奉 強(qiáng), 何華龍, 高添桃, 朱永霞, 張強(qiáng)勝, 劉志昊, 何 冰, 張力丹, 余洛汀*

(1. 華西醫(yī)院 藥物化學(xué)實(shí)驗(yàn)室、腫瘤中心，四川大學(xué)協(xié)同創(chuàng)新中心，四川 成都 610041；2. 成都師范學(xué)院 化學(xué)與生命科學(xué)學(xué)院，四川 成都 611130)

·研究論文·

1/2,5-二取代吲唑-3-甲酰胺衍生物的合成及其抗腫瘤活性

奉 強(qiáng)1,2, 何華龍1, 高添桃1, 朱永霞1, 張強(qiáng)勝1, 劉志昊1, 何 冰2, 張力丹1, 余洛汀1*

(1. 華西醫(yī)院 藥物化學(xué)實(shí)驗(yàn)室、腫瘤中心，四川大學(xué)協(xié)同創(chuàng)新中心，四川 成都 610041；2. 成都師范學(xué)院 化學(xué)與生命科學(xué)學(xué)院，四川 成都 611130)

以吲唑-3-羧酸為原料，依次經(jīng)溴代和酯化反應(yīng)制得中間體5-溴吲唑-3-羧酸甲酯(2)；2經(jīng)N-烷基化并原位水解生成1/2-取代-5-溴吲唑-3-羧酸(3)；3與吡啶酮甲胺類化合物經(jīng)縮合反應(yīng)制得酰胺(4)；4與芳基頻哪醇硼酸酯發(fā)生Suzuki偶聯(lián)反應(yīng)合成了14個(gè)新型二取代吲唑-3-甲酰吡啶酮甲胺衍生物(5a～5n)，收率26%～32%，其結(jié)構(gòu)經(jīng)1H NMR,13C NMR和HR-MS(ESI-TOF)表征。通過(guò)NOE差譜確證了取代基在吲唑氮原子上的取代位置。采用MTT法研究了5a～5n對(duì)人B淋巴瘤細(xì)胞(Ramos)、人黑色素瘤細(xì)胞(CHL-1, WM-266-4)和乳腺癌細(xì)胞(BT-474)的體外抗腫瘤活性。結(jié)果表明:5a,5b,5m對(duì)Ramos細(xì)胞、5a,5b,5l對(duì)WM-266-4細(xì)胞、5a,5b,5d,5e,5h,5j,5m,5n對(duì)CHL-1細(xì)胞和5a,5b,5d,5h,5m對(duì)BT-474細(xì)胞具有較好的抑制活性(IC50<10.0 μmol.L-1)。

吲唑-3-甲酸; 1/2,5-二取代吲唑-3-甲酰胺衍生物; Suzuki偶聯(lián)反應(yīng); 合成; 抗腫瘤活性

吲唑衍生物種類繁多，是一類重要的雜環(huán)化合物，在農(nóng)藥、醫(yī)藥領(lǐng)域有著廣泛用途[1-5]。吲唑-4-甲酰胺類化合物因其具有良好的抗腫瘤生物活性而得以開發(fā)研究。2013年美國(guó)北卡羅萊納州大學(xué)和結(jié)構(gòu)基因聯(lián)盟公司報(bào)道的抗腫瘤小分子抑制劑UNC1999[6]、比作用于其他組蛋白甲基轉(zhuǎn)移酶選擇性高1 000倍以上的選擇性EZH2抑制劑GSK343[7]以及抗腫瘤臨床藥物EPZ005687[8]，均含有1,6-二取代吲唑-4-甲酰胺結(jié)構(gòu)。

吡啶酮甲胺結(jié)構(gòu)在抗腫瘤藥物中廣泛存在[9]。在前期研究中本課題組以取代四氫異喹啉酮甲胺為原料合成了系列含吡啶酮結(jié)構(gòu)的新型苯甲酰胺衍生物，體外和大鼠實(shí)驗(yàn)結(jié)果表明：該類苯甲酰吡啶酮甲胺衍生物對(duì)黑色素瘤和乳腺癌的生長(zhǎng)具有很好的抑制作用[10-12]。

為滿足活性篩選和新藥研究開發(fā)的需要，依據(jù)藥物設(shè)計(jì)原理，本文對(duì)1,6-二取代吲唑-4-甲酰吡啶酮甲胺進(jìn)行結(jié)構(gòu)改造，合成系列1/2,5-二取代吲唑-3-甲酰吡啶酮胺。以吲唑-3-羧酸為原料，經(jīng)溴代反應(yīng)制得5-溴吲唑-3-羧酸(1)；1經(jīng)酯化反應(yīng)制得5-溴吲唑-3-羧酸甲酯(2)；2經(jīng)N-烷基化并原位水解生成1/2-取代-5-溴吲唑-3-羧酸(3)；3在縮合劑作用下與吡啶酮甲胺類化合物縮合制得酰胺(4)；4與芳基頻哪醇硼酸酯發(fā)生Suzuki偶聯(lián)反應(yīng)合成了14個(gè)新型二取代吲唑-3-甲酰吡啶酮甲胺衍生物(5a～5n, Scheme 1)，其結(jié)構(gòu)經(jīng)1H NMR,13C NMR和HR-MS(ESI-TOF)表征。通過(guò)NOE差譜確證了取代基在吲唑N原子上的取代位置，并采用MTT法對(duì)目標(biāo)化合物5a～5n進(jìn)行抑制人B淋巴瘤細(xì)胞(Ramos)、人黑色素瘤細(xì)胞(CHL-1， WM-266-4)和乳腺癌細(xì)胞(BT-474)體外抗腫瘤活性測(cè)試。

1 實(shí)驗(yàn)部分

1.1 儀器與試劑

Bruker-400 MHz型核磁共振儀(CDCl3為溶劑，TMS為內(nèi)標(biāo))；MicroTMQ-TOF型高分辨質(zhì)譜儀。

中間體1和2按文獻(xiàn)[13-14]方法制備；吡啶酮甲胺類化合物，純度≥97%(HPLC)；硼酸酯化合物，純度≥98%(HPLC)；硅膠，200～300目，青島海洋化工有限公司；其余所用試劑均為分析純，使用前未經(jīng)進(jìn)一步處理，直接使用。

1.25a～5n的合成(以5a為例)

(1) 6-溴-2-環(huán)己基吲唑羧酸(3a)的合成

在反應(yīng)瓶中加入214.7 g(57.6 mmol),無(wú)水碳酸鈉9.8 g(92.4 mmol)，溴代環(huán)己烷15.1 g(92.5 mmol)和DMF 80 mL，緩慢升溫至60 ℃，反應(yīng)24 h(TLC監(jiān)測(cè))。冷卻至室溫，倒入碎冰中，攪拌至冰塊融化，用乙酸乙酯(2×300 mL)萃取，合并萃取液，依次用水(400 mL)和飽和食鹽水(300 mL)洗滌，無(wú)水硫酸鈉干燥，減壓蒸除溶劑后經(jīng)硅膠柱層析(洗脫劑：石油醚 ∶乙酸乙酯=8 ∶1，V∶V)純化得白色固體組分A8.7 g和組分B5.0 g。

將組分A8.7 g溶于甲醇50 mL中，攪拌下一次性加入一水合氫氧化鋰2.0 g(48 mmol)的水(25 mL)溶液，升溫至50 ℃，反應(yīng)2 h(TLC監(jiān)測(cè))。減壓蒸除甲醇，殘余物用水(50 mL)稀釋，冷卻至0 ℃，小心加入稀鹽酸調(diào)至pH 2～3(析出白色固體)，陳化1 h。過(guò)濾，固體用甲醇重結(jié)晶得白色固體3a7.38 g，收率40%；1H NMR(400 MHz, DMSO-d6)δ:13.86(s, 1H), 8.18(d,J=1.8 Hz, 1H), 7.77(d,J=9.0 Hz, 1H), 7.45(dd,J=9.1 Hz, 1.9 Hz, 1H), 6.20～5.94(m, 1H), 1.89(d,J=12.2 Hz, 2H), 1.64(dd,J=38.0 Hz, 12.4 Hz, 2H), 1.48～0.99(m, 6H)；13C NMR(101 MHz, DMSO-d6)δ: 161.04, 145.27, 129.54, 124.84, 124.66, 123.82, 120.73, 117.93, 62.72, 32.57, 25.84, 25.00。

(2) 5-溴-2-環(huán)己基-2H-吲唑-3-甲酰(4,6-二甲基-2-氧代-1,2-二氫吡啶-3-甲基)甲胺(4a)的合成

在反應(yīng)瓶中加入3a3.23 g(10 mmol)和無(wú)水二甲亞砜50 mL，攪拌10 min，滴加N-甲基嗎啉5.06 g(50 mmol)，冰水浴冷卻下一次性加入EDCI 3.45 g(18 mmol)和HOAt 2.46 g(18 mmol)，控制溫度不高于20 ℃，攪拌反應(yīng)1 h。冷卻至5 ℃，加入4,6-二甲基吡啶酮-3-甲胺1.82 g(12 mmol)，加畢，自然升至室溫，反應(yīng)過(guò)夜(TLC監(jiān)測(cè))。反應(yīng)液倒入水/乙酸乙酯(300 mL,V/V=1/1)中，攪拌15 min后靜置分液，水層用乙酸乙酯(2×50 mL) 萃取，合并萃取液，依次用水100 mL和飽和食鹽水100 mL洗滌,無(wú)水硫酸鈉干燥，減壓蒸除溶劑得化合物4a粗品，經(jīng)硅膠柱層析[洗脫劑：以DCM/Pet(V/V=1/1)洗去小極性雜質(zhì)，再用DCM洗脫]純化得4a4.08 g，收率86%;1H NMR(400 MHz)δ: 12.35(s, 1H), 7.88(t,J=6.2 Hz, 1H), 7. 65(dd,J=8.8 Hz, 1.7 Hz, 1H), 7.51(d,J=1.7 Hz, 1H), 6.80(d,J=8.8 Hz, 1H), 5.96(s, 1H), 4.76(d,J=6.1 Hz, 2H), 4.62～4.40(m, 1H), 2.47(s, 3H), 2.39(s, 3H), 2.10～1.76(m, 6H), 1.46(m, 2H), 1.38～1.24(m, 2H);13C NMR(101 MHz, CDCl3)δ: 165.64, 157.38, 142.74, 136.72, 132.48, 127.00, 125.66, 123.32, 122.85, 119.80, 109.87, 109.73, 106.28, 65.79, 34.80, 32.04, 25.69, 25.34, 19.57, 18.64。

Scheme 1

(3) 2-環(huán)己基-5-[(6-嗎啡啉-4-基)吡啶-3-基]-2H-吲唑-3-甲酰(4,6-二甲基-2-氧代-1,2-二氫吡啶酮-3-基)甲胺(5a)的合成

氮?dú)獗Ｗo(hù)下，在100 mL三口瓶中加入二氧六環(huán)/水(V/V=5/1)36 mL，攪拌下依次加入4a0.474 g(1 mmol)、6-(嗎啉-4-基)吡啶-3-硼酸頻吶醇酯0.32 g(1.1mmol)和無(wú)水碳酸鈉0.212 g(2 mmol)，氮?dú)獬浞种脫Q,快速加入催化劑dppf·PdCl240 mg(0.05 mmol)，再次用氮?dú)獬浞种脫Q反應(yīng)體系，快速升溫至回流，反應(yīng)3 h(TLC監(jiān)測(cè))。減壓蒸除二氧六環(huán)，將反應(yīng)混合物轉(zhuǎn)移至燒杯中，加入50 mL水，攪拌10 min，用氯仿(3×30 mL)萃取，合并萃取液，依次用水(2×50 mL)和飽和食鹽水(50 mL)洗滌，無(wú)水硫酸鈉干燥，減壓蒸出溶劑得5a粗品，經(jīng)硅膠柱層析[洗脫劑：用DCM洗去小極性雜質(zhì)，再用DCM/甲醇(V/V=15/1)洗脫]純化得5a0.48 g。

用上述類似方法合成5b～5n。

5a: 收率89%;1H NMR(400 MHz)δ: 12.57(s, 1H, Pyridone-NH), 8.52(dd,J=8.0 Hz, 2.0 Hz, 2H, Indazole and Pyridine-ArH), 7.94(t,J=6.2 Hz, 1H, CONH), 7.86(dd,J=8.8, 2.6 Hz, 1H, Pyridine-ArH), 7.56(dd,J=8.8, 1.7 Hz, 1H, Indazole-ArH), 7.49(d,J=8.7 Hz, 1H, Indazole-ArH), 6.71(d,J=8.8 Hz, 1H, Pyridine-ArH), 5.96(s, 1H, Pyridone-ArH), 4.64(d,J=6.1 Hz, 2H), 4.48～4.30(m, 1H, Indazole-CH), 4.00～3.76(m, 4H), 3.55(dd,J=5.7 Hz, 4.0 Hz, 4H), 2.44(s, 3H), 2.35(s, 3H), 2.11～1.74(m, 6H), 1.46(m, 2H), 1.38～1.21(m, 2H);13C NMR(101 MHz)δ: 165.46, 162.50, 158.60, 150.64, 146.32, 142.67, 139.22, 137.15, 136.66, 132.84, 127.00, 125.49, 123.72, 122.58, 119.88, 109.83, 109.71, 106.76, 66.79, 58.77, 45.76, 34.84, 32.44, 25.66, 25.32, 19.75, 18.88; HR-MS(ESI-TOF)m/z: Calcd for C31H37N6O3{[M+H]+} 541.292 7, found 541.293 4。

2-環(huán)己基-5-[6-(4-甲基哌嗪-1-基)吡啶-3-基]-2H-吲唑-3-甲酰(4,6-二甲基-2氧代-1,2-二氫吡啶-3-基)甲胺(5b): 收率30.3%;1H NMR(400 MHz, CDCl3)δ: 11.86(s, 1H, Pyridone-NH), 8.50(dd,J=8.8 Hz, 2.1 Hz, 2H, Indazole-， Pyridine-ArH), 7.93(t,J=6.2 Hz, 1H, CONH), 7.84(dd,J=8.8 Hz, 2.6 Hz, 1H, Pyridine-ArH), 7.56(dd,J=8.8 Hz, 1.7 Hz, 1H, Indazole-ArH), 7.48(d,J=8.8 Hz, 1H, Indazole-ArH), 6.73(d,J=8.8 Hz, 1H, Pyridine-ArH), 5.94(s, 1H, Pyridone-ArH), 4.63(d,J=6.2 Hz, 2H), 4.40(m, 1H, Indazole-CH), 3.64(t,J=5.0 Hz, 4H), 2.59(t,J=5.0 Hz, 4H), 2.44(s, 3H), 2.39(s, 3H), 2.33(s, 3H), 2.12～1.87(m, 7H), 1.47(q,J=12.7 Hz, 2H);13C NMR(101 MHz)δ: 165.16, 162.52, 158.43, 150.58, 146.33, 142.33, 139.19, 137.11, 136.61, 132.94, 126.51, 125.47, 123.75, 122.76, 119.78, 109.80, 109.60, 106.92, 58.76, 54.82, 46.12, 45.18, 34.83, 32.44, 29.71, 25.67, 19.75, 18.95; HR-MS(ESI-TOF)m/z: Calcd for C32H40N7O2{[M+H]+} 554.324 3, found 554.321 5。

1-(4-甲氧基-1-丁基)-5-[(4-嗎啡啉-4-基)甲基吡啶基]-1H-吲唑-3-甲酰(1-甲基-3-氧-2,3,5,6,7,8-六氫喹啉-4-基)甲胺(5c): 收率31.2%;1H NMR(400 MHz)δ: 13.42(s, 1H, Pyridone-NH), 8.53(d,J=1.6 Hz, 1H, Indazole-ArH), 8.00(t,J=6.0 Hz, 1H, CONH), 7.61～7.51(m, 3H, Indazole-， benzene-ArH), 7.35(d,J=8.8 Hz, 1H, Indazole-ArH), 7.30(d,J=7.9 Hz, 2H, benzene-ArH), 4.60(d,J=6.0 Hz, 2H), 4.29(t,J=7.1 Hz, 2H, Indazole-CH2), 3.65(t,J=4.6 Hz, 4H), 3.45(s, 2H), 3.26(t,J=6.2 Hz, 2H), 3.18(s, 3H), 2.91(t,J=6.0 Hz, 2H), 2.38(dt,J=11.2 Hz, 5.1 Hz, 6H), 2.26(s, 3H), 1.91(t,J=7.2 Hz, 2H), 1.73～1.57(m, 4H), 1.47(dq,J=12.6 Hz, 6.4 Hz, 2H);13C NMR(101 MHz)δ: 163.97, 162.24, 150.95, 140.89, 140.20, 140.00, 137.83, 136.44, 135.53, 129.59, 127.31, 126.53, 123.57, 121.61, 120.77, 114.59, 109.46, 72.03, 67.02, 63.14, 58.59, 53.65, 49.28, 34.48, 26.92, 26.78, 25.04, 22.41, 22.34, 16.69; HR-MS(ESI-TOF)m/z: Calcd for C35H44N5O4{[M+H]+} 598.339 3, found 598.336 4。

1-環(huán)己基-5-[6-(4-甲基哌嗪-1-基)吡啶-3-基]-1H-吲唑-3-甲酰(1-甲基-3-氧-2,3,5,6,7,8-六氫喹啉-4-基)甲胺(5d): 收率30.7%;1H NMR(400 MHz)δ: 13.61(s, 1H, Pyridone-NH), 8.58(t,J=5.8 Hz, 1H, CONH), 8.50(d,J=2.4 Hz, 1H, Pyridine-ArH), 8.10(s, 1H, Indazole-ArH), 7.83(d,J=8.9 Hz, 1H, Indazole-ArH), 7.71(dd,J=8.8 Hz, 2.6 Hz, 1H, Pyridine-ArH), 7.50(dd,J=9.0 Hz, 1.4 Hz, 1H, Indazole-ArH), 6.39(d,J=8.8 Hz, 1H, Pyridine-ArH), 5.67～5.37(m, 1H, Indazole-CH), 4.68(d,J=5.7 Hz, 2H), 3.31(t,J=5.0 Hz, 4H), 2.94(t,J=6.3 Hz, 2H), 2.62(s, 2H), 2.27(t,J=5.3 Hz, 6H), 2.22(s, 3H), 2.16(dd,J=11.2 Hz, 3.4 Hz, 2H), 1.99～1.92(m, 2H), 1.90(s, 3H), 1.82～1.66(m, 5H), 1.64～1.50(m, 2H), 1.36(m, 1H);13C NMR(101 MHz)δ: 163.92, 159.54, 158.20, 150.38, 146.43, 145.96, 141.34, 135.72, 133.70, 127.37, 125.81, 125.08, 120.90, 120.35, 119.09, 114.99, 114.82, 106.51, 60.90, 54.54, 46.05, 45.05, 35.99, 33.68, 29.70, 27.52, 25.71, 25.41, 24.85, 22.25, 16.50; HR-MS(ESI-TOF)m/z: Calcd for C35H44N7O2{[M+H]+}594.355 6, found 594.352 6。

1-(4-甲氧基-1-丁基)-5-[(6-嗎啡啉-4-基)吡啶-3-基]-1H-吲唑-3-甲酰(4,6-二甲基-2-氧代-1,2-二氫吡啶酮-3-基)甲胺(5e): 收率28.9%;1H NMR(400 MHz)δ: 13.07(s, 1H, Pyridone-NH), 8.51(dd,J=4.7, 2.0 Hz, 2H, Indazole-， Pyridine-ArH), 8.05(t,J=6.1 Hz, 1H, CONH), 7.85(dd,J=8.8, 2.6 Hz, 1H, Pyridine-ArH), 7.57(dd,J=8.8 Hz, 1.7 Hz, 1H, Indazole-ArH), 7.44(d,J=8.7 Hz, 1H, Indazole-ArH), 6.71(d,J=8.8 Hz, 1H, Pyridine-ArH), 5.97(s, 1H, Pyridone-ArH), 4.64(d,J=6.0 Hz, 2H), 4.37(t,J=7.1 Hz, 2H, Indazole -CH), 3.91～3.75(m, 4H), 3.60～3.49(m, 4H), 3.35(t,J=6.2 Hz, 2H), 3.28(s, 3H), 2.44(s, 3H), 2.38(s, 3H), 2.06～1.91(m, 2H), 1.62～1.47(m, 2H);13C NMR(101 MHz)δ: 165.63, 162.29, 158.61, 150.50, 146.32, 143.00, 140.01, 137.57, 136.61, 132.89, 126.87, 125.89, 123.63, 122.34, 119.83, 109.78, 109.70, 106.75, 72.02, 66.77, 58.60, 49.30, 45.73, 34.95, 26.90, 26.77, 19.71, 18.81; HR-MS(ESI-TOF)m/z: Calcd for C30H37N6O4{[M+H]+} 545.287 6, found 545.288 6。

2-環(huán)己基-5-[4-(嗎啡啉-4-基)甲基]苯基-1H-吲唑-3-甲酰(4,6-二甲基-2-氧代-1,2-二氫吡啶酮-3-基)甲胺(5f): 收率31.7%;1H NMR(400 MHz)δ: 12.88(s, 1H, Pyridone-NH), 8.61(d,J=1.6 Hz, 1H, Indazole-ArH), 7.95(t,J=6.2 Hz, 1H, CONH), 7.66～7.60(m, 3H, Indazole-， benzene-ArH), 7.48(d,J=8.9 Hz, 1H, Indazole-ArH), 7.38(d,J=7.9 Hz, 2H, benzene-ArH), 5.96(s, 1H, Pyridone-ArH), 4.64(d,J=6.1 Hz, 2H), 4.49～4.31(m, 1H, Indazole-CH), 3.73(t,J=4.6 Hz, 4H), 3.54(s, 2H), 2.48(t,J=4.6 Hz, 4H), 2.44(s, 3H), 2.36(s, 3H), 2.03(dt,J=11.7 Hz, 4.2 Hz, 3H), 1.98～1.88(m, 3H), 1.78(dt,J=13.2 Hz, 3.3 Hz, 1H), 1.46(m, 2H), 1.37～1.19(m, 3H);13C NMR(101 MHz)δ: 165.59, 162.49, 150.71, 142.85, 140.10, 139.41, 137.33, 136.35, 135.54, 129.60, 127.34, 126.18, 123.63, 122.51, 120.76, 109.78, 109.58, 67.02, 63.15, 58.73, 53.64, 34.84, 32.45, 25.67, 25.33, 19.76, 18.84; HR-MS(ESI-TOF)m/z: Calcd for C33H40N5O3{[M+H]+} 554.313 1, found 554.313 2。

1-環(huán)己基-5-[4-(嗎啡啉-4-基)甲基]苯基-1H-吲唑-3-甲酰(1-甲基-3-氧-2,3,5,6,7,8-六氫喹啉-4-基)甲胺(5g): 收率30.8%;1H NMR(400 MHz)δ: 13.76(s, 1H, Pyridone-NH), 8.42(t,J=5.8 Hz, 1H, CONH), 8.07(s, 1H, Indazole-ArH), 7.77(d,J=9.0 Hz, 1H, Indazole-ArH), 7.45(d,J=9.0 Hz, 1H, Indazole-ArH), 7.37(d,J=7.7 Hz, 2H, benzene-ArH), 6.98(d,J=7.7 Hz, 2H, benzene-ArH), 5.48(tt,J=10.1 Hz, 5.0 Hz, 1H, Indazole-CH), 4.65(d,J=5.7 Hz, 2H), 3.39(t,J=4.5 Hz, 4H), 3.04(s, 2H), 2.88(t,J=6.3 Hz, 2H), 2.18(t,J=6.3 Hz, 2H), 2.10(q,J=5.2 Hz, 4.8 Hz, 8H), 1.88(d,J=13.4 Hz, 2H), 1.78(s, 3H), 1.74～1.58(m, 4H), 1.49(m, 2H), 1.29(m, 2H);13C NMR(101 MHz)δ: 164.07, 159.63, 150.20, 146.64, 141.57, 140.02, 136.48, 129.44, 127.66, 126.79, 126.08, 120.80, 120.44, 118.86, 116.46, 114.88, 66.73, 62.96, 60.96, 53.34, 35.89, 33.68, 27.55, 25.73, 25.41, 24.83, 22.29, 16.35; HR-MS(ESI-TOF)m/z: Calcd for C36H44N5O3{[M+H]+}594.344 4, found 594.342 8。

1-(4-甲氧基-1-丁基)-5-[6-(4-甲基哌嗪-1-基)吡啶-3-基]-1H-吲唑-3-甲酰(1-甲基-3-氧-2,3,5,6,7,8-六氫喹啉-4-基)甲胺(5h): 收率29.6%;1H NMR(400 MHz)δ: 13.32(s, 1H, Pyridone-NH), 8.43(d,J=1.6 Hz, 1H, Indazole-ArH), 8.41(d,J=2.6 Hz, 1H, Pyridine-ArH), 7.99(t,J=6.0 Hz, 1H, CONH), 7.75(dd,J=8.8 Hz, 2.6 Hz, 1H, Pyridine-ArH), 7.49(dd,J=8.8 Hz, 1.7 Hz, 1H, Indazole-ArH), 7.35(d,J=8.8 Hz, 1H, Indazole-ArH), 6.64(d,J=8.8 Hz, 1H, Pyridine-ArH), 4.59(d,J=6.0 Hz, 2H), 4.29(t,J=7.1 Hz, 2H, Indazole-CH2), 3.55(t,J=4.9 Hz, 4H), 3.25(t,J=6.2 Hz, 2H), 3.18(s, 3H), 2.91(t,J=6.0 Hz, 2H), 2.50(t,J=5.0 Hz, 4H), 2.37(t,J=6.0 Hz, 2H), 2.29(s, 3H), 2.25(s, 3H), 1.90(p,J=7.3 Hz, 2H), 1.78～1.56(m, 4H), 1.47(m, 2H);13C NMR(101 MHz)δ: 163.88, 162.26, 158.44, 151.00, 146.27, 140.88, 139.97, 137.60, 136.54, 132.94, 126.38, 125.84, 123.62, 121.56, 119.70, 114.61, 109.68, 106.92, 72.01, 58.58, 54.80, 49.27, 46.10, 45.16, 34.49, 27.35, 26.88, 26.76, 25.02, 22.38, 22.32, 16.68; HR-MS(ESI-TOF)m/z: Calcd for C34H44N7O3{[M+H]+}598.350 6, found 598.354 1。

1-(4-甲氧基-1-丁基)-5-[6-(4-甲基哌嗪-1-基)吡啶-3-基]-1H-吲唑-3-甲酰(4,6-二甲基-2-氧代-1,2-二氫吡啶酮-3-基)甲胺(5i): 收率28.1%;1H NMR(400 MHz)δ: 12.98(s, 1H, Pyridone-NH), 8.51～8.50(m, 1H, Indazole-ArH), 8.49(d,J=2.6 Hz, 1H, Pyridine-ArH), 8.04(t,J=6.1 Hz, 1H, CONH), 7.83(dd,J=8.8 Hz, 2.6 Hz, 1H, Pyridine-ArH), 7.57(dd,J=8.8 Hz, 1.7 Hz, 1H, Indazole-ArH), 7.43(d,J=8.7 Hz, 1H, Indazole-ArH), 6.72(d,J=8.8 Hz, 1H, Pyridine-ArH), 5.96(s, 1H, Pyridone-ArH), 4.63(d,J=6.1 Hz, 2H), 4.37(t,J=7.1 Hz, 2H, Indazole-CH2), 3.63(t,J=5.1 Hz, 4H), 3.35(t,J=6.2 Hz, 2H), 3.28(s, 3H), 2.58(t,J=5.0 Hz, 4H), 2.43(s, 3H), 2.37(s, 6H), 2.05～1.92(m, 2H), 1.64～1.46(m, 2H);13C NMR(101 MHz)δ: 165.57, 162.30, 158.45, 150.53, 146.28, 142.97, 139.97, 137.54, 136.56, 132.99, 126.38, 125.88, 123.62, 122.34, 119.70, 109.79, 109.68, 106.93, 72.02, 58.59, 54.81, 49.29, 46.12, 45.17, 34.94, 26.89, 26.76, 19.71, 18.81; HR-MS(ESI-TOF)m/z: Calcd for C31H40N7O3{[M+H]+} 558.319 3, found 558.317 3。

1-環(huán)己基-5-[6-(嗎啡啉-4-基)吡啶-3-基]-1H-吲唑-3-甲酰(1-甲基-3-氧-2,3,5,6,7,8-六氫喹啉-4-基)甲胺(5j): 收率29.4%;1H NMR(400 MHz)δ: 13.63(s, 1H, Pyridone-NH), 8.43(t,J=5.9 Hz, 1H, CONH), 8.41(d,J=2.5 Hz, 1H, Pyridine-ArH), 8.05(s, 1H, Indazole-ArH), 7.76(d,J=8.9 Hz, 1H, Pyridine-ArH), 7.67～7.56(m, 1H, Indazole-ArH), 7.41(dd,J=9.0 Hz, 1.5 Hz, 1H, Indazole-ArH), 6.22(d,J=8.7 Hz, 1H, Pyridine-ArH), 5.50(m, 1H, Indazole-CH), 4.61(d,J=5.8 Hz, 2H), 3.46(t,J=4.7 Hz, 4H), 3.04(t,J=4.8 Hz, 4H), 2.89(t,J=6.4 Hz, 2H), 2.23(t,J=6.3 Hz, 2H), 2.15～1.98(m, 4H), 1.86(s, 4H), 1.74～1.60(m, 6H), 1.49(m, 2H), 1.37～1.20(m, 1H);13C NMR(101 MHz)δ: 162.95, 158.49, 157.35, 149.35, 145.39, 145.01, 140.39, 134.78, 132.75, 130.46, 126.25, 125.42, 124.16, 119.81, 119.55, 118.14, 113.93(d,J=2.6 Hz), 105.30, 68.65, 65.35, 59.92, 44.41, 34.89, 32.63, 26.50, 24.70, 24.39, 23.85, 21.26, 15.48; HR-MS(ESI-TOF)m/z: Calcd for C34H41N6O3{[M+H]+} 581.324 0, found 581.322 9。

1-環(huán)戊基-5-[6-(4-甲基哌嗪-1-基)吡啶-3-基]-1H-吲唑-3-甲酰(1-甲基-3-氧-2,3,5,6,7,8-六氫喹啉-4-基)甲胺(5k): 收率29.1%;1H NMR(400 MHz)δ: 13.72(s, 1H, Pyridone-NH), 8.42(d,J=6.0 Hz, 1H, CONH), 8.40(d,J=2.3 Hz, 1H, Pyridine-ArH), 8.01(s, 1H, Indazole-ArH), 7.73(d,J=8.9 Hz, 1H, Indazole-ArH), 7.62(dd,J=8.8 Hz, 2.5 Hz, 1H, Pyridine-ArH), 7.41(d,J=9.0 Hz, 1H, Indazole-ArH), 6.25(d,J=8.7 Hz, 1H, Pyridine-ArH), 5.96(p,J=7.7 Hz, 1H, Indazole-CH), 4.59(d,J=5.7 Hz, 2H), 3.18(t,J=4.9 Hz, 4H), 2.88(t,J=6.3 Hz, 2H), 2.17(dt,J=9.8 Hz, 6.1 Hz, 10H), 2.13(s, 3H), 1.95(dq,J=13.4 Hz, 7.4 Hz, 2H), 1.80(s, 3H), 1.75～1.53(m, 6H);13C NMR(101 MHz)δ: 163.99, 159.62, 158.23, 150.35, 146.44, 145.95, 141.43, 135.66, 133.76, 128.04, 125.76, 125.10, 121.11, 120.41, 119.10, 114.98, 114.65, 106.42, 62.42, 54.53, 46.08, 45.01, 35.91, 33.77, 27.46, 24.93, 24.83, 22.27, 16.43; HR-MS(ESI-TOF)m/z: Calcd for C34H42N7O2{[M+H]+}580.340 0, found 580.339 3。

1-環(huán)戊基-5-[4-(嗎啡啉-4-基)甲基]苯基-1H-吲唑-3-甲酰(1-甲基-3-氧-2,3,5,6,7,8-六氫喹啉-4-基)甲胺(5l): 收率30.2%;1H NMR(400 MHz)δ: 13.88(s, 1H, Pyridone-NH), 8.34(t,J=5.9 Hz, 1H, CONH), 8.06(s, 1H, Indazole-ArH), 7.74(d,J=8.9 Hz, 1H, Indazole-ArH), 7.44(d,J=9.0 Hz, 1H, Indazole-ArH), 7.37(d,J=7.6 Hz, 2H, Benzene-ArH), 6.95(d,J=7.7 Hz, 2H, Benzene-ArH), 5.97(p,J=7.7 Hz, 1H, Indazole-CH), 4.64(d,J=5.8 Hz, 2H), 3.37(t,J=4.5 Hz, 4H), 3.00(s, 2H), 2.90(d,J=6.6 Hz, 2H), 2.28～2.13(m, 6H), 2.08(t,J=4.4 Hz, 4H), 2.00～1.89(m, 2H), 1.77(s, 3H), 1.73～1.56(m, 6H);13C NMR(101 MHz)δ: 163.08, 158.68, 149.17, 145.62, 140.58, 138.99, 135.43, 135.15, 128.41, 127.35, 125.74, 125.06, 119.97, 119.54, 117.82, 115.29, 113.82, 65.68, 61.92, 61.41, 52.28, 34.79, 32.77, 28.66, 26.44, 23.93, 23.79, 21.27, 15.25; HR-MS(ESI-TOF)m/z: Calcd for C35H42N5O3{[M+H]+}580.328 8, found 580.326 2。

1-(4-甲氧基-1-丁基)-5-[6-(嗎啡啉-4-基)吡啶-3-基]-1H-吲唑-3-甲酰(1-甲基-3-氧-2,3,5,6,7,8-六氫喹啉-4-基)甲胺(5m): 收率26.4%;1H NMR(400 MHz)δ: 13.18(s, 1H, Pyridone-NH), 8.53(d,J=1.6 Hz, 1H, Indazole-ArH), 8.51(d,J=2.5 Hz, 1H, Indazole-ArH), 8.06(t,J=6.1 Hz, 1H, CONH), 7.85(dd,J=8.8 Hz, 2.6 Hz, 1H, Indazole-ArH), 7.58(dd,J=8.7 Hz, 1.7 Hz, 1H, Indazole-ArH), 7.44(d,J=8.7 Hz, 1H, Indazole-ArH), 6.71(d,J=8.8 Hz, 1H, Indazole-ArH), 4.67(d,J=6.0 Hz, 2H), 4.38(t,J=7.1 Hz, 2H, Indazole-CH2), 3.86(t,J=4.8 Hz, 4H), 3.56(t,J=4.8 Hz, 4H), 3.35(t,J=6.2 Hz, 2H), 3.27(s, 3H), 3.00(t,J=6.0 Hz, 2H), 2.46(t,J=5.9 Hz, 2H), 2.34(s, 3H), 1.99(p,J=7.3 Hz, 2H), 1.75(tt,J=11.8 Hz, 6.5 Hz, 4H), 1.56(dq,J=12.7 Hz, 6.4 Hz, 2H);13C NMR(101 MHz)δ: 163.86, 162.27, 158.62, 150.99, 146.34, 140.71, 140.02, 137.65, 136.62, 132.87, 126.91, 125.86, 123.66, 121.68, 119.88, 114.57, 109.69, 106.75, 72.04, 66.79, 58.61, 49.30, 45.74, 34.49, 27.36, 26.91, 26.78, 25.05, 22.41, 22.33, 16.76; HR-MS(ESI-TOF)m/z: Calcd for C33H41N6O4{[M+H]+}585.318 9, found 585.320 1。

1-環(huán)己基-5-[6-(嗎啡啉-4-基)吡啶-3-基]-1H-吲唑-3-甲酰(1-甲基-3-氧-2,3,5,6,7,8-六氫喹啉-4-基)甲胺(5n): 收率28.7%;1H NMR(400 MHz)δ: 13.55(s, 1H, Pyridone-NH), 8.48(d,J=2.5 Hz, 1H, Pyridine-ArH), 8.40(t,J=5.9 Hz, 1H, CONH), 8.11(s, 1H, Indazole-ArH), 7.82(d,J=8.9 Hz, 1H, Indazole-ArH), 7.69(dd,J=8.8, 2.6 Hz, 1H, Pyridine-ArH), 7.47(dd,J=9.0, 1.6 Hz, 1H, Indazole-ArH), 6.29(d,J=8.8 Hz, 1H, Pyridine-ArH), 6.05(p,J=7.6 Hz, 1H, Indazole-CH), 4.67(d,J=5.9 Hz, 2H), 3.55(t,J=4.8 Hz, 4H), 3.11(t,J=4.8 Hz, 4H), 2.99(t,J=6.4 Hz, 2H), 2.38～2.19(m, 4H), 2.03(dt,J=10.6 Hz, 4.4 Hz, 2H), 1.95(s, 3H), 1.85～1.66(m, 4H);13C NMR(101 MHz)δ: 163.92, 159.63, 158.39, 150.36, 146.45, 146.05, 141.32, 135.84, 133.76, 128.00, 126.51, 125.20, 121.09, 120.84, 119.15, 114.89, 106.33, 66.39, 62.48, 45.43, 35.85, 33.76, 27.45, 24.92, 24.90, 22.31, 16.52; HR-MS(ESI-TOF)m/z: Calcd for C33H39N6O3{[M+H]+}567.308 4, found 567.310 6。

1.3 體外抗腫瘤活性測(cè)試

采用MTT法測(cè)試了5a～5n對(duì)人B淋巴瘤細(xì)胞(Ramos)、人黑色素瘤細(xì)胞(CHL-1, WM-266-4)及乳腺癌細(xì)胞(BT-474)的體外抗腫瘤活性。

收集處于對(duì)數(shù)生長(zhǎng)期的腫瘤細(xì)胞，將細(xì)胞重懸成濃度為1～3×103個(gè)/mL的細(xì)胞懸液，按照每孔100 μL細(xì)胞懸液將細(xì)胞接種到96孔板中，并置于37 ℃、 5%CO2細(xì)胞培養(yǎng)箱內(nèi)孵育過(guò)夜。次日，加入100 μL含有不同梯度濃度藥物的培養(yǎng)基處理，每個(gè)藥物濃度設(shè)置3個(gè)復(fù)孔，并用含有0.1%DMSO的培養(yǎng)基處理細(xì)胞作為陰性對(duì)照。將細(xì)胞培養(yǎng)板在細(xì)胞培養(yǎng)箱內(nèi)繼續(xù)孵育4 d后，每個(gè)孔內(nèi)加入20 μL 5 mg·mL-1MTT溶液，對(duì)于貼壁生長(zhǎng)的細(xì)胞，于37 ℃孵育1～4 h，形成甲臜結(jié)晶后棄去上清，再向每個(gè)孔內(nèi)加入150 μL DMSO溶解甲臜結(jié)晶；對(duì)于懸浮生長(zhǎng)的細(xì)胞，于37 ℃孵育24 h，形成甲臜后加入50 μL 20%的SDS溶液，放置過(guò)夜。最后，用酶標(biāo)儀檢測(cè)570 nm波長(zhǎng)下的吸光度值(A)(A值與活細(xì)胞數(shù)成正比)，并計(jì)算藥物對(duì)腫瘤細(xì)胞的生長(zhǎng)抑制率[抑制率/%=(A570對(duì)照組-A570實(shí)驗(yàn)組)/A570對(duì)照組×100%]。最后，用Graphpad Prism計(jì)算擬合藥物的生長(zhǎng)抑制曲線并計(jì)算半數(shù)抑制濃度值IC50。

2 結(jié)果與討論

2.1 合成

(1)3的合成

吲唑-N-烷基化反應(yīng)選擇性不強(qiáng)，常常同時(shí)發(fā)生于1-或2-氮原子上，生成兩種取代產(chǎn)物的混合物。本實(shí)驗(yàn)以DMF為溶劑，在碳酸鈉或碳酸鉀等無(wú)機(jī)堿作用下，2與鹵代烴于60～80 ℃反應(yīng)，生成1-或2-吲唑N-烷基化產(chǎn)物。經(jīng)柱層析純化分別得到1-， 2-N-烷基化產(chǎn)物，純度可達(dá)90%。經(jīng)堿性水解、酸化得到對(duì)應(yīng)酸。將所得酸用甲醇重結(jié)晶，能以約40%收率獲得吲唑2-烴基化羧酸3a和約20%收率得到吲唑1-烴基化羧酸3b。

(2)4的合成

羧酸與胺的縮合，可選擇的縮合試劑較多。本路線多種縮合試劑在毫摩爾規(guī)模反應(yīng)合成4時(shí)收率一般都較高，柱層析純化反應(yīng)收率能達(dá)到60～90%。在后期將反應(yīng)規(guī)模放大至10 g量時(shí)，發(fā)現(xiàn)以EDCI和HOAt為催化劑時(shí)反應(yīng)效果最好：副反應(yīng)少，反應(yīng)體系易于純化，經(jīng)簡(jiǎn)單洗滌后即以93%收率得到純度高于95%以上產(chǎn)品，無(wú)需再經(jīng)柱層析純化步驟即可用于下步反應(yīng)。

(3)3的結(jié)構(gòu)確證

3a和3b的1H NMR譜中，吲唑芳環(huán)上質(zhì)子峰的裂分形狀完全相同，氫質(zhì)子化學(xué)位移幾乎相等，差別僅在與吲唑氮原子相接的C—H上，雖然二者均顯示出多重峰，但前者吸收峰位于δ6.20～5.94,后者吸收峰位于δ5.23～5.47。

由于3a,3b分子中與吲唑氮原子相接的C—H與吲唑7-H距離差別很大，表現(xiàn)出較大NOE效應(yīng)差異，為此，通過(guò)NOE差譜(圖1)確定二者的結(jié)構(gòu)。激發(fā)該C—H，發(fā)現(xiàn)在3a中它與吲唑芳?xì)鋬H有極弱NOE效應(yīng)，而在3b中觀察到它與吲唑芳?xì)?-H具有強(qiáng)烈NOE效應(yīng)，故知在3a中取代烷基位于距吲唑芳?xì)?-H較遠(yuǎn)處，3b中取代烷基位于距吲唑芳?xì)?-H較近處，即3a中烷基在吲唑環(huán)N2上取代，3b中烷基在吲唑環(huán)N1上取代。

2.2 生物活性

以MTT法測(cè)定5a～5n體外對(duì)人B淋巴瘤細(xì)胞(Ramos)、人黑色素瘤細(xì)胞(CHL-1, WM-266-4)以及乳腺癌細(xì)胞(BT-474)的抑制作用，其IC50結(jié)果見表1。

由表1可知，當(dāng)吲唑氮原子上取代基為鏈狀取代基時(shí)，僅5m對(duì)Ramos,5e,5h,5m對(duì)CHL-1和5h對(duì)BT-474具有較好的腫瘤細(xì)胞抑制活性，最高達(dá)到7. 25 μmol·L-1(5m對(duì)CHL-1腫瘤細(xì)胞)；當(dāng)吲唑氮原子上取代基換為環(huán)狀烴基時(shí)，其抗腫瘤活性最高可提高近1倍(5h與5d對(duì)CHL-1腫瘤細(xì)胞抑制活性IC50值分別為8.73 μmol·L-1和4.29 μmol·L-1)。

δ

CompIC50/μmol·L-1RamosCHL-1WM-266-4BT-4745a9.023.083.777.115b3.190.990.740.865c26.317.7>3021.45d10.24.2925.15.605e14.87.8813.420.05f>3011.710.6>305g>30>30>30>305h10.18.7311.610.05i11.118.76.87>305j11.97.2214.0>305k15.211.214.720.65l31.316.70.31>305m9.307.2526.310.05m20.26.2016.7>30EPZ00568717.668>80>30

當(dāng)吡啶酮片段上取代基為兩甲基時(shí)，比其并環(huán)類似物活性要好一些，但5l對(duì)WM-266-4具有優(yōu)異的抑制活性，達(dá)到0.37 μmol·L-1；同時(shí)，當(dāng)吲唑環(huán)上取代芳基為哌嗪吡啶時(shí)，其抗腫瘤活性往往由于取代苯基和嗎啉吡啶，如5g對(duì)所給腫瘤細(xì)胞幾乎沒(méi)有抑制活性。5c和5f僅有中等抑制活性(5l對(duì)WM-266-4例外)。

當(dāng)將吲唑氮原子上取代基由1-改至2-時(shí)，對(duì)腫瘤細(xì)胞抗腫瘤活性普適性更廣，活性更高。如5a,5b對(duì)所給腫瘤細(xì)胞均有較好抗腫瘤活性，達(dá)到低濃度抗腫瘤活性。因此，在今后的進(jìn)一步結(jié)構(gòu)優(yōu)化中，應(yīng)當(dāng)保留吲唑N2上環(huán)狀取代基，改變吡啶酮結(jié)構(gòu)上取代基，探索5-芳雜環(huán)對(duì)藥物活性的影響，找到活性優(yōu)異的新藥分子。

綜上，所合成的14個(gè)化合物中，部分化合物體外抗腫瘤活性好于陽(yáng)性對(duì)照化合物EPZ005687，這說(shuō)明二取代吲唑-3-羧酸酰胺類化合物可以作為先導(dǎo)化合物進(jìn)一步研究。

以吲唑-3-甲酸為起始原料，合成了14個(gè)新型的1/2-烴基-5-芳香基二取代吲唑-3-甲酰吡啶酮甲胺衍生物(5a～5n)并用MTT法研究了他們對(duì)人B淋巴瘤細(xì)胞(Ramos)、人黑色素瘤細(xì)胞(CHL-1, WM-266-4)、乳腺癌細(xì)胞(BT-474)的體外抗腫瘤活性。結(jié)果表明：5a,5b,5m對(duì)Ramos具有較好的抑制活性；5a,5b,5l對(duì)WM-266-4具有較好的抑制活性；5a,5b,5d,5e,5h,5j,5m,5n對(duì)CHL-1具有較好的抑制活性；5a,5b,5d,5h,5m對(duì)BT-474具有較好的抑制活性；IC50值均小于10.0 μmol·L-1，優(yōu)于陽(yáng)性對(duì)照物EPZ005687，說(shuō)明這類1/2-烴基-5-芳香基二取代吲唑-3-甲酰吡啶酮甲胺類化合物可以作為先導(dǎo)化合物進(jìn)行進(jìn)一步的抗腫瘤活性研究。

[1] Jakupec M A, Reisner E, Keppler B K,etal. Redox-active antineoplastic ruthenium complexes with indazole:Correlation ofinVitropotency and reduction potential[J].J Med Chem,2005,48(8):2831-2837.

[2] Wong TW, Lee FY, Vite GD,etal. Preclinical antitumor activity of BMS-599626,a pan-HER Kinase inhibitor that inhibits HER1/HER2 homodimer and heterodimer Signaling[J].Clin Cancer Res,2006,12(20):6186-6193.

[3] Duan JX, Cai XH, Matteucci M,etal. Potent antitubulin tumor cell cytotoxins based on 3-aroyl indazoles[J].J Med Chem,2007,50(5):1001-1006.

[4] 龍偉,邱文革,何宏,等. 新型吲唑類PARP-1抑制劑的合成及其生物活性評(píng)價(jià)[J].合成化學(xué),2016,24(4):277-282.

[5] 周云鵬,何暢,王洋,等. 新型吲唑類化合物的合成及其抗腫瘤活性[J].合成化學(xué),2016,24(4):293-296.

[6] Konze K D, Ma A, Li F,etal. An orally bioavailable chemical probe of the lysine methyltransferases EZH2 and EZH1[J].ACS chemical biology,2013,8(6):1324-1334.

[7] Verma S K, Tian X, LaFrance L V,etal. Identification of potent,selective,cell-active inhibitors of the histone lysine methyltransferase EZH2[J].ACS medicinal chemistry letters,2012,3(12):1091-1096.

[8] Knutson S K, Warholic N M, Wigle T J,etal. Durable tumor regression in genetically altered malignant rhabdoid tumors by inhibition of methyltransferase EZH2[J].Proceedings of the National Academy of Sciences,2013,110(19):7922-7927.

[9] Diaz E, Machutta C A, Chen S,etal. Development and validation of reagents and assays for EZH2 peptide and nucleosome high-throughput screens[J].Journal of biomolecular screening,2012,17(10):1279-1292.

[10] Zhang L D, Song X J, Yu L T,etal. Design,synthesis and biological evaluation of novel 1-methyl-3-oxo-2,3,5,6,7,8-hexahydroisoquinolins as potential EZH2 inhibitors[J].RSC Adv,2015(5):25967-25978.

[11] Song X J, Gao T T, Yu L T,etal. Selective inhibition of EZH2 by ZLD1039 blocks H3K27methylation and leads to potent anti-tumor activity in breast cancer[J].Sci Rep,2016(6):24893.

[12] Gao T T, Zhang L D, Yu L T,etal. ZLD1122,a novel EZH2 and EZH1 small molecular inhibitor, blocks H3K27 methylation and diffuse large B cell lymphoma cell growth[J].RSC Adv,2016,(6):28512-28521.

[13] Schmidt A, Snovydovych B, Habeck T,etal.N-heterocyclic carbenes of 5-haloindazoles generated by decarboxylation of 5-haloindazolium-3-carboxylates[J].Eu J Org Chem,2007(29):4909-4916.

[14] Migliorini A, Oliviero C, Gasperi T,etal. The Suzuki reaction applied to the synthesis of novel pyrrolyl and thiophenyl indazoles[J].Molecules,2012,(17):4508-4521.

Synthesis and Antitumor Activities of 1/2,5-Disubstituted Indazole-3-carboxamide Derivatives

FENG Qiang1,2, HE Hua-long1, GAO Tian-tao1, ZHU Yong-xia1, ZHANG Qiang-sheng1, LIU Zhi-hao1, HE Bing2, ZHANG Li-dan1, YU Luo-ting1*

(1. Lab of Medicinal Chemistry, Cancer Center, West China Hospital, Sichuan University and Collaborative Innovation Center, Chengdu 610041, China； 2. College of Chemistry and Life Science, Chengdu Normal University, Chengdu 611130, China)

Fourteen novel disubsitituted indazole-3-carboxamide derivatives(5a～5n) were synthesized from indazole-3-carboxylic acidviabromination, esterification,N-alkylation, amidation and Suzuki-coupling reactions in overall yields of 26%～32%. These products were characterized by1H NMR,13C NMR and HR-MS(ESI-TOF). We also examined the1H-1H NOE difference spectroscopy of3aand3band confirmed the exact substitution position of R on the N of indazole. Theinvitroantitumor activities against human B lymphoma cells(Ramos)， human melanoma cells(CHL-1， WM-266-4) and breast cancer cells(BT-474) have been demonstrated by MTT assays. The results showed that5a,5band5mexhibited good inhibition against Ramos;5a,5band5lexhibited excellent inhibition against WM-266-4;5a,5b,5d,5e,5h,5j,5mand5nshowed advanced inhibition against CHL-1;5a,5b,5d,5hand5mexhibited good inhibition against BT-474 with IC50<10.0 μmol·L-1, respectively.

indazole-3-carboxylic acid; disubsitituted indazole-3-carboxamide derivative; Suzuki coupling reaction; synthesis; antitumor activity

2017-05-09；

： 2017-07-28

國(guó)家自然科學(xué)基金資助項(xiàng)目(81602398); 中國(guó)博士后基金資助項(xiàng)目(2016T90861)

奉強(qiáng)(1974-)，男，四川內(nèi)江人，講師，博士研究生，主要從事藥物合成化學(xué)研究。 E-mail： 592744998@qq.com

余洛汀，教授，博士生導(dǎo)師， Tel. 028-85502796, E-mail： yuluot@scu.edu.cn

O626.21； O626.32

： ADOI： 10.15952/j.cnki.cjsc.1005-1511.2017.09.17108