技术领域technical field
本发明涉及一种1，8-萘酰亚胺衍生物及其应用，属于医药技术领域。The invention relates to a 1,8-naphthalimide derivative and its application, belonging to the technical field of medicine.
背景技术Background technique
萘酰亚胺类衍生物具有独特的平面刚性结构，使其拥有较强的嵌入DNA的能力，越来越多的研究显示萘二甲酰亚胺及其衍生物具有很好的抗肿瘤活性，其对DNA分子具有较高的亲和力，能通过插入作用与DNA结合。Naphthalimide derivatives have a unique planar rigid structure, which enables them to have a strong ability to embed DNA. More and more studies have shown that naphthalimide and its derivatives have good anti-tumor activity. It has a high affinity for DNA molecules and can bind to DNA through intercalation.
已有的研究表明，1,8-萘酰亚胺衍生物具有重要的抗肿瘤活性，其衍生物氨萘菲特(amonafide)和米托萘胺(mitonafide)已进入II期临床试验阶段，但由于二者表现出较大的毒副作用，使得它们的应有受到了严重限制。因此，我们期望合成得到既具有显著生物活性、毒副作用更小的新型萘酰亚胺类化合物。目前尚未见有在1,8-萘酰亚胺荧光团的缺电子4位上引入芳基硫化物的相关报道。Existing studies have shown that 1,8-naphthalimide derivatives have important antitumor activity, and its derivatives, amonafide and mitonafide, have entered phase II clinical trials, but Due to the large toxic and side effects of the two, their application has been severely limited. Therefore, we expect to synthesize new naphthalimide compounds with significant biological activity and less toxic side effects. So far, there has been no report on the introduction of aryl sulfides into the electron-deficient 4-position of 1,8-naphthoimide fluorophores.
发明内容Contents of the invention
本发明要解决的技术问题是提供一种活性更好且活性更低的1，8-萘酰亚胺衍生物及其应用。The technical problem to be solved by the present invention is to provide a 1,8-naphthoimide derivative with better activity and lower activity and its application.
本发明涉及具有下述式(I)所示化合物或其药学上可接受的盐：The present invention relates to a compound represented by the following formula (I) or a pharmaceutically acceptable salt thereof:
上述式(I)所示化合物的化学名称为N-(2-N，N-二甲氨基)乙胺基-6-(4-氟苯亚磺酰基)-1,8-萘二甲酰亚胺，分子量为：410.46。The chemical name of the compound shown in the above formula (I) is N-(2-N, N-dimethylamino) ethylamino-6-(4-fluorophenylsulfinyl)-1,8-naphthalene dicarboxylic acid Amine, molecular weight: 410.46.
上述式(I)所示化合物的制备方法主要包括以下步骤：取如下式(II)所示化合物和N,N-二甲基乙二胺置于有机溶剂中反应，反应结束后回收溶剂，即得目标化合物粗品；The preparation method of the compound represented by the above formula (I) mainly includes the following steps: taking the compound represented by the following formula (II) and N,N-dimethylethylenediamine to react in an organic solvent, and recovering the solvent after the reaction, that is Obtain the target compound crude product;
本发明所述制备方法中，所述的有机溶剂具体可以是选自乙醇、甲醇、二氯甲烷(DCM)、N,N-二甲基甲酰胺(DMF)和石油醚(PE)中的一种或两种以上的组合。当有机溶剂的选择为上述两种以上物质的组合时，它们的配比可以为任意配比。所述有机溶剂的用量可根据需要确定，通常情况下，以1mmol的式(II)所示化合物为基准计算，所有参加反应的原料共用5-10mL的有机溶剂来溶解。In the preparation method of the present invention, the organic solvent may specifically be one selected from ethanol, methanol, dichloromethane (DCM), N,N-dimethylformamide (DMF) and petroleum ether (PE). one or a combination of two or more. When the choice of the organic solvent is a combination of the above two or more substances, their ratio can be any ratio. The amount of the organic solvent can be determined according to the needs. Usually, based on the calculation of 1 mmol of the compound represented by formula (II), all the raw materials participating in the reaction share 5-10 mL of the organic solvent to dissolve.
本发明所述制备方法中，所述的反应可以在不加热或加热条件下进行，优选在不加热条件下进行。反应是否完全可以通过TLC跟踪检测。In the preparation method of the present invention, the reaction can be carried out without heating or under heating conditions, preferably without heating. Whether the reaction is complete can be detected by TLC follow-up.
本发明所述制备方法中，式(II)所示化合物和N,N-二甲基乙二胺的用量比为化学计量比，在实际的实验操作中，通常取式(II)所示化合物和N,N-二甲基乙二胺的物质的量之比为：1：1.5-3。In the preparation method of the present invention, the amount ratio of the compound shown in formula (II) and N,N-dimethylethylenediamine is a stoichiometric ratio. In actual experimental operations, the compound shown in formula (II) is usually used The ratio of the amount of substance to N,N-dimethylethylenediamine is: 1:1.5-3.
本发明所述制备方法制得的是式(I)所示化合物的粗品，可采用现有常规的纯化方法对其进行纯化以提高式(I)所示化合物的纯度。通常采用硅胶柱层析来进行纯化，具体是将制得的目标化合物粗品经硅胶柱层析，用由体积比为5-20：1的乙酸乙酯(EA)和甲醇组成的洗脱剂洗脱，洗脱液蒸除溶剂，得到纯化后的目标化合物。The preparation method of the present invention produces the crude product of the compound represented by formula (I), which can be purified by existing conventional purification methods to improve the purity of the compound represented by formula (I). Silica gel column chromatography is usually used for purification. Specifically, the crude product of the target compound is subjected to silica gel column chromatography and washed with an eluent composed of ethyl acetate (EA) and methanol at a volume ratio of 5-20:1. The solvent was evaporated from the eluent to obtain the purified target compound.
本发明所述制备方法中涉及的原料式(II)所示化合物化学名称为6-(4-氟苯亚磺酰基)-1,8-萘二甲酰亚胺，可参考现有文献合成，也可自行设计路线合成。优选按以下方法进行制备：The chemical name of the compound represented by the raw material formula (II) involved in the preparation method of the present invention is 6-(4-fluorophenylsulfinyl)-1,8-naphthalimide, which can be synthesized with reference to existing literature, You can also design your own route synthesis. It is preferably prepared as follows:
1)取4-氯-1,8-萘二甲酸酐和对氟苯硫酚置于第一溶剂中，在体系呈碱性条件下于加热条件下反应，回收溶剂，所得反应物进行酸化，有沉淀析出，收集沉淀，得到化合物2；1) Take 4-chloro-1,8-naphthalene dicarboxylic anhydride and p-fluorothiophenol and place them in the first solvent, react under heating conditions when the system is alkaline, recover the solvent, and acidify the obtained reactant, There was precipitation, and the precipitate was collected to obtain compound 2;
2)取化合物2和间氯过氧苯甲酸(m-CBPA)置于第二溶剂中，于不加热条件下反应，反应物用饱和NaHCO3溶液洗涤后，用萃取剂萃取，收集有机相，回收溶剂，得到化合物3即式(II)所示化合物。2) Put compound 2 and m-chloroperoxybenzoic acid (m-CBPA) in the second solvent and react without heating. After the reactant is washed with saturated NaHCO3 solution, extract with extractant, collect the organic phase, and recover solvent to obtain compound 3, namely the compound represented by formula (II).
上述式(II)所示化合物制备方法的步骤1)中，所述的第一溶剂优选为N,N-二甲基甲酰胺；通过向体系中加入弱碱性物质(如碳酸氢钠或碳酸钠等)调节体系呈碱性，以有利于反应的进行；反应通常在40℃至第一溶剂的回流温度下进行，反应还优选在搅拌条件下进行，通过TLC跟踪检测反应是否完全；反应完成后，采用酸液(通常为稀盐酸溶液，如5v/v％盐酸溶液)中各体系中的碱性，使体系维持在pH值为7左右。该步骤所得的产物可经过进一步纯化(如重结晶，重结晶的溶剂可以是乙醇等常用溶剂)后再用于后序操作。In step 1) of the preparation method of the compound represented by the above formula (II), the first solvent is preferably N,N-dimethylformamide; by adding a weakly basic substance (such as sodium bicarbonate or carbonic acid Sodium, etc.) adjust the system to be alkaline to facilitate the reaction; the reaction is usually carried out at the reflux temperature of 40°C to the first solvent, and the reaction is preferably carried out under stirring conditions, and whether the reaction is completely detected by TLC; the reaction is completed Finally, use the alkalinity in each system in the acid solution (usually a dilute hydrochloric acid solution, such as a 5v/v% hydrochloric acid solution) to maintain the system at a pH of about 7. The product obtained in this step can be further purified (such as recrystallization, and the solvent for recrystallization can be a common solvent such as ethanol) before being used in subsequent operations.
上述式(II)所示化合物制备方法的步骤2)中，所述的第二溶剂优选为N,N-二甲基甲酰胺；反应通常在常温下进行，通过TLC跟踪检测反应是否完全；反应完成后，最好是采用饱和NaHCO3溶液洗涤除去体系中可能存在的酸；所述的萃取剂优选为二氯甲烷。该步骤所得产物为式(II)所示化合物的粗品，优选是将其经硅胶柱层析纯化(先用由乙酸乙酯和石油醚按1：2-50的体积比组成的混合溶剂洗脱，再用由二氯甲烷、乙酸乙酯和甲醇按9:3:1的体积比组成的混合溶剂洗脱，收集二氯甲烷、乙酸乙酯和甲醇组成的混合溶剂洗脱下来的洗脱液)后再用于后序操作。In step 2) of the preparation method of the compound represented by the above formula (II), the second solvent is preferably N,N-dimethylformamide; the reaction is usually carried out at room temperature, and whether the reaction is complete is tracked by TLC; the reaction After completion, it is best to use saturated NaHCO3 solution to wash and remove the acid that may exist in the system; the extractant is preferably dichloromethane. The product obtained in this step is the crude product of the compound shown in formula (II), and it is preferably purified by silica gel column chromatography (eluted with a mixed solvent consisting of ethyl acetate and petroleum ether in a volume ratio of 1:2-50) , and then eluted with a mixed solvent composed of dichloromethane, ethyl acetate and methanol at a volume ratio of 9:3:1, and collected the eluate eluted from the mixed solvent composed of dichloromethane, ethyl acetate and methanol ) for subsequent operations.
上述式(II)所示化合物制备方法各步骤中涉及的各种反应，原料的摩尔比为化学计量比。For the various reactions involved in each step of the preparation method of the compound represented by the above formula (II), the molar ratio of the raw materials is a stoichiometric ratio.
本发明还包括上述式(I)所示化合物或其药学上可接受的盐在制备抗肿瘤药物中的应用，特别是在制备抑制人肺癌细胞、膀胱癌细胞或乳腺癌细胞药物中的应用。The present invention also includes the application of the compound represented by the above formula (I) or a pharmaceutically acceptable salt thereof in the preparation of antitumor drugs, especially in the preparation of drugs inhibiting human lung cancer cells, bladder cancer cells or breast cancer cells.
本发明还提供一种药物组合物，该药物含有治疗上有效剂量的上述式(I)所示化合物或其药学上可接受的盐。The present invention also provides a pharmaceutical composition, which contains a therapeutically effective dose of the compound represented by the above formula (I) or a pharmaceutically acceptable salt thereof.
与现有技术相比，本发明提供了一种结构新颖的1，8-萘酰亚胺衍生物，申请人的体外试验表明，该衍生物具有显著生物活性，特别是对非小细胞肺癌细胞株NCI-H460、人膀胱癌细胞T24以及人乳腺癌细胞MCF-7均具有极为显著的抑制活性，而且对人正常细胞WI-38的毒副作用较氨萘非特更小，有望开发成靶向治疗药物。Compared with the prior art, the present invention provides a novel structure of 1,8-naphthoimide derivatives, the applicant's in vitro tests show that the derivatives have significant biological activity, especially for non-small cell lung cancer cells Strain NCI-H460, human bladder cancer cell T24 and human breast cancer cell MCF-7 all have extremely significant inhibitory activity, and the toxic and side effects on human normal cell WI-38 are less than that of nafide, and it is expected to be developed into a targeted therapy drug.
附图说明Description of drawings
图1为式(I)所示化合物(即空白组)作为荧光探针在激发波长405nm，发射波长498nm的荧光光谱谱图；Fig. 1 is compound (being blank group) shown in formula (I) as fluorescent probe at excitation wavelength 405nm, the fluorescence spectrogram of emission wavelength 498nm;
图2为式(I)所示化合物与GSSG结合(即实验2组)作为荧光探针在激发波长405nm，发射波长498nm的荧光光谱谱图；Fig. 2 is that compound shown in formula (I) is combined with GSSG (being experiment 2 groups) as fluorescent probe at excitation wavelength 405nm, the fluorescence spectrogram of emission wavelength 498nm;
图3为式(I)所示化合物与GSH结合(即实验1组)作为荧光探针在激发波长405nm，发射波长498nm的荧光光谱谱图。Fig. 3 is the fluorescence spectrum spectrogram of the compound shown in formula (I) combined with GSH (ie experiment 1 group) as a fluorescent probe at an excitation wavelength of 405nm and an emission wavelength of 498nm.
具体实施方式Detailed ways
下面结合具体实施例对本发明作进一步的详述，以更好地理解本发明的内容，但本发明并不限于以下实施例。The present invention will be further described below in conjunction with specific examples to better understand the content of the present invention, but the present invention is not limited to the following examples.
实施例1：式(II)所示化合物的制备Embodiment 1: the preparation of compound shown in formula (II)
按下述合成路线制备式(II)所示化合物：Prepare compound shown in formula (II) according to following synthetic route:
具体的制备方法包括以下步骤：Concrete preparation method comprises the following steps:
1)在电磁搅拌下依次向250mL的圆底烧瓶中加入4-氯-1,8-萘二甲酸酐(2.32g,10mmol)、DMF(30mL,28.35g)、NaHCO3(0.84g)和对氟苯硫酚(1.59mL,15mmol),加热回流反应10h(TLC监测反应,展开剂：VEA:VPE＝1:4)。冷却,减压除去溶剂,用5％的HCl溶液酸化,得到黄色沉淀,过滤,用乙醇重结晶得到1.62g化合物2,产率69.8％；1) Add 4-chloro-1,8-naphthalic anhydride (2.32g, 10mmol), DMF (30mL, 28.35g), NaHCO3 (0.84g) and para Fluorothiophenol (1.59mL, 15mmol), heated to reflux for 10h (reaction monitored by TLC, developer: VEA : VPE = 1:4). Cool, remove the solvent under reduced pressure, acidify with 5% HCl solution to obtain a yellow precipitate, filter, and recrystallize with ethanol to obtain 1.62g of compound 2, with a yield of 69.8%;
2)在电磁搅拌下向盛有DCM(10mL)溶剂的圆底烧瓶中依次加入化合物2(0.30g,0.93mmol)与1.5当量的m-CBPA(纯度85％,0.28g,1.40mmol)常温反应5h(TLC监测反应,展开剂:VEA:VPE＝1:2),用饱和NaHCO3溶液洗涤(4×15mL),二氯甲烷(DCM)萃取,收集有机相，干燥,过滤,减压除去溶剂,用硅胶柱层析纯化(先用由乙酸乙酯和石油醚按1：50的体积比组成的混合溶剂洗脱，再用由二氯甲烷、乙酸乙酯和甲醇按9:3:1的体积比组成的混合溶剂洗脱，收集二氯甲烷、乙酸乙酯和甲醇组成的混合溶剂洗脱下来的洗脱液),得到0.81g化合物3(淡黄色固体)即式(II)所示化合物。2) Add compound 2 (0.30g, 0.93mmol) and 1.5 equivalents of m-CBPA (purity 85%, 0.28g, 1.40mmol) to a round bottom flask filled with DCM (10mL) solvent and react at room temperature under electromagnetic stirring 5h (TLC monitoring reaction, developer: VEA : VPE = 1: 2), washed with saturated NaHCO3 solution (4×15 mL), extracted with dichloromethane (DCM), collected organic phase, dried, filtered, and reduced pressure Remove the solvent and purify by silica gel column chromatography (elution with a mixed solvent consisting of ethyl acetate and petroleum ether at a volume ratio of 1:50, and then dichloromethane, ethyl acetate and methanol at a ratio of 9:3: The volume ratio of 1 is eluted with a mixed solvent composed of dichloromethane, ethyl acetate and methanol, and the eluent eluted with a mixed solvent composed of dichloromethane, ethyl acetate and methanol) is obtained to obtain 0.81g of compound 3 (light yellow solid) that is represented by formula (II) shows the compound.
对本实施例所得化合物3进行核磁共振氢谱和核磁共振碳谱分析，数据分别如下所示：Compound 3 obtained in the present embodiment was analyzed by proton nuclear magnetic resonance spectrum and carbon nuclear magnetic resonance spectrum, and the data were as follows respectively:
1H NMR(500MHz,CDCl3)δ8.80(d,J＝7.7Hz,1H),8.68(d,J＝7.3Hz,1H),8.59(dd,J＝11.4,8.5Hz,2H),7.89(t,J＝8.4Hz,1H),7.72(dd,J＝8.7,5.0Hz,2H),7.15(t,J＝8.4Hz,2H).1H NMR (500MHz, CDCl3) δ8.80(d, J=7.7Hz, 1H), 8.68(d, J=7.3Hz, 1H), 8.59(dd, J=11.4, 8.5Hz, 2H), 7.89(t ,J=8.4Hz,1H),7.72(dd,J=8.7,5.0Hz,2H),7.15(t,J=8.4Hz,2H).
13C NMR(125MHz,CDCl3)δ164.8(d,J＝228.1Hz),159.6,159.5,149.7,139.2(d,J＝3.1Hz),133.4(d,J＝121.4Hz),130.5,129.5,128.7,128.1,128.0,127.7,124.1,120.6(d,J＝190.7Hz),117.4,117.3.13C NMR (125MHz, CDCl3) δ164.8(d, J=228.1Hz), 159.6, 159.5, 149.7, 139.2(d, J=3.1Hz), 133.4(d, J=121.4Hz), 130.5, 129.5, 128.7 ,128.1,128.0,127.7,124.1,120.6(d,J=190.7Hz),117.4,117.3.
因此，可确定上述淡黄色固体的结构如下述式(II)所示：Therefore, it can be determined that the structure of the above-mentioned pale yellow solid is shown in the following formula (II):
由此，确定其为式(II)所示化合物，化学名称为6-(4-氟苯亚磺酰基)-1,8-萘二甲酰亚胺。Therefore, it was confirmed that it was a compound represented by formula (II), and its chemical name was 6-(4-fluorophenylsulfinyl)-1,8-naphthalimide.
实施例2：式(I)所示化合物的制备Embodiment 2: the preparation of compound shown in formula (I)
在电磁搅拌下向盛有乙醇(20mL)的圆底烧瓶中依次加入式(II)所示化合物(0.27g,0.59mmol，按实施例1所述方法制得)和N,N-二甲基乙二胺(0.12mL,0.71mmol)，常温反应2h(TLC监测反应,展开剂:VEA:VMeOH＝4:1),减压除去溶剂,上硅胶柱层析(洗脱剂:VEA:VMeOH＝10:1),得到0.21g淡黄色固体，产率77.8％。Add the compound shown in formula (II) (0.27g, 0.59mmol, prepared according to the method described in Example 1) and N,N-dimethyl Ethylenediamine (0.12mL, 0.71mmol), reacted at room temperature for 2h (TLC monitoring reaction, developing solvent: VEA : VMeOH = 4: 1), removed the solvent under reduced pressure, and applied silica gel column chromatography (eluent: VEA :VMeOH =10:1), 0.21g of a light yellow solid was obtained, and the yield was 77.8%.
对本实施例所得淡黄色固体进行核磁共振氢谱、核磁共振碳谱和电喷雾质谱分析，数据分别如下所示：The light yellow solid obtained in this example was analyzed by proton nuclear magnetic resonance, carbon nuclear magnetic resonance and electrospray mass spectrometry, and the data were as follows:
1H NMR(500MHz,CDCl3)δ8.75(d,J＝7.7Hz,1H),8.62(d,J＝7.3Hz,1H),8.48(dd,J＝8.0,4.5Hz,2H),7.80(t,J＝7.6Hz,1H),7.69(dd,J＝8.7,5.0Hz,2H),7.12(t,J＝8.4Hz,2H),4.31(t,J＝6.8Hz,2H),2.64(t,J＝6.8Hz,2H),2.33(s,6H).1 H NMR (500MHz, CDCl3) δ8.75 (d, J = 7.7Hz, 1H), 8.62 (d, J = 7.3Hz, 1H), 8.48 (dd, J = 8.0, 4.5Hz, 2H), 7.80 ( t,J=7.6Hz,1H),7.69(dd,J=8.7,5.0Hz,2H),7.12(t,J=8.4Hz,2H),4.31(t,J=6.8Hz,2H),2.64( t,J=6.8Hz,2H),2.33(s,6H).
13C NMR(125MHz,CDCl3)δ:165.5,163.5,163.3,147.5,139.7(d,J＝3.2Hz),131.2(d,J＝125.0Hz),128.6,128.1(d,J＝17.3Hz),127.9(d,J＝9.1Hz),127.5,125.2,123.8,123.6,117.2,117.0,56.9,53.3,45.7,38.3.13 C NMR (125MHz, CDCl3) δ: 165.5, 163.5, 163.3, 147.5, 139.7 (d, J = 3.2Hz), 131.2 (d, J = 125.0Hz), 128.6, 128.1 (d, J = 17.3Hz), 127.9 (d, J=9.1Hz), 127.5, 125.2, 123.8, 123.6, 117.2, 117.0, 56.9, 53.3, 45.7, 38.3.
电喷雾质谱：ESI-MS m/z:411.1173[M+H]+.Electrospray mass spectrometry: ESI-MS m/z: 411.1173[M+H]+.
因此，可确定上述淡黄色固体即为目标产物N-(2-N，N-二甲氨基)乙胺基-6-(4-氟苯亚磺酰基)-1,8-萘二甲酰亚胺，其化学结构式如下式(I)所示：Therefore, it can be determined that the above light yellow solid is the target product N-(2-N,N-dimethylamino)ethylamino-6-(4-fluorobenzenesulfinyl)-1,8-naphthalene dicarboxylic acid Amine, its chemical structural formula is shown in following formula (I):
实验例1：式(I)所示化合物的荧光测定Experimental Example 1: Fluorescence Measurement of Compounds Shown in Formula (I)
本实验使用荧光分光光度计检测式(I)所示化合物作为荧光探针在激发波长405nm，发射波长498nm的荧光强度以及式(I)所示化合物作为荧光探针分别与GSH(还原型谷胱甘肽)、GSSG(氧化型谷胱甘肽)孵育后在同等条件下的的荧光强度。This experiment uses the compound shown in fluorescence spectrophotometer detection formula (I) as fluorescent probe at excitation wavelength 405nm, the fluorescence intensity of emission wavelength 498nm and the compound shown in formula (I) as fluorescent probe respectively with GSH (reduced glutathione Glycine), GSSG (oxidized glutathione) after incubation under the same conditions of fluorescence intensity.
实验分组情况为：The experimental groups are as follows:
实验组，包括2个小组，具体如下：The experimental group, including 2 groups, is as follows:
实验1组：即式(I)所示化合物+GSH，以式(I)所示化合物终浓度为5μmol/L，终浓度为10μmol/L，共孵育1h。Experiment 1 group: the compound represented by formula (I)+GSH, the final concentration of the compound represented by formula (I) was 5 μmol/L, and the final concentration was 10 μmol/L, and co-incubated for 1 h.
实验2组：即式(I)所示化合物+GSSG，以式(I)所示化合物终浓度为5μmol/L，GSSG终浓度为10μmol/L，共孵育1h。Experimental group 2: the compound represented by formula (I)+GSSG, the final concentration of the compound represented by formula (I) was 5 μmol/L, the final concentration of GSSG was 10 μmol/L, and co-incubated for 1 h.
空白组：即CON组，以式(I)所示化合物终浓度为5μmol/L。Blank group: the CON group, the final concentration of the compound represented by formula (I) was 5 μmol/L.
实验结果：式(I)所示化合物本身显示微弱荧光，与还原型谷胱甘肽反应后，荧光强度可增加40％，与氧化型谷胱甘肽反应后无明显变化。Experimental results: the compound represented by the formula (I) itself exhibits weak fluorescence. After reacting with reduced glutathione, the fluorescence intensity can increase by 40%, and there is no obvious change after reacting with oxidized glutathione.
各组测定结果如图1-3所示，其中图1为式(I)所示化合物的荧光光谱谱图，图2为实验2组(即式(I)所示化合物+GSSG)的荧光光谱谱图，图3为实验1组(即式(I)所示化合物+GSH)的荧光光谱谱图。Each group of measurement results is shown in Figures 1-3, wherein Figure 1 is the fluorescence spectrum spectrogram of the compound shown in the formula (I), and Figure 2 is the fluorescence spectrum of the experimental 2 groups (i.e. the compound+GSSG shown in the formula (I) Spectrum, Fig. 3 is the fluorescence spectrum spectrogram of experiment 1 group (ie compound+GSH shown in formula (I)).
为了充分说明本发明所述目标化合物(即式(I)所示化合物，N-(2-N，N-二甲氨基)乙胺基-6-(4-氟苯亚磺酰基)-1,8-萘二甲酰亚胺)在制药中的用途，申请人对其进行了体外抗肿瘤活性实验。In order to fully illustrate the target compound of the present invention (i.e. the compound shown in formula (I), N-(2-N, N-dimethylamino) ethylamino-6-(4-fluorobenzenesulfinyl)-1, 8-naphthalimide) in the purposes of pharmacy, the applicant has carried out in vitro antitumor activity experiment to it.
1、细胞株与细胞培养1. Cell lines and cell culture
本实验选用人肺癌细胞HCC-827、H1299、NCI-H460、A549，人胃癌细胞MGC-803、人膀胱癌细胞T24、人卵巢癌细胞SKOV3、人肝癌细胞株Hep G2、人乳腺癌细胞MCF-7以及人正常细胞WI-38、LO2共11种细胞株。In this experiment, human lung cancer cells HCC-827, H1299, NCI-H460, A549, human gastric cancer cell line MGC-803, human bladder cancer cell line T24, human ovarian cancer cell line SKOV3, human liver cancer cell line Hep G2, human breast cancer cell line MCF- 7 and human normal cells WI-38, LO2, a total of 11 cell lines.
HCC-827、H1299、NCI-H460、A549、人正常细胞LO2等肿瘤细胞株均培养在含10wt％小牛血清、100U/mL青霉素、100U/mL链霉素的RPMI-1640培养液内，置37℃含体积浓度5％CO2孵箱中培养；人胃癌细胞MGC-803、人膀胱癌细胞T24、人卵巢癌细胞SKOV3、人肝癌细胞株Hep G2、人乳腺癌细胞MCF-7以及人正常细胞WI-38细胞株则培养在含10wt％小牛血清、100U/mL青霉素、100U/mL链霉素的DMEM培养液内。Tumor cell lines such as HCC-827, H1299, NCI-H460, A549, and human normal cell LO2 were all cultured in RPMI-1640 culture medium containing 10wt% calf serum, 100U/mL penicillin, and 100U/mL streptomycin. Cultured in a 5% CO2 incubator at 37°C; human gastric cancer cell line MGC-803, human bladder cancer cell line T24, human ovarian cancer cell line SKOV3, human liver cancer cell line Hep G2, human breast cancer cell line MCF-7, and human normal cells The WI-38 cell line was cultured in DMEM medium containing 10wt% calf serum, 100U/mL penicillin, and 100U/mL streptomycin.
2、待测化合物的配制2. Preparation of test compounds
所用的目标化合物为按本发明实施例4制得，纯度≥99％,将其DMSO储液(浓度为0.001mol/L)通过培养基依次稀释成五个浓度梯度，分别为20、10、5、2.5、1.25μmol/L，其中助溶剂DMSO终浓度≤1％。首先测试20μmol/L的目标产物对于肿瘤细胞增殖的抑制率，视为初筛结果；再分别测试不同梯度浓度下目标产物对各种肿瘤细胞的增殖抑制程度，用以拟合计算半数抑制浓度，即IC50值。The target compound used was prepared according to Example 4 of the present invention, with a purity of ≥99%, and its DMSO stock solution (concentration of 0.001mol/L) was diluted into five concentration gradients successively through the medium, respectively 20, 10, 5 , 2.5, 1.25 μmol/L, wherein the final concentration of co-solvent DMSO ≤ 1%. First test the inhibitory rate of 20 μmol/L target product on the proliferation of tumor cells, which is regarded as the result of the preliminary screening; then test the degree of inhibition of the target product on the proliferation of various tumor cells at different gradient concentrations, and use it to fit and calculate the half inhibitory concentration. That is the IC50 value.
3、细胞生长抑制实验(MTT法)3. Cell growth inhibition test (MTT method)
(1)取对数生长期的细胞，经胰蛋白酶消化后，用含10％小牛血清的培养液配制成浓度为5000个/mL的细胞悬液，以每孔180μL接种于96孔培养板中，使待测细胞密度至1000～10000个/孔(边缘孔用无菌PBS填充)；(1) Take the cells in the logarithmic growth phase, digest them with trypsin, prepare a cell suspension with a concentration of 5000 cells/mL with culture medium containing 10% calf serum, inoculate 180 μL per well in a 96-well culture plate , make the density of cells to be tested to 1000-10000/well (edge wells are filled with sterile PBS);
(2)5％CO2，37℃孵育24h，至细胞单层铺满孔底，每孔加入一定浓度梯度的药物20μL，每个浓度梯度设5个复孔；(2) 5% CO2, incubate at 37°C for 24 hours, until the cell monolayer covers the bottom of the well, add 20 μL of drug with a certain concentration gradient to each well, and set 5 duplicate wells for each concentration gradient;
(3)5％CO2，37℃孵育48h，倒置显微镜下观察；(3) 5% CO2, incubate at 37°C for 48 hours, observe under an inverted microscope;
(4)每孔加入10μL的MTT溶液(5mg/mL PBS，即0.5％MTT)，继续培养4h；(4) Add 10 μL of MTT solution (5 mg/mL PBS, ie 0.5% MTT) to each well, and continue to incubate for 4 h;
(5)终止培养，小心吸去孔内培养液，每孔加入100μL DMSO充分溶解甲瓒沉淀，振荡器混匀后，在酶标仪用波长为570nm，参比波长为630nm测定各孔的光密度值；(5) Terminate the culture, carefully suck out the culture medium in the wells, add 100 μL DMSO to each well to fully dissolve the formazan precipitate, mix well with a shaker, and measure the light in each well with a microplate reader with a wavelength of 570nm and a reference wavelength of 630nm. density value;
(6)同时设置调零孔(培养基、MTT、DMSO)，对照孔(细胞、相同浓度的药物溶解介质、培养液、MTT、DMSO)。(6) At the same time, set zero adjustment wells (medium, MTT, DMSO) and control wells (cells, drug dissolution medium with the same concentration, culture medium, MTT, DMSO).
(7)根据测得的光密度值(OD值)，来判断活细胞数量，OD值越大，细胞活性越强。(7) According to the measured optical density value (OD value), the number of living cells is judged. The larger the OD value, the stronger the cell activity.
利用公式：Use the formula:
计算化合物对细胞生长的抑制率，进一步通过SPSS软件对五个浓度梯度的抑制率数据进行拟合，求出化合物对不同细胞株的半数抑制浓度(IC50值，单位μmol/L)，化合物对于不同肺细胞株的IC50值如表1所示，对胃癌、肝癌、乳腺癌等其他肿瘤细胞株和人正常细胞株的IC50值如表2所示。Calculate the inhibitory rate of the compound to cell growth, and further use SPSS software to fit the inhibitory rate data of five concentration gradients to obtain the half-maximum inhibitory concentration (IC50 value, unit μmol/L) of the compound to different cell lines. The IC50 values of different lung cell lines are shown in Table 1, and the IC50 values of gastric cancer, liver cancer, breast cancer and other tumor cell lines and human normal cell lines are shown in Table 2.
表1：本发明所述化合物对肺癌细胞株及其肺正常细胞株的IC50值(μM)Table 1: IC50 value (μ M) of compound of the present invention to lung cancer cell line and lung normal cell line thereof
表2：本发明所述化合物所述对其他肿瘤细胞株及其正常肝细胞株的IC50值(μM)Table 2: IC50 values (μM) of the compounds of the present invention to other tumor cell lines and their normal liver cell lines
注：“-”表示未检测。Note: "-" means not detected.
从体外抗肿瘤活性测试结果来看，本发明所述目标化合物对肿瘤细胞株具有较为显著的抑制作用，特别是对非小细胞肺癌细胞株NCI-H460、人膀胱癌细胞T24以及人乳腺癌细胞MCF-7的选择抑制作用更为明显，其中对非小细胞肺癌细胞株NCI-H460的抑制活性显著供优于氨萘非特，且其对人正常细胞WI-38的毒副作用较氨萘非特更小，有望开发成靶向治疗药物。From the test results of anti-tumor activity in vitro, the target compound of the present invention has a more significant inhibitory effect on tumor cell lines, especially on non-small cell lung cancer cell line NCI-H460, human bladder cancer cell T24 and human breast cancer cells. The selective inhibitory effect of MCF-7 is more obvious, and its inhibitory activity on non-small cell lung cancer cell line NCI-H460 is significantly better than that of damafetide, and its toxic side effects on human normal cell WI-38 are more severe than damafetide Small, it is expected to be developed into a targeted therapy drug.
综上所述，本发明所述的1,8-萘酰亚胺衍生物总体表现出了较好的体外抗肿瘤活性，同时对其进行荧光测定结果也显示该衍生物呈现良好的GSH指示作用，故有希望开发为抗肿瘤药物或指示剂。In summary, the 1,8-naphthoimide derivatives of the present invention generally exhibit good anti-tumor activity in vitro, and the results of fluorescence measurement also show that the derivatives have good GSH indicating effect , so it is expected to be developed as an antitumor drug or indicator.
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