新型去氫表雄酮C3酯類衍生物的合成及體外抗腫瘤活性評價

劉榮春, 任 博, 郭 聰, 湯江江

(西北農(nóng)林科技大學(xué) 化學(xué)與藥學(xué)院，陜西 楊凌 712100)

甾體是一類天然化合物，具有不同的生物學(xué)特性[1-4]，并且對各種疾病都有一定的治療作用，包括：腎上腺功能不全[5]、自身免疫性疾病[6]、心血管疾病[7]、真菌和微生物感染[8]。此外，許多不同的甾體衍生物均被報道具有抗癌作用[9-12]。去氫表雄酮(DHEA)也稱為脫氫表雄酮，是人類身上最豐富的循環(huán)類固醇激素[13]，DHEA及其硫酸酯還具有多種潛在的生物效應(yīng)，如抗衰老作用[14-15]、抗炎[16-17]、免疫調(diào)節(jié)[18]、抗病毒[19]、抗抑郁藥[20]和抗癌作用[21]。

Scheme 1

眾所周知，具有共軛體系和負(fù)電子特性的芳環(huán)被廣泛用于先導(dǎo)化合物的結(jié)構(gòu)修飾，它可以與目標(biāo)蛋白發(fā)生π-π相互作用并增加與目標(biāo)的親和力。研究發(fā)現(xiàn)芳香環(huán)中不同類型的取代基與電荷分布、化合物的性質(zhì)和抗腫瘤效力密切相關(guān)[22-23]，尤其是鹵素基團(tuán)，據(jù)報道它們顯著增強(qiáng)了生物活性并改善母體的代謝穩(wěn)定性[24-25]。Vosooghi等[26]已經(jīng)證明在去氫表雄酮的C16位置進(jìn)行修飾可以增強(qiáng)其抗腫瘤活性，Garrido等[27]研究表明，在去氫表雄酮的C3位置酯化后，可改進(jìn)其親脂性并表現(xiàn)出更強(qiáng)的穿透細(xì)胞膜能力，從而顯示出更強(qiáng)的生物學(xué)效應(yīng)。

基于此，合成了22個新的去氫表雄酮酯類衍生物，包括11個去氫表雄酮C3的酯類衍生物和11個(E)-16-(2-氯亞芐基)的C3酯類衍生物。進(jìn)一步研究的化合物的體外抗腫瘤活性，并發(fā)現(xiàn)一些具有更好抗腫瘤活性的甾體化合物，為新型甾體抗腫瘤藥物的研發(fā)提供參考。

1 實(shí)驗(yàn)部分

1.1 儀器與試劑

X-4型熔點(diǎn)儀；Bruker AV-400/500 MHz型超導(dǎo)核磁共振儀(TMS為內(nèi)標(biāo)); LCQ Advantage MAX 型質(zhì)譜儀。

所用溶劑均為分析純。

1.2 合成

(1)3的合成

依次稱取12.0 g(6.94 mmol)，二氯苯甲醛1.1 g(7.64 mmol)和NaOH 3.5 g(87.5 mmol)溶解于40 mL甲醇中，室溫?cái)嚢? h(TLC檢測)。將反應(yīng)混合物倒入冰水中，過濾最終的沉淀物。用冷水洗滌，減壓干燥，并在甲醇中結(jié)晶得白色固體3。

(2)2a～2k,4a～2k的合成

依次稱取10.29 g(1 mmol)、 EDCI 0.58 g(3 mmol)、 DMAP 0.06 g(0.5 mmol)和Et3N 0.3 g(3 mmol)溶解于5 mL無水二氯甲烷后，再加入相應(yīng)的酸(2 mmol)，室溫?cái)嚢? h(TLC檢測)。減壓除去溶劑，殘留物經(jīng)硅膠柱層析(洗脫劑：石油醚/乙酸乙酯=20/1,V/V)純化得相應(yīng)的酯2a～2l；使用3作為試劑，通過相同的方法制備4a～4k。

2a: 白色固體，收率90%, m.p.200～202 ℃;1H NMR(500 MHz, CDCl3)δ: 7.67(d,J=16.0 Hz, 1H), 7.55～7.49(m, 2H), 7.38(s, 3H), 6.42(d,J=16.0 Hz, 1H), 5.48～5.40(m, 1H), 4.75(dt,J=11.1 Hz, 6.1 Hz, 1H), 2.51～2.36(m, 3H), 2.09(dt,J=19.0 Hz, 9.1 Hz, 2H), 2.00～1.83(m, 4H), 1.66(m, 5H), 1.58～1.44(m, 2H), 1.35～1.25(m, 2H), 1.24～1.17(m, 1H), 1.08(s, 3H), 0.89(s, 3H);13C NMR(101 MHz, CDCl3)δ: 221.25, 166.50, 144.64, 140.04, 134.56, 130.33, 128.99, 128.16, 122.07, 118.67, 73.94, 51.80, 50.24, 47.65, 38.30, 37.08, 36.88, 35.98, 31.58, 31.51, 30.91, 27.93, 22.01, 20.45, 19.51, 13.68; ESI-MSm/z: Calcd for C28H34O3{[2M+Na]+} 859.50, found 859.01。

2b: 白色固體，收率90%, m.p.215～217 ℃;1H NMR(500 MHz, CDCl3)δ: 7.65(d,J=16.4 Hz, 1H), 7.42(d,J=8.0 Hz, 2H), 7.19(d,J=7.9 Hz, 2H), 6.38(d,J=16.0 Hz, 1H), 5.43(s, 1H), 4.74(dq,J=10.9 Hz, 5.6 Hz, 5.1 Hz, 1H), 2.51～2.41(m, 2H), 2.37(s, 3H), 2.15～2.06(m, 2H), 1.94(m,3H), 1.88～1.83(m, 1H), 1.68 m, 5H), 1.58～1.45(m, 2H), 1.35～1.28(m, 2H), 1.27～1.17(m, 2H), 1.08(s, 3H), 0.89(s, 3H);13C NMR(125 MHz, CDCl3)δ: 166.69, 144.65, 140.73, 140.12, 131.87, 129.73, 128.16, 122.03, 117.61, 73.84, 51.84, 50.29, 47.66, 38.34, 37.11, 36.91, 35.99, 31.62, 31.55, 30.93, 27.96, 22.02, 21.60, 20.47, 19.52, 13.69; ESI-MSm/z: Calcd for C29H36O3{[2M+Na]+} 887.54, found 887.78。

2c: 白色固體，收率50%, m.p.203～205 ℃;1H NMR(500 MHz, CDCl3)δ: 7.62(d,J=15.9 Hz, 1H), 7.47(d,J=8.7 Hz, 2H), 6.89(d,J=8.7 Hz, 2H), 6.29(d,J=15.9 Hz, 1H), 5.43(d,J=4.9 Hz, 1H), 4.73(tt,J=10.9 Hz, 4.9 Hz, 1H), 3.83(s, 3H), 2.44(m, 3H), 2.14～2.03(m, 2H), 1.93(m, 3H), 1.87～1.82(m, 1H), 1.71～1.63(m, 5H), 1.58～1.45(m, 2H), 1.34～1.15(m, 4H), 1.07(s, 3H), 0.88(s, 3H);13C NMR(125 MHz, CDCl3)δ: 166.83, 161.45, 144.31, 140.15, 129.80, 127.35, 122.00, 116.19, 114.44, 73.74, 55.49, 51.83, 50.29, 47.65, 38.35, 37.11, 36.90, 35.97, 31.61, 31.54, 30.92, 27.97, 22.01, 20.46, 19.50, 13.67; ESI-MSm/z: Calcd for C29H36O4{[2M+Na]+} 919.52, found 919.75。

2d: 白色固體，收率85%, m.p.158～160 ℃;1H NMR(500 MHz, CDCl3)δ: 7.58(d,J=16.0 Hz, 1H), 7.34～7.29(m, 1H), 7.24(d,J=7.1 Hz, 1H), 7.18(d,J=9.6 Hz, 1H), 7.07～7.01(m, 1H), 6.38(d,J=16.0 Hz, 1H), 5.41(d,J=5.0 Hz, 1H), 4.76～4.67(m, 1H), 2.47～2.33(m, 3H), 2.13～2.01(m, 2H), 1.96～1.80(m, 4H), 1.65(m 5H), 1.56～1.42(m, 2H), 1.27(m, 2H), 1.16(m, 1H), 1.04(s, 3H), 0.86(s, 3H);13C NMR(125 MHz, CDCl3)δ: 166.01, 162.05, 143.11, 139.91, 136.75, 130.48, 124.07, 122.05, 120.06, 117.17, 114.37, 74.07, 51.74, 50.19, 47.56, 38.18, 36.98, 36.80, 35.87, 31.51, 31.45, 30.83, 27.82, 21.91, 20.37, 19.40, 13.58; ESI-MSm/z: Calcd for C28H33O3F {[2M+Na]+} 895.52, found 895.39。

2e: 白色固體，收率85%, m.p.208～210 ℃;1H NMR(500 MHz, CDCl3)δ: 7.63(d,J=16.0 Hz, 1H), 7.53～7.47(m, 3H), 7.07(t,J=8.3 Hz, 2H), 6.34(d,J=16.0 Hz, 1H), 5.44(s, 1H), 4.74(dt,J=10.9 Hz, 5.8 Hz, 1H), 2.44(m, 3H), 2.09(dt,J=18.9 Hz, 9.1 Hz, 2H), 1.99～1.83(m, 4H), 1.66(m, 5H), 1.51(m, 2H), 1.30(m, 2H), 1.25～1.16(m, 1H), 1.07(s, 3H), 0.89(s, 3H);13C NMR(125 MHz, CDCl3)δ: 166.36, 163.00, 143.32, 140.09, 130.92, 130.05, 129.98, 122.11, 118.55, 116.24, 116.07, 74.04, 51.88, 50.34, 47.66, 38.34, 37.12, 36.92, 35.97, 31.65, 31.58, 30.94, 27.96, 22.02, 20.49, 19.51, 13.69; ESI-MSm/z: Calcd for C28H33O3F {[2M+Na]+} 895.48, found 895.82。

2f: 白色固體，收率82%, m.p.158～160 ℃;1H NMR(500 MHz, CDCl3)δ: 7.70～7.59(m, 5H), 6.49(d,J=16.0 Hz, 1H), 5.48～5.40(m, 1H), 4.76(dt,J=10.8 Hz, 5.6 Hz, 1H), 2.50～2.37(m, 3H), 2.09(dt,J=18.9 Hz, 9.1 Hz, 2H), 1.99～1.82(m, 4H), 1.73～1.62(m, 4H), 1.59～1.45(m, 2H), 1.25(m, 3H), 1.08(s, 3H), 0.89(s, 3H);13C NMR(125 MHz, CDCl3)δ: 165.90, 142.74, 139.99, 138.04, 131.98, 128.28, 126.00, 125.97, 125.05, 122.22, 121.38, 74.35, 51.88, 50.34, 47.66, 38.30, 37.10, 36.92, 35.97, 31.65, 31.58, 30.95, 27.94, 22.02, 20.49, 19.50, 13.69; ESI-MSm/z: Calcd for C29H33O3F3{[2M+Na]+}995.48, found 995.17。

2g: 白色固體，收率85%, m.p.182～184 ℃;1H NMR(500 MHz, CDCl3)δ: 8.07(d,J=16.0 Hz, 1H), 7.61(dd,J=7.5 Hz, 1.5 Hz, 1H), 7.43～7.39(m, 1H), 7.32～7.25(m, 2H), 6.42(d,J=16.0 Hz, 1H), 5.44(d,J=4.8 Hz, 1H), 4.76(tt,J=10.9 Hz, 5.0 Hz, 1H), 2.45(m, 3H), 2.09(dt,J=19.1 Hz, 9.0 Hz, 2H), 2.00～1.89(m, 3H), 1.86(d,J=12.8 Hz, 1H), 1.67(m, 5H), 1.51(m, 2H), 1.30(m, 2H), 1.21(m, 1H), 1.08(s, 3H), 0.89(s, 3H);13C NMR(125 MHz, CDCl3)δ: 166.11, 140.53, 140.15, 135.15, 133.02, 131.19, 130.40, 127.86, 127.29, 122.22, 121.51, 74.31, 51.95, 50.39, 47.76, 38.38, 37.19, 37.01, 36.08, 31.72, 31.65, 31.03, 28.02, 22.12, 20.57, 19.62, 13.79; ESI-MSm/z: Calcd for C28H33O3Cl{[2M+Na]+} 927.42, found 927.83。

2h: 白色固體，收率85%, m.p.173～175 ℃;1H NMR(500 MHz, CDCl3)δ: 7.59(d,J=16.0 Hz, 1H), 7.50(s, 1H), 7.38(d,J=7.2 Hz, 1H), 7.36～7.29(m, 2H), 6.41(d,J=16.0 Hz, 1H), 5.43(d,J=5.0 Hz, 1H), 4.74(tt,J=10.6 Hz, 5.0 Hz, 1H), 2.44(m, 3H), 2.08(m, 2H), 1.99～1.83(m, 4H), 1.72～1.62(m, 5H), 1.59～1.44(m, 2H), 1.29(m, 2H), 1.25～1.15(m, 1H), 1.07(s, 3H), 0.89(s, 3H);13C NMR(125 MHz, CDCl3)δ: 166.06, 142.99, 139.99, 136.45, 135.03, 130.25, 130.18, 127.90, 126.33, 122.15, 120.23, 74.18, 51.84, 50.29, 47.65, 38.28, 37.08, 36.89, 35.97, 31.61, 31.54, 30.92, 27.91, 22.01, 20.46, 19.50, 13.68; ESI-MSm/z: Calcd for C28H33O3Cl{[2M+Na]+}927.42, found 927.91。

2i: 白色固體，收率85%, m.p.185～187 ℃;1H NMR(500 MHz, CDCl3)δ: 7.62(d,J=16.0 Hz, 1H), 7.45(d,J=8.5 Hz, 2H), 7.36(d,J=8.5 Hz, 2H), 6.39(d,J=16.0 Hz, 1H), 5.44(d,J=5.0 Hz, 1H), 4.79～4.71(m, 1H), 2.50～2.37(m, 3H), 2.10(dt,J=19.0 Hz, 9.1 Hz, 2H), 1.94(m, 3H), 1.86(m, 1H), 1.73～1.63(m, 5H), 1.60～1.53(m, 1H), 1.49(m, 1H), 1.31(m, 2H), 1.25～1.16(m, 1H), 1.08(s, 3H), 0.89(s, 3H);13C NMR(125 MHz, CDCl3)δ: 166.49, 143.43, 140.27, 136.48, 133.37, 129.57, 129.54, 122.39, 119.59, 74.36, 52.09, 50.54, 47.91, 38.55, 37.34, 37.15, 36.22, 31.86, 31.80, 31.18, 28.18, 22.27, 20.72, 19.75, 13.93; ESI-MSm/z: Calcd for C28H33O3Cl{ [2M+Na]+}927.42, found 927.54。

2j: 白色固體，收率83%, m.p.191～193 ℃;1H NMR(500 MHz, CDCl3)δ: 7.59(d,J=16.0 Hz, 1H), 7.51(d,J=8.4 Hz, 2H), 7.38(d,J=8.4 Hz,2H), 6.40(d,J=16.0 Hz, 1H), 5.43(d,J=5.0 Hz, 1H), 4.74(tt,J=10.6 Hz, 5.0 Hz, 1H), 2.50～2.35(m, 3H), 2.09(dt,J=19.1 Hz, 9.1 Hz, 2H), 1.99～1.82(m, 4H), 1.72～1.64(m, 4H), 1.52(m, 2H), 1.35～1.23(m, 3H), 1.23～1.15(m, 1H), 1.07(s, 3H), 0.89(s, 3H);13C NMR(125 MHz, CDCl3)δ: 166.22, 143.24, 140.01, 133.54, 132.26(2C), 129.54(2C), 124.57, 122.14, 119.45, 74.13, 51.84, 50.29, 47.65, 38.30, 37.09, 36.90, 35.97, 31.61, 31.55, 30.93, 27.93, 22.02, 20.47, 19.50, 13.68; ESI-MSm/z: Calcd for C28H33O3Br{ [2M+Na]+}1015.32, found 1015.56。

2k: 白色固體，收率70%, m.p.149～151 ℃;1H NMR(500 MHz, CDCl3)δ: 7.31～7.26(m, 2H), 7.20(d,J=6.6 Hz, 3H), 5.40(d,J=5.1 Hz, 1H), 4.61(tt,J=10.9 Hz, 5.3 Hz, 1H), 2.95(t,J=7.8 Hz, 2H), 2.61(t,J=7.8 Hz, 2H), 2.46(dd,J=19.3 Hz, 8.7 Hz, 1H), 2.35～2.24(m, 2H), 2.14～2.04(m, 2H), 1.95(m, 1H), 1.89～1.80(m, 3H), 1.70～1.59(m, 4H), 1.55(m, 2H), 1.48(m, 1H), 1.29(m, 3H), 1.18～1.10(m, 1H), 1.04(s, 3H), 0.88(s, 3H);13C NMR(125 MHz, CDCl3)δ: 172.44, 140.69, 140.06, 128.57(2C), 128.45(2C), 126.34, 121.99, 73.89, 51.84, 50.27, 47.65, 38.18, 37.06, 36.87, 36.34, 35.98, 31.61, 31.55, 31.18, 30.91, 27.81, 22.02, 20.46, 19.48, 13.68; ESI-MSm/z: Calcd for C28H36O3{[2M+Na]+}863.54, found 863.01。

4a: 白色固體，收率85%, m.p.205～207 ℃;1H NMR(500 MHz, CDCl3)δ: 7.78(s, 1H), 7.67(d,J=16.0 Hz, 1H), 7.56～7.49(m, 3H), 7.45～7.41(m, 1H), 7.39～7.35(m, 3H), 7.33～7.27(m, 2H), 6.42(d,J=16.0 Hz, 1H), 5.43(d,J=3.9 Hz, 1H), 4.75(tt,J=10.5 Hz, 4.8 Hz, 1H), 2.75(dd,J=15.9 Hz, 5.7 Hz, 1H), 2.48～2.37(m, 3H), 2.18～2.11(m, 1H), 2.03～1.90(m, 3H), 1.82～1.53(m, 5H), 1.48～1.32(m, 2H), 1.27～1.17(m, 2H), 1.11(s, 3H), 1.00(s, 3H);13C NMR(125 MHz, CDCl3)δ: 208.78, 166.37, 144.55, 140.10, 138.17, 135.77, 134.50, 133.82, 130.24, 130.06, 129.91, 129.22, 128.90, 128.07, 126.60, 121.82, 118.61, 73.84, 50.23, 49.74, 47.54, 38.24, 36.94, 36.88, 31.56, 31.19, 30.95, 29.18, 27.85, 20.40, 19.47, 14.21; ESI-MSm/z: Calcd for C35H37O3Cl{[2M+Na]+}1103.48, found 1103.86。

4b: 白色固體，收率85%, m.p.197～199 ℃;1H NMR(500 MHz, CDCl3)δ: 7.78(s, 1H), 7.65(d,J=16.0 Hz, 1H), 7.56～7.51(m, 1H), 7.42(t,J=8.6 Hz, 3H), 7.30(q,J=6.3 Hz, 5.4 Hz, 2H), 7.18(d,J=7.8 Hz, 2H), 6.38(d,J=16.0 Hz, 1H), 5.43(d,J=4.0 Hz, 1H), 4.74(tt,J=10.5 Hz, 4.8 Hz, 1H), 2.75(dd,J=15.9 Hz, 5.9 Hz, 1H), 2.48～2.38(m, 3H), 2.36(s, 3H), 2.19～2.10(m, 1H), 2.03～1.90(m, 3H), 1.81～1.53(m, 5H), 1.48～1.32(m, 2H), 1.27～1.16(m, 2H), 1.11(s, 3H), 1.00(s, 3H);13C NMR(125 MHz,CDCl3)δ: 208.86, 166.64, 144.63, 140.71, 140.21, 138.25, 135.84, 133.89, 131.84, 130.13, 129.98, 129.71, 129.29, 128.13, 126.67, 121.85, 117.57, 73.79, 50.31, 49.81, 47.61, 38.32, 37.02, 36.95, 31.63, 31.26, 31.02, 29.25, 27.93, 21.57, 20.46, 19.54, 14.28; ESI-MSm/z: Calcd for C36H39O3Cl{[2M+Na]+}1131.52, found 1131.73。

4c: 白色固體，收率50%, m.p.209～211 ℃;1H NMR(500 MHz, CDCl3)δ: 7.78(s, 1H), 7.63(d,J=15.9 Hz, 1H), 7.56～7.51(m, 1H), 7.51～7.41(m, 3H), 7.29(dt,J=6.2 Hz, 2.8 Hz, 2H), 6.90(d,J=8.7 Hz, 2H), 6.29(d,J=15.9 Hz, 1H), 5.43(d,J=4.6 Hz, 1H), 4.74(dt,J=11.1 Hz, 5.9 Hz, 1H), 3.83(s, 3H), 2.75(dd,J=15.9 Hz, 5.3 Hz, 1H), 2.47～2.35(m, 3H), 2.20～2.11(m, 1H), 2.03～1.89(m, 3H), 1.84～1.53(m, 5H), 1.48～1.33(m, 2H), 1.28～1.17(m, 2H), 1.11(s, 3H), 1.00(s, 3H);13C NMR(125 MHz, CDCl3)δ: 208.94, 166.83, 161.46, 144.33, 140.30, 138.29, 135.89, 133.95, 130.17, 130.16, 130.00, 129.81, 129.36, 127.36, 126.69, 121.85, 116.20, 114.45, 73.74, 55.51, 50.36, 49.85, 47.66, 38.38, 37.07, 37.00, 31.67, 31.31, 31.06, 29.29, 27.98, 20.50, 19.57, 14.31; ESI-MSm/z: Calcd for C36H39O4Cl{[2M+Na]+}1163.50, found：1163.91。

4d: 白色固體，收率75%, m.p.203～205 ℃;1H NMR(500 MHz, CDCl3)δ: 7.78(s, 1H), 7.61(d,J=16.0 Hz, 1H), 7.56～7.50(m, 1H), 7.45～7.41(m, 1H), 7.37～7.32(m, 1H), 7.31～7.26(m, 3H), 7.21(d,J=9.5 Hz, 1H), 7.07(dt,J=8.0 Hz, 4.1 Hz, 1H), 6.41(d,J=16.0 Hz, 1H), 5.43(d,J=3.9 Hz, 1H), 4.74(tt,J=10.4 Hz, 4.8 Hz, 1H), 2.75(dd,J=15.8 Hz, 5.1 Hz, 1H), 2.47～2.37(m, 3H), 2.14(d,J=17.0 Hz, 1H), 2.03～1.89(m, 3H), 1.81～1.74(m, 1H), 1.74～1.63(m, 3H), 1.62～1.53(m, 1H), 1.47～1.33(m, 2H), 1.26～1.18(m, 2H), 1.11(s, 3H), 1.00(s, 3H);13C NMR(125 MHz, CDCl3)δ: 208.83, 166.05, 162.12, 143.20, 140.10, 138.24, 136.82, 135.85, 133.90, 130.50, 130.14, 129.98, 129.31, 126.67, 124.14, 121.97, 120.13, 117.24, 114.44, 74.12, 50.32, 49.82, 47.61, 38.28, 37.00, 36.95, 31.64, 31.27, 31.03, 29.25, 27.90, 20.47, 19.53, 14.28; ESI-MSm/z: Calcd for C35H36O3FCl{[2M+Na]+}1139.46, found 1139.35。

4e: 白色固體，收率75%, m.p.197～199 ℃;1H NMR(500 MHz, CDCl3)δ: 7.78(s, 1H), 7.62(d,J=16.0 Hz, 1H), 7.55～7.47(m, 3H), 7.46～7.40(m, 1H), 7.32～7.27(m, 2H), 7.06(t,J=8.5 Hz, 2H), 6.34(d,J=16.0 Hz, 1H), 5.42(d,J=4.0 Hz, 1H), 4.74(tt,J=10.4 Hz, 4.7 Hz, 1H), 2.75(dd,J=15.8 Hz, 5.8 Hz, 1H), 2.48～2.36(m, 3H), 2.19～2.10(m, 1H), 2.02～1.89(m, 3H), 1.81～1.53(m, 5H), 1.48～1.32(m, 2H), 1.28～1.21(m, 2H), 1.10(s, 3H), 1.00(s, 3H);13C NMR(125 MHz, CDCl3)δ:208.85, 166.32, 143.31, 140.15, 138.25, 135.86, 133.91, 130.86, 130.83, 130.14, 130.03, 129.98, 129.96, 129.33, 126.67, 121.93, 118.48, 118.46, 116.22, 116.04, 73.98, 50.32, 49.82, 47.62, 38.31, 37.01, 36.96, 31.64, 31.28, 31.03, 29.26, 27.92, 20.48, 19.54, 14.28. ESI-MSm/z: Calcd for C35H36O3FCl{[2M+Na]+}1139.46, found 1139.54。

4f: 白色固體，收率70%, m.p.164～166 ℃;1H NMR(400 MHz, CDCl3)δ: 7.78(s, 1H), 7.71～7.59(m, 5H), 7.56～7.51(m, 1H), 7.46～7.40(m, 1H), 7.33～.27(m, 2H), 6.49(d,J=16.0 Hz, 1H), 5.47～5.41(m, 1H), 4.76(tt,J=10.3 Hz, 5.4 Hz, 1H), 2.76(dd,J=15.8 Hz, 5.1 Hz, 1H), 2.48～2.37(m, 3H), 2.20～2.11(m, 1H), 2.05～1.89(m, 3H), 1.86～1.52(m, 6H), 1.49～1.32(m, 2H), 1.29～1.17(m, 1H), 1.11(s, 3H), 1.00(s, 3H);13C NMR(101 MHz, CDCl3)δ: 208.87, 165.88, 142.74, 140.05, 138.24, 137.97, 135.87, 133.90, 131.96, 130.16, 129.99, 129.35, 128.26, 126.68, 125.94, 122.05, 121.30, 74.29, 50.32, 49.83, 47.63, 38.27, 37.00, 36.96, 31.64, 31.27, 31.03, 29.27, 27.90, 20.48, 19.54, 14.29; ESI-MSm/z: Calcd for C35H36O3FCl{[2M+Na]+}1139.46, found 1139.51。

4g: 白色固體，收率75%, m.p.98～100 ℃;1H NMR(500 MHz, CDCl3)δ: 8.08(d,J=16.0 Hz, 1H), 7.79(s, 1H), 7.62(d,J=6.9 Hz, 1H), 7.57～7.52(m, 1H), 7.47～7.39(m, 2H), 7.34～7.27(m, 4H), 6.42(d,J=16.0 Hz, 1H), 5.48～5.40(m, 1H), 4.76(tt,J=10.5 Hz, 4.7 Hz, 1H), 2.76(dd,J=15.8 Hz, 5.6 Hz, 1H), 2.50～2.38(m, 3H), 2.16(d,J=16.9 Hz, 1H), 2.05～1.90(m, 3H), 1.84～1.54(m, 5H), 1.49～1.33(m, 2H), 1.29～1.18(m, 2H), 1.12(s, 3H), 1.01(s, 3H);13C NMR(125 MHz, CDCl3)δ: 208.85, 165.94, 140.38, 140.13, 138.24, 135.84, 135.00, 133.89, 132.86, 131.07, 130.26, 130.13, 129.97, 129.30, 127.73, 127.16, 126.67, 121.93, 121.36, 74.15, 50.30, 49.81, 47.61, 38.27, 37.00, 36.95, 31.64, 31.27, 31.03, 29.25, 27.89, 20.47, 19.54, 14.28; ESI-MSm/z: Calcd for C35H36O3Cl2{[2M+Na]+}1171.40, found 1171.37。

4h: 白色固體，收率75%, m.p.172～174 ℃;1H NMR(500 MHz, CDCl3)δ: 7.78(s, 1H), 7.59(d,J=16.0 Hz, 1H), 7.55～7.48(m, 2H), 7.45～7.41(m, 1H), 7.38(d,J=7.1 Hz, 1H), 7.35～7.27(m, 4H), 6.42(d,J=16.0 Hz, 1H), 5.43(d,J=4.2 Hz, 1H), 4.74(tt,J=10.5 Hz, 4.9 Hz, 1H), 2.75(dd,J=15.9 Hz, 5.5 Hz, 1H), 2.42(td,J=11.2 Hz, 9.8 Hz, 5.4 Hz, 3H), 2.14(d,J=16.9 Hz, 1H), 2.03～1.89(m, 3H), 1.83～1.53(m, 5H), 1.48～1.32(m, 2H), 1.28～1.16(m, 2H), 1.11(s, 3H), 1.00(s, 3H);13C NMR(125 MHz, CDCl3)δ: 208.84, 166.01, 142.97, 140.10, 138.24, 136.43, 135.86, 135.01, 133.91, 130.23, 130.15, 129.98, 129.33, 127.87, 126.67, 126.31, 121.97, 120.21, 74.14, 50.32, 49.82, 47.62, 38.28, 37.00, 36.96, 31.64, 31.27, 31.03, 29.26, 27.90, 20.48, 19.54, 14.29; ESI-MSm/z: Calcd for C35H36O3Cl2{[2M+Na]+}1171.40, found 1171.14。

4i: 白色固體，收率75%, m.p.201～203 ℃;1H NMR(500 MHz, CDCl3)δ: 7.78(s, 1H), 7.61(d,J=16.0 Hz, 1H), 7.55～7.51(m, 1H), 7.44(t,J=6.2 Hz, 3H), 7.35(d,J=8.4 Hz, 2H), 7.32～7.27(m, 2H), 6.39(d,J=16.0 Hz, 1H), 5.43(d,J=3.6 Hz, 1H), 4.74(tt,J=10.5 Hz, 4.8 Hz, 1H), 2.75(dd,J=15.8 Hz, 5.5 Hz, 1H), 2.46～2.38(m, 3H), 2.18～2.11(m, 1H), 2.03～1.90(m, 3H), 1.83～1.53(m, 5H), 1.48～1.33(m, 2H), 1.27～1.17(m, 2H), 1.11(s, 3H), 1.00(s, 3H);13C NMR(125 MHz, CDCl3)δ: 208.85, 166.19, 143.16, 140.13, 138.25, 136.22, 135.87, 133.92, 133.10, 130.15, 129.98, 129.34, 129.28, 126.67, 121.97, 119.31, 74.07, 50.33, 49.83, 47.63, 38.30, 37.01, 36.97, 31.65, 31.28, 31.04, 29.82, 29.27, 27.92, 20.49, 19.54, 14.29; ESI-MSm/z: Calcd for C35H36O3Cl2{[2M+Na]+}1171.20, found 1171.84。

4j: 白色固體，收率75%, m.p.199～201 ℃;1H NMR(400 MHz, CDCl3)δ: 7.78(s, 1H), 7.65～7.57(m, 1H), 7.56～7.48(m, 3H), 7.43(d,J=7.3 Hz, 1H), 7.38(d,J=8.0 Hz, 2H), 7.32～7.25(m, 2H), 6.41(d,J=16.0 Hz, 1H), 5.43(s, 1H), 4.74(dt,J=10.3 Hz, 5.3 Hz, 1H), 2.76(dd,J=15.8 Hz, 5.7 Hz, 1H), 2.48～2.37(m, 3H), 2.15(d,J=17.0 Hz, 1H), 2.05～1.89(m, 3H), 1.84～1.52(m, 5H), 1.48～1.32(m, 2H), 1.29～1.19(m, 2H), 1.11(s, 3H), 1.00(s, 3H);13C NMR(101 MHz, CDCl3)δ: 208.83, 166.15, 143.20, 140.07, 138.21, 135.84, 133.87, 133.49, 132.21, 130.13, 129.96, 129.50, 129.30, 126.66, 124.53, 121.95, 119.39, 74.06, 50.28, 49.79, 47.59, 38.27, 36.98, 36.93, 31.61, 31.24, 31.00, 29.24, 27.89, 20.45, 19.52, 14.27; ESI-MSm/z: Calcd for C35H36O3BrCl{[2M+Na]+}1259.30, found 1259.93。

4k: 白色固體，收率60%, m.p.161～163 ℃;1H NMR(400 MHz,CDCl3)δ: 7.78(s, 1H), 7.53(d,J=5.8 Hz, 1H), 7.43(d,J=7.0 Hz, 1H), 7.33～7.24(m, 4H), 7.20(d,J=6.9 Hz, 3H), 5.39(s, 1H), 4.66～4.56(m, 1H), 2.94(t,J=7.7 Hz, 2H), 2.74(dd,J=15.9 Hz, 5.8 Hz, 1H), 2.60(t,J=7.7 Hz, 2H), 2.47～2.24(m, 3H), 2.13(d,J=17.1 Hz, 1H), 1.99(d,J=12.5 Hz, 1H), 1.92～1.51(m, 7H), 1.47～1.20(m, 3H), 1.20～1.11(m, 1H), 1.07(s, 3H), 0.99(s, 3H);13C NMR(101 MHz, CDCl3)δ: 172.37, 140.64, 140.13, 138.22, 135.83, 133.87, 130.12, 129.96, 129.28, 128.54, 128.41, 126.66, 126.30, 121.80, 73.82, 50.26, 49.78, 47.59, 38.15, 36.95, 36.89, 36.30, 31.61, 31.23, 31.13, 30.98, 29.24, 27.76, 20.43, 19.49, 14.26; ESI-MSm/z: Calcd for C35H39O3Cl{[2M+Na]+}1107.52, found 1107.89。

1.3 生物活性

(1) 細(xì)胞系和細(xì)胞培養(yǎng)

Huh-7和MCF-7細(xì)胞在高糖的DMEM培養(yǎng)基中37 ℃和5% CO2下生長。每2 d更換一次培養(yǎng)基，用0.05%胰蛋白酶(含0.025% EDTA溶液)處理后傳代細(xì)胞。

(2) 體外抗腫瘤活性

使用SRB測定法測定化合物的細(xì)胞毒性。將3×103個細(xì)胞/孔接種到96孔培養(yǎng)板中，每孔加入100 μL細(xì)胞懸液，在CO2培養(yǎng)箱中于37 ℃孵育24 h。然后，將所有合成的化合物添加到細(xì)胞中，再孵育48 h。藥物處理48 h后，將細(xì)胞用10%(wt/V)的三氯乙酸固定并染色30 min，然后通過用1%(V/V)的乙酸反復(fù)洗滌去除多余染料。將結(jié)合蛋白的染料溶于pH 10.5的10 mM Tris堿中，并使用酶標(biāo)儀在570 nm下測定OD值。陽性對照使用一種臨床上用于治療多種惡性腫瘤的藥物依托泊苷(VP-16)。

(3) 細(xì)胞周期

將MCF-7細(xì)胞以2.5×105/孔種于6孔板中，培養(yǎng)24 h，然后分別用2d、依托泊苷和DMSO作為對照處理細(xì)胞24 h，收獲細(xì)胞后用70%乙醇于-20 ℃固定過夜，PBS洗滌細(xì)胞后，在37 ℃下用PI和1 mg/mL RNase染色40 min，最后用流式細(xì)胞儀檢測細(xì)胞周期。

表1 化合物對Huh-7細(xì)胞和MCF-7細(xì)胞的生長抑制率

2 結(jié)果與討論

2.1 合成

以去氫表雄酮(DHEA)為原料，通過和不同取代基肉桂酸在EDCI，DMAP和Et3N的條件下酯化得到11個新的DHEA酯類衍生物2a～2k，產(chǎn)率為50%～90%；進(jìn)一步，DHEA與相應(yīng)的苯甲醛通過羥醛縮合反應(yīng)得到關(guān)鍵中間產(chǎn)物3，只得到了(E)-異構(gòu)體的3，可能是因?yàn)镈HEA與芳香醛發(fā)生羥醛縮合反應(yīng)時，羰基的去屏蔽效應(yīng)使得乙烯基的trans構(gòu)型在COC=CH中更有利于結(jié)構(gòu)的穩(wěn)定；接著，3再與不同的肉桂酸在EDCI，DMAP和Et3N的條件下酯化得到11個新的含有(E)-16-(2-氯亞芐基)的酯類衍生物4a～4k，產(chǎn)率為50%～80%。在酯化反應(yīng)中加入Et3N可以使反應(yīng)更高效。

2.2 抗腫瘤活性

(1) 體外抗增殖活性

表1為2a～2k，4a～4k在50 μM濃度下對人乳腺癌細(xì)胞MCF-7、人肝癌細(xì)胞Huh-7的體外抑制活性。2a～2k，4a～4k均顯示有一定的抑制活性，其中2d對MCF-7細(xì)胞的抑制率最高，為63%，進(jìn)一步測定其IC50值為48 μM，4c對Huh-7細(xì)胞的抑制率最高，為46%， IC50值為55 μM。該結(jié)果表明去氫表雄酮C3酯類衍生物具有一定的抗癌活性。

(2) 細(xì)胞周期

為探究這些化合物的活性作用機(jī)制，使用FACS Calibur流式細(xì)胞儀分析化合物2d(25和50 μM)活性和陽性對照VP-16(10 μM)處理 24 h后的MCF-7細(xì)胞周期分布情況。細(xì)胞用碘化丙啶(PI)染色后上流式分析，結(jié)果如圖2A所示。

圖12d對MCF-7細(xì)胞周期阻滯的影響

VP-16處理的MCF-7細(xì)胞在G2/M期捕獲到了88.9%的細(xì)胞，而空白對照只能捕獲到7.5%，說明VP-16阻滯MCF-7細(xì)胞周期在G2/M期。而當(dāng)2d處理MCF-7細(xì)胞后，G0/G1期的細(xì)胞數(shù)隨2d濃度的增加而減少，而S期的細(xì)胞數(shù)相應(yīng)的增多。與空白對照(G0/G1： 60.0%)相比，25 μM處理組 G0/G1期細(xì)胞數(shù)減少為53.1%， 50 μM處理組 G0/G1期細(xì)胞數(shù)減少為46.5%，而S期細(xì)胞數(shù)相應(yīng)從32.5%增加到了41.0%(25 μM)和44.7%(50 μM)(圖1B)。說明去氫表雄酮C3酯類衍生物的抗癌活性與細(xì)胞周期阻滯有關(guān)。

以去氫表雄酮(DHEA)為原料，在C3位上與不同的酸反應(yīng)，以較高的產(chǎn)率得到了22個新的酯類衍生物，其結(jié)構(gòu)經(jīng)核磁和質(zhì)譜表征均正確。體外抗癌活性實(shí)驗(yàn)發(fā)現(xiàn)，DHEA對MCF-7和Huh-7細(xì)胞均沒有明顯抑制作用，但是酯類衍生物顯示了抑制活性，其中2d對MCF-7細(xì)胞的抑制率最高，4c對Huh-7細(xì)胞的抑制率最高。進(jìn)一步的研究發(fā)現(xiàn)，2d對MCF-7細(xì)胞周期分布、G0/G1期縮短、S期延長均有顯著影響。