新型3-五元碳環螺環氧化吲哚類化合物的合成及其抗人白血病細胞活性

陳智勇, 田民義, 楊 俊, 劉歡歡, 林 冰, 潘玉杰, 劉雄利

(貴州大學 貴州省中藥民族藥創制工程中心，貴州 貴陽 550025)

·研究論文·

新型3-五元碳環螺環氧化吲哚類化合物的合成及其抗人白血病細胞活性

陳智勇, 田民義, 楊 俊, 劉歡歡, 林 冰*, 潘玉杰, 劉雄利

(貴州大學 貴州省中藥民族藥創制工程中心，貴州 貴陽 550025)

以氧化吲哚與鄰芳基二甲醛為原料，經Knoevenagel縮合(或Michael，環化反應)，制得7個3-五元碳環螺環氧化吲哚(4a～4c,產率67%～86%,d/r值4 ∶1～10 ∶1)和4d～4g; 4d～4g與哌啶(或四氫吡咯)和多聚甲醛經胺甲基化反應，合成了4個3-五元碳環螺環氧化吲哚(5d～5g),產率55%～67%,d/r值10 ∶1～>20 ∶1,其結構經1H NMR,13C NMR和HR-MS(ESI-TOF)表征。采用MTT法研究了4a～4c和5d～5g對人白血病細胞(K562)的體外抗腫瘤活性。結果表明：4b, 5d和5f對K562抑制活性較好,IC50分別為29.3 μmol·L-1, 27.4 μmol·L-1和34.2 μmol·L-1，與陽性對照藥順鉑(26.8 μmol·L-1)相當。

氧化吲哚; 3-五元碳環螺環氧化吲哚類化合物; Knoevenagel縮合反應; 胺甲基化反應; 合成; 抗腫瘤活性

3-五元碳環螺環氧化吲哚骨架廣泛存在于天然產物和合成藥物分子中，吸引了諸多化學工作者和醫藥研發團隊的廣泛關注。如天然產物marcfortine A, B; paraherquamide A～E; notoamide A～N和sclerotiamide等具有抗炎、抗癌、抗微生物等多種藥理作用[1-4]。目前，已有文獻[5-11]報道了其合成方法。

本課題組主要從事3-季碳氧化吲哚骨架化合物的設計、合成及其生物活性的研究[12-14]。本文在課題組前期工作的基礎上，以氧化吲哚(1a～1g)與鄰芳基二甲醛(2, 2′)為原料，經Knoevenagel縮合(或Michael，環化反應)，制得7個3-五元碳環螺環氧化吲哚(4a～4c,產率67%～86%,d/r值4 ∶1～10 ∶1)和4d～4g; 4d～4g與哌啶[3或四氫吡咯(3′)]和多聚甲醛經胺甲基化反應，合成了4個3-五元碳環螺環氧化吲哚(5d～5g, Scheme 1),產率55%～67%,d/r值10 ∶1～>20 ∶1,其結構經1H NMR,13C NMR和HR-MS(ESI-TOF)表征。采用MTT法研究了4a～4c和5d～5g對人白血病細胞(K562)的體外抗腫瘤活性。

1 實驗部分

1.1 儀器與試劑

WRS-1B型數字熔點儀；Bruker-400 MHz型核磁共振儀(CD3Cl或DMSO-d6為溶劑，TMS為內標); MicroTMQ-TOF型高分辨質譜儀。

所用試劑均為分析純。

1.2 合成

(1) 4a～4c的合成(以4a為例)

攪拌下，在反應管中依次加入N-甲基-5-氟氧化吲哚(1a)148.5 mg(0.90 mmol), 2 40.2 mg(0.3 mmol), 10 mol%3和MeOH 6.0 mL，回流反應16 h。經硅膠柱層析[洗脫劑：A=V(石油醚)：V(乙酸乙酯)=4 ∶1～2 ∶1]純化得白色固體3-五元碳環螺環氧化吲哚(4a)115.1 mg。

用類似的方法合成白色固體4b和4c。

Scheme 1

4a: m.p.243.5～244.1 ℃;1H NMRδ(major+minor): 2.55(s, 3H, major), 2.69(s, 3H, major), 2.78(s, 0.8H, minor), 3.14(s, 0.8H, minor), 4.43～4.50(m, 1.3H, major+minor), 4.56～4.60(m, 1.3H, major+minor), 5.33～5.37(m, 1.3H, major+minor), 5.69～5.73(m, 1.3H, major+minor), 6.23(dd,J=8.2 Hz, 1.8 Hz, 1H, major), 6.59～6.62(m, 2.6H, major+minor), 6.81～6.86(m, 0.6H, minor), 6.93～7.08(m, 3.3H, major+minor), 7.36～7.56(m, 4H, major+minor), 7.70～7.76(m, 1.6H, major+minor)；13C NMRδ(major+minor): 25.7, 26.0, 26.8, 43.5, 50.2, 66.8, 79.1, 79.8, 108.0, 108.1, 108.2, 108.3, 113.1, 113.3, 114.3, 114.6, 114.7, 114.9, 115.0, 115.3, 123.5, 126.1, 126.5, 127.2, 127.6, 127.7, 128.7, 138.4, 140.6, 140.9, 143.1, 156.2, 157.0, 174.6, 178.1; HR-MS(ESI-TOF)m/z: Calcd for C26H20N2O3F2Na{[M+Na]+}469.134 0, found 469.134 2。

4b: m.p.223.8～224.2 ℃;1H NMRδ(major+minor): 1.92(s, 3.3H, major+minor), 1.98(s, 3.3H, major+minor), 4.59(s, 2.2H, major+minor), 4.69(d,J=16.0 Hz, 1H, major), 4.94(d,J=16.0 Hz, 1H, major), 5.45(d,J=8.1 Hz, 1H, major), 5.76～5.81(m, 1.1H, major+minor), 6.41(d,J=8.4 Hz, 1.1H, major+minor), 6.67(t,J=8.4 Hz, 1.1H, major+minor), 6.87～6.96(m, 5.5H, major+minor), 7.04～7.11(m, 3.3H, major+minor), 7.18～7.25(m, 6.6H, major+minor), 7.36(s, 3.3H, major+minor), 7.66～7.69(m, 0.3H, minor), 7.72～7.76(m, 1.3H, major+minor)；13C NMRδ: 18.4, 52.0, 65.3, 80.3, 118.4, 123.3, 125.5, 125.9, 126.2, 126.6, 127.3, 129.0, 132.0, 132.3, 138.3, 138.6, 138.8, 141.5, 143.9, 176.0, 179.9；HR-MS(ESI-TOF)m/z: Calcd for C40H34N2O3Na{[M+Na]+}613.246 7, found 613.246 9。

4c: m.p.188.4～188.9 ℃;1H NMRδ(major+minor): 3.93～4.03(m, 1.2H, major+minor), 4.14～4.25(m, 3.6H, major+minor), 4.43(d,J=16.0 Hz, 1.2H, major+minor), 4.71(d,J=16.4 Hz, 1.2H, major+minor), 4.93(d,J=16.1 Hz, 1.2H, major+minor), 5.07(d,J=16.2 Hz , 1.2H, major+minor), 6.02～6.14(m, 3.1H, major+minor), 6.55～6.57(m, 2H, major), 6.66～6.70(m, 1.2H, major+minor), 6.79～6.83(m, 1.2H, major+minor), 6.93～6.96(m, 1.2H, major+minor), 7.08～7.18(m, 5.7H, major+minor), 7.27～7.35(m, 6.2H, major+minor), 7.44～7.46(m, 2.4H, major+minor), 7.56(s, 1.2H, major+minor), 7.66～7.73(m, 2H, major), 8.04(s, 1H, major)；13C NMRδ(major+minor): 11.3, 14.0, 14.4, 19.1, 22.9, 23.7, 28.8, 30.5, 43.1, 48.3, 50.7, 65.5, 82.9, 124.6, 127.0, 127.7, 127.9, 128.9, 129.1, 132.0, 135.9, 136.6, 145.2, 176.0, 177.7; HR-MS(ESI-TOF)m/z: Calcd for C38H28N2O3F2Na{[M+Na]+}621.196 6, found 621.197 1。

(2) 5d～5g的合成(以5d為例)

攪拌下，在反應管中依次加入N-苯基氧化吲哚(1d) 188.2 mg(0.90 mmol), 2 40.2 mg(0.3 mmol), 10 mol%3和MeOH 6.0 mL，回流反應16 h。經硅膠柱層析[洗脫劑：V(正己烷) ∶V(乙酸乙酯)=4 ∶1～2 ∶1]純化得4d。加入3 51 mg(0.6 mmol)的EtOAc(6.0 mL)溶液和多聚甲醛36 mg(1.2 mmol),于70 ℃(浴溫)反應24 h。經硅膠柱層析(洗脫劑：A=3 ∶1～2 ∶1)純化得白色固體5d 104.2 mg。

用類似的方法合成白色固體5e, 5f和淡黃色油狀液體5g。

5d: m.p.133.6～134.4 ℃;1H NMRδ: 1.05～1.15(m, 6H), 1.77(d,J=8.1 Hz, 1H), 1.87(m, 2H), 2.25(m, 2H), 3.06～3.16(m, 2H), 4.14(s, 1H), 5.51(d,J=8.2 Hz, 1H), 5.96～5.98(m, 1H), 6.20(d,J=8.4 Hz, 1H), 6.67～6.75(m, 4H), 7.06～7.10(m, 2H), 7.30～7.40(m, 12H), 7.49～7.55(m, 5H);13C NMRδ: 24.0, 26.7, 29.8, 55.1, 56.6, 57.4, 65.1, 80.6, 108.9, 109.5, 121.7, 122.4, 122.6, 122.9, 124.6, 127.4, 127.7, 128.0, 128.3, 128.7, 129.0, 129.1, 129.3, 129.4, 130.3, 130.5, 134.6, 135.2, 140.6, 144.0, 145.6, 146.4, 174.8, 178.4; HR-MS(ESI-TOF)m/z: Calcd for C42H37N3O3Na{[M+Na]+}654.273 3, found 654.273 3。

5b: m.p.215.0～216.1 ℃;1H NMRδ(major+minor): 1.47～1.52(m, 5.5H, major+minor), 2.09～2.13(m, 2.2H, major+minor), 2.16(s, 3.3H, major+minor), 2.37(s, 3.3H, major+minor), 2.42～2.44(m, 2.2H, major+minor), 3.10～3.17(m, 1.1H, major+minor), 3.31～3.46(m, 1.1H, major+minor), 3.63(m, 1H, major), 4.56(s, 1.1H, major+minor), 4.93(d,J=16.1 Hz, 1.1H, major+minor), 5.04～5.13(m, 1.2H, major+minor), 5.49(d,J=12.4 Hz, 1H, major), 5.92～5.96(m, 1.1H, major+minor), 6.08～6.17(m, 1.1H, major+minor), 6.21～6.23(m, 0.3H, minor), 6.34(s, 1H, major), 6.42(m, 0.1H, minor), 6.56～6.59(m, 0.1H, minor), 6.66～6.71(m, 0.1H, minor), 6.79～6.84(m, 2.5H, major+minor), 7.10～7.25(m, 14.5H, major+minor), 7.32～7.64(m, 5.5H, major+minor), 8.23(s, 1.1H, major+minor)；13C NMRδ(major+minor): 21.2, 21.3, 21.4, 24.0, 43.0, 43.2, 52.2, 55.2, 56.4, 64.3, 67.5, 81.7, 107.7, 108.8, 123.2, 124.1, 125.7, 126.7, 127.0, 127.1, 127.4, 127.7, 128.3, 128.7, 129.4, 129.5, 130.1, 130.8, 135.7, 136.2, 137.8, 141.4, 141.7, 142.9, 177.5, 178.2; HR-MS(ESI-TOF)m/z: Calcd for C45H43N3O3Na{[M+Na]+}696.320 2, found 696.320 7。

5c: m.p.218.1～219.36 ℃;1H NMRδ: 1.54～1.57(m, 4H), 1.85(d,J=12.0 Hz, 1H), 2.04(s, 3H), 2.34～2.37(m, 5H), 2.48～2.51(m, 5H), 2.68(s, 3H), 3.43～3.54(m, 2H), 4.80(s, 1H), 5.52(d,J=12.4 Hz, 1H), 6.13～6.17(m, 2H), 6.27(d,J=8.2 Hz, 1H), 6.85(d,J=8.4 Hz, 2H), 7.21(s, 1H), 7.46～7.50(m, 2H), 7.76(s, 1H), 7.80～7.84(m, 1H), 7.97～7.99(m, 1H), 8.60(s, 1H);13C NMRδ: 21.1, 21.4, 24.2, 25.3, 25.9, 51.4, 54.9, 56.5, 63.8, 67.3, 81.02, 106.5, 107.5, 121.6, 124.1, 125.7, 125.8, 126.0, 127.6, 127.7, 127.9, 128.1, 129.1, 129.3, 129.7, 130.2, 130.9, 133.0, 133.5, 135.8, 141.7, 141.8, 142.6, 177.1, 177.8; HR-MS(ESI-TOF)m/z: Calcd for C37H37N3O3Na{[M+Na]+}594.273 3, found 594.273 5。

5d:1H NMRδ: 1.32～1.36(m, 4H), 2.03(s, 3H), 2.35(m, 7H), 2.50～2.55(m, 5H), 2.69(s, 3H), 3.01(d,J=16.4 Hz, 1H), 3.38(d,J=12.2 Hz, 1H), 4.82(s, 1H), 5.54(s, 1H), 6.14(d,J=8.1 Hz, 1H), 6.25(d,J=8.3 Hz, 1H), 6.85(d,J=8.2 Hz, 2H), 7.30(s, 1H), 7.46～7.51(m, 2H), 7.77～7.83(m, 2H), 7.98(d,J=8.7 Hz, 1H), 8.59(s, 1H);13C NMRδ: 11.4, 21.0, 21.4, 24.0, 25.2, 25.9, 26.5, 46.1, 50.6, 54.8, 56.8, 64.9, 67.3, 81.0, 106.4, 107.5, 121.6, 124.1, 125.7, 125.8, 126.0, 127.5, 128.0, 129.1, 129.2, 129.3, 130.0, 130.8, 133.0, 133.5, 136.1, 141.6, 142.0, 142.7, 177.2, 177.8; HR-MS(ESI-TOF)m/z: Calcd for C38H39N3O3Na{[M+Na]+}608.288 9, found 608.289 1。

1.3 體外抗人白血病細胞活性測試

采用MTT法[15-16]測試了4a～4c和5d～5g對人白血病細胞(K562)的體外抗腫瘤活性,以順鉑為陽性對照藥。

K562用RPMI-1640培養基(含10%胎牛血清，100 U·mL-1青霉素和100 U·mL-1鏈霉素)培養。細胞以每孔5 000個的濃度加至96孔中，于37 ℃在含5%CO2潮濕空氣的培養箱中培養24 h。依次將新配4a～4c和5d～5g的二甲基亞砜溶液以濃度梯度加入到各孔中，使孔中化合物的終濃度為6 μmol·L-1, 12 μmol·L-1, 25 μmol·L-1, 50 μmol·L-1和100 μmol·L-1。靜置48 h，每孔加入5 mg·mL-1MTT的磷酸鹽緩沖液10 μL，于37 ℃培養4 h。離心5 min除去未轉化的MTT，每孔加入二甲基亞砜150 μL。用酶標儀在波長490 nm處測定吸光度A490。半抑制濃度(IC50)由IBM-SPSS(19.0)軟件分析得到。

2 結果與討論

2. 1 合成

通過底物擴展，我們發現該反應的活性較高。第一步發生Knoevenagel縮合(Michael或環化反應)生成4, 16 h內基本反應完全(TLC檢測)。TLC顯示反應有少量副產物產生，但NMR和MS分析沒有發現有效的結構信息，因此可能為分解產物或聚合物。第二步胺甲基化反應生成5的反應活性也較高，以乙酸乙酯為溶劑，幾乎沒有羥基化副產物生成。

2.2 抗人白血病細胞活性

表1為4a～4c和5d～5g對K562的體外抗人白血病細胞活性。由表1可見，4b, 5d和5f對K562的抑制活性較好, IC50分別為29.3 μmol·L-1, 27.4 μmol·L-1和34.2 μmol·L-1，與陽性對照藥順鉑(26.8 μmol·L-1)相當,可作為先導化合物骨架進一步研究。

表1 4a～4c和5d～5g的體外抗人白血病細胞活性Table 1 In vitro activities of 4a～4c and 5d～5g against human leukemia cells

合成了7個新型的3-五元碳環螺環氧化吲哚類化合物(4a～4c和5d～5g)。并用MTT法研究了4a～4c和5d～5g對人白血病細胞(K562)的體外抑制活性。結果表明：4b, 5d和5f對K562抑制活性較好,IC50分別為29.3 μmol·L-1, 27.4 μmol·L-1和34.2 μmol·L-1，與陽性對照藥順鉑(26.8 μmol·L-1)相當,可作為先導化合物骨架進一步研究。其他相關藥理活性的研究正在進行中。

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Synthesis of Novel Five-membered Carbocyclic Spirooxindoles and Their Activities Against Human Leukemia Cells

CHEN Zhi-yong, TIAN Min-yi, YANG Jun, LIU Huan-huan,LIN Bing*, PAN Yu-jie, LIU Xiong-li

(Gui zhou Engineering Center for Innovative Traditional Chinese Medicine and Ethnic Medicine,Guizhou University, Guiyang 550025, China)

Seven novel five-membered carbocyclic spirooxindoles(4a～4c and 4d～4g) were prepared by knoevenagel condensation(or Michael, cyclization), using oxindole ando-aryldicarboxaldehyde as the materials. The yields andd/rof 4a～4c were 67%～86% and 4 ∶1～10 ∶1, respectively. Four five-membered carbocyclic spirooxindoles(5d～5g) were synthesized by the aminomethylation reaction of 4d～4g with piperidine(or pyrrdidine) and paraformaldehyde. The yields andd/rof 5d～5g were 55%～67% and 10 ∶1～>20 ∶1, respectively. The structures were characterized by1H NMR,13C NMR and HR-MS(ESI-TOF). Theinvitroantitumor activities against human leukemia cells(K562) were demonstrated by MTT assays. The results showed that 4b, 5d and 5f showed best activities equipotent than the positive control of Cisplatin(26.8 μmol·L-1), with IC50of 29.3 μmol·L-1, 27.4 μmol·L-1and 34.2 μmol·L-1, respectively.

oxindole; five-membered carbocyclic spirooxindole; Knoevenagel condensation; aminomethylation reaction; synthesis; antitumor activity

2016-10-16

國家自然科學基金地區基金資助項目(81560563, 81660576)； 貴州省教學改革創新項目(SJJG201423)； 研究生創新項目(黔教研合JG字[2016]06); 貴州省中藥現代化科技產業研究開發專項項目(黔科合中藥字【2011】5080號)

陳智勇(1988-)，男，漢族，貴州貴陽人，碩士研究生，主要從事天然活性物質的全合成及結構修飾的研究。 E-mail: 253259908@qq.com

林冰，副教授，碩士生導師, E-mail: nlin@gzu.edu.cn

O626.13; O623.7

A

10.15952/j.cnki.cjsc.1005-1511.2017.02.16260