CN102675134B - 含氮取代基姜黄素类似物及其药物用途 - Google Patents
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Abstract
Description
技术领域
本发明涉及一类新型含氮取代基姜黄素类似物及其药学上可接受的盐以及它们在抗肿瘤及神经退行性疾病方面的用途。
背景技术
姜黄素(Curcumin)是一种天然植物提取物,其主要存在于姜黄属植物如姜黄、郁金等的根茎中,其含量较高,易于提取,成本低廉。姜黄素生物活性多样,研究发现,姜黄素对多类肿瘤细胞具有较好的体外抗肿瘤活性,其既能预防/阻碍致癌物质诱发的癌变,又能直接诱导肿瘤细胞凋亡,还能抑制肿瘤的侵袭与迁移。近期也有研究报道,姜黄素能够有效抑制淀粉样蛋白(Aβ)聚集,同时具有一定的抗炎、抗氧化活性,因而具有治疗神经退行性疾病如阿尔茨海默病的潜在疗效。此外,作为一种广泛应用的食品添加剂,姜黄素具有极高的安全性,最新在台湾I期临床试验证明,给予患者8,000 mg/d的高剂量服用姜黄素,仍未见明显的毒副作用。然而,进一步的体内药效及药代动力学研究发现,姜黄素存在口服不吸收、作用靶向性差、生物利用度低等缺点,这极大地限制了其药用开发前景。
姜黄素
为了提高姜黄素的成药性,国内外研究人员以天然产物姜黄素为先导化合物,对其结构改造及衍生化做了大量的探索,概括而言主要有以下几个方面:1)酚羟基成酯/醚;2)庚烯二酮还原;3)活性亚甲基取代;4)芳环其他位置取代;等等。
综合文献调研发现,姜黄素分子中β-二酮结构及其酚羟基为抗肿瘤活性必需基团,而通过改造变换姜黄素分子中芳环上的取代基有可能获得活性及成药性更优的化合物。
发明内容
技术问题:本发明的主要目的是提供一种含氮取代基姜黄素类似物及其药物用途,本发明通过改变姜黄素的芳香环上的取代基,并在3’或(和)3’’位引入一个或多个含氮取代基,获得一类新型的姜黄素类似物,期望得到具有有效生物活性(包括抗肿瘤活性及抗神经退行性疾病活性)的化合物, 然后利用含氮取代基的碱性进一步制备药学上可接受的盐。
技术方案:本发明的一种含氮取代基姜黄素类似物,通过改变姜黄素的芳香环上的取代基,在3’ 和/或3’’ 位引入含氮取代基团,其结构通式如式(1):
式(1)
其中,R1为C1-6的烷基;R2为氢原子或C1-6的烷基;R3为氢原子、甲基或-CH2NX2,X为C1-6的烷基;R4为羟基、C1-6的烷氧基、C1-6的烷基或卤原子。
本发明的含氮取代基姜黄素类似物的制备方法为:
反应式(I)
各种不同取代的苯甲醛与等摩尔的仲胺及甲醛水溶液经Mannich反应得中间体2;将乙酰丙酮与B2O3溶于乙酸乙酯中,加热至70-90℃,搅拌反应,再加入中间体2和(n-BuO)3B,于70-90℃下继续搅拌,然后滴加正丁胺, 100℃下搅拌,再冷却至50℃,加入1N HCl水溶液,搅拌,结束反应,柱层析制备得关键中间体3,其中,R1为C1-6的烷基;R2为氢原子或C1-6的烷基;
中间体3和1.5当量的B2O3用乙酸乙酯溶解,加入1.4当量的3,4-取代的苯甲醛和1.5当量的(n-BuO)3B的乙酸乙酯溶液,70-90℃下搅拌;加入正丁胺, 80℃下搅拌;冷却至50℃,加入0.4N HCl水溶液,搅拌;冷却至室温,反应液用乙酸乙酯萃取,饱和NaCl洗涤,无水Na2SO4干燥,柱层析得产物4,其中,R3为氢原子、甲基或-CH2NX2(X为C1-6的烷基),R4为羟基、C1-6的烷氧基、C1-6的烷基或卤原子;
将产物4溶于二氯甲烷、乙酸乙酯、乙醚、或乙腈有机溶剂中,加入相应的1.5~3当量的酸或卤代烷的二氯甲烷、乙酸乙酯、乙醚、或乙腈溶液,反应制得药学上可接受的盐5。
所述的药学上可接受的盐是与氢卤酸所形成的盐,或与卤代烷所形成的季铵盐。
本发明含氮取代基姜黄素类似物的药物用途是,该含氮取代基姜黄素类似物用于抗肿瘤及神经退行性疾病,对人体肝癌细胞HepG2、人体乳腺癌细胞MCF-7、人体肺癌细胞A549以及人体结肠癌细胞HCT-116显示出良好的体外抑制活性,并具有高效的体外氧化自由基清除活性以及温和的体外Aβ聚集抑制活性。
有益效果:该类姜黄素类似物及其药学上可接受的盐中的一些代表化合物对人体肝癌细胞HepG2、人体乳腺癌细胞MCF-7、人体肺癌细胞A549以及人体结肠癌细胞HCT-116显示出了良好的体外抑制活性,部分化合物对所测试的四个肿瘤细胞株的抑制活性要优于阳性对照姜黄素,其中对A549和HCT-116肿瘤细胞株的抑制活性甚至要优于一线抗癌药物顺铂;该类化合物也显示了高效的体外氧化自由基清除活性,部分化合物对1,1-二苯基-2-三硝基苯肼(DPPH)氧化自由基清除能力(FRSA%)达到90%以上,对加尔万(galvinoxyl)氧化自由基清除能力达到50%以上,优于阳性对照姜黄素;同时,该类化合物对体外Aβ聚集也表现出了温和的抑制作用。此外,用HPLC法测定部分化合物的平衡溶解度及表观油水分配系数,发现其水溶性较之姜黄素有很大提高,最大的达2000倍以上;其表观油水分配系数大于1,表明其具有较好的亲油性,因而易于透过血脑屏障。上述药效学测试结果表明本发明所披露的姜黄素类似物具有抗肿瘤及神经退行性疾病的潜在疗效。
具体实施方式
本发明披露一类新型姜黄素类似物,通过改变姜黄素的芳香环上的取代基,在3’或(和)3’’位引入含氮取代基团,其结构通式如式(1):
式(1)
其中,R1为C1-6的烷基;R2为氢原子或C1-6的烷基;R3为氢原子、甲基或-CH2NX2,X为C1-6的烷基;R4为羟基、C1-6的烷氧基、C1-6的烷基或卤原子(F或Cl)。
本发明所说的药学上可接受的盐是与氢卤酸所形成的盐,如氯化物、溴化物或碘化物;或与卤代烷如碘甲烷所形成的季铵盐。
本发明还提供了式(1)所示化合物的合成方法,如反应式(I)所示:
反应式(I)
各种不同取代的苯甲醛与等摩尔的仲胺及甲醛水溶液经Mannich反应得中间体2;将乙酰丙酮与B2O3溶于乙酸乙酯中,加热至70-90℃,搅拌反应30min,再加入中间体2和(n-BuO)3B,于70-90℃下继续搅拌30min,然后滴加正丁胺, 100℃下搅拌2h,再冷却至50℃,加入1N HCl水溶液,搅拌30min,结束反应,柱层析制备得关键中间体3,其中,R1为C1-6的烷基;R2为氢原子或C1-6的烷基。
中间体3和B2O3用乙酸乙酯溶解,加入3,4-取代的苯甲醛和 (n-BuO)3B的乙酸乙酯溶液,70-90℃下搅拌30min;加入正丁胺, 80℃下搅拌1h;冷却至50℃,加入0.4N HCl水溶液,搅拌30min;冷却至室温,反应液用乙酸乙酯萃取,饱和NaCl洗涤,Na2SO4干燥,柱层析得产物4,其中,R3为氢原子、甲基或-CH2NX2,R4为羟基、C1-6的烷氧基、C1-6的烷基或卤原子(F或Cl)。
将产物4溶于二氯甲烷、乙酸乙酯、乙醚、或乙腈有机溶剂中,加入相应的1.5~3当量的酸(如盐酸)或卤代烷(如碘甲烷)的二氯甲烷、乙酸乙酯、乙醚、或乙腈溶液,反应制得药学上可接受的盐5。
本发明由下述实施例得到进一步说明,但这些说明并不限制本发明的实施。
实施例1. 3-二甲氨甲基-4-羟基苯甲醛(化合物2a)的制备
1.00 g(8 mmol) 4-羟基苯甲醛、1.12 g(8 mmol)二甲胺的水溶液(33%)、0.80 g(9.8 mmol)甲醛的水溶液(37%)在20 mL的甲醇中50℃下反应过夜。减压蒸去甲醇,残留液用乙酸乙酯萃取(15 mL×3),合并有机相,用无水Na2SO4干燥,浓缩,以乙酸乙酯/石油醚混合液为洗脱剂柱层析,得淡黄色油状物0.57 g,静置后凝固变为固体,收率39%,m.p.80~81℃。 1H NMR (CDCl3, 500MHz): δ 9.81 (s, 1H, CHO), 8.61 (s, 1H, OH), 7.71-7.69 (m, 1H, arom), 7.55-7.54 (d, 1H, arom), 6.92-6.90 (d, 1H, arom), 3.73 (s, 1H, CH 2), 2.36 (s, 6H, N(CH 3)2); IR (KBr): v 3421.4 (O-H), 2957.6, 2863.6 (C-H, CH3), 2729.5 (C-H, CHO), 1684.2 (C=O), 1595.3, 1494.3(arom) cm-1.
实施例2. 5-羟基-1-(3-二甲氨甲基-4-羟基)-1,4-戊二烯-3-酮(3a)的制备
0.79 g(7.92 mmol)乙酰丙酮和0.50 g(7.18 mmol) B2O3用乙酸乙酯(5 mL)溶解,于80℃下搅拌30min;再加入0.65 g(3.6 mmol)的2a和0.34 g(1.48 mmol) (n-BuO)3B,于80℃下继续搅拌30min;然后滴加0.10 g(1.43 mmol)的正丁胺, 100℃下搅拌2h;再冷却至50℃,加入1N HCl水溶液(10mL),搅拌30min。冷却至室温,反应液用乙酸乙酯萃取(25 mL×3),有机相用饱和NaCl洗涤(15 mL×2),无水Na2SO4干燥,以乙酸乙酯/石油醚混合液为洗脱剂柱层析得黄色固体0.13g,产率14%,m.p.105~106℃。1HNMR (CDCl3, 500MHz): δ 15.53 (s, 1H, CH=C-OH), 7.53-7.50 (d, 1H, arom-CH=CH), 7.38-7.36 (m, 1H, arom), 7.14-7.14 (d, 1H, arom), 6.83-6.81 (d, 1H, arom), 6.31-6.27 (d, 1H, CH=CH-CO), 5.60 (s, 1H, -CH=C-OH), 3.67 (s, 2H, CH 2), 2.35 (s, 6H, N(CH 3)2), 2.14 (s, 3H, CH 3 ); IR (KBr): v 1631.7 (C=C), 1585.2, 1498.5 (arom) cm-1, w 970.5, 829.2 (=C-H) cm-1; ESI-MS: 262.2 [M+H]+.
实施例3. 1-(3-二甲氨甲基-4-羟基苯基)-5-羟基-7-(4-羟基-3-甲氧基苯基)-1,4,6-戊三烯-3-酮(4a) 的制备
0.38 g(1.44 mmol)化合物3a和0.15 g(2.13 mmol) B2O3用乙酸乙酯(5 mL)溶解。加入4-羟基-3-甲氧基苯甲醛0.30 g(2.00 mmol)和0.49 g(2.11 mmol) (n-BuO)3B的5 mL乙酸乙酯溶液,80℃下搅拌30min;加入0.04 g(0.51 mmol)正丁胺, 80℃下继续搅拌1h;冷却至50℃,加入0.4N HCl水溶液(5 mL),再搅拌30min。冷却至室温,反应液用乙酸乙酯萃取(25 mL×3),饱和NaCl洗涤(15 mL×3),无水Na2SO4干燥,以乙酸乙酯/石油醚混合液为洗脱剂柱层析得黄色固体0.26g,产率46%,m.p.158~160℃。1H NMR (CDCl3, 300Hz): δ 7.63-7.58 (d, 2H, CH=CH), 7.44-7.44 (m, 1H, arom), 7.23-7.23 (d, 1H, arom), 7.13 (m, 2H, arom), 7.08-7.07 (d, 1H, arom); 6.97-6.88 (m, 2H, arom), 6.52-6.46 (m, 2H, CH=CH), 5.80 (s, 1H, -CH=C-OH), 3.96 (s, 3H, OCH 3 ), 3.75 (s, 2H, CH 2), 2.42 (s, 6H, N(CH 3)2); IR (KBr): v 3414.7 (O-H), 1625.6 (C=C), 1583.2, 1508.3 (arom) cm-1; ESI-MS: 396.1 [M+H]+.
实施例4. 1-(3-二甲氨甲基-4-羟基苯基)-5-羟基-7-(4-羟基苯基)-1,4,6-戊三烯-3-酮(4b)的制备
制备方法同实施例3,得暗红色固体,产率50%,m.p.164~166℃。1H NMR (DMSO, 300Hz): δ 7.61-7.49 (m, 6H), 6.84-6.67 (m, 5H), 6.05 (s, 1H, -CH=C-OH), 3.64 (s, 2H, CH 2), 2.27 (s, 6H, N(CH 3)2); IR (KBr): v 3355.3 (O-H), 1582.5, 1486.8 (arom) cm-1; ESI-MS: 366.2 [M+H]+.
实施例5. 1-(3-二甲氨甲基-4-羟基苯基)-5-羟基-7-(4-甲氧基苯基)-1,4,6-戊三烯-3-酮(4c)的制备
制备方法同实施例3,得橙色固体,产率42%, m.p.151~153℃。1H NMR (CDCl3, 300Hz): δ16.07 (s, 1H, CH=C-OH), 7.64-7.55 (m, 2H, CH=CH), 7.52-7.49 (d, 2H, arom), 7.43-7.40 (m, 1H, arom), 7.18 (d, 1H, arom), 6.93-6.90 (d, 2H, arom), 6.85-6.83 (d, 1H, arom), 6.51-6.43 (m, 2H, CH=CH), 5.76 (s, 1H, -CH=C-OH), 3.84 (s, 3H, OCH3), 3.69 (s, 2H, CH 2), 2.36 (s, 6H, N(CH 3)2); IR (KBr): v 1624.1 (C=O), 1593.7, 1504.0 (arom) cm-1; ESI-MS: 380.2 [M+H]+.
实施例6. 1-(3-二甲氨甲基-4-羟基苯基)-5-羟基-7-(3,4-二甲氧基苯基)-1,4,6-戊三烯-3-酮(4d)的制备
制备方法同实施例3,得橙红色固体,产率32%,m.p.129~131℃。1H NMR (CDCl3, 300Hz): δ 7.64-7.57 (m, 2H, CH=CH), 7.45-7.42 (dd, 1H, arom), 7.21-7.21 (d, 1H, arom), 7.17-7.14 (m, 1H, arom), 7.10-7.09 (d, 1H, arom), 6.91-6.85 (t, 2H, arom), 6.53-6.45 (m, 2H, CH=CH), 5.80 (s, 1H, -CH=C-OH),3.95 (s, 6H, OCH 3 ), 3.72 (s, 2H, CH 2), 2.37 (s, 6H, N(CH 3)2); IR (KBr): v 1621.6 (C=O), 1591.3, 1497.8 (arom) cm-1; ESI-MS: 410.25 [M+H]+.
实施例7. 1-(3-二甲氨甲基-4-羟基苯基)-5-羟基-7-(4-氟苯基)-1,4,6-戊三烯-3-酮(4e)的制备
制备方法同实施例3,得橙色固体,产率28%,m.p.171~174℃。1H NMR (CDCl3, 500Hz): 15.96 (s, 1H, CH=C-OH), 7.61-7.57 (m, 2H, CH=CH), 7.54-7.51 (m, 2H, arom), 7.42-7.41 (m, 1H, arom), 7.19 (s, 1H, arom), 7.09-7.06 (t, 2H, arom), 6.85-6.83 (d, 2H, arom), 6.53-6.44 (m, 2H, CH=CH), 5.77 (s, 1H, CH=C-OH), 3.69 (s, 2H, CH 2), 2.36 (s, 6H, N(CH 3)2); IR (KBr): v 1624.4 (C=O), 1590.0, 1505.5 (arom) cm-1; ESI-MS: 368.2 [M+H]+.
实施例8. 1-(3-二甲氨甲基-4-羟基苯基)-5-羟基-7-(3-羟基苯基)-1,4,6-戊三烯-3-酮(4f)的制备
制备方法同实施例3,得橙色固体,产率56%,m.p.164~166℃。1H NMR (DMSO, 300Hz): δ 7.70-7.59 (m, 4H), 7.36-7.31 (t, J=7.8Hz, 1H, arom), 7.24-7.22 (d, J=7.8Hz, 1H, arom), 7.15 (s, 1H, -CH=C-OH), 6.94-6.79 (m, 4H), 6.23 (s, 1H, -CH=C-OH),3.72 (s, 1H, CH 2), 2.36 (s, 6H, N(CH 3)2); IR (KBr): v 1626.8 (C=O), 1598.7, 1576.6, 1497.1, 1453.7 (arom) cm-1; ESI-MS: 366.1 [M+H]+.
实施例9. 1-(3-二甲氨甲基-4-羟基苯基)-5-羟基-7-(4-甲基苯基)-1,4,6-戊三烯-3-酮(4g)的制备
制备方法同实施例3,得橙色固体,产率32%,m.p. 166-169℃。1H NMR (CDCl3, 300Hz): δ 7.67-7.58(t, 2H, CH=CH), 7.46-7.42 (m, 4H, arom), 7.22-7.20 (d, 2H, arom), 6.97-6.95 (t, 1H, arom), 6.61-6.46 (m, 2H, CH=CH), 5.78 (s, 1H, -CH=C-OH), 3.80 (s, 1H, CH 2), 2.46 (s, 6H, N(CH 3)2), 2.38 (s, 3H, CH 3); IR (KBr): v 1620.8 (C=O), 1586.3, 1586.3, 1498.9 (arom) cm-1; ESI-MS: 364.3 [M+H]+.
实施例10. 1-(3-二甲氨甲基-4-羟基苯基)-5-羟基-7-(4-氯苯基)-1,4,6-戊三烯-3-酮(4h)的制备
制备方法同实施例3,得橙色固体,产率18%,m.p. 179-181℃。1H NMR (CDCl3, 300Hz): δ 7.63-7.55 (m, 2H, CH=CH), 7.49-7.46 (d, 2H, arom), 7.43-7.40 (m, 1H, arom), 7.3754-7.3472 (m, 2H, arom), 7.18 (s, 1H, arom), 6.85-6.82 (d, 1H, arom), 6.59-6.44 (m, 2H, CH=CH), 5.78 (s, 1H, -CH=C-OH),3.68 (s, 2H, CH 2), 2.35 (s, 6H, N(CH 3)2); IR (KBr): v 1627.0 (C=O), 1588.7, 1499.2 (arom) cm-1; ESI-MS: 384.2 [M+H]+.
实施例11. 1, 7-二(3-二甲氨甲基-4-羟基苯基)-5-羟基-1,4,6-戊三烯-3-酮(4i)的制备
由化合物2a与3a反应制得,制备方法同实施例3。产率15%,m.p. 119-121℃。1HNMR (CDCl3, 300MHz): δ 7.66-7.65 (br, 2H, -CH=CH), 7.62-7.59 (m, 2H, arom), 7.26-7.26 (d, 2H, arom), 6.88-6.87 (d, 2H, arom), 6.31-6.27 (br, 2H, CH=CH-CO), 5.60 (s, 1H, -CH=C-OH), 3.66 (s, 2H, CH 2), 2.38 (s, 6H, N(CH 3)2); IR (KBr): v 1630.6 (C=C), 1588.0, 1500.4 (arom) cm-1; ESI-MS: 423.2 [M+H]+.
实施例12. 1-(3-二甲氨甲基-4-羟基苯基)-5-羟基-7-(4-羟基-3-甲氧基苯基)-1,4,6-戊三烯-3-酮盐酸盐(5a)的制备
将化合物4a (0.4 g, 1 mmol)溶于15mL乙酸乙酯中,通入HCl气体2h,反应液浓缩至5 mL左右,残留液冷冻至-10℃,有固体析出,过滤得黄色固体0.38 g,产率87%, m.p. 145-147℃。1H NMR (DMSO, 300Hz): δ 11.02 (s, 1H, OH), 10.07 (s, 1H, OH), 9.71 (s, 1H, OH), 7.86 (s, 1H, arom), 7.68-7.65 (d, 1H, arom), 7.57-7.52 (dd, 2H, CH=CH), 7.33 (s, 1H, arom), 7.16-7.14 (d, 1H, arom), 7.08-7.05 (d, 1H, arom), 6.85-6.71 (m, 3H), 6.05 (s, 1H, -CH=C-OH),4.23 (s, 1H, CH2), 3.84 (s, 3H, OCH3), 2.73 (s, 6H, N(CH3)2); IR (KBr): v 1627.9 (C=O), 1593.7, 1511.0 (arom) cm-1.
实施例13. 1-(3-二甲氨甲基-4-羟基苯基)-5-羟基-7-(3-羟基苯基)-1,4,6-戊三烯-3-酮盐酸盐(5b)的制备
制备方法同实施例12,产率85%,m.p.172-174℃。1H NMR (DMSO, 500Hz): δ 7.87 (s, 1H, arom), 7.68-7.66 (m, 1H, arom), 7.60-7.51 (m, 2H, CH=CH), 7.25-7.22 (t, 1H, arom), 7.14-7.12 (d, 1H, arom), 7.08-7.06 (d, 2H, arom), 6.85-6.82 (m, 3H), 6.77-6.74 (d, 1H, CH=CH), 6.13 (s, 1H, -CH=C-OH),4.23 (s, 1H, CH 2), 2.74 (s, 6H, N(CH 3)2); IR (KBr): v 1629.0 (C=O), 1584.1, 1510.2 (arom) cm-1.
实施例14. 1-(3-三甲氨甲基-4-羟基苯基)-5-羟基-7-(4-羟基-3-甲氧基苯基)-1,4,6-戊三烯-3-酮碘盐(5c)的制备
将0.1 g (0.25 mmol)化合物4a溶解在5 mL乙腈中,加入0.11 g(0.75 mmol)CH3I,避光搅拌过夜。加入16 mL无水乙醚,有固体析出,过滤,干燥,得黄色固体0.08 g,产率62%,m.p.170-172℃。1H NMR (CDCl3, 500Hz): δ 7.12-7.02 (m, 4H), 6.69-6.64 (m, 3H), 6.55-6.54 (d, 1H, arom), 6.19-6.13 (t, 2H, CH=CH), 3.96 (s, 3H, OCH 3 ), 4.13 (s, 2H, CH 2), 3.65 (s, 3H, OCH 3 ), 2.96 (s, 6H, N(CH 3)2); IR (KBr): v 1627.8 (C=C), 1576.9, 1508.6 (arom) cm-1; ESI-MS: 566.1 [M+H2O+H]+.
实施例15. 典型化合物的体外抗肿瘤活性。
对一些典型化合物进行了体外抗肿瘤活性测定,被筛选的肿瘤细胞株有:人体肝癌细胞HepG2、人体乳腺癌细胞MCF-7、人体肺癌细胞A549和人体结肠癌细胞HCT-116, 选用姜黄素为阳性对照。实验采用CCK-8试剂盒法,具体操作如下:将选用的肿瘤细胞株(100微升5000个细胞)接种于96孔板上,同时加入10 μL受试化合物4‰DMSO水溶液(受试化合物浓度依次0.1,0.4,2,10,50 μg/mL)进行培养。然后每孔加入10 μL CCK-8溶液,在细胞培养箱内继续孵育0.5-4 h。在450nm波长处测定吸光度,并计算受试化合物IC50值。测试结果见表一。
表一、部分化合物的体外抗肿瘤活性
从测试结果可见,本发明化合物除4g外均显示了较好的体外抗肿瘤活性,其中化合物4a、4b和4f对所有四个肿瘤细胞株的抑制活性都要优于阳性对照姜黄素,并且化合物4a、4b和4c对A549、HCT-116肿瘤细胞株的抑制活性要优于抗癌一线药物顺铂。
实施例16. HPLC法测定典型化合物的平衡溶解度。
1.色谱条件的选择
姜黄素:反相C18色谱柱,流动相:甲醇-4%的冰醋酸水溶液(体积比为65:35),检测波长:428nm,流速:1.0mL/min,柱温:30℃,进样量:20 μL。
化合物5a、5b:反相C18色谱柱,流动相:甲醇-4%的冰醋酸水溶液(体积比为45:55),检测波长:428nm,流速:1.0mL/min,柱温:30℃,进样量:20 μL。
2.标准曲线的制备
分别称取姜黄素、5a和5b 各1 mg左右(准确质量为:0.78 mg姜黄素, 1.39 mg 5a, 1.16mg 5b)置于10 mL容量瓶中,用甲醇定容到刻度,摇匀,得质量浓度约为100 μg/mL的储备液(其中姜黄素:78 μg/mL, 5a:139 μg/mL,5b:116 μg/mL)。
精密量取储备液适量,置于10 mL容量瓶中,用甲醇稀释到刻度并摇匀,分别得到系列标准溶液,在上述的色谱条件下分别测定,记录峰面积。分别以浓度C(μg/mL)为横坐标,峰面积A为纵坐标进行线性回归,得到线性回归方程:
姜黄素: y = 203796x-185721,R2= 0.9988;
5a:y = 28530x-28804,R2= 0.9999;
5b:y =32938x-51298,R2= 0.9991。
3.平衡溶解度的测定
分别称取1 mg姜黄素两份于2 mL离心管中,加入1.5 mL水,用膜封好,将其嵌入装有适量水的10 mL试管中,用膜将口封好。用恒温水浴振荡器在37℃下振摇72 h,过膜,分别按照上述的液相条件进样20 μl,记录峰面积,带入标准曲线计算姜黄素在水溶液中的平衡溶解度。化合物5a、5b的方法同上。每组化合物平行测定2次,测试结果见表二。
姜黄素的水溶液在高效液相中没有出现相应的色谱峰,将1.17 μg/mL的姜黄素的甲醇溶液用甲醇稀释1000倍,测出用上述色谱条件姜黄素的检测限为0.0017μg/mL,故姜黄素在水中的平衡溶解度小于0.0017 μg/mL。
表二、化合物5a、5b的平衡溶解度
从结果可见,化合物5a和5b的平衡溶解度较之姜黄素有了显著的提升,其中5a的溶解度是姜黄素溶解度的2000倍以上。
实施例17. 体外抗氧化活性测试
1)体外DPPH氧化自由基清除能力测试
将DPPH(购于Sigma试剂公司)溶于乙醇配置得0.1 mM溶液,同时分别配置0.1、0.02、0.01、0.002、0.001 mM 5个浓度的受试化合物及姜黄素乙醇溶液,然后量取200 μL不同浓度的受试化合物溶液以及2 mL DPPH溶液加入至比色皿中,并用乙醇稀释至3 mL,在暗室中于30℃下静置40min,然后于517 nM波长下测量吸光度。空白对照组以1 mL乙醇加入2 mL DPPH溶液静置40min后测得。所有实验均平行测试3次。氧化自由基清除能力(FRSA%)以下述公式计算:
FRSA% = (A c - A s )/ A c *100%
其中,A c 为空白对照的实际吸光度,A s 为受试药物组试剂吸光度,实验结果见表三。
表三、部分化合物体外DPPH氧化自由基清除能力
从测试结果可见,化合物分子中酚羟基的个数对其氧化自由基清除活性有较大的影响,化合物4a、4b与姜黄素均具有两个酚羟基,因而活性最强,在0.1 mM浓度下有90%左右的清除率,即使在0.02 mM浓度下4a、4b也具有50%以上的清除率,优于该浓度下姜黄素的清除率。4f也含有两个酚羟基,但由于右侧酚羟基位于桥链的键位,活性不及4a、4b与姜黄素。其他化合物由于只含有1个酚羟基,因而只显现了温和的DPPH氧化自由基清除能力。
2)体外galvinoxyl氧化自由基清除能力测试
将galvinoxyl氧化自由基(购于Sigma试剂公司)溶于乙醇配置得0.1mM溶液,同时分别配置0.1、0.02、0.01、0.002、0.001 mM 5个浓度的受试化合物及姜黄素乙醇溶液,然后量取200 μL不同浓度的受试化合物溶液以及2 mL galvinoxyl溶液加入至比色皿中,并用乙醇稀释至3 mL,在暗室中于30℃下静置40min,然后于510 nM波长下测量吸光度。空白对照组以1 mL乙醇加入2 mL galvinoxyl溶液静置40min后测得。所有实验均平行测试3次。氧化自由基清除能力(FRSA%)以下述公式计算:
FRSA% = (A c - A s )/ A c *100%
其中,A c 为空白对照的实际吸光度,A s 为受试药物组试剂吸光度,实验结果见表四。
表四、部分化合物体外galvinoxyl氧化自由基清除能力
从测试结果可见,受试化合物清除galvinoxyl氧化自由基能力总体弱于其清除DPPH自由基能力。化合物4a、4b与姜黄素依旧显示了较强的活性,其中在0.1 mM浓度下4a、4b有50%以上的清除率,优于同浓度下姜黄素的活性。其他化合物也显现了一定的galvinoxyl氧化自由基清除能力,活性与阳性对照大致相当。
实施例18. 体外Aβ聚集抑制活性测试
具体测试方法如下:
(1) β-淀粉样蛋白母液配制:将Aβ (1-42)溶于100%的六氟异丙醇,配成 1mg/mL 母液,水浴超声10min,等分至doff管,真空或氮气将溶剂挥干,存于-20°C,临用前将其溶于DMSO,用pH7.4的PBS稀释至所需浓度。
(2) 实验分组:取2 μL Aβ母液加38 μl含有一定浓度乙醇的PBS,另取2 μL Aβ溶液加20 μL受试药和18 μL含有一定浓度乙醇的PBS,前者设为模型组,后者设为药物处理组,再取20 μL受试药加等量含一定浓度乙醇的PBS,将其设为药物对照组。
(3) Aβ老化:以上各组加入96孔黑板,于37°C条件下老化24h。
(4) 加入分子探针硫磺素T:各孔加入160μL 5 mM 硫磺素T,反应1min后,放入酶标仪中,在激发波长450nm和发射波长480nm处测定荧光强度,并计算药物对聚集Aβ的抑制率。
所有实验均平行测试3次。
各药物抑制率计算公式为:
药物处理组 / 药物对照组
模型组 / 溶剂对照
测试结果见表五。
表五、典型化合物体外Aβ聚集抑制活性
从测试结果可见,受试化合物显示了较好的Aβ聚集抑制活性,且总体活性要优于阳性对照姜黄素。化合物的活性与给药剂量未显示出相关性,在较低浓度下(如5 μM和50 μM)受试化合物的活性较高,且和姜黄素存在显著性差异。
实施例19. HPLC法测定表观油水分配系数
1)色谱条件的选择
姜黄素:反相C18色谱柱,流动相:甲醇-4%的冰醋酸水溶液(体积比为65:35),检测波长:428nm,流速:1.0 mL/min,柱温:30℃,进样量:20 μl。
化合物5a、5b:反相C18色谱柱,流动相:甲醇—4%的冰醋酸水溶液(体积比为45:55),检测波长:428nm,流速:1.0 mL/min,柱温:30℃,进样量:20 μl。
2)标准曲线的制备
分别称取姜黄素、5a和5b 各1 mg左右(准确质量为:0.78 mg姜黄素, 1.39 mg 5a, 1.16mg 5b)置于10 mL容量瓶中,用甲醇定容到刻度,摇匀,得质量浓度约为100 μg/mL的储备液(其中姜黄素:78 μg/mL, 5a:139 μg/mL,5b:116 μg/mL)。
精密量取储备液适量,置于10mL容量瓶中,用甲醇稀释到刻度并摇匀,分别得到系列标准溶液,在上述的色谱条件下分别测定,记录峰面积。分别以浓度C(μg/mL)为横坐标,峰面积A为纵坐标进行线性回归,得到线性回归方程:
姜黄素: y = 203796x-185721,R2= 0.9988;
化合物5a:y = 28530x-28804,R2= 0.9999;
化合物5b:y =32938x-51298,R2= 0.9991.
3)表观油水分配系数的测定
分别称取1mg 5a两份于10 mL具塞试管中,分别加入5 mL用正辛醇饱和的水溶液,涡旋5min之后,静置30min,过膜。准确量取0.5 mL过膜之后的溶液于10mL容量瓶中,用纯甲醇定容到刻度,摇匀,在上述的色谱条件下测定,记录峰面积,带入标准曲线计算质量浓度ρ0。
取2 mL过膜之后的溶液于10 mL具塞试管中,再加入2 mL用水饱和的正辛醇,涡旋5min,用恒温水浴振荡器于37℃下振摇24h。精确量取下层水相0.5 mL于10 mL容量瓶中,用纯甲醇定容到刻度,摇匀,在上述的色谱条件下测定,记录峰面积,带入标准曲线计算质量浓度ρequ。按照公式表观油水分配系数Papp=(ρ0-ρequ)/ρequ,计算表观油水分配系数。
5b的表观油水分配系数测定方法同上,测试结果见表六。
表六、部分化合物5a和5b表观油水分配系数
由上可见,化合物5a和5b的Papp均大于1,表明其具有较好的亲油性,因而易于透过血脑屏障。
Claims (3)
反应式(I)
反应物a与等摩尔的仲胺b及甲醛水溶液经Mannich反应得中间体2;将乙酰丙酮与B2O3溶于乙酸乙酯中,加热至70-90℃,搅拌反应,再加入中间体2和(n-BuO)3B,于70-90℃下继续搅拌,然后滴加正丁胺,100℃下搅拌,再冷却至50℃,加入1NHCl水溶液,搅拌,结束反应,柱层析制备得关键中间体3,其 中,R1为C1-6的烷基;R2为氢原子或C1-6的烷基;
中间体3和1.5当量的B2O3用乙酸乙酯溶解,加入1.4当量的反应物c和1.5当量的(n-BuO)3B的乙酸乙酯溶液,70-90℃下搅拌;加入正丁胺,80℃下搅拌;冷却至50℃,加入0.4NHCl水溶液,搅拌;冷却至室温,反应液用乙酸乙酯萃取,饱和NaCl洗涤,无水Na2SO4干燥,柱层析得产物4,其中,R3为氢原子或-CH2NX2,其中,X为C1-6的烷基,R4为羟基、C1-6的烷氧基、C1-6的烷基或卤原子。
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