in the present disclosure are not described again.

In addition, any combination of various embodiments of the present disclosure may be made, and the same should be considered as the disclosure of the present disclosure, as long as it does not depart from the spirit of the present disclosure.

Claims

1. A coumarin compound, wherein said compound has the structure of formula (i):

2. a method for preparing coumarin compound, wherein the coumarin compound is the compound of claim 1, and the preparation method comprises the following steps: 20mmol of 4-hydroxycoumarin and 10mmol of aromatic aldehyde synthetic reagent

Heating and refluxing in organic solvent, R is 3-OCH₃And separating out white solid particles, and recrystallizing the white solid particles to obtain the target product.

3. The method for preparing coumarin compounds according to claim 2, wherein the organic solvent is ethanol and the recrystallization is carried out in 95% by volume ethanol.

4. The use of the coumarins of claim 1 for the preparation of a medicament for the treatment of stroke.