Application of chalcone compounds in preparation of anti-ischemic stroke drugs

Abstract

The invention discloses an application of chalcone compounds in preparation of drugs for resisting cerebral arterial thrombosis. The chalcone compound disclosed by the invention can effectively improve primary cortical neuron injury induced by oxygen-glucose deprivation / reoxygenation, and can effectively improve brain injury of cerebral arterial thrombosis model animals, so that the effectiveness and applicability of the compound in treating cerebral arterial thrombosis are indicated, and the chalcone compound has a good application prospect.

Description

Technical Field

[0001] The invention belongs to the field of medicine, and particularly relates to a class of chalcone compounds and application thereof in preparing anti-ischemic stroke drugs. Background Art

[0002] Stroke is a common cerebrovascular disease worldwide. Its notable characteristics are high morbidity, disability and mortality. It can cause severe motor, sensory and language dysfunction, seriously affecting the patient's quality of life, and bringing huge economic burdens to the patient's family and society. Among them, ischemic stroke accounts for about 80% of the total number of stroke cases and is the most common type of stroke. At present, the clinical treatment of ischemic stroke is mainly intravenous thrombolysis and mechanical thrombectomy, supplemented by neurotrophic drugs, circulation-improving drugs and antiplatelet drugs. There is still a lack of effective therapeutic drugs for ischemic stroke. Therefore, the research and development of new anti-ischemic stroke drugs has obvious urgency and important clinical value.

[0003] Breadfruit tree [Artocarpus incisa (Thunb.) L] is a plant of the genus jackfruit of the Moraceae family, also known as jackfruit tree, pinang, etc. It is native to the Pacific Islands, India, and the Philippines. It is one of the famous tropical trees in the Malay Archipelago. It is also distributed in tropical areas of the Americas (such as Peru, Ecuador, etc.) and some provinces and cities in China (such as Hainan, Taiwan, etc.). Its leaves are often used for medicinal purposes. In some countries in South America, it is used as a traditional medicinal material to treat diseases such as diarrhea, abscesses, malaria, and tapeworm infections; its wood is light, soft, and coarse, and can be used as building materials; its fruit is rich in starch and is one of the common staple foods in tropical areas. Modern pharmacological studies have shown that breadfruit tree extracts have a wide range of pharmacological activities, such as antibacterial, antiviral, and anti-inflammatory. The present invention has discovered a series of chalcone compounds from breadfruit tree, and for the first time discovered that these chalcone compounds have potential anti-ischemic stroke effects. Summary of the invention

[0004] The primary purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art and provide a class of chalcone compounds for use in the preparation of anti-ischemic stroke drugs.

[0005] In order to achieve the above object, the purpose of the present invention is achieved through the following scheme: A chalcone compound is used in the preparation of an anti-ischemic stroke drug, wherein the compound has a structural formula shown in general formula I: ; or its tautomer, racemate, enantiomer or mixture thereof, or its pharmaceutically acceptable salt, wherein: R1, R4 and R6 are each independently selected from a hydrogen atom or a substituted or unsubstituted C10 unit, and an oxygen atom is present at the ortho position of the C10 unit, and the C10 unit is independently selected from: ; R2 is independently selected from a hydrogen atom, a hydroxyl group, or an oxygen atom connected to an ortho-C10 unit on the benzene ring to form a six-membered ring; R3 and R5 are each independently selected from a hydrogen atom or a carbon atom on the adjacent C10 unit on the benzene ring where it is located to form a six-membered ring; The α carbon and the β carbon are independently connected by a carbon-carbon single bond or a carbon-carbon double bond.

[0006] In a preferred experimental scheme of the present invention, the compound represented by the general formula I, wherein R3, R4, R5 and R6 are hydrogen atoms, the α carbon and the β carbon are connected in the form of a carbon-carbon single bond, has the structural formula represented by the general formula II: ; or its tautomers, racemates, enantiomers or mixtures thereof, or its pharmaceutically acceptable salts, wherein: R1, R2 and C10 units are as defined above.

[0007] In a preferred experimental scheme of the present invention, the compound represented by the general formula II includes, but is not limited to: ; or its tautomer, racemate, enantiomer or mixture form, or its pharmaceutically acceptable salt.

[0008] In a preferred experimental scheme of the present invention, the compound represented by the general formula I, wherein R1, R3 and R6 are hydrogen atoms, has the structural formula represented by the general formula III: ; or its tautomer, racemate, enantiomer or mixture thereof, or its pharmaceutically acceptable salt, wherein: R2, R4, R5, the connection mode between the α carbon and the β carbon and the C10 unit are as defined above.

[0009] In a preferred experimental scheme of the present invention, the compound represented by the general formula III includes, but is not limited to: ; or its tautomer, racemate, enantiomer or mixture form, or its pharmaceutically acceptable salt.

[0010] In a preferred experimental scheme of the present invention, the compound represented by the general formula I, wherein R1, R2, R3 and R4 are hydrogen atoms, has the structural formula represented by the general formula IV: ; or its tautomer, racemate, enantiomer or mixture form, or its pharmaceutically acceptable salt, wherein: R5, R6, the connection mode between α carbon and β carbon and C10 unit are as defined above.

[0011] In a preferred experimental scheme of the present invention, the compound represented by the general formula IV includes, but is not limited to: ; or its tautomer, racemate, enantiomer or mixture form, or its pharmaceutically acceptable salt.

[0012] The present invention relates to chalcone compounds represented by general formulas I, II, III and IV. The extraction and separation method thereof comprises the following steps: crushing breadfruit tree branches and leaves, cold-immersing and extracting with 95% (w / w) ethanol, concentrating the extract and then extracting with ethyl acetate, and separating and purifying the ethyl acetate extraction part by silica gel column chromatography, Sephadex LH-20, octadecyl bonded phase silica gel (ODS) column chromatography and preparative high performance liquid chromatography (PHPLC) to obtain the above-mentioned chalcone compounds 1-12.

[0013] The present invention relates to a pharmaceutical composition, which contains a therapeutically effective dose of a chalcone compound represented by general formula I, II, III and IV, or its tautomer, racemate, enantiomer, or mixture thereof, or its pharmaceutically acceptable salt, and one or more pharmaceutically acceptable carriers, diluents or excipients.

[0014] The present invention relates to the use of a chalcone compound represented by general formula I, II, III and IV, or its tautomer, racemate, enantiomer, or mixture thereof, or its pharmaceutically acceptable salt, or its pharmaceutical composition in the preparation of an anti-ischemic stroke drug containing a therapeutically effective dose.

[0015] A "pharmaceutical composition" means a mixture containing one or more compounds described herein or their physiologically / pharmaceutically acceptable salts or prodrugs together with other chemical components, as well as other components such as physiologically / pharmaceutically acceptable carriers and excipients.

[0016] "Pharmaceutically acceptable salts" refer to salts of the compounds of the present invention, which are safe and effective when used in mammals and have the desired biological activity.

[0017] The ischemic stroke models involved in the present invention include cell and animal models. The cell model is a neuronal oxygen glucose deprivation / reoxygenation (OGD / R) model, which is a classic cell model for studying ischemic stroke; the middle cerebral artery ischemia-reperfusion model and the middle cerebral artery distal thrombosis model. These two animal models can more comprehensively simulate different types of ischemic stroke patients in clinical practice.

[0018] The present invention has the following advantages and effects over the prior art: (1) The chalcone compounds 1 to 12 of the present invention have low cytotoxicity to primary neuronal cells.

[0019] (2) The present invention found that a class of chalcone compounds 1-12 derived from the breadfruit tree can protect mouse primary neuronal cells from oxygen-glucose deprivation / reoxygenation damage at relatively low concentrations, showing good neuroprotective activity and can be applied to the development of anti-ischemic stroke drugs.

[0020] (3) The present invention found that chalcone compound 3 derived from breadfruit tree can exert effective therapeutic effects on two ischemic stroke animal models and has a wide range of clinical applications.

[0021] (4) The present invention found that the chalcone compound 3 derived from the breadfruit tree can significantly improve the brain damage of ischemic stroke animals by administering the compound after modeling, which has important clinical significance. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The toxicity of chalcone compounds 1-12 on primary cortical neurons (data are expressed as mean±SEM, compared with the normal control group (Ctr), *P<0.05).

[0023] Figure 2 The results showed that chalcone compounds 1-12 could protect neurons against oxygen glucose deprivation / reoxygenation injury (data were expressed as mean ± SEM, ##P <0.01 compared with the normal control group (Ctr); *P <0.05, **P <0.01 compared with the OGD / R group).

[0024] Figure 3 It indicates that chalcone compound 3 improves brain damage in mice with middle cerebral artery ischemia-reperfusion model (data are expressed as mean ± SEM, compared with the Sham group, ###P <0.0001; compared with the MCAO group, *P<0.05); wherein: A is the TTC staining image (the cerebral infarction area is white); B is the statistical analysis of cerebral infarction volume; C is the statistical analysis of mNSS neurological function score.

[0025] Figure 4It indicates that chalcone compound 3 improves brain damage in mice with distal middle cerebral artery thrombosis model (data are expressed as mean ± SEM, compared with the Sham group, ###P<0.0001; compared with the ferric chloride (FeCl3) model group, *P<0.05, **P<0.01); wherein: A is magnetic resonance imaging (MRI) image; B is statistical analysis of cerebral infarction volume; C is statistical analysis of the proportion of cerebral infarction in the left cerebral cortex. Implementation

[0026] The structures of the compounds were determined by nuclear magnetic resonance (NMR) and / or mass spectrometry (MS). NMR shifts (δ) are given in 10-6 (ppm) units. NMR data were measured using Bruker AVANCE-400, Bruker AVANCE-500 and Bruker AVANCE-600 nuclear magnetic resonance spectrometers, with deuterated acetone (CD3COCD3), deuterated methanol (CD3OD) or deuterated chloroform (CDCl3) as the measuring solvent, and tetramethylsilane (TMS) as the internal standard; HR-ESI-MS data were measured using an Agilent 6210 LC / MSD TOF-MS mass spectrometer; analytical and preparative HPLC were measured using an Agilent 1260 high performance liquid chromatograph with an MWD detector. The GF254 thin layer silica gel prefabricated plate for TLC analysis was purchased from Yantai Institute of Chemical Industry; column chromatography silica gel (200–300 mesh) was provided by Qingdao Ocean Chemical Co., Ltd.; Sephadex LH-20 gel chromatography filler was produced by Amershanm Biosciences; and ODS column chromatography filler was produced by Merck.

[0027] The present invention is further described in detail below in conjunction with embodiments and drawings, but the embodiments of the present invention are not limited thereto.

[0028] Example 1 General extraction, separation and preparation method of chalcone compounds 1 to 12 (1) 10 kg of dried branches and leaves of breadfruit [Artocarpus incisa (Thunb.) L] were crushed into coarse powder, extracted with 95% (v / v) ethanol, and the extract was concentrated to dryness to obtain 1 kg of total extract. The total extract was suspended in water and extracted three times with an equal volume of ethyl acetate. The extracts were combined and the solvent was recovered under reduced pressure to obtain a dry ethyl acetate extract (421 g).

[0029] (2) The ethyl acetate extraction fraction obtained in (1) was subjected to silica gel column chromatography, and gradient elution was performed using a petroleum ether-ethyl acetate mixture as an eluent, and the elution was performed according to an elution gradient of petroleum ether and ethyl acetate volume ratios of 100:0, 100:1, 100:3, 20:1, 10:1, 5:1, 10:3, 2:1, 1:1 and 0:100. After thin layer chromatography (TLC) analysis, similar fractions were combined to obtain 8 main fractions (Fr.A-Fr.H). Fr.E (48 g) and Fr.G (95 g) were further subjected to silica gel column chromatography with a dichloromethane-methanol mixed solution as the eluent for gradient elution. The volume ratios were 100:0, 20:1, 10:1, 5:1, 2:1, and 1:1, respectively. Similar fractions were combined and analyzed by thin layer chromatography (TLC) to obtain 5 (Fr.EA–Fr.EE) and 6 (Fr.GA–Fr.GF) subfractions, respectively.

[0030] (3) The fraction Fr.EB (2.4 g) obtained in step (2) was concentrated and loaded onto a Sephadex LH-20 gel column. The column was eluted with a mobile phase of a 1:1 volume ratio of dichloromethane and methanol at a flow rate of 0.5 mL / min. The eluate was collected and analyzed by TLC. Similar fractions were combined to obtain a total of four sub-fractions, namely, Fr.EBA–Fr.EBD.

[0031] (4) The fraction Fr.GA (10 g) obtained in step (2) was concentrated and subjected to reverse ODS column chromatography using methanol-water as the eluent and eluted at a gradient of methanol:water volume ratios of 70:30, 80:20, 90:10, and 100:0. After analytical HPLC analysis, similar fractions were combined to obtain a total of 4 secondary fractions, namely Fr.GAA-Fr.GAD.

[0032] (5) The fraction Fr.GD (19.5 g) obtained in step (2) was concentrated and loaded onto a Sephadex LH-20 gel column using pure methanol as the eluent. Similar fractions were combined after TLC analysis to obtain a total of 6 fractions, namely Fr.GDA–Fr.GDF.

[0033] (6) The fraction Fr.EBB obtained in step (3) was concentrated and dissolved in methanol, and then separated and purified by reverse preparative HPLC using methanol-water with a volume ratio of 80:20 as the mobile phase and a flow rate of 5 mL / min to obtain compounds 1, 2, 4, 5 and 8.

[0034] (7) The fraction Fr.GAB obtained in step (4) was concentrated and dissolved in methanol, and then separated and purified by reverse preparative HPLC using methanol-water with a volume ratio of 85:15 as the mobile phase and a flow rate of 5 mL / min to obtain compounds 3 and 9~12.

[0035] (8) The fraction Fr.GDE obtained in step (5) was concentrated and dissolved in methanol, and then separated and purified by reverse preparative HPLC using acetonitrile-water in a volume ratio of 70:30 as the mobile phase at a flow rate of 5 mL / min to obtain compounds 6 and 7, respectively. Example 2 Structural Identification of Chalcone Compounds 1 to 12 The isolated chalcone compounds 1 to 12 were tested by nuclear magnetic resonance and high-resolution mass spectrometry, and the microspectral database search revealed that these compounds were all known compounds. Their physical and chemical data are as follows: Compound 1 (AC-5-1): 1H NMR (400 MHz, CD3COCD3) δ 12.85 (s, OH), 9.68 (s,OH), 8.41 (s, OH), 7.75 (d, J = 8.8 Hz, 1H), 7.03 (s, OH), 6.66 (d, J = 8.1Hz, 1H), 6.58 (d, J = 8.1 Hz, 1H), 6.41 (dd, J = 8.9, 2.4 Hz, 1H), 6.33 (d, J= 2.4 Hz, 1H), 5.18 (t, J = 6.5 Hz, 1H), 5.05 (d, J = 7.0 Hz, 1H), 3.44 (d, J= 6.7 Hz, 2H), 3.16 (m, 2H), 2.91 (m, 2H), 2.02 (m, 2H), 1.97 (m, 2H), 1.74(s, 3H), 1.59 (s, 3H), 1.53 (s, 3H); 13C NMR (100 MHz, CD3COCD3) δ 205.1,166.1, 165.4, 144.1, 143.4, 134.9, 133.4, 132.0, 131.5, 127.2, 125.0, 124.4,120.5, 113.7, 113.3, 108.7, 103.5, 40.3, 40.1, 27.9, 27.3, 25.7, 25.7, 17.6,16.3; HR-ESI-MS m / z 433.1985 [M + Na]+.

[0036] Compound 2 (sakenin C): 1H NMR (400 MHz, CD3COCD3) δ 12.84 (s, OH), 9.52(s, OH), 8.44 (s, OH), 7.84 (d, J = 8.9 Hz, 1H), 7.44 (s, OH), 6.66 (d, J =8.1 Hz, 1H), 6.58 (d, J = 8.1 Hz, 1H), 6.42 (dd, J = 8.8, 2.4 Hz, 1H), 6.33(d, J = 2.4 Hz, 1H), 5.22 (s, OH), 5.17 (s, 1H), 5.14 (m, 1H), 4.87 (s, 1H),4.44 (d, J = 9.2 Hz, 1H), 3.23 (m, 2H), 2.98 (m, 2H), 2.89 (m, 2H), 2.23 (m,2H), 2.18 (m, 2H),1.65 (s, 3H), 1.59 (s, 3H); 13C NMR (100 MHz, CD3COCD3) δ205.2, 166.4, 165.6, 153.2, 145.3, 144.6, 133.8, 132.1, 131.9, 126.4, 125.1,121.7, 114.0, 113.8, 109.4, 108.8, 103.6, 77.4, 40.4, 34.7, 32.6, 28.4, 27.5,25.9, 17.8; HR-ESI-MS m / z 425.1973 [M − H]−。

[0037] Compound 3 (3-[3,4-dihydroxy-2-[(2E)-7-methoxy-3,7-dimethyl-2-octen-1-yl]phenyl]-1-(2,4-dihydroxyphenyl)-1-propanone): 1H NMR (400 MHz, CD3COCD3) δ 12.85 (s, OH), 7.78 (d, J = 8.9 Hz, 1H), 6.65 (d, J = 8.1 Hz, 1H), 6.58 (d, J = 8.1 Hz, 1H), 6.43 (dd, J = 8.8, 2.4 Hz, 1H), 6.33 (d, J = 2.4 Hz, 1H), 5.18 (t, J = 1.3 Hz, 1H), 3.45 (d, J = 6.5 Hz, 2H), 3.17 (m, 2H), 3.05 (s, 3H), 2.92 (m, 2H), 1.94 (m, 2H), 1.74 (s, 3H), 1.38 (m, 2H), 1.37 (m, 2H), 1.04 (s, 6H); 13C NMR (100 MHz, CD3COCD3) δ 205.2, 166.2, 165.4, 144.2, 143.5, 135.1, 133.5, 132.1, 127.4, 124.5, 120.6, 113.8, 113.4, 108.7, 103.6, 74.6, 49.0, 40.8, 40.1, 40.0, 28.0, 25.8, 25.3, 22.7, 16.3; HR-ESI-MS m / z 443.4515 [M + H]+。

[0038] Compound 4 (3-[3,4-dihydro-8-hydroxy-2-methyl-2-(4-methyl-3-penten-1-yl)-2H-1-benzopyran-5-yl]-1-(2,4-dihydroxyphenyl)-1-propanone): 1H NMR (300 MHz, CDCl3) δ 12.75 (s, OH), 7.60 (d, J = 8.6 Hz, 1H), 6.71 (d, J = 8.1 Hz,1H), 6.61 (d, J = 8.1 Hz, 1H), 6.33 (m, 2H), 5.09 (m, 1H), 3.12 (m, 2H), 2.89(m, 2H), 2.68 (m, 2H), 2,08 (m, 2H), 1.84 (m, 3H), 1.65 (s, 3H), 1.63 (m,1H), 1.58 (s, 3H), 1.29 (s, 3H); 13C NMR (75 MHz, CDCl3) δ204.3, 165.5,162.9, 144.0, 141.1, 132.5, 132.0, 130.3, 124.3, 119.8, 119.6, 114.0, 111.9,108.0, 103.8, 76.4, 39.5, 38.9, 31.4, 26.8, 25.9, 24.1, 22.6, 19.7, 17.8; HR-ESI-MS m / z 433.1986 [M + Na]+。

[0039] Compound 5 (1-(2,4-dihydroxyphenyl)-3-[8-hydroxy-2-methyl-2-(4-methyl-3-penten-1-yl)-2H-1-benzopyran-5-yl]-1-propanone): 1H NMR (300 MHz, CDCl3) δ 12.81 (s, OH), 7.56 (d, J = 8.6 Hz, 1H), 6.73 (d, J = 8.2 Hz, 1H), 6.61 (d, J = 8.2 Hz, 1H), 6.55 (d, J = 10.2 Hz, 1H), 6.35 (m, 2H), 5.64 (d, J = 10.2 Hz, 1H), 5.09 (m, 1H), 3.10 (m, 2H), 2.98 (m, 2H), 2.09 (m, 2H), 1.73 (m, 2H), 1.67 (s, 3H), 1.57 (s, 3H), 1.39 (s, 3H); 13C NMR (75 MHz, CDCl3) δ 203.9, 165.4, 163.1, 143.2, 139.8, 132.5, 132.2, 130.3, 128.1, 124.0, 121.4, 119.6, 119.2, 114.7, 113.9, 108.1, 103.7, 79.0, 41.0, 39.9, 26.8, 26.3, 25.9, 23.0, 17.8; HR-ESI-MS m / z 409.2011 [M + H]+。

[0040] Compound 6 (xanthoangelol): 1H NMR (500 MHz, CDCl3) δ 13.87 (s, OH), 7.82 (d, J = 15.4 Hz, 1H), 7.72 (d, J = 8.9 Hz, 1H), 7.53 (d, J = 8.2 Hz, 2H), 7.45 (d, J = 15.4 Hz, 1H), 6.87 (d, J = 8.1 Hz, 2H), 6.43 (d, J = 8.8 Hz, 1H), 5.30 (m, 1H), 5.06 (m, 1H), 3.49 (d, J = 7.1 Hz, 2H), 2.10 (m, 4H), 1.83 (s, 3H), 1.67 (s, 3H), 1.59 (s, 3H); 13C NMR (125 MHz, CDCl3) δ 192.5, 164.0, 162.0, 158.4, 144.4, 139.8, 132.2, 130.7(◊2), 129.5, 127.7, 123.9, 121.2, 118.1, 116.2(◊2), 114.3, 114.1, 108.1, 39.8, 26.5, 25.8, 21.9, 17.8, 16.4; HR-ESI-MS m / z 393.2049 [M + H]+。

[0041] Compound 7 (3-(3,4-dihydroxyphenyl)-1-[3-[(2E)-3,7-dimethyl-2,6-octadien-1-yl]-2,4-dihydroxyphenyl]-1-propanone): 1H NMR (400 MHz, CD3OD) δ 7.56 (d, J = 8.8 Hz, 1H), 6.67 (m, 2H), 6.55 (dd, J = 8.0, 2.0 Hz, 1H), 6.36 (d, J = 8.8 Hz, 1H), 5.20 (m, 1H), 5.04 (m, 1H), 3.29 (d, J = 5.7 Hz, 2H), 3.15 (t, J = 7.7 Hz, 2H), 2.85 (t, J = 7.7 Hz, 2H), 2.04 (m, 2H), 1.94 (m, 2H), 1.76 (s, 3H), 1.71 (m, 1H), 1.59 (s, 3H), 1.54 (s, 3H), 1.46 (m, 1H); 13C NMR (100 MHz, CD3OD) δ 205.8, 164.1, 163.5, 146.2, 144.5, 135.5, 134.1, 132.3, 130.7, 125.4, 123.7, 120.6, 116.5, 116.5, 116.4, 113.8, 108.2, 40.9, 40.9, 31.5, 27.7, 25.8, 22.4, 17.7, 16.2; HR-ESI-MS m / z 411.1043 [M + H]+。

[0042] Compound 8 (3-geranyl-2,3′,4,4′-tetrahydroxychalcone): 1H NMR (400 MHz, CD3OD) δ 7.81 (d, J = 8.9 Hz, 1H), 7.72 (d, J = 15.3 Hz, 1H), 7.55 (d, J = 15.3 Hz, 1H), 7.18 (d, J = 2.1 Hz, 1H), 7.11 (dd, J = 8.2, 2.1 Hz, 1H), 6.82 (d, J = 8.2 Hz, 1H), 6.44 (d, J = 8.9 Hz, 1H), 5.23 (m, 1H), 5.05 (m, 1H), 3.33 (d, J = 6.3 Hz, 2H), 2.05 (m, 2H), 1.96 (m, 2H), 1.78 (s, 3H), 1.60 (s, 3H), 1.55 (s, 3H); 13C NMR (100 MHz, CD3OD) δ 193.7, 165.2, 163.6, 149.8, 146.9, 145.7, 135.5, 132.0, 130.3, 128.5, 125.5, 123.7, 123.5, 118.5, 116.6, 116.6, 115.8, 114.6, 108.2, 40.9, 27.7, 25.8, 22.4, 17.7, 16.3; HR-ESI-MS m / z 407.1863 [M − H]−。

[0043] Compound 9 (lespeol): 1H NMR (400 MHz, CDCl3) δ 7.82 (d, J = 15.3 Hz,1H), 7.71 (d, J = 8.9 Hz, 1H), 7.54 (d, J = 8.1 Hz, 2H), 7.43 (d, J = 15.4Hz, 1H), 6.88 (d, J = 8.1 Hz, 2H), 6.79 (d, J = 10.1 Hz, 1H), 6.37 (d, J =8.8 Hz, 1H), 5.54 (d, J = 10.2 Hz, 1H), 5.09 (m, 1H), 2.10 (m, 2H), 1.78 (m,1H), 1.69 (m, 1H), 1.67 (s, 3H), 1.57 (s, 3H), 1.43 (s, 3H); 13C NMR (100MHz, CDCl3) δ 192.2, 161.0, 160.3, 158.4, 144.4, 132.1, 130.8, 130.8(◊2),127.6, 127.1, 123.9, 117.8, 116.5, 116.18(◊2), 114.1, 109.3, 108.3, 80.5,41.8, 27.3, 25.8, 22.8, 17.8; HR-ESI-MS m / z 389.1755 [M − H]−。

[0044] Compound 10 (1-[5-(3,7-dimethyl-2,6-octadienyl)-2,4-dihydroxyphenyl]-3-(4-hydroxyphenyl)-1-propanone): 1H NMR (600 MHz, CDCl3) δ 12.79 (s, OH), 7.45(s, 1H), 7.08 (d, J = 5.6 Hz, 2H), 6.78 (d, J = 5.6 Hz, 2H), 6.39 (s, 1H),5.30 (t, J = 7.2 Hz, 1H), 5.09 (t, J = 7.0 Hz, 1H), 3.28 (d, J = 7.2 Hz, 2H),3.17 (t, J = 8.5, 6.9 Hz, 2H), 2.97 (t, J = 8.5, 6.9 Hz, 2H), 2.11 (m, 2H),2.08 (m, 2H), 1.74 (s, 3H), 1.68 (s, 3H), 1.59 (s, 3H); 13C NMR (150 MHz,CDCl3) δ 204.4, 163.4, 162.1, 154.1, 138.6, 132.9, 132.0, 131.5, 129.6(◊2),123.9, 121.5, 119.9, 115.6(◊2), 113.4, 103.7, 40.1, 39.8, 30.0, 28.5, 26.7,25.8, 17.8, 16.3; HR-ESI-MS m / z 395.2213 [M + H]+。

[0045] <h2 style=";text-align:left;direction:ltr">11-Methylphenidate (isoxanthoangelol) : 7.75 (d, J = 15.4 Hz, 1H), 7.70 (s, 1H), 7.56 (d, J = 8.7 Hz, 2H), 7.50 (d, J = 15.3 Hz, 1H), 6.82 (d, J = 8.7 Hz, 2H), 6.30 (s, 1H), 5.36 (m, 1H), 5.15 (m, 1H), 3.26 (d, J = 7.4 Hz, 2H), 2.17 (m, 2H), 2.07 (m, 2H), 1.73 (s, 3H), 1.62 (s, 3H), 1.56 (s, 3H); 13C NMR (125MHz, CD3OD) δ193.4, 165.8, 164.4, 161.5, 145.4, 137.6, 132.4, 131.8(◊2),131.7, 127.8, 125.3, 123.7, 121.8, 118.4, 116.9(◊2), 114.3, 103.3, 41.0,28.4, 28.3, 25.9, 17.8, 16.3; HR-ESI-MS m / z 393.2064 [M+H]+。<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0046] Compound 12 (isolespeol): 1H NMR (400 MHz, CDCl3) δ 7.84 (d, J = 15.4 Hz, 1H), 7.58 (d, J = 8.6 Hz, 2H), 7.48 (s, 1H), 7.43 (d, J = 15.4 Hz, 1H), 6.89(d, J = 8.6 Hz, 2H), 6.37 (s, 1H), 6.37 (d, J = 9.4 Hz, 1H), 5.53 (d, J =10.1 Hz, 1H), 5.08 (t, J = 7.2 Hz, 1H), 2.07 (m, 2H), 1.77 (m, 1H), 1.62 (s,3H),1.61 (m, 1H), 1.56 (s, 3H), 1.42 (s, 3H); 13C NMR (100 MHz, CDCl3) δ191.7, 166.7, 161.1, 158.1, 144.2, 132.2, 130.7(◊2), 127.9, 127.8, 127.5,123.8, 121.9, 118.0, 116.1(◊2), 114.2, 113.5, 104.6, 80.6, 42.0, 27.7, 25.8,22.8, 17.8; HR-ESI-MS m / z 391.1904 [M + H]+. Example 3 Toxicity of chalcone compounds 1-12 to primary cortical neurons Primary cortical neuron culture: embryos of 18-day-pregnant SD rats were obtained, and the brains were removed on ice. The cortex was quickly separated under a microscope and digested with trypsin at 37 °C for 20 minutes. Primary cortical neurons were extracted from the cortex and cultured in growth medium (Neurobasal medium + 2% B27 serum-free medium + 0.5% L-glutamine + 0.4% D-(+)-glucose + 100 U / mL of penicillin and 100 μg / mL of streptomycin). The culture dishes were pre-coated with poly-L-lysine (PLL). The cells adhered to the wall for 3-4 hours and then the medium was changed. Thereafter, half of the medium was changed every 3 days. The formal experiment began on the 8th day of culture.

[0047] On the 8th day of cell culture, primary cortical neuron cells in good condition were taken, and control groups and sample groups with different concentrations were set up, and different concentrations of chalcone compounds 1 to 12 were added. After 24 hours of culture, the culture medium was discarded, and growth medium containing 10% MTT (5 mg / mL, Sigama) was added to continue culture. After 3 hours, the culture medium was discarded, DMSO was added to dissolve the purple precipitate, and the absorbance value was detected at a wavelength of 490 nm using the American Boten SynergyHT multifunctional microplate reader to calculate the cell survival rate.

[0048] The experimental results are as follows Figure 1 As shown in the results, the chalcone compounds 1, 3 and 5 of the present invention have no significant toxic effects on primary cortical neurons below 25 μM, and other chalcone compounds have no obvious effect on the survival rate of primary cortical neurons below 50 μM, indicating that the chalcone compounds 1 to 12 have less toxicity to primary cortical neurons. Example 4 Chalcone compounds 1-12 can protect primary cortical neurons from oxygen-glucose deprivation / reoxygenation damage Formal experiments were started on the 8th day of primary cortical neuron culture. After pre-administration of different concentrations of chalcone compounds 1-12 for 2 hours, the culture medium was replaced with drug-containing sugar-free culture medium and placed in an oxygen-deficient chamber. 95% N2 and 5% CO2 were introduced for 10 minutes with the airflow rate controlled at 20-25 L / min. After ventilation, the cells were placed in a 37 °C constant temperature incubator. After 4 hours, the cells were taken out and replaced with normal culture medium containing the drug. After 24 hours of resugar and reoxygenation, the cell survival rate was detected by MTT experiment.

[0049] The experimental results are as follows Figure 2 As shown in the results, oxygen-glucose deprivation / reoxygenation can significantly reduce the survival rate of primary cortical neurons, and chalcone compounds 1 to 12 can significantly protect primary neurons against oxygen-glucose deprivation / reoxygenation injury in a dose-dependent manner. Among them, chalcone compounds 1, 3 and 5 can protect neurons against oxygen-glucose deprivation / reoxygenation injury at a dose of 1.5 to 3 μM, and other chalcone compounds can protect neurons against oxygen-glucose deprivation / reoxygenation injury at a dose of 5 to 10 μM, confirming that chalcone compounds 1 to 12 have the potential to treat ischemic stroke. Example 5 Chalcone compound 3 can effectively improve brain damage in ischemic stroke model mice The commonly used etiology of ischemic stroke in clinical practice mainly includes large artery atherosclerosis, cardioembolism, small artery occlusion and other etiologies, among which large artery atherosclerosis and cardioembolism are the main types. Nearly half of patients with ischemic stroke can receive thrombolytic therapy during clinical treatment, while the other half cannot receive reperfusion therapy. Therefore, for different types of ischemic stroke in clinical practice, this study intends to use two classic ischemic stroke mouse models: the middle cerebral artery ischemia-reperfusion model and the middle cerebral artery distal thrombosis model, to study the effect of chalcone compound 3 in improving ischemic stroke in mice.

[0050] 1) Chalcone compound 3 improves brain damage in mice with middle cerebral artery ischemia-reperfusion model The method of establishing the middle cerebral artery ischemia-reperfusion model (MCAO) is as follows: After the mice are anesthetized, the neck skin is disinfected with alcohol, and then the neck is cut open from the midline to separate the common carotid, external carotid, and internal carotid artery neck vessels. A small incision is made on the external carotid artery, and a suture is used to enter the internal carotid artery (ICA) through the external carotid artery to block the starting end of the middle cerebral artery (MCA) to cause focal cerebral ischemia. After 1.5 hours, the suture is pulled out to form blood reperfusion, resulting in a reperfusion injury model. This model has a reperfusion process, focuses on blood flow obstruction, and has no thrombosis. The mechanism is similar to clinical cardiogenic stroke.

[0051] 7-8 week old male C57BL / 6 mice were used to establish the cerebral artery ischemia-reperfusion model, and the drug was intraperitoneally injected once 1.5 hours after occlusion, and once a day for three days thereafter, for a total of 4 times. After administration, the mice were scored for mNSS neurological function (the lower the score, the more sound the function), and the brain was taken for 2,3,5-triphenyltetrazolium chloride (TTC) staining to calculate the cerebral infarction volume.

[0052] The experimental results are as follows Figure 3As shown in the results, the cerebral infarction volume of MCAO model mice increased significantly compared with the sham group (Sham group), while the cerebral infarction volume of MCAO mice after intraperitoneal injection of chalcone compound 3 (0.1 mg / kg and 0.2 mg / kg) was significantly reduced, showing a dose-dependent effect, and the therapeutic effect of the 0.2 mg / kg dose group was better than that of the positive control drug edaravone (3 mg / kg). The mNSS neurological function score results also showed that the neurological function of MCAO mice was significantly impaired. After administration of chalcone compound 3 (0.1 mg / kg and 0.2 mg / kg), the behavioral function of MCAO mice was significantly improved, while edaravone (3 mg / kg) had no significant effect on improving the mNSS neurological function score. The results show that chalcone compound 3 can significantly improve the cerebral infarction volume and neurological function damage in mice with middle cerebral artery ischemia-reperfusion model, and its therapeutic effect is better than that of the positive control drug edaravone. 2) Chalcone compound 3 improves brain damage in mice with distal middle cerebral artery thrombosis model The method of establishing the ferric chloride (FeCl3)-induced distal middle cerebral artery thrombosis model is to use the strong oxidant properties of ferric chloride solution to induce vascular endothelial cell damage, thereby causing platelet aggregation in the blood vessels to form thrombi, resulting in distal arterial embolism in the mouse brain. This model has no reperfusion process, but has thrombosis, and is a permanent infarction model, and its mechanism is similar to the clinical large artery atherosclerotic stroke.

[0053] 7-8 week old male C57BL / 6 mice were used to model the distal middle cerebral artery thrombosis induced by ferric chloride. The head skin was disinfected, the scalp was cut to separate the subcutaneous tissue, the temporalis muscle was cut and the skull was drilled to expose the distal end of the left middle cerebral artery (MCA). A 0.5 mm × 0.5 mm filter paper soaked in 10% FeCl3 solution was applied to the MCA distal artery bifurcation for 3 minutes. After removing the filter paper, the surface was cleaned with saline and sutured. The sham operation group was treated with saline instead of FeCl3 solution. The drug was intraperitoneally injected once 1.5 hours after modeling, and then once a day for 3 consecutive days, for a total of 4 times. After drug administration, the mice were analyzed for cerebral infarction volume using magnetic resonance imaging (MRI).

[0054] The experimental results are as follows Figure 4 As shown in the results, compared with the sham group (Sham group) mice, the cerebral infarction volume of the ferric chloride model mice was significantly increased, while intraperitoneal injection of chalcone compound 3 (0.1 mg / kg and 0.2 mg / kg) can significantly reduce the cerebral infarction volume of the ferric chloride model mice, among which the 0.1 mg / kg dose group had a statistically significant difference, indicating that chalcone compound 3 can significantly improve the cerebral infarction volume of ferric chloride-induced cerebral ischemia mice.

[0055] The above results indicate that low-dose chalcone compound 3, administered after modeling, has a significant therapeutic effect on the mouse middle cerebral artery ischemia-reperfusion model and the ferric chloride-induced middle cerebral artery distal thrombosis model, suggesting its effectiveness and applicability for the clinical treatment of ischemic stroke. It should be noted that the specific embodiments of the present invention are described in detail above, but they are only examples, and the present invention is not limited to the specific embodiments described above. For those skilled in the art, any equivalent modifications and substitutions made to the present invention are also within the scope of the present invention. Therefore, the equalization changes and modifications made without departing from the spirit and scope of the present invention should be included in the scope of the present invention.

Claims

1. Use of a chalcone compound in the preparation of an anti-ischemic stroke drug, wherein the compound has a structural formula shown in general formula I; ； or its tautomers, racemates, enantiomers or mixtures thereof, or its pharmaceutically acceptable salts, wherein: R1, R4 and R6 are each independently selected from a hydrogen atom or a substituted or unsubstituted C10 unit, and an oxygen atom is present at an adjacent position of the C10 unit, and the C10 unit is independently selected from: ； R2 is independently selected from a hydrogen atom, a hydroxyl group, or an oxygen atom connected to an ortho-C10 unit on the benzene ring to form a six-membered ring; R3 and R5 are each independently selected from a hydrogen atom or a carbon atom on the adjacent C10 unit on the benzene ring where it is located to form a six-membered ring; The α carbon and the β carbon are independently connected by a carbon-carbon single bond or a carbon-carbon double bond.

2. The use according to claim 1, characterized in that: The R3, R4, R5 and R6 are hydrogen atoms; the α carbon and the β carbon are connected in the form of a carbon-carbon single bond; the chalcone compound has a structural formula shown in Formula II: ； or its tautomer, racemate, enantiomer or mixture form, or its pharmaceutically acceptable salt, wherein: R1, R2 and C10 units are as defined in claim 1.

3. The use according to claim 2, wherein the chalcone compound is selected from: ； or its tautomer, racemate, enantiomer or mixture form, or its pharmaceutically acceptable salt.

4. The use according to claim 1, characterized in that: The R1, R3 and R6 are hydrogen atoms; the chalcone compound has a structural formula shown in Formula III: ； Or its tautomer, racemate, enantiomer or mixture form, or its pharmaceutically acceptable salt, wherein: R2, R4, R5, the connection mode between α carbon and β carbon and C10 unit are as defined in claim 1.

5. The use according to claim 4, wherein the chalcone compound is selected from: ； or its tautomer, racemate, enantiomer or mixture form, or its pharmaceutically acceptable salt.

6. The use according to claim 1, characterized in that: The R1, R2, R3 and R4 are hydrogen atoms; the chalcone compound has a structural formula shown in Formula IV: ； Or its tautomer, racemate, enantiomer or mixture form, or its pharmaceutically acceptable salt, wherein: R5, R6, the connection mode between α carbon and β carbon and C10 unit are as defined in claim 1.

7. The use according to claim 6, wherein the chalcone compound is selected from: ； or its tautomer, racemate, enantiomer or mixture form, or its pharmaceutically acceptable salt.

8. A pharmaceutical composition comprising a chalcone compound represented by general formula I, II, III and IV according to any one of claims 1 to 7, or its tautomer, racemate, enantiomer or mixture thereof, or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers, diluents or excipients.

9. Use of the chalcone compounds represented by general formula I, II, III and IV according to any one of claims 1 to 7 or their tautomers, racemates, enantiomers or mixtures thereof, or the pharmaceutical composition according to claim 8 in the preparation of anti-stroke drugs.

10. The use of the anti-stroke drug according to claim 9, characterized in that: The type of stroke is ischemic stroke.

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