陳湊喜, 李學(xué)強(qiáng),2, 馮 雷, 李天才, 周學(xué)章, 代 靜, 孫 健

(1. 寧夏大學(xué) 化學(xué)化工學(xué)院,寧夏 銀川 750021; 2. 寧夏天然藥物工程技術(shù)研究中心,寧夏 銀川 750021; 3. 西北大學(xué) 化學(xué)與材料科學(xué)學(xué)院,陜西 西安 710069; 4. 西部特色生物資源保護(hù)與利用教育部重點(diǎn)實(shí)驗(yàn)室,寧夏 銀川 750021)

18β-甘草次酸(9)作為甘草的主要藥理成分，具有抗炎[1]、抗氧化[2,3]，抗?jié)僛4]、保肝[5]、抗病毒[6,7]、抗癌[8～10]等活性；以芳香酸和1,3,4-噁二唑?yàn)槟负说碾s環(huán)化合物因其具有生物及藥理活性，被廣泛應(yīng)用于醫(yī)藥和農(nóng)藥制備[11～13]。

根據(jù)生物電子等排和活性亞結(jié)構(gòu)拼接理論[14]，本文嘗試以18β-甘草次酸-30-哌嗪(10)為先導(dǎo)化合物，通過(guò)酰胺鍵將不同結(jié)構(gòu)的雜環(huán)酸(1a～1g和2h～2n)與其串聯(lián)拼接，合成了14個(gè)含不同雜環(huán)的新型甘草次酸多酰胺衍生物(11a～11n),收率78.6%～92.3%,其結(jié)構(gòu)經(jīng)1H NMR,13C NMR和IR表征。

11i和11n的初步抑菌活性測(cè)試結(jié)果表明,雜環(huán)酰胺結(jié)構(gòu)對(duì)甘草次酸衍生物的抗結(jié)核活性具有明顯的增效作用。

1 實(shí)驗(yàn)部分

1．1 儀器與試劑

X-24型數(shù)字顯示顯微熔點(diǎn)儀(溫度未校正);Bruker Avance 400 MHz型核磁共振儀(CDCl3為溶劑,TMS為內(nèi)標(biāo));島津FTIR-8400S型紅外光譜儀(KBr壓片)。

9,純度99.6%,寧夏啟元藥業(yè)有限公司;1-乙基-3-二甲基氨基丙基)碳亞胺鹽酸鹽(EDCl), 1-羥基苯并三氮唑(HOBt),上海共價(jià)化學(xué)科技有限公司；其余所用試劑均為分析純或化學(xué)純，所用溶劑使用前均采用標(biāo)準(zhǔn)方法處理；常規(guī)色譜柱硅膠,青島海洋化工廠。

人結(jié)核分支桿菌標(biāo)準(zhǔn)株H37Rv(ATCC 27294)和牛結(jié)核分支桿菌標(biāo)準(zhǔn)株(ATCC 19210),上海市結(jié)核重點(diǎn)實(shí)驗(yàn)室惠贈(zèng)；Middlebrook 7H9肉湯,BD Difco公司；Alamar Blue solution, Trek Diagnostics公司；Tween-80,普洛麥格(北京)生物技術(shù)有限公司；HP 400GS智能人工CO2氣候培養(yǎng)箱。

1.2 合成

(1)2的合成(以2h為例)[15]

在三口瓶中加入苯甲酸(1a或3h) 200 mmol, 乙醇100 mL，催化量濃硫酸，攪拌下回流分水反應(yīng)得芳香甲酸乙酯(4h)，產(chǎn)率87%。

在三口瓶中加入4h100 mmol，乙醇80 mL，稍過(guò)量的水合肼，攪拌下回流反應(yīng)5 h～9 h。冷卻至室溫，抽濾，濾餅用乙醇重結(jié)晶得苯甲酰肼(5h)，產(chǎn)率90%。

將5h40 mmol和KOH 44 mmol溶于適量絕對(duì)無(wú)水乙醇，攪拌下緩慢滴加稍過(guò)量的CS2，滴畢，回流反應(yīng)至無(wú)H2S氣體產(chǎn)生為止。蒸去乙醇得苯酰肼基二硫代甲酸鉀(6h)，產(chǎn)率92%。

將6h30 mmol研成粉末，激烈攪拌下分批加至稀鹽酸(粉末質(zhì)量5倍量體積)中(控制溫度在0 ℃～5 ℃)，待其全部溶解后反應(yīng)10 min。慢慢倒入碎冰(粉末質(zhì)量50倍量體積)中，用氨水調(diào)節(jié)至pH 2～3(析出大量黃色晶體)，抽濾，濾餅用水洗滌，50 %乙醇重結(jié)晶，干燥得2-巰基-5-苯基-1,3,4-噁二唑(7h)，產(chǎn)率82%。

將7h20 mmol和KOH 40 mmol溶于混合溶劑[60 mL,V(MeOH) ∶V(H2O)=60 ∶40]中，加入氯乙酸20 mmol，攪拌下回流反應(yīng)3 h～5 h。冷卻，減壓蒸除多余的甲醇，殘余物用冰水(50 mL)稀釋,過(guò)濾,濾液用冷稀鹽酸調(diào)節(jié)至pH 2～3(產(chǎn)生大量沉淀)，抽濾，濾餅用水洗滌，50%乙醇多次重結(jié)晶，干燥得白色針狀晶體2h，收率83%。

用類似方法合成2i～2n，表征數(shù)據(jù)與文獻(xiàn)[15]值吻合。

(2)10的合成

在反應(yīng)瓶中加入絕對(duì)無(wú)水二氯甲烷50 mL, 9 1.0 g(2.12 mmo1), EDCl 767 mg(4 mmol)和HOBT 290 mg(2.12 mmol),攪拌使其完全溶解;滴加三乙胺1 mL，滴畢，于室溫活化2 h；加入無(wú)水哌嗪360 mg(4 mmol)，反應(yīng)至終點(diǎn)(TLC跟蹤)。傾入冰水(30 mL)中，用二氯甲烷(2×15 ml)萃取，合并萃取液,用飽和食鹽水洗滌，無(wú)水Na2SO4干燥，過(guò)濾，濾液濃縮后經(jīng)硅膠柱層析[洗脫劑: A=V(乙酸乙酯) ∶V(甲醇)=4 ∶1]分離得白色固體10，收率80.6%, m.p.244 ℃～246 ℃;1H NMRδ: 5.68(s, 1H, 12-H), 3.83(s, 3H, 31,34-H), 3.23(dd,J=5.6 Hz, 10.8 Hz, 1H, 3-H), 3.11(s, 3H, 32,33-H), 2.76(d,J=13.6 Hz, 1H, 18-H), 2.34(s, 1H, 9-H), 2.19～1.17(m, 16H, 2,6,7,15,16,19,21,22-H), 1.36(s, 3H, 27-H), 1.22(s, 3H, 30-H), 1.13(s, 3H, 25-H), 1.12(s, 3H, 26-H), 1.06～0.97(m, 2H, 1-H), 1.05(s, 3H, 23-H), 0.82(s, 3H, 24-H), 0.80(s, 3H, 28-H), 0.72～0.69(m, 1H, 5-H);13C NMRδ: 200.27(C11), 174.09(C30), 169.58(C13), 128.60(C12), 78.71(C3), 61.81(C9), 54.94(C5), 48.06(C18), 48.06(C31), 48.06(C34), 45.30(C8), 44.87(C33), 43.89(C32), 43.86(C20), 43.31(C14), 39.17(C19), 39.17(C4), 39.14(C1), 37.71(C22), 37.11(C10), 33.01(C7), 32.81(C17), 31.79(C21), 29.69(C28), 28.42(C29), 28.10(C23) 27.31(C2), 26.72(C16), 26.40(C15), 23.13(C27), 18.71(C26), 17.48(C6), 16.41(C25), 15.60(C24); IRν: 3 445(N-H), 3 347(O-H), 2 926, 2 920, 2 845, 1 742, 1 652(N-C=O), 1 525, 1 4 56, 1 382, 1 211, 1 054 cm-1。

(3)11的合成(以11a為例)

在三口燒瓶中將1a140 mmol， EDCl 32.2 mg(170 mmol)和HOBt 18.9 mg(140 mmol)溶于絕對(duì)無(wú)水二氯甲烷(5 mL)中，攪拌下滴加三乙胺0.1 mL，滴畢，于室溫反應(yīng)2 h;加入10 140 mmol，反應(yīng)至終點(diǎn)(TLC跟蹤)。傾入冰水(10 mL)中，用二氯甲烷(2×15 ml)萃取，合并萃取液,用無(wú)水Na2SO4干燥，濃縮后經(jīng)硅膠柱層析(梯度洗脫劑:A=2 ∶1～7 ∶1)純化得11a。

表 1 合成11的實(shí)驗(yàn)結(jié)果Table 1 Experimental results of synthesizing 11

用類似方法合成11b～11n。合成11的實(shí)驗(yàn)結(jié)果見(jiàn)表1， NMR數(shù)據(jù)見(jiàn)表2。

11h: IRν: 3 469, 2 930, 2 867, 2 362, 2 246, 1 742, 1 651, 1 558, 1 468, 1 411, 1 285, 1 189, 1 071, 1 017, 912, 774, 732 cm-1。

11i: IRν: 3 472, 2 937, 2 868, 2 355, 2 244, 1 646, 1 467, 1 405, 1 283, 1 188, 1 087, 1 012, 914, 840, 730 cm-1。

11j: IRν: 3 478, 2 931, 2 867, 2 362, 2 246, 1 651, 1 553, 1 483, 1 409, 1 251, 1 185, 1 126, 1 017, 913, 732 cm-1。

11k: IRν: 3 478, 2 931, 2 867, 2 362, 2 246, 1 651, 1 553, 1 483, 1 409, 1 251, 1 185, 1 126, 1 017, 913, 732 cm-1。

11l: IRν: 3 489, 2 932, 2 867, 2 481, 2 121, 1 967, 1 647, 1 521, 1 462, 1 346, 1 285, 1 186, 1 074, 1 016, 916, 858, 733 cm-1。

11m: IRν: 3 467, 2 929, 2 866, 2 245, 1 651, 1 614, 1 503, 1 469, 1 413, 1 259, 1 173, 1 071, 1 017, 994, 911, 839, 732 cm-1。

11n: IRν: 3 464, 2 928, 2 866, 2 244, 1 650, 1 460, 1 413, 1 370, 1 284, 1 209, 1 173, 1 070, 1 017, 912, 887, 732 cm-1。

1.3 抑菌活性測(cè)試

在平行條件下對(duì)比測(cè)試9,10,11i和11n對(duì)H37Rv, ATCC 27294和ATCC 19210的抑菌活性。在96孔細(xì)胞培養(yǎng)板外圍孔中加蒸餾水200 μL, Middlebrook 7H9肉湯100 μL 和接種菌懸液(包括無(wú)藥對(duì)照孔)100 μL。分別將藥液100 μL注入每排第一個(gè)孔，然后依次進(jìn)行系列二倍稀釋，每孔DMSO的最終體積濃度0.5%。同時(shí)設(shè)置1 ∶10和1 ∶100菌懸液的稀釋對(duì)照，用作10%和1%細(xì)菌污染檢測(cè)。檢測(cè)范圍200 μg·mL-1～3.12 μg·mL-1。

表 2 11的NMR數(shù)據(jù)Table 2 NMR data of 11

續(xù)表2

Comp1H NMR δ(J/Hz)13C NMR δ11c7.30～7.28(m, 1H), 7.26(s, 2H), 7.17～7.15(d, J=8.4, 1H), 5.68(s, 1H), 3.80～3.45(m, 8H), 3.22(dd, J=4.4, 10.4, 1H), 2.79(t, J=7.2, 1H), 2.35(s, 1H), 2.29～1.12(m, 16H), 1.36(s, 3H), 1.23(s, 3H), 1.12(s, 3H), 1.09(s, 3H), 1.05～0.97(m, 2H), 1.00(s, 3H), 0.82(s, 3H), 0.78(s, 3H), 0.72～0.68(m, 1H)200.03, 174.25, 169.14, 147.33, 130.71, 127.01, 125.93, 123.80, 122.96, 78.78, 61.81, 54.96, 52.19, 48.13, 47.33, 45.29, 43.91, 43.29, 39.37, 37.72, 37.09, 33.47, 32.83, 31.79, 30.04, 29.70, 29.66, 29.37, 28.41, 28.10, 24.47, 23.16, 22.70, 22.63, 21.03, 19.75, 18.69, 17.4911d7.39～7.36(m, 4H), 5.68(s, 1H), 3.67～3.33(m, 8H), 3.22(dd, J=5.6, 10.4, 1H), 2.79(t, J=7.2, 1H), 2.35(s, 1H), 2.33～1.13(m, 16H), 1.36(s, 3H), 1.22(s, 3H), 1.12(s, 3H), 1.05(s, 3H), 1.04～0.94(m, 2H), 1.00(s, 3H), 0.82(s, 3H), 0.80(s, 3H), 0.71～0.68(m, 1H)200.09, 174.33, 169.56, 136.25, 130.98, 128.84, 61.83, 54.98, 50.84, 48.25, 43.80, 43.20, 39.17, 39.14, 37.79, 37.71, 37.12, 33.23, 32.84, 32.79, 32.01, 31.81, 31.22, 29.69, 28.56, 28.41, 28.11, 28.04, 27.31, 27.05, 26.73, 26.43, 23.51, 23.16, 18.7111e7.51～7.50(m, J=2.4, 1H), 7.06～7.05(m, J=4.4, 1H), 6.51～6.50(m, J=5.6, 1H), 5.69(s, 1H), 3.82～3.66(m, 8H), 3.23(dd, J=5.6, 11.2, 1H), 2.79(t, J=7.6, 1H), 2.35(s, 1H), 2.31～1.12(m, 16H), 1.38(s, 3H), 1.25(s, 3H), 1.13(s, 3H), 1.12(s, 3H), 1.05～0.98(m, 2H), 1.01(s, 3H), 0.82(s, 3H), 0.80(s, 3H), 0.72～0.69(m, 1H)200.10, 174.31, 169.32, 159.25, 147.61, 143.97, 128.64, 117.12, 111.49, 61.82, 54.98, 50.78, 48.10, 45.30, 43.92, 43.31, 38.95, 37.74, 37.12, 33.08, 32.84, 31.90, 29.35, 28.11, 27.04, 26.42, 23.16, 22.68, 18.42, 17.50, 16.38, 15.59, 14.1211f7.41～7.38(d, J=11.6, 2H), 6.94～6.91(d, J=11.6, 2H), 5.68(s, 1H), 3.82～3.63(m, 8H), 3.22(dd, J=5.6, 11.2, 1H), 2.78(t, J=7.2, 1H), 2.34(s, 1H), 2.30～1.12(m, 16H), 1.36(s, 3H), 1.23(s, 3H), 1.12(s, 3H), 1.05(s, 3H), 1.04～0.97(m, 2H), 1.00(s, 3H), 0.82(s, 3H), 0.81(s, 3H), 0.71～0.68(m, 1H)200.09, 174.30, 170.66, 169.32, 161.07, 132.15, 127.12, 124.83, 113.88, 61.82, 55.39, 54.98, 50.84, 48.15, 45.31, 43.96, 43.20, 40.93, 39.14, 37.12, 32.84, 32.01, 31.80, 29.70, 28.56, 28.11, 27.31, 26.43, 23.16, 18.71, 17.50, 16.38, 15.5811g8.29～8.33(d, J=13.2, 2H), 7.62～7.59(d, J=13.2, 2H), 5.66(s, 1H), 3.85～3.53(m, 8H), 3.23(dd, J=4.4, 11.2, 1H), 2.79(t, J=7.2, 1H), 2.33(s, 1H), 2.31～1.13(m, 16H), 1.38(s, 3H), 1.25(s, 3H), 1.13(s, 3H), 1.12(s, 3H), 1.06～0.97(m, 2H), 1.01(s, 3H), 0.83(s, 3H), 0.80(s, 3H), 0.71～0.68(m, 1H)200.12, 174.39, 169.30, 168.24, 148.60, 141.26, 128.59, 128.20, 124.05, 61.85, 54.98, 50.83, 48.25, 47.63, 45.32, 43.73, 42.37, 39.14, 37.12, 33.30, 31.92, 29.36, 28.11, 27.05, 26.43, 23.16, 22.69, 21.42, 17.49, 16.3811h8.02(d, J=1.2, 2H), 7.53～7.47(m, 3H), 5.68(s, 1H), 4.37(s, 2H), 3.80～3.63(m, 8H), 3.22(dd, J=5.2, 10.8, 1H), 2.78(t, J=7.2, 1H), 2.33(s, 1H), 2.31～1.13(m, 16H), 1.36(s, 3H), 1.24(s, 3H), 1.12(s, 3H), 1.11(s, 3H), 1.04～0.97(m, 2H), 1.00(s, 3H), 0.81(s, 3H), 0.79(s, 3H), 0.71～0.68(m, 1H)200.02, 174.31, 169.19, 166.03, 165.11, 163.87, 136.86, 131.79, 129.07, 128. 65, 128.50, 126.73, 126.19, 123.42, 113.79, 78.76, 61.81, 54.95, 48.06, 46.19, 45.28, 43.96, 43.90, 43. 29, 42.41, 39.13, 37.72, 37.09, 36.44, 34.13, 33.01, 32.83, 31.80, 28.40, 28.10, 27.31, 27.06, 26.71, 26. 40, 23.17, 18.69, 17.49, 16.38, 15.59

續(xù)表2

Comp1H NMR δ(J/Hz)13C NMR δ11i7.94～7.91(m, 2H), 7.48～7.46(m, 2H), 5.66(s, 1H), 4.37(s, 2H), 3.80～3.62(m, 8H), 3.21(dd, J=5.2, 10.8, 1H), 2.76 (d, J=13.6, 1H), 2.32(s, 1H), 2.30～1.15(m, 16H), 1.35(s, 3H), 1.23(s, 3H), 1.14(s, 3H), 1.12(s, 3H), 1.04～0.97(m, 2H), 0.99(s, 3H), 0.81(s, 3H), 0.79(s, 3H), 0.70～0.67(m, 1H)200.04, 174.32, 169.22, 165.18, 164.99, 164.17, 138.05, 129.47, 129.43, 128.62, 128.49, 127.98, 126.18, 121.89, 78.73, 61.80, 54.94, 48.07, 46.16, 45.28, 44.86, 43.95, 43.86, 43.29, 42.64, 42.41, 39.15, 39.13, 37.71, 37.09, 36.55, 33.03, 32.82, 31.79, 29.68, 28.40, 28.10, 27.30, 27.05, 26.70, 26.40, 23.16, 18.69, 17.48, 16.38, 15.60, 14.2611j9.87～9.72(br, 1H), 7.72～7.70(m, 1H), 7.45～7.41(m, 1H), 7.11～7.09(m, 1H), 7.02～6.98(m, 1H), 5.67(s, 1H), 4.39(s, 2H), 3.77～3.61(m, 8H), 3.22(dd, J=5.6, 10.8, 1H), 2.77(d, J=13.6, 1H), 2.32(s, 1H), 2.30～1.13(m, 16H), 1.36(s, 3H), 1.24(s, 3H), 1.12(s, 3H), 1.11(s, 3H), 1.05～0.97(m, 2H), 1.00(s, 3H), 0.81(s, 3H), 0.80(s, 3H), 0.71～0.68(m, 1H)200. 06, 174.35, 169.21, 165.38, 164.75, 163.31, 157.17, 133.76, 128.65, 126.53, 120.05, 117.50, 78.77, 61.81, 54.95, 48.09, 46.14, 45.29, 43.97, 43.86, 43.29. 42.43, 39.13, 37.71, 37.09, 36.84, 33.06, 32.82, 31.81, 28.41, 28.10, 27.31, 27.07, 26.70, 26.40, 23.18, 18.69, 17.49, 16.38, 15.3811k9.09～8.88(br, 1H), 7.78(d, J=8.8, 2H), 6.91(d, J=8.4, 2H), 5.69(s, 1H), 4.24(q, J=12.6, 29.0, 2H), 3.79～3.56(m, 8H), 3.23(dd, J=5.2, 10.4, 1H), 2.76(d, J=13.2, 1H), 2.35(s, 1 H), 2.34～1.18(m, 16H), 1.36(s, 3H), 1.24(s, 3H), 1.17(s, 3H), 1.16(s, 3H), 1.04～0.97(m, 2H), 1.00(s, 3H), 0.81(s, 3H), 0.80(s, 3H), 0.71～0.68(m, 1H)200.06, 174.35, 169.21, 165.38, 164.75, 163.31, 157.17, 133.76, 128.65, 126.53, 120.05, 117.50, 107.77, 78.77, 61.81, 54.95, 48.09, 46.14, 45.29, 43.97, 43.86, 43.29, 42.43, 39.13, 37.71, 37.09, 36.84, 33.06, 32.82, 31.81, 28.41, 28.10, 27.31, 27.07, 26.70, 26.40, 23.18, 18.69, 17.49, 16.38, 15.3811l8.37(d, J=8.8, 2H), 8.20(d, J=8.8, 2H), 5.67(s, 1H), 4.41(s, 2H), 3.79～3.63(m, 8H), 3.21(dd, J=5.4, 10.6, 1H), 2.77(d, J=13.2, 1H), 2.33(s, 1H), 2.26～1.17(m, 16H), 1.36(s, 3H), 1.24(s, 3H), 1.12(s, 3H), 1.11(s, 3H), 1.05～0.97(m, 2H), 1.00(s, 3H), 0.82(s, 3H), 0.80(s, 3H), 0.71～0.68(m, 1H)200.06, 174.35, 169.23, 165.63, 164.77, 164.23, 149.53, 128.87, 128.63, 127.63, 124.43, 78.77, 61.83, 54.96, 48.15, 46.20, 45.30, 43.97, 43.82, 43.30, 42.48, 39.17, 39.13, 37.71, 37.10, 36.81, 33.15, 32.83, 31.82, 29.69, 28.41, 28.10, 27.31, 27.08, 26.72, 26.42, 23.18, 22.70. 18.70, 17.49, 16.38, 15.5811m7.92(d, J=8.4, 2H), 6.98(d, J=8.4, 2H), 5.67(s, 1H), 4.35(s, 2H), 3.87(s, 3H), 3.76～3.62(m, 8H), 3.21(dd, J=5.6, 10.6, 1H), 2.77(d, J=13.2, 1H), 2.33(s, 1H), 2.28～1.17(m, 16H), 1.36(s, 3H), 1.23(s, 3H), 1.12(s, 3H), 1.11(s, 3H), 1.04～0.97(m, 2H), 0.99(s, 3H), 0.81(s, 3H), 0.79(s, 3H), 0.70～0.67(m, 1H)200.01, 174.30, 169.18, 166.00, 165.20, 163.06, 162.37, 128.66, 128.53, 115.92, 114.50, 78.77, 61.81, 55.47, 54.96, 48.05, 46.21, 45.28, 43.95, 43.91, 43.29, 42.41, 39.13, 37.72, 37.09, 36.36, 33.01, 32.83, 31.80, 29.70, 28.40, 28.10, 27.31, 27.07,26.71, 26.41, 23.18, 18.69, 17.49, 16.38, 15.58

續(xù)表2

Comp1H NMR δ(J/Hz)13C NMR δ11n7.56(d, J=1.6, 1H), 7.12(d, J=3.6, 1H), 6.57(q, J=1.8, 3.4, 1H), 5.68(s, 1H), 4.40(s, 2H), 3.79～3.65(m, 8H), 3.22(dd, J=5.6, 10.8, 1H), 2.77(d, J=13.6, 1H), 2.33(s, 1H), 2.28～1.14(m, 16H), 1.36(s, 3H), 1.24(s, 3H), 1.13(s, 3H), 1.11(s, 3H), 1.04～0.97(m, 2H), 1.00(s, 3H), 0.81(s, 3H), 0.80(s, 3H), 0.71～0.68(m, 1H)200.03, 174.29, 169.21, 165.58, 163.30, 145.04, 145.01, 128.65, 112.52, 111.53, 78.78, 61.80, 54.96, 48.05, 46.27, 45.29, 43.95, 43.29, 42.39, 39.13, 37.72, 37.09, 36.58, 33.00, 32.83, 31.80, 28.40, 28.10, 27.31, 27.06, 26.72, 26.41, 23.17, 18.69, 17.49, 16.38, 15.58

將培養(yǎng)板置于密封袋中，在5%CO2氣氛下于37 ℃孵育5 d;在對(duì)照孔中加入Alamar blue solution 10 μL和高壓滅菌10 %Tween-80 12 μL，于37 ℃孵育24 h。若對(duì)照組變?yōu)榉奂t色，說(shuō)明菌體生長(zhǎng)，即為陽(yáng)性孔，可將染料加到平板剩余孔中，于37 ℃再孵育24 h。粉紅色記錄為陽(yáng)性生長(zhǎng)孔。最低抑菌濃度(MIC)定義為阻止顏色變?yōu)榉奂t色的最低藥物濃度。MIC值測(cè)定重復(fù)3次，結(jié)果見(jiàn)表3。

表 3 化合物的抗結(jié)核菌活性Table 3 Anti-tuberculosis activities of compounds

2 結(jié)果與討論

在11的合成過(guò)程中，多步酰胺化反應(yīng)均在EDCl和HOBt高效酰胺鍵形成促進(jìn)劑催化下進(jìn)行,這樣不僅有效地克服了10中五環(huán)三萜骨架的空間位阻影響，而且反應(yīng)時(shí)間短、后處理簡(jiǎn)便、可重復(fù)性強(qiáng)、收率高。

在抗結(jié)核藥物的研發(fā)過(guò)程中，普遍面臨藥物副作用大、易產(chǎn)生耐藥性等缺陷。特別是近年來(lái)耐藥及多重耐藥菌株的出現(xiàn),更是對(duì)現(xiàn)有的臨床用抗結(jié)核藥物提出了嚴(yán)峻挑戰(zhàn)。為此，我們隨機(jī)選取11i和11n測(cè)試其相關(guān)抗結(jié)核菌活性，初步研究發(fā)現(xiàn)11i和11n對(duì)ATCC 27294和ATCC 19210均有不同程度的抑制效果，其中11i對(duì)ATCC 27294的MIC僅為22 μg·mL-1(具有抗菌活性的化合物MIC ≤200 μg·mL-1)。

盡管相對(duì)商業(yè)化藥物異煙肼(MIC=0.025 μg·mL-1)和利福平(MIC=0.10 μg·mL-1)，11i和11n的MIC還是偏高，但是實(shí)驗(yàn)過(guò)程發(fā)現(xiàn)它們對(duì)正常肺細(xì)胞在所測(cè)藥物濃度范圍內(nèi)基本沒(méi)有毒性，而且在后續(xù)試驗(yàn)中持續(xù)表現(xiàn)出良好的抑菌效果，未出現(xiàn)顯著耐藥現(xiàn)象。因此，我們認(rèn)為通過(guò)這樣的結(jié)構(gòu)修飾改造得到的11對(duì)于抑制結(jié)核分支桿菌活性具有一定的功效，具備發(fā)展成為新型治療結(jié)核病藥物的潛力。這將為新型作用機(jī)制下的抗結(jié)核藥物研發(fā)提供很好的思路，對(duì)于抗結(jié)核中藥現(xiàn)代化研究也具有重要的理論指導(dǎo)意義，值得進(jìn)一步研究與探討。

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