含二氟甲基的螺[環(huán)丙烷?1,3′?吲哚啉]?2′?酮類化合物和藥物用途的制作方法

本發(fā)明涉及化學(xué)技術(shù)領(lǐng)域，尤其是一種含二氟甲基的螺[環(huán)丙烷-1,3'-吲哚啉]-2'-酮類化合物和藥物用途。

背景技術(shù)：

螺環(huán)氧化吲哚結(jié)構(gòu)單元廣泛存在于許多天然分子和臨床藥物分子中，在已發(fā)現(xiàn)的含有3,3'-螺環(huán)結(jié)構(gòu)的氧化吲哚類化合物中，很多都具有較高的生物活性，諸如抗腫瘤、抗病毒、抗hiv、抗氧化性等。其中，含有氧化吲哚螺環(huán)環(huán)丙烷結(jié)構(gòu)單元的分子更加引人注目，由于該類化合物可以作為黃體酮受體拮抗劑、rsv抑制劑、激酶抑制劑、抗腫瘤藥物等。

在另一方面，二氟甲基(-cf2h)，作為一個重要的含氟基團，含有一個弱酸化的c-h鍵，是羥基、巰基及羥甲基的電子等排體，能夠調(diào)節(jié)分子的生物活性、代謝穩(wěn)定性以及親脂性等，在醫(yī)藥、農(nóng)藥等領(lǐng)域起著與其他氟烷基同樣重要的作用。因此，在分子中引入二氟甲基可賦予其諸多優(yōu)良性能，同時引入或保持了生物靶點的關(guān)鍵識別元素。這些決定了其在生物醫(yī)藥、生物成像等領(lǐng)域的巨大潛力和科研價值。

鑒于氧化吲哚螺環(huán)環(huán)丙烷化合物具有潛在的生物活性以及二氟甲基在藥物化學(xué)中的重要作用，因此，以二氟甲基重氮甲烷為砌塊，通過[3+2]反應(yīng)和縮環(huán)反應(yīng)策略與3-烯基氧化吲哚反應(yīng)，合成含有二氟甲基基團的3,3'-氧化吲哚螺環(huán)環(huán)丙烷可能會產(chǎn)生一系列結(jié)構(gòu)和活性上有意義的新化合物，它們的合成可以為含二氟甲基的螺[環(huán)丙烷-1,3'-吲哚啉]-2'-酮類化合物在藥物發(fā)現(xiàn)和成藥性評價中的應(yīng)用建立物質(zhì)基礎(chǔ)。

技術(shù)實現(xiàn)要素：

本發(fā)明的目的在于提供一種含二氟甲基的螺[環(huán)丙烷-1,3'-吲哚啉]-2'-酮類化合物及其制備防治腫瘤疾病藥物的應(yīng)用，該類化合物是一類重要的藥物活性分子，對藥物篩選和制藥行業(yè)具有重要的應(yīng)用價值，并且該類分子的合成具有反應(yīng)條件溫和、操作簡單、底物普適性好的優(yōu)點。

本發(fā)明是這樣實現(xiàn)的：原位生成的二氟甲基重氮甲烷與靛紅衍生的3-烯基氧化吲哚在無催化劑條件下，在有機溶劑中進行[3+2]反應(yīng)和縮環(huán)反應(yīng)，獲得含二氟甲基的螺[環(huán)丙烷-1,3'-吲哚啉]-2'-酮類化合物。該類化合物具有如通式(ⅰ)所示的結(jié)構(gòu)：

式中，r¹為烷基、烷氧基或鹵素，r²為烷基，r³為烷基、芳基或?；?/p>

所述的有機溶劑為二氯甲烷、三氯甲烷、四氯化碳、乙腈、四氫呋喃、苯、甲苯、二甲苯、均三甲苯、1,4-二氧六環(huán)、乙二醇二甲醚、乙二醇二乙醚或甲基叔丁基醚。

原位生成的二氟甲基重氮甲烷與靛紅衍生的3-烯基氧化吲哚的反應(yīng)溫度為25-130℃，反應(yīng)時間為1-50小時。

所述的含二氟甲基的螺[環(huán)丙烷-1,3'-吲哚啉]-2'-酮類化合物在制備防治腫瘤疾病藥物的應(yīng)用。

本發(fā)明的反應(yīng)原理如下：

其中，r¹，r²，r³和有機溶劑如上所述。

通過采用上述技術(shù)方案，二氟乙胺(1)放置于反應(yīng)瓶中，依次加入有機溶劑、亞硝酸叔丁酯和乙酸，并在70℃條件下反應(yīng)10分鐘，停止加熱，即可原位得到二氟甲基重氮甲烷(2)。然后將靛紅衍生的3-烯基氧化吲哚(3)加入反應(yīng)瓶中，經(jīng)過[3+2]反應(yīng)和縮環(huán)反應(yīng)，合成了一系列含二氟甲基的螺[環(huán)丙烷-1,3'-吲哚啉]-2'-酮類化合物(4)。該類化合物含有潛在生物活性的二氟甲基基團和3,3'-氧化吲哚螺環(huán)環(huán)丙烷骨架，可以為藥物發(fā)現(xiàn)和成藥性評價建立物質(zhì)基礎(chǔ)，具有重要的應(yīng)用價值。本發(fā)明原料易于獲得，可以在各種有機溶劑中進行，具有反應(yīng)條件溫和、操作簡單、底物普適性好的優(yōu)點。

附圖說明

附圖1為本發(fā)明的實施例化合物4a的單晶-x射線衍射結(jié)構(gòu)。

附圖2為本發(fā)明的實施例化合物4g的單晶-x射線衍射結(jié)構(gòu)。

附圖3為本發(fā)明的實施例化合物4r的單晶-x射線衍射結(jié)構(gòu)。

附圖4-6為本發(fā)明的實施例化合物4a的核磁共振譜圖。

具體實施方式

下面結(jié)合實施例及附圖進一步介紹本發(fā)明，但本發(fā)明不僅限于下述實施例，可以預(yù)見本領(lǐng)域技術(shù)人員在結(jié)合現(xiàn)有技術(shù)的情況下，實施情況可能產(chǎn)生種種變化。

本發(fā)明的實施例1：含二氟甲基的螺[環(huán)丙烷-1,3'-吲哚啉]-2'-酮(4)的制備；化合物4a：在圓底燒瓶中，依次加入二氯甲烷(15ml)，二氟乙胺(1,97.3mg,1.2mmol)，亞硝酸叔丁酯(148.5mg,1.44mmol)，乙酸(14.5mg,0.24mmol)，在70℃條件下反應(yīng)10分鐘，停止加熱，即可原位制備獲得二氟甲基重氮甲烷(2)。再加入靛紅衍生的3-烯基氧化吲哚(3a，92.5mg,0.4mmol)，并在室溫下充分攪拌48小時，濃縮，再加入甲苯(15ml)，120℃條件下反應(yīng)2小時，薄層色譜檢測反應(yīng)完畢后，硅膠柱層析(300-400目)分離(石油醚：乙酸乙酯＝5：1)，得到淺黃色固體103.9mg，產(chǎn)率88％，熔點：88.9–90.0℃，核磁共振和高分辨質(zhì)譜測試分析結(jié)果如下：94:6trans/cis(determinedby¹⁹fnmranalysis)；¹hnmr(400mhz,cdcl3)δ(major)7.36-7.31(m,2h),7.06(t,j＝7.8hz,1h),6.91(d,j＝7.8hz,1h),6.40(td,j＝6.0,56.0hz,1h),4.19-4.11(m,2h),3.28(s,3h),2.99-2.95(m,2h),1.22(t,j＝7.2hz,3h)；¹³cnmr(100mhz,cdcl3)δ(major+minor)173.1,172.3,167.4,166.8,144.8,144.2,132.5,128.7,128.6,124.3,124.0,122.9,122.8,113.4(t,j＝236.3hz),108.9,108.5,108.2,62.0,61.9,35.8(d,j＝8.6hz),34.9(d,j＝9.7hz),34.6(t,j＝33.7hz),33.3(t,j＝33.9hz),32.8(d,j＝7.9hz),27.0,27.9,25.9,14.2,14.1；¹⁹fnmr(376mhz,cdcl3)δ(major+minor)-111.04(abd,j＝54.2,293.9hz,major),-112.94(abd,j＝55.4,297.4hz,minor),-114.43(abd,j＝54.1,297.6hz,minor),-115.88(abd,j＝54.1,293.4hz,major)；hrms(esi-tof)calcd.forc15h16f2no3[m+h]⁺:296.1093；found:296.1101.

附圖中單晶-x射線衍射結(jié)構(gòu)圖和核磁共振譜圖由現(xiàn)有技術(shù)軟件生成，本領(lǐng)域技術(shù)人員可以通過圖中的特點即可了解各化合物屬性，對于圖中的數(shù)字可能因提交原因不是很清晰，但不影響本發(fā)明的完全公開。

通過實施例制備的化合物4b～4s的制備方法同化合物4a，投料比與化合物4a相同，可得到化合物4b～4s，反應(yīng)產(chǎn)率見表1，但需強調(diào)的是本發(fā)明的化合物不限于表1所表示的內(nèi)容。

表1為含二氟甲基的螺[環(huán)丙烷-1,3'-吲哚啉]-2'-酮類化合物的化學(xué)結(jié)構(gòu)

表1：

本實施例制備化合物4b：紅色油狀物，產(chǎn)率91％；核磁共振和高分辨質(zhì)譜測試分析結(jié)果如下：93:7trans/cis(determinedby¹⁹fnmranalysis)；¹hnmr(400mhz,cdcl3)δ(major+minor)7.18(s,0.07h),7.16(s,0.93h),7.12(d,j＝7.9hz,0.93h),6.83(d,j＝7.9hz,0.07h),6.79(d,j＝7.9hz,0.93h),6.68(d,j＝7.9hz,0.07h),6.40(td,j＝7.2,55.8,hz,1h),4.36-4.30(m,0.14h),4.24-4.07(m,1.86h),3.25(d,j＝1.6hz,2.79h),3.20(d,j＝1.6hz,0.21h),2.99-2.90(m,1.86h),2.86-2.83(m,0.14h),2.34(s,0.21h),2.32(s,2.79h),1.39-1.35(m,0.21h),1.24-1.20(m,2.79h)；¹³cnmr(100mhz,cdcl3)δ(major+minor)172.2,167.6,166.8(d,j＝2.0hz),165.9,141.8,132.9,132.4,129.4,128.9,125.0,124.0,123.6,122.2,113.4(t,j＝236.3hz),108.2,61.9,61.2,35.9(d,j＝8.6hz),34.8(d,j＝9.7hz),34.5(t,j＝33.7hz),26.9,26.3,21.4,21.3,21.2,14.3,14.2；¹⁹fnmr(376mhz,cdcl3)δ(major+minor)-111.03(abdd,j＝5.9,54.7,294.3hz,major),-112.87(abdd,j＝8.6,55.9,297.2hz,minor)-114.51(abdd,j＝7.1,53.6,297.3hz,minor),-115.84(abdd,j＝7.0,56.1,294.6hz,major)；hrms(esi-tof)calcd.forc16h18f2no3[m+h]⁺:310.1249；found:310.1253.

本實施例制備化合物4c：橙色油狀物，產(chǎn)率81％；核磁共振和高分辨質(zhì)譜測試分析結(jié)果如下：>99:1trans/cis(determinedby¹⁹fnmranalysis)；¹hnmr(400mhz,cdcl3)δ7.16(dd,j＝2.6,8.6hz,1h),7.02(td,j＝2.6,8.6hz,1h),6.79(dd,j＝8.0,8.6hz,1h),6.38(td,j＝7.4,55.2hz,1h),4.25-4.09(m,2h),3.26(s,3h),3.02-3.01(m,1h),2.96-2.89(m,1h),1.23(t,j＝7.2hz,3h)；¹³cnmr(100mhz,cdcl3)δ172.0,166.7(d,j＝2.0hz,1c),159.2(d,j＝239.2hz,1c),140.2(d,j＝1.8hz,1c),125.6(d,j＝9.5hz,1c),114.9(d,j＝23.6hz,1c),113.2(t,j＝236.5hz,1c),111.4(d,j＝26.8hz,1c),108.9(d,j＝8.3hz,1c),62.2,35.9(dd,j＝2.0,8.6hz,1c),34.9(d,j＝4.5hz,1c),34.9(t,j＝28.9hz,1c),27.0,14.1；¹⁹fnmr(376mhz,cdcl3)δ-111.07(abddd,j＝1.6,5.9,54.7,295.3hz,1f),-116.01(abdd,j＝7.2,55.6,295.2hz,1f),-119.47(td,j＝4.1,8.8hz,1f)；hrms(esi-tof)calcd.forc15h15f3no3[m+h]⁺:314.0999；found:314.1010.

本實施例制備化合物4d：橙色油狀物，產(chǎn)率71％；核磁共振和高分辨質(zhì)譜測試分析結(jié)果如下：>99:1trans/cis(determinedby¹⁹fnmranalysis)；¹hnmr(400mhz,cdcl3)δ(major)7.37(d,j＝2.0hz,1h),7.02(dd,j＝2.0,8.4hz,1h),6.82(d,j＝8.4hz,1h),6.37(td,j＝7.2,55.2hz,1h),4.26-4.10(m,2h),3.26(s,3h),3.02-2.91(m,2h),1.24(t,j＝7.2hz,3h)；¹³cnmr(100mhz,cdcl3)δ(major+minor)172.3,166.7,145.3,134.7,125.3,124.0,112.7,122.3,113.2(t,j＝236.6hz),109.7,109.3,62.2,35.6(d,j＝8.6hz),34.9(d,j＝8.6hz),34.7(t,j＝34.0hz),27.2,27.0,14.2；¹⁹fnmr(376mhz,cdcl3)δ(major)-111.06(abdd,j＝5.6,54.5,295.4hz,major,1f),-116.01(abdd,j＝7.0,55.8,295.6hz,major,1f)；hrms(esi-tof)calcd.forc15h15clf2no3[m+h]⁺:330.0703；found:330.0708.

本實施例制備化合物4e：黃色油狀物，產(chǎn)率85％；核磁共振和高分辨質(zhì)譜測試分析結(jié)果如下：97:3trans/cis(determinedby¹⁹fnmranalysis)；¹hnmr(400mhz,cdcl3)δ(major)7.49(s,1h),7.44(dd,j＝1.9,8.4hz,1h),6.77(d,j＝8.4hz,1h),6.36(td,j＝7.2,55.3hz,1h),4.26-4.10(m,2h),3.25(s,3h),3.01-2.93(m,2h),1.24(t,j＝7.2hz,3h)；¹³cnmr(100mhz,cdcl3)δ(major)171.7,166.5,143.2,131.5,126.2,125.9,115.5,113.1(t,j＝236.7hz,1c),109.9,62.2,35.5(d,j＝8.6hz,1c),35.1(d,j＝9.5hz,1c),35.0(t,j＝34.0hz,1c),27.0,14.2；¹⁹fnmr(376mhz,cdcl3)δ(major+minor)-111.02(abdd,j＝3.7,54.6,295.3hz,major),-112.80(abdd,j＝8.2,55.9,298.9hz,minor),-114.42(abdd,j＝7.2,53.4,298.9hz,minor),-115.98(abdd,j＝6.7,55.8,295.7hz,major)；hrms(esi-tof)calcd.forc15h15brf2no3[m+h]⁺:374.0198；found:374.0204.

本實施例制備化合物4f：黃色油狀物，產(chǎn)率80％；核磁共振和高分辨質(zhì)譜測試分析結(jié)果如下：97:3trans/cis(determinedby¹⁹fnmranalysis)；¹hnmr(400mhz,cdcl3)δ(major)7.29(s,1h),7.20(d,j＝8.2hz,1h),6.89(d,j＝8.4hz,1h),6.38(td,j＝7.0,55.2hz,1h),4.20-4.11(m,2h),3.28(s,3h),3.03-2.94(m,2h),1.23-1.19(m,3h)；¹³cnmr(100mhz,cdcl3)δ(major)172.1,166.5,144.8,142.9,125.5,121.9,120.6(q,j＝255.2hz,1c),117.3,113.1(t,j＝236.2hz),108.9,62.3,35.7(d,j＝8.6hz),35.1(d,j＝9.5hz),34.9(t,j＝34.1hz),27.0,14.0；¹⁹fnmr(376mhz,cdcl3)δ(major+minor)-58.54(s,3f),-111.14(abdd,j＝5.6,54.6,295.2hz,major),-113.01(abdd,j＝8.3,55.9,299.7hz,minor),-114.46(abdd,j＝7.4,53.4,299.3hz,minor),-116.14(abdd,j＝7.1,55.6,295.6hz,major)；hrms(esi-tof)calcd.forc16h15f5no4[m+h]⁺:380.0916；found:380.0922.

本實施例制備化合物4g：黃色固體，產(chǎn)率92％；熔點:102.0–103.8℃；核磁共振和高分辨質(zhì)譜測試分析結(jié)果如下：89:11trans/cis(determinedby¹⁹fnmranalysis)；¹hnmr(400mhz,cdcl3)δ(major+minor)7.39(d,j＝8.2hz,0.11h),7.28(d,j＝8.2hz,0.89h),7.05(d,j＝1.9hz,0.11h),7.02(dd,j＝1.8,8.2hz,0.89h),6.94(d,j＝1.8hz,0.11h),6.90(d,j＝1.8hz,0.89h),6.37(td,j＝7.1,55.2hz,1h),4.34-4.29(m,0.22h),4.23-4.07(m,1.78h),3.26(s,2.67h),3.22(s,0.33h)3.00-2.93(m,1.78h),2.65-2.57(m,0.22h),1.36(t,j＝7.2hz,0.33h),1.22(t,j＝7.2hz,2.67h)；¹³cnmr(100mhz,cdcl3)δ(major+minor)172.3,166.7,145.3,134.7,125.3,124.0,122.7,122.6,122.3,113.2(t,j＝236.6hz),109.7,109.3,62.2,62.1,35.6(d,j＝8.6hz),35.0(d,j＝8.6hz),34.7(t,j＝34.0hz),32.9(d,j＝7.6hz),27.2,27.0,14.2,14.1；¹⁹fnmr(376mhz,cdcl3)δ(major+minor)-111.03(abdd,j＝5.6,54.6,295.2hz,major),-113.05(abdd,j＝8.2,55.8,298.8hz,minor),-114.40(abdd,j＝7.4,53.6,298.7hz,minor),-115.96(abdd,j＝7.0,55.6,295.3hz,major)；hrms(esi-tof)calcd.forc15h15clf2no3[m+h]⁺:330.0703；found:330.0713.

本實施例制備化合物4h：黃色固體，產(chǎn)率83％；熔點:103.7–105.2℃；核磁共振和高分辨質(zhì)譜測試分析結(jié)果如下：92:8trans/cis(determinedby¹⁹fnmranalysis)；¹hnmr(400mhz,cdcl3)δ(major+minor)7.32(d,j＝8.2hz,0.08h),7.22(d,j＝8.2hz,0.92h),7.18-7.16(m,1h),7.09(d,j＝1.6hz,0.08h),7.04(d,j＝1.6hz,0.92h),6.37(td,j＝7.2,55.1hz,1h),4.32-4.28(m,0.16h),4.23-4.06(m,1.84h),3.26(s,2.76h),3.21(s,0.24h),3.00-2.91(m,1.84h),2.65-2.57(m,0.16h),1.22(t,j＝7.2hz,2.76h),0.95(t,j＝7.3hz,0.24h)；¹³cnmr(100mhz,cdcl3)δ(major+minor)172.2,166.7,145.4,125.6,125.5,124.3,122.9,122.4,113.1(t,j＝236.6,hz),112.4,112.1,62.1,35.6(d,j＝8.7hz),35.0(d,j＝9.6hz),34.7(t,j＝33.9hz),27.1,27.0,14.2；¹⁹fnmr(376mhz,cdcl3)δ(major+minor)-111.03(abdd,j＝3.9,54.6,295.1hz,major),-113.05(abdd,j＝6.6,55.8,298.6hz,minor),-114.40(abdd,j＝7.1,55.5,298.8hz,minor),-115.96(abdd,j＝6.5,55.6,295.3hz,major)；hrms(esi-tof)calcd.forc15h15brf2no3[m+h]⁺:374.0198；found:374.0210.

本實施例制備化合物4i：黃色固體，產(chǎn)率96％；熔點:83.3–85.0℃；核磁共振和高分辨質(zhì)譜測試分析結(jié)果如下：92:8trans/cis(determinedby¹⁹fnmranalysis)；¹hnmr(400mhz,cdcl3)δ(major+minor)7.05-7.03(m,0.16h),6.98-6.92(m,1.84h),6.90-6.87(m,0.92h),6.84-6.81(m,0.08h),6.40(td,j＝7.2hz,1h),4.34-4.28(m,0.16h),4.22-4.04(m,1.84h),3.86(s,3h),3.55(s,2.76h),3.50(s,0.24h),2.99-2.83(m,1.84h),2.61-2.53(m,0.16h),1.36(t,j＝7.1hz,0.24h),1.21(t,j＝7.2hz,2.76h)；¹³cnmr(100mhz,cdcl3)δ(major+minor)172.5,166.7,146.0,145.6,132.1,125.6,123.3,122.9,121.4,116.7,115.3,113.4(t,j＝236.3hz),112.7,112.5,62.0,61.9,61.3,56.1,36.0(d,j＝8.6hz),35.3(d,j＝8.6hz),34.8(t,j＝33.8hz),33.1(d,j＝8.7hz),30.6,30.4,14.3,14.2；¹⁹fnmr(376mhz,cdcl3)δ(major+minor)-111.12(abdd,j＝5.8,54.8,294.4hz,major),-112.85(abdd,j＝8.3,56.0,297.4hz,minor),-114.53(abdd,j＝7.2,53.5,297.3hz,minor),-116.05(abdd,j＝7.0,55.8,294.3hz,major)；hrms(esi-tof)calcd.forc16h18f2no4[m+h]⁺:326.1198；found:326.1202.

本實施例制備化合物4j：黃色固體，產(chǎn)率75％；熔點:96.0–97.5℃；核磁共振和高分辨質(zhì)譜測試分析結(jié)果如下：90:10trans/cis(determinedby¹⁹fnmranalysis)；¹hnmr(400mhz,cdcl3)δ(major+minor)7.24(d,j＝7.8hz,0.10h),7.13(d,j＝7.4hz,0.90h),7.07-7.02(m,1h),6.99-6.95(m,0.90h),6.81-6.79(m,0.10h),6.38(td,j＝6.9,55.2hz,1h),4.23-4.07(m,2h),3.50(s,3h),3.01(d,j＝7.4hz,0.90h),2.97-2.92(m,0.90h),2.87(d,j＝9.3hz,0.10h),2.65-2.57(m,0.10h),1.22(t,j＝7.0hz,3h)；¹³cnmr(100mhz,cdcl3)δ(major+minor)172.0,170.9,167.2,166.5,147.9(d,j＝242.3hz),130.7(d,j＝23.9hz),126.8(d,j＝3.7hz),123.3(d,j＝6.6hz),120.1(d,j＝3.4hz),118.7(d,j＝3.3hz),116.9,116.7(d,j＝18.9hz),113.2(t,j＝236.6hz),62.1,62.0,36.0(d,j＝12.0hz),35.5(d,j＝9.6hz),35.0(t,j＝33.9hz),33.3(d,j＝7.9hz),29.7(d,j＝6.1hz),29.5(d,j＝6.0hz),14.2,14.1；¹⁹fnmr(376mhz,cdcl3)δ(major+minor)-111.09(abddd,j＝1.3,5.7,54.7,295.3hz,major),-112.99(abddd,j＝1.7,8.3,55.9,298.6hz,minor),-114.60(abdd,j＝7.3,53.5,298.5hz,minor),-116.13(abdd,j＝6.8,55.3,295.3hz,major),(-135.65)–(-135.69)(m,minor),(-136.40)–(-136.45)(m,major)；hrms(esi-tof)calcd.forc15h15f3no3[m+h]⁺:314.0999；found:314.1004.

本實施例制備化合物4k：白色固體，產(chǎn)率90％；熔點:117.0–118.7℃；核磁共振和高分辨質(zhì)譜測試分析結(jié)果如下：93:7trans/cis(determinedby¹⁹fnmranalysis)；¹hnmr(400mhz,cdcl3)δ(major+minor)7.34(d,j＝7.8hz,0.07h),7.29(d,j＝1.2hz,0.07h),7.26-7.22(m,1.86h),6.95(t,j＝8.0hz,1h),6.38(td,j＝7.3hz,1h),4.21-4.08(m,2h),3.67(s,0.21h),3.66(s,2.79h),3.01(d,j＝7.8hz,0.93h),2.98-2.92(m,0.93h),2.88(d,j＝9.3hz,0.07h),2.64-2.60(m,0.07h),1.22(t,j＝7.2hz,3h)；¹³cnmr(100mhz,cdcl3)δ(major+minor)172.7,166.4,140.0,131.2,131.0,126.7,123.5,123.4,122.6,121.3,116.1,113.2(t,j＝236.7hz),62.2,62.1,35.8(d,j＝9.6hz),35.6(d,j＝8.6hz),35.1(t,j＝33.9hz),33.5(d,j＝7.6hz),30.8,30.5,14.2；¹⁹fnmr(376mhz,cdcl3)δ(major+minor)-111.07(abddd,j＝2.3,5.4,54.6,295.5hz,major),-112.87(abdd,j＝8.2,56.1,299.2hz,minor)-114.52(abddd,j＝3.0,7.6,53.4,299.1hz,minor),-116.08(abddd,j＝2.3,7.1,55.7,295.4hz,major)；hrms(esi-tof)calcd.forc15h14f2nnao3[m+na]⁺:352.0523；found:352.0532.

本實施例制備化合物4l：淺棕色固體，產(chǎn)率97％；熔點:104.3–105.7℃；核磁共振和高分辨質(zhì)譜測試分析結(jié)果如下：89:11trans/cis(determinedby¹⁹fnmranalysis)；¹hnmr(400mhz,cdcl3)δ(major+minor)7.45(dd,j＝1.0,8.2hz,0.11h),7.42(dd,j＝1.0,8.2hz,0.89h),7.39(d,j＝8.8hz,0.11h),7.27(d,j＝7.6hz,0.89h),6.90(t,j＝7.9hz,1h),6.38(td,j＝7.2,55.2hz,1h),4.23-4.06(m,2h),3.68(s,0.33h),3.66(s,2.67h),3.02(d,j＝7.6hz,0.89h),2.98-2.91(m,0.89h),2.88(d,j＝9.4hz,0.11h),2.66-2.58(m,0.11h),1.21(t,j＝7.2hz,3h)；¹³cnmr(100mhz,cdcl3)δ(major+minor)173.8,172.8,167.1,166.4,141.4,134.5,134.3,127.0,125.2,123.8,123.7,123.1,121.8,113.2(t,j＝236.6hz),103.2,102.8,62.2,62.1,35.9(d,j＝9.6hz),35.5(d,j＝8.7hz),35.0(t,j＝34.0hz),33.6(d,j＝8.1hz),31.1,30.7,14.2；¹⁹fnmr(376mhz,cdcl3)δ(major+minor)-111.06(abdd,j＝4.3,54.6,295.3hz,major),-112.28(abdd,j＝6.8,56.4,299.2hz,minor),-114.49(abdd,j＝7.2,53.5,299.2hz,minor),-116.05(abdd,j＝6.6,54.6,295.3hz,major)；hrms(esi-tof)calcd.forc15h15brf2no3[m+h]⁺:374.0198；found:374.0202.

本實施例制備化合物4m：黃色固體，產(chǎn)率60％；熔點:118.2–119.3℃；核磁共振和高分辨質(zhì)譜測試分析結(jié)果如下：>99:1trans/cis(determinedby¹⁹fnmranalysis)；¹hnmr(400mhz,cdcl3)δ7.35-7.31(m,2h),7.08-7.04(m,1h),6.91(d,j＝7.5hz,1h),6.40(td,j＝7.2,55.6hz,1h),3.70(s,3h),3.28(s,3h),3.02-2.94(m,2h)；¹³cnmr(100mhz,cdcl3)δ172.3,167.3,144.2,128.7,124.0,123.0(d,j＝2.7hz),122.0113.3(t,j＝236.4hz),108.6,52.8,35.9(d,j＝8.5hz),34.7(d,j＝9.5hz),34.6(t,j＝33.7hz),26.9；¹⁹fnmr(376mhz,cdcl3)δ-111.09(abdd,j＝5.3,54.9,295.1hz,1f),-115.91(abdd,j＝5.9,55.5,294.2hz,1f)；hrms(esi-tof)calcd.forc14h14f2no3[m+h]⁺:282.0936；found:282.0945.

本實施例制備化合物4n：淺黃色固體，產(chǎn)率87％；熔點:63.2–64.0℃；核磁共振和高分辨質(zhì)譜測試分析結(jié)果如下：>99:1trans/cis(determinedby¹⁹fnmranalysis)；¹hnmr(400mhz,cdcl3)δ7.36-7.31(m,2h),7.06(t,j＝7.7hz,1h),6.91(d,j＝7.8hz,1h),6.41(td,j＝7.1,55.3hz,1h),4.12-3.98(m,2h),3.29(s,3h),3.02-2.95(m,2h)1.62-1.59(m,2h)0.86(t,j＝7.4hz,3h)；¹³cnmr(100mhz,cdcl3)δ172.4,166.9,144.2,128.7,124.1,122.9,122.8,113.4(t,j＝236.4hz,1c),108.6,67.6,35.9(d,j＝8.6hz,1c),34.9(d,j＝9.6hz,1c),34.7(t,j＝33.7hz,1c),26.9,21.9,10.3；¹⁹fnmr(376mhz,cdcl3)δ-111.04(abdd,j＝5.6,55.0,294.6hz,1f),-115.89(abdd,j＝7.0,55.6,294.4hz,1f)；hrms(esi-tof)calcd.forc16h18f2no3[m+h]⁺:310.1249；found:310.1255.

本實施例制備化合物4o：淺黃色固體，產(chǎn)率99％；熔點:80.3–82.0℃；核磁共振和高分辨質(zhì)譜測試分析結(jié)果如下：87:13trans/cis(determinedby¹⁹fnmranalysis)；¹hnmr(400mhz,cdcl3)δ(major+minor)7.38-7.29(m,2h),7.08-7.02(m,1h),6.90(d,j＝8.2hz,0.87h),6.79(d,j＝7.8hz,0.13h),6.38(td,j＝7.2,55.7hz,1h),3.29(s,2.61h),3.23(s,0.39h),2.95-2.87(m,1.74h),2.81-2.78(m,0.13h),2.60-2.49(m,0.13h),1.41(s,1.17h),1.38(s,7.83h)；¹³cnmr(100mhz,cdcl3)δ(major+minor)172.6,165.7,144.1,132.2,128.7,128.6,128.5,125.0,124.2,124.1,122.9,122.7(d,j＝5.9hz),113.5(t,j＝236.2hz),108.9,108.5,108.2,82.9,35.9(d,j＝9.0hz),35.6(d,j＝9.0hz),34.3(t,j＝33.6hz),33.8(d,j＝8.0hz),28.2,28.1,26.9；¹⁹fnmr(376mhz,cdcl3)δ(major+minor)-110.97(abdd,j＝5.2,54.5,293.8hz,major),-112.66(abdd,j＝8.8,56.1,296.4hz,minor),-115.20(abdd,j＝6.9,53.5,296.3hz,minor),-115.90(abdd,j＝6.7,55.6,293.7hz,major)；hrms(esi-tof)calcd.forc17h20f2no3[m+h]⁺:324.1406；found:324.1415.

本實施例制備化合物4p：黃色固體，產(chǎn)率93％；熔點:70.3–72.3℃；核磁共振和高分辨質(zhì)譜測試分析結(jié)果如下：85:15trans/cis(determinedby¹⁹fnmranalysis)；¹hnmr(400mhz,cdcl3)δ(major+minor)7.47(d,j＝7.8hz,0.15h),7.37(d,j＝7.8hz,0.85h),7.32(t,j＝7.8hz,1h),7.04(t,j＝6.0hz,0.85h),6.97(d,j＝8.0hz,0.15h),6.93(d,j＝7.8hz,0.85h),6.83(t,j＝8.2hz,0.15h),6.40(td,j＝8.0,56.0hz,1h),4.35-4.30(m,0.3h),4.25-4.07(m,1.7h),3.86-3.80(q,j＝7.2hz,2h),2.95(d,j＝7.5hz,0.85h),2.97-2.92(m,0.85h),2.85(d,j＝9.4hz,0.15h),2.65-2.56(m,0.15h),1.39-1.35(m,0.9h),1.32-1.21(m,5.1h)；¹³cnmr(100mhz,cdcl3)δ(major+minor)172.8,171.9,167.5,166.9,143.3,132.5,129.1,128.7,128.6,124.6,124.3,123.2,122.8,122.7,122.6,122.5,113.4(t,j＝236.5hz),109.0,108.7,108.4,62.0,61.3,35.9(d,j＝9.0hz),35.7,35.5,34.7(d,j＝9.6hz),34.6(t,j＝33.8hz),32.8(d,j＝7.8hz),14.3,14.2,12.9,12.8；¹⁹fnmr(376mhz,cdcl3)δ(major+minor)-111.06(abddd,j＝1.8,5.7,54.7,294.5hz,major),-113.01(abddd,j＝1.7,8.3,55.9,297.6hz,minor),-114.45(abdd,j＝7.3,53.7,297.6hz,minor),-113.01(abdd,j＝7.3,55.1,294.3hz,major)；hrms(esi-tof)calcd.forc16h18f2no3[m+h]⁺:310.1249；found:310.1252.

本實施例制備化合物4q：橙色油狀物，產(chǎn)率99％；核磁共振和高分辨質(zhì)譜測試分析結(jié)果如下：85:15trans/cis(determinedby¹⁹fnmranalysis)；¹hnmr(400mhz,cdcl3)δ(major+minor)7.50(d,j＝7.6hz,0.15h),7.39(d,j＝7.6hz,0.85h),7.36-7.20(m,6h),7.07-7.01(m,1h),6.87(d,j＝7.8hz,0.15h),6.81(d,j＝7.8hz,0.85h),6.47(td,j＝7.4,55.2hz,1h),5.08-4.90(m,2h),4.38-4.33(m,0.30h),4.27-4.09(m,1.70h),3.13-3.11(m,0.85h),3.07-3.00(m,0.85h),2.98-2.95(m,0.15h),2.74-2.66(m,0.15h),1.39(t,j＝7.2hz,0.45h),1.25(t,j＝7.2hz,2.55h)；¹³cnmr(100mhz,cdcl3)δ(major+minor)173.4,172.5,167.4,166.8,144.0,143.3,135.4,132.5,129.0,128.9,128.7,128.6,127.9,127.8,127.4,127.3,124.5,124.1,123.0,122.9,122.8,1227,113.5(t,j＝236.5hz),109.9,109.5,62.1,62.0,44.6,44.4,35.90(d,j＝9.0hz),35.0(d,j＝9.7hz),34.9(t,j＝33.8hz),33.5(t,j＝34.1hz),32.9(d,j＝7.7hz),14.1,14.2；¹⁹fnmr(376mhz,cdcl3)δ(major+minor)-110.88(abdd,j＝5.5,54.7,294.6hz,major),-112.87(abdd,j＝8.3,55.9,297.8hz,minor)-114.44(abdd,j＝7.3,53.7,297.6hz,minor),-112.87(abdd,j＝7.6,55.7,294.5hz,major)；hrms(esi-tof)calcd.forc21h20f2no3[m+h]⁺:372.1406；found:372.1418.

本實施例制備化合物4r：黃色油狀物，產(chǎn)率94％；核磁共振和高分辨質(zhì)譜測試分析結(jié)果如下：92:8trans/cis(determinedby¹⁹fnmranalysis)；¹hnmr(400mhz,cdcl3)δ(major+minor)7.57-7.54(m,2h),7.46-6.43(m,4h),7.26(t,j＝7.8hz,1h),7.10(t,j＝7.8hz,1h),6.89(d,j＝7.8hz,1h),6.44(td,j＝7.0,55.2hz,1h),4.30-4.12(m,2h),3.13-3.03(m,1.84h),2.97(d,j＝9.4hz,0.08h),2.76-2.68(m,0.08h),1.40(t,j＝7.2hz,0.24h),1.27(t,j＝7.2hz,2.76h)；¹³cnmr(100mhz,cdcl3)δ(major+minor)172.7,171.9,167.4,166.8,144.2,134.1,134.0,129.8,128.7,128.6,128.5,126.8,126.7,124.6,123.9,123.3,123.2,113.3(t,j＝236.5hz),110.2,109.9,62.1,62.0,36.1(d,j＝8.5hz),35.4(d,j＝9.6hz),35.2(t,j＝33.9hz),33.3(d,j＝7.9hz),14.2,14.1；¹⁹fnmr(376mhz,cdcl3)δ(major+minor)-110.83(abdd,j＝3.6,54.7,295.2hz,major),-115.18(abdd,j＝8.0,55.9,298.4hz,minor),-114.42(abdd,j＝7.0,53.6,298.2hz,minor),-115.94(abdd,j＝6.8,55.4,294.9hz,major)；hrms(esi-tof)calcd.forc20h18f2no3[m+h]⁺:358.1249；found:328.1253.

本實施例制備化合物4s：淺黃色固體，產(chǎn)率70％；熔點:98.7–100.2℃；核磁共振和高分辨質(zhì)譜測試分析結(jié)果如下：87:13trans/cis(determinedby¹⁹fnmranalysis)；¹hnmr(400mhz,cdcl3)δ(major+minor)7.99(d,j＝8.2hz,0.13h),7.89(d,j＝8.2hz,0.87h),7.44(d,j＝7.8hz,0.13h),7.38-7.32(m,1.87h),7.19-7.13(m,1h),6.36(td,j＝7.0,55.3hz,1h),4.23-4.08(m,2h),3.04-3.00(m,1.74h),2.90(d,j＝9.4hz,0.13h),2.66-2.61(m,0.13h),1.65(s,7.83h),1.59(s,1.17h),1.27-1.19(m,3h)；¹³cnmr(100mhz,cdcl3)δ(major+minor)171.3,166.2,148.8,140.3,129.0,125.0,124.7,122.9,122.5,115.2,113.0(t,j＝236.8hz),85.3,62.2,36.8(d,j＝9.6hz),36.3(d,j＝9.6hz),35.6(t,j＝34.1hz),28.2,14.2；¹⁹fnmr(376mhz,cdcl3)δ(major+minor)-115.69(abd,j＝54.6,296.2hz,major),-112.80(abd,j＝52.2,303.1hz,minor),-114.78(abd,j＝53.3,297.6hz,minor),-115.99(abd,j＝55.7,296.2hz,major)；hrms(esi-tof)calcd.forc19h22f2no5[m+h]⁺:382.1461；found:382.1466.

本發(fā)明的式(1)化合物具有重要的生物活性，體外對人前列腺(pc-3)和人肺癌細胞(a549)共兩株腫瘤細胞的細胞毒性試驗表明：此類式(1)所示的含二氟甲基的螺[環(huán)丙烷-1,3'-吲哚啉]-2'-酮類化合物對腫瘤細胞生長具有抑制作用，有可能發(fā)展成為新的防治腫瘤藥物。

本發(fā)明的式(1)化合物或其可藥用鹽及其溶劑化物可以與藥學(xué)上常用的輔料或載體結(jié)合，制備得到具有腫瘤細胞生長抑制活性從而可以用于防治腫瘤的藥物組合物。上述各類藥物組合物可以是固體形式，如片劑、膠囊劑、顆粒劑、粉劑；也可以是液體形式，如注射劑、懸浮劑和乳劑等。除此之外，還可以采用現(xiàn)代制藥界所公知的控釋劑或緩釋劑或納米劑。

本發(fā)明的式(1)化合物或其可藥用鹽及其溶劑化物可以與現(xiàn)已上市的抗腫瘤藥物如鉑類藥物順鉑(ddp)、脫氧胞昔類藥物吉西他濱(gemcitabine,gemzar,健擇)、紫杉醇(paclitaxel)、長春花堿類藥物失碳長春花堿(vinorebine,nvb諾維本)、喜樹堿類藥物伊立替康(irinatecan、cpt-11)、足葉乙甙(etoposide)等聯(lián)合使用，制備得到具有腫瘤生長抑制活性的細胞毒性組合物，可用于治療腫瘤疾病。該類藥物組合物可以是固體形式，如片劑、膠囊劑、顆粒劑、粉劑；也可以是液體形式，如注射劑、懸浮劑和乳劑等。除此之外，還可以采用現(xiàn)代制藥界所公知的控釋劑或緩釋劑或納米劑。

藥理實施例1：化合物4f、4l、4o或4p對pc-3細胞的細胞毒性

pc-3(人前列腺癌)細胞用rpmi-1640培養(yǎng)基培養(yǎng)，培養(yǎng)基中含10％的胎牛血清，100u/ml青霉素及100u/ml的鏈霉素。細胞以每孔5000個細胞的濃度加入到96孔中，在37℃含5％co2潮濕空氣的培養(yǎng)箱中培養(yǎng)24小時。

細胞存活率的測定用改良mtt法。細胞經(jīng)過24小時的孵育后，分別將新配的化合物4f、4l、4o或4p的二甲基亞砜溶液以濃度梯度加入到各孔中，使孔中化合物最終濃度分別為6.25μmol/l,12.5μmol/l,25μmol/l,50μmol/l和100μmol/l。48小時后，每孔加入10μlmtt(5mg/ml)的磷酸鹽緩沖液，再繼續(xù)在37℃培養(yǎng)4小時后，離心5分鐘除去未轉(zhuǎn)化的mtt，每孔中加入150μl二甲基亞砜。以溶解還原的mtt晶體甲臜(formazan)，用酶標儀在490nm波長測定od值。其中化合物4f、4l、4o或4p對pc-3細胞半抑制濃度ic50由spss軟件(19版本)分析得到?；衔?f對pc-3腫瘤細胞的ic50為72.1μmol/l；化合物4l對pc-3腫瘤細胞的ic50為80.8μmol/l；化合物4o對pc-3腫瘤細胞的ic50為25.7μmol/l；化合物4p對pc-3腫瘤細胞的ic50為81.8μmol/l；而陽性對照順鉑對pc-3腫瘤細胞的ic50為23.7μmol/l。

實驗結(jié)論：pc-3細胞是測試化合物對腫瘤細胞的細胞毒性的有效工具和評價指標。本實驗表明此類式(1)所示的含二氟甲基的螺[環(huán)丙烷-1,3'-吲哚啉]-2'-酮類化合物對pc-3細胞具有一定的細胞毒性，有可能發(fā)展成新的具有抗腫瘤作用的藥物。

藥理實施例2：化合物4n、4o、4q或4r對a549細胞的細胞毒性

a549(人肺癌細胞)用dmem培養(yǎng)基培養(yǎng)，培養(yǎng)基中含10％的胎牛血清，100u/ml的青霉素和100u/ml鏈霉素。細胞以每孔4000個細胞的濃度加入到96孔中，在37℃含5％co2潮濕空氣的培養(yǎng)箱中培養(yǎng)24小時。

細胞存活率的測定用改良mtt法。具體方法如藥理實施例1?；衔?n對a549腫瘤細胞的ic50為89.9μmol/l；化合物4o對a549腫瘤細胞的ic50為36.8μmol/l；化合物4q對a549腫瘤細胞的ic50為87.9μmol/l；化合物4r對a549腫瘤細胞的ic50為62.4μmol/l；而陽性對照順鉑對a540腫瘤細胞的ic50為23.0μmol/l。

實驗結(jié)論：a549細胞是測試化合物對腫瘤細胞的細胞毒性的有效工具和評價指標。本實驗表明此類式(1)所示的含二氟甲基的螺[環(huán)丙烷-1,3'-吲哚啉]-2'-酮類化合物對a549細胞具有一定的細胞毒性，有可能發(fā)展成新的具有抗腫瘤作用的藥物。

從以上藥理實施例中我們可以看出該類化合物對這兩株腫瘤細胞都顯示有一定的細胞毒性?？梢娺@些化合物具有開發(fā)成為抗腫瘤藥物的潛力，值得繼續(xù)深入研究。