WO2021164155A1 - Cryptotanshinone derivative, preparation method therefor and application thereof in resisting neuroinflammation and neuroprotection - Google Patents

Cryptotanshinone derivative, preparation method therefor and application thereof in resisting neuroinflammation and neuroprotection Download PDF

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WO2021164155A1
WO2021164155A1 PCT/CN2020/096101 CN2020096101W WO2021164155A1 WO 2021164155 A1 WO2021164155 A1 WO 2021164155A1 CN 2020096101 W CN2020096101 W CN 2020096101W WO 2021164155 A1 WO2021164155 A1 WO 2021164155A1
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cryptotanshinone
formula
compound
derivative
elegans
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王长云
邵长伦
武京帅
管华诗
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中国海洋大学
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    • C07—ORGANIC CHEMISTRY
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    • C07J73/00—Steroids in which the cyclopenta[a]hydrophenanthrene skeleton has been modified by substitution of one or two carbon atoms by hetero atoms
    • C07J73/001—Steroids in which the cyclopenta[a]hydrophenanthrene skeleton has been modified by substitution of one or two carbon atoms by hetero atoms by one hetero atom
    • C07J73/003—Steroids in which the cyclopenta[a]hydrophenanthrene skeleton has been modified by substitution of one or two carbon atoms by hetero atoms by one hetero atom by oxygen as hetero atom
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
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    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/02—Oxygen as only ring hetero atoms
    • C12P17/04—Oxygen as only ring hetero atoms containing a five-membered hetero ring, e.g. griseofulvin, vitamin C

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  • the invention belongs to the field of medicine, and specifically relates to a cryptotanshinone derivative, a preparation method thereof, and application in anti-neural inflammation and neuroprotection.
  • Neurodegenerative diseases are universal diseases caused by the loss of neuron and/or myelin sheath function, including Alzheimer’s disease (AD), Parkinson’s disease (PD), amyotrophic lateral sclerosis (ALS) ) And Huntington's disease (HD), which are very harmful brain diseases, and have gradually become a worldwide health care problem.
  • the etiology mainly includes oxidative stress, mitochondrial dysfunction, neurotoxin and immune inflammation. So far, the treatment of these diseases is still limited to the control and alleviation of the manifestation, and there is no drug to treat the disease fundamentally. Therefore, it is urgent to develop safe, effective and reliable neurodegenerative disease drugs. Many evidences indicate that inflammation plays an important role in the occurrence and development of neurodegenerative diseases, especially Alzheimer's disease.
  • the nerve cells of the body Under the stimulation of inflammatory factors (lipopolysaccharide, ⁇ -amyloid, etc.), the nerve cells of the body produce a large amount of NO as a signal molecule to participate in the occurrence of various neuroinflammatory diseases. At the same time, it activates the inflammatory factors IL6, IL1 ⁇ and TNF ⁇ to further activate the nerve cells. Inflammation pathway. Therefore, the development of safe and effective nerve cell NO and inflammatory factor production inhibitors can be expected to become a candidate drug for the treatment of neuroinflammatory diseases.
  • inflammatory factors lipopolysaccharide, ⁇ -amyloid, etc.
  • the present invention relates to the preparation of three cryptotanshinone oxidation products at the C-3 position by a biotransformation method, which has significant anti-neuro-inflammatory activity and neuroprotective effect, and can obviously inhibit the release of NO and inflammatory factors IL1 ⁇ , IL6 and TNF ⁇ in nerve cells, and Inhibit the expression of iNOS, COX-2 and TLR4 in cells, exert anti-neuro-inflammatory effects by inhibiting the phosphorylation of JNK, ERK and p38 in the MAPK pathway, and at the same time alleviate the death of nerve cells induced by high concentration of glutamate, which is beneficial to protection Nerve cells perform normal physiological functions.
  • the present invention provides a cryptotanshinone derivative or a pharmaceutically acceptable salt thereof, characterized in that the cryptotanshinone derivative has the structure shown in formula I:
  • Another embodiment of the present invention provides a preparation method of the above formula I compound, which is characterized in that it comprises the following steps:
  • the medium is a PDA solid medium, which is made into a test tube slope when used.
  • the culture temperature is 25-30°C, and the culture time is 5-7 days to obtain the filamentous fungus C.elegans AS3.2028;
  • the filamentous fungus C.elegans AS3.2028 obtained in step (1) was cultured with a modified Chashi liquid medium on a shaker, and the modified Chashi liquid medium contained glucose 1.5%, sucrose 1.5%, peptone 0.5%, and phosphoric acid. Dipotassium hydrogen 0.1%, magnesium sulfate 0.05%, potassium chloride 0.05%, ferrous sulfate 0.001%, the rest is water, the above percentages are all by weight; the cultivation temperature is 25-30°C, the shaker rotation speed is 180rpm, and the culture After 24 hours, the amplified culture medium of the filamentous fungus C.elegans AS3.2028 was obtained.
  • the cryptotanshinone (substrate) was added to the expansion medium of the filamentous fungus C.elegans AS3.2028 obtained in step (2), and cultured in a shaker at 28°C and a shaker speed of 180 rpm for 72 hours to obtain a transformant ;
  • the stationary phase used for the normal phase silica gel column chromatographic separation in step (4) 100-400 mesh silica gel, and the mobile phase: 30%-90% ethyl acetate-petroleum ether mixed solvent; the HPLC separation uses The stationary phase: ODS chromatographic column (Kromasil, 5 ⁇ m, 150 ⁇ 10mm), mobile phase: 55%-60% methanol-water mixed solvent; the above-mentioned mixed solvent percentages are all volume percentages.
  • Another embodiment of the present invention provides the use of the cryptotanshinone derivative of the above formula I structure or a pharmaceutically acceptable salt thereof in the preparation of an anti-neuro-inflammatory drug.
  • Figure 1 shows the inhibition of the expression levels of iNOS, COX-2 and TLR4 by the compound of formula I.
  • 1 the substrate cryptotanshinone
  • 2 the compound of formula I
  • 3 the compound of formula I
  • 4 the compound of formula I 3.
  • A, B, C western bolttting detection of compound action of 1 ⁇ M, 3 ⁇ M and 10 ⁇ M; gray scale analysis of cytokine expression of compound action of D, E, F: 1 ⁇ M, 3 ⁇ M and 10 ⁇ M, *p ⁇ 0.05,**p ⁇ 0.01 ,***p ⁇ 0.001, compared with the LPS treatment group.
  • Figure 3 is an immunofluorescence image of compound 1 of formula I inhibiting NF- ⁇ B p65.
  • the filamentous fungus C.elegans AS3.2028 obtained in step (1) was cultured with a modified Chashi liquid medium on a shaker, and the modified Chashi liquid medium contained glucose 1.5%, sucrose 1.5%, peptone 0.5%, and phosphoric acid. Dipotassium hydrogen 0.1%, magnesium sulfate 0.05%, potassium chloride 0.05%, ferrous sulfate 0.001%, the rest is water, the above percentages are weight percentages; culture temperature is 28°C, shaker rotation speed is 180rpm, culture time is 24h , To obtain the expansion medium of the filamentous fungus C.elegans AS3.2028;
  • step (2) Add cryptotanshinone to the expansion medium of the filamentous fungus C.elegans AS3.2028 obtained in step (2) (the final concentration of cryptotanshinone is 0.10 mg/mL), at 28°C, and the shaker rotates at 180 rpm Incubate on a shaker for 72 hours to obtain transformants;
  • step (3) Take the transformant (10L) obtained in step (3), separate the transformant from the bacteria, extract the transformant 3 times with ethyl acetate, combine the extracts and concentrate under reduced pressure to obtain the transformant extract.
  • step (3) Take the transformant (10L) obtained in step (3), separate the transformant from the bacterial cells, extract the transformant twice with ethyl acetate, combine the extracts and concentrate under reduced pressure to obtain the transformant extract.
  • the inhibition of NO production by the compound of formula I was evaluated using microglia BV-2 as an evaluation model.
  • the experiment was carried out in a 96-well plate, adding different concentrations of the compound of formula I, the substrate cryptotanshinone, the positive control quercetin and the blank control DMSO, Then add 1 ⁇ g/mL LPS to each well and incubate for 24h.
  • the amount of NO production was detected by Griess reaction. 50 ⁇ L of cell supernatant from each well was transferred to a new 96-well plate, the same amount of Griess reagent was added and mixed, the absorbance was measured at 540nm, and the amount of NO production and inhibition rate were calculated.
  • the experiment was repeated 3 times, and the results are expressed as mean ⁇ SD, as shown in Table 1.
  • the anti-neuro-inflammatory activity of the substrate cryptotanshinone is 2.5 times higher than that of the positive control quercetin, while the anti-neuro-inflammatory activity of formula I compound 1, formula I compound 2 and formula I compound 3 are 6.7 times, 2.3 times and 20.6 times higher than that of the substrate. Times. This shows that the anti-inflammatory activity of cryptotanshinone can be significantly improved by the method of biotransformation.
  • Table 1 The inhibitory activity of the compound of formula I of the present invention on LPS-induced NO production in BV2 cells
  • Nerve cells BV-2 were cultured in a 6-well plate, 10 ⁇ M compound of formula I and the substrate cryptotanshinone were added, after incubation at 37°C for 1h, 1 ⁇ g/mL LPS was added to each well and no LPS was added as a control, cultured for 16h, centrifuged at 4000rpm for 5min, Collect the supernatant in a new 1.5 mL sterile centrifuge tube and repeat three times.
  • the release of neuroinflammatory factors was determined using Lianke ELSIA kit, IL1 ⁇ (EK201B-01), IL6 (EK206-01) and TNF ⁇ (EK282-01).
  • Terminating the reaction Add 100 ⁇ l of 2M sulfuric acid to each reaction well, and the color changes from blue to yellow.
  • LPS stimulates the production of nerve cells BV-2 and releases the inflammatory factors IL1 ⁇ , IL6 and TNF ⁇ .
  • Both the compound of formula I and cryptotanshinone can significantly inhibit the release of inflammatory factors, and the inhibitory activity of the compound of formula I is stronger (Table 2).
  • LPS group IL1 ⁇ , 70.05 ⁇ 7.71pg/mL, IL6, 377.77 ⁇ 39.02pg/mL, TNF ⁇ , 759.44 ⁇ 45.55pg/mL; control group without LPS: IL1 ⁇ , 7.77 ⁇ 0.96pg/mL, IL6, 1.27 ⁇ 0.12pg/mL, TNF ⁇ , 70.84 ⁇ 9.93pg/mL.
  • BV-2 cells were seeded in a 6-well culture plate, incubated with the compound for 1 h, and then incubated with LPS (1 ⁇ g/mL) for 16 h.
  • the protein concentration was determined with Pierce-Rapid-Gold-BCA protein detection kit.
  • Each quantitative protein sample was electrophoresed with 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and then transferred to a polyvinylidene fluoride (PVDF) membrane.
  • the membrane was blocked with 5% (W/V) skim milk in TBST buffer (tri-buffered saline containing 0.1% Tween 20) for 2 h, and then the membrane was incubated with primary antibody overnight at 4°C.
  • BV-2 cells were seeded in a 6-well culture plate, incubated with the compound for 1 h, and then incubated with LPS (1 ⁇ g/mL) for 16 h.
  • the cell slide was treated with 4% paraformaldehyde and 0.2% Triton X-100 (PBS). Then, it was blocked with 5% bovine serum albumin (PBS) for 1 hour, and incubated with antibody NF- ⁇ B p65 at 4°C overnight, and then an antibody labeled with Alexa Fluor 594 was added and incubated for 1 hour. After staining with DAPI, wash and seal the cover glass, and take pictures with a fluorescence microscope. The results show that LPS can activate the production and localization of NF- ⁇ B p65, while compound 1 of formula I can significantly inhibit the production and localization of NF- ⁇ B p65 ( Figure 3).

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Abstract

The present invention relates to a cryptotanshinone derivative, a preparation method therefor and an application thereof in resisting neuroinflammation and neuroprotection. The cryptotanshinone derivative has the structure as shown in formula I: (I). The present invention provides three novel anti-neuroinflammatory and neuroprotective agents, characterized in: using the cryptotanshinone derivative having the structure as shown in formula I or salts thereof in the present invention as an effective component for preventing or treating neurodegenerative disorders related to neuroinflammation such as Alzheimer's disease and parkinsonism.

Description

一种隐丹参酮衍生物及其制备方法与在抗神经炎症和神经保护中的应用Cryptotanshinone derivative, preparation method thereof and application in anti-neuritis and neuroprotection 技术领域Technical field
本发明属于医药领域，具体涉及一种隐丹参酮衍生物及其制备方法与在抗神经炎症和神经保护中的应用。The invention belongs to the field of medicine, and specifically relates to a cryptotanshinone derivative, a preparation method thereof, and application in anti-neural inflammation and neuroprotection.
背景技术Background technique
中药丹参具有显著的活血化瘀、通心包络等功效，以多种形式应用于临床，如注射液、滴丸、片剂等。其主要的药效物质为水溶性的丹酚酸和脂溶性的丹参酮类化合物。在丹参酮类成分中，丹参酮IIA已被开发为治疗心绞痛、冠心病和心肌梗死的药物。另外，有研究报道丹参中另一丹参酮类化合物隐丹参酮具有良好的抗炎和抗氧化活性，尤其在治疗神经退行性疾病方面有着潜在的应用前景。神经退行性疾病是由神经元和(或)其髓鞘的功能丧失所致的普遍性疾病，包括阿茨海默症(AD)，帕金森症(PD)，肌萎缩侧索硬化症(ALS)和亨廷顿舞蹈症(HD)等，是危害甚大的脑部病变，已逐渐成为世界性的卫生保健难题。其病因主要包括氧化应激、线粒体功能障碍、神经性毒素及免疫炎症。到目前为止，对这些疾病的治疗仍局限于表征的控制和缓解，尚无从根本上治疗疾病的药物。因此，开发安全、有效、可靠的神经退行性疾病药物十分迫切。许多证据表明，炎症在神经退行性疾病尤其是阿尔兹海默症的发生和发展中发挥着重要的作用。在炎症因子(脂多糖、β样淀粉蛋白等)刺激下，机体神经细胞产生大量的NO作为信号分子参与多种神经炎症疾病的发生，同时激活炎症因子IL6、IL1β及TNFα，进一步激活神经细胞的炎症通路。因此，开发安全有效的神经细胞NO及炎症因子产生抑制剂即能够有望成为治疗神经炎症疾病候选药物。The traditional Chinese medicine Danshen has significant effects of promoting blood circulation, removing blood stasis, and dredging the heart envelope. It is used clinically in many forms, such as injections, dripping pills, and tablets. The main medicinal substances are water-soluble salvianolic acid and fat-soluble tanshinone compounds. Among the tanshinone components, tanshinone IIA has been developed as a drug for the treatment of angina pectoris, coronary heart disease and myocardial infarction. In addition, studies have reported that cryptotanshinone, another tanshinone compound in Danshen, has good anti-inflammatory and antioxidant activities, and has potential application prospects especially in the treatment of neurodegenerative diseases. Neurodegenerative diseases are universal diseases caused by the loss of neuron and/or myelin sheath function, including Alzheimer’s disease (AD), Parkinson’s disease (PD), amyotrophic lateral sclerosis (ALS) ) And Huntington's disease (HD), which are very harmful brain diseases, and have gradually become a worldwide health care problem. The etiology mainly includes oxidative stress, mitochondrial dysfunction, neurotoxin and immune inflammation. So far, the treatment of these diseases is still limited to the control and alleviation of the manifestation, and there is no drug to treat the disease fundamentally. Therefore, it is urgent to develop safe, effective and reliable neurodegenerative disease drugs. Many evidences indicate that inflammation plays an important role in the occurrence and development of neurodegenerative diseases, especially Alzheimer's disease. Under the stimulation of inflammatory factors (lipopolysaccharide, β-amyloid, etc.), the nerve cells of the body produce a large amount of NO as a signal molecule to participate in the occurrence of various neuroinflammatory diseases. At the same time, it activates the inflammatory factors IL6, IL1β and TNFα to further activate the nerve cells. Inflammation pathway. Therefore, the development of safe and effective nerve cell NO and inflammatory factor production inhibitors can be expected to become a candidate drug for the treatment of neuroinflammatory diseases.
本发明涉及通过生物转化的方法制备三个隐丹参酮C-3位氧化产物，具有显著的抗神经炎症活性和神经保护作用，能明显抑制神经细胞中NO及炎症因 子IL1β、IL6及TNFα释放，并抑制细胞内iNOS，COX-2及TLR4的表达，通过抑制MAPK途径中JNK,ERK及p38的磷酸化而发挥抗神经炎症作用，同时缓解高浓度谷氨酸盐诱导的神经细胞死亡，有利于保护神经细胞行使正常的生理功能。The present invention relates to the preparation of three cryptotanshinone oxidation products at the C-3 position by a biotransformation method, which has significant anti-neuro-inflammatory activity and neuroprotective effect, and can obviously inhibit the release of NO and inflammatory factors IL1β, IL6 and TNFα in nerve cells, and Inhibit the expression of iNOS, COX-2 and TLR4 in cells, exert anti-neuro-inflammatory effects by inhibiting the phosphorylation of JNK, ERK and p38 in the MAPK pathway, and at the same time alleviate the death of nerve cells induced by high concentration of glutamate, which is beneficial to protection Nerve cells perform normal physiological functions.
发明内容Summary of the invention
本发明提供一种隐丹参酮衍生物或其药学上可接受的盐，其特征在于所述隐丹参酮衍生物具有式I所示结构：The present invention provides a cryptotanshinone derivative or a pharmaceutically acceptable salt thereof, characterized in that the cryptotanshinone derivative has the structure shown in formula I:
Figure PCTCN2020096101-appb-000001
Figure PCTCN2020096101-appb-000001
本发明的另一实施方案提供上述式Ⅰ化合物的制备方法，其特征在于包括如下步骤：Another embodiment of the present invention provides a preparation method of the above formula I compound, which is characterized in that it comprises the following steps:
(1)丝状真菌Cunninghamella elegans AS3.2028的培养(1) Cultivation of filamentous fungus Cunninghamella elegans AS3.2028
培养基为PDA固体培养基，使用时制成试管斜面，培养温度为25-30℃，培养时间5-7天，得丝状真菌C.elegans AS3.2028；The medium is a PDA solid medium, which is made into a test tube slope when used. The culture temperature is 25-30°C, and the culture time is 5-7 days to obtain the filamentous fungus C.elegans AS3.2028;
(2)丝状真菌C.elegans AS3.2028的扩增(2) Amplification of the filamentous fungus C.elegans AS3.2028
用改良的察氏液体培养基摇床培养步骤(1)得到的丝状真菌C.elegans AS3.2028，所述改良的察氏液体培养基含有葡萄糖1.5％、蔗糖1.5％、蛋白胨0.5％、磷酸氢二钾0.1％、硫酸镁0.05％、氯化钾0.05％、硫酸亚铁0.001％，其余为水，上述百分含量均为重量百分比；培养温度为25-30℃，摇床转速180rpm，培养时间24h，得丝状真菌C.elegans AS3.2028的扩增培养液。The filamentous fungus C.elegans AS3.2028 obtained in step (1) was cultured with a modified Chashi liquid medium on a shaker, and the modified Chashi liquid medium contained glucose 1.5%, sucrose 1.5%, peptone 0.5%, and phosphoric acid. Dipotassium hydrogen 0.1%, magnesium sulfate 0.05%, potassium chloride 0.05%, ferrous sulfate 0.001%, the rest is water, the above percentages are all by weight; the cultivation temperature is 25-30°C, the shaker rotation speed is 180rpm, and the culture After 24 hours, the amplified culture medium of the filamentous fungus C.elegans AS3.2028 was obtained.
(3)隐丹参酮的生物转化(3) Biotransformation of cryptotanshinone
将隐丹参酮(底物)添加到步骤(2)得到的丝状真菌C.elegans AS3.2028的扩增培养液中，于28℃，摇床转速180rpm的条件下摇床培养72h，得转化物；The cryptotanshinone (substrate) was added to the expansion medium of the filamentous fungus C.elegans AS3.2028 obtained in step (2), and cultured in a shaker at 28°C and a shaker speed of 180 rpm for 72 hours to obtain a transformant ；
(4)本发明式Ⅰ化合物的分离纯化(4) Separation and purification of the compound of formula I of the present invention
将步骤(3)得到的转化物中的转化液和菌体分离，转化液用乙酸乙酯萃取2-3次，合并萃取液、减压浓缩得到转化液浸膏，先进行正相硅胶柱色谱分离、再进行高效液相色谱分离转化产物即得式Ⅰ化合物(化合物1-3)。Separate the transformation solution and the bacteria in the transformation obtained in step (3), extract the transformation solution with ethyl acetate for 2-3 times, combine the extracts and concentrate under reduced pressure to obtain the transformation solution extract, and perform normal phase silica gel column chromatography first After separation and high performance liquid chromatography to separate the conversion product, the compound of formula I (compound 1-3) can be obtained.
步骤(3)中隐丹参酮的添加量优选添加后隐丹参酮的终浓度为0.05-0.20mg/mL；The addition amount of cryptotanshinone in step (3) is preferably the final concentration of cryptotanshinone after addition is 0.05-0.20mg/mL;
步骤(4)中所述正相硅胶柱色谱分离采用的固定相：100～400目硅胶，流动相：30％–90％的乙酸乙酯-石油醚混合溶剂；所述高效液相色谱分离采用的固定相：ODS色谱柱(Kromasil,5μm,150×10mm)，流动相：55％–60％的甲醇-水混合溶剂；上述混合溶剂百分比均为体积百分比。The stationary phase used for the normal phase silica gel column chromatographic separation in step (4): 100-400 mesh silica gel, and the mobile phase: 30%-90% ethyl acetate-petroleum ether mixed solvent; the HPLC separation uses The stationary phase: ODS chromatographic column (Kromasil, 5μm, 150×10mm), mobile phase: 55%-60% methanol-water mixed solvent; the above-mentioned mixed solvent percentages are all volume percentages.
本发明的另一实施方案提供上述式I结构的隐丹参酮衍生物或其药学上可接受的盐在制备抗神经炎症药物中的应用。Another embodiment of the present invention provides the use of the cryptotanshinone derivative of the above formula I structure or a pharmaceutically acceptable salt thereof in the preparation of an anti-neuro-inflammatory drug.
本发明的另一实施方案提供上述式I结构的隐丹参酮衍生物或其药学上可接受的盐在制备神经保护药物中的应用。Another embodiment of the present invention provides the use of the cryptotanshinone derivative of the above formula I structure or a pharmaceutically acceptable salt thereof in the preparation of neuroprotective drugs.
本发明的另一实施方案提供一种药物组合物，其特征在于所述药物组合物以上述式I结构的隐丹参酮衍生物或其药学上可接受的盐作为有效成分。还任选包括其他抗神经炎症药物和/或神经保护药物。还可包括药学上可接受的辅料(例如药学上可接受的载体、稀释剂或赋形剂)。Another embodiment of the present invention provides a pharmaceutical composition, characterized in that the pharmaceutical composition uses the cryptotanshinone derivative of the above formula I structure or a pharmaceutically acceptable salt thereof as an active ingredient. Optionally, other anti-neuro-inflammatory drugs and/or neuroprotective drugs are also included. It may also include pharmaceutically acceptable excipients (e.g., pharmaceutically acceptable carriers, diluents or excipients).
附图说明Description of the drawings
图1是式Ⅰ化合物抑制iNOS,COX-2及TLR4的表达水平，注：1：底物隐丹参酮，2：式Ⅰ化合物1，3：式Ⅰ化合物2，4：式Ⅰ化合物3。A,B,C:1μM、3μM和10μM化合物作用的western boltting检测；D,E,F:1μM、3μM和10μM 化合物作用的细胞因子表达的灰度分析，*p<0.05,**p<0.01,***p<0.001，与LPS处理组相比。Figure 1 shows the inhibition of the expression levels of iNOS, COX-2 and TLR4 by the compound of formula I. Note: 1: the substrate cryptotanshinone, 2: the compound of formula I 1, 3: the compound of formula I, 4: the compound of formula I 3. A, B, C: western bolttting detection of compound action of 1 μM, 3 μM and 10 μM; gray scale analysis of cytokine expression of compound action of D, E, F: 1 μM, 3 μM and 10 μM, *p<0.05,**p<0.01 ,***p<0.001, compared with the LPS treatment group.
图2是式Ⅰ化合物1抑制JNK,ERK及p38的磷酸化检测，注：A：JNK、ERK和p38磷酸化检测；B，C，D：JNK、ERK和p38磷酸化的灰度分析。*p<0.05,**p<0.01,***p<0.001，与LPS处理组相比。Figure 2 is the detection of the inhibition of the phosphorylation of JNK, ERK and p38 by compound 1 of formula I. Note: A: JNK, ERK and p38 phosphorylation detection; B, C, D: Grayscale analysis of the phosphorylation of JNK, ERK and p38. *p<0.05, **p<0.01, ***p<0.001, compared with LPS treatment group.
图3是式Ⅰ化合物1抑制NF-κB p65的免疫荧光图。Figure 3 is an immunofluorescence image of compound 1 of formula I inhibiting NF-κB p65.
图4是式Ⅰ化合物的神经保护活性，注：Control：DMSO，Glu：5mM的谷氨酸钠。1：底物隐丹参酮，2：式Ⅰ化合物1，3：式Ⅰ化合物2，4：式Ⅰ化合物3。*p<0.05,**p<0.01,***p<0.001，与Glu处理组相比。Figure 4 shows the neuroprotective activity of the compound of formula I. Note: Control: DMSO, Glu: 5mM sodium glutamate. 1: Substrate cryptotanshinone, 2: Formula I compound 1, 3: Formula I compound 2, 4: Formula I compound 3. *p<0.05, **p<0.01, ***p<0.001, compared with Glu treatment group.
具体实施方式Detailed ways
为了便于对本发明的进一步理解，下面提供的实施例对其做了更详细的说明。但是这些实施例仅为了更好的理解发明而并非用来限定本发明的范围或实施原则，本发明的实施方式不限于以下内容。In order to facilitate a further understanding of the present invention, the following embodiments provide a more detailed description thereof. However, these examples are only for a better understanding of the invention and are not used to limit the scope or implementation principles of the invention, and the embodiments of the invention are not limited to the following content.
实施例1Example 1
式Ⅰ化合物制备Formula I compound preparation
(1)丝状真菌C.elegans AS3.2028的培养(1) Cultivation of filamentous fungus C.elegans AS3.2028
培养基为PDA固体培养基，使用时制成试管斜面，培养温度为28℃，培养时间7天，得丝状真菌C.elegans AS3.2028；The medium is a PDA solid medium, which is made into a test tube slope when used, the culture temperature is 28°C, and the culture time is 7 days to obtain the filamentous fungus C.elegans AS3.2028;
(2)丝状真菌C.elegans AS3.2028的扩增(2) Amplification of the filamentous fungus C.elegans AS3.2028
用改良的察氏液体培养基摇床培养步骤(1)得到的丝状真菌C.elegans AS3.2028，所述改良的察氏液体培养基含有葡萄糖1.5％、蔗糖1.5％、蛋白胨0.5％、磷酸氢二钾0.1％、硫酸镁0.05％、氯化钾0.05％、硫酸亚铁0.001％，其余为水，上述百分含量均为重量百分比；培养温度为28℃，摇床转速180rpm，培养时间24h，得丝状真菌C.elegans AS3.2028的扩增培养液；The filamentous fungus C.elegans AS3.2028 obtained in step (1) was cultured with a modified Chashi liquid medium on a shaker, and the modified Chashi liquid medium contained glucose 1.5%, sucrose 1.5%, peptone 0.5%, and phosphoric acid. Dipotassium hydrogen 0.1%, magnesium sulfate 0.05%, potassium chloride 0.05%, ferrous sulfate 0.001%, the rest is water, the above percentages are weight percentages; culture temperature is 28℃, shaker rotation speed is 180rpm, culture time is 24h , To obtain the expansion medium of the filamentous fungus C.elegans AS3.2028;
(3)隐丹参酮的生物转化(3) Biotransformation of cryptotanshinone
将隐丹参酮添加到步骤(2)得到的丝状真菌C.elegans AS3.2028的扩增培养液中(隐丹参酮的终浓度为0.10mg/mL)，于28℃，摇床转速180rpm的条件下摇床培养72h，得转化物；Add cryptotanshinone to the expansion medium of the filamentous fungus C.elegans AS3.2028 obtained in step (2) (the final concentration of cryptotanshinone is 0.10 mg/mL), at 28°C, and the shaker rotates at 180 rpm Incubate on a shaker for 72 hours to obtain transformants;
(4)本发明式Ⅰ化合物的分离纯化(4) Separation and purification of the compound of formula I of the present invention
取步骤(3)所得的转化物(10L)，将转化液和菌体分离，转化液用乙酸乙酯萃取3次，合并萃取液、减压浓缩得到转化液浸膏，先进行正相硅胶柱色谱分离(固定相：300～400目硅胶，流动相：30％–90％的乙酸乙酯-石油醚混合溶剂)、再进行高效液相色谱分离(固定相：ODS色谱柱(Kromasil,5μm,150×10mm)，流动相：55％的甲醇-水混合溶剂)即得式Ⅰ化合物1(6.2mg，橘黄色粉末)，式Ⅰ化合物2(56.0mg)和式Ⅰ化合物3(17.8mg)。Take the transformant (10L) obtained in step (3), separate the transformant from the bacteria, extract the transformant 3 times with ethyl acetate, combine the extracts and concentrate under reduced pressure to obtain the transformant extract. First, perform a normal phase silica gel column Chromatographic separation (stationary phase: 300-400 mesh silica gel, mobile phase: 30%-90% ethyl acetate-petroleum ether mixed solvent), and then high performance liquid chromatography (stationary phase: ODS chromatography column (Kromasil, 5μm, 150×10 mm), mobile phase: 55% methanol-water mixed solvent) to obtain formula I compound 1 (6.2 mg, orange powder), formula I compound 2 (56.0 mg) and formula I compound 3 (17.8 mg).
式Ⅰ化合物1结构确证数据： 1H NMR(CDCl 3,500MHz)δ:7.65(1H,d,J＝8.1Hz,H-6),7.62(1H,d,J＝8.1Hz,H-7),4.93(1H,d,J＝9.6Hz,H-16a),4.41(1H,dd,J＝9.6,6.1Hz,H-16b),3.67(2H,m,H-1),3.63(1H,m,H-15),2.65(2H,m,H-2),1.48(3H,s,H-18),1.47(3H,s,H-19),1.38(3H,d,J＝6.1Hz,H-17). 13C NMR(CDCl 3,125MHz)δ:212.5(C,C-3),184.4(C,C-11),175.6(C,C-12),170.2(C,C-14),150.3(C,C-5),141.6(C,C-10),131.8(CH,C-6),128.4(C,C-9),126.9(C,C-8),123.8(CH,C-7),119.0(C,C-13),81.6(CH 2,C-16),48.5(C,C-4),36.3(CH 2,C-2),34.7(CH,C-15),27.1(CH 3,C-19),27.0(CH 3,C-18),25.9(CH 2,C-1),18.8(CH 3,C-17).HRESIMS m/z 311.1273[M+H] +(calcd for C 19H 19O 4,311.1278).[α] 20 D-33.9(c 0.05,MeOH)；UV(MeOH)λ max(logε)209(1.47),264(0.93)nm；IR(KBr)ν max 3741,3445,1645,1558cm -1. Structure confirmation data of compound 1 of formula I: 1 H NMR (CDCl 3 , 500MHz) δ: 7.65 (1H, d, J = 8.1 Hz, H-6), 7.62 (1H, d, J = 8.1 Hz, H-7) ,4.93(1H,d,J=9.6Hz,H-16a), 4.41(1H,dd,J=9.6,6.1Hz,H-16b), 3.67(2H,m,H-1),3.63(1H, m,H-15), 2.65(2H,m,H-2), 1.48(3H,s,H-18), 1.47(3H,s,H-19), 1.38(3H,d,J=6.1Hz ,H-17). 13 C NMR(CDCl 3 ,125MHz)δ:212.5(C,C-3),184.4(C,C-11),175.6(C,C-12),170.2(C,C- 14), 150.3 (C, C-5), 141.6 (C, C-10), 131.8 (CH, C-6), 128.4 (C, C-9), 126.9 (C, C-8), 123.8 ( CH, C-7), 119.0 (C, C-13), 81.6 (CH 2, C-16), 48.5 (C, C-4), 36.3 (CH 2, C-2), 34.7 (CH, C -15),27.1(CH 3 ,C-19),27.0(CH 3 ,C-18),25.9(CH 2 ,C-1),18.8(CH 3 ,C-17).HRESIMS m/z 311.1273[ M+H] + (calcd for C 19 H 19 O 4 ,311.1278).[α] 20 D -33.9(c 0.05,MeOH); UV(MeOH)λ max (logε)209(1.47),264(0.93) nm; IR(KBr)ν max 3741,3445,1645,1558cm -1 .
式Ⅰ化合物2结构确证数据： 1H NMR(CDCl 3,500MHz)δ:7.66(1H,d,J＝8.1Hz,H-6),7.51(1H,d,J＝8.1Hz,H-7),4.90(1H,d,J＝9.6Hz,H-16a),4.37(1H,dd,J＝9.6,6.1Hz,H-16b),3.78(1H,m,H-3),3.59(1H,m,H-15),3.42(1H,m,H-1a),3.27(1H,m,H-1b),2.04(1H,m,H-2a),1.94(1H,m,H-2b),1.36(3H,d, J＝6.1Hz,H-17),1.31(3H,s,H-19),1.31(3H,d,J＝6.1Hz,H-18). 13C NMR(CDCl 3,125MHz)δ:183.9(C,C-11),175.3(C,C-12),170.7(C,C-14),151.2(C,C-5),142.2(C,C-10),132.9(CH,C-6),127.7(C,C-9),126.5(C,C-8),122.9(CH,C-7),118.4(C,C-13),81.5(CH 2,C-16),73.8(CH,C-3),40.0(C,C-4),34.5(CH,C-15),29.4(CH 3,C-19),26.2(CH 2,C-2),25.9(CH 2,C-1),25.3(CH 3,C-18),18.7(CH 3,C-17).HRESIMS m/z 313.2[M+H] +. Structure confirmation data of compound 2 of formula I: 1 H NMR (CDCl 3 , 500MHz) δ: 7.66 (1H, d, J = 8.1 Hz, H-6), 7.51 (1H, d, J = 8.1 Hz, H-7) , 4.90(1H,d,J=9.6Hz,H-16a), 4.37(1H,dd,J=9.6,6.1Hz,H-16b), 3.78(1H,m,H-3),3.59(1H, m, H-15), 3.42 (1H, m, H-1a), 3.27 (1H, m, H-1b), 2.04 (1H, m, H-2a), 1.94 (1H, m, H-2b) ,1.36(3H,d, J=6.1Hz,H-17),1.31(3H,s,H-19),1.31(3H,d,J=6.1Hz,H-18). 13 C NMR(CDCl 3 , 125MHz) δ: 183.9 (C, C-11), 175.3 (C, C-12), 170.7 (C, C-14), 151.2 (C, C-5), 142.2 (C, C-10), 132.9(CH,C-6),127.7(C,C-9),126.5(C,C-8),122.9(CH,C-7),118.4(C,C-13),81.5(CH 2 , C-16), 73.8 (CH, C-3), 40.0 (C, C-4), 34.5 (CH, C-15), 29.4 (CH 3 , C-19), 26.2 (CH 2 , C-2 ),25.9(CH 2 ,C-1),25.3(CH 3 ,C-18),18.7(CH 3 ,C-17).HRESIMS m/z 313.2[M+H] + .
式Ⅰ化合物3结构确证数据： 1H NMR(CDCl 3,500MHz)δ:7.66(1H,d,J＝8.1Hz,H-6),7.53(1H,d,J＝8.1Hz,H-7),4.90(1H,d,J＝9.5Hz,H-16a),4.37(1H,dd,J＝9.3,6.0Hz,H-16b),3.79(1H,m,H-3),3.60(1H,m,H-15),3.46(1H,m,H-1a),3.28(1H,m,H-1b),2.06(1H,m,H-2a),1.93(1H,m,H-2b),1.37(3H,d,J＝6.1Hz,H-17),1.36(3H,s,H-19),1.34(3H,d,J＝6.1Hz,H-18). 13C NMR(CDCl 3,125MHz)δ:184.0(C,C-11),175.4(C,C-12),170.6(C,C-14),151.1(C,C-5),142.1(C,C-10),132.8(CH,C-6),127.8(C,C-9),126.6(C,C-8),123.0(CH,C-7),118.5(C,C-13),81.5(CH 2,C-16),73.9(CH,C-3),40.0(C,C-4),34.6(CH,C-15),29.3(CH 3,C-19),26.3(CH 2,C-2),25.9(CH 2,C-1),25.3(CH 3,C-18),18.8(CH 3,C-17).HRESIMS m/z 313.2[M+H] +. Structure confirmation data of compound 3 of formula I: 1 H NMR (CDCl 3 , 500MHz) δ: 7.66 (1H, d, J = 8.1 Hz, H-6), 7.53 (1H, d, J = 8.1 Hz, H-7) ,4.90(1H,d,J=9.5Hz,H-16a), 4.37(1H,dd,J=9.3,6.0Hz,H-16b), 3.79(1H,m,H-3), 3.60(1H, m,H-15), 3.46 (1H, m, H-1a), 3.28 (1H, m, H-1b), 2.06 (1H, m, H-2a), 1.93 (1H, m, H-2b) ,1.37(3H,d,J=6.1Hz,H-17),1.36(3H,s,H-19),1.34(3H,d,J=6.1Hz,H-18). 13 C NMR(CDCl 3 , 125MHz) δ: 184.0 (C, C-11), 175.4 (C, C-12), 170.6 (C, C-14), 151.1 (C, C-5), 142.1 (C, C-10), 132.8(CH,C-6),127.8(C,C-9),126.6(C,C-8),123.0(CH,C-7),118.5(C,C-13),81.5(CH 2 , C-16), 73.9 (CH, C-3), 40.0 (C, C-4), 34.6 (CH, C-15), 29.3 (CH 3 , C-19), 26.3 (CH 2 , C-2 ),25.9(CH 2 ,C-1),25.3(CH 3 ,C-18),18.8(CH 3 ,C-17).HRESIMS m/z 313.2[M+H] + .
实施例2Example 2
式Ⅰ化合物制备Formula I compound preparation
(1)丝状真菌C.elegans AS3.2028的培养(1) Cultivation of filamentous fungus C.elegans AS3.2028
培养基为PDA固体培养基，使用时制成试管斜面，培养温度为25℃，培养时间5天，得丝状真菌C.elegans AS3.2028；The medium is a PDA solid medium, which is made into a test tube slope when used, the culture temperature is 25°C, and the culture time is 5 days to obtain the filamentous fungus C.elegans AS3.2028;
(2)丝状真菌C.elegans AS3.2028的扩增(2) Amplification of the filamentous fungus C.elegans AS3.2028
用改良的察氏液体培养基摇床培养步骤(1)得到的丝状真菌C.elegans AS3.2028，所述改良的察氏液体培养基含有葡萄糖1.5％、蔗糖1.5％、蛋白胨0.5％、磷酸氢二钾0.1％、硫酸镁0.05％、氯化钾0.05％、硫酸亚铁0.001％，其 余为水，上述百分含量均为重量百分比；培养温度为30℃，摇床转速180rpm，培养时间24h，得丝状真菌C.elegans AS3.2028的扩增培养液；The filamentous fungus C.elegans AS3.2028 obtained in step (1) was cultured with a modified Chashi liquid medium on a shaker, and the modified Chashi liquid medium contained glucose 1.5%, sucrose 1.5%, peptone 0.5%, and phosphoric acid. Dipotassium hydrogen 0.1%, magnesium sulfate 0.05%, potassium chloride 0.05%, ferrous sulfate 0.001%, the rest is water, the above percentages are all by weight; culture temperature is 30℃, shaker rotation speed is 180rpm, culture time is 24h , To obtain the expansion medium of the filamentous fungus C.elegans AS3.2028;
(3)隐丹参酮的生物转化(3) Biotransformation of cryptotanshinone
将隐丹参酮添加到步骤(2)得到的丝状真菌C.elegans AS3.2028的扩增培养液中(隐丹参酮的终浓度为0.05mg/mL)，于28℃，摇床转速180rpm的条件下摇床培养72h，得转化物；Add cryptotanshinone to the expansion medium of the filamentous fungus C.elegans AS3.2028 obtained in step (2) (the final concentration of cryptotanshinone is 0.05 mg/mL), at 28°C, and the shaker rotates at 180 rpm Incubate on a shaker for 72 hours to obtain transformants;
(4)本发明式Ⅰ化合物的分离纯化(4) Separation and purification of the compound of formula I of the present invention
取步骤(3)所得的转化物(10L)，将转化液和菌体分离，转化液用乙酸乙酯萃取2次，合并萃取液、减压浓缩得到转化液浸膏，先进行正相硅胶柱色谱分离(固定相：200～300目硅胶，流动相：30％–90％的乙酸乙酯-石油醚混合溶剂)、再进行高效液相色谱分离(固定相：ODS色谱柱(Kromasil,5μm,150×10mm)，流动相：60％的甲醇-水混合溶剂)即得式Ⅰ化合物1(2.9mg，橘黄色粉末)，式Ⅰ化合物2(22.4mg)和式Ⅰ化合物3(8.3mg)。Take the transformant (10L) obtained in step (3), separate the transformant from the bacterial cells, extract the transformant twice with ethyl acetate, combine the extracts and concentrate under reduced pressure to obtain the transformant extract. First, perform a normal phase silica gel column Chromatographic separation (stationary phase: 200-300 mesh silica gel, mobile phase: 30%-90% ethyl acetate-petroleum ether mixed solvent), and then high performance liquid chromatography (stationary phase: ODS chromatography column (Kromasil, 5μm, 150×10mm), mobile phase: 60% methanol-water mixed solvent) to obtain formula I compound 1 (2.9 mg, orange powder), formula I compound 2 (22.4 mg) and formula I compound 3 (8.3 mg).
实施例3Example 3
本发明式Ⅰ化合物抑制NO产生活性测试Test for inhibiting NO production activity of the compound of formula I of the present invention
式Ⅰ化合物的抑制NO产生以小胶质细胞BV-2为评价模型，实验在96孔板中进行，添加不同浓度的式Ⅰ化合物、底物隐丹参酮、阳性对照槲皮素及空白对照DMSO，然后每孔中添加1μg/mL的LPS孵育24h。通过Griess反应检测NO产生量，每孔中吸取50μL的细胞上清液转移到新的96孔板中，加入等量的Griess试剂混匀，540nm下检测吸光度，计算NO产生量及抑制率。实验重复3次，结果以均值±SD表示，如表1所示。底物隐丹参酮的抗神经炎症活性比阳性对照槲皮素高2.5倍，而式Ⅰ化合物1，式Ⅰ化合物2和式Ⅰ化合物3的抗神经炎症活性比底物高6.7倍，2.3倍和20.6倍。这表明通过生物转化的方法能使得隐丹参酮的抗炎活性明显提高The inhibition of NO production by the compound of formula I was evaluated using microglia BV-2 as an evaluation model. The experiment was carried out in a 96-well plate, adding different concentrations of the compound of formula I, the substrate cryptotanshinone, the positive control quercetin and the blank control DMSO, Then add 1μg/mL LPS to each well and incubate for 24h. The amount of NO production was detected by Griess reaction. 50μL of cell supernatant from each well was transferred to a new 96-well plate, the same amount of Griess reagent was added and mixed, the absorbance was measured at 540nm, and the amount of NO production and inhibition rate were calculated. The experiment was repeated 3 times, and the results are expressed as mean±SD, as shown in Table 1. The anti-neuro-inflammatory activity of the substrate cryptotanshinone is 2.5 times higher than that of the positive control quercetin, while the anti-neuro-inflammatory activity of formula I compound 1, formula I compound 2 and formula I compound 3 are 6.7 times, 2.3 times and 20.6 times higher than that of the substrate. Times. This shows that the anti-inflammatory activity of cryptotanshinone can be significantly improved by the method of biotransformation.
表1本发明的式Ⅰ化合物对LPS诱导BV2细胞产NO的抑制活性Table 1 The inhibitory activity of the compound of formula I of the present invention on LPS-induced NO production in BV2 cells
Figure PCTCN2020096101-appb-000002
Figure PCTCN2020096101-appb-000002
实施例4Example 4
本发明式Ⅰ化合物抑制炎症因子IL1β、IL6及TNFα释放的活性测试Activity test of the compound of formula I of the present invention in inhibiting the release of inflammatory factors IL1β, IL6 and TNFα
神经细胞BV-2培养于6孔板中，添加10μM式Ⅰ化合物、底物隐丹参酮，37℃孵育1h后，每孔添加1μg/mL LPS并设置未加LPS为对照培养16h，4000rpm离心5min，收集上清液于新的1.5mL无菌离心管，重复三次。Nerve cells BV-2 were cultured in a 6-well plate, 10μM compound of formula I and the substrate cryptotanshinone were added, after incubation at 37°C for 1h, 1μg/mL LPS was added to each well and no LPS was added as a control, cultured for 16h, centrifuged at 4000rpm for 5min, Collect the supernatant in a new 1.5 mL sterile centrifuge tube and repeat three times.
神经炎症因子释放测定采用联科ELSIA试剂盒，IL1β(EK201B-01)、IL6(EK206-01)及TNFα(EK282-01)。The release of neuroinflammatory factors was determined using Lianke ELSIA kit, IL1β (EK201B-01), IL6 (EK206-01) and TNFα (EK282-01).
(1)加样：加适当稀释的待检样品100μl于上述已包被的反应孔中。(同时做空白孔，倍比稀释的标准品孔，以阴性对照孔及阳性对照孔作为质控点)。(1) Add sample: add 100μl of the appropriately diluted sample to be tested into the above-mentioned coated reaction well. (At the same time, make blank wells, standard wells with multiple dilutions, and use negative control wells and positive control wells as quality control points).
(2)温育：用封板膜封板后置37℃孵育1-2h。(2) Incubation: After sealing the plate with a sealing film, incubate at 37°C for 1-2h.
(3)洗涤：弃去液体，每孔加入300uL洗液，浸泡1-2min，在吸水纸上拍干，重复3遍。(3) Washing: Discard the liquid, add 300uL lotion to each hole, soak for 1-2min, pat dry on absorbent paper, and repeat 3 times.
(4)加抗体：于各孔中加稀释好的生物素化抗体工作液100μl。(4) Add antibody: add 100μl of diluted biotinylated antibody working solution to each well.
(5)温育：用封板膜封板后置37℃孵育1h。(5) Incubation: Seal the plate with a sealing film and incubate at 37°C for 1 hour.
(6)洗涤：同步骤3。(6) Washing: same as step 3.
(7)加酶结合物：于各孔中加稀释好的酶结合物工作液100μl。(7) Add enzyme conjugate: add 100μl of diluted enzyme conjugate working solution to each well.
(8)温育：用封板膜封板后置37℃避光孵育30min。(8) Incubation: After sealing the plate with a sealing film, incubate at 37°C and avoid light for 30 minutes.
(9)洗涤：同步骤3。(9) Washing: same as step 3.
(10)加显色底物：于各孔中加入TMB底物溶液100μl，37℃避光反应10～30min，直到倍比稀释的标准品孔出现明显的颜色梯度为止。(10) Add chromogenic substrate: add 100μl of TMB substrate solution to each well, and react at 37°C in the dark for 10-30 minutes, until a clear color gradient appears in the wells diluted by multiples.
(11)终止反应：于各反应孔中加入2M硫酸100μl，颜色由蓝色变为黄色。(11) Terminating the reaction: Add 100 μl of 2M sulfuric acid to each reaction well, and the color changes from blue to yellow.
(12)结果测定：10min内，在酶标仪上，于450nm处，以空白对照孔调零后测各孔OD值。(12) Result determination: Within 10 minutes, measure the OD value of each well after zeroing the blank control well at 450nm on the microplate reader.
LPS刺激神经细胞BV-2产生释放了炎症因子IL1β、IL6及TNFα。式Ⅰ化合物和隐丹参酮均能显著抑制炎症因子的释放，而且式Ⅰ化合物的抑制活性更强(表2)。LPS stimulates the production of nerve cells BV-2 and releases the inflammatory factors IL1β, IL6 and TNFα. Both the compound of formula I and cryptotanshinone can significantly inhibit the release of inflammatory factors, and the inhibitory activity of the compound of formula I is stronger (Table 2).
表2本发明的式Ⅰ化合物抑制炎症因子的活性(IC 50±SD(μM)) Table 2 The compound of formula I of the present invention inhibits the activity of inflammatory factors (IC 50 ± SD (μM))
Figure PCTCN2020096101-appb-000003
Figure PCTCN2020096101-appb-000003
注：LPS组:IL1β,70.05±7.71pg/mL,IL6,377.77±39.02pg/mL,TNFα,759.44±45.55pg/mL；无LPS的对照组:IL1β,7.77±0.96pg/mL,IL6,1.27±0.12pg/mL,TNFα,70.84±9.93pg/mL。Note: LPS group: IL1β, 70.05±7.71pg/mL, IL6, 377.77±39.02pg/mL, TNFα, 759.44±45.55pg/mL; control group without LPS: IL1β, 7.77±0.96pg/mL, IL6, 1.27 ±0.12pg/mL, TNFα, 70.84±9.93pg/mL.
实施例5Example 5
本发明式Ⅰ化合物1抑制炎症因子iNOS、COX-2和TLR4的表达及JNK、ERK和p38的磷酸化水平检测The compound 1 of formula I of the present invention inhibits the expression of inflammatory factors iNOS, COX-2 and TLR4 and the phosphorylation level detection of JNK, ERK and p38
BV-2细胞接种于6孔培养板中，与化合物共孵育1h，再与LPS(1μg/mL)共孵育16h。用Pierce-Rapid-Gold-BCA蛋白检测试剂盒测定蛋白浓度。用10％十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)对每个定量的蛋白样品进行电泳，然后转移到聚偏二氟乙烯(PVDF)膜上。用含5％(W/V)脱脂乳在TBST缓冲液(含0.1％吐温20的三缓冲盐水)中封闭膜2h，然后在4℃下用一级抗体孵育膜过夜。TBST洗涤三次后，在室温下与相应的二级抗体孵育2小时，最后用化学发光成像仪(GE-Amersham成像仪600，美国)检测，并使用Image J密度成像软件(国立卫生研究院，MD)进行量化。结果如图1，式Ⅰ化合物均能显著抑制iNOS、COX-2和TLR4的表达，从而发挥抗炎效果。并且式Ⅰ化合物1还能抑制JNK、ERK和p38的磷酸化并呈现浓度依赖关系(图2)，说明 式Ⅰ化合物1主要是通过TLR4/MAPK通路发挥抗神经炎症作用。BV-2 cells were seeded in a 6-well culture plate, incubated with the compound for 1 h, and then incubated with LPS (1 μg/mL) for 16 h. The protein concentration was determined with Pierce-Rapid-Gold-BCA protein detection kit. Each quantitative protein sample was electrophoresed with 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and then transferred to a polyvinylidene fluoride (PVDF) membrane. The membrane was blocked with 5% (W/V) skim milk in TBST buffer (tri-buffered saline containing 0.1% Tween 20) for 2 h, and then the membrane was incubated with primary antibody overnight at 4°C. After TBST was washed three times, it was incubated with the corresponding secondary antibody for 2 hours at room temperature, and finally detected with a chemiluminescence imager (GE-Amersham Imager 600, USA) and Image J density imaging software (National Institutes of Health, MD) ) For quantification. The results are shown in Figure 1. The compounds of formula I can significantly inhibit the expression of iNOS, COX-2 and TLR4, thereby exerting an anti-inflammatory effect. In addition, compound 1 of formula I can inhibit the phosphorylation of JNK, ERK and p38 in a concentration-dependent relationship (Figure 2), indicating that compound 1 of formula I mainly exerts an anti-neuro-inflammatory effect through the TLR4/MAPK pathway.
实施例6Example 6
本发明式Ⅰ化合物1抑制NF-κB p65的效果检测Detection of the effect of compound 1 of formula I of the present invention on inhibiting NF-κB p65
BV-2细胞接种于6孔培养板中，与化合物共孵育1h，再与LPS(1μg/mL)共孵育16h。细胞爬片采用4％多聚甲醛和0.2％Triton X-100(PBS)处理。然后，用5％牛血清白蛋白(PBS)封闭1h，并在4℃条件下与抗体NF-κB p65孵育过夜，然后添加标记有Alexa Fluor 594的抗体孵育1h。用DAPI染色后，清洗并密封盖玻片，荧光显微镜拍照。结果表明，LPS能够激活NF-κB p65的产生和定位，而式Ⅰ化合物1能够明显抑制NF-κB p65的产生和定位(图3)。BV-2 cells were seeded in a 6-well culture plate, incubated with the compound for 1 h, and then incubated with LPS (1 μg/mL) for 16 h. The cell slide was treated with 4% paraformaldehyde and 0.2% Triton X-100 (PBS). Then, it was blocked with 5% bovine serum albumin (PBS) for 1 hour, and incubated with antibody NF-κB p65 at 4°C overnight, and then an antibody labeled with Alexa Fluor 594 was added and incubated for 1 hour. After staining with DAPI, wash and seal the cover glass, and take pictures with a fluorescence microscope. The results show that LPS can activate the production and localization of NF-κB p65, while compound 1 of formula I can significantly inhibit the production and localization of NF-κB p65 (Figure 3).
实施例7Example 7
本发明式Ⅰ化合物的神经保护活性测试The neuroprotective activity test of the compound of formula I of the present invention
式Ⅰ化合物的神经保护活性采用高浓度谷氨酸钠诱导的小鼠海马神经元细胞HT-22细胞毒性为评价模型。该实验在96孔板中进行，首先加入1μM和0.1μM式Ⅰ化合物、底物隐丹参酮和空白对照DMSO，预处理1h。每孔中添加5mM的谷氨酸钠，37℃孵育12h。采用MTT法评价细胞存活率。实验重复3次，采用Graphpad prism 7计算细胞存活率。结果如图4所示，在1μM和0.1μM浓度下，式Ⅰ化合物与底物隐丹参酮有着明显的神经保护活性。The neuroprotective activity of the compound of formula I adopts the cytotoxicity of mouse hippocampal neuron cells HT-22 induced by high concentration of sodium glutamate as an evaluation model. The experiment was carried out in a 96-well plate. First, 1 μM and 0.1 μM of formula I compound, substrate cryptotanshinone and blank control DMSO were added, and pretreated for 1 h. Add 5mM sodium glutamate to each well and incubate at 37°C for 12h. The MTT method was used to evaluate the cell survival rate. The experiment was repeated 3 times, and Graphpad prism 7 was used to calculate the cell survival rate. The results are shown in Figure 4. At concentrations of 1 μM and 0.1 μM, the compound of formula I and the substrate cryptotanshinone have obvious neuroprotective activity.
4讨论4 Discussion
本发明提供一个新的抗神经炎症剂和神经保护剂，其特征在于以本发明的隐丹参酮C-3位羰基化或羟基化产物或其盐作为有效成分，用于预防或治疗与神经炎症相关的神经退行性疾病，如阿尔兹海默症及帕金森综合征。且原料隐丹参酮来源广泛，菌体可大规模培养，均不受资源限制，因此应用前景广阔。The present invention provides a new anti-neuro-inflammatory agent and neuroprotective agent, which is characterized in that the C-3 position carbonylation or hydroxylation product of cryptotanshinone of the present invention or its salt is used as an effective ingredient for the prevention or treatment of neuroinflammation-related Of neurodegenerative diseases, such as Alzheimer’s disease and Parkinson’s syndrome. In addition, the raw material cryptotanshinone has a wide range of sources, and the bacteria can be cultured on a large scale without being restricted by resources, so the application prospect is broad.

Claims (10)

  1. 一种隐丹参酮衍生物或其药学上可接受的盐，其特征在于所述隐丹参酮衍生物具有式I所示结构：A cryptotanshinone derivative or a pharmaceutically acceptable salt thereof, characterized in that the cryptotanshinone derivative has the structure shown in formula I:
    Figure PCTCN2020096101-appb-100001
    Figure PCTCN2020096101-appb-100001
  2. 权利要求1所述的式Ⅰ化合物的制备方法，其特征在于包括如下步骤：The method for preparing the compound of formula I according to claim 1, characterized in that it comprises the following steps:
    (1)丝状真菌Cunninghamella elegans AS3.2028的培养(1) Cultivation of filamentous fungus Cunninghamella elegans AS3.2028
    培养基为PDA固体培养基，使用时制成试管斜面，培养温度为25-30℃，培养时间5-7天，得丝状真菌C.elegans AS3.2028；The medium is a PDA solid medium, which is made into a test tube slope when used. The culture temperature is 25-30°C, and the culture time is 5-7 days to obtain the filamentous fungus C.elegans AS3.2028;
    (2)丝状真菌C.elegans AS3.2028的扩增(2) Amplification of the filamentous fungus C.elegans AS3.2028
    用改良的察氏液体培养基摇床培养步骤(1)得到的丝状真菌C.elegans AS3.2028，所述改良的察氏液体培养基含有葡萄糖1.5％、蔗糖1.5％、蛋白胨0.5％、磷酸氢二钾0.1％、硫酸镁0.05％、氯化钾0.05％、硫酸亚铁0.001％，其余为水，上述百分含量均为重量百分比；培养温度为25-30℃，摇床转速180rpm，培养时间24h，得丝状真菌C.elegans AS3.2028的扩增培养液。The filamentous fungus C.elegans AS3.2028 obtained in step (1) was cultured with a modified Chashi liquid medium on a shaker, and the modified Chashi liquid medium contained glucose 1.5%, sucrose 1.5%, peptone 0.5%, and phosphoric acid. Dipotassium hydrogen 0.1%, magnesium sulfate 0.05%, potassium chloride 0.05%, ferrous sulfate 0.001%, the rest is water, the above percentages are all by weight; the cultivation temperature is 25-30°C, the shaker rotation speed is 180rpm, and the culture After 24 hours, the amplified culture medium of the filamentous fungus C.elegans AS3.2028 was obtained.
    (3)隐丹参酮的生物转化(3) Biotransformation of cryptotanshinone
    将隐丹参酮添加到步骤(2)得到的丝状真菌C.elegans AS3.2028的扩增培养液中，于28℃，摇床转速180rpm的条件下摇床培养72h，得转化物；The cryptotanshinone was added to the expansion culture solution of the filamentous fungus C.elegans AS3.2028 obtained in step (2), and cultured on a shaker at 28° C. and a shaker speed of 180 rpm for 72 hours to obtain a transformant;
    (4)本发明式Ⅰ化合物的分离纯化(4) Separation and purification of the compound of formula I of the present invention
    将步骤(3)得到的转化物中的转化液和菌体分离，转化液用乙酸乙酯萃取3次，合并萃取液、减压浓缩得到转化液浸膏，先进行正相硅胶柱色谱分离、再进行高效液相色谱分离即得式Ⅰ化合物。Separate the conversion solution and the bacteria in the conversion product obtained in step (3), extract the conversion solution 3 times with ethyl acetate, combine the extracts and concentrate under reduced pressure to obtain the conversion solution extract, which is separated by normal phase silica gel column chromatography, After separation by high performance liquid chromatography, the compound of formula I can be obtained.
  3. 权利要求2所述的方法，其特征在于所述步骤(3)中隐丹参酮的添加量优选添加后终浓度为0.05-0.20mg/mL。The method according to claim 2, characterized in that the amount of cryptotanshinone added in the step (3) is preferably 0.05-0.20 mg/mL after addition.
  4. 权利要求2-3任一项所述的方法，其特征在于所述步骤(4)中所述正相硅胶柱色谱分离采用的固定相：100～400目硅胶，流动相：30％–90％的乙酸乙酯-石 油醚混合溶剂；所述高效液相色谱分离采用的固定相：ODS色谱柱(Kromasil,5μm,150×10mm)，流动相：50％–60％的甲醇-水混合溶剂；上述混合溶剂百分比均为体积百分比。The method of any one of claims 2-3, characterized in that the stationary phase used for the normal phase silica gel column chromatographic separation in the step (4): 100-400 mesh silica gel, mobile phase: 30%-90% The mixed solvent of ethyl acetate-petroleum ether; the stationary phase used in the high-performance liquid chromatography separation: ODS chromatography column (Kromasil, 5μm, 150×10mm), mobile phase: 50%-60% methanol-water mixed solvent; The above-mentioned mixed solvent percentages are all volume percentages.
  5. 权利要求1所述的式I结构的隐丹参酮衍生物或其药学上可接受的盐在制备抗神经炎症药物中的应用。The use of the cryptotanshinone derivative with the structure of formula I according to claim 1 or a pharmaceutically acceptable salt thereof in the preparation of an anti-neuro-inflammatory drug.
  6. 权利要求1所述的式I结构的隐丹参酮衍生物或其药学上可接受的盐在制备神经保护药物中的应用。The use of the cryptotanshinone derivative with the structure of formula I or a pharmaceutically acceptable salt thereof according to claim 1 in the preparation of neuroprotective drugs.
  7. 一种药物组合物，其特征在于所述药物组合物以权利要求1所述的式I结构的隐丹参酮衍生物或其药学上可接受的盐作为有效成分。A pharmaceutical composition, characterized in that the pharmaceutical composition uses the cryptotanshinone derivative of the formula I described in claim 1 or a pharmaceutically acceptable salt thereof as an active ingredient.
  8. 权利要求7所述的药物组合物，其特征在于所述药物组合物还任选包括其他抗神经炎症药物和/或神经保护药物。The pharmaceutical composition according to claim 7, characterized in that the pharmaceutical composition optionally further comprises other anti-neuro-inflammatory drugs and/or neuroprotective drugs.
  9. 权利要求7-8任一项所述的药物组合物，其特征在于所述药物组合物还可包括药学上可接受的辅料。The pharmaceutical composition according to any one of claims 7-8, characterized in that the pharmaceutical composition can also include pharmaceutically acceptable excipients.
  10. 权利要求9所述的药物组合物，其特征在于所述药学上可接受的辅料选自药学上可接受的载体、稀释剂或赋形剂。The pharmaceutical composition of claim 9, wherein the pharmaceutically acceptable excipients are selected from pharmaceutically acceptable carriers, diluents or excipients.
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113307731A (en) * 2021-06-10 2021-08-27 广州中大南沙科技创新产业园有限公司 Cryptotanshinone derivative, preparation method thereof and application thereof in resisting myocardial fibrosis

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111269283B (en) * 2020-02-18 2021-01-15 中国海洋大学 Cryptotanshinone derivative, preparation method thereof and application thereof in anti-neuritis and neuroprotection
CN112999231A (en) * 2021-03-01 2021-06-22 杭州百晓生物技术有限公司 Application of medicine in reducing side effect of anesthetic

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102603861A (en) * 2012-02-25 2012-07-25 中国科学院昆明植物研究所 Tanshinone derivatives, medicine compositions thereof, and purposes thereof in medicine
CN111269283A (en) * 2020-02-18 2020-06-12 中国海洋大学 Cryptotanshinone derivative, preparation method thereof and application thereof in anti-neuritis and neuroprotection

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102603861A (en) * 2012-02-25 2012-07-25 中国科学院昆明植物研究所 Tanshinone derivatives, medicine compositions thereof, and purposes thereof in medicine
CN111269283A (en) * 2020-02-18 2020-06-12 中国海洋大学 Cryptotanshinone derivative, preparation method thereof and application thereof in anti-neuritis and neuroprotection

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
GUANGHONG CUI, DUAN LIXIN, JIN BAOLONG, QIAN JUN, XUE ZHEYONG, SHEN GUOAN, SNYDER JOHN, HUGH, SONG JINGYUAN, CHEN SHILIN, HUANG LU: "Functional divergence of diterpene syntheses in the medicinal plant Salvia miltiorrhiza", PLANT PHYSIOLOGY, AMERICAN SOCIETY OF PLANT PHYSIOLOGISTS, ROCKVILLE, MD, USA, vol. 169, 1 November 2015 (2015-11-01), Rockville, Md, USA, pages 1607 - 1618, XP055292937, ISSN: 0032-0889, DOI: 10.1104/pp.15.00695 *
MAIONE FRANCESCO; PICCOLO MARIALUISA; DE VITA SIMONA; CHINI MARIA GIOVANNA; CRISTIANO CLAUDIA; DE CARO CARMEN; LIPPIELLO PELLEGRIN: "Down regulation of pro-inflammatory pathways by tanshinone IIA and cryptotanshinone in a non-genetic mouse model of Alzheimer’s disease", PHARMACOLOGICAL RESEARCH., ACADEMIC PRESS, LONDON., GB, vol. 129, 20 November 2017 (2017-11-20), GB, pages 482 - 490, XP085353387, ISSN: 1043-6618, DOI: 10.1016/j.phrs.2017.11.018 *
SUN JIANG-HAO, YANG MIN, MA XIAO-CHI, KANG JIE, HAN JIAN, GUO DE-AN: "Microbial biotransformation of cryptotanshinone by Cunninghamella elegans and its application for metabolite identification in rat bile", JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH, GORDON AND BREACH, CHUR, CH, vol. 11, no. 6, 1 June 2009 (2009-06-01), CH, pages 482 - 489, XP055838567, ISSN: 1028-6020, DOI: 10.1080/10286020902877754 *
ZHU BOFENG, LI XIAOQIN;WU JINGSHUAI;YOU SONG;ZHOU LINA: "The Primary and Second Hydroxylation of Sclareol by Cunninghamella Elegans AS3.2028", SHENYANG YAOKE DAXUE XUEBAO - JOURNAL OF SHENYANG PHARMACEUTICAL UNIVERSITY., CHINESE ELECTRONIC PERIODICAL SERVICES, LIAONING, CN, vol. 35, no. 4, 1 April 2018 (2018-04-01), LIAONING, CN, pages 325 - 328, XP055838571, ISSN: 1006-2858, DOI: 10.14066 /j.cnki.cn21-1349 /r.2018.04.013 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113307731A (en) * 2021-06-10 2021-08-27 广州中大南沙科技创新产业园有限公司 Cryptotanshinone derivative, preparation method thereof and application thereof in resisting myocardial fibrosis

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