CN113683552A - 一种色胺水杨酸类化合物及其制备和应用 - Google Patents
一种色胺水杨酸类化合物及其制备和应用 Download PDFInfo
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- CN113683552A CN113683552A CN202011093086.9A CN202011093086A CN113683552A CN 113683552 A CN113683552 A CN 113683552A CN 202011093086 A CN202011093086 A CN 202011093086A CN 113683552 A CN113683552 A CN 113683552A
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Abstract
Description
技术领域
本发明属于医药色胺水杨酸化合物技术领域,具体涉及一种N-苯基的色胺水杨酸类化合物及其制备和应用。
背景技术
胶质瘤,是成人最常见的原发性脑瘤,居颅内肿瘤发病率第一位,且致死率高居颅内肿瘤首位。目前,胶质瘤治疗手段仍主要以手术切除为主,结合放疗和化疗进行治疗。然而,由于胶质瘤属于脑内肿瘤,血脑屏障的存在限制了绝大多数抗肿瘤药物的应用,目前临床上传统化疗方法主要以烷化剂为主,如细胞毒类药物(替莫唑胺等)。由于这些已有的药物存在毒性大,选择性差,存在耐药性,不能显著提高患者的生存期等问题,因此,寻找高效低毒的新型抗胶质瘤药物势在必行。
色胺水杨酸类衍生物,以往的研究中报道该类衍生物细胞实验和动物模型上均具有显著的抗神经炎症、抗癫痫等重要药理作用。但对于该类衍生物对抗肿瘤尤其是抗胶质瘤的应用研究尚属空白。特别是N-苯基结构取代的色胺水杨酸类衍生物模板分子13具有较高的细胞毒性和COX-2抑制作用,但对其结构改造和在肿瘤细胞尤其是胶质瘤细胞的应用目前没有报道。因此本发明以N-苯基结构取代的色胺水杨酸类衍生物(13)为模板分子进行结构改造, 设计开发了一类或化合物,该类化合物具有良好的血脑屏障透过性的新型COX-2抑制剂用于靶向治疗胶质瘤。。
发明内容
本发明所要解决的技术问题是针对现有技术中的缺点而提供一种以N-苯基的色胺水杨酸(13)为模版分子,对其进行不同的修饰改造得到系列衍生物,进行抗肿瘤尤其是胶质瘤生物活性研究,结果表明,该化合物具有很好的抗胶质瘤肿瘤活性,具有抗肿瘤药物开发潜力,可以用来制备抗肿瘤药物。
本发明的另一目的是提供上述N-苯基的色胺水杨酸类化合物的制备方法。
本发明的还有一目的是提供上述N-苯基的色胺水杨酸类化合物的应用。
一种N-苯基色胺水杨酸类化合物,以N-苯基色胺水杨酸(13)为模版分子,对其进行不同的修饰改造得到的,其结构通式为:
其中:R1为卤素F或 Cl或Br或I、烷基、杂烷烃、氨基、硝基的一种或几种;R1在苯环上的取代位置为2位、3位、4位,取代基数量为单取代或多取代;
R2为卤素或烷基的一种,其中卤素为F或 Cl或Br或I;
n为2-5;
其中R1为1-4个碳原子低级烷基链;或为甲氧基、三氟甲基、N-N二甲基、氨基、硝基的一种或几种。
上述N-苯基色胺水杨酸化合物,具体结构式为:
上述N-苯基色胺水杨酸类化合物的制备方法,其合成路线如下:
上述N-苯基色胺水杨酸类化合物的制备方法,其工艺步骤如下:
(1)氩气条件下,邻碘苯甲酸类化合物与氨类化合物Ⅰ在溶解了K2CO3、铜粉的 DMF中,反应生成化合物Ⅱ;
(2)将化合物II、EDCI·HCl、HOBT溶解在二氯甲烷中,在室温下加入Et3N和色胺反应得到化合物III。
上述N-苯基色胺水杨酸类化合物的制备方法,其具体步骤如下:
(1)氩气条件下,邻碘苯甲酸类化合物与氨类化合物Ⅰ在溶解了K2CO3、铜粉的 DMF中,其中邻碘苯甲酸类化合物:氨类化合物Ⅰ:K2CO3:铜的摩尔比为1:1.2:1:0.001,邻碘苯甲酸类化合物在DMF中的浓度为0.5 mmol/ mL,在100 摄氏度的条件下加热12 小时后冷却至室温,用水进行萃取后用水稀释和2.0N HCl酸化,pH达到2,过滤除胺得到化合物Ⅱ;
(2)将化合物II(1.0 eq),EDCI·HCl(1.2 eq),HOBT(1.1 eq)溶解在二氯甲烷(3mL)中,并在室温下加入Et3N和色胺,搅拌8小时,其中化合物II:EDCI·HCl:HOBT:Et3N:色胺摩尔比为1:1.2:1.1:2.5:1,化合物II在二氯甲烷的浓度为0.5 mmol/ mL,通过TLC检测反应完成后减压旋转蒸发除去反应液,然后将得到的残余物通过硅胶快速柱色谱纯化得到化合物III。
上述N-苯基色胺水杨酸类化合物是抑制多种人肿瘤细胞株,优选人胶质瘤细胞株U87MG、U251、C6等的增殖、迁移、侵袭、愈合;诱导人胶质瘤细胞株U87MG、U251、C6等的凋亡,诱导人胶质瘤细胞株U87MG、U251、C6等的G2/M期周期阻滞,可用于治疗肿瘤,尤其是用于胶质瘤治疗。
上述N-苯基色胺水杨酸类化合物的应用,其为一种COX-2抑制剂,在胶质瘤动物模型有效。
本发明公开了一类新型N-苯基色胺酸类化合物,本化合物合成路线绿色环保、简洁高效、原料易得,通过体内外实验证在体外抗肿瘤活性表明,该类化合物表现出良好的抗各类人肿瘤活性,优选胶质瘤,具有抗肿瘤药物开发潜力,可以用来制备抗肿瘤药物,给恶性胶质瘤的治疗提供了新思路,有助于开发新的高效低毒的抗胶质瘤药物。
附图说明
图1 为本发明N-苯基色胺酸类化合物对多种人胶质瘤细胞株的抗增殖作用图;
图2 为本发明化合物 NP16显著减少U251、U87MG细胞增殖细胞比例与Control组相比较图,*p< 0.05;
图3 a、图3 b为本发明化合物 NP16显著增加U251、U87MG细胞G2/M期细胞比例与Control组相比较图,*p< 0.05;
图4为本发明化合物NP16显著诱导U251、U87MG细胞凋亡的发生,并诱导凋亡蛋白的表达增加,下调抗凋亡蛋白的表达与control组相比较图,*p< 0.05,**p< 0.01;
图5为本发明化合物NP16显著诱导U251、U87MG细胞活性氧的爆发与control组相比较图,*p< 0.05,**p< 0.01;
图6为本发明化合物NP16显著抑制U251、U87MG细胞侵袭迁移的发生,并下调金属基质蛋白酶MMP-9表达与control组相比较图,*p< 0.05,**p< 0.01;
图7a、图7b、图7c为本发明化合物NP16显著增加造模成功的大鼠胶质瘤原位模型的SD大鼠的生存期,显著改善大鼠胶质瘤原位模型的记忆探索行为,显著改善胶质瘤对周围正常阻滞的侵袭与control组相比较图I,*p< 0.05,**p< 0.01;
图7d为本发明化合物NP16显著增加造模成功的大鼠胶质瘤原位模型的SD大鼠的生存期,显著改善大鼠胶质瘤原位模型的记忆探索行为,显著改善胶质瘤对周围正常阻滞的侵袭与control组相比较图Ⅱ,*p< 0.05,**p< 0.01;
图7e、图7f、图7g为本发明化合物NP16显著增加造模成功的大鼠胶质瘤原位模型的SD大鼠的生存期,显著改善大鼠胶质瘤原位模型的记忆探索行为,显著改善胶质瘤对周围正常阻滞的侵袭与control组相比较图Ⅲ,*p< 0.05,**p< 0.01;
图7h、图7i、图7j为本发明化合物NP16显著增加造模成功的大鼠胶质瘤原位模型的SD大鼠的生存期,显著改善大鼠胶质瘤原位模型的记忆探索行为,显著改善胶质瘤对周围正常阻滞的侵袭与control组相比较图Ⅳ,*p< 0.05,**p< 0.01;
图7k为本发明化合物NP16显著增加造模成功的大鼠胶质瘤原位模型的SD大鼠的生存期,显著改善大鼠胶质瘤原位模型的记忆探索行为,显著改善胶质瘤对周围正常阻滞的侵袭与control组相比较图Ⅴ,*p< 0.05,**p< 0.01;
图8 为本发明化合物NP16经腹腔注射后(60 mg/kg)的大鼠脑脊液分布图,(A)NP16 加入到空白脑脊液的质谱图,(B)化合物NP16经腹腔注射后(60 mg/kg)在大鼠脑脊液质谱图,黑色箭头表示检测到的目标分子量的质子化分子离子和钠加和离子。
具体实施方式:
实施例1
本发明N-苯基色胺水杨酸化合物的合成路线图:
具体步骤如下:
(1)化合物Ⅱ的制备:氩气条件下,把邻碘苯甲酸(1.0 mmol, 1.0 eq)、氨基类化合物(1.2 mmol, 1.2 eq)、K2CO3 (1.0 mmol, 1.0 eq)、铜粉(0.001 mmol, 0.1% eq)溶解于 2 mL DMF 中,在100 摄氏度的条件下加热12 小时,之后冷却到室温,之后用水进行萃取,用水稀释深绿色固体,并用2.0N HCl酸化,直到pH达到2,大量黑色固体沉淀出来,将悬浮液过滤,用过量的水洗涤以除去胺,然后将滤饼溶解在二氯甲烷中,通过减压旋转蒸发除去溶剂,并用硫酸钠干燥并浓缩,无需进一步纯化即可获得粗产物邻氨基苯甲酸衍生物。
(2)化合物III的制备:将化合物II(1.0 eq)、EDCI·HCl(1.2 eq)、HOBT(1.1 eq)溶解在二氯甲烷(3 mL)中,并在室温下加入Et3N(2.5 eq)和色胺(1.0 eq),搅拌8小时,通过TLC检测反应完成后,通过减压旋转蒸发除去反应液,然后将得到的残余物通过硅胶快速柱色谱纯化,得到所需的固体产物,两步的总产率为50-90%。
实施例2
一种N-苯基色胺水杨酸类化合物,为
N-(2-(1H-indol-3-yl)ethyl)-2-((2-fluorophenyl)amino)benzamide (NP1),
white solid (78% yield), 1H NMR (400 MHz, CDCl3) δ 9.34 (s, 1H), 8.20(s, 1H), 7.60 (d, J = 7.8 Hz, 1H), 7.34 (dd, J = 13.3, 4.9 Hz, 2H), 7.24 (dd,J = 8.3, 1.3 Hz, 1H), 7.22 – 7.19 (m, 2H), 7.18 – 7.15 (m, 1H), 7.12 – 7.06(m, 2H), 7.04 (dd, J = 10.9, 4.4 Hz, 1H), 6.97 (d, J = 1.5 Hz, 1H), 6.96 –6.91 (m, 1H), 6.71 – 6.65 (m, 1H), 6.28 (s, 1H), 3.73 (q, J = 6.6 Hz, 2H),3.04 (t, J = 6.6 Hz, 2H).13C NMR (101 MHz, CDCl3) δ 169.30, 154.91 (d, J =244.9 Hz), 144.42, 136.32, 131.98, 129.60 (d, J = 11.3 Hz), 127.43, 127.13,124.13 (d, J = 3.7 Hz), 122.81 (d, J = 7.4 Hz), 122.16, 122.12, 121.20 (d, J= 1.7 Hz), 119.40, 119.05, 118.60, 118.45, 115.96 (d, J = 19.5 Hz), 115.39,112.62, 111.29, 39.95, 25.06. HRMS (ESI+) Calcd for C23H20FN3O [M + H]+374.1663, found 374.1667。
实施例3
一种N-苯基色胺水杨酸类化合物,为
4.3.2 N-(2-(1H-indol-3-yl)ethyl)-2-((2-iodophenyl)amino)benzamide
(NP2)
white solid (80% yield), 1H NMR (400 MHz, Acetone-d 6 ) 10.05 (s, 1H),9.89 (s, 1H), 7.98 (s, 1H), 7.89 (dd, J = 7.9, 1.4 Hz, 1H), 7.66 (s, 1H),7.64 (d, J = 0.9 Hz, 1H), 7.39 (dd, J = 12.5, 4.6 Hz, 2H), 7.35 – 7.28 (m,2H), 7.24 (s, 1H), 7.22 (d, J = 1.4 Hz, 1H), 7.12 – 7.07 (m, 1H), 7.03 – 6.98(m, 1H), 6.85 – 6.80 (m, 1H), 6.77 (m, J = 7.8, 1.5 Hz, 1H), 3.72 (dd, J =13.2, 7.2 Hz, 2H), 3.10 (t, J = 7.3 Hz, 2H).13C NMR (101 MHz, Acetone-d 6 ) δ169.52, 144.97, 144.14, 140.65, 137.50, 132.29, 129.66, 129.02, 128.43,124.41, 123.23, 121.93, 120.75, 120.16, 119.57, 119.26, 119.19, 116.58,113.22, 111.97, 92.26, 41.05, 25.91. HRMS (ESI+) Calcd for C23H20IN3O [M + H]+482.0724, found 482.0726。
实施例4
一种N-苯基色胺水杨酸类化合物,为
4.3.3 N-(2-(1H-indol-3-yl)ethyl)-2-(o-tolylamino)benzamide (NP3)
white solid (89% yield), 1H NMR (400 MHz, CDCl3) δ 9.21 (s, 1H), 8.09(s, 1H), 7.64 (d, J = 7.9 Hz, 1H), 7.37 (d, J = 8.1 Hz, 1H), 7.31 (d, J = 7.8Hz, 1H), 7.24 (d, J = 2.3 Hz, 1H), 7.21 (d, J = 2.4 Hz, 2H), 7.20 – 7.16 (m,2H), 7.16 – 7.12 (m, 2H), 7.12 – 7.07 (m, 1H), 7.05 (d, J = 2.2 Hz, 1H), 6.99(t, J = 7.4 Hz, 1H), 6.67 – 6.60 (m, 1H), 6.21 (d, J = 5.3 Hz, 1H), 3.77 (q,J = 6.4 Hz, 2H), 3.09 (t, J = 6.6 Hz, 2H), 2.30 (s, 3H).13C NMR (101 MHz,CDCl3) δ 169.54, 145.98, 139.83, 136.34, 132.03, 130.97, 130.89, 127.33,127.16, 126.40, 123.00, 122.25, 122.12, 120.93, 119.52, 118.70, 117.84,117.25, 115.17, 112.84, 111.28, 39.87, 25.18, 18.09. HRMS (ESI+) Calcd forC24H23N3O [M + H]+ 370.1914, found 370.1918。
实施例5
一种N-苯基色胺水杨酸类化合物,为
4.3.4 N-(2-(1H-indol-3-yl)ethyl)-2-((2-aminophenyl)amino)benzamide
(NP4)
yellow solid (51% yield), 1H NMR (400 MHz, CDCl3) δ 8.93 (s, 1H), 8.24(s, 1H), 7.63 (d, J = 7.7 Hz, 1H), 7.34 (d, J = 8.0 Hz, 1H), 7.22 (s, 1H),7.20 – 7.16 (m, 2H), 7.16 – 7.10 (m, 3H), 7.04 (d, J = 7.6 Hz, 1H), 7.01 (s,1H), 6.75 (dd, J = 16.1, 7.8 Hz, 2H), 6.66 (d, J = 8.3 Hz, 1H), 6.57 (t, J =7.4 Hz, 1H), 6.29 (s, 1H), 3.75 (dd, J = 12.1, 6.0 Hz, 2H), 3.07 (t, J = 6.4Hz, 2H).13C NMR (101 MHz, CDCl3) δ 169.61, 147.40, 142.66, 136.32, 132.35,127.12, 126.85, 126.52, 126.27, 122.16, 119.43, 118.73, 118.64, 116.65,116.40, 115.87, 114.44, 112.72, 111.30, 39.89, 25.16. HRMS (ESI+) Calcd forC23H22N4O [M + H]+ 371.1866, found 371.1869。
实施例6
一种N-苯基色胺水杨酸类化合物,为
4.3.5 N-(2-(1H-indol-3-yl)ethyl)-2-((3-fluorophenyl)amino)benzamide
(NP5)
white solid (81% yield), 1H NMR (400 MHz, Acetone-d 6 ) δ 10.05 (s, 1H),9.99 (s, 1H), 8.02 (s, 1H), 7.65 (dd, J = 7.9, 1.6 Hz, 2H), 7.45 (dd, J =8.3, 0.9 Hz, 1H), 7.39 (d, J = 8.1 Hz, 1H), 7.38 – 7.32 (m, 1H), 7.32 – 7.26(m, 1H), 7.21 (d, J = 2.3 Hz, 1H), 7.14 – 7.08 (m, 1H), 7.04 – 7.00 (m, 1H),7.00 – 6.98 (m, 1H), 6.97 (t, J = 2.3 Hz, 1H), 6.87 – 6.81 (m, 1H), 6.73 –6.66 (m, 1H), 3.72 (td, J = 7.3, 6.0 Hz, 2H), 3.09 (t, J = 7.3 Hz, 2H).13C NMR(101 MHz, Acetone-d 6 ) δ 169.76, 164.31 (d, J = 242.6 Hz), 144.97 (d, J = 10.5Hz), 144.55, 137.50, 132.45, 131.44 (d, J = 10.0 Hz), 129.09, 128.40, 123.21,121.96, 120.83, 119.72, 119.28, 119.16, 116.75, 115.41 (d, J = 2.5 Hz),113.17, 112.00, 108.28 (d, J = 21.5 Hz), 105.83 (d, J = 24.6 Hz), 41.07,25.89. HRMS (ESI+) Calcd for C23H20FN3O [M + H]+ 374.1663, found 374.1665。
实施例7
一种N-苯基色胺水杨酸类化合物,为
4.3.6 N-(2-(1H-indol-3-yl)ethyl)-2-((3-chlorophenyl)amino)benzamide
(NP6)
white solid (80% yield), 1H NMR (400 MHz, Acetone-d 6 ) δ 10.05 (s, 1H),9.96 (s, 1H), 8.03 (s, 1H), 7.68 – 7.62 (m, 2H), 7.44 – 7.39 (m, 1H), 7.38 –7.37 (m, 1H), 7.34 (dd, J = 8.3, 1.3 Hz, 1H), 7.29 (t, J = 8.0 Hz, 1H), 7.23(t, J = 2.1 Hz, 1H), 7.21 (d, J = 2.3 Hz, 1H), 7.15 – 7.07 (m, 2H), 7.01 (td,J = 7.5, 0.9 Hz, 1H), 6.96 (m, J = 7.9, 1.9, 0.8 Hz, 1H), 6.87 – 6.81 (m,1H), 3.74 – 3.67 (m, 2H), 3.08 (t, J = 7.3 Hz, 2H). 13C NMR (101 MHz, Acetone- d 6 ) δ 169.72, 144.57, 144.52, 137.49, 135.10, 132.46, 131.41, 129.11, 128.40,123.21, 121.95, 121.73, 120.87, 119.77, 119.27, 119.15, 118.96, 118.01,116.69, 113.17, 111.99, 41.06, 25.89. HRMS (ESI+) Calcd for C23H20ClN3O [M+H]+390.1368, found 390.1364。
实施例8
一种N-苯基色胺水杨酸类化合物,为
4.3.7 N-(2-(1H-indol-3-yl)ethyl)-2-((3-bromophenyl)amino)benzamide
(NP7)
white solid (78% yield), 1H NMR (400 MHz, Acetone-d 6 ) δ 10.05 (s, 1H),9.95 (s, 1H), 8.02 (s, 1H), 7.68 – 7.61 (m, 2H), 7.38 (m, J = 16.6, 8.3, 1.1Hz, 4H), 7.25 (d, J = 8.0 Hz, 1H), 7.21 (d, J = 2.4 Hz, 1H), 7.18 (dd, J =7.0, 1.8 Hz, 1H), 7.11 (d, J = 0.7 Hz, 1H), 7.09 (dd, J = 7.0, 1.0 Hz, 1H),7.04 – 6.98 (m, 1H), 6.87 – 6.81 (m, 1H), 3.70 (dd, J = 13.3, 7.2 Hz, 2H),3.08 (t, J = 7.3 Hz, 2H).13C NMR (101 MHz, Acetone-d 6 ) δ 169.70, 144.74,144.51, 137.50, 132.46, 131.71, 129.11, 128.41, 124.69, 123.22, 121.94,121.91, 120.88, 119.80, 119.26, 119.15, 118.44, 116.67, 113.17, 111.99,41.06, 25.89. HRMS (ESI+) Calcd for C23H20BrN3O [M + H]+ 434.0863, found434.0866。
实施例9
一种N-苯基色胺水杨酸类化合物,为
4.3.8 N-(2-(1H-indol-3-yl)ethyl)-2-(m-tolylamino)benzamide (NP8)
white solid (86% yield), 1H NMR (400 MHz, CDCl3) δ 9.23 (s, 1H), 8.17(s, 1H), 7.62 (d, J = 7.9 Hz, 1H), 7.37 – 7.30 (m, 2H), 7.22 (dd, J = 6.8,3.2 Hz, 2H), 7.19 (d, J = 3.5 Hz, 1H), 7.18 – 7.14 (m, 1H), 7.12 (dd, J =11.0, 3.9 Hz, 1H), 6.98 (d, J = 7.1 Hz, 3H), 6.81 (d, J = 7.4 Hz, 1H), 6.65(t, J = 7.2 Hz, 1H), 6.25 (s, 1H), 3.73 (q, J = 6.5 Hz, 2H), 3.05 (t, J = 6.6Hz, 2H), 2.30 (s, 3H).13C NMR (101 MHz, CDCl3) δ 169.45, 145.14, 141.45,139.11, 136.32, 131.93, 129.00, 127.43, 127.15, 123.03, 122.16, 122.14,121.16, 119.44, 118.69, 118.64, 117.82, 117.34, 115.61, 112.70, 111.29,39.92, 25.13, 21.44. HRMS (ESI+) Calcd for C24H23N3O [M + H]+ 370.1914, found370.1911。
实施例10
一种N-苯基色胺水杨酸类化合物,为
4.3.9 N-(2-(1H-indol-3-yl)ethyl)-2-((4-fluorophenyl)amino)benzamide
(NP9)
white solid (73% yield), 1H NMR (400 MHz, Acetone-d 6 ) δ 10.06 (s, 1H),9.88 (s, 1H), 7.98 (s, 1H), 7.68 – 7.60 (m, 2H), 7.38 (d, J = 8.1 Hz, 1H),7.29 (dd, J = 8.4, 1.4 Hz, 1H), 7.25 (dd, J = 5.6, 3.5 Hz, 2H), 7.21 (dd, J =10.4, 1.9 Hz, 2H), 7.14 – 7.10 (m, 2H), 7.10 – 7.07 (m, 1H), 7.04 – 6.98 (m,1H), 6.76 – 6.70 (m, 1H), 3.70 (td, J = 7.3, 5.9 Hz, 2H), 3.08 (t, J = 7.3Hz, 2H).13C NMR (101 MHz, Acetone-d 6 ) δ 169.94, 160.25, 157.87, 146.63,138.86, 138.84, 137.53, 132.55, 128.98, 128.43, 123.37, 123.29, 123.22,121.96, 119.27, 119.18, 118.96, 118.27, 116.64, 116.42, 114.89, 113.24,112.00, 41.02, 25.98. HRMS (ESI+) Calcd for C23H20FN3O [M + H]+ 374.1663, found374.1660。
实施例11
一种N-苯基色胺水杨酸类化合物,为
4.3.10 N-(2-(1H-indol-3-yl)ethyl)-2-((4-chlorophenyl)amino)benzamide
(NP10)
white solid (75% yield), 1H NMR (400 MHz, Acetone-d 6 ) δ 10.05 (s, 1H),9.95 (s, 1H), 8.00 (s, 1H), 7.68 – 7.61 (m, 2H), 7.39 (d, J = 8.1 Hz, 1H),7.34 (m, J = 4.1, 2.0 Hz, 2H), 7.32 – 7.28 (m, 2H), 7.23 – 7.18 (m, 3H), 7.14– 7.08 (m, 1H), 7.05 – 6.99 (m, 1H), 6.79 (m, J = 8.0, 6.9, 1.5 Hz, 1H), 3.75– 3.68 (m, 2H), 3.09 (t, J = 7.3 Hz, 2H).13C NMR (101 MHz, Acetone-d 6 ) δ169.83, 145.20, 141.66, 137.49, 132.47, 129.86, 129.06, 128.39, 126.37,123.19, 121.95, 121.59, 120.11, 119.27, 119.16, 115.91, 113.18, 111.99,41.04, 25.91. HRMS (ESI+) Calcd for C23H20ClN3O [M + H]+ 390.1368, found390.1372。
实施例12
一种N-苯基色胺水杨酸类化合物,为
4.3.11 N-(2-(1H-indol-3-yl)ethyl)-2-((4-bromophenyl)amino)benzamide
(NP11)
white solid (76% yield), 1H NMR (400 MHz, Acetone-d 6 ) δ 10.05 (s, 1H),9.94 (s, 1H), 8.01 (s, 1H), 7.64 (dd, J = 7.9, 1.5 Hz, 2H), 7.45 – 7.41 (m,2H), 7.41 – 7.35 (m, 2H), 7.34 – 7.29 (m, 1H), 7.21 (d, J = 2.2 Hz, 1H), 7.17– 7.13 (m, 2H), 7.13 – 7.07 (m, 1H), 7.05 – 6.99 (m, 1H), 6.83 – 6.77 (m,1H), 3.72 (dd, J = 13.3, 7.2 Hz, 2H), 3.09 (t, J = 7.3 Hz, 2H).13C NMR (101MHz, Acetone-d 6 ) δ 169.79, 144.98, 142.14, 137.47, 132.80, 132.45, 129.06,128.38, 123.19, 121.95, 121.81, 120.27, 119.28, 119.26, 119.15, 116.04,113.64, 113.16, 111.98, 41.04, 25.90. HRMS (ESI+) Calcd for C23H20BrN3O [M+H]+434.0863, found 484.0865。
实施例13
一种N-苯基色胺水杨酸类化合物,为
4.3.12 N-(2-(1H-indol-3-yl)ethyl)-2-(p-tolylamino)benzamide (NP12)
pale yellow solid (88% yield), 1H NMR (400 MHz, CDCl3) δ 9.23 (s, 1H),8.22 (s, 1H), 7.60 (d, J = 7.8 Hz, 1H), 7.30 (d, J = 8.1 Hz, 1H), 7.22 (dd, J= 8.7, 1.1 Hz, 1H), 7.20 – 7.14 (m, 3H), 7.10 (dd, J = 7.9, 0.7 Hz, 1H), 7.05(dd, J = 9.5, 3.2 Hz, 4H), 6.93 (d, J = 2.1 Hz, 1H), 6.63 – 6.56 (m, 1H),6.28 (t, J = 5.5 Hz, 1H), 3.70 (q, J = 6.5 Hz, 2H), 3.02 (t, J = 6.7 Hz, 2H),2.29 (s, 3H).13C NMR (101 MHz, CDCl3) δ 169.53, 145.84, 138.63, 136.27,132.10, 131.99, 129.74, 127.42, 127.10, 122.13, 122.06, 121.30, 119.34,118.57, 117.82, 117.31, 114.83, 112.57, 111.28, 39.92, 25.07, 20.73. HRMS(ESI+) Calcd for C24H23N3O [M + H]+ 370.1914, found 370.1919。
实施例14
一种N-苯基色胺水杨酸类化合物,为
4.3.13 N-(2-(1H-indol-3-yl)ethyl)-2-((4-methoxyphenyl)amino)benzamide
(NP13)
yellow solid (89% yield), 1H NMR (400 MHz, CDCl3) δ 9.20 (s, 1H), 8.19(s, 1H), 7.63 (d, J = 7.8 Hz, 1H), 7.35 (d, J = 8.1 Hz, 1H), 7.22 (dd, J =6.0, 5.0 Hz, 1H), 7.17 (d, J = 7.6 Hz, 2H), 7.14 – 7.10 (m, 3H), 7.07 – 7.03(m, 1H), 7.01 (d, J = 2.2 Hz, 1H), 6.89 – 6.82 (m, 2H), 6.60 – 6.54 (m, 1H),6.25 (s, 1H), 3.78 (s, 3H), 3.74 (dd, J = 12.6, 6.6 Hz, 2H), 3.07 (t, J = 6.6Hz, 2H).13C NMR (101 MHz, CDCl3) δ 169.59, 155.79, 147.14, 136.33, 134.16,132.14, 127.31, 127.16, 124.34, 122.18, 122.12, 119.45, 118.66, 116.79,116.65, 114.48, 114.12, 112.78, 111.28, 55.44, 39.91, 25.17. HRMS (ESI+)Calcd for C24H23N3O2 [M + H]+ 386.1863, found 386.1866。
实施例15
一种N-苯基色胺水杨酸类化合物,为
4.3.14 N-(2-(1H-indol-3-yl)ethyl)-2-((4-(trifluoromethyl)phenyl)
amino)benzamide (NP14)
white solid (72% yield), 1H NMR (400 MHz, CDCl3) δ 9.50 (s, 1H), 8.09(s, 1H), 7.63 (d, J = 7.9 Hz, 1H), 7.49 (d, J = 8.3 Hz, 2H), 7.43 (d, J = 8.3Hz, 1H), 7.38 (d, J = 7.8 Hz, 1H), 7.30 (t, J = 7.8 Hz, 1H), 7.25 (s, 1H),7.23 (d, J = 3.4 Hz, 1H), 7.19 (d, J = 8.6 Hz, 2H), 7.15 – 7.10 (m, 1H), 7.05(s, 1H), 6.79 (t, J = 7.5 Hz, 1H), 6.24 (s, 1H), 3.76 (dd, J = 12.2, 6.0 Hz,2H), 3.09 (t, J = 6.5 Hz, 2H).13C NMR (101 MHz, CDCl3) δ 169.17, 145.05,143.17, 136.35, 131.98, 127.53, 127.15, 126.52 (q, J = 3.5 Hz), 122.21 (d, J= 25.6 Hz), 120.47, 119.63 (d, J = 8.4 Hz), 118.66, 118.52, 118.00, 116.82,112.73, 111.31, 40.01, 25.09. HRMS (ESI+) Calcd for C24H20F3N3O [M + H]+424.1631, found 424.1635。
实施例16
一种N-苯基色胺水杨酸类化合物,为
4.3.15 N-(2-(1H-indol-3-yl)ethyl)-2-((3,4,5-trimethoxyphenyl)amino)
benzamide (NP16)
yellow solid (86% yield), 1H NMR (400 MHz, CDCl3) δ 9.31 (s, 1H), 8.29(s, 1H), 7.63 (d, J = 7.9 Hz, 1H), 7.37 (d, J = 8.1 Hz, 1H), 7.29 (d, J = 8.2Hz, 1H), 7.20 (t, J = 8.2 Hz, 3H), 7.12 (t, J = 7.5 Hz, 1H), 7.04 (d, J = 1.6Hz, 1H), 6.65 (t, J = 7.4 Hz, 1H), 6.42 (s, 2H), 6.28 (t, J = 5.3 Hz, 1H),3.82 (s, 3H), 3.78 (s, 6H), 3.76 – 3.72 (m, 2H), 3.08 (t, J = 6.6 Hz, 2H).13CNMR (101 MHz, CDCl3) δ 169.46, 153.59, 145.66, 137.53, 136.36, 133.38,132.08, 127.43, 127.14, 122.18, 122.15, 119.45, 118.62, 118.05, 117.60,115.24, 112.67, 111.31, 98.59, 60.99, 55.96, 39.98, 25.15. HRMS (ESI+) Calcdfor C26H7N3O4 [M + H]+ 446.2074, found 446.2078。
实施例17
一种N-苯基色胺水杨酸类化合物,为
4.3.16 N-(2-(1H-indol-3-yl)ethyl)-2-((2-chlorophenyl)amino)benzamide
(NP17)
white solid (78% yield), 1H NMR (400 MHz, Acetone-d 6 ) δ 10.11 (s, 1H),10.07 (s, 1H), 8.02 (s, 1H), 7.65 (t, J = 7.1 Hz, 2H), 7.54 – 7.48 (m, 1H),7.46 (dd, J = 8.0, 1.2 Hz, 1H), 7.38 (m, J = 5.4, 2.7 Hz, 2H), 7.36 – 7.31(m, 1H), 7.28 – 7.24 (m, 1H), 7.22 (s, 1H), 7.09 (dd, J = 11.1, 3.9 Hz, 1H),7.01 (d, J = 7.1 Hz, 1H), 6.99 – 6.92 (m, 1H), 6.89 – 6.82 (m, 1H), 3.72 (dd,J = 13.3, 7.1 Hz, 2H), 3.09 (t, J = 7.3 Hz, 2H).13C NMR (101 MHz, Acetone-d 6 )δ 169.62, 144.15, 139.71, 137.47, 132.29, 130.71, 129.08, 128.40, 128.21,124.65, 123.22, 122.76, 121.92, 121.31, 119.85, 119.24, 119.14, 119.13,116.65, 113.16, 111.97, 41.05, 39.11, 25.87. HRMS (ESI+) Calcd for C23H20ClN3O[M + H]+ 390.1368, found 390.1371。
实施例18
一种N-苯基色胺水杨酸类化合物,为
4.3.17 N-(2-(1H-indol-3-yl)ethyl)-2-((2-bromophenyl)amino)benzamide
(NP18)
white solid (79% yield), 1H NMR (400 MHz, Acetone-d 6 ) δ 10.07 (s, 1H),10.03 (s, 1H), 8.03 (s, 1H), 7.68 – 7.61 (m, 3H), 7.51 – 7.45 (m, 1H), 7.38(d, J = 8.2 Hz, 1H), 7.36 – 7.32 (m, 2H), 7.32 – 7.26 (m, 1H), 7.22 (s, 1H),7.09 (t, J = 7.2 Hz, 1H), 7.00 (t, J = 7.1 Hz, 1H), 6.92 – 6.88 (m, 1H), 6.84(dd, J = 11.1, 4.9 Hz, 1H), 3.72 (dd, J = 13.3, 7.0 Hz, 2H), 3.09 (t, J = 7.3Hz, 2H).13C NMR (101 MHz, Acetone-d 6 ) δ 169.55, 144.26, 141.05, 137.47,134.02, 132.27, 129.07, 128.82, 128.40, 123.33, 123.21, 121.91, 121.29,119.84, 119.54, 119.24, 119.15, 116.71, 115.29, 113.17, 111.97, 41.05, 25.87.HRMS (ESI+) Calcd for C23H20BrN3O [M + H]+ 434.0863, found 434.0859。
实施例19
一种N-苯基色胺水杨酸类化合物,为
4.3.18 N-(2-(1H-indol-3-yl)ethyl)-2-(benzylamino)benzamide (N4)
yellow solid (83% yield), 1H NMR (400 MHz, CDCl3) δ 8.07 (d, J = 5.2Hz, 2H), 7.66 (d, J = 7.9 Hz, 1H), 7.38 (dd, J = 11.0, 7.7 Hz, 3H), 7.32 (t,J = 7.5 Hz, 2H), 7.22 (dd, J = 12.3, 3.9 Hz, 2H), 7.19 (s, 1H), 7.18 – 7.12(m, 2H), 7.09 (d, J = 2.2 Hz, 1H), 6.59 (d, J = 8.3 Hz, 1H), 6.50 (t, J = 7.5Hz, 1H), 6.15 (s, 1H), 4.41 (d, J = 5.6 Hz, 2H), 3.76 (dd, J = 12.5, 6.4 Hz,2H), 3.09 (t, J = 6.6 Hz, 2H).13C NMR (101 MHz, CDCl3) δ 169.73, 149.37,139.16, 136.35, 132.61, 128.57, 127.19, 127.15, 127.04, 126.94, 122.29,122.08, 119.55, 118.76, 115.36, 114.83, 113.03, 112.02, 111.26, 47.05, 39.74,25.27. HRMS (ESI+) Calcd for C24H23N3O [M + H]+ 370.1914, found 370.1918。
实施例20
一种N-苯基色胺水杨酸类化合物,为
4.3.19 N-(2-(1H-indol-3-yl)ethyl)-2-((3,4-dimethoxyphenethyl)amino)
benzamide (N6)
yellow solid (85% yield), 1H NMR (400 MHz, CDCl3) δ 8.26 (s, 1H), 7.70– 7.60 (m, 2H), 7.35 (d, J = 8.1 Hz, 1H), 7.24 (d, J = 3.5 Hz, 1H), 7.22 –7.17 (m, 1H), 7.16 – 7.08 (m, 2H), 6.97 (s, 1H), 6.79 (d, J = 3.7 Hz, 3H),6.67 (d, J = 8.4 Hz, 1H), 6.48 (t, J = 7.4 Hz, 1H), 6.14 (s, 1H), 3.87 (s,3H), 3.82 (s, 3H), 3.71 (dd, J = 12.3, 6.3 Hz, 2H), 3.35 (d, J = 4.1 Hz, 2H),3.04 (t, J = 6.5 Hz, 2H), 2.89 (t, J = 7.2 Hz, 2H).13C NMR (101 MHz, CDCl3) δ169.67, 149.27, 148.69, 147.36, 136.33, 132.60, 132.05, 127.25, 127.14,122.11, 120.53, 119.37, 118.63, 115.20, 114.54, 112.77, 111.98, 111.43,111.26, 111.14, 55.78, 55.71, 44.90, 39.74, 35.16, 25.20. HRMS (ESI+) Calcdfor C27H29N3O3 [M + H]+ 444.2282, found 444.2286。
实施例21
一种N-苯基色胺水杨酸类化合物,为
4.3.20 N-(2-(1H-indol-3-yl)ethyl)-2-((4-hydroxybutyl)amino)benzamide
(N8)
pale yellow solid (76% yield), 1H NMR (400 MHz, Acetone-d 6 ) δ 10.07(s, 1H), 8.00 (s, 1H), 7.73 (s, 1H), 7.65 (d, J = 7.9 Hz, 1H), 7.51 (dd, J =7.9, 1.4 Hz, 1H), 7.38 (d, J = 8.1 Hz, 1H), 7.27 – 7.23 (m, 1H), 7.22 (dd, J= 5.1, 1.9 Hz, 1H), 7.13 – 7.07 (m, 1H), 7.05 – 6.99 (m, 1H), 6.69 (d, J =8.2 Hz, 1H), 6.52 – 6.44 (m, 1H), 3.71 – 3.65 (m, 3H), 3.62 (dd, J = 11.4,5.9 Hz, 2H), 3.18 (dd, J = 12.0, 6.6 Hz, 2H), 3.06 (t, J = 7.3 Hz, 2H), 1.70(m, J = 10.5, 8.1, 4.5, 1.5 Hz, 4H).13C NMR (101 MHz, Acetone-d 6 ) δ 170.30,150.70, 137.52, 132.94, 128.55, 128.43, 123.16, 121.92, 119.23, 119.19,115.94, 114.58, 113.34, 111.98, 111.75, 43.17, 40.78, 31.16, 26.35, 26.09.HRMS (ESI+) Calcd for C21H25N3O2 [M + H]+ 352.2020, found 352.2023。
实施例22
一种N-苯基色胺水杨酸类化合物,为
4.3.21 N-(2-(1H-indol-3-yl)ethyl)-2-(4-methylphenylsulfonamido)
benzamide (N12)
yellow solid (79% yield), 1H NMR (400 MHz, CDCl3) δ 10.90 (s, 1H),8.13 (s, 1H), 7.70 (d, J = 8.1 Hz, 2H), 7.65 (d, J = 8.3 Hz, 1H), 7.58 (d, J= 7.9 Hz, 1H), 7.40 (d, J = 8.1 Hz, 1H), 7.34 (t, J = 7.8 Hz, 1H), 7.23 (t, J= 7.6 Hz, 1H), 7.17 (d, J = 8.2 Hz, 2H), 7.12 (dd, J = 7.5, 4.2 Hz, 2H), 7.04(s, 1H), 6.93 (t, J = 7.6 Hz, 1H), 6.11 (s, 1H), 3.67 (q, J = 6.3 Hz, 2H),3.02 (t, J = 6.6 Hz, 2H), 2.31 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 168.16,143.51, 136.58, 136.37, 132.46, 129.49, 127.23, 127.08, 126.48, 123.27,122.39, 122.10, 121.34, 121.00, 119.65, 118.56, 112.53, 111.38, 40.08, 25.03,21.49. HRMS (ESI+) Calcd for C24H23N3O3S [M + H]+ 434.1533, found 434.1536。
实施例23
一种N-苯基色胺水杨酸类化合物,为
4.3.22 N-(2-(1H-indol-3-yl)ethyl)-2-(pyridin-3-ylamino)benzamide
(NZ1)
white solid (68% yield), 1H NMR (400 MHz, DMSO-d 6 ) δ 10.82 (s, 1H),9.73 (s, 1H), 8.72 (t, J = 5.4 Hz, 1H), 8.44 (s, 1H), 8.17 (s, 1H), 7.65 (d,J = 7.8 Hz, 1H), 7.58 (dd, J = 7.2, 3.2 Hz, 2H), 7.33 (m, J = 18.4, 11.0, 4.8Hz, 4H), 7.19 (s, 1H), 7.06 (t, J = 7.5 Hz, 1H), 6.97 (t, J = 7.4 Hz, 1H),6.89 (t, J = 7.5 Hz, 1H), 3.54 (dd, J = 13.7, 6.7 Hz, 2H), 2.96 (t, J = 7.4Hz, 2H).13C NMR (101 MHz, DMSO-d 6 ) δ 168.99, 143.76, 142.81, 141.88, 136.70,132.29, 129.32, 127.72, 125.89, 123.09, 121.38, 120.55, 119.52, 118.72,118.69, 115.82, 112.27, 111.84, 40.55, 25.42. HRMS (ESI+) Calcd for C22H20N4O[M + H]+ 357.1710, found 357.1713。
实施例24
一种N-苯基色胺水杨酸类化合物,为
4.3.23 N-(2-(1H-indol-3-yl)ethyl)-2-((2-methylpyridin-3-yl)amino)
benzamide (NZ2)
white solid (74% yield), 1H NMR (400 MHz, Acetone-d 6 ) δ 10.09 (s, 1H),9.92 (s, 1H), 8.13 (dd, J = 4.7, 1.3 Hz, 1H), 8.05 (s, 1H), 7.68 (d, J = 1.6Hz, 1H), 7.66 (d, J = 1.7 Hz, 1H), 7.63 (s, 1H), 7.40 – 7.36 (m, 1H), 7.34 –7.28 (m, 1H), 7.23 (d, J = 2.3 Hz, 1H), 7.18 – 7.15 (m, 1H), 7.12 (dd, J =8.6, 0.9 Hz, 1H), 7.08 (dd, J = 8.1, 1.1 Hz, 1H), 7.00 (m, J = 8.0, 7.1, 1.0Hz, 1H), 6.82 – 6.76 (m, 1H), 3.73 (m, J = 7.3, 5.9 Hz, 2H), 3.10 (t, J = 7.2Hz, 2H), 2.50 (s, 3H).13C NMR (101 MHz, Acetone-d 6 ) δ 169.94, 151.28, 145.66,143.16, 137.53, 136.94, 132.55, 129.08, 128.44, 126.33, 123.24, 122.19,121.94, 119.72, 119.26, 119.16, 118.91, 115.50, 113.21, 112.00, 41.05, 25.93,21.15. HRMS (ESI+) Calcd for C23H22N4O [M + H]+ 371.1866, found 371.1862。
实施例25
一种N-苯基色胺水杨酸类化合物,为
4.3.24 N-(2-(1H-indol-3-yl)ethyl)-2-((6-methylpyridin-3-yl)amino)
benzamide (NZ3)
yellow solid (69% yield), 1H NMR (400 MHz, CDCl3) δ 9.39 (s, 1H), 8.36(d, J = 17.0 Hz, 2H), 7.65 (d, J = 7.8 Hz, 1H), 7.45 – 7.36 (m, 2H), 7.25 –7.19 (m, 3H), 7.16 (dd, J = 11.4, 8.0 Hz, 2H), 7.12 – 7.07 (m, 2H), 6.69 (t,J = 7.4 Hz, 1H), 6.33 (s, 1H), 3.77 (q, J = 6.3 Hz, 2H), 3.11 (t, J = 6.5 Hz,2H), 2.52 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 169.35, 152.04, 145.27, 142.52,136.38, 135.47, 128.95, 127.49, 127.17, 123.37, 122.26, 122.14, 119.53,118.67, 118.25, 118.19, 114.56, 112.76, 111.79, 111.32, 40.00, 25.15, 23.57.HRMS (ESI+) Calcd for C23H22N4O [M + H]+ 371.1866, found 371.1868。
实施例26
一种N-苯基色胺水杨酸类化合物,为
4.3.25 N-(2-(1H-indol-3-yl)ethyl)-2-((6-chloropyridin-3-yl)amino)
benzamide (NZ4)
pale yellow solid (62% yield), 1H NMR (400 MHz, CDCl3) δ 9.52 (s, 1H),8.25 (s, 1H), 8.22 (d, J = 2.7 Hz, 1H), 7.64 (d, J = 7.9 Hz, 1H), 7.45 (dd, J= 8.5, 2.8 Hz, 1H), 7.38 (d, J = 8.1 Hz, 1H), 7.28 (d, J = 8.1 Hz, 1H), 7.24– 7.19 (m, 3H), 7.13 (t, J = 7.4 Hz, 1H), 7.08 (d, J = 1.6 Hz, 1H), 6.76 (t,J = 7.5 Hz, 1H), 6.35 (s, 1H), 3.77 (dd, J = 12.5, 6.3 Hz, 2H), 3.10 (t, J =6.6 Hz, 2H).13C NMR (101 MHz, CDCl3) δ 169.15, 143.91, 143.29, 141.55, 137.46,136.36, 132.25, 129.68, 127.59, 127.14, 124.24, 122.30, 122.11, 119.56,119.32, 119.27, 118.63, 115.14, 112.69, 111.33, 40.06, 25.10. HRMS (ESI+)Calcd for C22H19ClN4O [M + H]+ 391.1320, found 391.1324。
实施例27
一种N-苯基色胺水杨酸类化合物,为
4.3.26 N-(2-(1H-indol-3-yl)ethyl)-2-((5-methylpyridin-3-yl)amino)
benzamide (NZ5)
yellow solid (67% yield), 1H NMR (400 MHz, CDCl3) δ 9.39 (s, 1H), 8.50(d, J = 11.9 Hz, 1H), 8.28 (d, J = 1.6 Hz, 1H), 8.04 (s, 1H), 7.63 (d, J =7.9 Hz, 1H), 7.37 (d, J = 8.1 Hz, 1H), 7.29 (s, 1H), 7.27 (d, J = 1.9 Hz,1H), 7.25 (s, 1H), 7.24 – 7.21 (m, 1H), 7.21 – 7.18 (m, 1H), 7.15 – 7.09 (m,1H), 7.06 (d, J = 2.1 Hz, 1H), 6.75 – 6.70 (m, 1H), 6.46 (d, J = 5.4 Hz, 1H),3.76 (q, J = 6.5 Hz, 2H), 3.10 (t, J = 6.6 Hz, 2H), 2.28 (s, 3H).13C NMR (101MHz, CDCl3) δ 169.30, 144.40, 143.59, 139.56, 137.86, 136.39, 133.54, 132.11,127.58, 127.47, 127.17, 122.18, 119.45, 119.11, 118.73, 118.63, 115.32,112.67, 111.33, 40.05, 25.13, 18.33. HRMS (ESI+) Calcd for C23H22N4O [M + H]+371.1866, found 371.1863。
实施例28
一种N-苯基色胺水杨酸类化合物,为
4.3.27 N-(2-(1H-indol-3-yl)ethyl)-2-((5-bromopyridin-3-yl)amino)
benzamide (NZ6)
yellow solid (69% yield), 1H NMR (400 MHz, Acetone-d 6 ) δ 10.06 (s,1H), 10.01 (s, 1H), 8.44 (d, J = 1.9 Hz, 1H), 8.20 (d, J = 1.3 Hz, 1H), 8.09(s, 1H), 7.79 (t, J = 2.1 Hz, 1H), 7.70 – 7.66 (m, 1H), 7.63 (d, J = 7.9 Hz,1H), 7.46 – 7.41 (m, 1H), 7.40 – 7.38 (m, 1H), 7.38 – 7.36 (m, 1H), 7.21 (d,J = 2.2 Hz, 1H), 7.09 (dd, J = 9.6, 5.5 Hz, 1H), 7.00 (dd, J = 11.0, 3.9 Hz,1H), 6.94 – 6.89 (m, 1H), 3.71 (dd, J = 13.3, 7.2 Hz, 2H), 3.08 (t, J = 7.3Hz, 2H).13C NMR (101 MHz, Acetone-d 6 ) δ 169.54, 143.64, 143.09, 140.95,140.32, 137.51, 132.62, 129.21, 128.41, 127.19, 123.25, 121.94, 121.61,120.73, 119.25, 119.14, 116.90, 113.14, 112.00, 41.10, 25.87. HRMS (ESI+)Calcd for C22H19N4O [M + H]+ 435.0815, found 435.0818。
实施例29
一种N-苯基色胺水杨酸类化合物,为
4.3.28 N-(2-(1H-indol-3-yl)ethyl)-2-(quinoxalin-6-ylamino)benzamide
(NZ7)
yellow solid (76% yield), 1H NMR (400 MHz, CDCl3) δ 9.82 (s, 1H), 8.65(s, 1H), 8.65 (s, 1H), 8.53 (s, 1H), 7.93 (d, J = 9.0 Hz, 1H), 7.76 (s, 1H),7.63 (dd, J = 12.9, 8.2 Hz, 2H), 7.48 (d, J = 9.1 Hz, 1H), 7.34 (d, J = 8.1Hz, 1H), 7.32 – 7.26 (m, 2H), 7.19 (t, J = 7.5 Hz, 1H), 7.10 (t, J = 7.4 Hz,1H), 7.04 (s, 1H), 6.80 (t, J = 7.5 Hz, 1H), 6.48 (s, 1H), 3.76 (q, J = 6.2Hz, 2H), 3.09 (t, J = 6.5 Hz, 2H).13C NMR (101 MHz, CDCl3) δ 169.15, 145.00,144.47, 143.66, 142.52, 141.86, 139.17, 136.33, 132.00, 130.18, 127.56,127.10, 125.21, 122.18, 122.14, 120.92, 120.30, 119.44, 118.57, 117.43,112.54, 111.30, 110.66, 40.06, 25.04. HRMS (ESI+) Calcd for C25H21N5O [M + H]+408.1819, found 408.1822。
实施例30
一种N-苯基色胺水杨酸类化合物,为
4.3.29 N-(2-(1H-indol-3-yl)ethyl)-2-(quinolin-8-ylamino)benzamide
(NZ8)
yellow solid (77% yield), 1H NMR (400 MHz, DMSO-d 6 ) δ 10.84 (s, 1H),10.79 (s, 1H), 8.91 (dd, J = 4.2, 1.6 Hz, 1H), 8.70 (t, J = 5.6 Hz, 1H), 8.30(dd, J = 8.3, 1.6 Hz, 1H), 7.76 (d, J = 8.1 Hz, 1H), 7.66 – 7.61 (m, 2H),7.57 (dd, J = 8.2, 4.3 Hz, 2H), 7.50 – 7.41 (m, 2H), 7.41 – 7.34 (m, 2H),7.21 (d, J = 2.1 Hz, 1H), 7.10 – 7.04 (m, 1H), 6.96 (m, J = 7.8, 3.6, 1.7 Hz,2H), 3.58 (dd, J = 13.3, 7.1 Hz, 2H), 2.99 (t, J = 7.4 Hz, 2H). 13C NMR (101MHz, DMSO-d 6 ) δ 168.79, 148.69, 139.45, 139.13, 136.69, 136.66, 131.77,129.36, 129.20, 127.77, 127.48, 123.62, 123.08, 122.50, 121.37, 120.08,118.74, 118.68, 118.35, 117.45, 112.35, 111.82, 110.01, 40.58, 25.45. HRMS(ESI+) Calcd for C26H22N4O [M + H]+ 407.1866, found 407.1863。
实施例31
一种N-苯基色胺水杨酸类化合物,为
4.3.30 N-(2-(1H-indol-3-yl)ethyl)-2-(cyclopropylamino)benzamide (NH1)
yellow solid (63% yield), 1H NMR (400 MHz, CDCl3) δ 8.14 (s, 1H), 7.70(s, 1H), 7.63 (d, J = 7.8 Hz, 1H), 7.36 (d, J = 8.1 Hz, 1H), 7.29 (t, J = 7.7Hz, 1H), 7.21 (t, J = 7.5 Hz, 1H), 7.12 (dd, J = 12.9, 6.0 Hz, 3H), 7.02 (s,1H), 6.53 (t, J = 7.5 Hz, 1H), 6.11 (s, 1H), 3.72 (q, J = 6.3 Hz, 2H), 3.05(t, J = 6.6 Hz, 2H), 2.40 (s, 1H), 0.74 (q, J = 6.2 Hz, 2H), 0.56 – 0.48 (m,2H).13C NMR (101 MHz, CDCl3) δ 169.64, 150.14, 136.35, 132.41, 127.17, 126.94,122.21, 122.07, 119.47, 118.70, 115.24, 115.14, 113.06, 112.91, 111.26,39.76, 25.22, 24.31, 7.37. HRMS (ESI+) Calcd for C20H21N3O [M + H]+ 320.1757,found 320.1755。
实施例32
一种N-苯基色胺水杨酸类化合物,为
4.3.31 N-(2-(1H-indol-3-yl)ethyl)-2-(cyclopentylamino)benzamide (NH3)
yellow solid (68% yield), 1H NMR (400 MHz, CDCl3) δ 8.20 (s, 1H), 7.62(d, J = 7.8 Hz, 2H), 7.35 (d, J = 8.1 Hz, 1H), 7.22 (m, J = 14.7, 7.5 Hz,2H), 7.11 (dd, J = 14.5, 7.5 Hz, 2H), 7.00 (s, 1H), 6.69 (d, J = 8.4 Hz, 1H),6.44 (t, J = 7.5 Hz, 1H), 6.12 (s, 1H), 3.84 – 3.75 (m, 1H), 3.71 (q, J = 6.3Hz, 2H), 3.05 (t, J = 6.6 Hz, 2H), 1.99 (td, J = 12.5, 7.1 Hz, 2H), 1.72 (dd,J = 12.3, 6.6 Hz, 2H), 1.64 – 1.50 (m, 4H).13C NMR (101 MHz, CDCl3) δ 169.84,149.26, 136.34, 132.51, 127.26, 127.15, 122.14, 122.10, 119.41, 118.67,114.80, 114.03, 112.86, 112.37, 111.26, 53.64, 39.72, 33.34, 25.19, 24.08.HRMS (ESI+) Calcd for C22H25N3O [M + H]+ 348.2070, found 348.2072。
实施例33
一种N-苯基色胺水杨酸类化合物,为
4.3.32 N-(2-(1H-indol-3-yl)ethyl)-2-(cyclohexylamino)benzamide (NH4)
yellow solid (75% yield), 1H NMR (400 MHz, CDCl3) δ 8.29 (s, 1H), 7.66(d, J = 7.7 Hz, 2H), 7.39 (d, J = 8.1 Hz, 1H), 7.25 (dd, J = 15.0, 7.7 Hz,2H), 7.18 – 7.12 (m, 2H), 7.03 (s, 1H), 6.72 (d, J = 8.4 Hz, 1H), 6.46 (t, J= 7.4 Hz, 1H), 6.18 (s, 1H), 3.76 (q, J = 6.4 Hz, 2H), 3.35 (s, 1H), 3.09 (t,J = 6.6 Hz, 2H), 2.10 – 2.00 (m, 2H), 1.79 (dd, J = 9.1, 3.6 Hz, 2H), 1.70 –1.59 (m, 1H), 1.46 – 1.27 (m, 6H).13C NMR (101 MHz, CDCl3) δ 169.86, 148.74,136.33, 132.51, 127.48, 127.14, 122.12, 122.08, 119.35, 118.64, 114.66,113.87, 112.79, 112.00, 111.27, 50.55, 39.72, 32.75, 25.83, 25.18, 24.73.HRMS (ESI+) Calcd for C23H27N3O [M + H]+ 362.2227, found 362.2224。
实施例 34
MTT肿瘤药物敏感试验的方法学研究
精密称定目标化合物,分别稀释至浓度为100 mmol/L作为母液,过滤除菌,4℃密封保存备用。
预先培养各种肿瘤细胞(HePG2、MGC-803、BGC-823、SGC-7901、AGS、A549、MCF-7、HGC-27、HCT116、HL-60)或正常细胞(GES-1)生长至对数生长期,再把对数生长期的贴壁细胞用胰蛋白酶消化液消化后(悬浮细胞不需要消化),每孔100μl细胞悬浮液,每孔1000-10000个细胞,接种于96孔板中,周围一圈孔加入100μl的PBS缓冲液;
37℃细胞培养箱(5%CO2,90%H2O)中培养至细胞贴壁;之后加入含待测目标化合物的培养液,实验组加入按浓度梯度稀释(终浓度为100、50、25、12.5、6.25μmol/L)的待测化合物,每孔加入100μl的待测样品,每个浓度均设置三个平行对照孔;阳性对照组加入同体积同浓度梯度的5-FU溶液,空白对照组加入100μl培养液,继续培养72h;
每孔加入10μl的MTT溶液,继续培养4h;之后终止培养,吸去孔内培养液;
每孔加入150μl 的DMSO,震荡使结晶甲瓒充分溶解(悬浮细胞用三联液溶解,放置12 h);
置于酶标仪中570 nm下测定吸光度值,根据公式计算出目标化合物的抑制率,再用软件计算出IC50的值。结果见表1和表2,结果表明N-苯基色胺水杨酸类化合物具有显著的抗癌细胞增殖作用。
表 1.代表性化合物抗8种人癌细胞的增殖抑制作用
表 2.代表性化合物胶质瘤细胞的增殖抑制作用和COX-2抑制作用
实施例35
肿瘤细胞周期检测实验
细胞周期是指连续分裂细胞从一次有丝分裂结束到下一次有丝分裂结束所经历的整个过程。在这个过程中,细胞遗传物质复制并加倍,且在分裂结束时平均分配到两个子细胞中去。细胞周期又可以分为间期和有丝分裂期,细胞间期常划分为休眠期(G0),DNA 合成前期(G1),DNA 合成期(S),DNA 合成后期 (G2),整个周期可表示为 G1→S→G2→M。DNA周期检测可用来反应细胞周期的各个期的状况,即细胞增殖状况。利用细胞内 DNA 能够和荧光染料(如碘化丙啶 PI)结合的特性,细胞各个时期其 DNA 量不同从而结合的荧光染料不同,流式细胞仪检测的荧光强度也不一样。
具体方法为:1、用NP16 (2, 4,8 μM) 处理胶质瘤细胞U251和U87MG,同时设立阴性对照组,48小时后消化并收集细胞。 2、用 PBS 洗涤细胞一次,1500rpm,5min 离心收集,调整细胞浓度为 1×106/ml,取1mL 单细胞悬液。3、制备的单细胞悬液离心后,去除上清,在细胞中加入70%预冷乙醇 500uL固定2小时至过夜,4℃保存,染色前用PBS洗去固定液;如需要,细胞悬液可用200目细胞筛网过滤一次。4、细胞沉淀中加 100µl RNase A 溶液,重悬细胞,37℃水浴 30min。5、再加入 400µlPI 染色液混匀,4℃避光孵育 30min。6、 上机检测,记录激发波长 488nm 处红色荧光。结果见附图2,表明化合物NP16 对胶质瘤细胞U251和U87MG有较强的G2/M 周期阻滞作用。
实施例36
肿瘤细胞凋亡检测实验
Annexin V-FITC细胞凋亡检测试剂盒(Annexin V-FITC Apoptosis DetectionKit)是用FITC标记的重组人Annexin V来检测细胞 凋亡时出现在细胞膜表面的磷脂酰丝氨酸的一种细胞凋亡检测试剂盒。可以使用流式细胞仪、荧光显微镜或其它荧光检测设备进行检测。 Annexin是一类广泛分布于真核细胞细胞浆内钙离子依赖的磷脂结合蛋白,参与细胞内的信号转导。但仅Annexin-V被报道可以调控一些PKC的活性。 Annexin V选择性结合磷脂酰丝氨酸(phosphatidylserine,简称PS)。磷脂酰丝氨酸主要分布在细胞膜内侧,即与细胞浆相邻的 一侧。在细胞发生凋亡的早期,不同类型的细胞都会把磷脂酰丝氨酸外翻到细胞表面,即细胞膜外侧。磷脂酰丝氨酸暴露 到细胞表面后会促进凝血和炎症反应。而Annexin V和外翻到细胞表面的磷脂酰丝氨酸结合后可以阻断磷脂酰丝氨酸的促凝血和促炎症反应活性。 用带有绿色荧光的荧光探针FITC标记的Annexin V,即Annexin V-FITC,就可以用流式细胞仪或荧光显微镜非常简单而直接地检测到磷脂酰丝氨酸的外翻这一细胞凋亡的重要特征。 此外,碘化丙啶(Propidium Iodide, PI)染色液可以染色坏死细胞或凋亡晚期丧失细胞膜完整性的细胞, 呈现红色荧光。对于坏死细胞,由于细胞膜的完整性已经丧失,Annexin V-FITC可以进入到细胞浆内,与位于细胞膜内侧的磷脂酰丝氨酸结合,从而也使坏死细胞呈现绿色荧光于贴壁细胞的消化后检测:
具体操作步骤为:a. 用NP16 处理6孔板中的U251和U87MG 48h. 之后把细胞培养液吸出至一合适离心管内,PBS洗涤贴壁细胞一次,加入适量胰酶细胞消化液(可含有EDTA)消化细胞。室温 孵育至轻轻吹打可以使贴壁细胞吹打下来时,吸除胰酶细胞消化液。需避免胰酶的过度消化。注意:对于贴壁细胞,胰 酶消化步骤很关键。胰酶消化时间如果过短,细胞需要用力吹打才能脱落,容易造成细胞膜的损伤,从而导致细胞坏死 的假阳性;消化时间如果过长,同样易造成细胞膜损伤而出现细胞坏死的假阳性,甚至会影响细胞膜上磷脂酰丝氨酸与 Annexin V-FITC的结合从而干扰对于细胞凋亡的检测。同时,胰酶细胞消化液中应尽量不含EDTA,因为EDTA可能会影 响Annexin V与磷脂酰丝氨酸的结合。b. 加入步骤a中收集的细胞培养液,把细胞轻轻吹打下来,转移到离心管内,1000g离心5分钟,弃上清,收集细胞,用PBS 轻轻重悬细胞并计数。注意:加入步骤a中的细胞培养液非常重要,一方面可以收集已经悬浮的发生凋亡或坏死的细胞, 另一方面细胞培养液中的血清可以有效抑制或中和残留的胰酶。残留的胰酶会消化并降解后续加入的Annexin V-FITC, 导致染色失败。 c. 取5-10万重悬的细胞,1000g离心5分钟,弃上清,加入195µl Annexin V-FITC结合液轻轻重悬细胞。d. 加入5µl Annexin V-FITC,轻轻混匀。e. 加入10µl碘化丙啶染色液,轻轻混匀。f. 室温(20-25ºC) 避光孵育10-20分钟,随后置于冰浴中。可以使用铝箔进行避光。孵育过程中可以重悬细胞2-3次以改善染色效果。g.之后立即进行上机检测,Annexin V-FITC为绿色荧光,碘化丙啶(PI)为红色荧光。结果见附图4,结果显示NP16显著诱导U251和U87MG的凋亡。
实施例37
对胶质瘤瘤细胞的活性氧诱导检测
活性氧检测试剂盒是利用荧光探针DCFH-DA进行活性氧检测的。DCFH-DA本身没有荧光,可以自由穿过细胞膜,进入细胞内后,可以被细胞内的酯酶水解生成DCFH。而DCFH不能通透细胞膜,从而使探针很容易被装载到细胞内。细胞内的活性氧可以氧化无荧光的DCFH生成有荧光的DCF。检测DCF的荧光就可以知道细胞内活性氧的水平。活性氧检测试剂盒[9]。荧光法检测线粒体产生和释放的反应性氧化物(ROS)。
A. 把细胞培养液吸出至一合适离心管内,PBS洗涤贴壁细胞一次,加入适量胰酶细胞消化液(可含有EDTA) 消化细胞。室温孵育至轻轻吹打可以使贴壁细胞吹打下来时,吸除胰酶细胞消化液。需避免胰酶的过度消化。
B. 加入步骤2a中收集的细胞培养液,稍混匀,转移到离心管内,1000g离心5分钟,弃上清,收集细胞,用PBS轻轻重悬细胞并计数。注意:加入步骤2a中的细胞培养液一方面可以收集已经悬浮的发生凋亡或坏死的细胞,另一方面细胞培养液中的血清可以有效抑制或中和残留的胰酶;残留的胰酶会消化并降解后续加入的Annexin V-PE导致染色失败。
C. 取5-10万重悬的细胞,1000g离心5分钟,弃上清,加入1DCF-H (10 μM)结合液轻轻重悬细胞。
D.室温 (20-25℃)避光孵育10-20分钟,随后置于冰浴中。可以使用铝箔进行避光。孵育过程中可以重悬细胞2-3次以改善标记效果。如果用于荧光显微镜下检测,结果见附图5,结果显示 显著诱导胶质瘤ROS 的产生。
实施例38
胶质瘤细胞侵袭和迁移实验
Matrigel 是从小鼠EHS肉瘤中提取的基质成分,含有LN、IV型胶原、接触蛋白和肝素硫酸多糖,铺在无聚乙烯吡硌烷酮的聚碳酸酯滤膜上,能在DMEM培养基中重建形成膜结构,这种膜结构与天然基质膜结构极为相似。
滤膜孔径一般为8um,而且膜孔都被Matrigel覆盖,细胞不能自由穿过,必须分泌水解酶,并通过变形运动才能穿过这种铺有Martrigel 的滤膜,这与体内情况较为相似。
铺有Martrigel的滤膜放在以Blind Well腔或MICS腔上下室之间,铺有Martrigel面朝向上室,在下室中加有趋化剂,如一定浓度的LN、FN或小鼠3T3条件培养液或人睾丸上皮成纤维细胞培养液,上室加入重悬的U87MG, U251细胞,具有侵袭能力的细胞在趋化剂15%的血清诱导下开始穿膜运动。细胞穿膜所用的时间与Martrigel的用量有关,选择25ugMartrigell铺膜,16小时后观察结果较为合适。穿过滤膜的细胞多数粘附在滤膜下表面,可用棉签将上表面的细胞拭去,然后用甲醇固定滤膜,1%结晶紫染色,迁移实验不加Matrigel,其余步骤与侵袭相同。结果见图6。
实施例39
对C6胶质瘤原位模型的作用
建立脑胶质瘤动物模型的方法有很多种,目前应用最广泛的是大鼠C6 胶质瘤模型。该模型通过立体定向的方式将C6 胶质瘤细胞植入大鼠右侧尾状核,具有成瘤快,成瘤率高,实验周期短,重复性好,没有明显的颅外生长和转移等优点,便于各种实验的研究。本实验将建立胶质肿瘤模型后的大鼠分为建模后1 周、建模后2 周、建模后3周三组,利用MRI、病理诊断等技术,验证移植瘤的生长情况。并采用免疫组化,HE 染色,Western blot和RT-PCR 等方法,研究化合物NP16在大鼠C6胶质肿瘤增殖过程中的表达变化,探讨其在胶质肿瘤增殖过程中的作用。
C6 细胞系及其移植瘤模型被广泛应用于脑胶质瘤的实验研究。C6 细胞是经N-亚硝基甲脲 (N-nitrosomethylurea)诱导的非纯种Wistar 大鼠脑胶质瘤细胞,是目前国际上通用的胶质瘤细胞系。当使用SD大鼠进行造模时,由于同种异体性导致的宿主排斥反应,C6模型不能实现100%的成功率。当C6 细胞移植于新生大鼠成瘤后,其组织学特性与人类脑胶质母细胞瘤(星形细胞肿瘤,WHO Ⅳ级)病理特征一致,当其移植于大鼠脑内时,具有较好的组织相容性,能在大鼠脑内成功接种建立胶质瘤模型,并能稳定生长,具有胶质瘤特异性标志物GFAP 等的特征。李维方等通过MRI 检查发现C6 胶质瘤模型建立成功后仅1 周肿瘤已基本形成,随接种时的延长,其肿瘤体积不断增大,逐渐产生压迫及颅内高压症状。接种2周时大鼠即有明显的颅内高压症状,2 周后即有死亡,3 周时肿瘤鼠大多处于濒危状态。提示部分大鼠的C6 胶质瘤可能因自身免疫活性较高而自然消亡。
研究发现,注入鼠脑的细胞悬液体积和细胞量均影响此模型的成功率,并证实最适合的接种体积为10μL,细胞数为106个/mL。这为本实验接种细胞的量和密度提供了参考。胶质瘤细胞在鼠脑内变化过程大致可分为三期,即①胶质瘤细胞在脑组织内分散、聚集期;②胶质瘤细胞增殖期; ③肿瘤组织形成期; 前两个阶段一般在1周左右出现, 肿瘤形成期多数在荷瘤两周前后。
1 实验动物
健康成年SD大鼠,全部为雄性,体质量 190-220g,由兰州牧药研究所实验动物中心提供,符合清洁级标准。在实验饲养和实验中,遵守实验动物管理与保护的有关准则。(2)细胞培养大鼠C6 星形胶质瘤细胞于10%胎牛血清、青霉素100U/mL、链霉素50U/mL 的DMEM培养基中、5%二氧化碳、37℃条件下培养传代生长,在细胞生长处于对数生长期大约80%时,以0.25%胰酶消化,收集消化液离心后去上清液,PBS 液洗涤两次后以DMSM液配制成细胞悬液待接种,待接种的细胞活力应>90%,接种的细胞量配制成1×106/10 μL 备用。之后分装于500 μL的离心管中;用保温箱的冰袋保存运输。(冰袋温度以-20摄氏度为宜;-80摄氏度会使细胞冻住,存活率降低)。
2 模型制作
SD 大鼠腹腔内注射10%水合氯醛 (5 g,50 mL;0.6 mL/200g 体重的水合氯醛)后,固定于脑立体定位仪上,用剪刀剪去大鼠头顶部毛发,纵向切开头皮,用止血钳和剪刀剪开多层筋膜;暴露前囟颅骨标志begama 点(在两耳中点旁边);用10mL注射器刮点做标记;之后用脑立体定位仪定位begama点前1mm,右旁开3mm 处用颅骨钻钻一骨孔,用注射器再次刮取做标记;用电钻钻孔1 mm;快钻开时;用注射器稍微挑掉一层薄膜(注意不要穿破硬膜)。用5μL 微量注射器抽取细胞悬液5μL(吸取之前一定要弹散细胞,否则聚团导致注射细胞数降低),垂直固定于立体定位仪上(设置参数:mode: infuse only; syringe:Hamilton 5.00 μL; RATES: I:0.001 ml/min,TARGET: No Target No target set)经骨孔处进针(注意针头不能沾染碘酒,否则细胞存活率降低),针头锐性刺破硬膜,进针至硬脑膜下5mm,后退1mm,于5 min 内将细胞悬液注入右侧尾状核区 (1μL/min),注射完毕后按stop, reset后;留针5min,缓慢退针,骨蜡封闭骨孔,1 号丝线缝合头皮切口,碘酒涂抹伤口。对照组大鼠以相同剂量的生理盐水,同样方法注入相同部位。实验进行后要观察C6 细胞的活力,再次保证模型的成功;
给药需要灭菌的注射器,防止大鼠感染;造模后使用灭菌的垫料防止感染。对于状态不好的大鼠,可以给双抗(青霉素-链霉素:是40万U/Kg,q.d.连用三日;手术时撒在伤口缝合处,之后前三天每天两次肌注青链霉素,剂量大点没关系,一支青霉素80万单位打一次)。
3. MRI 影像学观察
分别于建模后1 周、2 周、3 周对大鼠进行头颅MRI 检查,活体观察肿瘤的生长情况。MRI 常规扫描:层厚2mm,层间距0mm,FOV,矩阵,2NEX. SE 序列,T1WI: TR/TE=800ms/26ms,T2WI: TR/TE=3000ms/80ms,增强扫描:尾静脉注射造影剂GD-DTAP(0.2-0.3ml/Kg)后,TR/TE=800ms/26ms。
4. 开场实验
1、实验开始之前,先确认实验箱是清洁,无味道的,尤其要注意清理实验箱底部上一次实验测试动物遗留下来的粪便、尿液等。
2、实验人员在软件中设置好相应的参数,记录好动物的编号、日期、状态等信息;
3、将实验动物从饲养笼中轻轻取出,注意背向实验者;
4、将实验动物迅速放置于实验箱的中央区域,并立即离开;
5、打开动物行为学分析软件,自动记录动物在箱体内的活动,实验时间通常为15分钟;
6、实验结束后,将实验动物放入事先准备好的其他饲养笼内;
7、用酒精喷洒仪器装置除味,并用纸巾檫干。
四、实验指标
区域的划分:区域通常分为四角、四周及中央区域,软件自动统计这些区域内实验动物的活动路程、滞留时间及进入次数、平均速度;这里需要注意,关于区域的划分有很多不同的方式,也有很多实验者将箱体底部划分成9宫格、16宫格或者25宫格。
1、水平运动总距离--反应大鼠的运动情况。例如抑郁大鼠水平运动距离将大大减少,多躲避于旷场一角不动。
2、中央区运进入总次数--反应大鼠的焦虑情况。抗焦虑药物可以在不改变一般运动情况的前提下增加大鼠的中央区进入次数。抑郁大鼠中央区进入次数也会大幅度下降。
3、中央区运滞留时间--反应大鼠的焦虑情况。抗焦虑药物可以在不改变一般运动情况的前提下增加大鼠的中央区滞留时间。抑郁大鼠中央区滞留时间也会大幅度下降。
4、直立次数--反应大鼠垂直水平活动情况,主要反应大鼠的探究行为。抑郁大鼠对外界环境的好奇性或探究兴趣下降,所以表现出直立次数显著下降。
5、粪便数量--反应大鼠的焦虑情况。但是由于粪便数受影响因素多并不能充分反映出大鼠的焦虑情况。
6. 轨迹图热图:自发活动行为的复杂表现:这一类行为可以更加客观的表现动物的自发性行为,比如修饰、转圈、蜷缩、急冲、甩头、躯体伸长等,如果软件支持自动测试这些指标,可以一并统计
3.4 肿瘤组织的获取:将大鼠分别于MRI 检查后取材,用于病理检验及免疫组化实验的组织:在各相应时间点用10%水合氯醛(350mg/kg)腹腔注射麻醉动物,打开胸腔暴露其心脏,经左心室插管至升主动脉起始处,剪开右心耳,用注射器进行灌流固定:首先用0.9%的生理盐水(normal saline,NS)(4 ℃)约200 ml 快速冲洗血液,用4%多聚甲醛(Paraformaldehyde, PFA. 0.1M PBS 配置,pH 7.4)300-400 ml 作灌注固定,直到大鼠发生四肢抽搐。术毕,迅速取出肿瘤组织。放入 4% PFA中后固定 24 h (4 ℃)。对照组大鼠以相同方式处理后,取右脑尾状核区组织作为对照。用于Western blot 及RT-PCR 实验的组织:将各实验组的大鼠麻醉后,冰上直接断头快速取出肿瘤组织,用EP 管分装后置-80℃保存备用。取右脑尾状核周围组织作为对照。
经上述实验证实,NP16 对C6胶质瘤原位模型具有显著抑制侵袭的作用,显著改善大鼠的焦虑行为,显著延长大鼠的生存期,结果见附图7。
实施例40
脑脊液含量测定
SD大鼠腹腔注射60mg/kg的NP16, 2 h后通过延髓取脑脊液100μL, LC-MS 分析其中NP16 的含量,同时制备空白脑脊液,加入NP16 的标准品,结果见附图8。
Claims (9)
2.根据权利要求1所述的一种色胺水杨酸化合物,其特征在于:R1为1-4个碳原子低级烷基链;或为甲氧基、三氟甲基、N-N二甲基、氨基、硝基的一种或几种。
5.根据权利要求4所述的一种色胺水杨酸类化合物的制备方法,其特征在于工艺步骤如下:
(1)氩气条件下,邻碘苯甲酸类化合物与氨类化合物Ⅰ在溶解了K2CO3 、铜粉的 DMF 中,反应生成化合物Ⅱ;
(2)将化合物II、EDCI·HCl、HOBT溶解在二氯甲烷中,在室温下加入Et3N和色胺反应得到化合物III。
6.根据权利要求5所述的一种色胺水杨酸类化合物的制备方法,其特征在于具体步骤如下:
(1)氩气条件下,邻碘苯甲酸类化合物与氨类化合物Ⅰ在溶解了K2CO3 、铜粉的 DMF 中,其中邻碘苯甲酸类化合物:氨类化合物Ⅰ:K2CO3 :铜的摩尔比为1:1.2:1:0.001,邻碘苯甲酸类化合物在DMF中的浓度为0.5 mmol/ mL,在100 摄氏度的条件下加热12 小时后冷却至室温,用水进行萃取后用水稀释和2.0N HCl酸化,pH达到2,过滤除胺得到化合物Ⅱ;
(2)将化合物II(1.0 eq),EDCI·HCl(1.2 eq),HOBT(1.1 eq)溶解在二氯甲烷(3 mL)中,并在室温下加入Et3N和色胺,搅拌8小时,其中化合物II:EDCI·HCl:HOBT:Et3N:色胺摩尔比为1:1.2:1.1:2.5:1,化合物II在二氯甲烷的浓度为0.5 mmol/ mL,通过TLC检测反应完成后减压旋转蒸发除去反应液,然后将得到的残余物通过硅胶快速柱色谱纯化得到化合物III。
7.根据上述任一权利要求所述的一种色胺水杨酸类化合物的应用,其特征在于用于治疗肿瘤。
8.根据权利要求7所述的一种色胺水杨酸类化合物的应用,其特征在于为COX-2抑制剂。
9.根据权利要求7或8所述的一种色胺水杨酸类化合物的应用,其特征在于用于胶质瘤治疗。
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