CN102584763B - 4-methyl-7-hydroxy-8-(1-hydroxyethyl) coumarin as well as preparation method and application thereof - Google Patents

Abstract

Translated fromChinese

本发明公开了一种4-甲基-7-羟基-8-(1-羟基乙基)香豆素及其制备方法与应用。本发明所提供的化合物结构式如式I所示。其是由4-甲基-7-羟基-8-乙酰基香豆素与NaBH₄反应得到的。该化合物的应用包括以下两方面：1、在制备预防和/或治疗神经细胞损伤的产品中的应用；2、在制备预防和/或治疗神经系统疾病的产品中的应用。体外药效实验证明，本发明的式I化合物对SH-SY5Y神经细胞损伤具有明显的保护作用。因此，该化合物在老年性痴呆、帕金森病等与氧自由基有一定关系的神经系统疾病领域具有重要意义，在临床应用中具有广阔前景。

(式I)。The invention discloses a 4-methyl-7-hydroxyl-8-(1-hydroxyethyl)coumarin, a preparation method and application thereof. The structural formula of the compound provided by the present invention is shown in Formula I. It is obtained from the reaction of 4-methyl-7-hydroxy-8-acetylcoumarin with_NaBH4 . The application of the compound includes the following two aspects: 1. Application in preparation of products for preventing and/or treating nerve cell damage; 2. Application in preparation of products for preventing and/or treating nervous system diseases. In vitro pharmacodynamic experiments prove that the compound of formula I of the present invention has obvious protective effect on SH-SY5Y nerve cell injury. Therefore, the compound is of great significance in the field of neurological diseases related to oxygen free radicals, such as senile dementia and Parkinson's disease, and has broad prospects in clinical application.

(Formula I).

Description

Translated fromChinese

4-甲基-7-羟基-8-(1-羟基乙基)香豆素及其制备方法与应用4-methyl-7-hydroxyl-8-(1-hydroxyethyl)coumarin and its preparation method and application

技术领域technical field

本发明涉及4-甲基-7-羟基-8-(1-羟基乙基)香豆素及其制备方法与应用。The invention relates to 4-methyl-7-hydroxyl-8-(1-hydroxyethyl)coumarin and its preparation method and application.

背景技术Background technique

阿尔茨海默病(Alzheimer’s disease，AD)，又称老年痴呆症，它是一种以记忆力丧失和认知功能障碍为主要特点的老年性脑神经退行性病变，表现为大脑皮层获得性高级功能的全面障碍，包括记忆、感觉、运动、语言功能等的进行性损害。老年痴呆症的发病机制尚不清楚，许多学者从不同角度提出了多种病因假说，如基因学说、胆碱能学说、β淀粉样蛋白学说、能量代谢学说、雌激素学说、炎性病变、自由基氧化损伤、基因缺陷与突变等，其中，氧自由基学说与上述部分学说又有关联。近年来越来越多的证据表明，多种神经退行性病变与氧化应激密切相关，并认为氧化应激在AD发生、发展中起着关键作用。H₂O₂是一种重要的活性氧成分，在体内可转变成细胞毒性极强的·OH，是一种膜易透性氧化剂，在许多神经元细胞培养模型中引起了细胞损伤。它参与许多神经系统疾病的发病机制，常被用作神经细胞氧化损伤的诱导剂。香豆素类化合物具有一定的抗氧化、抑制血小板凝聚、抗菌、抗炎、抗病毒、抗艾滋病和抗肿瘤等广泛生物活性。Alzheimer's disease (AD), also known as senile dementia, is an senile cranial neurodegenerative disease characterized by memory loss and cognitive dysfunction, manifested as the acquisition of advanced functions in the cerebral cortex comprehensive impairment of memory, sensory, motor, and language functions. The pathogenesis of senile dementia is still unclear, and many scholars have proposed a variety of etiological hypotheses from different angles, such as genetic theory, cholinergic theory, β-amyloid protein theory, energy metabolism theory, estrogen theory, inflammatory lesions, free Radical oxidative damage, gene defects and mutations, etc., among which, the theory of oxygen free radicals is related to some of the above theories. In recent years, more and more evidences have shown that a variety of neurodegenerative diseases are closely related to oxidative stress, and it is believed that oxidative stress plays a key role in the occurrence and development of AD. H₂ O₂ , an important reactive oxygen species, can be transformed into OH in vivo, which is extremely cytotoxic, a membrane-permeable oxidant, and has caused cell damage in many neuronal cell culture models. It is involved in the pathogenesis of many neurological diseases and is often used as an inducer of oxidative damage to nerve cells. Coumarin compounds have a wide range of biological activities such as anti-oxidation, inhibition of platelet aggregation, anti-bacterial, anti-inflammatory, anti-virus, anti-AIDS and anti-tumor.

发明内容Contents of the invention

本发明的目的是提供4-甲基-7-羟基-8-(1-羟基乙基)香豆素及其制备方法。The object of the present invention is to provide 4-methyl-7-hydroxyl-8-(1-hydroxyethyl)coumarin and its preparation method.

本发明所提供的4-甲基-7-羟基-8-(1-羟基乙基)香豆素，其结构式如式I所示：4-methyl-7-hydroxyl-8-(1-hydroxyethyl)coumarin provided by the present invention has a structural formula as shown in formula I:

(式I)(Formula I)

其制备方法如下：Its preparation method is as follows:

使式II所示的4-甲基-7-羟基-8-乙酰基香豆素与NaBH₄进行反应，得到式I所示4-甲基-7-羟基-8-(1-羟基乙基)香豆素。4-methyl-7-hydroxyl-8-acetylcoumarin shown in formula II is reacted with NaBH to obtain₄ -methyl-7-hydroxyl-8-(1-hydroxyethyl ) coumarin.

(式II)(Formula II)

本发明的再一个目的是提供式I所示4-甲基-7-羟基-8-(1-羟基乙基)香豆素的应用。Another object of the present invention is to provide the application of 4-methyl-7-hydroxyl-8-(1-hydroxyethyl)coumarin shown in formula I.

本发明所提供的式I化合物的应用包括以下两方面：1、在制备预防和/或治疗神经细胞损伤的产品中的应用；2、在制备预防和/或治疗神经系统疾病的产品中的应用。The application of the compound of formula I provided by the present invention includes the following two aspects: 1. Application in the preparation of products for the prevention and/or treatment of nerve cell damage; 2. Application in the preparation of products for the prevention and/or treatment of nervous system diseases .

所述神经细胞损伤具体可为神经细胞氧化损伤。The nerve cell damage can specifically be nerve cell oxidative damage.

所述神经系统疾病具体可为与氧自由基有关的神经系统疾病，包括：阿尔茨海默病、帕金森病等。The nervous system disease can specifically be a nervous system disease related to oxygen free radicals, including: Alzheimer's disease, Parkinson's disease and the like.

所述产品可为药品和/或保健品。The product can be medicine and/or health product.

本发明还保护一种预防和/或治疗神经细胞损伤的产品，其有效成分为式I所示的化合物。The present invention also protects a product for preventing and/or treating nerve cell damage, the active ingredient of which is the compound represented by formula I.

以式(I)所示化合物为有效成分制备的预防和/或治疗神经系统疾病的药物，也属于本发明的保护范围。Drugs for the prevention and/or treatment of nervous system diseases prepared with compounds represented by formula (I) as active ingredients also belong to the protection scope of the present invention.

所述药物可通过注射、喷射、滴鼻、滴眼、渗透、吸收、物理或化学介导的方法导入机体如肌肉、皮内、皮下、静脉、粘膜组织；或是被其他物质混合或包裹后导入机体。The drug can be introduced into the body through injection, spray, nasal drop, eye drop, penetration, absorption, physical or chemical mediated methods such as muscle, intradermal, subcutaneous, vein, mucosal tissue; or after being mixed or wrapped by other substances Import the body.

需要的时候，在上述药物中还可以加入一种或多种药学上可接受的载体。所述载体包括药学领域常规的稀释剂、赋形剂、填充剂、粘合剂、湿润剂、崩解剂、吸收促进剂、表面活性剂、吸附载体、润滑剂等。When necessary, one or more pharmaceutically acceptable carriers can also be added to the above drugs. The carrier includes conventional diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption promoters, surfactants, adsorption carriers, lubricants and the like in the pharmaceutical field.

用式I化合物制备的预防和/或治疗神经系统疾病的药物可以制成注射液、片剂、粉剂、颗粒剂、胶囊、口服液、膏剂、霜剂等多种形式。上述各种剂型的药物均可以按照药学领域的常规方法制备。The medicine for preventing and/or treating nervous system diseases prepared by the compound of formula I can be made into various forms such as injection, tablet, powder, granule, capsule, oral liquid, ointment, and cream. The above-mentioned medicines in various dosage forms can be prepared according to conventional methods in the field of pharmacy.

体外药效实验证明，本发明的化合物4-甲基-7-羟基-8-(1-羟基乙基)香豆素对SH-SY5Y神经细胞损伤具有明显的保护作用。因此，本发明化合物在老年性痴呆、帕金森病等与氧自由基有一定关系的神经系统疾病领域具有重要意义，在临床应用中具有广阔前景。The in vitro pharmacodynamic experiment proves that the compound 4-methyl-7-hydroxyl-8-(1-hydroxyethyl)coumarin of the present invention has obvious protective effect on SH-SY5Y nerve cell injury. Therefore, the compound of the present invention is of great significance in the field of neurological diseases related to oxygen free radicals such as senile dementia and Parkinson's disease, and has broad prospects in clinical application.

附图说明Description of drawings

图1为4-甲基-7-羟基-8-(1-羟基乙基)香豆素的合成路线图；Fig. 1 is the synthetic route figure of 4-methyl-7-hydroxyl-8-(1-hydroxyethyl) coumarin;

图2为4-甲基-7-羟基-8-(1-羟基乙基)香豆素的晶体结构图；Fig. 2 is the crystal structure figure of 4-methyl-7-hydroxyl-8-(1-hydroxyethyl) coumarin;

图3为不同浓度的H₂O₂对SH-SY5Y神经细胞的损伤作用；Figure 3 shows the damage effect of different concentrations of H₂ O₂ on SH-SY5Y nerve cells;

图4为4-甲基-7-羟基-8-(1-羟基乙基)香豆素对H₂O₂损伤SH-SY5Y细胞的保护作用。Figure 4 shows the protective effect of 4-methyl-7-hydroxy-8-(1-hydroxyethyl)coumarin on SH-SY5Y cells damaged by H₂ O₂ .

具体实施方式Detailed ways

下面通过具体实施例对本发明进行说明，但本发明并不局限于此。The present invention will be described below through specific examples, but the present invention is not limited thereto.

下述实施例中所述实验方法，如无特殊说明，均为常规方法；所述试剂和生物材料，如无特殊说明，均可从商业途径获得。The experimental methods described in the following examples, unless otherwise specified, are conventional methods; the reagents and biological materials, unless otherwise specified, can be obtained from commercial sources.

实施例1、化合物4-甲基-7-羟基-8-(1-羟基乙基)香豆素的制备及鉴定Embodiment 1, preparation and identification of compound 4-methyl-7-hydroxyl-8-(1-hydroxyethyl)coumarin

化合物合成路线如图1所示。The synthesis route of the compound is shown in Figure 1.

一、制备方法1. Preparation method

称取4-甲基-7-羟基-8-乙酰基香豆素2.18g(10mmol)，无水乙醇30mL置于100mL三口烧瓶中，在50℃条件下，加入1.5g(20mmol)NaBH₄反应1h后，再加入1.5g(20mmol)NaBH₄继续反应2h，反应毕。冷却至室温后加入适量丙酮和水，然后用10％盐酸调节pH＝5，静置过夜，抽滤，干燥得粗品，用95％的乙醇重结晶得无色透明柱状晶体，产率80％(1.76g)。Weigh 2.18g (10mmol) of 4-methyl-7-hydroxy-8-acetylcoumarin, put 30mL of absolute ethanol in a 100mL three-necked flask, and add 1.5g (20mmol) of NaBH₄ to react at 50°C After 1h, 1.5g (20mmol) NaBH₄ was added to continue the reaction for 2h, and the reaction was completed. After cooling to room temperature, add an appropriate amount of acetone and water, then adjust the pH=5 with 10% hydrochloric acid, let stand overnight, filter with suction, and dry to obtain a crude product, which is recrystallized with 95% ethanol to obtain a colorless transparent columnar crystal with a yield of 80% ( 1.76g).

二、鉴定2. Identification

该化合物的结构信息：Structural information for this compound:

¹H NMR：400MHz CDCl₃，δ7.72(s，1H)，6.09～6.11(m，1H)，5.14～1.16(m，1H)，2.44(s，3H)，1.43(d，J＝6.4Hz，3H).¹ H NMR: 400MHz CDCl₃ , δ7.72(s, 1H), 6.09～6.11(m, 1H), 5.14～1.16(m, 1H), 2.44(s, 3H), 1.43(d, J=6.4Hz , 3H).

LC/MS(ESI)m/z 221[M+H]⁺，243[M+Na]⁺LC/MS(ESI) m/z 221[M+H]⁺ , 243[M+Na]⁺

三、晶体结构测定3. Determination of crystal structure

选取一粒尺寸大小为0.5mm×0.3mm×0.3mm的单晶，置于Bruker SMART CCD1000单晶衍射仪上，采用单色M_oK_α

辐射，在298(2)K温度下，以

方式扫描，在2.37≤θ≤25.01°范围内共收集到衍射点8405个，其中独立衍射点1900个(R_int＝0.1147)，全部1900个独立衍射点被用于结构精修，采用直接法确认全部非氢原子，并且被各向异性地精修。全部氢原子采用几何加氢法得到，并且采用刚性模型处理。结构分析表明，该晶体属于单斜晶系，P2₁/c空间群，晶胞参数：a＝0.79609(9)nm，b＝1.7166(2)nm，c＝0.78586(9)nm，β＝91.857(1)°，V＝21.073.3(2)nm³，Z＝4，D_c＝1.363g·cm^-3，F(000)＝464，吸收系数μ＝0.103mm^-1，残差因子R₁＝0.0419，wR₂＝0.1147[I＞2σ(I)]，S＝1.067。最终差值电子云图中最大残余正峰和最大残余负峰分别为Δρ_max＝169e·nm^-3和Δρ_min＝-209e·nm^-3。晶体结构解析和精修采用SHELXS-97和SHELXL-97程序完成(参考文献Sheldrick，G M.Acta Cryst.A.2008，64(1)：112)。Select a single crystal with a size of 0.5mm×0.3mm×0.3mm, place it on a Bruker SMART CCD1000 single crystal diffractometer, and use monochromatic M_o K_α

radiation, at a temperature of 298(2)K, with

A total of 8405 diffraction points were collected in the range of 2.37≤θ≤25.01°, including 1900 independent diffraction points (R_int =0.1147), and all 1900 independent diffraction points were used for structure refinement and confirmed by the direct method All non-hydrogen atoms and are anisotropically refined. All hydrogen atoms were obtained by geometric hydrogenation and treated with a rigid model. Structural analysis shows that the crystal belongs to monoclinic system, P2₁ /c space group, unit cell parameters: a=0.79609(9)nm, b=1.7166(2)nm, c=0.78586(9)nm, β=91.857 (1)°, V=21.073.3(2)nm³ , Z=4, D_c =1.363g·cm^-3 , F(000)=464, absorption coefficient μ=0.103mm^-1 , residual factor R₁ =0.0419, wR₂ =0.1147 [I>2σ(I)], S=1.067. The maximum residual positive peak and maximum residual negative peak in the final difference electron nephogram are Δρ_max =169e·nm^-3 and Δρ_min =-209e·nm^-3 , respectively. Crystal structure elucidation and refinement were performed using SHELXS-97 and SHELXL-97 programs (reference Sheldrick, G M. Acta Cryst. A. 2008, 64(1): 112).

实施例2、4-甲基-7-羟基-8-(1-羟基乙基)香豆素的药效试验Drug efficacy test ofembodiment 2, 4-methyl-7-hydroxyl-8-(1-hydroxyethyl)coumarin

4-甲基-7-羟基-8-(1-羟基乙基)香豆素在体外对SH-SY5Y神经细胞损伤的保护作用Protective effect of 4-methyl-7-hydroxy-8-(1-hydroxyethyl)coumarin on SH-SY5Y nerve cell injury in vitro

采用体外细胞培养的方法，建立SH-SY5Y神经细胞过氧化氢(H₂O₂)损伤模型。本实验选用的细胞模型SH-SY5Y细胞(American Type Culture Collection，VA，USA)来源于人的神经母细胞瘤，其形态与正常神经元相似，呈锥形而且具有明显的轴突，某些生理功能亦与正常神经元有相似之处，是国际上目前研究神经细胞功能较好的一种细胞模型。实验发现，200μM的H₂O₂能够产生明显的SH-SY5Y神经细胞损伤作用(图3)。在之后的研究中，应用该浓度作为损伤浓度，通过观察细胞形态，测定细胞存活率(MTS法)，检测化合物4-甲基-7-羟基-8-(1-羟基乙基)香豆素对SH-SY5Y神经细胞损伤的保护作用。即，将SH-SY5Y细胞按每1mL含5×10⁴个细胞接种至96孔板，细胞生长汇合率达60％～70％时，空白对照和模型组换成含5％胎牛血清的DMEM培养液，药物组换为含试验化合物0.2，2，20，200μmol·L^-1的5％胎牛血清DMEM培养液，每个浓度设定平行的6个孔，继续培养24h后，加入终浓度为200μmol·L^-1的H₂O₂，继续培养1h后，每孔加MTS 10μL，置于37℃，5％CO₂细胞培养箱中孵育3h，振荡后于波长490nm处测定吸光度(Absorbance)。应用Graphpad Prism 4统计处理软件进行统计分析，结果以“均值±标准差”表示，采用方差分析(ANOVA)和t检验进行比较，P＜0.05认为具有统计学差异。同模型组比较，化合物能显著地减轻H₂O₂引起的SH-SY5Y神经细胞的损伤，明显提高细胞的存活率(图4)。The hydrogen peroxide (H₂ O₂ ) injury model of SH-SY5Y nerve cells was established by means of in vitro cell culture. The cell model SH-SY5Y cell (American Type Culture Collection, VA, USA) used in this experiment is derived from human neuroblastoma, its shape is similar to normal neurons, it is cone-shaped and has obvious axons, and some physiological The function is also similar to that of normal neurons, and it is a good cell model for studying the function of nerve cells in the world. Experiments found that 200 μM H₂ O₂ can produce obvious damage to SH-SY5Y nerve cells ( FIG. 3 ). In subsequent studies, this concentration was used as the injury concentration, and the cell viability was determined by observing the cell morphology (MTS method), and the compound 4-methyl-7-hydroxyl-8-(1-hydroxyethyl)coumarin was detected Protective effect on SH-SY5Y nerve cell injury. That is, SH-SY5Y cells were inoculated into a 96-well plate with 5×¹⁰⁴ cells per 1 mL, and when the cell growth confluence reached 60% to 70%, the blank control and model groups were replaced with DMEM containing 5% fetal bovine serum Culture medium, the drug group was replaced with 5% fetal bovine serum DMEM culture medium containing test compounds 0.2, 2, 20, 200 μmol^L-1 , 6 parallel wells were set for each concentration, and after continuing to culture for 24 hours, the final concentration was added 200 μmol·L^-1 H₂ O₂ , continue culturing for 1 hour, add 10 μL of MTS to each well, place in a 37°C, 5% CO₂ cell culture incubator and incubate for 3 hours, measure the absorbance (Absorbance) at a wavelength of 490 nm after shaking . Graphpad Prism 4 statistical processing software was used for statistical analysis, and the results were expressed as "mean ± standard deviation". Analysis of variance (ANOVA) and t-test were used for comparison, and P<0.05 was considered to be statistically different. Compared with the model group, the compound can significantly reduce the damage of SH-SY5Y nerve cells caused by H₂ O₂ , and significantly increase the survival rate of cells (Figure 4).

结果显示，4-甲基-7-羟基-8-(1-羟基乙基)香豆素对SH-SY5Y神经细胞损伤具有明显的保护作用。The results showed that 4-methyl-7-hydroxy-8-(1-hydroxyethyl)coumarin had obvious protective effect on SH-SY5Y nerve cell injury.

Claims (4)

Translated fromChinese

1.式Ⅰ所示的化合物在制备预防和/或治疗神经细胞损伤的产品中的应用；1. The application of the compound represented by formula I in the preparation of products for preventing and/or treating nerve cell damage;

2.根据权利要求1所述的应用，其特征在于：所述神经细胞损伤为神经细胞氧化损伤；所述产品为药品和/或保健品。2. The application according to claim 1, characterized in that: the nerve cell damage is nerve cell oxidative damage; the product is medicine and/or health care product.3.权利要求1中式Ⅰ所示化合物在制备预防和/或治疗神经系统疾病的产品中的应用。3. The use of the compound represented by formula I in claim 1 in the preparation of products for the prevention and/or treatment of nervous system diseases.4.根据权利要求3所述的应用，其特征在于：所述神经系统疾病为与氧自由基有关的神经系统疾病；所述产品为药品和/或保健品。4. The application according to claim 3, characterized in that: the nervous system disease is a nervous system disease related to oxygen free radicals; the product is a medicine and/or a health care product.

Images