CN102786484B - Ligustrazine methanoyl cinnamic acid derivative and preparation method thereof and application - Google Patents

Abstract

Translated fromChinese

本发明涉及川芎嗪甲酰氧基肉桂酸类衍生物及其制备方法与应用，具有如下结构通式：其中R为氢、羟基、卤素原子取代基或甲氧基中的一种或多种组合，R′为-H、-CH₃或-C₂H₅。本发明的川芎嗪甲酰氧基肉桂酸类衍生物可作为抗血小板聚集药物或血管内皮细胞保护药物应用。川芎嗪甲酰氧基肉桂酸类衍生物中的肉桂酸基团具有确切的抗血小板聚集活性和抗氧化活性，且在体内能以相对较快的速度水解产生酚羟基，以发挥抗氧化作用。另外川芎嗪甲酰氧基肉桂酸类衍生物不具有钙通道阻滞剂的副作用，且不会产生胺类物质。The present invention relates to ligustrazine formyloxycinnamic acid derivatives and their preparation method and application, having the following general structural formula: wherein R is one or more combinations of hydrogen, hydroxyl, halogen atom substituent or methoxy , R' is -H, -CH₃ or -C₂ H₅ . The ligustrazine formyloxycinnamic acid derivatives of the invention can be used as anti-platelet aggregation drugs or vascular endothelial cell protection drugs. The cinnamic acid group in ligustrazine formyloxycinnamic acid derivatives has exact anti-platelet aggregation activity and antioxidant activity, and can be hydrolyzed at a relatively fast speed in vivo to produce phenolic hydroxyl groups to exert antioxidant effects. In addition, the ligustrazine formyloxycinnamic acid derivatives do not have the side effects of calcium channel blockers, and do not produce amine substances.

Description

Translated fromChinese

川芎嗪甲酰氧基肉桂酸类衍生物及其制备方法与应用Ligustrazine formyloxycinnamic acid derivatives and preparation method and application thereof

技术领域technical field

本发明涉及川芎嗪衍生物，特别涉及川芎嗪甲酰氧基肉桂酸类衍生物及其制备方法和应用，属于抗心脑血管疾病药物技术领域。The invention relates to ligustrazine derivatives, in particular to ligustrazine formyloxycinnamic acid derivatives and their preparation method and application, belonging to the technical field of anti-cardiovascular and cerebrovascular disease drugs.

背景技术Background technique

心脑血管疾病是严重危害人类健康的一种疾病，具有“高发病率、高致残率、高死亡率、高复发率，多并发症”即“四高一多”的特点。据统计，全世界由各种心脑血管疾病导致的死亡人数高居各种死亡病因的首位。在我国，心脑血管疾病发病率不断升高，每年由心脑血管疾病导致的死亡数近300万，在严重威胁人类健康的同时，也给社会带来沉重的经济负担。因此，研究开发心脑血管药物仍是药物研究的重要内容之一。Cardiovascular and cerebrovascular disease is a disease that seriously endangers human health. It has the characteristics of "high incidence, high disability rate, high mortality rate, high recurrence rate, and multiple complications", that is, "four highs and one high". According to statistics, the number of deaths caused by various cardiovascular and cerebrovascular diseases in the world ranks first among various causes of death. In my country, the incidence of cardiovascular and cerebrovascular diseases continues to rise, and the number of deaths caused by cardiovascular and cerebrovascular diseases is nearly 3 million every year. While seriously threatening human health, it also brings a heavy economic burden to society. Therefore, the research and development of cardiovascular and cerebrovascular drugs is still one of the important contents of drug research.

中药川芎是伞形科植物川芎的干燥根茎，具有开郁燥湿、祛风止痛等功效。川芎嗪是从川芎中分离出的生物碱，为其主要有效成分，化学结构为2,3,5,6-四甲基吡嗪(Tetramethylpyrazine,TMP)，结构式如下所示：The traditional Chinese medicine Chuanxiong is the dried rhizome of the Umbelliferae plant Chuanxiong. Ligustrazine is an alkaloid isolated from Ligusticum chuanxiong. Its main active ingredient is 2,3,5,6-tetramethylpyrazine (Tetramethylpyrazine, TMP). The structural formula is as follows:

川芎嗪具有扩张血管、抗血小板聚集、清除自由基等多种多样的药理作用，因此在临床上广泛用于缺血性脑血管疾病和冠状动脉粥样硬化等疾病的治疗。然而川芎嗪在体内药代动力学研究显示，其生物利用度低、代谢快、半衰期短等特点使其应用受到限制（参见程先超等，川芎嗪的结构改造及修饰，药学进展，2005，6(29)，241-246）。因此以川芎嗪为先导化合物，对其进行结构修饰和改造，对研发新一代川芎嗪类心脑血管药物具有重要的意义。Ligustrazine has a variety of pharmacological effects such as dilating blood vessels, anti-platelet aggregation, and scavenging free radicals, so it is widely used clinically in the treatment of ischemic cerebrovascular diseases and coronary atherosclerosis. However, studies on the pharmacokinetics of ligustrazine in vivo have shown that its low bioavailability, fast metabolism, and short half-life limit its application (see Cheng Xianchao et al., Structural transformation and modification of ligustrazine, Advances in Pharmacy, 2005, 6( 29), 241-246). Therefore, using ligustrazine as a lead compound to modify and transform its structure is of great significance for the development of a new generation of ligustrazine cardiovascular and cerebrovascular drugs.

根据药物设计中的拼合原理，本课题组曾将川芎嗪与带有酚羟基的肉桂酸类取代基结合，得到一系列川芎嗪肉桂酸酚醚类衍生物，并对其进行了ADP诱导的血小板聚集的抑制活性实验和保护血管内皮细胞过氧化损伤的活性实验，发现了部分有较高抗血小板聚集活性和对血管内皮细胞具有较好的保护活性的化合物。（参见HongfeiChen,GuoningLi,PengZhan,XinyongLiu,Ligustrazinederivatives.Part5:Design,synthesisandbiologicalevaluationofnovelligustrazinyloxy-cinnamicacidderivativesaspotentcardiovascularagents,EuropeanJournalofMedicinalChemistry，2011,46,5609-5615）在此基础上，对川芎嗪进行进一步结构修饰，设计合成了川芎嗪甲酰氧基肉桂酸类衍生物，以期发现新一代川芎嗪类心脑血管药物。According to the combination principle in drug design, our research group combined ligustrazine with cinnamic acid substituents with phenolic hydroxyl groups to obtain a series of ligustrazine cinnamic acid phenolic ether derivatives, and performed ADP-induced platelet The aggregation inhibitory activity experiment and the activity experiment of protecting vascular endothelial cells from peroxidative damage have found some compounds with higher anti-platelet aggregation activity and better protective activity on vascular endothelial cells. (See HongfeiChen, GuoningLi, PengZhan, XinyongLiu, Ligustrazine derivatives. Part5: Design, synthesis and biological evaluation of novelligustrazinyloxy-cinnamic acid derivative saspotent cardiovascular agents, European Journal of Medicinal Chemistry, 2011, 46, 5609-5615) Oxycinnamic acid derivatives, in order to discover a new generation of ligustrazine cardiovascular and cerebrovascular drugs.

发明内容Contents of the invention

本发明是提供一种川芎嗪甲酰氧基肉桂酸类衍生物及其制备方法和应用，该类衍生物具有更好的抗血小板聚集、保护血管内皮细胞的功能。The invention provides a ligustrazine formyloxycinnamic acid derivative and its preparation method and application. The derivative has better functions of resisting platelet aggregation and protecting vascular endothelial cells.

本发明采取的技术方案为：The technical scheme that the present invention takes is:

川芎嗪甲酰氧基肉桂酸类衍生物，具有如下结构通式：Ligustrazine formyloxycinnamic acid derivatives have the following general structural formula:

其中R为氢、羟基、卤素原子取代基或甲氧基中的一种或多种组合，R′为-H、-CH₃或-C₂H₅。Wherein R is one or more combinations of hydrogen, hydroxyl, halogen substituent or methoxy, and R' is -H, -CH₃ or -C₂ H₅ .

所述的川芎嗪甲酰氧基肉桂酸类衍生物，优选：The ligustrazine formyloxycinnamic acid derivatives are preferably:

(E)-3-(4-((3,5,6-三甲基吡嗪-2-羰基)氧)苯基)丙烯酸乙酯，(E)-3-(3-((3,5,6-三甲基吡嗪-2-羰基)氧)苯基)丙烯酸，(E)-3-(2-((3,5,6-三甲基吡嗪-2-羰基)氧)苯基)丙烯酸，(E)-3-(4-甲氧基-3-((3,5,6-三甲基吡嗪-2-羰基)氧)苯基)丙烯酸，(E)-3-(3-羟基-4-((3,5,6-三甲基吡嗪-2-羰基)氧)苯基)丙烯酸，(E)-3-(3-羟基-4-((3,5,6-三甲基吡嗪-2-羰基)氧)苯基)丙烯酸乙酯。(E)-3-(4-((3,5,6-trimethylpyrazine-2-carbonyl)oxy)phenyl)ethyl acrylate, (E)-3-(3-((3,5 ,6-trimethylpyrazine-2-carbonyl)oxy)phenyl)acrylic acid, (E)-3-(2-((3,5,6-trimethylpyrazine-2-carbonyl)oxy)benzene Base) acrylic acid, (E)-3-(4-methoxy-3-((3,5,6-trimethylpyrazine-2-carbonyl)oxy)phenyl)acrylic acid, (E)-3- (3-Hydroxy-4-((3,5,6-trimethylpyrazine-2-carbonyl)oxy)phenyl)acrylic acid, (E)-3-(3-Hydroxy-4-((3,5 ,6-Trimethylpyrazine-2-carbonyl)oxy)phenyl)ethyl acrylate.

上述川芎嗪甲酰氧基肉桂酸类衍生物的制备方法，包括步骤如下：The preparation method of the above-mentioned ligustrazine formyloxycinnamic acid derivatives comprises the following steps:

（1）取川芎嗪溶于水中，30-40°C条件下搅拌缓慢加入KMnO₄的水溶液，反应10-30h，TLC监测反应完全后，向反应液中加入亚硫酸氢钠饱和水溶液至高锰酸钾紫红色完全褪去，过滤混合液，滤渣用热水（30-40°C）清洗，合并滤液，浓盐酸调解pH=2-3，乙酸乙酯萃取，无水硫酸钠干燥，减压蒸干溶剂得3,5,6-三甲基吡嗪-2-甲酸；(1) Dissolve Ligustrazine in water, stir slowly at 30-40°C and add KMnO₄ aqueous solution, react for 10-30h, after TLC monitors that the reaction is complete, add saturated aqueous solution of sodium bisulfite to permanganate to the reaction solution The potassium purple color faded completely, filtered the mixture, washed the filter residue with hot water (30-40°C), combined the filtrates, adjusted the pH to 2-3 with concentrated hydrochloric acid, extracted with ethyl acetate, dried over anhydrous sodium sulfate, and evaporated to dryness under reduced pressure Solvent to get 3,5,6-trimethylpyrazine-2-carboxylic acid;

（2）取3,5,6-三甲基吡嗪-2-甲酸溶于无水二氯甲烷，冰浴条件下加入草酰氯，搅拌反应0.5-1h，TLC监测至反应完全后，减压蒸除剩余溶剂和草酰氯，得到3,5,6-三甲基吡嗪-2-甲酰氯；(2) Dissolve 3,5,6-trimethylpyrazine-2-carboxylic acid in anhydrous dichloromethane, add oxalyl chloride under ice-bath conditions, stir for 0.5-1h, monitor by TLC until the reaction is complete, then depressurize Evaporate the remaining solvent and oxalyl chloride to obtain 3,5,6-trimethylpyrazine-2-formyl chloride;

（3）将取代苯甲醛溶于吡啶，加入丙二酸和哌啶，于120°C下反应1-2h，TLC监测反应完毕后，蒸除吡啶，残余物加水溶解，以HCl调pH为2-3，乙酸乙酯萃取，有机层干燥，过滤，蒸除溶剂，得取代肉桂酸；(3) Dissolve the substituted benzaldehyde in pyridine, add malonic acid and piperidine, and react at 120°C for 1-2 hours. After the reaction is monitored by TLC, evaporate the pyridine, dissolve the residue in water, and adjust the pH to 2 with HCl -3, extraction with ethyl acetate, drying the organic layer, filtering, and evaporating the solvent to obtain substituted cinnamic acid;

（4）取步骤（2）的3,5,6-三甲基吡嗪-2-甲酰氯溶于无水二氯甲烷中，室温搅拌下加入取代肉桂酸、三乙胺，20-30°C反应0.5-1h，TLC监测反应完全后，蒸除剩余溶剂，残留物溶于乙酸乙酯，分别用水、饱和NaCl水溶液洗涤，有机层用无水硫酸钠干燥，过滤、浓缩得粗品，经快速柱分离，乙醇重结晶得纯品川芎嗪甲酰氧基肉桂酸；(4) Dissolve the 3,5,6-trimethylpyrazine-2-formyl chloride in step (2) in anhydrous dichloromethane, add substituted cinnamic acid and triethylamine under stirring at room temperature, 20-30° C was reacted for 0.5-1h. After the reaction was completed by TLC monitoring, the remaining solvent was evaporated, the residue was dissolved in ethyl acetate, washed with water and saturated NaCl aqueous solution respectively, the organic layer was dried with anhydrous sodium sulfate, filtered and concentrated to obtain a crude product, which was subjected to rapid Column separation, ethanol recrystallization to obtain pure Ligustrazine formyloxycinnamic acid;

（5）取川芎嗪甲酰氧基肉桂酸溶于乙醇溶液中，冰浴条件下逐滴加入氯化亚砜，反应液加热回流24h，TLC监测至反应完全，蒸除剩余溶剂，残余物溶于乙酸乙酯，分别用水、饱和氯化钠水溶液洗涤，有机层由无水硫酸钠干燥，过滤，浓缩得粗品，快速柱分离，正己烷重结晶得产品。(5) Dissolve ligustrazine formyloxycinnamic acid in ethanol solution, add thionyl chloride drop by drop under ice bath condition, heat the reaction solution to reflux for 24 hours, monitor by TLC until the reaction is complete, evaporate the remaining solvent, and dissolve the residue In ethyl acetate, washed with water and saturated aqueous sodium chloride solution, the organic layer was dried over anhydrous sodium sulfate, filtered, concentrated to obtain a crude product, separated by flash column, and recrystallized from n-hexane to obtain the product.

上述制备方法中：In the above-mentioned preparation method:

步骤（1）所述的川芎嗪与KMnO₄的摩尔比为1:1~4，优选1:4，川芎嗪与水的质量/体积比为1：25，g/mL。The molar ratio of ligustrazine to KMnO₄ in step (1) is 1:1-4, preferably 1:4, and the mass/volume ratio of ligustrazine to water is 1:25, g/mL.

步骤（2）中3,5,6-三甲基吡嗪-2-甲酸与草酰氯的摩尔比为1:1~2，优选1:1，3,5,6-三甲基吡嗪-2-甲酸与无水二氯甲烷的质量/体积比为1:25，g/mL。The molar ratio of 3,5,6-trimethylpyrazine-2-carboxylic acid to oxalyl chloride in step (2) is 1:1~2, preferably 1:1, 3,5,6-trimethylpyrazine- The mass/volume ratio of 2-formic acid to anhydrous dichloromethane is 1:25, g/mL.

步骤（3）中取代苯甲醛、丙二酸和哌啶的摩尔比为1:1:0.1，取代苯甲醛与吡啶的质量/体积比为1:25，g/mL。取代苯甲醛是：4-羟基苯甲醛、3-羟基苯甲醛、2-羟基苯甲醛、3-甲氧基-4-羟基苯甲醛、3-羟基-4-甲氧基苯甲醛、2-羟基-3-甲氧基苯甲醛、2-羟基-4-甲氧基苯甲醛、2-羟基-5-甲氧基苯甲醛、4-羟基-3,5-二甲氧基苯甲醛、3,4-二羟基苯甲醛、2-羟基-5-氯苯甲醛、或3,4,5-三羟基苯甲醛。In step (3), the molar ratio of substituted benzaldehyde, malonic acid and piperidine is 1:1:0.1, and the mass/volume ratio of substituted benzaldehyde to pyridine is 1:25, g/mL. Substituted benzaldehydes are: 4-hydroxybenzaldehyde, 3-hydroxybenzaldehyde, 2-hydroxybenzaldehyde, 3-methoxy-4-hydroxybenzaldehyde, 3-hydroxy-4-methoxybenzaldehyde, 2-hydroxybenzaldehyde -3-methoxybenzaldehyde, 2-hydroxy-4-methoxybenzaldehyde, 2-hydroxy-5-methoxybenzaldehyde, 4-hydroxy-3,5-dimethoxybenzaldehyde, 3, 4-dihydroxybenzaldehyde, 2-hydroxy-5-chlorobenzaldehyde, or 3,4,5-trihydroxybenzaldehyde.

步骤（4）中3,5,6-三甲基吡嗪-2-甲酰氯、取代肉桂酸、三乙胺的摩尔比为1:1:3，3,5,6-三甲基吡嗪-2-甲酰氯与无水二氯甲烷的质量/体积比为1:25-30，g/mL。In step (4), the molar ratio of 3,5,6-trimethylpyrazine-2-formyl chloride, substituted cinnamic acid and triethylamine is 1:1:3, 3,5,6-trimethylpyrazine The mass/volume ratio of -2-formyl chloride to anhydrous dichloromethane is 1:25-30, g/mL.

步骤（5）中川芎嗪甲酰氧基肉桂酸与氯化亚砜的摩尔比为1:1.2，川芎嗪甲酰氧基肉桂酸与乙醇的质量/体积比为1:30，g/mL。In step (5), the molar ratio of ligustrazine formyloxycinnamic acid to thionyl chloride is 1:1.2, and the mass/volume ratio of ligustrazine formyloxycinnamic acid to ethanol is 1:30, g/mL.

川芎嗪甲酰氧基肉桂酸类衍生物的合成路线如下：The synthetic route of ligustrazine formyloxycinnamic acid derivatives is as follows:

上述反应式中，其中R为H、OH、卤原子或甲氧基或这些取代基的自由组合，R′为H或C₂H₅。试剂：(i)丙二酸，吡啶，哌啶，120°C，(ii)KMnO₄，H₂O，35°C，(iii)草酰氯，二氯甲烷，冰浴，(iv)Et₃N，二氯甲烷，rt，(v)EtOH，SOCl₂，回流。In the above reaction formula, wherein R is H, OH, halogen atom or methoxy group or a free combination of these substituents, R' is H or C₂ H₅ . Reagents: (i) malonic acid, pyridine, piperidine, 120°C, (ii) KMnO₄ , H₂ O, 35°C, (iii) oxalyl chloride, dichloromethane, ice bath, (iv) Et₃ N, dichloromethane, rt, (v) EtOH,_SOCl2 , reflux.

表1.合成的川芎嗪甲酰氧基肉桂酸类衍生物Table 1. Synthesized ligustrazine formyloxycinnamic acid derivatives

本发明的川芎嗪甲酰氧基肉桂酸类衍生物可作为抗血小板聚集药物或血管内皮细胞保护药物应用。具体地说，作为抗血小板聚集药物或血管内皮细胞保护药物用于制备抗心脑血管疾病药物。在活性试验中表现出了良好的活性，证明该类化合物具有进一步研发的价值，可作为抗心脑血管疾病的先导化合物加以利用。The ligustrazine formyloxycinnamic acid derivatives of the invention can be used as anti-platelet aggregation drugs or vascular endothelial cell protection drugs. Specifically, it is used as an anti-platelet aggregation drug or a vascular endothelial cell protection drug for preparing anti-cardiovascular and cerebrovascular disease drugs. Good activity was shown in the activity test, which proves that this type of compound has the value of further research and development, and can be used as a lead compound for anti-cardiovascular and cerebrovascular diseases.

一种抗心脑血管疾病药物组合物，由本发明的川芎嗪甲酰氧基肉桂酸类衍生物和药学上可接受的辅料组成。An anti-cardiovascular and cerebrovascular disease pharmaceutical composition is composed of the tetramethylpyrazine formyloxycinnamic acid derivatives of the present invention and pharmaceutically acceptable auxiliary materials.

与现有技术比本发明的有益效果是：Compared with prior art, the beneficial effects of the present invention are:

与现有技术相比，本发明的川芎嗪酸是从川芎嗪由KMnO₄进行直接氧化得到。而现有技术路线则是将川芎嗪经单氮氧化，Boekelheide重排，水解，氧化的过程而制得川芎嗪酸。其中单氮氧化，水解反应两步反应耗时较长，而Boekelheide重排需要高温无水，反应条件苛刻，总反应收率低。本发明将川芎嗪直接以KMnO₄氧化，通过控制反应温度及反应时间，一步得到川芎嗪酸，收率较高，可达60%以上。Compared with the prior art, the ligustrazinic acid of the present invention is obtained from the direct oxidation of ligustrazine by KMnO₄ . The prior art route is to prepare Ligustrazine through the processes of mononitrogen oxidation, Boekelheide rearrangement, hydrolysis and oxidation. Among them, the two-step reaction of nitrogen oxidation and hydrolysis takes a long time, while Boekelheide rearrangement requires high temperature and no water, the reaction conditions are harsh, and the overall reaction yield is low. The present invention directly oxidizes ligustrazine with KMnO₄ , and obtains ligustrazinic acid in one step by controlling the reaction temperature and reaction time, and the yield is higher, up to more than 60%.

与川芎嗪酰基哌嗪和川芎嗪酰胺相比，川芎嗪甲酰氧基肉桂酸类衍生物是将川芎嗪甲酰基与各种肉桂酸结合而得到的化合物。川芎嗪酰基哌嗪是用哌嗪环将川芎嗪与乙酰水杨酰基，烟酰基等拼合而成，而川芎嗪酰胺则是将川芎胺与水杨酰基，2-吡啶酰基等拼合而成。与这两类化合物中的基团相比，川芎嗪甲酰氧基肉桂酸类衍生物中的肉桂酸基团具有确切的抗血小板聚集活性和抗氧化活性，且在体内能以相对较快的速度水解产生酚羟基，以发挥抗氧化作用。另外川芎嗪甲酰氧基肉桂酸类衍生物不具有钙通道阻滞剂的副作用，且不会产生胺类物质。Compared with ligustrazinyl piperazine and ligustrazinamide, ligustrazine formyloxycinnamic acid derivatives are compounds obtained by combining ligustrazine formyl with various cinnamic acids. Ligustrazinyl piperazine is formed by combining ligustrazine with acetylsalicyloyl, nicotinyl, etc. with a piperazine ring, while ligustrazinamide is formed by combining ligustramine, salicylyl, 2-pyridyl, etc. Compared with the groups in these two types of compounds, the cinnamic acid group in ligustrazine formyloxycinnamic acid derivatives has exact anti-platelet aggregation activity and antioxidant activity, and can be in vivo in a relatively fast Speed hydrolysis produces phenolic hydroxyl groups to exert antioxidant effects. In addition, the ligustrazine formyloxycinnamic acid derivatives do not have the side effects of calcium channel blockers, and do not produce amine substances.

具体实施方式detailed description

下面结合实施例进一步说明。Below in conjunction with embodiment further illustrate.

实施例1.3,5,6-三甲基吡嗪-2-甲酰氯的制备The preparation of embodiment 1.3,5,6-trimethylpyrazine-2-formyl chloride

取川芎嗪（13.6g，0.1mol）溶于水，缓慢加入KMnO₄（63.2g，0.4mol）的水溶液，于35°C搅拌反应24h。TLC监测至反应完全，向反应液中加入亚硫酸氢钠饱和水溶液至高锰酸钾紫红色完全褪去，过滤混合液，滤渣用热水（30-40°C）清洗，合并滤液，浓盐酸调解pH=2-3，乙酸乙酯萃取，无水硫酸钠干燥，减压蒸干溶剂。固体以乙酸乙酯重结晶，得3,5,6-三甲基吡嗪-2-甲酸，产率67%。取0.1mol3,5,6-三甲基吡嗪-2-甲酸溶于无水二氯甲烷，冰浴条件下加入0.1mol草酰氯，搅拌反应0.5h，TLC监测至反应完全后，减压蒸除剩余溶剂和草酰氯，得到3,5,6-三甲基吡嗪-2-甲酰氯。Ligustrazine (13.6 g, 0.1 mol) was dissolved in water, an aqueous solution of KMnO₄ (63.2 g, 0.4 mol) was slowly added, and the reaction was stirred at 35°C for 24 hours. TLC monitoring until the reaction is complete, add a saturated aqueous solution of sodium bisulfite to the reaction solution until the purple color of potassium permanganate completely fades, filter the mixed solution, wash the filter residue with hot water (30-40°C), combine the filtrates, adjust the pH with concentrated hydrochloric acid =2-3, extracted with ethyl acetate, dried over anhydrous sodium sulfate, and evaporated to dryness under reduced pressure. The solid was recrystallized from ethyl acetate to obtain 3,5,6-trimethylpyrazine-2-carboxylic acid with a yield of 67%. Take 0.1 mol of 3,5,6-trimethylpyrazine-2-carboxylic acid and dissolve it in anhydrous dichloromethane, add 0.1 mol of oxalyl chloride under ice-bath conditions, stir for 0.5 h, monitor by TLC until the reaction is complete, evaporate under reduced pressure After removing the remaining solvent and oxalyl chloride, 3,5,6-trimethylpyrazine-2-carbonyl chloride was obtained.

实施例2.(E)-3-(4-((3,5,6-三甲基吡嗪-2-羰基)氧)苯基)丙烯酸(F1)的制备Example 2. Preparation of (E)-3-(4-((3,5,6-trimethylpyrazine-2-carbonyl)oxy)phenyl)acrylic acid (F1)

取反式4-羟基肉桂酸(0.33g,2mmol)，溶于10mL二氯甲烷，加入三乙胺(0.51g,6mmol)，搅拌5min，加入3,5,6-三甲基吡嗪-2-甲酰氯(0.37g,2mmol)，室温下搅拌反应2h。TLC监测反应完毕后，减压蒸除溶剂，残余物加入50mL乙酸乙酯，水洗，有机层以无水硫酸钠干燥，过滤，蒸除溶剂，将得到的粗品快速柱分离(乙酸乙酯：环己烷=1：5)，乙醇重结晶得产物，产率63%，mp:212-214°C。¹H-NMR(600MHz,CDCl₃,δppm):12.46(s,1H,COOH)，7.82(d，2H，Ar-H，J=8.4Hz)，7.65(d，1H，Ar-CH=C，J=15.6Hz)，7.35(d，2H，Ar-H，J=7.8Hz),6.57(d,1H,C=CH-C=O,J=15.6Hz),2.72(s,3H,CH₃),2.57(s,3H,CH₃),2.55(s,3H,CH₃).IR(KBr，cm^-1):3400(OH),2925(CH₃),1743,1693(C=O),1628(C=C),1599,1583,1506(C=N,C=C),1164(C-O).ESI-MS:313.3(M+H)⁺,calcd.forC₁₇H₁₆N₂O₄312.32.Take trans 4-hydroxycinnamic acid (0.33g, 2mmol), dissolve it in 10mL of dichloromethane, add triethylamine (0.51g, 6mmol), stir for 5min, add 3,5,6-trimethylpyrazine-2 -Formyl chloride (0.37g, 2mmol), stirred at room temperature for 2h. After the TLC monitoring reaction was completed, the solvent was evaporated under reduced pressure, the residue was added 50mL ethyl acetate, washed with water, the organic layer was dried with anhydrous sodium sulfate, filtered, and the solvent was evaporated, and the obtained crude product was separated by a fast column (ethyl acetate:cyclo Hexane=1:5), the product was obtained by recrystallization from ethanol, the yield was 63%, mp: 212-214°C.¹ H-NMR (600MHz, CDCl₃ , δppm): 12.46(s, 1H, COOH), 7.82(d, 2H, Ar-H, J=8.4Hz), 7.65(d, 1H, Ar-CH =C, J=15.6Hz), 7.35(d, 2H, Ar-H, J=7.8Hz), 6.57(d,1H, C=CH -C=O, J=15.6Hz), 2.72(s,3H,_CH3 ),2.57(s,3H,CH₃ ),2.55(s,3H,CH₃ ).IR(KBr,cm^-1 ):3400(OH),2925(CH₃ ),1743,1693(C=O) ,1628(C=C),1599,1583,1506(C=N,C=C),1164(CO).ESI-MS:313.3(M+H)⁺ ,calcd.forC₁₇ H₁₆ N₂ O₄ 312.32.

实施例3.(E)-3-(3-((3,5,6-三甲基吡嗪-2-羰基)氧)苯基)丙烯酸(F2)的制备Example 3. Preparation of (E)-3-(3-((3,5,6-trimethylpyrazine-2-carbonyl)oxy)phenyl)acrylic acid (F2)

取3-羟基肉桂酸(0.33g,2mmol)，溶于10mL二氯甲烷，加入三乙胺(0.51g,6mmol)，搅拌5min，加入3,5,6-三甲基吡嗪-2-甲酰氯(0.37g,2mmol)，室温下搅拌反应2h。TLC监测反应完毕后，减压蒸除溶剂，残余物加入50mL乙酸乙酯，水洗，有机层以无水硫酸钠干燥，过滤，蒸除溶剂，将得到的粗品快速柱分离(乙酸乙酯：环己烷=1：5)，乙醇重结晶得产物，产率54%,mp:208-210°C.¹H-NMR(600MHz,CDCl₃,δppm):12.50(s,1H,COOH),7.68-7.61(m，3H，Ar-H，Ar-CH=C)，7.54(t，1H，Ar-H)，7.36(dd，1H，Ar-H，J₁=1.2Hz，J₂=7.8Hz)，6.63(d，1H,C=CH-C=O,J=16.2Hz),2.73(s,3H,CH₃),2.57(s,3H,CH₃),2.55(s,3H,CH₃).IR(KBr，cm^-1):3450(OH),2966,2852(CH₃),1736,1692(C=O),1631(C=C),1580,1539(C=N,C=C),1172(C-O).ESI-MS:313.4(M+H)⁺,calcd.forC₁₇H₁₆N₂O₄312.32.Take 3-hydroxycinnamic acid (0.33g, 2mmol), dissolve it in 10mL of dichloromethane, add triethylamine (0.51g, 6mmol), stir for 5min, add 3,5,6-trimethylpyrazine-2-formazine Acid chloride (0.37g, 2mmol), stirred at room temperature for 2h. After the TLC monitoring reaction was completed, the solvent was evaporated under reduced pressure, the residue was added 50mL ethyl acetate, washed with water, the organic layer was dried with anhydrous sodium sulfate, filtered, and the solvent was evaporated, and the obtained crude product was separated by a fast column (ethyl acetate:cyclo Hexane=1:5), the product was recrystallized from ethanol, yield 54%, mp: 208-210°C.¹ H-NMR (600MHz, CDCl₃ , δppm): 12.50 (s, 1H, COOH), 7.68 -7.61(m, 3H, Ar-H, Ar-CH =C), 7.54(t, 1H, Ar-H), 7.36(dd, 1H, Ar-H, J₁ =1.2Hz, J₂ =7.8Hz ), 6.63(d,1H,C=CH -C=O,J=16.2Hz), 2.73(s,3H,CH₃ ), 2.57(s,3H,CH₃ ), 2.55(s,3H,CH₃ ).IR(KBr, cm^-1 ): 3450(OH), 2966, 2852(CH₃ ), 1736, 1692(C=O), 1631(C=C), 1580, 1539(C=N,C= C), 1172 (CO). ESI-MS: 313.4 (M+H)⁺ , calcd. for C₁₇ H₁₆ N₂ O₄ 312.32.

实施例4.(E)-3-(2-((3,5,6-三甲基吡嗪-2-羰基)氧)苯基)丙烯酸(F3)的制备Example 4. Preparation of (E)-3-(2-((3,5,6-trimethylpyrazine-2-carbonyl)oxy)phenyl)acrylic acid (F3)

取2-羟基肉桂酸(0.33g,2mmol)，溶于10mL二氯甲烷，加入三乙胺(0.51g,6mmol)，搅拌5min，加入3,5,6-三甲基吡嗪-2-甲酰氯(0.37g,2mmol)，室温下搅拌反应2h。TLC监测反应完毕后，减压蒸除溶剂，残余物加入50mL乙酸乙酯，水洗，有机层以无水硫酸钠干燥，过滤，蒸除溶剂，将得到的粗品快速柱分离(乙酸乙酯：环己烷=1：5)，乙醇重结晶得产物，产率57%,mp:204-206°C.¹H-NMR(600MHz,CDCl₃,δppm):12.48(s,1H,COOH),7.94(d,1H,Ar-H,J=7.8Hz),7.67(d,1H,Ar-CH=C,J=15.6Hz),7.56-7.53(t,1H,Ar-H),7.42-7.38(m,2H,Ar-H),6.75(d,1H,C=CH-C=O,J=15.6Hz),2.71(s,3H,CH₃),2.58(s,3H,CH₃),2.50(s,3H,CH₃).IR(KBr,cm^-1):3450(OH),2980,2929(CH₃),1733,1692(C=O),1627(C=C),1577,1543(C=N,C=C),1166(C-O).ESI-MS:313.4(M+H)⁺,calcd.forC₁₇H₁₆N₂O₄312.32.Take 2-hydroxycinnamic acid (0.33g, 2mmol), dissolve it in 10mL of dichloromethane, add triethylamine (0.51g, 6mmol), stir for 5min, add 3,5,6-trimethylpyrazine-2-formazine Acid chloride (0.37g, 2mmol), stirred at room temperature for 2h. After the TLC monitoring reaction was completed, the solvent was evaporated under reduced pressure, the residue was added 50mL ethyl acetate, washed with water, the organic layer was dried with anhydrous sodium sulfate, filtered, and the solvent was evaporated, and the obtained crude product was separated by a fast column (ethyl acetate:cyclo Hexane=1:5), the product was recrystallized from ethanol, yield 57%, mp: 204-206°C.¹ H-NMR (600MHz, CDCl₃ , δppm): 12.48 (s, 1H, COOH), 7.94 (d,1H,Ar-H,J=7.8Hz),7.67(d,1H,Ar-CH =C,J=15.6Hz),7.56-7.53(t,1H,Ar-H),7.42-7.38( m,2H,Ar-H),6.75(d,1H,C=CH -C=O,J=15.6Hz),2.71(s,3H,CH₃ ),2.58(s,3H,CH₃ ),2.50 (s,3H,CH₃ ).IR(KBr,cm^-1 ):3450(OH),2980,2929(CH₃ ),1733,1692(C=O),1627(C=C),1577,1543 (C=N,C=C),1166(CO).ESI-MS:313.4(M+H)⁺ ,calcd.for C₁₇ H₁₆ N₂ O₄ 312.32.

实施例5.(E)-3-(3-甲氧基-4-((3,5,6-三甲基吡嗪-2-羰基)氧)苯基)丙烯酸(F4)的制备Example 5. Preparation of (E)-3-(3-methoxy-4-((3,5,6-trimethylpyrazine-2-carbonyl)oxy)phenyl)acrylic acid (F4)

取3-甲氧基-4-羟基肉桂酸(0.38g,2mmol)，溶于11.4mL二氯甲烷，加入三乙胺(0.51g,6mmol)，搅拌5min，加入3,5,6-三甲基吡嗪-2-甲酰氯(0.37g,2mmol)，室温下搅拌反应2h。TLC监测反应完毕后，减压蒸除溶剂，残余物加入50mL乙酸乙酯，水洗，有机层以无水硫酸钠干燥，过滤，蒸除溶剂，将得到的粗品快速柱分离(乙酸乙酯：环己烷=1：5)，乙醇重结晶得产物，产率61%,mp:214-218°C.¹H-NMR(600MHz,CDCl₃,δppm):12.44(s,1H,COOH)，7.63(d，1H，Ar-CH=C，J=15.6Hz)，7.56(s，1H，Ar-H)，7.34(d，1H，Ar-H，J=7.8Hz)，7.30(d,1H,Ar-H,J=7.8Hz),6.65(d,1H,C=CH-C=O,J=16.2Hz),3.85(s,3H,OCH₃),2.70(s,3H,CH₃),2.56(s,3H,CH₃),2.51(s,3H,CH₃).IR(KBr,cm^-1):3422(OH),2924,2852(CH₃),1745,1696(C=O),1630(C=C),1591(C=N,C=C),1152(C-O).ESI-MS:343.4(M+H)⁺,calcd.forC₁₈H₁₈N₂O₅342.35.Take 3-methoxy-4-hydroxycinnamic acid (0.38g, 2mmol), dissolve it in 11.4mL of dichloromethane, add triethylamine (0.51g, 6mmol), stir for 5min, add 3,5,6-trimethyl Pyrazine-2-carbonyl chloride (0.37g, 2mmol), stirred at room temperature for 2h. After the TLC monitoring reaction was completed, the solvent was evaporated under reduced pressure, the residue was added 50mL ethyl acetate, washed with water, the organic layer was dried with anhydrous sodium sulfate, filtered, and the solvent was evaporated, and the obtained crude product was separated by a fast column (ethyl acetate:cyclo Hexane=1:5), the product was recrystallized from ethanol, yield 61%, mp: 214-218°C.¹ H-NMR (600MHz, CDCl₃ , δppm): 12.44 (s, 1H, COOH), 7.63 (d, 1H, Ar-CH =C, J=15.6Hz), 7.56(s, 1H, Ar-H), 7.34(d, 1H, Ar-H, J=7.8Hz), 7.30(d, 1H, Ar-H,J=7.8Hz),6.65(d,1H,C=CH -C=O,J=16.2Hz),3.85(s,3H,OCH₃ ),2.70(s,3H,CH₃ ), 2.56(s,3H,CH₃ ),2.51(s,3H,CH₃ ).IR(KBr,cm^-1 ):3422(OH),2924,2852(CH₃ ),1745,1696(C=O) ,1630(C=C),1591(C=N,C=C),1152(CO).ESI-MS:343.4(M+H)⁺ ,calcd.for C₁₈ H₁₈ N₂ O₅ 342.35.

实施例6.(E)-3-(4-甲氧基-3-((3,5,6-三甲基吡嗪-2-羰基)氧)苯基)丙烯酸(F5)的制备Example 6. Preparation of (E)-3-(4-methoxy-3-((3,5,6-trimethylpyrazine-2-carbonyl)oxy)phenyl)acrylic acid (F5)

取3-羟基-4-甲氧基肉桂酸(0.38g,2mmol)，溶于11.4mL二氯甲烷，加入三乙胺(0.51g,6mmol)，搅拌5min，加入3,5,6-三甲基吡嗪-2-甲酰氯(0.37g,2mmol)，室温下搅拌反应2h。TLC监测反应完毕后，减压蒸除溶剂，残余物加入50mL乙酸乙酯，水洗，有机层以无水硫酸钠干燥，过滤，蒸除溶剂，将得到的粗品快速柱分离(乙酸乙酯：环己烷=1：5)，乙醇重结晶得产物，产率64%,mp:202-204°C.¹H-NMR(600MHz,CDCl₃,δppm):12.36(s,1H,COOH),7.68-7.61(m,3H,Ar-H,Ar-CH=C),7.24(d,1H,Ar-H,J=8.4Hz),6.48(d,1H,C=CH-C=O,J=16.2Hz),3.84(s,3H,OCH₃),2.71(s,3H,CH₃),2.57-2.49(m,6H,CH₃×2).IR(KBr,cm^-1):3488(OH),2957,2927,2848(CH₃),1753,1720(C=O),1636(C=C),1610(C=N,C=C),1161(C-O).ESI-MS:343.5(M+H)⁺,calcd.forC₁₈H₁₈N₂O₅342.35.Take 3-hydroxy-4-methoxycinnamic acid (0.38g, 2mmol), dissolve it in 11.4mL of dichloromethane, add triethylamine (0.51g, 6mmol), stir for 5min, add 3,5,6-trimethyl Pyrazine-2-carbonyl chloride (0.37g, 2mmol), stirred at room temperature for 2h. After the TLC monitoring reaction was completed, the solvent was evaporated under reduced pressure, the residue was added 50mL ethyl acetate, washed with water, the organic layer was dried with anhydrous sodium sulfate, filtered, and the solvent was evaporated, and the obtained crude product was separated by a fast column (ethyl acetate:cyclo Hexane=1:5), the product was recrystallized from ethanol, yield 64%, mp: 202-204°C.¹ H-NMR (600MHz, CDCl₃ , δppm): 12.36 (s, 1H, COOH), 7.68 -7.61(m,3H,Ar-H,Ar-CH =C),7.24(d,1H,Ar-H,J=8.4Hz),6.48(d,1H,C=CH -C=O,J= 16.2Hz), 3.84(s,3H,OCH₃ ),2.71(s,3H,CH₃ ),2.57-2.49(m,6H,CH₃ ×2).IR(KBr,cm^-1 ):3488(OH ), 2957, 2927, 2848 (CH₃ ), 1753, 1720 (C=O), 1636 (C=C), 1610 (C=N, C=C), 1161 (CO).ESI-MS: 343.5 ( M+H)⁺ ,calcd.for C₁₈ H₁₈ N₂ O₅ 342.35.

实施例7.(E)-3-(4-((3,5,6-三甲基吡嗪-2-羰基)氧)苯基)丙烯酸乙酯(F′1)的制备Example 7. Preparation of (E)-3-(4-((3,5,6-trimethylpyrazine-2-carbonyl)oxy)phenyl)ethyl acrylate (F'1)

取(E)-3-(4-((3,5,6-三甲基吡嗪-2-羰基)氧)苯基)丙烯酸(0.62g,2mmol)溶于乙醇中，冰浴条件下缓慢滴加2.4mmol氯化亚砜，滴毕加热回流24h。TLC监测至反应完全，蒸除剩余溶剂，残余物溶于乙酸乙酯，分别用水、饱和氯化钠水溶液洗涤，有机层由无水硫酸钠干燥，过滤，浓缩得粗品。快速柱分离，正己烷重结晶，产率81%,mp:77-80°C.¹H-NMR(600MHz,CDCl₃,δppm):7.82(d,2H,Ar-H,J=8.4Hz),7.65(d,1H,Ar-CH=C,J=15.6Hz),7.35(d,2H,Ar-H,J=7.8Hz),6.57(d,1H,C=CH-C=O,J=15.6Hz),4.23(q,2H,OCH₂),2.72(s,3H,CH₃),2.57(s,3H,CH₃),2.55(s,3H,CH₃),1.30(t,3H,CH₃).IR(KBr,cm^-1):3063(C=C-H),2988,2929(CH₃),1747,1712(C=O),1642(C=C),1601,1541,1509(C=N,C=C),1169(C-O).ESI-MS:341.4(M+H)⁺,calcd.forC₁₉H₂₀N₂O₄340.37.Dissolve (E)-3-(4-((3,5,6-trimethylpyrazine-2-carbonyl)oxy)phenyl)acrylic acid (0.62g, 2mmol) in ethanol, and slowly Add 2.4 mmol thionyl chloride dropwise, and heat to reflux for 24 h after dropping. TLC monitored that the reaction was complete, and the remaining solvent was distilled off. The residue was dissolved in ethyl acetate, washed with water and saturated aqueous sodium chloride solution, and the organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product. Fast column separation, n-hexane recrystallization, yield 81%, mp: 77-80°C.¹ H-NMR (600MHz, CDCl₃ , δppm): 7.82 (d, 2H, Ar-H, J=8.4Hz) ,7.65(d,1H,Ar-CH =C,J=15.6Hz),7.35(d,2H,Ar-H,J=7.8Hz),6.57(d,1H,C=CH -C=O,J =15.6Hz), 4.23(q,2H,OCH₂ ),2.72(s,3H,CH₃ ),2.57(s,3H,CH₃ ),2.55(s,3H,CH₃ ),1.30(t,3H ,CH₃ ).IR(KBr,cm^-1 ):3063(C=CH ),2988,2929(CH₃ ),1747,1712(C=O),1642(C=C),1601,1541,1509 (C=N,C=C),1169(CO).ESI-MS:341.4(M+H)⁺ ,calcd.for C₁₉ H₂₀ N₂ O₄ 340.37.

实施例8.(E)-3-(3-((3,5,6-三甲基吡嗪-2-羰基)氧)苯基)丙烯酸乙酯(F′2)的制备Example 8. Preparation of (E)-3-(3-((3,5,6-trimethylpyrazine-2-carbonyl)oxy)phenyl)ethyl acrylate (F'2)

取(E)-3-(3-((3,5,6-三甲基吡嗪-2-羰基)氧)苯基)丙烯酸(0.62g,2mmol)溶于乙醇中，冰浴条件下缓慢滴加2.4mmol氯化亚砜，滴毕加热回流24h。TLC监测至反应完全，蒸除剩余溶剂，残余物溶于乙酸乙酯，分别用水、饱和氯化钠水溶液洗涤，有机层由无水硫酸钠干燥，过滤，浓缩得粗品。快速柱分离，正己烷重结晶，产率81%,mp:87-91°C.¹H-NMR(600MHz,CDCl₃,δppm):7.68-7.61(m，3H，Ar-H，Ar-CH=C)，7.54(t，1H，Ar-H)，7.36(dd，1H，Ar-H，J₁=1.2Hz，J₂=7.8Hz),6.63(d,1H,C=CH-C=O,J=16.2Hz),2.73(s,3H,CH₃),4.23(q,2H,OCH₂),2.57(s,3H,CH₃),2.55(s,3H,CH₃),1.30(t,3H,CH₃).IR(KBr,cm^-1):3061(C=C-H),2977,2927(CH₃),1731,1716(C=O),1637(C=C),1604,1583,1541(C=N,C=C),1169(C-O).ESI-MS:341.4(M+H)⁺,calcd.forC₁₉H₂₀N₂O₄340.37.Dissolve (E)-3-(3-((3,5,6-trimethylpyrazine-2-carbonyl)oxy)phenyl)acrylic acid (0.62g, 2mmol) in ethanol, and slowly Add 2.4 mmol thionyl chloride dropwise, and heat to reflux for 24 h after dropping. TLC monitored that the reaction was complete, and the remaining solvent was distilled off. The residue was dissolved in ethyl acetate, washed with water and saturated aqueous sodium chloride solution, and the organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product. Flash column separation, n-hexane recrystallization, yield 81%, mp: 87-91°C.¹ H-NMR (600MHz, CDCl₃ , δppm): 7.68-7.61 (m, 3H, Ar-H, Ar-CH =C), 7.54(t, 1H, Ar-H), 7.36(dd, 1H, Ar-H, J₁ =1.2Hz, J₂ =7.8Hz), 6.63(d, 1H, C=CH -C= O,J=16.2Hz),2.73(s,3H,CH₃ ),4.23(q,2H,OCH₂ ),2.57(s,3H,CH₃ ),2.55(s,3H,CH₃ ),1.30( t,3H,CH₃ ).IR(KBr,cm^-1 ):3061(C=CH ),2977,2927(CH₃ ),1731,1716(C=O),1637(C=C),1604, 1583,1541(C=N,C=C),1169(CO).ESI-MS:341.4(M+H)⁺ ,calcd.for C₁₉ H₂₀ N₂ O₄ 340.37.

实施例9.(E)-3-(2-((3,5,6-三甲基吡嗪-2-羰基)氧)苯基)丙烯酸乙酯(F′3)的制备Example 9. Preparation of (E)-3-(2-((3,5,6-trimethylpyrazine-2-carbonyl)oxy)phenyl)ethyl acrylate (F′3)

取(E)-3-(2-((3,5,6-三甲基吡嗪-2-羰基)氧)苯基)丙烯酸(0.62g,2mmol)溶于乙醇中，冰浴条件下缓慢滴加2.4mmol氯化亚砜，滴毕加热回流24h。TLC监测至反应完全，蒸除剩余溶剂，残余物溶于乙酸乙酯，分别用水、饱和氯化钠水溶液洗涤，有机层由无水硫酸钠干燥，过滤，浓缩得粗品。快速柱分离，正己烷重结晶，产率81%,mp:77-79°C.¹H-NMR(600MHz,CDCl₃,δppm):12.48(s，1H，COOH)，7.94(d，1H，Ar-H，J=7.8Hz)，7.67(d，1H，Ar-CH=C，J=15.6Hz)，7.56-7.53(t,1H,Ar-H),7.42-7.38(m,2H,Ar-H),6.75(d,1H,C=CH-C=O,J=15.6Hz),4.23(q,2H,OCH₂),2.71(s,3H,CH₃),2.58(s,3H,CH₃),2.50(s,3H,CH₃),1.30(t,3H,CH₃).IR(KBr,cm^-1):3080(C=C-H),2988,2957(CH₃),1745,1705(C=O),1629(C=C),1601,1577,1541(C=N,C=C),1151(C-O).ESI-MS:341.4(M+H)⁺,calcd.forC₁₉H₂₀N₂O₄340.37.Dissolve (E)-3-(2-((3,5,6-trimethylpyrazine-2-carbonyl)oxy)phenyl)acrylic acid (0.62g, 2mmol) in ethanol, and slowly Add 2.4 mmol thionyl chloride dropwise, and heat to reflux for 24 h after dropping. TLC monitored that the reaction was complete, and the remaining solvent was distilled off. The residue was dissolved in ethyl acetate, washed with water and saturated aqueous sodium chloride solution, and the organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product. Flash column separation, n-hexane recrystallization, yield 81%, mp: 77-79°C.¹ H-NMR (600MHz, CDCl₃ , δppm): 12.48(s, 1H, COOH), 7.94(d, 1H, Ar-H, J=7.8Hz), 7.67(d, 1H, Ar-CH =C, J=15.6Hz), 7.56-7.53(t, 1H, Ar-H), 7.42-7.38(m, 2H, Ar -H),6.75(d,1H,C=CH -C=O,J=15.6Hz),4.23(q,2H,OCH₂ ),2.71(s,3H,CH₃ ),2.58(s,3H, CH₃ ),2.50(s,3H,CH₃ ),1.30(t,3H,CH₃ ).IR(KBr,cm^-1 ):3080(C=CH ),2988,2957(CH₃ ),1745, 1705(C=O),1629(C=C),1601,1577,1541(C=N,C=C),1151(CO).ESI-MS:341.4(M+H)⁺ ,calcd.forC₁₉ H₂₀ N₂ O₄ 340.37.

实施例10.(E)-3-(3-甲氧基-4-((3,5,6-三甲基吡嗪-2-羰基)氧)苯基)丙烯酸乙酯(F′4)的制备Example 10. (E)-3-(3-methoxy-4-((3,5,6-trimethylpyrazine-2-carbonyl)oxy)phenyl)ethyl acrylate (F'4) preparation of

取(E)-3-(3-甲氧基-4-((3,5,6-三甲基吡嗪-2-羰基)氧)苯基)丙烯酸(0.68g,2mmol)溶于乙醇中，冰浴条件下缓慢滴加2.4mmol氯化亚砜，滴毕加热回流24h。TLC监测至反应完全，蒸除剩余溶剂，残余物溶于乙酸乙酯，分别用水、饱和氯化钠水溶液洗涤，有机层由无水硫酸钠干燥，过滤，浓缩得粗品。快速柱分离，正己烷重结晶，产率81%,mp:107-110°C.¹H-NMR(600MHz,CDCl₃,δppm):7.70(d,1H,Ar-CH=C,J=16.2Hz),7.61(s,1H,Ar-H),7.38(d,1H,Ar-H,J=7.8Hz),7.31(d,1H,Ar-H,J=8.4Hz),6.77(d,1H,C=CH-C=O,J=15.6Hz),4.23(q,2H,OCH₂),3.86(s,3H,OCH₃),2.70(s,3H,CH₃),2.56(s,3H,CH₃),2.51(s,3H,CH₃),1.30(t,3H,CH₃).IR(KBr,cm^-1):3064(C=C-H),2983,2928(CH₃),1737,1703(C=O),1676(C=C),1601,1510(C=N,C=C),1158(C-O).ESI-MS:371.4(M+H)⁺,calcd.forC₂₀H₂₂N₂O₅370.40.Dissolve (E)-3-(3-methoxy-4-((3,5,6-trimethylpyrazine-2-carbonyl)oxy)phenyl)acrylic acid (0.68g, 2mmol) in ethanol 2.4 mmol thionyl chloride was slowly added dropwise under ice bath conditions, and heated to reflux for 24 h after dropping. TLC monitored that the reaction was complete, and the remaining solvent was distilled off. The residue was dissolved in ethyl acetate, washed with water and saturated aqueous sodium chloride solution, and the organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product. Flash column separation, n-hexane recrystallization, yield 81%, mp: 107-110°C.¹ H-NMR (600MHz, CDCl₃ , δppm): 7.70(d, 1H, Ar-CH =C, J=16.2 Hz),7.61(s,1H,Ar-H),7.38(d,1H,Ar-H,J=7.8Hz),7.31(d,1H,Ar-H,J=8.4Hz),6.77(d, 1H,C=CH -C=O,J=15.6Hz),4.23(q,2H,OCH₂ ),3.86(s,3H,OCH₃ ),2.70(s,3H,CH₃ ),2.56(s, 3H,CH₃ ),2.51(s,3H,CH₃ ),1.30(t,3H,CH₃ ).IR(KBr,cm^-1 ):3064(C=CH ),2983,2928(CH₃ ), 1737,1703(C=O),1676(C=C),1601,1510(C=N,C=C),1158(CO).ESI-MS:371.4(M+H)⁺ ,calcd.forC₂₀ H₂₂ N₂ O₅ 370.40.

川芎嗪甲酰氧基肉桂酸类衍生物的活性筛选试验：Activity Screening Test of Ligustrazine Formyloxycinnamic Acid Derivatives:

对川芎嗪甲酰氧基肉桂酸类衍生物进行了抗血小板聚集试验，实验步骤如下：Ligustrazine formyloxycinnamic acid derivatives were tested for anti-platelet aggregation, and the experimental steps were as follows:

家兔心脏取血，以3.8%的枸椽酸钠抗凝(v/v血：抗凝剂=9：1)，静置30min后，将其1000rpmin离心5min后取上清液即得富含血小板血浆(PRP)，将剩余部分以3000rpmin离心15min后取上清液即得贫血小板血浆(PPP)。用PPP将PRP调数为3×10¹¹/L备用(显微镜下用计数板计数)。制备好的PRP应在2h内用完。Blood was taken from the heart of rabbits, anticoagulated with 3.8% sodium citrate (v/v blood:anticoagulant=9:1), after standing for 30min, centrifuged at 1000rpm for 5min, and the supernatant was obtained to obtain the supernatant. For platelet plasma (PRP), the remaining part was centrifuged at 3000 rpm for 15 minutes and the supernatant was taken to obtain platelet poor plasma (PPP). Use PPP to adjust the PRP to 3×10¹¹ /L for later use (count with a counting plate under a microscope). The prepared PRP should be used up within 2 hours.

取90μLPRP，加入96孔板中，加入5μL待测药品(终浓度400,200,100,50μM)，37℃温育振摇5min。将96孔板置于酶标仪上，快速震摇10S，570nm下测定吸光度，每30S测定一次，连续测3次，记各孔三次吸光度均值为A₀。之后加入5μLADP(工作浓度5μM)，继续每30S测定一次吸光度直至吸光度不再变化，记平行孔的吸光度均值为A。Take 90 μL PRP, add it to a 96-well plate, add 5 μL of the drug to be tested (final concentration 400, 200, 100, 50 μM), incubate and shake at 37°C for 5 minutes. Place the 96-well plate on a microplate reader, shake quickly for 10S, and measure the absorbance at 570nm, once every 30S, and measure 3 times continuously, record the average value of the absorbance of each well three times as A₀ . Then add 5μLADP (working concentration 5μM), continue to measure the absorbance every 30S until the absorbance no longer changes, record the average absorbance of the parallel wells as A.

结果OD_570nm以表示，t检验分析试验组与对照组之间是否有显著性差异，按以下公式计算血小板聚集率(aggregationrate,AR)：AR=(AbsPRP-Abssample)/(AbsPRP-AbsPPP)。同时计算药物对血小板聚集的抑制率(aggregationinhibitionrate,AIR)：AIR=[1-(给药管聚集百分率/对照管聚集百分率)]×100%。用直线回归法求出药物的半数抑制浓度IC₅₀。Results OD_570nm with The t test was used to analyze whether there was a significant difference between the test group and the control group, and the platelet aggregation rate (aggregation rate, AR) was calculated according to the following formula: AR=(AbsPRP-Abssample)/(AbsPRP-AbsPPP). At the same time, the inhibition rate (aggregation inhibition rate, AIR) of the drug on platelet aggregation was calculated: AIR=[1-(aggregation percentage of administration tube/aggregation percentage of control tube)]×100%. The half inhibitory concentration IC₅₀ of the drug was obtained by linear regression method.

以奥扎格雷（ozagrel），氯吡格雷（clopidogrel）为阳性对照，其活性列于表2中。由表2可以看出，化合物F′1,F2,F3显示出了明显的抗血小板聚集活性，IC₅₀分别达到了24.4,26.4和9.59μM，活性强于对照药奥扎格雷（IC₅₀=144μM），其中化合物F3活性最强，接近对照药氯吡格雷（IC₅₀=7.57μM）。Take ozagrel (ozagrel) and clopidogrel (clopidogrel) as positive controls, and their activities are listed in Table 2. It can be seen from Table 2 that the compounds F′1, F2, and F3 showed obvious anti-platelet aggregation activity, and the IC₅₀ reached 24.4, 26.4 and 9.59 μM respectively, which was stronger than that of the control drug ozagrel (IC₅₀ =144 μM ), among which compound F3 has the strongest activity, which is close to that of the control drug clopidogrel (IC₅₀ =7.57μM).

表2.川芎嗪甲酰氧基肉桂酸类衍生物的血小板聚集抑制率Table 2. Platelet aggregation inhibition rate of ligustrazine formyloxycinnamic acid derivatives

IC₅₀：化合物对血小板聚集的半数抑制浓度IC₅₀ : the half inhibitory concentration of the compound on platelet aggregation

对川芎嗪甲酰氧基肉桂酸类衍生物还进行了血管内皮细胞损伤保护试验，实验步骤如下：Ligustrazine formyloxycinnamic acid derivatives were also tested for protection against vascular endothelial cell damage, and the experimental steps were as follows:

Ea.hy926细胞在含10%小牛血清的RPMI-1640培养液中培养24h后，在正常组中加入不含H₂O₂和药物的正常培养液，在模型组中加入浓度为0.15mmol.L^-1H₂O₂的培养液，在保护组中加入含不同浓度药物且含终浓度为0.15mmol.L^-1H₂O₂的培养液，继续培养12h，然后每孔加入0.01mLMTT溶液(5mg.mL^-1)，37℃培养4h，倾去上清液，每孔加入二甲基亚砜0.lmL，放置10min。30min内在全自动酶标仪上于570nm处测定吸光度值(A_570nm)，计算Ea.hy926细胞增殖百分率。After Ea.hy926 cells were cultured in RPMI-1640 medium containing 10% calf serum for 24 hours, normal medium without H₂ O₂ and drugs was added to the normal group, and the concentration of 0.15mmol was added to the model group. L^-1 H₂ O₂ culture solution, in the protection group, add the culture solution containing different concentrations of drugs and the final concentration of 0.15mmol.L^-1 H₂ O₂ , continue to cultivate for 12h, and then add 0.01mL MTT solution to each well (5 mg.mL^-1 ), cultured at 37°C for 4 h, poured off the supernatant, added 0.1 mL of dimethyl sulfoxide to each well, and left for 10 min. Measure the absorbance value (A_570nm ) at 570nm on an automatic microplate reader within 30min, and calculate the proliferation percentage of Ea.hy926 cells.

结果OD_570nm以表示，t检验分析试验组与对照组之间是否有显著性差异，按下式计算细胞增殖率P(%)。Results OD_570nm with Indicates that the t test is used to analyze whether there is a significant difference between the test group and the control group, and the cell proliferation rate P (%) is calculated according to the following formula.

$\mathrm{<span><span>P</span>P</span>}<span><span>(</span>(</span><span><span>\%</span>\%</span><span><span>)</span>)</span><span><span>=</span>=</span>\frac{\mathrm{<span><span>OD</span>OD</span>}<span><span>-</span>-</span>\mathrm{<span><span>ODa</span>O\; Da</span>}}{{\mathrm{<span><span>OD</span>OD</span>}}_{\mathrm{<span><span>b</span>b</span>}}<span><span>-</span>-</span>\mathrm{<span><span>ODa</span>O\; Da</span>}}<span><span>\&times;</span>\&times;</span>\mathrm{<span><span>100</span>100</span>}<span><span>\%</span>\%</span>$

(OD-保护组OD值，ODa-模型组OD值，OD_b-正常组OD值)(OD-protected group OD value, ODa-model group OD value, OD_b -normal group OD value)

以硫辛酸（lipoicacid）和丁基羟基茴香醚（BHA）为对照，其活性列于表3中，并。由表3可以看出，化合物F5，F10和F′10显示出了明显的血管内皮细胞损伤保护活性，EC₅₀分别达到了8.84,2.23和1.71μM，活性强于对照药硫辛酸和BHA（EC₅₀分别为68.0μM和111μM），其中化合物F′10活性最强，达到了硫辛酸的40倍。Taking lipoic acid (lipoicacid) and butylated hydroxyanisole (BHA) as controls, their activities are listed in Table 3, and. It can be seen from Table 3 that compounds F5, F10 and F'10 showed obvious protective activity against vascular endothelial cell injury, with EC₅₀ reaching 8.84, 2.23 and 1.71 μM, respectively, which were stronger than the control drugs lipoic acid and BHA (EC₅₀ are 68.0μM and 111μM respectively), and compound F′10 has the strongest activity, which is 40 times that of lipoic acid.

表3.川芎嗪甲酰氧基肉桂酸类化合物对EA.hy926细胞增殖率及其EC₅₀Table 3. Ligustrazine formyloxycinnamic acid compounds on the proliferation rate and EC₅₀ of EA.hy926 cells

Claims (8)

Translated fromChinese

1.川芎嗪甲酰氧基肉桂酸类衍生物，具有如下结构通式：1. Ligustrazine formyloxycinnamic acid derivatives have the following general structural formula:其中inR选自-H，-OH，-Cl，-Br，-OCH₃；R′选自-H或-C₂H₅。R is selected from -H, -OH, -Cl, -Br, -OCH₃ ; R' is selected from -H or -C₂ H₅ .2.权利要求1所述的川芎嗪甲酰氧基肉桂酸类衍生物的制备方法，其特征是，包括步骤如下：2. the preparation method of Ligustrazine formyloxycinnamic acid derivatives as claimed in claim 1, is characterized in that, comprises steps as follows:(1)取川芎嗪溶于水中，30-40℃条件下搅拌缓慢加入KMnO₄的水溶液，反应10-30h，TLC监测反应完全后，向反应液中加入亚硫酸氢钠饱和水溶液至高锰酸钾紫红色完全褪去，过滤混合液，滤渣用热水清洗，合并滤液，浓盐酸调节pH＝2-3，乙酸乙酯萃取，无水硫酸钠干燥，减压蒸干溶剂得3,5,6-三甲基吡嗪-2-甲酸；(1) Dissolve ligustrazine in water, stir slowly at 30-40°C and add KMnO₄ aqueous solution, react for 10-30h, after TLC monitors that the reaction is complete, add saturated aqueous solution of sodium bisulfite to potassium permanganate to the reaction solution The purple color faded completely, filtered the mixed solution, washed the filter residue with hot water, combined the filtrate, adjusted the pH to 2-3 with concentrated hydrochloric acid, extracted with ethyl acetate, dried over anhydrous sodium sulfate, and evaporated the solvent under reduced pressure to obtain 3,5,6- Trimethylpyrazine-2-carboxylic acid;(2)取3,5,6-三甲基吡嗪-2-甲酸溶于无水二氯甲烷，冰浴条件下加入草酰氯，搅拌反应0.5-1h，TLC监测至反应完全后，减压蒸除剩余溶剂和草酰氯，得到3,5,6-三甲基吡嗪-2-甲酰氯；(2) Dissolve 3,5,6-trimethylpyrazine-2-carboxylic acid in anhydrous dichloromethane, add oxalyl chloride under ice-bath conditions, stir for 0.5-1h, monitor by TLC until the reaction is complete, then depressurize Evaporate the remaining solvent and oxalyl chloride to obtain 3,5,6-trimethylpyrazine-2-formyl chloride;(3)将取代苯甲醛溶于吡啶，加入丙二酸和哌啶，于120℃下反应1-2h，TLC监测反应完毕后，蒸除吡啶，残余物加水溶解，以HCl调pH为2-3，乙酸乙酯萃取，有机层干燥，过滤，蒸除溶剂，得取代肉桂酸；(3) Dissolve the substituted benzaldehyde in pyridine, add malonic acid and piperidine, and react at 120°C for 1-2h. After the reaction is monitored by TLC, evaporate the pyridine, dissolve the residue in water, and adjust the pH to 2- 3. Extract with ethyl acetate, dry the organic layer, filter, and evaporate the solvent to obtain substituted cinnamic acid;(4)取步骤(2)的3,5,6-三甲基吡嗪-2-甲酰氯溶于无水二氯甲烷中，室温搅拌下加入取代肉桂酸、三乙胺，20-30℃反应0.5-1h，TLC监测反应完全后，蒸除剩余溶剂，残留物溶于乙酸乙酯，分别用水、饱和NaCl水溶液洗涤，有机层用无水硫酸钠干燥，过滤、浓缩得粗品，经快速柱分离，乙醇重结晶得纯品川芎嗪甲酰氧基肉桂酸；(4) Dissolve the 3,5,6-trimethylpyrazine-2-formyl chloride in step (2) in anhydrous dichloromethane, add substituted cinnamic acid and triethylamine under stirring at room temperature, 20-30°C After reacting for 0.5-1h, TLC monitors that the reaction is complete, distill off the remaining solvent, dissolve the residue in ethyl acetate, wash with water and saturated NaCl aqueous solution respectively, and dry the organic layer with anhydrous sodium sulfate, filter and concentrate to obtain a crude product, which is passed through a flash column Separation, ethanol recrystallization to obtain pure Ligustrazine formyloxycinnamic acid;(5)取川芎嗪甲酰氧基肉桂酸溶于乙醇溶液中，冰浴条件下逐滴加入氯化亚砜，反应液加热回流24h，TLC监测至反应完全，蒸除剩余溶剂，残余物溶于乙酸乙酯，分别用水、饱和氯化钠水溶液洗涤，有机层由无水硫酸钠干燥，过滤，浓缩得粗品，快速柱分离，正己烷重结晶得产品。(5) Dissolve ligustrazine formyloxycinnamic acid in an ethanol solution, add thionyl chloride dropwise under ice bath conditions, heat the reaction solution to reflux for 24 hours, monitor by TLC until the reaction is complete, evaporate the remaining solvent, and dissolve the residue In ethyl acetate, washed with water and saturated aqueous sodium chloride solution, the organic layer was dried over anhydrous sodium sulfate, filtered, concentrated to obtain the crude product, separated by flash column, and recrystallized from n-hexane to obtain the product.3.根据权利要求2所述的川芎嗪甲酰氧基肉桂酸类衍生物的制备方法，其特征是，步骤(1)所述的川芎嗪与KMnO₄的摩尔比为1:1～4，川芎嗪与水的质量/体积比为1：25，g/mL。3. the preparation method of ligustrazine formyloxycinnamic acid derivatives according to claim 2, is characterized in that, ligustrazine described in step (1) and KMnO_The mol ratio is 1:1～4, The mass/volume ratio of Ligustrazine to water is 1:25, g/mL.4.根据权利要求2所述的川芎嗪甲酰氧基肉桂酸类衍生物的制备方法，其特征是，步骤(2)中3,5,6-三甲基吡嗪-2-甲酸与草酰氯的摩尔比为1:1～2，3,5,6-三甲基吡嗪-2-甲酸与无水二氯甲烷的质量/体积比为1:25，g/mL。4. the preparation method of ligustrazine formyloxycinnamic acid derivatives according to claim 2, is characterized in that, in step (2), 3,5,6-trimethylpyrazine-2-formic acid and grass The molar ratio of acid chloride is 1:1~2, and the mass/volume ratio of 3,5,6-trimethylpyrazine-2-carboxylic acid to anhydrous dichloromethane is 1:25, g/mL.5.根据权利要求2所述的川芎嗪甲酰氧基肉桂酸类衍生物的制备方法，其特征是，步骤(3)中取代苯甲醛、丙二酸和哌啶的摩尔比为1:1:0.1，取代苯甲醛与吡啶的质量/体积比为1:25，g/mL。5. the preparation method of ligustrazine formyloxycinnamic acid derivative according to claim 2, is characterized in that, in step (3), the mol ratio of substituting benzaldehyde, malonic acid and piperidine is 1:1 :0.1, the mass/volume ratio of substituted benzaldehyde and pyridine is 1:25, g/mL.6.根据权利要求2所述的川芎嗪甲酰氧基肉桂酸类衍生物的制备方法，其特征是，步骤(4)中3,5,6-三甲基吡嗪-2-甲酰氯、取代肉桂酸、三乙胺的摩尔比为1:1:3，3,5,6-三甲基吡嗪-2-甲酰氯与无水二氯甲烷的质量/体积比为1:25-30，g/mL。6. the preparation method of ligustrazine formyloxycinnamic acid derivatives according to claim 2 is characterized in that, in step (4), 3,5,6-trimethylpyrazine-2-formyl chloride, The molar ratio of substituted cinnamic acid and triethylamine is 1:1:3, and the mass/volume ratio of 3,5,6-trimethylpyrazine-2-carbonyl chloride to anhydrous dichloromethane is 1:25-30 , g/mL.7.根据权利要求2所述的川芎嗪甲酰氧基肉桂酸类衍生物的制备方法，其特征是，步骤(5)中川芎嗪甲酰氧基肉桂酸与氯化亚砜的摩尔比为1:1.2，川芎嗪甲酰氧基肉桂酸与乙醇的质量/体积比为1:30，g/mL。7. the preparation method of ligustrazine formyloxycinnamic acid derivative according to claim 2, is characterized in that, in step (5), the mol ratio of ligustrazine formyloxycinnamic acid and sulfur oxychloride is 1:1.2, the mass/volume ratio of ligustrazine formyloxycinnamic acid to ethanol is 1:30, g/mL.8.权利要求1所述的川芎嗪甲酰氧基肉桂酸类衍生物在制备抗血小板聚集药物或血管内皮细胞保护药物中的应用。8. The application of the ligustrazine formyloxycinnamic acid derivatives according to claim 1 in the preparation of anti-platelet aggregation drugs or vascular endothelial cell protection drugs.