CN116751244A - α,β-不饱和羰基类18β-甘草次酸衍生物及其制备方法与应用 - Google Patents

α,β-不饱和羰基类18β-甘草次酸衍生物及其制备方法与应用 Download PDF

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CN116751244A
CN116751244A CN202310589748.9A CN202310589748A CN116751244A CN 116751244 A CN116751244 A CN 116751244A CN 202310589748 A CN202310589748 A CN 202310589748A CN 116751244 A CN116751244 A CN 116751244A
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glycyrrhetinic acid
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怀其勇
李伊
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Shandong University
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Abstract

本发明属于生物医药技术领域，涉及α,β‑不饱和羰基类18β‑甘草次酸衍生物及其制备方法与应用。其化学结构式如下所示：其中，n为4或5，R1为一取代或二取代的苯基，苯基的取代基团为卤素、三氟甲基、甲基和/或硝基。研究表明本发明提供的α,β‑不饱和羰基类18β‑甘草次酸衍生物具有较高的抗肿瘤活性。

Description

α,β-不饱和羰基类18β-甘草次酸衍生物及其制备方法与应用
技术领域
本发明属于生物医药技术领域，涉及α,β-不饱和羰基类18β-甘草次酸衍生物及其制备方法与应用。
背景技术
公开该背景技术部分的信息仅仅旨在增加对本发明的总体背景的理解，而不必然被视为承认或以任何形式暗示该信息构成已经成为本领域一般技术人员所公知的现有技术。
18β-甘草次酸(18β-glycyrrhetinic acid，18β-GA)属于齐墩果烷型五环三萜类化合物，是传统中药甘草中甘草酸的主要活性成分。它同样拥有丰富的药理活性，包括抗肿瘤作用在内的抗炎、抗病毒、保肝活性等。但该类化合物本身具有水溶性差和生物利用度低等缺陷，限制了它作为抗肿瘤制剂的使用，另外其生物活性与临床使用的药物仍有差距。
发明内容
为了解决现有技术的不足，本发明的目的是提供α,β-不饱和羰基类18β-甘草次酸衍生物及其制备方法与应用，研究表明本发明提供的α,β-不饱和羰基类18β-甘草次酸衍生物具有较高的抗肿瘤活性。
为了实现上述目的，本发明的技术方案为：
一方面，一种α,β-不饱和羰基类18β-甘草次酸衍生物，其化学结构式如下所示：
其中，n为4或5，R1为一取代或二取代的苯基，苯基的取代基团为卤素、三氟甲基、甲基和/或硝基。
在一些实施例中，R1为2-氯-5硝基苯基、4-氟-3-甲基苯基、3-三氟甲基苯基、对三氟甲基苯基、对氟苯基、对氯苯基、对溴苯基或对碘苯基。
在一些实施例中，R1为2-氯-5硝基苯基、对三氟甲基苯基或对氟苯基。
在一些实施例中，n为4，R1为2-氯-5硝基苯基；n为5，R1为对三氟甲基苯基或对氟苯基。
另一方面，一种上述α,β-不饱和羰基类18β-甘草次酸衍生物的制备方法，以18β-甘草次酸(18β-GA)作为原料按照如下反应路线进行反应的步骤；
其中，R1、n如上所述。
具体地，18β-甘草次酸与氧化剂进行羟基氧化反应获得化合物2，化合物与取代苯甲醛(R1CHO)进行羟醛缩合反应获得化合物3，化合物3与1,4-二溴丁烷或1,5-二溴戊烷进行取代反应获得化合物4，化合物4与三苯基膦进行季膦化反应获得化合物5，即α,β-不饱和羰基类18β-甘草次酸衍生物。
更为具体地，氧化剂为氯铬酸吡啶鎓盐(PCC)。
更为具体地，羟基氧化反应的温度为0℃至室温。本发明所述室温为室内环境的温度，一般为15～30℃。
更为具体地，羟醛缩合反应中，添加氢氧化钾或氢氧化钠，在40～50℃条件下进行反应。
更为具体地，取代反应在室温条件下进行。
更为具体地，季膦化反应的温度为75～85℃。
更为具体地，试剂及反应条件：a.氯铬酸吡啶鎓盐(PCC)，硅胶，CH2Cl2(DCM)，0℃至室温，2d；b.氢氧化钾(KOH)，取代苯甲醛，无水乙醇(dry CH3CH2OH)，46℃,1d；c.1,4-二溴丁烷或1,5-二溴戊烷，DMF，室温，7h；d.三苯基膦(TPP)，无水乙腈(dry CH3CN)，80℃，回流3d。
第三方面，一种药物组合物，包括上述α,β-不饱和羰基类18β-甘草次酸衍生物或其药学上可接受的盐。
本发明所述的药学上可接受的盐包括盐酸盐、硫酸盐、苯磺酸盐、醋酸盐等。
第四方面，一种药物制剂，包括上述α,β-不饱和羰基类18β-甘草次酸衍生物或上述药物组合物和药学上可接受的辅料。
本发明所述的药学上可接受的辅料为赋形剂和/或载体，其中，所述载体包括血清蛋白、聚乙二醇、缓冲液、氧化铝、卵磷脂等；所述赋形剂包括粘合剂、填充剂、缓冲剂、pH调节剂、乳化剂、稳定剂等。
第五方面，一种上述α,β-不饱和羰基类18β-甘草次酸衍生物、药物组合物或药物制剂在制备抗肿瘤药物中的应用。
具体地，肿瘤包括肝癌、结肠癌、神经母细胞瘤。更为具体地，肿瘤细胞为HepG2、HCT-116、SH-SY5Y。
本发明的有益效果为：
本发明以18β-GA为结构改造的母核，通过羟醛缩合反应向18β-GA的C-2位引入环外的α,β-不饱和羰基结构。同时，在C-30位的羧基引入靶向肿瘤细胞线粒体的烷基三苯基膦阳离子基团(Triphenylphosphonium cation，TPP+)。一方面，具有多种取代基的苯甲醛通过羟醛缩合反应与18β-GA母核结合，形成了环外的α,β-不饱和羰基结构；另一方面，亲脂性阳离子，即本研究的烷基TPP+的引入可以进一步提高衍生物的选择性，降低对正常体细胞的毒副作用。经过实验表明，本发明提供的α,β-不饱和羰基类18β-甘草次酸衍生物具有较高的抗肿瘤活性。其中，部分α,β-不饱和羰基类18β-甘草次酸衍生物对HepG2、HCT-116、SH-SY5Y的生长抑制活性更加显著，IC50值范围分别是1.52～3.46μM、1.58～1.66μM和1.60～2.18μM。
具体实施方式
为了使得本领域技术人员能够更加清楚地了解本发明的技术方案，以下将结合具体的实施例详细说明本发明的技术方案。
实施例
化合物结构及合成路线
设计、合成了5a-5p共16个α,β-不饱和羰基修饰的18β-GA衍生物。相关结构及合成路线如下所示。
试剂及反应条件：a.氯铬酸吡啶鎓盐(PCC)，硅胶，CH2Cl2(DCM)，0℃至室温，2d；b.氢氧化钾(KOH)，取代苯甲醛，无水乙醇(dry CH3CH2OH)，46℃,1d；c.1,4-二溴丁烷或1,5-二溴戊烷，DMF，室温，7h；d.三苯基膦(TPP)，无水乙腈(dry CH3CN)，80℃，回流3d。
合成步骤：
(1)18β-GA衍生物2的合成：
将18β-GA(5mmol)，200～300目的硅胶(称取质量与PCC的质量相当)以及30mL的DCM加入150mL的圆底烧瓶内。在0℃搅拌条件下，通过加料漏斗将PCC(7.5mmol)缓慢倒入反应液中。室温下搅拌反应2d，TLC监测反应进程。减压抽滤除去体系内的硅胶固体。DCM稀释反应液，有机相经去离子水水洗3次，饱和食盐水水洗3次。无水硫酸钠处理有机相，静置10min。减压抽滤、旋蒸，转移并浓缩粗产品。正向硅胶柱层析分离纯化，流动相为正己烷：乙酸乙酯＝6:1→4:1，该柱层析的流动相内需加入冰乙酸，比例为每100mL流动相加入1mL的冰乙酸。
(2)18β-GA衍生物3a-3p的合成
将化合物2(1mmol)，氢氧化钾(2mmol)以及无水乙醇溶剂25mL加入到50mL的圆底烧瓶内，46℃下搅拌活化1h。之后将取代的苯甲醛(2mmol)加入反应体系内，46℃下搅拌反应1d，TLC监测反应进程。待反应结束后，在搅拌条件下逐滴滴加稀盐酸溶液，调节反应液的pH值为5～6，此时原本澄清均一的溶液内有白色固体析出。乙酸乙酯稀释反应液，有机相经去离子水水洗3次，饱和食盐水水洗3次。无水硫酸钠处理有机相，静置10min。减压抽滤、旋蒸，转移并浓缩粗产品。正向硅胶柱层析分离纯化，流动相为正己烷：乙酸乙酯＝60:1→40:1→20:1或10:1，该流动相内同样需加入冰乙酸，比例同上述(1)。
其中，取代的苯甲醛分别为2-氯-5-硝基苯甲醛、4-氟-3-甲基苯甲醛、4-三氟甲基苯甲醛、3-三氟甲基苯甲醛、4-氟苯甲醛、4-氯苯甲醛、4-溴苯甲醛、4-碘苯甲醛。
(3)18β-GA衍生物4a-4p的合成
将化合物3a-3p(1mmol)，无水碳酸钾(6mmol)以及6mL的DMF加入到50mL的圆底烧瓶内，室温下搅拌活化反应30min。之后将1,4-二溴丁烷或1,5-二溴戊烷加入反应体系内，室温下搅拌7h，TLC监测反应进程。DCM稀释反应液，有机相经去离子水水洗3次，饱和食盐水水洗3次，以除去DMF以及水溶性杂质。无水硫酸钠处理有机相，静置10min。减压抽滤、旋蒸，转移并浓缩粗产品。正向硅胶柱层析分离纯化，流动相为正己烷：乙酸乙酯＝20:1→10:1。
(4)18β-GA衍生物5a-5p的合成
将化合物4a-4p(1mmol)、三苯基膦(4mmol)以及无水乙腈10mL加入到25mL的圆底烧瓶内，80℃下回流搅拌3d，TLC监测反应进程。该反应液无需经过后处理，直接减压旋蒸、浓缩得粗产品。正向硅胶柱层析分离纯化，流动相为二氯甲烷：甲醇＝100:1→80:1→50:1→30:1→20:1。
衍生物5a-5p的命名及结构表征
2-((2-chloro-5-nitrophenyl)methylene)-3,11-dioxo-12-en-30-(4-(triphenylphos-phonio)butoxycarbonyl)-18β-glycyrrhetinic acid bromide
化合物5a：熔点：157.0–158.2℃；产率：36％；产物状态：白色固体；1HNMR(400MHz,CDCl3)δ8.18–7.97(m,2H,Ph-H at C-2),7.82–7.63(m,15H,PPh3-H),7.57–7.49(m,2H,Ph-Hat C-2and C＝CH at C-2),5.44(s,1H,H-12),4.10(m,2H,COOCH2),3.84(dt,2H,Ph3PCH2,J＝16.3,5.2Hz),2.42(s,1H,H-9),2.30–0.81(m,41H,CH and CH2 in pentacyclicskeleton or alkyl chains),1.33(s,3H,CH3),1.17(s,9H,3×CH3),1.09(s,3H,CH3),0.92(s,3H,CH3),0.70(s,3H,CH3).13C NMR(101MHz,CDCl3)δ206.05,198.56,175.86,169.95,145.75,137.65,135.72,134.75,133.40(d,J c,p＝9.9Hz),131.60,130.20(d,J c,p＝12.7Hz),127.97,124.70,123.53,117.79(d,J c,p＝86.3Hz),58.67,53.51,47.95,45.69,43.08,40.75,37.37,36.33,31.47,31.20,30.62,29.38,28.91,28.35,27.97,26.14,25.98,22.98,22.50,21.62,19.14,18.93,17.84,14.93.
2-((2-chloro-5-nitrophenyl)methylene)-3,11-dioxo-12-en-30-(5-(triphenylphos-phonio)pentoxycarbonyl)-18β-glycyrrhetinic acid bromide
化合物5b：熔点：142.0–143.3℃；产率：35％；产物状态：白色固体；1HNMR(400MHz,CDCl3)δ7.80–7.60(m,15H,PPh3-H),7.31(s,1H,C＝CH at C-2),7.06–6.91(m,1H,Ph-H atC-2),6.61(d,2H,Ph-H at C-2,J＝19.8Hz),5.52(s,1H,H-12),4.09(t,2H,COOCH2,J＝6.0Hz),3.81–3.75(m,2H,Ph3PCH2),2.42(s,1H,H-9),2.23–0.72(m,31H,CH and CH2 inpentacyclic skeleton or alkyl chains),1.31(s,3H,CH3),1.12(s,9H,3×CH3),1.09(s,3H,CH3),0.92(s,3H,CH3),0.69(s,3H,CH3).13C NMR(101MHz,CDCl3)δ207.11,198.68,175.97,169.91,156.43,136.04,135.87,134.73(d,J c,p＝2.8Hz),133.29(d,J c,p＝10.5Hz),130.18(d,J c,p＝12.6Hz),128.72,127.93,117.69(d,J c,p＝86.0Hz),115.49,106.10,100.88,77.05,62.74,59.06,56.26,53.09,47.75,45.47,44.62,43.56,42.97,40.75,37.41,36.14,31.41,30.54,28.83,28.28,27.93,26.09,25.89,23.07,22.35,19.08,17.74,15.32.
2-((4-fluoro-3-methylphenyl)methylene)-3,11-dioxo-12-en-30-(4-(triphenylpho s-phonio)butoxycarbonyl)-18β-glycyrrhetinic acid bromide
化合物5c：熔点：150.1–151.5℃；产率：38％；产物状态：白色固体；1HNMR(400MHz,CDCl3)δ7.84–7.57(m,15H,PPh3-H),7.33(s,1H,C＝CH at C-2),7.21(d,2H,Ph-H at C-2,J＝6.6Hz),6.83(t,1H,Ph-H at C-2,J＝9.2Hz),5.52(s,1H,H-12),4.12–4.05(m,2H,COOCH2),3.85–3.73(m,2H,Ph3PCH2),2.46(s,1H,H-9),2.18(s,3H,CH3),2.08–0.81(m,23H,CH and CH2 in pentacyclic skeleton or alkyl chains),1.33(s,3H,CH3),1.13(s,3H,CH3),1.07(s,9H,3×CH3),0.93(s,3H,CH3),0.69(s,3H,CH3).13C NMR(101MHz,CDCl3)δ207.14,198.86,175.88,169.92,135.86,134.74(d,J c,p＝2.8Hz),133.86,133.31(d,J c,p＝10.1Hz),130.17(d,J c,p＝12.7Hz),128.71,128.63,128.02,124.59,117.68(d,J c,p＝86.0Hz),114.73,77.05,62.73,58.95,52.94,47.91,45.04,44.62,43.93,43.58,43.00,40.76,37.37,35.91,31.43,31.06,30.55,29.29,28.31,27.93,26.10,25.92,22.94,22.21,19.19,18.94,17.69,15.02,14.25.
2-((4-fluoro-3-methylphenyl)methylene)-3,11-dioxo-12-en-30-(5-(triphenylpho s-phonio)pentoxycarbonyl)-18β-glycyrrhetinic acid bromide
化合物5d：熔点：128.9–130.7℃；产率：10％；产物状态：白色固体；1HNMR(400MHz,CDCl3)δ7.93–7.59(m,17H,PPh3-H and Ph-H at C-2),7.39(s,1H,C＝CH at C-2),6.89(td,1H,Ph-H at C-2,J＝11.4,10.4,6.7Hz),5.62(s,1H,H-12),4.13–3.98(m,2H,COOCH2),3.90–3.77(m,2H,Ph3PCH2),2.54(s,1H,H-9),2.23(s,3H,CH3),2.10–1.42(m,38H,CH and CH2 in pentacyclic skeleton or alkyl chains),1.40(s,3H,CH3),1.19(s,3H,CH3),1.14(s,3H,CH3),1.13(s,3H,CH3),1.11(s,3H,CH3),1.08(s,3H,CH3),0.80(s,3H,CH3).13C NMR(101MHz,CDCl3)δ207.34,199.16,176.11,135.98,134.76(d,J c,p＝2.7Hz),134.36,133.68,133.51(d,J c,p＝10.0Hz),131.60,130.23(d,J c,p＝12.6Hz),128.14,118.08(d,Jc,p＝86.0Hz),77.05,63.80,59.11,53.07,48.44,45.13,44.83,43.80,43.23,41.09,36.06,31.21,29.49,28.18,26.17,23.07,22.31,19.40,17.87.
2-((3-trifluoromethylphenyl)methylene)-3,11-dioxo-12-en-30-(4-(triphenylphos-phonio)butoxycarbonyl)-18β-glycyrrhetinic acid bromide
化合物5e：熔点：145.7–147.0℃；产率：35％；产物状态：白色固体；1HNMR(400MHz,CDCl3)δ7.82–7.57(m,17H,PPh3-H and Ph-H at C-2),7.51–7.36(m,3H,Ph-H at C-2and C＝CH at C-2),5.54(s,1H,H-12),4.16–4.07(m,2H,COOCH2),3.86–3.74(m,2H,Ph3PCH2),2.47(s,1H,H-9),2.43–0.76(m,27H,CH and CH2 in pentacyclic skeleton or alkylchains),1.34(s,3H,CH3),1.17–1.07(s,12H,4×CH3),0.94(s,3H,CH3),0.70(s,3H,CH3).13C NMR(101MHz,CDCl3)δ207.01,198.77,175.96,169.97,136.30,135.49,134.77(d,J c,p＝2.8Hz),133.35(d,J c,p＝10.1Hz),132.31,130.21(d,J c,p＝12.6Hz),128.63,117.74(d,Jc,p＝85.9Hz),77.05,62.76,58.89,53.07,47.92,45.24,44.65,43.61,37.41,36.03,31.47,31.09,29.22,28.35,27.95,22.97,22.27,19.21,17.73,15.09,15.09.
2-((3-trifluoromethylphenyl)methylene)-3,11-dioxo-12-en-30-(5-(triphenylphos-phonio)pentoxycarbonyl)-18β-glycyrrhetinic acid bromide
化合物5f：熔点：136.5–137.6℃；产率：39％；产物状态：白色固体；1HNMR(400MHz,CDCl3)δ7.83–7.77(m,1H,Ph-H at C-2),7.74–7.52(m,15H,PPh3-H),7.47–7.28(m,4H,Ph-Hat C-2and C＝CH at C-2),5.54(s,1H,H-12),4.03–3.88(m,2H,COOCH2),3.80–3.50(m,2H,Ph3PCH2),2.49(s,1H,H-9),2.45–0.76(m,28H,CH and CH2 in pentacyclic skeletonor alkyl chains),1.36(s,3H,CH3),1.14(s,3H,CH3),1.09(s,3H,CH3),1.07(s,3H,CH3),1.05(s,3H,CH3),1.02(s,3H,CH3),0.73(s,3H,CH3).13C NMR(101MHz,CDCl3)δ206.95,198.90,175.93,170.47,136.10,135.34,134.68(d,J c,p＝3.3Hz),133.42,133.22(d,J c,p＝10.0Hz),132.24,130.42,130.10(d,J c,p＝12.4Hz),128.81,127.81,127.12,117.74(d,J c,p＝86.0Hz),77.05,63.51,58.79,52.87,48.35,45.06,44.62,44.07,43.62,43.07,40.84,37.22,35.86,31.46,30.97,30.64,29.27,28.33,28.04,27.95,26.14,25.93,22.86,22.14,19.17,17.67,14.99.
2-((4-trifluoromethylphenyl)methylene)-3,11-dioxo-12-en-30-(4-(triphenylphos-phonio)butoxycarbonyl)-18β-glycyrrhetinic acid bromide
化合物5g：熔点：158.2–159.7℃；产率：40％；产物状态：白色固体；1HNMR(400MHz,CDCl3)δ7.81–7.59(m,15H,PPh3-H),7.54–7.44(m,4H,Ph-H at C-2),7.38(s,1H,C＝CH atC-2),5.52(s,1H,H-12),4.14–4.06(m,2H,COOCH2),3.88–3.75(m,2H,Ph3PCH2),2.45(s,1H,H-9),2.39–1.35(m,32H,CH and CH2 in pentacyclic skeleton or alkyl chains),1.32(s,3H,CH3),1.14(s,3H,CH3),1.12–1.06(s,9H,3×CH3),0.92(s,3H,CH3),0.69(s,3H,CH3).13C NMR(101MHz,CDCl3)δ206.97,198.90,175.82,170.25,139.09,135.88,134.74,133.32(d,J c,p＝10.0Hz),130.16(d,J c,p＝12.4Hz),129.90,127.94,124.89,117.67(d,J c,p＝86.0Hz),77.05,62.66,58.86,53.00,47.95,45.21,44.60,43.94,43.56,43.03,35.94,31.43,31.02,30.54,29.19,28.30,27.92,26.07,25.90,22.93,22.22,19.14,15.03.
2-((4-trifluoromethylphenyl)methylene)-3,11-dioxo-12-en-30-(5-(triphenylphos-phonio)pentoxycarbonyl)-18β-glycyrrhetinic acid bromide
化合物5h：熔点：148.4–149.7℃；产率：37％；产物状态：白色固体；1HNMR(400MHz,CDCl3)δ7.87–7.60(m,15H,PPh3-H),7.55–7.44(m,4H,Ph-H at C-2),7.39(s,1H,C＝CH atC-2),5.61(s,1H,H-12),4.12–3.96(m,2H,COOCH2),3.81–3.71(m,2H,Ph3PCH2),2.51(s,1H,H-9),2.33–1.41(m,32H,CH and CH2 in pentacyclic skeleton or alkyl chains),1.37(s,3H,CH3),1.18(s,3H,CH3),1.12(s,6H,2×CH3),1.07(s,6H,2×CH3),0.77(s,3H,CH3).13C NMR(101MHz,CDCl3)δ207.26,199.15,176.00,170.75,139.08,136.06,134.75(d,J c,p＝2.6Hz),134.65,133.53,133.37(d,J c,p＝10.2Hz),130.19(d,J c,p＝12.4Hz),128.38,127.99,125.03,124.99,117.89(d,J c,p＝86.1Hz),77.05,63.68,58.96,52.98,48.30,45.21,44.71,43.70,43.18,40.92,37.35,36.00,31.55,31.07,30.73,29.34,28.43,28.09,26.88,26.22,26.04,22.99,22.20,19.26,17.76,15.15.
2-((4-fluorophenyl)methylene)-3,11-dioxo-12-en-30-(4-(triphenylphosphonio)butoxycarbonyl)-18β-glycyrrhetinic acid bromide
化合物5i：熔点：150.2–151.4℃；产率：32％；产物状态：白色固体；1HNMR(400MHz,CDCl3)δ7.91–7.58(m,15H,PPh3-H),7.51–7.32(m,3H,Ph-H at C-2and C＝CH at C-2),7.05–6.83(m,2H,Ph-H at C-2),5.53(s,1H,H-12),4.16–4.04(m,2H,COOCH2),3.83(td,2H,Ph3PCH2,J＝12.4,5.7Hz),2.47(s,1H,H-9),2.26–0.81(m,25H,CH and CH2 inpentacyclic skeleton or alkyl chains),1.33(s,3H,CH3),1.17–1.04(s,12H,4×CH3),0.94(s,3H,CH3),0.70(s,3H,CH3).13C NMR(101MHz,CDCl3)δ207.11,198.95,175.88,170.10,135.54,134.77,134.75,133.36(d,J c,p＝9.9Hz),131.95,131.87,130.19(d,J c,p＝12.5Hz),128.02,117.74(d,J c,p＝85.8Hz),115.21,114.99,77.05,62.77,58.99,52.92,45.05,44.64,44.09,43.60,43.05,40.80,37.37,35.90,31.47,31.09,30.58,29.34,28.33,27.95,26.12,25.95,22.95,22.22,19.21,18.99,17.70,15.05.
2-((4-fluorophenyl)methylene)-3,11-dioxo-12-en-30-(5-(triphenylphosphonio)pentoxycarbonyl)-18β-glycyrrhetinic acid bromide
化合物5j：熔点：134.5–135.8℃；产率：41％；产物状态：白色固体；1HNMR(400MHz,CDCl3)δ7.85–7.59(m,16H,PPh3-H and Ph-H at C-2),7.44–7.40(m,2H,Ph-H at C-2and C＝CH at C-2),7.00–6.93(m,2H,Ph-H at C-2),5.62(s,1H,H-12),4.10–3.97(m,2H,COOCH2),3.90–3.73(m,2H,Ph3PCH2),2.54(s,1H,H-9),2.32–1.45(m,34H,CH and CH2 inpentacyclic skeleton or alkyl chains),1.40(s,3H,CH3),1.19(s,3H,CH3),1.14(s,3H,CH3),1.12(s,3H,CH3),1.10(s,3H,CH3),1.07(s,3H,CH3),0.79(s,3H,CH3).13C NMR(101MHz,CDCl3)δ207.32,199.22,176.06,135.56,134.75(d,J c,p＝2.9Hz),133.48(d,Jc,p＝10.0Hz),132.22,132.13,130.21(d,J c,p＝12.5Hz),128.07,118.00(d,J c,p＝86.0Hz),115.41,115.19,63.79,59.09,52.95,48.48,45.08,44.79,44.44,43.78,43.22,41.04,37.38,35.98,31.62,31.15,30.81,29.53,28.49,28.16,26.31,26.12,23.03,22.25,19.36,17.83,15.16.
2-((4-chlorophenyl)methylene)-3,11-dioxo-12-en-30-(4-(triphenylphosphonio)butoxycarbonyl)-18β-glycyrrhetinic acid bromide
化合物5k：熔点：141.4–143.2℃；产率：33％；产物状态：白色固体；1HNMR(400MHz,CDCl3)δ7.85–7.62(m,15H,PPh3-H),7.41–7.31(m,3H,Ph-H at C-2and C＝CH at C-2),7.21(d,2H,Ph-H,J＝8.3Hz),5.53(s,1H,H-12),4.15–4.08(m,2H,COOCH2),3.95–3.81(m,2H,Ph3PCH2),2.47(s,1H,H-9),2.29–0.84(m,33H,CH and CH2 in pentacyclic skeletonor alkyl chains),1.35(s,3H,CH3),1.16(s,3H,CH3),1.10(s,9H,3×CH3),0.96(s,3H,CH3),0.72(s,3H,CH3).13C NMR(101MHz,CDCl3)δ207.05,198.93,175.91,170.15,135.39,134.77(d,J c,p＝2.3Hz),134.32,134.11,133.87,133.46(d,J c,p＝10.0Hz),131.26,130.23(d,J c,p＝12.5Hz),128.32,117.84(d,J c,p＝85.6Hz),77.05,59.02,53.04,48.07,45.17,44.14,43.66,43.11,40.88,37.41,35.98,31.53,31.15,29.38,28.39,28.03,26.01,23.01,22.29,19.26,19.04,17.76,15.11.
2-((4-chlorophenyl)methylene)-3,11-dioxo-12-en-30-(5-(triphenylphosphonio)pentoxycarbonyl)-18β-glycyrrhetinic acid bromide
化合物5l：熔点：148.0–149.7℃；产率：21％；产物状态：白色固体；1HNMR(400MHz,CDCl3)δ7.84–7.60(m,15H,PPh3-H),7.40–7.32(m,3H,Ph-H at C-2and C＝CH at C-2),7.23(d,2H,Ph-H,J＝8.4Hz),5.61(s,1H,H-12),4.09–3.95(m,2H,COOCH2),3.88–3.64(m,2H,Ph3PCH2),2.53(s,1H,H-9),2.49–0.81(m,38H,CH and CH2 in pentacyclic skeletonor alkyl chains),1.39(s,3H,CH3),1.18(s,3H,CH3),1.12(s,6H,2×CH3),1.09(s,3H,CH3),1.06(s,3H,CH3),0.78(s,3H,CH3).13C NMR(101MHz,CDCl3)δ207.27,199.20,176.04,170.71,135.26,134.77,134.75,134.30,134.03,133.41(d,J c,p＝10.1Hz),131.44,130.20(d,J c,p＝12.5Hz),128.41,128.03,117.91(d,J c,p＝85.6Hz),63.76,59.03,52.91,48.42,45.09,44.75,43.74,43.20,40.99,37.36,35.96,31.58,31.10,30.77,29.46,28.46,28.14,26.26,26.08,23.01,22.22,19.32,17.79,15.16.
2-((4-bromophenyl)methylene)-3,11-dioxo-12-en-30-(4-(triphenylphosphonio)butoxycarbonyl)-18β-glycyrrhetinic acid bromide
化合物5m：熔点：125.0–126.9℃；产率：34％；产物状态：白色固体；1H NMR(400MHz,CDCl3)δ7.85–7.57(m,15H,PPh3-H),7.39–7.20(m,4H,Ph-H at C-2and C＝CH atC-2),7.05–6.88(m,1H,Ph-H at C-2),5.51(s,1H,H-12),4.10(dd,2H,COOCH2,J＝11.4,5.4Hz),3.83–3.73(m,2H,Ph3PCH2),2.45(s,1H,H-9),2.22–0.92(m,28H,CH and CH2 inpentacyclic skeleton or alkyl chains),1.32(s,3H,CH3),1.14(s,3H,CH3),1.10(s,9H,3×CH3),0.94(s,3H,CH3),0.70(s,3H,CH3).13C NMR(101MHz,CDCl3)δ207.06,198.95,178.54,170.19,137.28,134.78,133.54(d,J c,p＝10.2Hz),131.63,130.26(d,J c,p＝12.6Hz),117.50,62.90,48.12,45.24,43.14,40.89,37.44,36.03,31.57,30.69,29.38,28.43,26.24,26.07,23.06,22.34,17.81,15.15.
2-((4-bromophenyl)methylene)-3,11-dioxo-12-en-30-(5-(triphenylphosphonio)pentoxycarbonyl)-18β-glycyrrhetinic acid bromide
化合物5n：熔点：145.6–147.3℃；产率：24％；产物状态：白色固体；1HNMR(400MHz,CDCl3)δ7.86–7.58(m,15H,PPh3-H),7.40–7.25(m,5H,Ph-H at C-2and C＝CH at C-2),5.60(s,1H,H-12),4.06–3.93(m,2H,COOCH2),3.79–3.64(m,2H,Ph3PCH2),2.51(s,1H,H-9),2.29–1.41(m,32H,CH and CH2in pentacyclic skeleton or alkyl chains),1.37(s,3H,CH3),1.16(s,3H,CH3),1.10(s,6H,2×CH3),1.07(s,3H,CH3),1.04(s,3H,CH3),0.76(s,3H,CH3).13C NMR(101MHz,CDCl3)δ207.24,119.15,175.99,170.71,134.76(d,J c,p＝2.9Hz),134.42,134.40,133.34(d,J c,p＝10.0Hz),131.62,131.31,130.18(d,J c,p＝12.6Hz),127.95,122.35,117.81(d,J c,p＝86.1Hz),77.05,63.70,58.98,52.84,48.36,45.05,44.71,43.69,43.15,40.93,31.53,31.04,29.41,28.42,28.07,26.21,26.03,22.97,22.17,19.26,17.75,15.12.
2-((4-Iodophenyl)methylene)-3,11-dioxo-12-en-30-(4-(triphenylphosphonio)butoxycarbonyl)-18β-glycyrrhetinic acid bromide
化合物5o：熔点：142.3–143.9℃；产率：20％；产物状态：白色固体；1HNMR(400MHz,CDCl3)δ7.83–7.61(m,15H,PPh3-H),7.57–7.51(m,2H,Ph-H at C-2),7.30(s,1H,C＝CH atC-2),7.14–7.07(m,2H,Ph-H at C-2),5.50(s,1H,H-12),4.12–4.07(m,2H,COOCH2),3.91–3.75(m,2H,Ph3PCH2),2.45(s,1H,H-9),2.22–1.36(m,30H,CH and CH2 in pentacyclicskeleton or alkyl chains),1.32(s,3H,CH3),1.13(s,3H,CH3),1.09(s,6H,2×CH3),1.05(s,3H,CH3),0.94(s,3H,CH3),0.70(s,3H,CH3).13C NMR(101MHz,CDCl3)δ206.97,198.86,175.82,170.14,137.15,136.83,135.42,135.01,134.75(d,J c,p＝3.0Hz),134.57,133.39(d,J c,p＝10.2Hz),130.18(d,J c,p＝12.5Hz),130.01,127.96,117.71(d,Jc,p＝86.1Hz),62.70,58.93,52.96,48.02,45.11,44.62,44.06,43.59,43.04,40.80,37.34,35.91,31.46,31.06,30.58,29.28,28.33,27.97,26.10,25.94,22.96,22.23,19.18,18.97,17.69,15.04.
2-((4-Iodophenyl)methylene)-3,11-dioxo-12-en-30-(5-(triphenylphosphonio)
pentoxycarbonyl)-18β-glycyrrhetinic acid bromide
化合物5p：熔点：138.8–140.1℃；产率：31％；产物状态：白色固体；1H NMR(400MHz,CDCl3)δ7.80–7.54(m,17H,PPh3-H and Ph-H at C-2),7.28(s,1H,C＝CH at C-2),7.11(d,2H,Ph-H at C-2,J＝8.0Hz),5.58(s,1H,H-12),4.04–3.94(m,2H,COOCH2),3.78–3.66(m,2H,Ph3PCH2),2.49(s,1H,H-9),2.44–0.78(m,34H,CH and CH2 inpentacyclic skeleton or alkyl chains),1.35(s,3H,CH3),1.15(s,3H,CH3),1.12–1.05(s,9H,3×CH3),1.02(s,3H,CH3),0.75(s,3H,CH3).13C NMR(101MHz,CDCl3)δ207.20,199.10,175.94,170.69,137.22,135.24,134.90,134.74(d,J c,p＝2.8Hz),134.58,133.31(d,J c,p＝10.0Hz),131.68,130.16(d,J c,p＝12.5Hz),129.97,127.91,117.76(d,J c,p＝85.9Hz),63.66,58.94,52.78,48.30,45.00,44.66,44.38,43.65,43.11,40.89,37.27,35.87,31.48,30.99,30.68,29.37,28.39,28.05,26.16,25.99,22.95,22.12,19.21,17.70,15.09.
体外细胞毒活性检测：
细胞种类、细胞培养与测试流程：
测试细胞包含3种人类癌细胞和1种人类正常肝细胞，它们分别为人结肠癌细胞HCT-116，人神经母细胞瘤细胞SH-SY5Y、人肝癌细胞HepG2和人体正常肝细胞QSG-7701。测试化合物包括16个α,β-不饱和羰基修饰的18β-GA衍生物以及阳性对照药物阿霉素。
MTT试验用于检测化合物的细胞毒活性。将所有细胞系以3～4×103细胞/孔的密度接种在96孔板上，于37℃和5％的CO2条件下过夜。当细胞贴壁且生长状态良好后，用指定浓度的测试化合物处理细胞48小时，同时设置三个具有相同浓度的平行组。阴性对照组内加入相同浓度的DMSO。随后，向每个孔中加入20μL的MTT(5mg/mL)，继续孵育4小时。抽泵吸取并丢弃上清液，加入150μL的DMSO。接下来通过微板读取器在570nm处测量光密度值(OD)。IC50软件(Prism 5.0)分析细胞存活情况，计算三个独立平行实验的平均值。MTT法细胞毒活性测试结果如表1所示。
表1MTT法细胞毒活性测试结果
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1.IC50值：即半数抑制浓度；在本实验中指当50％的测试细胞被抑制时对应化合物的浓度；所有测试结果均为mean±SD(n＝3)。
2.阿霉素Doxorubicin，阳性对照药物。
本实施例利用MTT法测试了16个18β-GA衍生物对3种人类癌细胞系，分别是人结肠癌细胞HCT-116、人神经母细胞瘤细胞SH-SY5Y和人肝癌细胞HepG2，以及1种人体正常肝细胞QSG-7701的抗增殖活性。结果表明，所有相关的衍生物均显示出较高的细胞毒活性，IC50值的范围为1.52～5.21μM。但所有衍生物的细胞毒活性略逊于或低于阳性对照药物阿霉素。其中，化合物5a的细胞毒活性最佳，对HCT-116细胞的IC50值为1.52μM，可能提示18β-GA的C-2位苯环上的取代基为2-Cl-5-NO2时，对改善衍生物的细胞毒活性有利。
本实施例的结果表明，本发明的衍生物中烷基TPP+侧链的碳原子数量对衍生物的活性有影响在一定的范围内，碳原子数量越多，衍生物的细胞毒活性越高，如化合物5g和5h，以及化合物5i和5j的烷基TPP+侧链中的碳原子数量对细胞毒活性的影响显著。当n＝5时，普遍显示出更低的IC50值。
另外，衍生物C-2位环外的苯环取代基对细胞毒活性的影响。从整体来看，当苯环上具有吸电子取代基时，对化合物的细胞毒活性有利，如5-NO2和4-CF3取代基，化合物5a和5h的IC50值范围分别是1.52～3.46μM和1.58～1.66μM。同时，基团的吸电子能力越强，化合物的细胞毒活性越高，如4位卤素取代基的活性整体低于化合物5a和5h。同样地，4位卤素取代的化合物基本符合该规律，活性大致是4-F>4-Cl>4-Br>4-I。其中4-F取代基的化合物5j活性最佳，IC50值的范围为1.60～2.18μM。
总结
本实施例将具有α，β-不饱和羰基的活性基团引入到18β-GA的结构母核中，设计并合成了16个结构新颖的α,β-不饱和羰基修饰的三苯基膦类18β-GA衍生物。利用MTT法测试了以上衍生物对3种人类癌细胞系，分别是HepG2、HCT-116、SH-SY5Y以及1种人体正常肝细胞QSG-7701的细胞毒活性。结果显示，衍生物5a、5h和5j对HCT-116、SH-SY5Y和HepG2的生长抑制活性更加显著，IC50值范围分别是1.52～3.46μM、1.58～1.66μM和1.60～2.18μM。构效关系显示，18β-GA C-2位引入环外的α，β-不饱和羰基的活性基团可以显著提高抑制活性，另外C-2位苯环上的取代基对衍生物的活性至关重要，即取代基的吸电子作用越强，吸电子基团的数量越多，衍生物的活性越高。
以上所述仅为本发明的优选实施例而已，并不用于限制本发明，对于本领域的技术人员来说，本发明可以有各种更改和变化。凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。

Claims (10)

1.一种α,β-不饱和羰基类18β-甘草次酸衍生物，其特征是，其化学结构式如下所示：
其中，n为4或5，R1为一取代或二取代的苯基，苯基的取代基团为卤素、三氟甲基、甲基和/或硝基。
2.如权利要求1所述的α,β-不饱和羰基类18β-甘草次酸衍生物，其特征是，R1为2-氯-5硝基苯基、4-氟-3-甲基苯基、3-三氟甲基苯基、对三氟甲基苯基、对氟苯基、对氯苯基、对溴苯基或对碘苯基。
3.如权利要求1所述的α,β-不饱和羰基类18β-甘草次酸衍生物，其特征是，R1为2-氯-5硝基苯基、对三氟甲基苯基或对氟苯基。
4.如权利要求1所述的α,β-不饱和羰基类18β-甘草次酸衍生物，其特征是，n为4，R1为2-氯-5硝基苯基；n为5，R1为对三氟甲基苯基或对氟苯基。
5.一种权利要求1所述的α,β-不饱和羰基类18β-甘草次酸衍生物的制备方法，其特征是，以18β-甘草次酸作为原料按照如下反应路线进行反应的步骤；
其中，R1、n如权利要求1所述。
6.如权利要求5所述的α,β-不饱和羰基类18β-甘草次酸衍生物的制备方法，其特征是，18β-甘草次酸与氧化剂进行羟基氧化反应获得化合物2，化合物与R1CHO进行羟醛缩合反应获得化合物3，化合物3与1,4-二溴丁烷或1,5-二溴戊烷进行取代反应获得化合物4，化合物4与三苯基膦进行季膦化反应获得化合物5，即α,β-不饱和羰基类18β-甘草次酸衍生物；
优选地，氧化剂为氯铬酸吡啶鎓盐；
优选地，羟基氧化反应的温度为0℃至室温；
优选地，羟醛缩合反应中，添加氢氧化钾或氢氧化钠，在40～50℃条件下进行反应；
优选地，取代反应在室温条件下进行；
优选地，季膦化反应的温度为75～85℃。
7.一种药物组合物，其特征是，包括权利要求1～4任一所述的α,β-不饱和羰基类18β-甘草次酸衍生物或其药学上可接受的盐。
8.一种药物制剂，其特征是，包括权利要求1～4任一所述的α,β-不饱和羰基类18β-甘草次酸衍生物或权利要求7所述的药物组合物和药学上可接受的辅料。
9.一种权利要求1～4任一所述的α,β-不饱和羰基类18β-甘草次酸衍生物、权利要求7所述的药物组合物或权利要求8所述的药物制剂在制备抗肿瘤药物中的应用。
10.如权利要求9所述的应用，其特征是，肿瘤包括肝癌、结肠癌、神经母细胞瘤。
CN202310589748.9A 2023-05-24 2023-05-24 α,β-不饱和羰基类18β-甘草次酸衍生物及其制备方法与应用 Pending CN116751244A (zh)

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