本發(fā)明屬藥物制備技術(shù)領(lǐng)域，涉及一種Dolastatin 10衍生物，具體涉及一種Dolastatin 10衍生物I、其制備方法及其應(yīng)用。

背景技術(shù)：

Dolastatin 10具有顯著的抗腫瘤活性，但因毒性強(qiáng)、副作用大而使其應(yīng)用受到限制。該化合物通過抑制微管蛋白和微管依賴性的GTP水解，導(dǎo)致細(xì)胞周期終止和細(xì)胞凋亡。Pfizer的研究人員通過合成一系列dolastatin 10的類似物，對(duì)其與微管的結(jié)合方式進(jìn)行了研究，共結(jié)晶復(fù)合物顯示該化合物結(jié)合與微管α,β-亞基的交界處，N-端靠近長(zhǎng)春花堿結(jié)合區(qū)域，C-端靠近GTP/GDP交換結(jié)合位點(diǎn)。除了具有微管抑制作用外，研究還發(fā)現(xiàn)，dolastatin10和其類似物還具有抗血管生成作用。以auristatin-PE和auristatin-PYE為代表的一系列C-端修飾化合物與dolastatin 10相比雖然毒性減弱，但臨床實(shí)驗(yàn)中表現(xiàn)出明顯的副作用導(dǎo)致它們依然未能獲得臨床使用。Dolastatin 10和其合成類似物的臨床應(yīng)用受到了限制。dolastatin 10的研究帶來了突破性的進(jìn)展得益于Miyazaki等人的研究成果。他們發(fā)現(xiàn)去除N-端dolavalin中的一個(gè)甲基獲得的去甲基類似物6活性與dolastatin 10相比僅有微弱降低。Senter等人利用這一成果，通過連接鏈將去甲基類似物與單抗相連，設(shè)計(jì)了ADC。這一設(shè)計(jì)理念為，發(fā)現(xiàn)了具有高活性和可臨床使用的ADC

因此，Dolastatin 10衍生物及其組合物的顯著抗腫瘤活性，發(fā)展其作為新型的抗癌藥物或與其它抗癌藥物聯(lián)合應(yīng)用的新藥，將具有很大的市場(chǎng)，亦將產(chǎn)生較大的社會(huì)效益和經(jīng)濟(jì)效益。

技術(shù)實(shí)現(xiàn)要素：

本發(fā)明所要解決的技術(shù)問題在于，提供一種與現(xiàn)有技術(shù)完全不同的，具有抗腫瘤作用的dolastatin 10衍生物I及其制備方法和應(yīng)用。本發(fā)明的dolastatin 10衍生物I為一種新型的具有抗腫瘤活性的化合物，為研發(fā)抗癌藥物打開了一個(gè)新的研究方向，拓展了dolastatin 10衍生物抗癌藥物的研究范圍。

本發(fā)明提供了如下式的dolastatin 10衍生物I；

其中，R₁獨(dú)立地優(yōu)選自氫、鹵素或C₁～C₆的烷氧基或烷基。所述的C₁～C₆的烷氧基或烷基優(yōu)選甲氧基和異丙基。

其中，R₂優(yōu)選自氫、鹵素或C₁～C₆的烷氧基。

所述的R₁、R₂和R₃獨(dú)立地優(yōu)選自氫或鹵素。所述的R₁、R₂和R₃獨(dú)立地更優(yōu)選自氟或氯，且R₁、R₂和R₃不同時(shí)為氫。

所述的dolastatin 10衍生物I優(yōu)選如下情況：

R1、R2、R3為氫；

或者，R1為氟，R2、R3為氫；

或者，R1為氫，R2為氟以及R3為氫；

或者，R1為氫，R2為氯以及R3為氟；

或者，R1為氫，R2為氫以及R3為氯；

或者，R1為異丙基，R2為氫以及R3為氫；

或者，R1為甲氧基，R2為氫以及R3為氯；

或者，R1為氫，R2為氫以及R3為氟；

或者，R1為氫，R2為氫以及R3為叔丁基；

或者，R1為氫，R2為氫以及R3為苯基。

本發(fā)明還提供了一種dolastatin 10衍生物I的制備方法，

其中，R₁、R₂和R₃均如上所述。

所述的縮合反應(yīng)可為本領(lǐng)域常規(guī)的此類縮合反應(yīng)本發(fā)明特別優(yōu)選如下反應(yīng)條件：

所述的溶劑優(yōu)選極性非質(zhì)子性溶劑。所述的極性非質(zhì)子性溶劑優(yōu)選四氫呋喃、吡啶、乙腈、二氯甲烷、三氯甲烷、N,N-二甲基甲酰胺、N,N-二甲基乙酰胺、N-甲基吡咯烷酮和二甲亞砜中的一種或多種。

所述的縮合劑可為本領(lǐng)域此類反應(yīng)常用的脫水劑，優(yōu)選二環(huán)己基碳二亞胺(DCC)、1-乙基-3-[3-(二甲胺基)丙基]碳二亞胺鹽酸鹽(EDCI)、1-羥基苯并三唑(HOBt)、N,N'-羰基二咪唑(CDI)、O-(7-氮雜苯并三唑-1-基)-N,N,N’,N’-四甲基脲六氟磷酸鹽(HATU)、苯并三氮唑-N,N,N',N'-四甲基脲六氟磷酸酯(HBTU)、甲基膦酸酐、乙基膦酸酐、正丙基膦酸酐、丙膦酸環(huán)酸酐(T3P)和苯并三唑基磷酸二乙酯中的一種或多種，更優(yōu)選1-乙基-3-[3-(二甲胺基)丙基]碳二亞胺鹽酸鹽和/或1-羥基苯并三唑。

所述的堿可為有機(jī)堿和/或無機(jī)堿。所述的有機(jī)堿優(yōu)選吡啶、三乙胺、N,N-二甲胺基吡啶和二異丙基乙胺(DIEA)中的一種或多種。

所述的縮合反應(yīng)的反應(yīng)溫度優(yōu)選-15℃～100℃，更優(yōu)選0℃～30℃

所述的縮合反應(yīng)的進(jìn)程可通過TLC或HPLC進(jìn)行監(jiān)測(cè)，一般以化合物II消失時(shí)作為反應(yīng)的終點(diǎn)，優(yōu)選反應(yīng)1～120小時(shí)反應(yīng)結(jié)束，更優(yōu)選反應(yīng)24～48小時(shí)反應(yīng)結(jié)束。

所述的縮合反應(yīng)結(jié)束后還可包括后處理過程，以進(jìn)一步純化得到的dolastatin 10衍生物I。所述的后處理過程可為本領(lǐng)域常規(guī)的后處理過程，優(yōu)選包括如下步驟：

本發(fā)明還提供了dolastatin 10在制備抗腫瘤藥物中的應(yīng)用。

在不違背本領(lǐng)域常識(shí)的基礎(chǔ)上，上述各優(yōu)選條件，可任意組合，即得本 發(fā)明各較佳實(shí)例。

本發(fā)明所用試劑和原料均市售可得。

本發(fā)明的積極進(jìn)步效果在于：本發(fā)明提供了一種與現(xiàn)有技術(shù)完全不同的，具有抗腫瘤效果的dolastatin 10衍生物I，為鬼臼衍生物類抗癌藥物的研發(fā)提供了一個(gè)新方向。

具體實(shí)施方式

下面通過實(shí)施例的方式進(jìn)一步說明本發(fā)明，但并不因此將本發(fā)明限制在所述的實(shí)施例范圍之中。下列實(shí)施例中未注明具體條件的實(shí)驗(yàn)方法，按照常規(guī)方法和條件，或按照商品說明書選擇。本發(fā)明實(shí)施例涉及的化合物：

實(shí)施例1 (S)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N-((3R,4S,5S)-3-methoxy-1-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-(3-phe nylazetidin-1-yl)propyl)pyrrolidin-1-yl)-5-methyl-1-oxoheptan-4-yl)-N,3-dim ethylbutanamide 1的制備

A：tert-butyl 3-phenylazetidine-1-carboxylate

將化合物5(400mg,1.0mmol,1.00eq)和苯硼酸(244mg,2.0mmol,2.00eq)溶于干燥1,2-二氧六環(huán)中，加入Cs2CO3(650mg,2.0mmol,2.00eq)，110℃反應(yīng)24小時(shí)；恢復(fù)室溫，加入二氯甲烷，飽和碳酸氫鈉溶液洗滌，分出有機(jī)層，飽和食鹽水洗，無水硫酸鈉干燥，過濾蒸干，乙酸乙酯/石油醚(1:10)為洗脫劑進(jìn)行柱層析，得無色油狀物1A 180mg，收率77％；

B：tert-butyl(S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-(3-phenylazetidin-1-yl)propyl)pyrrolidine-1-carboxylate

化合物1A(22mg,0.1mmol)溶于二氯甲烷(1mL)，加入1mL三氟乙酸，室溫反應(yīng)半小時(shí)后，蒸出溶劑，油泵干燥兩小時(shí)后，以干燥四氫呋喃溶解，加入二異丙基乙胺(40uL,0.27mmol,3.00eq)，pyAop(70mg,0.14mmol,1.5eq)，化合物8(25mg,0.1mmol,1.00eq)，室溫反應(yīng)18小時(shí)。蒸干溶劑，乙酸乙酯/石油醚(1:1)為洗脫劑進(jìn)行柱層析，得無色油狀物1B 30mg，收率81％

制備(S)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N-((3R,4S,5S)-3-methoxy-1-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-(3-phe nylazetidin-1-yl)propyl)pyrrolidin-1-yl)-5-methyl-1-oxoheptan-4-yl)-N,3-dim ethylbutanamide

化合1B(30mg,0.07mmol,1.00eq)和化合物11(34mg,0.07mmol,1.00eq)分別溶于二氯甲烷，以三氟乙酸脫除保護(hù)基，蒸干溶劑，油泵干燥；以干燥四氫呋喃溶解，加入二異丙基乙胺(30uL,0.21mmol,3.00eq),pyAOP(54mg,0.11mmol,1.5eq)，室溫反應(yīng)24小時(shí)。蒸干溶劑，以二氯甲烷/甲醇(40:1～20:1)為洗脫劑進(jìn)行柱層析，得化合物23mg，收率43％。

¹H NMR(400MHz,CDCl₃)7.40-7.36(m,2H),7.31-7.27(m,3H),6.93(d,J＝9.1Hz,1H),[4.87(t,J＝7.2Hz)and 4.79(t,J＝7.8Hz),1H],4.66-4.59(m,1H),4.54-4.46(m,1H),4.45-4.40(m,1H),4.37-4.32(m,1H),4.28-4.23(m,1H),4.21-3.77(m,11H),[3.43(s)and 3.41(s),3H],[3.36(s)and 3.35(s),3H],[3.34(s)and 3.30(s),3H],3.14(d,J＝6.2Hz,1H),3.03-3.02(m,2H),2.63-0.79(m,36H).¹³C NMR(150MHz,CDCl₃)174.4,147.1,173.8,173.7,173.1,171.2,170.2,169.9,161.8,161.7,160.1,129.2,129.1,129.1,129.0,128.8,128.4,128.2,128.1,127.9,127.7,127.7,124.5,124.4,116.0,115.8,115.7,115.6,115.5,115.5,86.4,86.2,82.6,82.4,78.3,77.8,76.0,61.9,61.8,60.5,60.4,59.4,59.2,59.0,58.1,57.9,57.8,56.5,56.4,56.2,56.0,55.8,53.8,53.6,53.4,53.2,47.7,47.5,46.6,46.5,42.6,39.4,38.8,38.8,37.7,37.4,35.8,33.2,33.1,32.3,31.8,30.9,28.4,27.8,27.7,27.3,26.2,26.0,25.7,25.0,24.9,24.6,23.6,23.5,20.1,19.8,19.5,17.9,17.8,15.8,15.4,14.8,14.6,13.7,13.5,10.8,10.7,10.3.MS 714.0(M+H)⁺-50.200(MeOH,0.50)。

實(shí)施例2(S)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(3-(2-fluorophenyl)azetidin-1-yl)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)-N,3-dimethylbutanamide(2)的制備

制備過程同實(shí)施例1；

¹H NMR(400 MHz,CDCl3)δ7.41–7.31(m,1H),7.25–7.20(m,1H),7.16(dt,J＝8.7,4.4 Hz,1H),7.04(m,1H),4.33(t,J＝8.1 Hz,2H),4.08–4.02(m,2H),4.01–3.95(m,1H),1.46(s,9H).

1H NMR(400 MHz,Chloroform-d)δ7.27-7.10(m,1H),6.96-6.94(m,1H),6.83(d,J＝1.7 Hz,1H),4.50-4.43(m,1H),4.35-4.31(m,1H),4.27-4.24(m,1H),4.19-4.08(m,2H),3.97-3.82(m,3H),3.81(s,3H),3.78-3.74(m,1H),3.26-3.22(m,1H),1.95(s,3H),1.92-1.68(m,4H),1.52(s,3H),1.44(d,J＝7.8 Hz,3H).

¹³C NMR(150 MHz,Chloroform-d)δ174.4,154.6,129.0,128.1,128.0,128.0,124.5,115.8,84.2,84.1,82.1,79.9,79.1,61.2,60.8,59.5,59.3,58.9,56.4,53.5,53.3,47.0,46.7,39.2,38.4,28.7,28.6,27.8,27.3,27.4,26.2,26.1,25.7,24.7,24.6,24.3,14.7,14.6,13.8.

¹H NMR(400 MHz,CDCl₃)7.36-7.28(m,1H),7.09-6.95(m,4H),[4.87(t,J＝7.5 Hz)and 4.79(t,J＝7.8 Hz),1H],4.66-4.60(m,1H),4.57-4.50(m,1H),4.45-4.41(m,1H),4.38-4.34(m,1H),4.27-4.22(m,13H),[3.43(s)and 3.41(s),3H],[3.36(s)and 3.35(s),3H],[3.33(s)and 3.29(s),3H],2.63-0.79(m,34H)； ¹³C NMR(150 MHz,CDCl₃)δ174.6,174.3,173.9,173.3,171.7,170.4,169.9, 169.5,142.1,141.91,141.3,129.1,129.1,128.9,127.5,127.3,126.8,126.7,126.7,126.5,86.5,86.3,82.7,82.6,77.3,77.1,76.9,62.00,60.6,60.5,59.53,59.34,59.1,58.3,58.1,58.0,57.9,57.8,57.6,57.6,55.94,55.2,55.15,55.04,54.8,53.9,53.8,47.8,47.6,46.8,46.7,46.7,45.9,43.00,39.71,39.0,37.9,37.64,36.03,33.59,33.44,33.3,33.3,33.2,33.1,32.5,32.06,31.09,29.8,27.8,26.47,26.18,25.88,25.28,25.1,24.8,24.5,23.7,22.8,20.30,19.9,19.8,19.7,18.1,17.92,16.03,15.64,14.9,14.2,13.8,13.6,11.1,10.9,10.9,10.4.MS 731.9(M+H)+-35.200(MeOH,0.50)。

實(shí)施例3

(S)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(3-(3-fluorophenyl)azetidin-1-yl)-1-methoxy-2-methyl-3-oxopr opyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)-N,3-dimethylbu tanamide(3)的制備

制備過程同實(shí)施例1；

1H NMR(400MHz,CDCl3)8.63(s,1H),7.36-6.99(m,5H),[4.88-4.84(m)and 4.80-4.74(m),1H],4.70-4.63(m,1H),4.58-4.54(m,1H),4.50-4.34(m,2H),4.29-3.68(m,10H),3.53-3.45(m,2H),[3.43(s)and 3.39(s),3H],[3.36(s)and3.35(s),3H],[3.12(s)and 3.02(s),3H],2.78-0.80(m,34H)

¹³C NMR(150MHz,CDCl₃)δ173.1,169.4,150.4,122.0,82.5,78.1,77.3,77.1,76.9,61.9,60.5,59.6,59.4,59.1,58.4,58.2,58.0,56.7,54.4,54.3,53.9,53.6,47.8,47.6,46.8,45.9,42.6,42.1,39.8,39.5,37.6,33.6,33.3,32.6,32.5,32.0,31.5,31.0,30.3,29.8,27.0,26.5,26.1,25.8,25.1,24.8,24.5,23.7,20.1,19.8,19.6,18.2,18.1,17.6,16.0,13.6,13.4,11.8,11.1,10.9-41.400(MeOH,0.50)。

實(shí)施例4

(S)-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(3-(3-chloro-4-fluorophenyl)azetid in-1-yl)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-meth yl-1-oxoheptan-4-yl)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N,3-di methylbutanamide(4)的制備

制備過程同實(shí)施例1；

1H NMR(400MHz,Chloroform-d)δ7.39–7.34(m,1H),7.21–7.15(m,1H),7.12(t,J＝7.8Hz,1H),4.33(t,J＝8.7Hz,2H),3.95–3.87(m,2H),3.73–3.63(m,1H),1.47(s,9H)；13C NMR(151MHz,Chloroform-d)δ156.4,139.5,129.1,126.6,126.6,117.3,117.0,116.8,114.8,80.0,56.6,32.8,29.8,28.5.

1H NMR(400MHz,Chloroform-d)δ7.36–7.31(m,1H),7.15–7.11(m,2H),4.58-4.55(m,1H),4.44-4.33(m,1H),4.19-3.57(m,6H),3.45(s,3H),3.27-3.25(m,1H),2.50-2.41(m,1H),1.98-1.74(m,4H),1.52(s,9H),1.46(d,J＝6.8Hz,3H).

13C NMR(151MHz,DMSO-d6)δ173.3,156.9,155.3,153.3,140.2,129.1,128.8,127.6,127.5,127.4,127.4,119.5,117.1,117.0,116.9,116.8,83.4,83.7,82.0,81.8,78.5,78.23,60.48,60.2,58.8,58.2,56.9,56.6,54.3,46.6,46.3,40.0,38.2,37.4,31.4,28.9,28.2,25.6,25.1,24.1,23.6,14.2,13.8,13.6.

1H NMR(400MHz,CDCl3)7.37-7.35(m,2H),7.16-7.10(m,2H),[4.88-4.87(m)and 4.79-4.77(m),1H],4.64-4.60(m,1H),4.52-4.46(m,1H),4.48-4.41(m,1H),4.35-4.33(m,1H),4.21-4.19(m,2H),4.18-4.01(m,3H),3.89-3.76(,2H),[3.49(s)and 3.43(s),3H],[3.39(s)and 3.35(s),3H],[3.13(s)and 3.02(s),3H],2.63-0.79(m,36H)；13C NMR(151MHz,DMSO-d6)δ175.5,174.5,174.3,173.9,173.2,171.3,170.0,158.1,156.4,138.8,129.9,129.8,129.0,128.9,128.8,128.7,126.5,126.2,121.3,117.0,116.9,116.8,86.3,86.1,82.5,82.3,61.8,60.6,59.5,59.2,59.0,58.2,58.1,57.8,57.7,57.4,57.1,55.9,55.2,55.0,54.8,54.6,53.9,47.8,46.7,42.7,39.5,38.8,37.6,35.9,33.4,32.5,32.4,29.7,29.5,29.2,27.6,27.2,26.3,26.0,25.7,25.4,25.0,24.4,22.6,20.8,20.0,19.5,18.2,15.8,14.1,10.9；13C NMR(151MHz,CDCl3)δ174.5,174.1,173.9,173.7,173.3,171.3,170.3,170.2,169.5,158.1,134.0,133.9,133.7,133.1,128.1,128.0,127.7,127.25,127.15,120.7,120.6,111.5,111.4,111.3,111.3,86.3,82.5,82.4,78.9,78.15,77.37,77.16,76.9,76.23,61.9,61.9,60.8,60.7,59.6,59.4,59.1,58.2,57.9,57.8,57.6,57.0,56.3,56.1,55.9,55.80,55.7,54.1,53.8,53.0,52.9,52.8,52.7,47.9,,46.8,46.7,45.9,42.8,39.48,39.10,39.03,38.94,37.7,37.6,35.9,33.5,33.1,33.0,32.5,32.0,31.0,29.8,29.0,28.9,28.6,27.8,26.3,26.1,25.8,25.6,25.1,25.0,24.8,24.5,23.7,20.8,20.2,20.1,19.9,19.8,19.6,18.1,18.0,17.9,17.9,17.9,16.7,16.5,16.2,15.6,14.9,14.7,14.1,12.4,11.0,10.8,10.8,10.4.MS 76.0(M+H)+-37.600(MeOH,0.50)。

實(shí)施例5

(S)-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(3-(4-chlorophenyl)azetidin-1-yl)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxo heptan-4-yl)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N,3-dimethylb utanamide(5)的制備

制備過程同實(shí)施例1；

1H NMR(400MHz,Chloroform-d)δ7.23(d,J＝8.1Hz,2H),7.15(d,J＝8.1Hz,2H),4.25(t,J＝8.7Hz,2H),3.85(t,J＝8.6,5.8Hz,2H),3.64-3.57(m,1H),1.39(s,9H)

1H NMR(400MHz,CDCl3)7.33-7.32(m,2H),7.24-7.22(m,3H),4.58-4.53(m,1H),4.45-4.40(m,1H),4.36-4.34(m,1H),4.20-3.78(m,6H),3.46(s,3H),3.33-3.25(m,1H),2.50-2.39(m,1H),1.95-1.71(m,4H),1.52(s,9H),1.45(d,J＝6.8Hz,3H)；¹³C NMR(151MHz,Chloroform-d)δ174.6,154.5,133.1,129.0,128.2,128.0,84.3,84.1,82.6,79.9,79.1,61.1,60.8,59.5,59.3,58.8,57.6,57.6,55.9,54.9,54.8,47.0,46.7,39.6,39.3,38.6,38.5,32.2,29.7,28.7,28.6,26.3,24.2,14.6.

1H NMR(400MHz,CDCl3)7.36-7.31(m,2H),7.24-7.20(m,2H),7.06(brs,1H),[4.86(t,J＝7.2Hz)and 4.78(t,J＝7.8Hz),1H],4.67-4.60(m,1H),4.56-4.50(m,1H),4.43(t,J＝9.5Hz,1H),4.34(t,J＝9.5Hz,1H),4.21-3.74(m,11H),[3.43(s)and 3.41(s),3H],[3.36(s)and 3.35(s),3H],[3.13(s)and 3.02(s), 3H],2.63-0.79(m,36H)；¹³C NMR(150MHz,CDCl₃)δ174.6,174.3,174.0,173.3,171.6,170.3,169.9,140.6,140.3,140.0,139.8,133.3,133.0,129.29,129.2,129.10,128.2128.0,87.1,86.4,86.2,82.6,77.3,77.1,76.9,76.41,61.9,60.5,59.36,59.1,58.4,58.0,57.73,57.5,55.2,54.9,53.9,47.6,45.9,42.3,40.1,39.7,39.2,38.9,37.8,36.0,33.3,32.9,32.7,32.0,29.9,29.7,29.4,27.8,26.4,25.8,25.2,24.8,24.3,22.8,20.219.68,18.116.015.12,14.90,14.46,14.2,13.713.110.9,10.4MS 770.1(M+Na)+；-33.400(MeOH,0.50)。

實(shí)施例6

(S)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(3-(2-isopropylphenyl)azetidin-1-yl)-1-methoxy-2-methyl-3-ox opropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)-N,3-dimethy lbutanamide(6)的制備

制備過程同實(shí)施例1；

¹H NMR(400MHz,CDCl3)7.65(d,J＝7.6Hz,2H),7.20(d,J＝7.5Hz,2H),7.07(brs,1H),[4.88-4.84(m)and 4.80-4.75(m),1H],4.58-4.54(m,1H),4.50-4.32(m,3H),4.21-3.76(m,10H),3.52-3.46(m,2H),[3.43(s)and 3.41(s),3H],3.39-3.36(m,2H),[3.33(s)and 3.28(s),3H],[3.13(s)and 3.09(s),3H],3.03-0.79(m,35H)；¹³C NMR(150MHz,CDCl₃)δ174.6,174.3,174.0,173.3,171.6,169.9,140.6,140.0,139.8,133.0,129.29,129.10,128.2,87.1,86.4,86.2,82.6,77.3,77.1,76.9,76.41,61.9,60.5,59.36,59.1,58.4,58.0,57.73,57.5,55.2,54.9,53.9,47.6,45.9,42.3,40.1,39.7,39.2,38.9,37.8,36.0,33.3,32.9,32.7,32.0,29.9,29.7,29.4,27.8,26.4,25.8,25.2,24.8,24.3,22.8,20.219.68,18.116.015.12,14.90,14.46,14.2,13.713.110.9,10.4MS 756.0(M+H)+； -30.400(MeOH,0.50)。

實(shí)施例7

(S)-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(3-(4-chloro-2-methoxyphenyl)aze tidin-1-yl)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-m ethyl-1-oxoheptan-4-yl)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N,3-dimethylbutanamide(7)的制備

制備過程同實(shí)施例1；

1H NMR(400MHz,Chloroform-d)δ6.94(dd,J＝8.1,1.8Hz,1H),6.83(d,J＝1.7Hz,1H),4.25(t,J＝8.4Hz,2H),4.04–3.86(m,3H),3.80(s,3H),1.45(s,9H).

1H NMR(400MHz,Chloroform-d)δ7.27-7.10(m,1H),6.96-6.94(m,1H),6.83(d,J＝1.7Hz,1H),4.50-4.43(m,1H),4.35-4.31(m,1H),4.27-4.24(m,1H),4.19-4.08(m,2H),3.97-3.82(m,3H),3.81(s,3H),3.78-3.74(m,1H),3.26-3.22(m,1H),1.95(s,3H),1.92-1.68(m,4H),1.52(s,3H),1.44(d,J＝7.8Hz,3H).

13C NMR(151MHz,Chloroform-d)δ174.41,158.12,154.65,133.83,128.15,128.02,120.67,111.42,84.21,84.10,82.59,79.96,79.22,61.30,60.91,59.55,59.41,59.01,58.90,56.06,55.74,55.70,52.95,52.79,52.72,47.11,46.79,39.15,38.66,28.79,28.76,26.33,26.12,25.87,24.63,24.31,14.76,14.61,13.88.

¹H NMR(400MHz,CDCl₃)7.15-6.84(m,4H),[4.88-4.87(m)and4.79-4.77(m),1H],4.54-4.50(m,1H),4.45-4.41(m,1H),4.35-4.33(m,1H),4.35-4.33(m,1H),4.23-4.21(m,1H),4.18-4.13(m,4H),3.99-3.76(m,6H),[3.44(s)and 3.43(s),3H],[3.35(s)and 3.33(s),3H],[3.31(s)and 3.29(s),3H],2.63-0.80(m,39H)；

¹³C NMR(150MHz,DMSO-d6)δ175.5,174.5,174.3,173.9,173.2,171.3,170.0,158.1,156.4,138.8,129.9,129.8,129.0,128.9,128.8,128.7,126.5,126.2,121.3,117.0,116.9,116.8,86.3,86.1,82.5,82.3,61.8,60.6,59.5,59.2,59.0,58.2,58.1,57.8,57.7,57.4,57.1,55.9,55.2,55.0,54.8,54.6,53.9,47.8,46.7,42.7,39.5,38.8,37.6,35.9,33.4,32.5,32.4,29.7,29.5,29.2,27.6,27.2,26.3,26.0,25.7,25.4,25.0,24.4,22.6,20.8,20.0,19.5,18.2,15.8,14.1,10.9；13C NMR(151MHz,CDCl3)δ174.5,174.1,173.9,173.7,173.3,171.3,170.3,170.2,169.5,158.1,134.0,133.9,133.7,133.1,128.1,128.0,127.7,127.25,127.15,120.7,120.6,111.5,111.4,111.3,111.3,86.3,82.5,82.4,78.9,78.15,77.37,77.16,76.9,76.23,61.9,61.9,60.8,60.7,59.6,59.4,59.1,58.2,57.9,57.8,57.6,57.0,56.3,56.1,55.9,55.80,55.7,54.1,53.8,53.0,52.9,52.8,52.7,47.9,,46.8,46.7,45.9,42.8,39.48,39.10,39.03,38.94,37.7,37.6,35.9,33.5,33.1,33.0,32.5,32.0,31.0,29.8,29.0,28.9,28.6,27.8,26.3,26.1,25.8,25.6,25.1,25.0,24.8,24.5,23.7,20.8,20.2,20.1,19.9,19.8,19.6,18.1,18.0,17.9,17.9,17.9,16.7,16.5,16.2,15.6,14.9,14.7,14.1,12.4,11.0,10.8,10.8,10.4.MS 778.0(M+H)+-42.955(MeOH,0.44)。

實(shí)施例8

(S)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N-((3R,4S,5S)-1-((S)-2-(( 1R,2R)-3-(3-(4-fluorophenyl)azetidin-1-yl)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)-N,3-dimethylbutana mide(8)制備

¹H NMR(400MHz,CDCl₃)7.15-6.84(m,4H),[4.88-4.87(m)and4.79-4.77(m),1H],4.54-4.50(m,1H),4.45-4.41(m,1H),4.35-4.33(m,1H),4.35-4.33(m,1H),4.23-4.21(m,1H),4.18-4.13(m,4H),3.99-3.76(m,6H),[3.44(s)and 3.43(s),3H],[3.35(s)and 3.33(s),3H],[3.31(s)and 3.29(s),3H],2.63-0.80(m,39H)；

¹³C NMR(150MHz,DMSO-d6)δ175.5,174.5,174.3,173.9,173.2,171.3,170.0,158.1,156.4,138.8,129.9,129.8,129.0,128.9,128.8,128.7,126.5,126.2,121.3,117.0,116.9,116.8,86.3,86.1,82.5,82.3,61.8,60.6,59.5,59.2,59.0,58.2,58.1,57.8,57.7,57.4,57.1,55.9,55.2,55.0,54.8,54.6,53.9,47.8,46.7,42.7,39.5,38.8,37.6,35.9,33.4,32.5,32.4,29.7,29.5,29.2,27.6,27.2,26.3,26.0,25.7,25.4,25.0,24.4,22.6,20.8,20.0,19.5,18.2,15.8,14.1,10.9；¹³C NMR(150MHz,CDCl3)δ174.5,174.1,173.9,173.7,173.3,171.3,170.3,170.2,169.5,158.1,134.0,133.9,133.7,133.1,128.1,128.0,127.7,127.25,127.15,120.7,120.6,111.5,111.4,111.3,111.3,86.3,82.5,82.4,78.9,78.15,77.37,77.16,76.9,76.23,61.9,61.9,60.8,60.7,59.6,59.4,59.1,58.2,57.9,57.8,57.6,57.0,56.3,56.1,55.9,55.80,55.7,54.1,53.8,53.0,52.9,52.8,52.7,47.9,,46.8,46.7,45.9,42.8,39.48,39.10,39.03,38.94,37.7,37.6,35.9,33.5,33.1,33.0,32.5,32.0,31.0,29.8,29.0,28.9,28.6,27.8,26.3,26.1,25.8,25.6,25.1,25.0,24.8,24.5,23.7,20.8,20.2,20.1,19.9,19.8,19.6,18.1,18.0,17.9,17.9,17.9,16.7,16.5,16.2,15.6,14.9,14.7,14.1,12.4,11.0,10.8,10.8,10.4.MS 778.0(M+H)+-40.955(MeOH,0.44)。

實(shí)施例9

(S)-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(3-(4-(tert-butyl)phenyl)azetidin-1 -yl)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N,3-dimet hylbutanamide(9)的制備

1H NMR(400MHz,Chloroform-d)δ7.38(d,J＝8.3Hz,2H),7.25(d,J＝8.1Hz,2H),4.31(t,J＝8.6Hz,2H),4.10–3.87(m,2H),3.72(m,1H),1.47(s,9H),1.32(s,9H).

1H NMR(400MHz,Chloroform-d)δ7.38(d,J＝8.3Hz,2H),7.25(d,J＝8.1Hz,2H),4.55-4.51(m,1H),4.43-4.32(m,1H),4.12-3.76(m,8H),3.46(s,3H),2.48-2.39(m,1H),1.98-1.74(m,4H),1.52(s,9H),1.32(s,9H),1.24(d,J＝6.4Hz,3H).

1H NMR(400MHz,CDCl3)7.36-7.31(m,2H),7.24-7.20(m,2H),7.06(brs,1H),[4.86(t,J＝7.2Hz)and 4.78(t,J＝7.8Hz),1H],4.67-4.60(m,1H),4.56-4.50(m,1H),4.43(t,J＝9.5Hz,1H),4.34(t,J＝9.5Hz,1H),4.21-3.74(m,11H),[3.43(s)and 3.41(s),3H],[3.36(s)and 3.35(s),3H],[3.13(s)and 3.02(s),3H],2.63-0.79(m,36H)；¹³C NMR(150MHz,CDCl₃)δ174.6,174.3,174.0, 173.3,171.6,170.3,169.9,140.6,140.3,140.0,139.8,133.3,133.0,129.29,129.2,129.10,128.2128.0,87.1,86.4,86.2,82.6,77.3,77.1,76.9,76.41,61.9,60.5,59.36,59.1,58.4,58.0,57.73,57.5,55.2,54.9,53.9,47.6,45.9,42.3,40.1,39.7,39.2,38.9,37.8,36.0,33.3,32.9,32.7,32.0,29.9,29.7,29.4,27.8,26.4,25.8,25.2,24.8,24.3,22.8,20.219.68,18.116.015.12,14.90,14.46,14.2,13.713.110.9,10.4；MS 770.1(M+Na)+；-33.400(MeOH,0.50)。

實(shí)施例10

(S)-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(3-([1,1'-biphenyl]-4-yl)azetidin-1-yl)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N,3-dimeth ylbutanamide(10)的制備

1H NMR(400MHz,CDCl3)7.58(d,J＝7.8Hz,4H),7.44(t,J＝7.5Hz,2H),7.40-7.33(m,3H),4.61-4.56(m,1H),4.48-4.37(m,1H),4.23-4.06(m,2H),3.91-3.78(m,3H),3.46(s,3H),3.28-3.26(m,1H),2.49-2.42(m,1H),2.08-1.92(m,6H),1.53(s,9H),1.25(t,J＝6.4Hz,3H)；13C NMR(150MHz,Chloroform-d)δ156.58,141.42,140.83,140.10,128.93,128.88,127.57,127.45,127.36,127.29,127.17,79.74,33.37,28.58.

1H NMR(400MHz,CDCl3)7.58(d,J＝8.4Hz,4H),7.43(t,J＝7.5Hz,2H),7.37-7.35(m,3H),6.94(brs,1H),[4.89-4.85(m)and 4.80-4.76(m),1H], 4.69-4.62(m,1H),4.58-4.52(m,1H),4.48-4.46(m,1H),4.42-4.35(m,1H),4.30-4.27(m,1H),4.22-4.19(m,4H),[3.43(s)and 3.42(s),3H],[3.36(s)and3.34(s),3H],[3.15(s)and 3.02(s),3H],2.78-0.81(m,42H)；13C NMR(400MHz,CDCl3)13C NMR(150MHz,Chloroform-d)δ156.58,141.42,140.83,140.10,128.93,128.88,127.57,127.45,127.36,127.29,127.17,79.74,33.37,28.58.

¹H NMR(400MHz,CDCl₃)7.58(d,J＝7.8Hz,4H),7.44(t,J＝7.5Hz,2H),7.40-7.33(m,3H),4.36(t,J＝8.6Hz,2H),4.02(J＝8.6Hz,2H),3.81-3.75(m,1H),1.48(s,3H)；¹³C NMR(400MHz,CDCl₃)13C NMR(150MHz,Chloroform-d)δ156.58,141.42,140.83,140.10,128.93,128.88,127.57,127.45,127.36,127.29,127.17,79.74,52.9,52.8,52.7,47.9,,46.8,46.7,45.9,42.8,39.48,39.10,39.03,38.94,37.7,37.6,35.9,33.5,33.1,33.0,32.5,32.0,31.0,29.8,29.0,28.9,28.6,27.8,26.3,26.1,25.8,25.6,25.1,25.0,24.8,24.5,23.7,20.8,20.2,20.1,19.9,19.8,19.6；MS 790.0(M+H)+；-23.636(MeOH,0.11)。

實(shí)施例11

體外腫瘤細(xì)胞增殖抑制活性篩選

選用不同的腫瘤細(xì)胞株對(duì)所合成的10個(gè)化合物進(jìn)行了體外抗腫瘤活性篩選。

實(shí)驗(yàn)材料

細(xì)胞株：SU-DHL-6，MCF-7,HCT 116,HeLa；

培養(yǎng)基：DMEM細(xì)胞培養(yǎng)基(GIBCO)：含10％滅活新生小牛血清(上 海賽達(dá)生物藥業(yè)有限公司)；L-谷氨酰氨(進(jìn)口分裝，SANGON)；丙酮酸鈉；GIBCO雙抗，用于KB細(xì)胞；

1640細(xì)胞培養(yǎng)基：含10％滅活新生小牛血清，GIBCO雙抗，用于K562、L1210細(xì)胞；

IMDM細(xì)胞培養(yǎng)基：含20％滅活胎牛血清，GIBCO雙抗；用于HL-60細(xì)胞；

藥物配制方法：樣品制備：將樣品溶解于二甲亞砜中，得到濃度為10mg/ml的溶液。再用磷酸鹽緩沖液(PBS)作梯度稀釋，得到濃度分別為1000μg/ml、100μg/ml、10μg/ml、1μg/ml、0.1μg/ml、0.01μg/ml的稀釋樣品；

腫瘤細(xì)胞離體培養(yǎng)

1、對(duì)于沉淀細(xì)胞，采用步驟3-7，對(duì)于懸浮細(xì)胞采用步驟4-7；

2、將細(xì)胞從液氮中取出，在37℃水浴中迅速解凍，細(xì)胞在無菌操作臺(tái)中移入10ml無菌離心管中加6ml相應(yīng)細(xì)胞培養(yǎng)基，1000轉(zhuǎn)/分離心5分鐘；

3、加入3.0mL Trypsin-EDTA溶液，顯微鏡下觀察直至分層；

4、加入8.0mL生長(zhǎng)基質(zhì)，小心移除細(xì)胞；

5、無菌離心管中加6ml相應(yīng)細(xì)胞培養(yǎng)基，1000轉(zhuǎn)/分離心5分鐘，棄去上清液；隔日，自培養(yǎng)箱中取出細(xì)胞，棄去細(xì)胞瓶中DMEM細(xì)胞培養(yǎng)基，加入PBS(pH7.4)2-3ml晃動(dòng)清洗，倒掉PBS溶液后再重復(fù)一次清洗，在培養(yǎng)瓶中加入3-5滴0.25％胰蛋白酶溶液晃動(dòng)均勻，加蓋置于37℃細(xì)胞培養(yǎng)箱內(nèi)3分鐘左右，于顯微鏡下觀察發(fā)現(xiàn)細(xì)胞自培養(yǎng)瓶壁上脫離，加DMEM細(xì)胞培養(yǎng)基2ml，滴管吹打使細(xì)胞完全脫離瓶壁后，分別移入2個(gè)干凈培養(yǎng)瓶中，加入DMEM細(xì)胞培養(yǎng)基5-6ml吹打均勻，置于37℃細(xì)胞培養(yǎng)箱內(nèi)；懸浮細(xì)胞取出1－2ml懸浮液，分別移入2個(gè)干凈培養(yǎng)瓶中，K562、L1210加入1640細(xì)胞培養(yǎng)基5-6ml、HL-60加入IMDM細(xì)胞培養(yǎng)基，置于37℃細(xì)胞培養(yǎng)箱內(nèi)；

6、隔日，重復(fù)步驟③。在整個(gè)培養(yǎng)過程中，貼壁細(xì)胞不允許生長(zhǎng)過密，懸浮細(xì)胞始終保持對(duì)數(shù)生長(zhǎng)期；

MTT法測(cè)定14個(gè)化合物對(duì)不同瘤株的體外增殖抑制作用

收獲對(duì)數(shù)生長(zhǎng)期細(xì)胞，計(jì)數(shù)后調(diào)整濃度至20×10⁴/mL，96孔板，每孔加90μL細(xì)胞懸液，藥物按比例稀釋后10μL/孔加樣，計(jì)算終濃度，混勻后37℃、5％CO₂培養(yǎng)箱中培養(yǎng)48小時(shí)后，加5mg/ml MTT(PBS)溶液20μL/孔，37℃、5％CO₂培養(yǎng)箱中再培養(yǎng)4小時(shí)后，加溶解液(10％SDS，5％異丁醇，0.02M HCl)100μL/孔，37℃、5％CO₂培養(yǎng)箱中放置過夜，次日觀察各孔中沉淀完全溶解即可用酶標(biāo)儀在570nm測(cè)定OD值。

進(jìn)行回歸，計(jì)算IC₅₀。