技术领域technical field
本发明属于生物医药技术领域，具体涉及一种二酮衍生物及其制备方法与应用。The invention belongs to the technical field of biomedicine, and specifically relates to a diketone derivative and a preparation method and application thereof.
背景技术Background technique
阿尔茨海默症(Alzheimer’s disease，AD)是一种退行性脑神经系统疾病，属于最常见的老年痴呆症的一种。它的临床表现分为社交能力障碍、记忆力逐渐减退、认知功能(时间空间定向能力)减弱、思考理解行为衰退、视空间功能以及日常自理能力丧失、性格淡漠孤僻等等一些症状，晚期患者可危及生命。流行病学研究结果表明，老年痴呆已成为发达国家仅次于癌症、中风、心脏病之后引起老年人死亡的第4位死因。随着患者数量的急剧增长，给家庭和社会造成了沉重的负担。由于AD发病隐蔽性强，病程缓慢，逐渐发展且不可逆，等根据临床症状可以做出诊断时，往往患者基本己处于中晚期，发病后15-25年由于多种并发症而导致死亡。现有的治疗方案只能起到缓解症状，对于疾病均不能取得明显的治疗疗效和改善作用。Alzheimer's disease (AD) is a degenerative brain nervous system disease, which is one of the most common senile dementias. Its clinical manifestations include social impairment, memory loss, cognitive function (time-spatial orientation ability) weakening, thinking and understanding behavior decline, loss of visuospatial function and daily self-care ability, apathy and withdrawn personality, and other symptoms. life threatening. Epidemiological research results show that senile dementia has become the fourth cause of death of the elderly after cancer, stroke and heart disease in developed countries. With the rapid increase in the number of patients, it has caused a heavy burden on families and society. Due to the strong concealment of the onset of AD, the slow course of the disease, gradual development and irreversibility, when the diagnosis can be made based on clinical symptoms, the patients are often in the middle and late stages, and die due to various complications 15-25 years after the onset. Existing treatment schemes can only alleviate the symptoms, but cannot achieve obvious curative effect and improvement for the disease.
AD是一种发病因素比较多样的疾病，主要是由于遗传基因、衰老和外界环境因素共同的作用后所诱发的。它的病理表现如下：(1)Tau蛋白过度的磷酸化在细胞内聚集形成神经纤维缠结(NFT)，(2)β-淀粉样肽段(Aβ)，细胞外的自聚形成的具有细胞毒性的Aβ低聚物(soluble oligomers)和原纤维(fibrils)，(3)胆碱能神经的损伤而导致的胆碱能传导障碍，(4)神经突触的缺失，(5)神经炎症和细胞内金属离子浓度过高，(6)氧化应激和线粒体功能的紊乱等等。AD is a disease with various pathogenic factors, which is mainly induced by the combined effects of genetics, aging and external environmental factors. Its pathological manifestations are as follows: (1) excessive phosphorylation of Tau protein aggregates in cells to form neurofibrillary tangles (NFT); Toxic Aβ oligomers (soluble oligomers) and fibrils (fibrils), (3) cholinergic conduction disorders caused by damage to cholinergic nerves, (4) loss of synapses, (5) neuroinflammation and Intracellular metal ion concentration is too high, (6) oxidative stress and mitochondrial dysfunction and so on.
过去十几年里，抗AD药物的研究大都集中在A级联假说相关的策略上，但最近，以Aβ为靶点的部分药物在三期临床试验中却以失败告终，因而“神经元纤维缠结”在AD病中的作用被人们广泛认可。tau蛋白作为阿尔茨海默氏病靶点药物的研究,近年来受到越来越多的关注。In the past ten years, most of the research on anti-AD drugs has focused on strategies related to the A cascade hypothesis, but recently, some drugs targeting Aβ have failed in phase III clinical trials, thus "neuron fibers The role of "tangles" in AD disease is widely recognized. The study of tau protein as a target drug for Alzheimer's disease has received more and more attention in recent years.
Tau蛋白是一种分布在中枢神经系统内的低分子量含磷糖蛋白。在神经元中,Tau蛋白是高度可溶且丰富存在，它对于微管蛋白的稳定起着至关重要的作用，尤其是在突触中。Tau蛋白的过度磷酸化将会导致不溶性成对螺旋丝的形成，从而进一步会构成神经元纤维的缠结。AD患者脑内Tau蛋白磷酸化水平为正常人的3～4倍，一般认为过度磷酸化的Tau蛋白会部分或完全丧失其活性,它的细胞毒性作用是由于自身诱导凋亡所产生的，并在Aβ参与的神经细胞凋亡中起主要作用。Tau protein is a low molecular weight phosphorous glycoprotein distributed in the central nervous system. In neurons, Tau protein is highly soluble and abundant, and it plays a crucial role in the stabilization of tubulin, especially at synapses. Hyperphosphorylation of tau leads to the formation of insoluble paired helical filaments, which further form tangles of neuronal fibers. The phosphorylation level of Tau protein in the brain of AD patients is 3 to 4 times that of normal people. It is generally believed that hyperphosphorylated Tau protein will partially or completely lose its activity, and its cytotoxic effect is due to self-induced apoptosis, and It plays a major role in the apoptosis of nerve cells involved in Aβ.
微管蛋白与Tau蛋白的结合能力是通过丝、苏氨酸介导的磷酸化途径来调控的，是调节Tau蛋白与微管亲和力的最重要的机制。Tau蛋白异常过度磷酸化是AD等神经系统病变脑神经元降解的重要初始步骤，磷酸化后的Tau蛋白与微管蛋白的结合能力明显下降，丧失了促进微管组装的功能。使微管蛋白游离出来，影响微管的功能和结构，引起神经元功能紊乱和退行性变,最终会导致神经功能障碍和神经元死亡。The binding ability of tubulin to Tau protein is regulated by the phosphorylation pathway mediated by silk and threonine, which is the most important mechanism for regulating the affinity between Tau protein and microtubules. Abnormal hyperphosphorylation of Tau protein is an important initial step in the degradation of brain neurons in neurological diseases such as AD. The binding ability of phosphorylated Tau protein to tubulin is significantly reduced, and the function of promoting microtubule assembly is lost. Make tubulin free, affect the function and structure of microtubules, cause neuronal dysfunction and degeneration, and eventually lead to neuronal dysfunction and neuron death.
以tau蛋白为靶点治疗阿尔茨海默氏病的研究目前尚处在初级阶段，绝大多数药物小分子还处在理论设计阶段，只有一小部分进入二期临床试验，例如甲基蓝(MTC)，这一吩噻嗪类化合物能够阻止tau-tau之间的相互作用，它不仅能够部分地分解孤立的双螺旋纤维丝(PHFs)，还能影响tau蛋白的聚合过程。目前这类药物的研究已进入二期临床阶段。The research on treating Alzheimer's disease with tau protein as a target is still in its infancy. Most of the small drug molecules are still in the theoretical design stage, and only a small part has entered the second phase of clinical trials, such as methylene blue ( MTC), a phenothiazine compound that prevents tau-tau interactions, not only partially disintegrates isolated double-helical filaments (PHFs), but also affects the polymerization process of tau protein. At present, the research of this kind of drug has entered the second clinical stage.
另外，研究表明，外来的微管稳定剂能够补偿Tau蛋白功能的缺失，调节微管的动态稳定性，使其恢复正常的介导轴突运输的功能，因而保护神经细胞。因此，微管蛋白稳定剂为治疗AD症和相关Tau蛋白病变提供了另一条新的途径。目前这类药物对AD的治疗作用已经在动物实验上得到了证实。In addition, studies have shown that external microtubule stabilizers can compensate for the loss of Tau protein function, regulate the dynamic stability of microtubules, restore the normal function of mediating axonal transport, and thus protect nerve cells. Therefore, tubulin stabilizers provide another new avenue for the treatment of AD and related tauopathies. At present, the therapeutic effect of these drugs on AD has been confirmed in animal experiments.
发明内容Contents of the invention
根据上述AD病理机制，本发明的首要目的在于提供一种二酮衍生物。所述的二酮衍生物包括含异吲哚-1,3-二酮衍生物,含吡咯并吡啶-5,7-二酮衍生物,吡咯并吡嗪-5,7-二酮衍生物，所述的二酮衍生物为能作用tau蛋白和微管蛋白双靶点的小分子，既对tau蛋白聚合具有抑制作用又对微管蛋白起稳定作用,用于治疗阿尔茨海默氏病。According to the above pathological mechanism of AD, the primary purpose of the present invention is to provide a diketone derivative. The diketone derivatives include isoindole-1,3-dione derivatives, pyrrolopyridine-5,7-dione derivatives, pyrrolopyrazine-5,7-dione derivatives, The diketone derivative is a small molecule capable of acting on the dual targets of tau protein and tubulin, which not only inhibits tau protein polymerization but also stabilizes tubulin, and is used for treating Alzheimer's disease.
本发明的另一目的在于提供上述二酮衍生物的制备方法。Another object of the present invention is to provide a preparation method of the above-mentioned diketone derivatives.
本发明的再一目的在于提供上述二酮衍生物的应用。应用于制备治疗阿茨海默症的药物。Another object of the present invention is to provide the application of the above-mentioned diketone derivatives. Applied in the preparation of medicines for treating Alzheimer's disease.
本发明的目的通过下述技术方案实现：一种二酮衍生物，所述衍生物的结构式如下式Ⅰ示：The object of the present invention is achieved through the following technical solutions: a diketone derivative, the structural formula of which is shown in Formula I below:
其中：X为C或N；Y为C或N；Among them: X is C or N; Y is C or N;
R1为氢、氯、溴或噻吩中的一种；R1 is one of hydrogen, chlorine, bromine or thiophene;
R2为氢、异丙基或甲氧基中的一种；R2 is one of hydrogen, isopropyl or methoxy;
上述如式Ⅰ所示的二酮衍生物的制备方法，包括如下步骤：在碱性三乙胺和羧基活化试剂，(2-氧代-恶唑-3-基)膦酸二苯酯(DPPOx)条件下，酸酐化合物与苯胺衍生物反应生成如式Ⅰ的二酮衍生物，所用溶剂为乙腈或水，其中R1，R2与权利要求1中相同。The preparation method of above-mentioned diketone derivative shown in formula I comprises the steps: in basic triethylamine and carboxyl activation reagent, (2-oxo-oxazol-3-yl) phosphonic acid diphenyl ester (DPPOx ) conditions, the anhydride compound reacts with the aniline derivative to form the diketone derivative of formula I, the solvent used is acetonitrile or water, wherein R1 and R2 are the same as those in claim 1.
所述的酸酐化合物为的合成方法为：从3-溴喹啉出发，经高锰酸钾氧化开环再缩合得到酸酐3。The synthesis method of the acid anhydride compound is as follows: starting from 3-bromoquinoline, the acid anhydride 3 is obtained through oxidation and ring opening of potassium permanganate and condensation.
上述的二酮衍生物还包括由式Ⅰ所示的衍生物在醇溶液中与盐酸、醋酸、三氟乙酸制备获得的相应盐酸盐、醋酸盐、三氟乙酸盐。The above-mentioned diketone derivatives also include the corresponding hydrochloride, acetate, and trifluoroacetate prepared from the derivative shown in formula I with hydrochloric acid, acetic acid, and trifluoroacetic acid in alcohol solution.
上述的二酮衍生物的应用，应用于制备治疗阿茨海默症的药物。The application of the above-mentioned diketone derivatives is applied to the preparation of drugs for treating Alzheimer's disease.
上述的式Ⅰ二酮衍生物的制备方法，具体由反应式I或反应式II所述的方法制备得到：The preparation method of the above-mentioned diketone derivatives of formula I is specifically prepared by the method described in reaction formula I or reaction formula II:
(1)化合物1的制备：如反应式I所示,首先从商品化的5-溴异苯并呋喃-1,3-二酮化合物，呋喃并[3,4-b]吡啶-5,7-二酮或呋喃并[3,4-b]吡嗪-5,7-二酮出发，在碱性三乙胺和羧基活化试剂(2-氧代-恶唑-3-基)膦酸二苯酯(DPPOx)条件下，酸酐首先开环，羧基活化试剂中的磷负电子进攻开环后羧酸的羰基碳形成中间体，苯胺上的氨基进攻另外一个羰基上的碳，之后进行关环，得到我们所要的化合物；该反应使用乙腈作反应溶剂，回流2.5小时，经过层析柱层析纯化后即可得到一系列的化合物1，化合物1即为式1化合物；随后,化合物1于噻吩-2-基硼酸发生铃木偶联反应,得到化合物2，化合物2即为式2化合物。(1) Preparation of compound 1: as shown in Reaction Formula I, firstly, from the commercialized 5-bromoisobenzofuran-1,3-dione compound, furo[3,4-b]pyridine-5,7 -diketone or furo[3,4-b]pyrazine-5,7-dione, in basic triethylamine and carboxyl activating reagent (2-oxo-oxazol-3-yl)phosphonic acid di Under the condition of phenyl ester (DPPOx), the acid anhydride first opens the ring, and the phosphorus negative electrons in the carboxyl activating reagent attack the carbonyl carbon of the carboxylic acid after ring opening to form an intermediate, and the amino group on the aniline attacks the carbon on another carbonyl, and then the ring is closed , to obtain the compound we want; this reaction uses acetonitrile as the reaction solvent, reflux for 2.5 hours, and a series of compound 1 can be obtained after column chromatography purification, and compound 1 is the compound of formula 1; subsequently, compound 1 is dissolved in thiophene -2-ylboronic acid undergoes Suzuki coupling reaction to obtain compound 2, which is the compound of formula 2.
(2)化合物5的制备：如反应式II所示，首先从商品化的3-溴喹啉出发，加入高锰酸钾将其氧化开环形成5-溴吡啶-2,3-二羧酸3；二羧酸化合物3再由苯磺酸条件下进行缩合得到酸酐4，之后采用步骤(1)在羧基活化试剂条件下的反应方法，或者将化合物3溶于冰乙酸中，加入苯胺衍生物直接回流反应12～24小时，所得产物经柱层析分离，得到化合物5，化合物5即为式5化合物。(2) Preparation of compound 5: as shown in Reaction Formula II, first starting from commercialized 3-bromoquinoline, adding potassium permanganate to oxidize and open it to form 5-bromopyridine-2,3-dicarboxylic acid 3; dicarboxylic acid compound 3 is condensed under the condition of benzenesulfonic acid to obtain acid anhydride 4, then adopt the reaction method of step (1) under the condition of carboxyl activating reagent, or dissolve compound 3 in glacial acetic acid, and add aniline derivative Directly reflux the reaction for 12-24 hours, and the obtained product is separated by column chromatography to obtain compound 5, which is the compound of formula 5.
本发明相对于现有技术具有如下的优点及效果：Compared with the prior art, the present invention has the following advantages and effects:
本发明的二酮衍生物不仅tau蛋白自聚集具有较好的抑制活性，而且对对微管蛋白聚合具有很强的稳定作用。因而,通过前者的作用，能阻止神经元纤维缠结的形成，以防止微管蛋白解聚，再通过后者的作用，进一步调节微管的动态稳定性，使其恢复正常的介导轴突运输的功能，因而保护神经细胞而达到治疗阿茨海默症的目的。The diketone derivative of the present invention not only has better inhibitory activity on self-aggregation of tau protein, but also has strong stabilizing effect on tubulin polymerization. Therefore, through the former function, it can prevent the formation of neurofibrillary tangles to prevent the depolymerization of tubulin, and through the latter function, it can further regulate the dynamic stability of microtubules so that it can restore the normal mediation of axons. Transport function, thus protecting nerve cells and achieving the purpose of treating Alzheimer's disease.
附图说明Description of drawings
图1为表1中第一部分化合物增加微管蛋白聚合率的结果图；Fig. 1 is the result figure that the first part compound in table 1 increases tubulin polymerization rate;
图2为表1中第二部分化合物增加微管蛋白聚合率的结果图。FIG. 2 is a graph showing the results of the second part of the compounds in Table 1 increasing the polymerization rate of tubulin.
具体实施方式detailed description
下面结合实施例及附图对本发明作进一步详细的描述，但本发明的实施方式不限于此。The present invention will be further described in detail below in conjunction with the embodiments and the accompanying drawings, but the embodiments of the present invention are not limited thereto.
实施例1Example 1
合成5-溴-2-(2,6-二异丙基-苯基)-异吲哚-1,3-二酮1aSynthesis of 5-bromo-2-(2,6-diisopropyl-phenyl)-isoindole-1,3-dione 1a
称取4-溴邻苯二甲酸酐(0.67mmol,1eq)和化合物2,6-二异丙基苯胺(0.80mmol,1.2eq)于25mL圆底瓶中，将其溶解在乙腈中(3mL)后，加入参照文献(Tetrahedron,1982,39(20):3253)制备的DPPOx(1.34mmol,2eq)，Et3N(0.18mL,1.29mmol,1.9eq)，反应在回流的情况下持续2.5h，用TLC监测反应至原料完全消失后，将反应液旋干，将反应物重新溶于乙酸乙酯中后分别用HCl(30mL,3M)和NaHCO3(30mL,3M)水洗、萃取。再将萃取液收集干燥后旋干，用柱层析的方法提纯，得到产物1a，白色固体，熔点:165℃，收率92％。Weigh 4-bromophthalic anhydride (0.67mmol, 1eq) and compound 2,6-diisopropylaniline (0.80mmol, 1.2eq) in a 25mL round bottom bottle, dissolve it in acetonitrile (3mL) Finally, DPPOx (1.34mmol, 2eq) prepared by reference (Tetrahedron, 1982,39(20):3253), Et3 N (0.18mL, 1.29mmol, 1.9eq) was added, and the reaction continued for 2.5h under reflux , the reaction was monitored by TLC until the raw materials disappeared completely, the reaction solution was spin-dried, and the reactant was redissolved in ethyl acetate, washed with HCl (30mL, 3M) and NaHCO3 (30mL, 3M) respectively, and extracted. The extract was collected and dried, spin-dried, and purified by column chromatography to obtain product 1a, a white solid, melting point: 165° C., yield 92%.
1H NMR(400MHz,CDCl3)δ8.11(d,J＝3.8Hz,1H),7.96(dd,J＝7.9,1.4Hz,1H),7.84(d,J＝7.9Hz,1H),7.46(t,J＝7.8Hz,1H),7.30(d,J＝7.8Hz,2H),2.67(hept,J＝6.8Hz,2H),1.17(s,12H)；ESI-MS m/z:386.1[M+H]+。1 H NMR (400MHz, CDCl3 ) δ8.11 (d, J=3.8Hz, 1H), 7.96 (dd, J=7.9, 1.4Hz, 1H), 7.84 (d, J=7.9Hz, 1H), 7.46 (t, J=7.8Hz, 1H), 7.30(d, J=7.8Hz, 2H), 2.67(hept, J=6.8Hz, 2H), 1.17(s, 12H); ESI-MS m/z: 386.1 [M+H]+ .
实施例2Example 2
合成5-溴-2-(3,4,5-三甲氧基-苯基)-异吲哚-1,3-二酮1bSynthesis of 5-bromo-2-(3,4,5-trimethoxy-phenyl)-isoindole-1,3-dione 1b
化合物4-溴邻苯二甲酸酐(0.67mmol)和化合物3,4,5-三甲氧基苯胺(0.80mmol)，参照实施例1操作过程，分离得化合物1b，白色固体，熔点：200℃，Compound 4-bromophthalic anhydride (0.67mmol) and compound 3,4,5-trimethoxyaniline (0.80mmol) were isolated according to the operation process of Example 1 to obtain compound 1b, a white solid, melting point: 200°C,
收率88％。Yield 88%.
1H NMR(400MHz,CDCl3)δ8.09(d,J＝0.8Hz,1H),7.94(dd,J＝7.9,1.3Hz,1H),7.82(d,J＝7.9Hz,1H),6.63(s,2H),3.89(s,3H),3.88(s,6H)；ESI-MS m/z:392.1[M+H]+。1 H NMR (400MHz, CDCl3 ) δ8.09 (d, J=0.8Hz, 1H), 7.94 (dd, J=7.9, 1.3Hz, 1H), 7.82 (d, J=7.9Hz, 1H), 6.63 (s,2H), 3.89(s,3H), 3.88(s,6H); ESI-MS m/z: 392.1 [M+H]+ .
实施例3Example 3
合成6-(2,6-二异丙基-苯基)吡咯并(3,4-b)吡啶-5,7-二酮1cSynthesis of 6-(2,6-diisopropyl-phenyl)pyrrolo(3,4-b)pyridine-5,7-dione 1c
化合物2,3-吡啶二羧酸酐(0.67mmol)和化合物2,6-二异丙基苯胺(0.80mmol)，参照实施例1操作过程，分离得化合物1c，白色固体，熔点:170℃，收率56％。Compound 2,3-pyridinedicarboxylic acid anhydride (0.67mmol) and compound 2,6-diisopropylaniline (0.80mmol) were separated according to the operation process of Example 1 to obtain compound 1c, a white solid, melting point: 170°C, and obtained rate 56%.
1H NMR(400MHz,DMSO)δ9.11(dd,J＝5.0,1.4Hz,1H),8.46(dd,J＝7.7,1.4Hz,1H),7.92(dd,J＝7.7Hz,1H),7.54(t,J＝7.7Hz,1H),7.37(d,J＝7.7Hz,2H),2.73(hept,J＝6.8Hz,2H),1.07(d,J＝7.7Hz,12H)；ESI-MS m/z:309.2[M+H]+。1 H NMR (400MHz, DMSO) δ9.11 (dd, J = 5.0, 1.4Hz, 1H), 8.46 (dd, J = 7.7, 1.4Hz, 1H), 7.92 (dd, J = 7.7Hz, 1H), 7.54(t, J=7.7Hz, 1H), 7.37(d, J=7.7Hz, 2H), 2.73(hept, J=6.8Hz, 2H), 1.07(d, J=7.7Hz, 12H); ESI- MS m/z: 309.2 [M+H]+ .
实施例4Example 4
合成6-(3,4,5-三甲氧基-苯基)-吡咯[3,4-b]吡啶-5,7-二酮1dSynthesis of 6-(3,4,5-trimethoxy-phenyl)-pyrrolo[3,4-b]pyridine-5,7-dione 1d
化合物2,3-吡啶二羧酸酐(0.67mmol)和化合物3,4,5-三甲氧基苯胺(0.80mmol)，参照实施例1操作过程，分离得化合物1d，白色固体，熔点:255℃，收率54％。Compound 2,3-pyridinedicarboxylic acid anhydride (0.67mmol) and compound 3,4,5-trimethoxyaniline (0.80mmol) were isolated according to the operation process of Example 1 to obtain compound 1d, a white solid, melting point: 255°C, Yield 54%.
1H NMR(400MHz,DMSO)δ9.05(dd,J＝4.9,1.3Hz,1H),8.39(dd,J＝7.7,1.3Hz,1H),7.86(dd,J＝7.7Hz,1H),6.83(s,2H),3.77(s,6H),3.73(s,3H)；ESI-MS m/z:315.1[M+H]+。1 H NMR (400MHz, DMSO) δ9.05 (dd, J = 4.9, 1.3Hz, 1H), 8.39 (dd, J = 7.7, 1.3Hz, 1H), 7.86 (dd, J = 7.7Hz, 1H), 6.83 (s, 2H), 3.77 (s, 6H), 3.73 (s, 3H); ESI-MS m/z: 315.1 [M+H]+ .
实施例5Example 5
合成6-(2,6-二异丙基-苯基)吡咯[3,4-b]吡嗪-5,7-二酮1eSynthesis of 6-(2,6-diisopropyl-phenyl)pyrrolo[3,4-b]pyrazine-5,7-dione 1e
称取2,3-吡嗪二酸酐(0.67mmol)和化合物2,6-二异丙基苯胺(0.80mmol),参照实施例1操作过程，分离得化合物1e，黄色固体，熔点:200℃，产率69％。Weigh 2,3-pyrazine diacid anhydride (0.67mmol) and compound 2,6-diisopropylaniline (0.80mmol), refer to the operation process of Example 1, and isolate compound 1e, a yellow solid, melting point: 200°C, Yield 69%.
1H NMR(400MHz,CDCl3)δ9.05(s,2H),7.52(t,J＝7.7Hz,1H),7.47(d,J＝7.7Hz,2H),2.67(hept,J＝7.8Hz,2H),1.07(d,J＝7.7Hz,12H),1.19(s,12H)；ESI-MS m/z:310.2[M+H]+。1 H NMR (400MHz, CDCl3 ) δ9.05(s, 2H), 7.52(t, J=7.7Hz, 1H), 7.47(d, J=7.7Hz, 2H), 2.67(hept, J=7.8Hz , 2H), 1.07 (d, J = 7.7 Hz, 12H), 1.19 (s, 12H); ESI-MS m/z: 310.2 [M+H]+ .
实施例6Example 6
合成6-(3,4,5-三甲氧基-苯基)-吡咯[3,4-b]吡嗪-5,7-二酮1fSynthesis of 6-(3,4,5-trimethoxy-phenyl)-pyrrolo[3,4-b]pyrazine-5,7-dione 1f
化合物2,3-吡嗪二酸酐(0.67mmol)和化合物3,4,5-三甲氧基苯胺(0.80mmol)，参照实施例1操作过程，分离得化合物1f，黄色固体，熔点:270℃，收率83％。Compound 2,3-pyrazine diacid anhydride (0.67mmol) and compound 3,4,5-trimethoxyaniline (0.80mmol) were isolated according to the operation process of Example 1 to obtain compound 1f, a yellow solid, melting point: 270°C, Yield 83%.
1H NMR(400MHz,DMSO)δ＝9.09(s,2H),6.80(s,2H),3.78(s,6H),3.74(s,3H)；ESI-MS m/z:316.1[M+H]+。1 H NMR (400MHz, DMSO) δ=9.09(s,2H),6.80(s,2H),3.78(s,6H),3.74(s,3H); ESI-MS m/z:316.1[M+H ]+ .
实施例7Example 7
合成2-(2,6-二异丙基苯基)-5-(噻吩-2-基)异吲哚-1,3-二酮2aSynthesis of 2-(2,6-diisopropylphenyl)-5-(thien-2-yl)isoindole-1,3-dione 2a
将化合物5-溴-2-(2,6-二异丙基-苯基)-异吲哚-1,3-二酮(0.13mmol,1eq)和2-噻吩硼酸(0.25mmol,2eq)溶于2mL的二氧六环中，加入Pd(Ph3P)4(0.013mmol,0.1eq)和Na2CO3(0.38mmol,3eq)，然后再滴加200μL的水，完毕，混合液在100℃下回流3个小时。TLC板跟踪反应直至反应原料消失后，用2厘米厚的硅藻土层对混合液进行过滤，收集滤液，然后用乙酸乙酯对滤液进行3次萃取，合并有机相干燥并旋干得到粗产物。再采用柱层析的方法对粗产物进行提纯，得到化合物2a，白色固体，熔点：176℃℃，收率76％。Compound 5-bromo-2-(2,6-diisopropyl-phenyl)-isoindole-1,3-dione (0.13mmol, 1eq) and 2-thiopheneboronic acid (0.25mmol, 2eq) were dissolved In 2mL of dioxane, add Pd(Ph3 P)4 (0.013mmol, 0.1eq) and Na2 CO3 (0.38mmol, 3eq), and then add 200μL of water dropwise, complete, the mixture is at 100 Reflux for 3 hours at °C. TLC plate followed the reaction until the reaction raw materials disappeared, filtered the mixed solution with a 2 cm thick diatomaceous earth layer, collected the filtrate, then extracted the filtrate three times with ethyl acetate, combined the organic phases to dry and spin dry to obtain the crude product . The crude product was purified by column chromatography to obtain compound 2a, a white solid, melting point: 176°C, yield 76%.
1H NMR(400MHz,CDCl3)δ8.18(s,1H),8.20(s,1H),8.01(d,J＝8.0Hz,1H),7.97(d,J＝8.0Hz,1H),7.53(d,J＝3.5Hz,1H),7.46(m,2H),7.31(d,J＝8.0Hz,2H),7.17(t,J＝7.8Hz,1H),2.71(hept,J＝7.8Hz,2H),1.19(s,6H),1.17(s,6H)；ESI-MS m/z:390.2[M+H]+。1 H NMR (400MHz, CDCl3 )δ8.18(s,1H),8.20(s,1H),8.01(d,J=8.0Hz,1H),7.97(d,J=8.0Hz,1H),7.53 (d, J=3.5Hz, 1H), 7.46(m, 2H), 7.31(d, J=8.0Hz, 2H), 7.17(t, J=7.8Hz, 1H), 2.71(hept, J=7.8Hz ,2H), 1.19(s,6H), 1.17(s,6H); ESI-MS m/z: 390.2[M+H]+ .
实施例8Example 8
合成5-(噻吩-2-基)-2-(3,4,5-三甲氧基苯基)异吲哚-1,3-二酮2bSynthesis of 5-(thiophen-2-yl)-2-(3,4,5-trimethoxyphenyl)isoindole-1,3-dione 2b
化合物5-溴-2-(3,4,5-三甲氧基-苯基)-异吲哚-1,3-二酮(0.13mmol)和2-噻吩硼酸(0.25mmol)，参照实施例7的操作过程，分离得到化合物2b，白色固体，熔点：239℃，收率74％。Compound 5-bromo-2-(3,4,5-trimethoxy-phenyl)-isoindole-1,3-dione (0.13mmol) and 2-thiophene boronic acid (0.25mmol), refer to Example 7 During the operation, the compound 2b was isolated as a white solid, melting point: 239°C, and the yield was 74%.
1H NMR(400MHz,CDCl3)δ8.17(s,1H),8.0(d,J＝8.0Hz,1H),7.94(d,J＝8.0Hz,1H),7.55(d,J＝7.8Hz,1H),7.46(d,J＝8.0Hz,1H),7.18(t,J＝8.0Hz,1H),6.66(s,2H),3.90(s,3H),3.89(s,6H)；ESI-MS m/z:396.1[M+H]+。1 H NMR (400MHz, CDCl3 ) δ8.17(s, 1H), 8.0(d, J=8.0Hz, 1H), 7.94(d, J=8.0Hz, 1H), 7.55(d, J=7.8Hz ,1H),7.46(d,J=8.0Hz,1H),7.18(t,J=8.0Hz,1H),6.66(s,2H),3.90(s,3H),3.89(s,6H); - MS m/z: 396.1 [M+H]+ .
实施例9Example 9
合成3-溴-6-(2,6-二异丙基-苯基)-吡咯并(3,4-b)吡啶-5,7-二酮5a1Synthesis of 3-bromo-6-(2,6-diisopropyl-phenyl)-pyrrolo(3,4-b)pyridine-5,7-dione 5a1
将100mL的沸水加入到的三溴喹啉(2g)中，然后将1.5g的KMnO4分批次加入到反应液中，搅拌，将温度升至100℃后保持2h。发现反应液慢慢有紫色变成黑色，用TLC监测反应至原料完全消失后，反应液用硅藻土过滤，得到黄色的滤液。将滤渣用沸水清洗后过滤，得到滤液，集中滤液，用浓盐酸将其pH调至酸性(pH＝3-4)。然后将反应液旋干后，用真空泵干燥后，得到白色固体，将其溶于丙酮中过滤，滤液旋干得到化合物2,固体颗粒，熔点:158℃，收率69％。100 mL of boiling water was added to tribromoquinoline (2 g), and then 1.5 g of KMnO4 was added to the reaction solution in batches, stirred, and the temperature was raised to 100° C. and kept for 2 h. It was found that the reaction solution gradually changed from purple to black, and the reaction was monitored by TLC until the raw materials disappeared completely, then the reaction solution was filtered with diatomaceous earth to obtain a yellow filtrate. The filter residue was washed with boiling water and then filtered to obtain a filtrate. The filtrate was pooled and its pH was adjusted to acidic (pH=3-4) with concentrated hydrochloric acid. Then the reaction solution was spin-dried and dried with a vacuum pump to obtain a white solid, which was dissolved in acetone and filtered, and the filtrate was spin-dried to obtain compound 2, solid particles, melting point: 158°C, yield 69%.
1H NMR(400MHz,DMSO)δ8.89(d,J＝2.1Hz,1H),8.42(d,J＝2.2Hz,1H).1 H NMR (400MHz, DMSO) δ8.89(d, J=2.1Hz, 1H), 8.42(d, J=2.2Hz, 1H).
在冰浴的条件下将SO2Cl2(4mL)缓慢滴加到装有化合物2(200mg,0.82mmol)的烧瓶中，完毕之后，将反应加热至100℃，经过30min，将反应液旋干，得到化合物3,呈灰色固体,熔点:138℃，收率82％。Slowly add SO2 Cl2 (4 mL) dropwise to the flask containing compound 2 (200 mg, 0.82 mmol) in an ice bath. After completion, the reaction was heated to 100° C., and after 30 min, the reaction solution was spin-dried , to obtain compound 3 as a gray solid, melting point: 138°C, yield 82%.
1H NMR(400MHz,DMSO)δ9.30(d,J＝1.9Hz,1H),8.95(d,J＝1.9Hz,1H).1 H NMR (400MHz, DMSO) δ9.30(d, J=1.9Hz, 1H), 8.95(d, J=1.9Hz, 1H).
化合物3(100mg,0.44mmol,1eq)与2,6-二异丙基苯胺(0.53mmol,1.2eq),参照实施例1操作过程，分离得化合物4a1,呈白色固体,熔点:146℃，收率48％。Compound 3 (100mg, 0.44mmol, 1eq) and 2,6-diisopropylaniline (0.53mmol, 1.2eq) were separated according to the operation process of Example 1 to obtain compound 4a1 as a white solid, melting point: 146°C. rate 48%.
1H NMR(400MHz,DMSO)δ9.25(d,J＝2.0Hz,1H),8.79(d,J＝2.0Hz,1H),7.53(t,J＝7.7Hz,1H),7.35(d,J＝7.7Hz,2H),2.76(hept,J＝7.6Hz,2H),1.07(d,J＝7.6Hz,12H)；ESI-MS m/z:387.1[M+H]+。1 H NMR (400MHz, DMSO) δ9.25(d, J=2.0Hz, 1H), 8.79(d, J=2.0Hz, 1H), 7.53(t, J=7.7Hz, 1H), 7.35(d, J = 7.7 Hz, 2H), 2.76 (hept, J = 7.6 Hz, 2H), 1.07 (d, J = 7.6 Hz, 12H); ESI-MS m/z: 387.1 [M+H]+ .
实施例8Example 8
合成3-溴-6-(3,4,5-三甲氧基-苯基)-吡咯[3,4-b]吡啶-5,7-二酮5a2Synthesis of 3-bromo-6-(3,4,5-trimethoxy-phenyl)-pyrrolo[3,4-b]pyridine-5,7-dione 5a2
化合物3(0.44mmol)和化合物3,4,5-三甲氧基苯胺(0.53mmol)，参照实施例7操作过程，分离得化合物4a2，白色固体，熔点:225℃，总收率36％。Compound 3 (0.44mmol) and compound 3,4,5-trimethoxyaniline (0.53mmol) were isolated according to the procedure in Example 7 to obtain compound 4a2, a white solid, melting point: 225°C, and a total yield of 36%.
1H NMR(400MHz,DMSO)δ9.20(d,J＝2.0Hz,1H),8.74(d,J＝2.0Hz,1H),6.80(s,2H),3.77(s,6H),3.73(s,3H)；ESI-MS m/z:393.1[M+H]+。1 H NMR (400MHz, DMSO) δ9.20(d, J=2.0Hz, 1H), 8.74(d, J=2.0Hz, 1H), 6.80(s, 2H), 3.77(s, 6H), 3.73( s, 3H); ESI-MS m/z: 393.1 [M+H]+ .
体外微管蛋白聚合抑制活性测试方法:In vitro tubulin polymerization inhibitory activity test method:
微管蛋白聚合是通过比浊测定法在384孔板检测的。在4℃和缓冲液(80mM PIPES,pH 7.0,1mM EGTA,1mM MgCl2)中，微管蛋白(4mg/mL)与各种化合物(1μM)预培养30min，之后，加入1mM GTP，检测样品快速升温到37℃。然后采用恒温控制分光光度计(SpectramaxParadigm,Molecular Devices,San Francisco,CA,USA),每分钟定期测量在340nm处的吸光度变化。测试中，紫杉醇和诺考达唑作为稳定剂和破坏剂对照使用。Tubulin polymerization was detected by nephelometric assay in 384-well plates. At 4°C and buffer (80mM PIPES, pH 7.0, 1mM EGTA, 1mM MgCl2 ), tubulin (4mg/mL) was pre-incubated with various compounds (1μM) for 30min, after which 1mM GTP was added, and the sample was detected quickly The temperature was raised to 37°C. The absorbance change at 340 nm was then measured periodically every minute using a thermostatically controlled spectrophotometer (Spectramax Paradigm, Molecular Devices, San Francisco, CA, USA). In the test, paclitaxel and nocodazole were used as stabilizer and destroyer controls.
体外Tau蛋白聚合抑制活性的硫磺素-S(ThS)测试方法:Thioflavin-S (ThS) test method for Tau protein polymerization inhibitory activity in vitro:
在37℃下，在384孔板中，Tau蛋白(5μM)与肝素(2.5μM)，乙酸铵(50mM)和各种化合物(5μM)一齐培养24小时。之后，加入硫磺素-S到最终浓度为20μM。采用荧光分光光度计测定(Spectramax Paradigm,Molecular Devices,San Francisco,CA,USA)荧光值。发射波长在440nm激发波长在520nm。Tau protein (5 μM) was incubated with heparin (2.5 μM), ammonium acetate (50 mM) and various compounds (5 μM) in a 384-well plate at 37° C. for 24 hours. Afterwards, Thioflavin-S was added to a final concentration of 20 μM. Fluorescence values were measured with a spectrofluorometer (Spectramax Paradigm, Molecular Devices, San Francisco, CA, USA). The emission wavelength is at 440nm and the excitation wavelength is at 520nm.
依照上述方法测试所得部分化合物的活性结果列表如下：According to the above method test the activity results of some of the compounds obtained are listed as follows:
表1：Tau蛋白自聚集抑制率和微管蛋白聚合增加率Table 1: Tau protein self-aggregation inhibition rate and tubulin polymerization increase rate
从表1及图1-2可见，除其中化合物1b,2a,2b稍差一些之外，极大分化合物都比紫杉醇有更强的稳定微管蛋白聚合的能力,特别是化合物4a1对微管蛋白聚合有非常强的稳定作用。部分化合物(1e,1f)对微管蛋白聚合具有较好的稳定作用，同时对tau自聚集具有一定程度的抑制活性，特别是化合物1e，有望成为多靶点抗阿茨海默症药物的先导化合物。It can be seen from Table 1 and Figure 1-2 that, except for compounds 1b, 2a, and 2b, which are slightly inferior, most of the compounds have stronger ability to stabilize tubulin polymerization than paclitaxel, especially compound 4a1 on tubulin Polymerization has a very strong stabilizing effect. Some compounds (1e, 1f) have a good stabilizing effect on tubulin polymerization, and at the same time have a certain degree of inhibitory activity on tau self-aggregation, especially compound 1e, which is expected to become the leader of multi-target anti-Alzheimer's disease drugs compound.
上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受上述实施例的限制，其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本发明的保护范围之内。The above-mentioned embodiment is a preferred embodiment of the present invention, but the embodiment of the present invention is not limited by the above-mentioned embodiment, and any other changes, modifications, substitutions, combinations, Simplifications should be equivalent replacement methods, and all are included in the protection scope of the present invention.
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