Musk ketone derivatives, processes for their preparation and use thereof

Abstract

The application belongs to the technical field of medicine, and particularly relates to a muscone derivative, a preparation method and application thereof. The muscone derivative cyclopentadecanedione monoxime has obvious advantages over muscone and other derivatives in improving the MCAO rat mNSS score, microcirculation blood flow, reducing the cerebral infarction rate, the cortical and hippocampal neuron necrosis rate, and the brain water content, and increasing the SOD and CAT contents of the rat brain tissue which are abnormally reduced due to ischemia, and is a potential effective drug for cerebral ischemia-reperfusion injury.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of medicine, and particularly relates to a muscone derivative, a preparation method and application thereof. BACKGROUND

[0002] Muscone is one of the active ingredients extracted from the dry secretion musk of mature male Mosochus Bersxoxskii Fleror or Moshumoschiferus L, and its scientific name is 3-methylcyclopentadecanone. It is the main flavor component of musk, oily liquid, and has a special fragrance. It has the effects of aromatizing, dredging meridians, activating collaterals, and relieving swelling and pain. It is a world-renowned medicinal material. It is clinically used for stroke, angina pectoris, vascular headache, sciatica, and contusion injury.

[0003] Muscone is the core pharmacodynamic component of musk and the only quality control standard of musk. It can rapidly pass through the blood-brain barrier into the brain. Many traditional Chinese medicine compounds using musk as the monarch drug for treating ischemic stroke use muscone as the only index for detecting the brain activity of musk. Due to its special structure (hydrophobic pocket) and material properties (high fat solubility), muscone can pass through the blood-brain barrier into the brain tissue and quickly reach the peak value. Compared with other organs, muscone is accumulated in the brain tissue for a long time, decays slowly, and is more stable. The existing studies show that different doses of muscone have excitatory or inhibitory effects on the central nervous system. Artificially synthesized muscone can affect the function of platelet contractile protein, significantly prolong the coagulation time of rabbits, and has an anti-tumor effect. It can destroy cancer cells in vitro and significantly inhibit the cell respiration of animal tumor tissue. Muscone has a significant protective effect on hypoxia and anoxia and reoxygenation injury of neuroblastoma cells, suggesting that muscone may be used for acute treatment of stroke. However, due to the slow metabolism of muscone in the brain, it has potential toxic and side effects. So far, there are few reports on the related functions of safe and effective muscone derivatives in the treatment of anti-cerebral ischemia-reperfusion injury.

[0004] Modern pharmacology shows that muscone has anti-cerebral ischemia, anti-angina pectoris, and anti-inflammatory effects, and shows a wide range of biological activities. After structural modification, it is expected to obtain a new drug with higher activity. Therefore, it is necessary to study the drug property of muscone as a single clinical drug.

[0005] The present application screens derivatives with high activity and good efficacy through in vitro cell models, comprehensive evaluation of in vivo pharmacodynamics, and mechanism research. Subsequently, pharmacokinetics and safety research will be carried out to complete the preliminary drug property evaluation. SUMMARY

[0006] In order to solve the above problems, the present application provides a muscone derivative, a preparation method and application thereof in preparation of a medicine for preventing or treating cerebral ischemia-reperfusion injury, and indicates that the muscone derivative cyclopentadecanedione monoxime has the effects of improving the mNSS score, microcirculation blood flow of MCAO rats, reducing the cerebral infarction rate, the cortical and hippocampal neuron necrosis rate and the brain water content, and is an effective medicine for preventing or treating cerebral ischemia-reperfusion injury.

[0007] The present application provides one of the following technical solutions:

[0008] A muscone derivative, comprising a compound of the following structural formula or a pharmaceutically acceptable salt thereof:

[0009] Chemical name: cyclopentadecanedione monoxime.

[0010] The present application provides the second of the following technical solutions:

[0011] Application of the muscone derivative as described above in preparation of a medicine for preventing or treating cerebral ischemia-reperfusion injury.

[0012] Preferably, the medicine is a medicine preparation prepared by taking the muscone derivative as an active ingredient.

[0013] The present application provides the third of the following technical solutions:

[0014] A medicine for preventing or treating cerebral ischemia-reperfusion injury, the medicine containing an effective treatment amount of an active ingredient and a pharmaceutically acceptable pharmaceutical adjuvant; the active ingredient is a muscone pharmaceutical derivative, and the muscone pharmaceutical derivative is cyclopentadecanedione monoxime.

[0015] The present application provides the fourth of the following technical solutions:

[0016] The preparation method of the muscone derivative as described above, and the synthesis method comprises the following steps:

[0017] (1) cyclopentadecanone and sodium nitrite are taken, tetrahydrofuran (THF) and water are added, concentrated hydrochloric acid is slowly added dropwise under ice bath, and the reaction is continued under ice bath stirring;

[0018] (2) after the reaction is completed, the mixed solution is poured into a separatory funnel, the organic phase is washed with ice water in several times, and ethyl acetate is extracted in several times;

[0019] (3) the organic phase is taken, anhydrous MgSO4 is added for drying, and the solvent is removed by suction filtration and rotary evaporation to obtain cyclopentadecanedione monoxime crude product; and the cyclopentadecanedione monoxime pure product is eluted.

[0020] Preferably, the specific reaction steps of step (1) are as follows: take 5 mmol of cyclopentadecanone 1.142 g, 5 mmol of sodium nitrite 0.235 g, add 10 ml of THF and 0.5 ml of water, slowly drop 3 ml of concentrated hydrochloric acid under ice bath, continue to stir under ice bath, and carry out the reaction.

[0021] Preferably, the ice bath stirring time is 5-7h.

[0022] Preferably, thin layer chromatography is used to detect the reaction process of step (1).

[0023] Preferably, the amount of ice water used in step (2) is 25 ml, and the organic phase is washed twice; the amount of ethyl acetate used is 10 ml, and the extraction is carried out twice.

[0024] Cyclopentadecanedione monoxime is prepared by silica gel column chromatography separation, and the eluent is petroleum ether: ethyl acetate at 15:1.

[0025] The beneficial effects of the present application include but are not limited to:

[0026] The pharmacodynamic study results of the present application show that the cyclopentadecanedione monoxime of muscone derivative has the effects of obviously improving the mNSS score, microcirculation blood flow of MCAO rats, reducing the infarction rate, cortical and hippocampal neuron necrosis rate, brain water content, and increasing the SOD and CAT contents of the abnormal reduced brain tissue due to ischemia, which indicates that it is a potential drug for treating cerebral ischemia-reperfusion injury, safe and effective, and has a wide application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0027] The drawings described herein are used to provide further understanding of the present application, and form a part of the present application. The schematic embodiments of the present application and their descriptions are used to explain the present application, and do not constitute an improper limitation on the present application. In the drawings:

[0028] Figure 1 It is the hydrogen spectrum of muscone derivative of the present application;

[0029] Figure 2 It is the influence of muscone and its derivatives on the safety of PC12 cells;

[0030] Figure 3 It is the influence of muscone and its derivatives on the activity of oxygen-glucose deprivation PC12 cells;

[0031] Figure 4 It is the influence of muscone and its derivatives on the cerebral infarction rate of MCAO rats;

[0032] Figure 5 It is the influence of muscone and its derivatives on the cerebral neuron necrosis of MCAO rats;

[0033] Figure 6 The effect of muscone and derivatives on the EB exudation of the brain of MCAO rats.

[0034] wherein, Figure 4 The effect of muscone and derivatives on the infarction rate of the brain of MCAO rats;

[0035] Figure 5 The upper row shows the effect of muscone and derivatives on the necrosis rate of hippocampal neurons of MCAO rats, and the lower row shows the effect of muscone and derivatives on the necrosis rate of cortical neurons of MCAO rats; each row from left to right is the effect of the sham operation group, the model group, the muscone group, the No. 4 derivative group, the No. 6 derivative group, and the No. 8 derivative group;

[0036] Figure 6 The effect of muscone and derivatives on the EB exudation of the brain of MCAO rats. DETAILED DESCRIPTION

[0037] To clearly illustrate the technical features of the present scheme, the present application will be described in detail below with specific embodiments, combined with the drawings. The scope of the present application is not limited to the following examples. Those skilled in the art can understand that various changes and modifications can be made to the present application without departing from the spirit and scope of the present application.

[0038] The instruments, reagents, materials, etc. involved in the following examples, if not specifically stated, are conventional instruments, reagents, materials, etc. that already exist in the prior art, which can be obtained through regular commercial channels.

[0039] I. The chemical structures of muscone and its derivatives are as shown in Table 1.

[0040] Table 1

[0041]

[0042]

[0043]

[0044] The synthesis method of No. 4 derivative in Table 1, cyclopentadecanedione monoxime, is as follows:

[0045] (1) Take 5 mmol of cyclopentadecanone 1.142 g and 5 mmol of sodium nitrite 0.235 g, add 10 ml of THF and 0.5 ml of water, slowly drop 3 ml of concentrated hydrochloric acid with a dropping funnel under ice bath, continue to stir for about 6 h under ice bath. The reaction process is detected by thin layer chromatography (TLC).

[0046] (2) After the reaction is completed, pour the mixed solution into a separatory funnel, take 25 ml of ice water, and wash the organic phase twice, and take 10 ml of ethyl acetate (twice) for extraction.

[0047] (3) Take the organic phase, add anhydrous MgSO4 for drying, and extract by suction filtration and rotary evaporation to remove the solvent to obtain the crude cyclopentadecanone monoxime. By silica gel column chromatography separation, with petroleum ether: ethyl acetate (15:1) as the eluent, the pure cyclopentadecanone monoxime in white powder can be obtained.

[0048] The muscone derivative obtained above is used for subsequent cytological screening and pharmacodynamic experiments.

[0049] Two, safety and activity screening of muscone and its derivatives in "one" on PC12 cells

[0050] CCK8 method is used to evaluate the toxicity of different concentrations of muscone and its derivatives on normal nerve cells (PC12), and the safe concentration range is selected. The PC12 cell oxygen glucose deprivation reperfusion model is established, and the model is evaluated by lactate dehydrogenase (LDH) method. The pharmacodynamic activity screening is carried out in the safe concentration range. The influence of the screened derivative on the oxidative stress injury of oxygen glucose deprivation PC12 cells is detected by ELISA: superoxide dismutase (SOD), malondialdehyde (MDA), catalase (CAT), glutathione peroxidase (GSH-Px), reactive oxygen species (ROS).

[0051] (I) Materials and methods

[0052] 1. Cell line

[0053] PC-12 (highly differentiated) cell strain, purchased from Shanghai Qida Biological Technology Co., Ltd.

[0054] 2. Experimental instruments

[0055] High-speed refrigerated centrifuge: model JW-3021HR, Anhui Jiwen Instrument and Equipment Co., Ltd.

[0056] -80℃ refrigerator: model CryoCube F740i, EPPendorf Company, Germany.

[0057] Electronic balance: model TLE204 / 02, Mettler-Toledo Instruments (Shanghai) Co., Ltd.

[0058] Electric heating constant temperature water bath: model HWS-26, Shanghai Bilang Company.

[0059] Inverted phase contrast, inverted fluorescence microscope: model IX73, OlymPus Company, Japan.

[0060] Cell incubator: Model IP610, Thermo, USA.

[0061] Cell counter: Model JSY-SC-021H, BodBoge.

[0062] SynergyHTX multifunctional microplate reader: Model Epoch2, BioTek, USA.

[0063] 3. Experimental reagents and consumables

[0064] RPMI-1640 complete medium: Batch No. 202210, Shanghai Qida Biological Technology Co., Ltd.

[0065] Trypsin digestion solution: Batch No. 2307001, Beijing Solarbio Technology Co., Ltd.

[0066] Dimethyl sulfoxide (DMSO): Batch No. 1121E0328, Beijing Solarbio Technology Co., Ltd.

[0067] Cell freezing solution: Batch No. 202210, Shanghai Qida Biological Technology Co., Ltd.

[0068] RPMI-1640 basal medium (without sugar): Batch No. 202210, Shanghai Qida Biological Technology Co., Ltd.

[0069] Edaravone: Batch No. M2251-01, Abmole, USA.

[0070] ELISA kit: Batch No. 202111, Jiangsu Jingmei Biological Engineering Co., Ltd.

[0071] PBS buffer: Batch No. M101R15147A, Shanghai Yuanye Biological Technology Co., Ltd.

[0072] CCK8 kit: Batch No. CR2207041, Wuhan Saivier Biological Technology Co., Ltd.

[0073] 4. Routine culture of cell lines

[0074] 4.1 Cell recovery

[0075] (1) Preheat the water bath to 37℃, take the cell freezing tube from the-80℃ refrigerator and put it into the water bath for continuous shaking to melt it quickly.

[0076] (2) Disinfect the cell freezing tube with 75% alcohol and transfer it to the clean bench. Use the pipette to suck the cell solution in the freezing tube into the centrifuge tube, balance the centrifuge, and centrifuge at 1000 r / min for 5 min.

[0077] (3) Discard the supernatant, resuspend the cells in 2 ml RPMI-1640 complete medium in the centrifuge tube, inoculate the cell liquid into the T25 cell culture bottle to which 6 ml complete medium has been added in advance, label the cell generation number and the recovery date. After observing the cell state under a microscope, place the cell culture bottle in a 37°C, 5% CO2 incubator. Check whether the cells are adherent on the second day.

[0078] 4.2 Cell subculture

[0079] (1) When the cells grow to a confluence rate of about 90%, discard the culture medium, add 1 ml trypsin digestion solution to digest the cells, and observe the cell morphology under a microscope. When the cell gap increases and the cells gradually become round and begin to change from adherent to suspended state, add complete medium to terminate the digestion.

[0080] (2) Suck the liquid in the culture bottle, and repeatedly and gently blow the adherent cells at the bottom of the culture bottle until all the cells are in a suspended state.

[0081] (3) Suck the suspension in the culture bottle into a centrifuge tube, balance the centrifuge, centrifuge at 1000 r / min for 5 min.

[0082] (4) Discard the supernatant, add complete medium to resuspend the cells, count and subculture into the culture bottle, label the cell name and subculture time, and place in a 37°C, 5% CO2 incubator.

[0083] 4.3 Cell cryopreservation

[0084] (1) Collect the logarithmic growth phase cells in a centrifuge tube, balance the centrifuge, centrifuge at 1000 r / min for 5 min.

[0085] (2) Discard the supernatant, add cryopreservation solution to adjust the cell density to about 2×10 6 / ml, then suck 1.5 ml of cell liquid into a cryopreservation tube, label the cell generation number and the cryopreservation time.

[0086] (3) Place the cell cryopreservation tube in a programmed cooling box and place it in a -80°C refrigerator.

[0087] 5. Preparation of complete medium containing muscone and its derivatives and edaravone

[0088] (1) According to the formula V = M / C·M, calculate the volume of medium required to configure the corresponding concentration according to the molecular mass of each muscone and its derivative.

[0089] (2) Weigh 10 mg of muscone and its derivatives / edaravone using an electronic balance and place it in a 1.5 ml EP tube. After disinfecting the EP tube with 75% alcohol, place it in a laminar flow hood and add 80 μl of DMSO to the EP tube to dissolve the muscone and its derivatives / edaravone and mix thoroughly.

[0090] (3) Add the dissolved muscone and its derivatives / edaravone from the EP tube to a 50ml centrifuge tube, set the final concentration to 500μmol / L according to the formula, and add an appropriate amount of complete culture medium.

[0091] (4) Using a drug concentration of 500 μmol / L, according to the formula V1C1=V2C2, prepare culture media with concentrations of 400 μmol / L, 300 μmol / L, 200 μmol / L, 150 μmol / L, 100 μmol / L, 50 μmol / L and 1 μmol / L in sequence.

[0092] 6. The effect of muscone and its derivatives on the safety of PC12 cells was detected using the CCK-8 assay.

[0093] (1) On the first day, collect PC12 cells in the logarithmic growth phase, resuspend them in complete culture medium, count them, and adjust the cell density to 2 × 10⁻⁶. 4 / ml, seeded at 100μl / well in a 96-well plate, and set up a blank zeroing well, with 3 replicates per group.

[0094] (2) On the second day (24 hours later), remove the culture medium, add 100 μl of complete culture medium to each well, and place the cells in an incubator (5% CO2, 37°C) for culture.

[0095] (3) On the third day, remove the 96-well plate containing cells from the incubator, aspirate the old culture medium, and add 100 μl of pre-prepared complete culture medium containing muscone and its derivatives / edaravone at concentrations of 500, 400, 300, 200, 100, and 1 μmol / L, respectively, setting up 3 replicates. Incubate at 37℃ and 5% CO2 for 24 h.

[0096] (4) On the fourth day, add 10 μl of CCK-8 solution to each well, wrap it with tin foil to protect it from light, disinfect it with 75% alcohol, and then incubate it in a 37°C, 5% CO2 incubator for 2 hours.

[0097] (5) Preheat the microplate reader, measure the OD value at 450 nm, set the temperature to 37℃, and plot the OD values. The growth rate formula is: [(absorbance of experimental wells - absorbance of blank wells) / (absorbance of control wells - absorbance of blank wells)] × 100%.

[0098] 7. Establishment of an oxygen-glucose deprivation model in PC12 cells

[0099] (1) Collect PC12 cells in the logarithmic growth phase, resuspend with basic medium and count, adjust the cell density to 3 x 10 4 / ml, inoculate in 96-well plates at 100 μl / well, set blank zero holes, and set 3 replicate holes for each group.

[0100] (2) Place the cells in a three-gas incubator (94% N2+5% CO2+1% O2, 37°C) for culture.

[0101] (3) After 2h of culture, re-oxygenate and re-sugar the cells, replace the medium with complete medium, and place in a 37°C, 5% CO2 incubator for 24h of culture.

[0102] (4) Evaluate the PC12 cell oxygen-glucose deprivation model by using the LDH method: measure the LDH content in the cell culture medium after 2h of oxygen-glucose deprivation by using the LDH method, and compare with the LDH content in the culture medium of synchronous normal cells.

[0103] 8. Detect the effect of muscone and its derivatives on the activity of oxygen-glucose deprivation PC12 cells by using the CCK-8 method

[0104] (1) Collect PC12 cells in the logarithmic growth phase, resuspend with complete medium and count, adjust the cell density to 3 x 10 4 / ml, inoculate in 96-well plates at 100 μl / well, set blank zero holes, and set 3 replicate holes for each group.

[0105] (2) After 24h, remove the medium, wash twice with PBS, add 100 μl of sugar-free complete medium to each well, and place in a three-gas incubator (94% N2+5% CO2+1% O2, 37°C) for culture.

[0106] (3) After 2h of culture, re-oxygenate and re-sugar the cells, remove the medium, wash twice with PBS, replace 100 μl of complete medium in each well, and place in a 37°C, 5% CO2 incubator for 24h of culture.

[0107] (4) After 24h, remove the old medium from the 96-well plate containing the cells, and add the previously prepared complete medium containing muscone and its derivatives / edaravone to the wells, at concentrations of 200, 150, 100, and 50 μmol / L, respectively, and set 3 replicate holes. 100 μl / well, place in a 37°C, 5% CO2 incubator for 24h of culture.

[0108] (5) On the fourth day, add 10 μl of CCK-8 solution to each well, wrap with tin foil to avoid light, sterilize with 75% alcohol, and place in a 37°C, 5% CO2 incubator for 2h of incubation.

[0109] (6) Enzyme plate preheating, detection of OD value at 450 nm, temperature setting at 37℃, according to OD value to draw a chart. Increase value rate formula: [(experimental hole absorbance-blank hole absorbance) / (control hole absorbance-blank hole absorbance)]*100%.

[0110] 9. Effects of muscone and derivatives on oxidative damage of PC12 cells under oxygen-glucose deprivation

[0111] (1) Collect cells in logarithmic growth phase, resuspend with complete culture medium and count, adjust cell density to 3*10 4 / ml, inoculate in 96-well plates at 100ul / well, set blank zero hole, set 3 duplicate holes in each group.

[0112] (2) After 24h, remove culture medium, wash with PBS twice, add 100ul of sugar-free complete culture medium to each hole, and place the cells in a three-gas incubator (94% N2+5% CO2+1% O2, 37℃) for culture.

[0113] (3) After 2h of culture, the cells are resugared and reoxygenated, the culture medium is removed, washed with PBS twice, 100ul of complete culture medium is added to each hole, and placed in a 37℃, 5% CO2 incubator for culture for 24h.

[0114] (4) After 24h on the third day, remove the old culture medium from the 96-well plate containing the cells in the incubator, and add the previously prepared complete culture medium containing muscone and its derivatives / edaravone in sequence, with a concentration of 100umol / L, set 3 duplicate holes. 100ul / hole, placed in a 37℃, 5% CO2 incubator for culture for 24h.

[0115] (5) On the fourth day, take the cell culture medium, centrifuge at 12000r*5min, take the supernatant, and place it in a-20℃ refrigerator for testing.

[0116] (6) Dilution of standard: dilute the standard and standard diluent in a ratio of 1:1.

[0117] (7) Sample addition:

[0118] Blank hole: no sample, biotin-labeled antibody, streptavidin, only color reagent A&B and stop solution are added, and the rest of the steps are the same.

[0119] Standard hole: add 50ul of standard, 50ul of streptomycin (biotin antibody has been integrated in the standard, so no additional biotin antibody is needed).

[0120] Sample hole: add 40ul of sample, then add 10ul of antibody and 50ul of streptavidin, cover the plate with sealing film, gently shake and mix, incubate at 37℃ for 30min.

[0121] (8) Carefully remove the sealing film, discard the liquid, spin dry, add enough washing solution to each well, discard after standing for 30 seconds, repeat 5 times, and pat dry.

[0122] (9) Add 50 μl of enzyme-labeled reagent to each well, except for the blank wells. Incubate as before. Wash as before.

[0123] (10) Add 50 μl of color developing agent A to each well first, then add 50 μl of color developing agent B, mix gently, and develop color at 37°C for 30 minutes in the dark.

[0124] (11) Take out the enzyme-labeled plate, quickly add 50 μl of stop solution, and measure the results immediately after adding the stop solution.

[0125] (12) After 10 minutes, measure the OD value of each well at 450 nm.

[0126] (13) Calculation: B = standard OD value, B0 = standard 0 point OD value. Draw a standard curve with B / B0% value as the vertical coordinate (Y) and the standard concentration as the horizontal coordinate (X). The index content of the sample is converted to the corresponding concentration according to its OD value from the standard curve.

[0127] 10. Statistical method

[0128] Data were processed by SPSS 26.0 statistical software. After normality test, the data were normally distributed, and the mean ± standard deviation Description. Pairwise comparison between groups was performed by one-way ANOVA, and LSD method was used for variance homogeneity, and Tamhan's T2 method was used for variance heterogeneity. P < 0.05 was considered statistically significant.

[0129] (ii) Results

[0130] 1. Effect of muscone and its derivatives on the safety of PC12 cells

[0131] The effect of muscone and its derivatives on the safety of PC12 cells was observed, and the results are shown in Table 2, Figure 2 .

[0132] Table 2 Effect of muscone and its derivatives on the safety of PC12 cells

[0133]

[0134] From the above Table 2 and Figure 2It can be seen that the cell activity reaches the highest when the drug concentration is 100-200 μmol / L, and the cell activity decreases when the drug concentration is higher than 200 μmol / L. When the drug concentration reaches 500 μmol / L, the cells in all groups except edaravone group die. Therefore, the safe concentration range is selected to be within 200 μmol / L.

[0135] According to the above results, 10 μmol / L, 20 μmol / L, 50 μmol / L, 100 μmol / L, 150 μmol / L and 200 μmol / L are selected for the next step of screening the activity of drugs on oxygen-glucose deprivation PC12 cells.

[0136] 2. PC12 cell oxygen-glucose deprivation model evaluation

[0137] The LDH content of PC12 cell oxygen-glucose deprivation model is evaluated, and the results are shown in Table 3.

[0138] Table 3 LDH content of normal cell group and oxygen-glucose deprivation group

[0139]

[0140] Note: * P < 0.05 compared with the normal group.

[0141] The results in Table 3 show that the LDH release amount of oxygen-glucose deprivation cells is significantly higher than that of normal cells, indicating that the model is successfully replicated.

[0142] 3. Influence of muscone and its derivatives on the activity of oxygen-glucose deprivation PC12 cells

[0143] The influence of muscone and its derivatives on the activity of oxygen-glucose deprivation PC12 cells is observed, and the results are shown in Table 4, Figure 3 .

[0144] Table 4 Influence of muscone and its derivatives on the activity of oxygen-glucose deprivation PC12 cells

[0145]

[0146] The results show that the cell activity is the highest when the drug concentration is 100 μmol / L, and the cell activity decreases as the drug concentration increases. The 50 and 100 μmol / L concentrations are selected for ranking the drug activity, and the No. 4 derivative is the highest, and the No. 8 and No. 6 derivatives have higher drug activity at both concentrations.

[0147] 4. Influence of muscone and its derivatives on the oxidative damage of oxygen-glucose deprivation PC12 cells

[0148] The effects of muscone and its derivatives on oxidative damage of PC12 cells under oxygen-glucose deprivation (OGD) were observed, and the results were shown in Table 5. Compared with the sham operation group, the MDA and ROS of the model group were significantly increased (P<0.05), and the SOD and GSH-Px were significantly decreased (P<0.001). Compared with the model group, the MDA of the muscone group and the derivative groups were significantly decreased (P<0.01), and the SOD and GSH-Px were significantly increased (P<0.001); the MDA of the edaravone group was significantly decreased (P<0.01), and the SOD was significantly increased (P<0.05). Compared with the edaravone group, the SOD and GSH-Px of the muscone group and the derivative groups were significantly increased (P<0.01).

[0149] Table 5 Effects of muscone and its derivatives on oxidative damage of PC12 cells under OGD

[0150]

[0151] Note: * P<0.05 compared with the sham operation group, ** P<0.01 compared with the sham operation group, *** P<0.001 compared with the sham operation group; ▲▲ P<0.01 compared with the model group, ▲▲▲ P<0.001 compared with the model group; ## P<0.01 compared with the edaravone group, ### P<0.001 compared with the edaravone group.

[0152] (III) Conclusion

[0153] The safety screening on normal PC12 cells showed that the safe concentration range of the above muscone derivatives was 0-200 μmol / L. Based on the trend of the effects of the derivatives on PC12 cells under OGD, the concentration of 100 μmol / L was selected for activity screening, and the activity of the No. 4 derivative was the highest. The No. 4, No. 6 and No. 8 derivatives could significantly reduce the abnormal increase of the MDA content of PC12 cells caused by OGD, and the effect was better than that of muscone and edaravone; at the same time, the SOD and GSH-Px contents which were abnormally decreased were significantly increased, and the effect was better than that of edaravone.

[0154] III. Animal pharmacodynamic experiment of muscone and its derivatives

[0155] The rat middle cerebral artery occlusion cerebral ischemia-reperfusion model was established. After 7 days of intervention with muscone and derivatives No. 4, No. 6 and No. 8, the rat neurological behavior was evaluated by mNSS, and the cerebral blood flow was detected by pial microcirculation. TTC staining was used to detect the cerebral infarction rate, paraffin section and HE staining were used to detect the ischemic cortical and hippocampal neuronal necrosis rate. The wet and dry weight method was used to detect the water content of brain tissue specimens, and the tail vein injection of Evans blue was used to detect the passage rate. The brain tissue was taken to detect the oxidative stress indicators: ELISA was used to detect the content of superoxide dismutase (SOD), malondialdehyde (MDA), catalase (CAT), glutathione peroxidase (GSH-Px) and reactive oxygen species (ROS) in brain tissue.

[0156] (I) Materials and methods

[0157] 1. Experimental animals

[0158] 126 SPF level SD rats, male, weight (200±20) g, purchased from Beijing Vito Lihua Experimental Animal Technology Co., Ltd., and the experimental animal quality certificate license number is SCXK (Jing) 2016-0011. The feeding environment is SPF level, natural lighting according to the day-night rhythm, feeding in IVC independent air supply cage, room temperature 22-26℃, relative humidity 40%-70% RH, Co60 irradiation feed and pure water feeding.

[0159] 2. Experimental instruments

[0160] Electric heating constant temperature drying oven: Model GZY-DH, Ningbo Medical Instrument No. 2 Factory.

[0161] Electric heating constant temperature incubator: Model DHP-360, Beijing Yongguangming Medical Instrument Factory.

[0162] Research microscope: American optical instrument company.

[0163] Image Pro Plus image processing system: American Media Cybernetics Company.

[0164] Constant temperature water bath: Model HH series, Jiangsu Jintan Zhongda Instrument Factory.

[0165] Full-automatic high-speed refrigerated centrifuge: Model GL-20A, Hunan Centrifuge Factory.

[0166] IVC independent ventilation cage system: Model BCR-RI01-25-C12 / PSU, Xinhua Medical.

[0167] Multi-channel small animal anesthetizing machine: Model R550IE, Shenzhen Ruivode.

[0168] Multifunctional enzyme label instrument: model SynergyHTX, USA BioTek.

[0169] Laser speckle blood flow monitoring video system: model PSI-ZR, Sweden PERIMED.

[0170] Inverted fluorescence microscope: model DM2500, Germany Leica.

[0171] Table type high speed refrigerated centrifuge: model D3024R, Beijing Dragonlab.

[0172] Decolorization shaker: model TSY-B, Wuhan Servicebio.

[0173] 3. Experimental reagent

[0174] Cord: batch number 2038 / A5, Beijing Xilong Technology Co., Ltd.

[0175] Isoflurane: batch number 20120701, Shenzhen Ruivode Life Science Co., Ltd.

[0176] Artificial musk: batch number 201101, Shandong Medicine Co., Ltd.

[0177] Musk ketone: batch number 2003001, Shandong Hongjitang Pharmaceutical Group Co., Ltd.

[0178] Tween 80: batch number 20200706, Jinan Qiguang Trade Co., Ltd.

[0179] Evans blue: batch number M13D10L103636, Qingdao Haosai Technology Co., Ltd.

[0180] Trichloroacetaldehyde: batch number 2005272, Shenguo Bioengineering (Shanghai) Co., Ltd.

[0181] Paraformaldehyde: batch number 70110900, Shandong Suopu Business and Trade Co., Ltd.

[0182] ELISA kit: batch number 202111, Jiangsu Jingmei Biological Engineering Co., Ltd.

[0183] 4. Model replication

[0184] Rats were anesthetized with isoflurane inhalation, fixed on the operating table in supine position, and a median incision was made on the neck under the anatomical microscope. The subcutaneous fascia, fat and muscle were gently separated. The left common carotid artery, external carotid artery, internal carotid artery and vagus nerve were exposed in the carotid triangle. The above-mentioned arteries were carefully separated to avoid pulling or stimulating the vagus nerve. The distal end of the external carotid artery was ligated, and the proximal end and the common carotid artery were ligated with a slipknot. The internal carotid artery was clamped with a microartery clamp. A "V"-shaped vascular incision was made between the two ligation lines of the external carotid artery with mosquito scissors, and a thread plug was inserted. After fixing the thread plug with the slipknot of the external carotid artery, the artery clamp on the internal carotid artery was loosened. The inserted thread plug was pushed from the external carotid artery to the internal carotid artery using microforceps, and finally turned into the middle cerebral artery. When resistance was felt, the thread plug was stopped. The depth of the thread plug was about 19-20 mm. The thread knot on the external carotid artery was tightened to fix the thread plug, and the excess length of the thread plug was cut off to prevent it from being pulled out after the animal woke up. Then the subcutaneous tissue and skin were sutured layer by layer. After blocking the blood flow for 2 hours, the rats were anesthetized again, and the thread plug was pulled out under the microscope. The slipknot of the common carotid artery was untied to restore the cerebral blood flow, and the incision was sutured.

[0185] Sham operation rat preparation: the depth of the thread plug inserted in the sham operation group was about 5 mm, and the rest of the operation was the same as the preparation of the MCAO model.

[0186] 5. Animal grouping, administration and treatment

[0187] After the animals were awake for 30 minutes after modeling, the behavior score was evaluated, and the animals were grouped according to the mNSS score. The model group, muscone group, No. 4, No. 6 and No. 8 derivative groups were divided into 21 animals each, and the animals were administered the next day. The muscone group and the derivative groups were respectively administered muscone, No. 4, No. 6 and No. 8 derivatives (0.0005 g / kg / d of crude drug amount, diluted with 1% Tween 80) by gavage, 1 time / day, for 7 consecutive days. Another 21 animals were taken as the sham operation group. The model group and the sham operation group were administered with the same amount of normal saline by gavage.

[0188] 6. Animal sacrifice

[0189] After 7 days of administration, the mNSS behavior score was evaluated, 6 animals were first detected for arachnoid microcirculation blood flow, then were euthanized with excessive isoflurane and the brain was taken, the wet and dry weight method was used to detect the water content of the brain tissue specimen, 3 animals were randomly taken from each group for TTC staining, 3 animals were taken for paraffin section after polyoxymethylene perfusion to detect the cerebral infarction rate and neuron necrosis rate, another 3 animals were taken for Evans blue detection of blood-brain barrier permeability, and 6 animals were taken for oxidative damage index detection.

[0190] 7. mNSS score

[0191] The total score of each evaluation was 18 points. The more severe the impairment of neural function, the higher the score. The following table 6:

[0192] Table 6

[0193]

[0194]

[0195] 8. Detection of pia mater microcirculation

[0196] After anesthesia, the rat's scalp was cut in the middle of the skin, the tissue on the skull was scraped off, and a skull window of about 5mm*5mm was opened on both sides of the parietal bone to expose the pia mater. A small amount of glycerol was applied to the skull window, and the laser probe of the laser speckle blood flow analyzer was fixed above the skull window. After 10 minutes of stabilization, the average blood flow of the pia mater under the skull window was recorded for 1 minute. The blood flow value per unit area of the affected side and the healthy side was subtracted to obtain the difference in pia mater blood flow per unit area on both sides.

[0197] 9. TTC staining to detect cerebral infarction volume

[0198] The rat was anesthetized by isoflurane inhalation, decapitated, and cut into 5 coronal sections of 2mm thickness using a sectioning trough. The brain slices were then quickly placed in a 2% TTC solution, protected from light, and incubated at 37°C for 30 minutes, with a turn every 7-8 minutes during the incubation period. The samples were then fixed and stored in a PBS buffer containing 40g / L paraformaldehyde. ImageJ software was used to analyze and calculate the cerebral infarction volume. Cerebral infarction volume = (normal side brain tissue volume - infarction side normal brain tissue volume) / normal side brain tissue volume x 100%.

[0199] 10. HE staining to detect cortical and hippocampal neuron necrosis rate

[0200] The conventional 5μm coronal section was deparaffinized with xylene, washed with gradient alcohol to water, stained with hematoxylin, washed with concentrated ammonia water to return blue, and stained with eosin, and then mounted with neutral resin. Under high-power field of an optical microscope (40x), the rat's ischemic cortex and hippocampal CA1 region were observed, and cells with dense staining, karyopyknosis, and karyorrhexis were counted as positive cells. Five different fields were selected from each section, and the percentage of positive cells in each field was calculated. The average value was taken as the cortical and hippocampal neuron necrosis rate, respectively.

[0201] 11. Brain water content detection

[0202] After the rat's brain was removed, the wet weight of the brain tissue was immediately measured on an electronic balance. Then the brain tissue was baked at 110°C for 24 hours or more until the weight was constant (the difference between two measurements was less than 0.2mg), and the dry weight of the brain tissue was measured. Brain water content (%) = (wet weight - dry weight) / wet weight x 100%.

[0203] 12. Blood-brain barrier permeability

[0204] 1 hour before the animals were sacrificed, 2% Evans blue 4 ml / kg was injected into the tail vein. After 1 hour of circulation, the animals were anesthetized and perfused with 500 ml of normal saline until the right atrium flowed with colorless clear liquid. The brain was removed.

[0205] 12.1 Qualitative detection of Evans blue

[0206] The frozen sections were taken 10 μm behind the optic chiasm. The sections were washed with PBS for 3 times, 5 min each time. Then the sections were mounted with a fluorescence quenching agent containing DAPI staining agent and observed under a fluorescence microscope.

[0207] 12.2 Quantitative detection of Evans blue

[0208] About 3 mm of the left brain tissue behind the optic chiasm was taken, weighed, and placed in 50% trichloroacetic acid (1 ml / 100 mg), homogenized, centrifuged (10000 r / min, 20 min), and the supernatant was diluted with absolute ethanol at a ratio of 1:3. The sample was taken and added to a 96-well plate. The content of Evans blue (EB) was detected using an enzyme label meter at an absorption of 620 nm. The content of EB in the brain tissue was calculated according to the standard curve of EB solution, and was expressed as the content per gram of tissue.

[0209] 13. Detection of the content of oxidative damage indicators in brain tissue by enzyme-linked immunoassay

[0210] 13.1 The ischemic brain tissue was homogenized with ice normal saline under ice bath to prepare 10% brain tissue homogenate. The homogenate was centrifuged at 3000 r / min for 15 min at 4°C, and the supernatant was taken and stored in a refrigerator at -20°C for testing.

[0211] 13.2 The standard was diluted at a ratio of 1:1.

[0212] 13.3 Sample addition:

[0213] 13.3.1 Blank well: The blank control well was not added with sample, biotin-labeled antibody, streptavidin, but was only added with color developing agent A & B and stop solution, and the rest of the steps were the same.

[0214] 13.3.2 Standard well: 50 μl of standard was added, and 50 μl of streptavidin (the biotin antibody was integrated in the standard in advance, so it was not added).

[0215] 13.3.3 Test sample well: 40 μl of sample was added, and then 10 μl of antibody and 50 μl of streptavidin were added. The plate was covered with a sealing film, gently shaken, and incubated at 37°C for 30 min.

[0216] 13.4 The sealing film was carefully removed, and the liquid was discarded. The well was filled with washing liquid, and after 30 seconds, the washing liquid was discarded. The above steps were repeated 5 times, and the well was tapped dry.

[0217] 13.5 Add 50 μl of enzyme-labeled reagent to each well except the blank wells. Incubate as before. Wash as before.

[0218] 13.6 Add 50 μl of color developing reagent A to each well first, then add 50 μl of color developing reagent B, mix gently, and develop color at 37°C for 30 minutes in the dark.

[0219] 13.7 Take out the enzyme-labeled plate, quickly add 50 μl of stop solution, and measure the results immediately after adding the stop solution.

[0220] 13.8 Measure the OD values of each well at 450 nm after 10 minutes.

[0221] 13.9 Calculation: B = standard OD value, B0 = standard 0-point OD value. Draw a standard curve with B / B0% value as the vertical coordinate (Y) and standard concentration as the horizontal coordinate (X). The index content of the sample is converted to the corresponding concentration according to its OD value from the standard curve.

[0222] 14. Statistical method

[0223] Apply SPSS 26.0 statistical software to process data. After normality test, the data meet normal distribution, and the mean ± standard deviation Description. Pairwise comparison between groups uses one-way ANOVA, and LSD method is used for variance equality, and Tamhan's T2 method is used for variance inequality analysis. P < 0.05 is considered to have significant difference.

[0224] (ii) Results

[0225] 1. Effects of muscone and derivatives on brain injury in MCAO rats

[0226] 1.1 mNSS score

[0227] Observe the effect of muscone and derivatives on the mNSS score of MCAO rats. The results are shown in Table 7.

[0228] Table 7 Effects of muscone and derivatives on the mNSS score of MCAO rats

[0229]

[0230] Note: *** P < 0.001 compared with the sham operation group at the same time point; ▲▲▲ P < 0.001 compared with the model group at the same time point.

[0231] After 30 min of modeling, the mNSS scores of rats in each group were significantly higher than those in the sham operation group

[0232] (P<0.001). On the 7th day, compared with the sham operation group, the mNSS scores of rats in each group were significantly increased (P<0.001); compared with the model group, the mNSS scores of rats in the muscone group and the No. 4 derivative group were significantly decreased (P<0.01), and the mNSS scores of rats in the No. 6 and No. 8 derivative groups had a decreasing trend, but there was no significant difference; compared with the muscone group, the mNSS scores of rats in the No. 4 derivative group had a decreasing trend, but there was no significant difference.

[0233] 1.2 Microcirculation blood flow

[0234] The effects of muscone and the No. 4, No. 6 and No. 8 derivatives on the microcirculation blood flow of the brain of MCAO rats were observed, and the results are shown in Table 8.

[0235] Table 8 Effects of muscone and the No. 4, No. 6 and No. 8 derivatives on the microcirculation blood flow of the brain of MCAO rats

[0236]

[0237] Note: *** P<0.001 compared with the sham operation group; ▲▲ P<0.01 compared with the model group.

[0238] Compared with the sham operation group, the difference in the bilateral leptomeningeal microcirculation blood flow of rats in each group was significantly increased (P<0.001); compared with the model group, the difference in the bilateral leptomeningeal microcirculation blood flow of rats in the muscone group and the No. 4 derivative group was significantly decreased (P<0.05), and the bilateral leptomeningeal microcirculation blood flow of rats in the No. 6 and No. 8 derivative groups had a decreasing trend, but there was no significant difference.

[0239] 1.3 Cerebral infarction rate

[0240] The effects of muscone and the No. 4, No. 6 and No. 8 derivatives on the cerebral infarction rate of MCAO rats were observed, and the results are shown in Table 9 and Figure 4 .

[0241] Table 9 Effects of muscone and the No. 4, No. 6 and No. 8 derivatives on the cerebral infarction rate of MCAO rats

[0242]

[0243] Note: ** P<0.01 compared with the sham operation group; ▲ P<0.05 compared with the model group.

[0244] Compared with the sham operation group, the cerebral infarction rate of rats in each group was significantly increased (P<0.05); compared with the model group, the cerebral infarction rate of rats in the muscone group and the No. 4 derivative group was significantly decreased (P<0.05), and the cerebral infarction rate of rats in the No. 6 and No. 8 derivative groups was slightly decreased without significant difference; compared with the muscone group, the cerebral infarction rate of rats in the No. 4 derivative group had a decreasing trend, but there was no significant difference.

[0245] 1.4 Cortical neuron necrosis rate

[0246] The effects of muscone and the No. 4, No. 6 and No. 8 derivatives on the cortical neuron necrosis rate of MCAO rats were observed, and the results are shown in Table 10, Figure 5 .

[0247] Table 10 Effects of muscone and the No. 4, No. 6 and No. 8 derivatives on the cortical neuron necrosis rate of MCAO rats

[0248]

[0249] Note: *** P<0.001 compared with the sham operation group; ▲ P<0.05 compared with the model group.

[0250] Compared with the sham operation group, the cortical neuron necrosis rate of rats in each group was significantly increased (P<0.001); compared with the model group, the cortical neuron necrosis rate of rats in the No. 4 derivative group was significantly decreased (P<0.05), and the cortical neuron necrosis rate of rats in the No. 6 and No. 8 derivative groups had a decreasing trend, but there was no significant difference; compared with the muscone group, the cortical neuron necrosis rate of rats in the No. 4 derivative group had a decreasing trend, but there was no significant difference.

[0251] 1.5 Hippocampal neuron necrosis rate

[0252] The effects of muscone and the No. 4, No. 6 and No. 8 derivatives on the hippocampal neuron necrosis rate of MCAO rats were observed, and the results are shown in Table 11, Figure 5 .

[0253] Table 11 Effects of muscone and the No. 4, No. 6 and No. 8 derivatives on the hippocampal neuron necrosis rate of MCAO rats

[0254]

[0255] Note: *** P<0.001 compared with the sham operation group; ▲▲ P<0.01 compared with the model group, ▲▲▲ P<0.001 compared with the model group.

[0256] Compared with the sham operation group, the hippocampal neuron necrosis rate of each group of rats was significantly increased (P<0.001); compared with the model group, the hippocampal neuron necrosis rate of the muscone group and the No. 4 derivative group was significantly reduced (P<0.01), and there was no significant difference in the hippocampal neuron necrosis rate between the No. 6 and No. 8 derivative groups; compared with the muscone group, the hippocampal neuron necrosis rate of the No. 4 derivative group had a decreasing trend, but there was no significant difference.

[0257] 1.6 Brain water content

[0258] The effects of muscone and the No. 4, No. 6 and No. 8 derivatives on the brain water content of MCAO rats were observed, and the results are shown in Table 12.

[0259] Table 12 Effects of muscone and the No. 4, No. 6 and No. 8 derivatives on the brain water content of MCAO rats

[0260]

[0261] Note: ** P<0.01 compared with the sham operation group; ▲ P<0.05 compared with the model group.

[0262] Compared with the sham operation group, the brain water content of the model group of rats was significantly increased (P<0.01); compared with the model group, the brain water content of the muscone group and the No. 4 derivative group was significantly decreased (P<0.05), and the brain water content of the No. 6 and No. 8 derivative groups was not significantly decreased.

[0263] 1.7 EB content

[0264] The effects of muscone and the No. 4, No. 6 and No. 8 derivatives on the brain tissue EB content of MCAO rats were observed, and the results are shown in Table 13, Figure 6 .

[0265] Table 13 Effects of muscone and the No. 4, No. 6 and No. 8 derivatives on the brain tissue EB content of MCAO rats

[0266]

[0267] Note: *** P<0.001 compared with the sham operation group.

[0268] Compared with the sham operation group, the EB content of each group of rats was significantly increased (P<0.001); compared with the model group, the EB content of the muscone group and the No. 4, No. 6 and No. 8 derivative groups had a decreasing trend, but there was no significant difference.

[0269] 2. Effects of muscone and derivatives on oxidative damage of brain tissue of MCAO / R rats

[0270] The effects of muscone and its derivatives on oxidative damage in MCAO rats were observed, and the results are shown in Table 14.

[0271] Table 14 Effects of muscone and No. 4 derivative on oxidative stress in MCAO rats

[0272]

[0273] Note: * P < 0.05 compared with the sham operation group, ** P < 0.01 compared with the sham operation group, *** P < 0.001 compared with the sham operation group; ▲▲ P < 0.01 compared with the model group, ▲▲▲ P < 0.001 compared with the model group.

[0274] Compared with the sham operation group, the brain tissue MDA and ROS of the model group rats were significantly increased, and the SOD, CAT and GSH-Px were significantly decreased (P < 0.05); the brain tissue CAT and GSH-Px of the muscone group rats were significantly decreased (P < 0.05); the brain tissue MDA and ROS of the No. 4 derivative group rats were significantly increased, and the GSH-Px was significantly decreased (P < 0.05); the brain tissue MDA and ROS of the No. 6 derivative group rats were significantly increased, and the SOD, CAT and GSH-Px were significantly decreased (P < 0.05). X X X X Compared with the model group, the brain tissue SOD and CAT of the muscone group and the No. 4 derivative group rats were significantly increased (P < 0.01). Compared with the muscone group, the CAT of the No. 4 derivative group had a trend of increase, but there was no significant difference.

[0275] (III) Conclusion

[0276] Muscone No. 4 derivative cyclopentadecanedione monoxime can significantly improve the mNSS score, microcirculation blood flow of MCAO rats, reduce the infarction rate of brain tissue, the rate of cortical and hippocampal neuron necrosis, and the brain water content, and significantly increase the SOD and CAT contents in the brain tissue of rats due to ischemia. The improvement of the derivative on the mNSS score of MCAO rats, the infarction rate of brain tissue, the rate of cortical and hippocampal neuron necrosis, and the CAT content of brain tissue is better than that of muscone and other derivatives.

[0277] ​​​The above merely illustrates the embodiments of the present application, and the protection scope of the present application is not limited to these specific embodiments, but determined by the claims of the present application. The present application can have various changes and modifications for those skilled in the art. Any modification, equivalent replacement, improvement, etc. within the technical thought and principle of the present application shall be included in the protection scope of the present application.

Claims

1. Use of muscone derivatives in the preparation of a medicament for preventing or treating cerebral ischemia-reperfusion injury, the muscone derivative being a compound of the following structural formula or a pharmaceutically acceptable salt thereof: ###0001### the medicament containing an effective therapeutic amount of the active ingredient and pharmaceutically acceptable pharmaceutical adjuvants; the active ingredient being the muscone derivative of the structural formula in claim 1. 。 2. A medicament for preventing or treating cerebral ischemia-reperfusion injury, characterized by, 2. The muscone derivative of claim 1, wherein the muscone derivative is a compound of the following structural formula: ###0002### 3. The muscone derivative of claim 1, wherein the muscone derivative is a compound of the following structural formula: ###0003### 4. The muscone derivative of claim 1, wherein the muscone derivative is a compound of the following structural formula: ###0004### 5. The muscone derivative of claim 1, wherein the muscone derivative is a compound of the following structural formula: ###0005### 6. The muscone derivative of claim 1, wherein the muscone derivative is

Images