姜 毅,易春蝶,張丹妮,尹俊林,曾廣智,樊保敏
(1.云南民族大學 民族藥資源化學國家民委-教育部重點實驗室,云南 昆明 650500;2 云南民族大學 化學與環(huán)境學院,云南 昆明 650500)
自進入21世紀以來,癌癥日趨成為人類健康的最大威脅之一,也成為眾多醫(yī)藥學家研究的焦點[1].在早期,以順鉑,環(huán)磷酰胺等為代表的細胞毒性藥物曾經(jīng)是癌癥患者的首選藥物.之后以紫杉醇[2-3]為代表的天然產(chǎn)物藥物也成為了眾多患者的福音.藤黃酸作為從我國傳統(tǒng)中藥藤黃中分離出的有效成分,以其良好的抗癌活性與廣闊的發(fā)展前景受到眾多研究人員的關注.
藤黃酸(gambogic acid,GA)是一種具有獨特橋環(huán)呫噸酮骨架的天然化合物(見圖1),提取自藤黃科植物藤黃樹分泌的樹脂(中藥藤黃)[4].研究表明其具有優(yōu)秀的抗癌活性[5-6],可以通過誘導腫瘤細胞凋亡[7]、抑制腫瘤細胞增殖[8]、誘導分化[9]和抗腫瘤轉(zhuǎn)移[10]等作用達到抗癌的效果.藤黃酸在發(fā)揮抗癌活性的同時,對于造血系統(tǒng)和白細胞的影響較小.為了克服藤黃酸水溶性差,血管刺激強,穩(wěn)定性差等缺點,研究人員對其進行了多種結構修飾.Zhang等[11]的研究表明9-位,10-位的雙鍵是藤黃酸的關鍵活性基團,對于誘導腫瘤細胞凋亡的活性具有重要作用;對30-羧基與6-酚羥基的酯化和酰胺化等改造基本不影響藤黃酸的腫瘤細胞毒活性,在30-位引入某些基團反而會提高活性.Wang等[12]對GA 衍生物的研究中發(fā)現(xiàn),針對C-35,C-39 位甲基修飾,C-32,C-33 和C-37,C-38 雙鍵改造,可以提高GA 的抗腫瘤活性.
氨基酸是構成蛋白質(zhì)的基本單位,而蛋白質(zhì)是人體細胞的重要組成部分,具有構建細胞,調(diào)節(jié)代謝,提供能量等作用.動物體內(nèi)有20種必需氨基酸,根據(jù)其側鏈性質(zhì)的不同,可分為四大類:非極性氨基酸、極性氨基酸、酸性氨基酸和堿性氨基酸.在藥物分子中引入不同種類的氨基酸,可以改變其水溶性和代謝速率等多種特性,同時多個氨基酸相連而成的多肽還具有特異性識別等作用[13].為了得到更具發(fā)展前景的藤黃酸藥物,我們對藤黃酸30位進行酰胺化,通過向其引入不同種類的氨基酸,合成得到了多個藤黃酸衍生物,并對其進行了體外抗腫瘤活性篩選,希望得到具有更好發(fā)展前景的分子.
以藤黃酸為原料,將氨基酸甲酯通過酰胺化法與藤黃酸30位羧酸相連接.其中疏水性氨基酸大多數(shù)直接相連接,親水性氨基酸先通過二氯樹脂保護側鏈再與藤黃酸相連,隨后脫掉保護基以暴露活性側鏈.合成路線見圖2.試劑和反應條件分別為:(a)DMAP,EDCI,CH2Cl2,DIEA,r.t.,35%~57% (b)HBTU,Amino acid-Resin,DIEA,DMF,r.t.;(c)CH2Cl2,CF3COOH,r.t.,28%~49%.
BrukerAvance 400、BrukerAvanceIII HD 600型核磁共振儀(CDCl3或CD4O為溶劑);Agilent 6420型質(zhì)譜儀(ESI-MS);Molecular Devices SpectraMax i3x型多功能酶標儀;Thermo Scientific 3425型二氧化碳培養(yǎng)箱.藤黃酸原料購自瑞芬思生物科技有限公司;氨基酸原料購自薩恩化學技術(上海)有限公司;保護氨基酸購自淘普生物科技有限公司.DMEM培養(yǎng)基、胎牛血清、谷氨酰胺購自Biological Industries公司;三(羥甲基)氨基甲烷購自生工生物工程(上海)股份有限公司;磺酰羅丹明B購自上海阿拉丁生化科技股份有限公司;三氯乙酸購自薩恩化學技術(上海)有限公司;冰乙酸購自天津富寧精細化工有限公司.實驗所用試劑均為分析純.
在反應管中加入 10 mL 的 CH2Cl2,然后加入 50 mg 藤黃酸以及 10 mg 甘氨酸甲酯,38.11 mg(2.5 eq)EDCl,4.8 mg(0.5 eq)DMAP,39.4 mmL (3 eq)DIEA.常溫下攪拌 4 h.用 1N鹽酸水溶液與 10 mL 蒸餾水分別洗滌3次.收集有機相,用無水硫酸鈉干燥并旋干,硅膠柱層析用V(二氯甲烷)∶V(甲醇)=90 ∶10.產(chǎn)物為黃色固體,產(chǎn)率43%.1H NMR (CDCl3,400 MHz)δ∶1.28 (s,3H,19-H),1.30~1.43 (m,3H,24-H),1.45 (s,3H,40-H),1.55 (s,3H,35-H),1.69 (s,3H,29-H),1.74 (s,3H,25-H),1.80 (s,3H,34-H),1.98~2.07 (m,2H,36-H),2.28~2.35 (dd,J=4.48 Hz,J=13.44 Hz,1H,21a-H),2.54 (d,J=9.76 Hz,1H,22-H),2.66~2.75 (m,1H,32-H),2.93(d,J=32 Hz,2H,26-H),3.16~3.24 (m,1H,31b-H),3.27~3.36 (m,1H,31a-H),3.46 ~ 3.51 (m,1H,11-H),3.73 (m,3H,Gly-OMe),3.97 (s,1H,Gly-OMe),3.98 (s,1H,Gly-OMe),5.0~5.08 (m,1H,37-H),6.62 (d,J=12 Hz,1H,4-H),6.77~6.83 (m,1H,27-H),7.56 (d,J=8 Hz,1H,10-H),8.0 (s,1H,NH),12.86 (s,1H,6-OH);13C NMR (CDCl3,100 MHZ)δ:17.6 (C-34),18.1 (C-40),18.4 (C-39),21 (C-31),22.6 (C-36),25.2 (C-21),25.6 (C-35),25.7 (C-39),27.9 (C-19),28.8 (C-24),28.9 (C-25),29.9 (C-26),40.8 (C-Gly),42.2 (C-20),46.9 (C-11),48.9 (C-22),5.23 (C-Gly),58.4 (C-23),83.6 (C-2),84 (C-13),90.9 (C-14),100.39 (C-7),102.7 (C-5),107.7 (C-17),115.7 (C-4),123.6 (C-32),124.8 (C-37),126.3 (C-3),121.9 (C-28),131.8 (C-38),131.9 (C-33),133 (C-9),134 (C-10),135 (C-27),157.4 (C-16),157.6 (C-6),161.6 (C-18),170.2 (C-30),178.9 (C-8),204 (C-12);ESI-MS (m/z):700.1[M+H]+.
操作同2.2,產(chǎn)物為黃色固體,產(chǎn)率61%.1H NMR (CDCl3,400 MHz)δ:1.28 (s,3H,19-H),1.30~1.43(m,3H,24-H),1.30~1.43 (m,3H,Ala-H),1.45 (s,3H,40-H),1.56 (s,3H,35-H),1.64 (s,3H,39-H),1.66 (s,3H,24H),1.69 (s,3H,29-H),1.73 (s,3H,25-H),1.80 (s,3H,34-H),1.99 ~ 2.08 (m,2H,36-H),2.30 (dd,J=5.6 Hz,J=13.2 Hz,1H,21a-H),2.54(d,J=10 Hz,1H,22-H),2.75~2.85(m,1H,32-H),2.93 (d,J=31.6 Hz,2H,26-H),3.12~3.24 (m,1H,31b-H),3.25~3.36 (m,1H,31a-H),3.42~3.52 (m,1H,11-H),3.73 (m,3H,Ala-OMe),4.44~4.54 (m,1H,Ala-OMe),5.0~5.08 (m,1H,37-H),5.0~5.08 (m,1H,3-H),5.43~5.45 (m,1H,32-H),6.07 (t,1H,32-H),6.67 (d,J=12 Hz,1H,4-H),6.64~6.83 (m,1H,27-H),7.56 (d,J=8 Hz,1H,10-H),8.0 (s,1H,NH),12.89 (s,1H,6-OH);13C NMR (CDCl3,100 MHZ)δ:17.6 (C-34),17.7 (C-Ala),18.1 (C-40),21 (C-31),22.6 (C-36),25.6 (C-35),25.7 (C-39),25.2 (C-21),27.9 (C-19),28.8 (C-24),28.9 (C-25),29.9 (C-26),42.2 (C-20),46.9 (C-11),47.8 (C-Ala),48.9 (C-22),5.23 (C-Ala),53.4 (C-23),83.6 (C-2),84 (C-13),90.9 (C-14),100.39 (C-7),102.7 (C-5),107.7 (C-17),115.7 (C-4),123.6 (C-32),124(C-37),126.3 (C-3),121.9 (C-28),131.8 (C-38),131.9 (C-33),133 (C-9),134 (C-10),135 (C-27),157.4 (C-16),157.6 (C-6),161.6 (C-18),173.4 (C-30),178.9 (C-8),204 (C-12);ESI-MS (m/z):714.1[M+H]+.
操作同2.2,產(chǎn)物為黃色固體,產(chǎn)率44%.1H NMR (CDCl3,400 MHz)δ:0.89~0.95 (M,6H,Val-H),1.25 (s,3H,19-H),1.28 (s,3H,24-H),1.44 (s,3H,40-H),1.56 (s,3H,35-H),1.64 (s,3H,39-H),1.66 (s,3H,24H),1.70 (s,3H,29-H),1.74 (s,3H,25-H),1.82 (s,3H,34-H),1.99~2.07 (m,2H,36-H),2.31(dd,J=6 Hz,J=12 Hz,1H,21a-H),2.52~2.55(m,1H,22-H),2.57~2.66 (m,1H,32-H),3.18~3.29 (m,1H,31b-H),3.43~3.51 (m,1H,11-H),3.70(m,3H,Val-OMe),4.40~4.51(m,1H,Ala-OMe),5.02~5.13 (m,1H,37-H),5.02~5.13 (m,1H,3-H),5.43~5.45 (m,1H,32-H),5.45~5.47 (m,1H,4-H),6.67(d,J=12 Hz,1H,4-H),6.64~6.83 (m,1H,27-H),7.56 (d,J=8 Hz,1H,10-H),8.0 (s,1H,NH),12.89 (s,1H,6-OH);13C NMR (CDCl3,100 MHZ)δ:17.6 (C-34),17.7 (C-Ala),18.1 (C-40),21 (C-31),22.6(C-36),25.6(C-35),25.7(C-39),25.2 (C-21),27.9(C-19),28.8(C-24),28.9(C-25),29.9(C-26),42.2(C-20),46.9(C-11),47.8(C-Ala),48.9(C-22),52.3(C-Ala),53.4(C-23),83.6(C-2),84(C-13),90.9(C-14),100.39(C-7),102.7(C-5),107.7(C-17),115.7(C-4),123.6(C-32),124.8(C-37),126.3(C-3),121.9(C-28),131.8(C-38),131.9(C-33),133(C-9),134(C-10),135(C-27),157.4 (C-16),157.6 (C-6),161.6 (C-18),172 (C-30),178.9(C-8),205 (C-12);ESI-MS(m/z):742.1[M+H]+.
操作同2.2,產(chǎn)物為黃色固體,產(chǎn)率38%.1H NMR (CDCl3,400 MHz)δ:0.90 (t,3H,Ile-CH3),1.10~1.26(m,3H,Ile-CH3),1.27(s,3H,19-H),1.30~1.43(m,3H,24-H),1.45 (s,3H,40-H),1.55(s,3H,35-H),1.69 (s,3H,29-H),1.74(s,3H,25-H),1.80(s,3H,34-H),1.98~2.07(m,2H,36-H),2.28~2.34(dd,J=4.0 Hz,J=13.6 Hz,1H,21a-H),2.54(d,J=7.2 Hz,1H,22-H),2.56~2.64(m,1H,32-H),3.23~3.32(m,2H,31-H),3.45~3.48(m,1H,11-H),3.69(s,3H,Ile-OMe),4.50(dd,J=4.4 HzJ=7.6 Hz,1H,Ile-OMe),5.0~5.08(m,1H,3-H),6.67(d,J=10.4 Hz,1H,4-H),6.76(d,J=12Hz,1H,27-H),7.56(d,J=7.2 Hz,1H,10-H);13C NMR(CDCl3,100 MHZ)δ:11.5 (C-Ile),15.7 (C-Ile),17.6(C-34),18.2(C-40),21.2(C-31),22.7(C-36),25.6(C-35),25.7(C-39),25.2(C-21),27.9(C-19),28.8(C-24),28.9(C-25),29.9(C-26),37.4(C-Gly),42.2(C-20),46.9(C-11),48.9(C-22),52.3(C-Ile),56.4(C-23),83.2(C-2),83.8(C-13),90.9(C-14),100.53(C-7),102.7(C-5),107.7(C-17),115.7(C-4),122.1(C-28),123.6(C-32),124.8(C-37),125.4(C-3),131.7(C-38),131.9(C-33),132.7(C-9),134.2(C-10),136.2(C-27),157.4(C-16),157.6(C-6),161.6(C-18),172(C-30),178.9(C-8),205.5(C-12);ESI-MS m/z:756.[M+H]+
操作同2.2,產(chǎn)物為黃色固體,產(chǎn)率29%.1H NMR (CDCl3,400 MHz)δ:1.32~1.36 (m,3H,24-H),1.38~1.44 (m,3H,40-H),1.55 (s,3H,35-H),1.69 (s,3H,29-H),1.66~1.72 (m,3H,25-H),1.74 (s,3H,34-H),2.01~2.04 (m,2H,36-H),2.26~2.40 (m,1H,21a-H),2.57~2.69 (m,1H,22-H),2.66~2.75 (m,1H,32-H),3.07~3.27 (m,2H,31-H),3.49 (s,3H,Phe-OMe),3.7~3.76 (m,2H,Phe-CH2),3.98 (s,1H,Phe-OMe),5.0~5.08 (m,1H,37-H),6.62~6.69 (m,1H,4-H),6.77~6.83 (m,1H,27-H),7.04~7.24 (m,5H,Phe),7.49~7.58 (m,1H,10-H),8.0 (s,1H,NH),12.7~12.8 (m,1H,6-OH);13C NMR (CDCl3,100 MHZ)δ:17.6 (C-34),18.1 (C-40),20.8 (C-31),22.7 (C-36),25.6 (C-35),25.7 (C-39),27.2 (C-19),28.4 (C-24),29.3 (C-25),29.8 (C-26),39.7 (C-Phe),41.7 (C-20),46.8 (C-11),49.0 (C-22),51.2 (C-Phe),52.5 (C-Phe),55.3 (C-23),82 (C-2),88.9 (C-14),108.6 (C-17),115.7 (C-4),123.8 (C-32),127.3 (C-Phe),128.9 (C-37),129.3 (C-Phe),132 (C-33),136 (C-27),155.8 (C-16),157.5 (C-6),161.3 (C-18),173.7 (C-30);ESI-MS (m/z):790.2[M+H]+.
操作同2.2,產(chǎn)物為黃色固體,產(chǎn)率32%.1H NMR (CDCl3,600 MHz)δ:1.27(s,3H,19-H),1.34 (s,3H,24-H),1.45(s,3H,40-H),1.54(s,3H,35-H),1.58(s,3H,29-H),1.65(s,3H,25-H),1.90~1.98(m,2H,36-H),2.23~2.28(dd,J=5.4 Hz,J=12 Hz,1H,21a-H),2.51 (d,J=9.24 Hz,1H,22-H),2.73~2.81 (m,1H,32-H),3.10~3.16(m,1H,31b-H),3.23~3.29(m,1H,31a-H),3.59~3.66 (m,1H,Ser-H),3.74(s,3H,Ser-H),3.79(dd,1H,J=12 Hz,J=3 Hz,Ser-OMe),3.84~3.80 (m,1H,Ser-OMe),5.4(d,J=12 Hz,1H,4-H),6.87~5.92(m,1H,27-H),7.47(d,J=6.6 Hz,1H,10-H);13C NMR (CDCl3,150 MHZ)δ:17.4(C-34),17.9(C-40),20.7(C-39),22.4(C-31),22.6(C-36),24.7(C-35),25.3(C-21),25.5(C-39),27.1(C-19),28.8(C-24),29.2(C-25),29.5(C-26),42.1(C-20),46.7(C-11),49.1(C-22),52.2(C-Ser),59.9(C-23),83.4(C-2),83.8(C-13),91.1(C-14),100.2(C-7),102.6(C-5),107.7(C-17),115.7(C-4),123.5(C-32),124.7(C-37),126.43(C-3),131.6 (C-38),131.8(C-33),132.8(C-9),133.9(C-10),135.4(C-27),157.4(C-16),161.5(C-18),178.7(C-8);ESI-MS (m/z):730.1[M+H]+.
操作同2.2,產(chǎn)物為黃色固體,產(chǎn)率36%.1H NMR(CDCl3,400 MHz)δ:1.29(s,3H,19-H),1.34(s,3H,24-H),1.37~1.44(s,3H,40-H),1.54(s,3H,35-H),1.62(s,3H,39-H),1.65(s,3H,24H),1.71(s,3H,29-H),1.73(s,3H,25-H),1.86(s,3H,34-H),1.99~2.06(m,2H,36-H),2.31(dd,J=4 Hz,J=14.8 Hz,1H,21a-H),2.54(d,J=9.6 Hz,1H,22-H),2.77~2.86(m,1H,32-H),2.87~2.95(m,2H,26-H),3.12~3.24(m,1H,31b-H),3.25~3.36(m,1H,31a-H),3.43~3.47(m,1H,11-H),3,49(s,2H,Tyr-OMe),3.73(m,3H,Tyr-OMe),4.98~5.12 (m,1H,37-H),4.98~5.12(m,1H,3-H),6.33(t,1H,32-H),6.61~6.66(m,1H,4-H),6.75~6.87(m,2H,Tyr-OMe),6.92~7.07(m,1H,27-H),7.11~7.22(m,2H,Tyr-OMe),7.51(d,J=7.6 Hz,1H,10-H),8.0(s,1H,NH),12.83(s,1H,6-OH);13C NMR (CDCl3,100 MHZ)δ:17.6 (C-34),18.1(C-40),21(C-31),22.6 (C-36),25.6(C-35),25.7(C-39),25.2(C-21),27.9(C-19),28.8(C-24),28.9(C-25),29.9(C-26),42.0(C-20),46.8(C-11),49.0(C-22),50.9(C-Tyr),52.1(C-Tyr),55.8(C-23),83.4(C-2),83.8(C-13),91.1(C-14),100.46(C-7),102.6 (C-5),107.7 (C-17),115.9(C-4),121.6(C-32),122.2(C-Tyr),124.5(C-37),130(C-Tyr),131.6(C-38),131.8(C-33),133.4(C-9),134.4(C-10),135.4(C-27),149.5(C-Tyr),157.5(C-16),157.6(C-6),161.6(C-18),175.2(C-30),178.9(C-8),203.6(C-12);ESI-MS(m/z):806.1[M+H]+.
操作同2.2,產(chǎn)物為黃色固體,產(chǎn)率55%.1H NMR (CDCl3,400 MHz)δ:1.26(s,3H,19-H),1.28~1.38(m,3H,24-H),1.42(s,3H,40-H),1.55(s,3H,35-H),1.62(s,3H,29-H),1.65(s,3H,25-H),1.72(s,3H,34-H),1.97~2.06(m,2H,36-H),2.17~2.29(m,1H,21a-H),2.39~2.50(m,1H,22-H),2.60~2.71(m,1H,32-H),3.14~3.24(m,1H,31b-H),3.24~3.33(m,1H,31a-H),3.14-3.33(m.2H,Trp-OMe),3.49(s,1H,11-H),3.64(s,3H,Trp-OMe),5.01~5.08(m,1H,37-H),6.66(d,J=10 Hz,1H,4-H),6.77~6.95(m,1H,27-H),7.0~1.5(m,5H,Trp-OMe),7.5~7.61(m,1H,10-H),8.0(s,1H,NH),12.86(s,1H,6-OH);13C NMR(CDCl3,100 MHZ)δ:17.6(C-34),18.2(C-40),18.3(C-39),21(C-31),22.8(C-36),25.8(C-35),25.2(C-21),27.9(C-19),28.8(C-24),28.9(C-25),29.9(C-26),42.2(C-20),46.9(C-11),48.9(C-22),5.23(C-Trp),58.4(C-23),83.6(C-2),84(C-13),91(C-14),100.7(C-7),102.9(C-5),107.8(C-17),111.3(C-Trp),116.0(C-4),118.8(C-Trp),119.5(C-Trp),121.9(C-28),122.3(C-Trp),122.9(C-Trp),123.9(C-32),124.8(C-37),126.6(C-3),127.6(C-Trp),131.8(C-38),131.9(C-33),132.7(C-9),133.7(C-10),136.2(C-27),157.4(C-16),157.6(C-6),161.6(C-18),172.2(C-30),204.5(C-12);ESI-MS(m/z):829.2[M+H]+.
取 0.25 g 二氯樹脂,加入 2 mL CH2Cl2浸泡 0.5 h 進行活化,過濾并用 2 mL DMF 洗滌3次.加入 100 mg保護氨基酸Fmoc-Lys(Boc)-OH與 0.5 mL DIEA,加入 2 mL DMF 作為溶劑常溫反應 2 h,過濾收集固體,之后加入 0.5 mL 哌啶與 1.5 mL DMF 反應 0.5 h,再次過濾,洗滌并收集固體,加入 50 mg 藤黃酸,40 mg HBTU,0.5 mL DIEA,常溫反應 2 h,過濾后向固體中加入含25% 三氟乙酸的DCM,常溫反應 4 h,過濾收集濾液,濃縮后葡聚糖凝膠柱層析分離產(chǎn)物,得到黃色固體,產(chǎn)率49%.1H NMR(CDCl3,400 MHz)δ:1.28(s,3H,19-H),1.30~1.43(m,3H,24-H),1.44(s,3H,40-H),1.54(s,3H,35-H),1.56(s,2H,Lys-NH2),1.57(s,2H,Lys),1.60(s,3H,39-H),1.62~1.64 (m,3H,24H),1.67(s,3H,29-H),1.68(s,3H,25-H),1.73~1.82(m,2H,36-H),2.26~2.27(m,1H,21a-H),2.50~2.55(m,1H,22-H),2.50~2.55(m,2H,Lys),2.79~2.83(m,1H,32-H),2.89~2.95(m,2H,26-H),3.13~3.19(m,2H,31-H),3.35~3.38(m,1H,11-H),4.96~5.04(m,1H,37-H),4.96~5.04(m,1H,3-H),6.55(dd,J=4.2 Hz,J=10.8 1H,4-H),7.46~7.49(m,1H,10-H);13C NMR(CDCl3,150 MHZ)δ:17.0(C-34),19.7(C-40),21.4(C-31),21.6(C-36),22.4(Lys-C),24.4(C-39),24.6(C-21),27.4(C-19),28.7(C-24),28.9(C-25),29.1(Lys),32.7(Lys-C1),41.8(C-20),48.8(C-22),83.3(C-2),83.8(C-13),90.9(C-14),100.2(C-7),102.4(C-5),107.4(C-17),115.4(C-4),123.6(C-32),124.7(C-37),131.8(C-38),131.3(C-33),133(C-9),136.4 (C-27),157.2(C-16),157.7(C-6),161.5(C-18),171.9(C-30),179.6(C-8);ESI-MS(m/z):757.1[M+H]+.
操作同2.10,產(chǎn)物為黃色固體,產(chǎn)率28%.1H NMR(CDCl3,600 MHz)δ:1.33(s,3H,19-H),1.30~1.43(m,3H,24-H),1.44(s,3H,40-H),1.53(s,3H,35-H),1.57(s,3H,29-H),1.64(s,3H,25-H),1.69(s,3H,34-H),1.88~1.96(m,2H,36-H),2.22~2.26(dd,J=4.2 Hz,J=13.2 Hz,1H,21a-H),2.47~2.51(m,1H,22-H),2.72~2.78(m,1H,32-H),2.83~2.93 (m,2H,26-H),3.07~3.12(m,1H,His-C1),3.22~3.28(m,1H,31b-H),3.33~3.38 (m,1H,31a-H),3.42~3.47(m,1H,11-H),3.90~3.96(m,1H,His-C),4.93~4.99(m,1H,37-H),5.39(d,J=10.8 Hz,1H,32-H),5.89(t,1H,32-H),6.52(d,J=10 Hz,1H,4-H),7.42~7.47(m,1H,10-H),7.79(s,1H,NH),8.61~8.71(m,1H,His-C),12.86(s,1H,6-OH);13C NMR (CDCl3,150 MHZ)δ:17.6(C-34),18.1(C-40),20.9(C-31),22.8(C-36),25.2(C-21),25.7(C-39),27.8(C-19),28.9(C-25),29.9(C-26),42.2(C-20),46.9(C-11),48.9(C-22),83.6 (C-2),84(C-13),90.9(C-14),102.7(C-5),107.7(C-17),115.7(C-4),123.6(C-32),124.8(C-37),125.3(C-3),121.9(C-28),131.6(C-38),131.9(C-33),133.3(C-9),134.6(C-10),135.4(C-27),137.4(C-His),157.4(C-16),157.5(C-6),161.1(C-18),168.2(C-30),179.2(C-8),203.7(C-12);ESI-MS (m/z):766.1 [M+H]+.
用sulfurhodamine B(SRB)法檢測合成所得化合物對4種人源腫瘤細胞株(人肺癌細胞A549,人黑色素瘤細胞 A375,人肝癌細胞 HepG2,人胃癌細胞 BGC823)的體外抗腫瘤活性,并以藤黃酸為對照化合物.結果表明,所有修飾后的衍生物針對不同的細胞系表現(xiàn)出了不同的抗癌活性,并且在針對于特定細胞系時,抗癌活性均獲得了保持(表1).
表1 不同化合物對4種腫瘤細胞株的半數(shù)抑制濃度(IC50) (μmol·L-1)
1)細胞培養(yǎng) A549、A375、BGC823和HeGP2 細胞使用含10%胎牛血清的DMEM培養(yǎng)基,培養(yǎng)在37 ℃,含5%CO2的細胞培養(yǎng)箱內(nèi).
2)細胞細胞毒性(SRB)實驗 細胞鋪入96孔板,分別設定空白對照組、陰性對照組、陽性對照組和實驗組,每個樣品設置至少3個復孔.培養(yǎng) 24 h后,分別加入梯度稀釋的樣品,繼續(xù)培養(yǎng) 48 h,之后加入50% 三氯乙酸溶液固定 1 h.棄固定液、洗板、晾干之后加入含1%醋酸的0.4% SRB染色液.洗板、晾干之后加入10 mmol/L Tris溶液溶解染料,在 515 nm 波長下檢測其吸光度并計算樣品對細胞生長的抑制作用.
20種常用氨基酸中選擇了10種有代表性的氨基酸來進行反應,分別包括非極性氨基酸,水溶性性氨基酸和堿性氨基酸.在使用含硫氨基酸進行反應時發(fā)現(xiàn)其產(chǎn)物并不穩(wěn)定,所以沒有對含硫氨基酸的衍生物進行檢測.同時在合成含胍基的精氨酸-藤黃酸衍生物與酸性氨基酸-藤黃酸衍生物過程中,產(chǎn)生了大量的副產(chǎn)物.故我們沒有對這兩類氨基酸衍生物進行合成與研究,只選取了10種氨基酸進行反應.
在合成極性氨基酸衍生物11、12時借助了多肽合成思路,利用二氯樹脂作為氨基酸C端的保護基減少了副產(chǎn)物,同時得到具有暴露的極性基團的產(chǎn)物.由于中間產(chǎn)物全部連接在樹脂上,只需過濾即可去除雜質(zhì),也可以方便地使用茚三酮顯色反應來監(jiān)測反應進程.使用硅膠柱層析對非極性氨基酸衍生物進行純化,在純化11、12、13時,發(fā)現(xiàn)較為嚴重的吸附與拖尾,產(chǎn)品損失過大同時難以得到純品,所以改用葡聚糖凝膠柱,利用產(chǎn)物與雜質(zhì)分子質(zhì)量的不同進行分離,去除了吸附的效果,得到了純度較好的產(chǎn)物.
如表1所示,在合成的多種藤黃酸-氨基酸衍生物中,發(fā)現(xiàn)含有非極性側鏈的衍生物活性較好,如化合物2、3、4,它們顯示出比藤黃酸對照品更低的半數(shù)抑制濃度,表明合成的這幾種衍生物具有更強的體外抗癌活性.同時較小的側鏈對生物活性保持更有利,推測原因可能是由于非極性側鏈與細胞融合更好,更易透過細胞膜,同時也減緩其代謝速率,保證其作用時間.含有共軛基團與極性基團的衍生物活性降低,如7~12,其針對幾種癌細胞的體外細胞毒活性較之藤黃酸對照品,均有不同程度的下降.此外,修飾后的衍生物對于不同的細胞,其活性表現(xiàn)出了明顯的差異性,在針對A375與HepG2 2種細胞株的活性測試中,衍生物均體現(xiàn)了較好的生物活性,而針對BGC823細胞株的活性則有下降.而在針對A549細胞株的測試中,具有極性基團的衍生物普遍活性下降,其IC50大多提高10倍以上,這表明這一類衍生物針對A549細胞株的細胞毒活性幾乎完全喪失.藤黃酸衍生物在針對不同細胞系表現(xiàn)出不同作用強度的現(xiàn)象值得我們繼續(xù)深入研究.