本發(fā)明屬于藥物合成技術(shù)領(lǐng)域,涉及一類L-對(duì)硝基苯丙氨酰胺二肽衍生物,還涉及其制備方法以及在制藥中的應(yīng)用。
背景技術(shù):
糖尿病(diabetes mellitus,DM)是由于機(jī)體胰島素絕對(duì)或相對(duì)不足以及細(xì)胞對(duì)胰島素敏感性降低而引起的內(nèi)分泌代謝疾病,現(xiàn)已成為全球發(fā)病率和死亡率最高的三大非傳染性疾病之一,是威脅人類健康的重大社會(huì)問(wèn)題。目前,糖尿病治療藥物種類繁多,類型各異,但長(zhǎng)期使用會(huì)出現(xiàn)程度不等的肝毒性、體重增加、水腫和消化不良等副作用,而且一般不能有效改善糖尿病伴發(fā)的脂代謝紊亂。因此,研究開發(fā)高效低毒的新穎抗糖尿病藥物,仍然是糖尿病防治工作中的重要任務(wù)。
過(guò)氧化物酶體增殖物激活受體(PPAR)是核受體超家族的一員,與許多生理和病理過(guò)程如細(xì)胞分化、脂質(zhì)代謝、糖尿病、肥胖、動(dòng)脈粥樣硬化、腫瘤抑制和能量平衡等密切相關(guān),其激動(dòng)劑被成功地應(yīng)用于2型糖尿病的治療。
技術(shù)實(shí)現(xiàn)要素:
有鑒于此,本發(fā)明的目的在于在現(xiàn)有技術(shù)的基礎(chǔ)上,進(jìn)一步研究開發(fā)高效低毒的新穎抗糖尿病藥物。
經(jīng)研究,本發(fā)明提供如下技術(shù)方案:
1.L-對(duì)硝基苯丙氨酰胺二肽衍生物,具有以下結(jié)構(gòu)式:
式中,R1為H,R2為甲基、乙基、正丙基、異丙基、正丁基、2-(乙酰氧基)乙基、環(huán)丙基、環(huán)戊基、環(huán)己基、甲氧基、2-(二甲基氨基)乙基或3-(二甲基氨基)丙基;
或者,R1和R2都為甲基;
或者,R1和R2都為乙基;
或者,-NR1R2為嗎啉基、四氫吡咯基、哌啶基、4-甲基哌嗪基或4-芐基哌嗪基。
優(yōu)選的,R1為H,R2為乙基、正丙基、異丙基、正丁基、環(huán)丙基、環(huán)戊基、環(huán)己基、甲氧基或2-(二甲基氨基)乙基;
或者,R1和R2都為甲基;
或者,R1和R2都為乙基;
或者,-NR1R2為4-甲基哌嗪基或4-芐基哌嗪基。
更優(yōu)選的,R1為H,R2為甲氧基;或者,R1和R2都為乙基;或者,-NR1R2為4-甲基哌嗪基。
最優(yōu)選的,R1和R2都為乙基。
2.所述L-對(duì)硝基苯丙氨酰胺二肽衍生物的制備方法,包括以下步驟:
A.將L-對(duì)硝基苯丙氨酸進(jìn)行氨基保護(hù),制得中間體I;
B.將中間體I與化合物R1R2NH偶聯(lián),制得中間體II;
C.將中間體II脫除氨基保護(hù)基,制得中間體III;
D.將中間體III與Fmoc-Val-OH偶聯(lián),制得L-對(duì)硝基苯丙氨酰胺二肽衍生物;
所述中間體I和中間體II結(jié)構(gòu)式中X為氨基保護(hù)基,所述化合物R1R2NH、中間體II和中間體III結(jié)構(gòu)式中R1和R2的定義與L-對(duì)硝基苯丙氨酰胺二肽衍生物結(jié)構(gòu)式中R1和R2的定義相同。
優(yōu)選的,所述L-對(duì)硝基苯丙氨酰胺二肽衍生物的制備方法,包括以下步驟:
A.將L-對(duì)硝基苯丙氨酸與(Boc)2O在含水有機(jī)溶劑中、堿作用下反應(yīng),制得中間體I;所述有機(jī)溶劑為乙醚、丙酮、四氫呋喃、二氧六環(huán)、N,N-二甲基甲酰胺、二氯甲烷和氯仿中的任一種或多種混合;所述堿為碳酸鈉、碳酸鉀、碳酸氫鈉、碳酸氫鉀、三乙胺、三甲胺、吡啶和N,N’-二異丙基乙胺中的任一種或多種混合;
B.將中間體I與化合物R1R2NH在有機(jī)溶劑中、堿以及偶聯(lián)劑和偶聯(lián)助劑作用下偶聯(lián),制得中間體II;所述有機(jī)溶劑為四氫呋喃、二氧六環(huán)、N,N-二甲基甲酰胺、二氯甲烷、氯仿和乙二醇二甲醚中的任一種或多種混合;所述堿為N,N’-二異丙基乙胺(DIPEA)、三乙胺、三甲胺、吡啶、4-二甲胺基吡啶和2,6-二甲基吡啶中的任一種或多種混合;所述偶聯(lián)劑為N,N'-二環(huán)己基碳二亞胺(DCC)、N,N'-二異丙基碳二亞胺(DIC)、1-(3-二甲氨基丙基)-3-乙基碳二亞胺鹽酸鹽(EDCI)、六氟磷酸苯并三唑-1-氧基三(二甲氨基)磷(BOP)、六氟磷酸苯并三唑-1-基-氧基三吡咯烷基磷(PyBOP)、苯并三氮唑-N,N,N',N'-四甲基脲六氟磷酸酯(HBTU)和O-苯并三氮唑-N,N,N',N'-四甲基脲四氟硼酸酯(TBTU)中的任一種或多種混合;所述偶聯(lián)助劑為1-羥基苯并三氮唑(HOBt)、6-氯-1-羥基苯并三氮唑(Cl-HOBt)以及1-羥基-7-偶氮苯并三氮唑(HOAt)中的任一種或多種混合;
C.將中間體II在有機(jī)溶劑中、酸性試劑作用下脫除N-Boc保護(hù)基,制得中間體III;所述有機(jī)溶劑為甲醇、乙醇、二氯甲烷、氯仿、四氫呋喃、乙酸乙酯、乙醚、甲丁醚、異丙醚和丙醚中的任一種或多種混合;所述酸性試劑為三氟醋酸、濃鹽酸、二氯亞砜、氯化氫和溴化氫中的任一種或多種混合;
D.將中間體III與Fmoc-Val-OH在有機(jī)溶劑中、堿以及偶聯(lián)劑和偶聯(lián)助劑作用下偶聯(lián),制得L-對(duì)硝基苯丙氨酰胺二肽衍生物;所述有機(jī)溶劑為四氫呋喃、二氧六環(huán)、N,N-二甲基甲酰胺、二氯甲烷、氯仿和乙二醇二甲醚中的任一種或多種混合;所述堿為DIPEA、三乙胺、三甲胺、吡啶、4-二甲胺基吡啶和2,6-二甲基吡啶中的任一種或多種混合;所述偶聯(lián)劑為DCC、DIC、EDCI、BOP、PyBOP、HBTU和TBTU中的任一種或多種混合;所述偶聯(lián)助劑為HOBt、Cl-HOBt和HOAt中的任一種或多種混合。
更優(yōu)選的,步驟A中所述有機(jī)溶劑為丙酮,堿為碳酸鈉;步驟B中所述有機(jī)溶劑為四氫呋喃,堿為DIPEA,偶聯(lián)劑為DCC,偶聯(lián)助劑為HOBt;步驟C中所述有機(jī)溶劑為二氯甲烷,酸性試劑為三氟醋酸;步驟D中所述有機(jī)溶劑為二氯甲烷,堿為DIPEA,偶聯(lián)劑為EDCI,偶聯(lián)助劑為HOBt。
3.所述L-對(duì)硝基苯丙氨酰胺二肽衍生物在制備抗糖尿病藥物中的應(yīng)用。
進(jìn)一步,所述抗糖尿病藥物為PPAR激動(dòng)劑。
本發(fā)明的有益效果在于:本發(fā)明設(shè)計(jì)合成了一類新穎的L-對(duì)硝基苯丙氨酰胺二肽衍生物,產(chǎn)物的化學(xué)結(jié)構(gòu)經(jīng)1H NMR,13C NMR和HR MS確認(rèn)。體外抗糖尿病活性測(cè)試結(jié)果表明,大部分化合物都具有PPAR激動(dòng)活性,其中化合物TM2-B8、TM2-B15和TM2-B18相對(duì)于羅格列酮的PPRE激動(dòng)率分別達(dá)到270.96%、108.53%和132.76%,EC50分別為0.4380μg/mL、1.9534μg/mL和6.8027μg/mL,這些化合物都可以用于制備抗糖尿病的藥物,在糖尿病治療領(lǐng)域具有潛在的應(yīng)用前景。
具體實(shí)施方式
為了使本發(fā)明的目的、技術(shù)方案和有益效果更加清楚,下面將對(duì)本發(fā)明的優(yōu)選實(shí)施例進(jìn)行詳細(xì)的描述。
一、L-對(duì)硝基苯丙氨酰胺二肽衍生物的合成
1、中間體I的合成
冰浴下,向反應(yīng)瓶中加入L-對(duì)硝基苯丙氨酸20mmol和飽和Na2CO3水溶液50mL,冰浴攪拌至溶解,緩慢滴入溶有(Boc)2O 24mmol的丙酮溶液10mL,攪拌1h后轉(zhuǎn)入35-55℃水浴中攪拌反應(yīng),保持反應(yīng)液pH為8~9,薄層色譜(TLC)監(jiān)測(cè)直至反應(yīng)結(jié)束。在pH約為10的條件下,將反應(yīng)液用乙醚萃取(30mL×2),用3N HCl調(diào)節(jié)水相pH至3~5,再用乙酸乙酯(EA)萃取(30mL×3),收集有機(jī)相,用飽和NaCl水溶液20mL洗滌,無(wú)水Na2SO4干燥,減壓蒸餾除去EA,得到中間體I(米白色固體)5.23g,收率83.4%。
2、中間體II的合成
冰浴下,向反應(yīng)瓶中加入中間體I 3~4mmol、HOBt 4.5~6mmol、無(wú)水四氫呋喃(THF)15~20mL、DIPEA 4.2~6mmol和DCC 5.4~7.2mmol,攪拌活化,加入經(jīng)過(guò)干燥處理后的R1R2NH/THF溶液7.5~10mL,控溫反應(yīng),TLC監(jiān)測(cè)直至反應(yīng)結(jié)束。將反應(yīng)液減壓蒸餾除去THF,加入EA 60~80mL和水40~60mL,攪拌10min,抽濾,濾餅用EA洗滌(15mL×3),洗液和濾液合并后,依次用10%檸檬酸水溶液(40mL×2)、0.5mol/L NaHCO3水溶液(40mL×2)、飽和NaCl水溶液(40mL)和水(20mL)洗滌,無(wú)水Na2SO4干燥,減壓蒸餾除去EA,柱層析純化(洗脫劑為體積比5:1~1:2的石油醚-乙酸乙酯混合液),得到中間體II。實(shí)驗(yàn)結(jié)果見(jiàn)表1。
表1中間體II的合成結(jié)果
3、中間體III的合成
向反應(yīng)瓶中加入中間體II 1~3.4mmol和二氯甲烷(DCM)2~8mL,攪拌得澄清液,冰浴下滴加三氟乙酸(TFA)1~4mL(DCM與TFA的體積比為2:1),滴畢轉(zhuǎn)入常溫下反應(yīng),TLC監(jiān)測(cè)反應(yīng)直至結(jié)束。將反應(yīng)液旋蒸盡可能除去TFA,殘余液在冰浴下加入冰乙醚15~50mL,迅速析出白色固體,冰柜中靜置2~3h后抽濾,濾餅用冰乙醚洗滌(10mL×3),真空干燥,得到中間體III。實(shí)驗(yàn)結(jié)果見(jiàn)表2。
表2中間體III的合成結(jié)果
4、L-對(duì)硝基苯丙氨酰胺二肽衍生物TM2-B的合成及表征
向反應(yīng)瓶中加入Fmoc-Val-OH 1.3mmol、HOBt 1.8~2.6mmol、DCM 6~10mL和DIPEA2.6mmol,冰浴攪拌30min,加入EDCI 1.8~2.6mmol,繼續(xù)攪拌活化1h,TLC檢測(cè)活性酯生成情況,再加入中間體III 1.0mmol,控溫22~28℃攪拌反應(yīng),TLC監(jiān)測(cè)直至反應(yīng)結(jié)束。向反應(yīng)液中加入EA 40mL,依次用10%檸檬酸水溶液(30mL×2)、0.5mol/L NaHCO3水溶液(30mL×2)、飽和NaCl水溶液(20mL)及水(10mL)洗滌,無(wú)水Na2SO4干燥,減壓蒸餾除去EA,柱層析(洗脫劑為體積比6:1~1:2的石油醚-乙酸乙酯混合液),得到目標(biāo)化合物TM2-B。實(shí)驗(yàn)結(jié)果見(jiàn)表3。
表3目標(biāo)化合物TM2-B的合成結(jié)果
產(chǎn)物結(jié)構(gòu)表征數(shù)據(jù)如下:
TM2-B1:m.p.192.1~193.9℃;1H NMR(600MHz,DMSO-d6)δ:8.09(d,J=8.6Hz,2H,H-11),7.95-7.88(m,3H,H-15,H-17),7.72(t,J=7.9Hz,2H,H-14),7.48(d,J=8.6Hz,2H,H-10),7.44-7.38(m,3H,H-13,H-2),7.33-7.30(m,3H,H-12,H-16),4.65-4.50(m,1H,H-9),4.36-4.11(m,3H,H-7,H-6),3.85-3.72(m,1H,H-8),3.09(dd,J=13.5,5.0Hz,1H,H-5),2.93(dd,J=13.5,9.4Hz,1H,H-4),2.57(d,J=4.5Hz,2H,H-5),1.85(dt,J=13.0,6.6Hz,1H,H-3),0.74(dd,J=19.5,6.8Hz,6H,H-1);HR MS formula:C30H32N4O6,calcd:544.5983,found:544.2223.
TM2-B2:m.p.208.6~210.4℃;1H NMR(600MHz,DMSO-d6)δ:8.10(m,3H,H-11,H-16),7.97(s,1H,H-17),7.90(d,J=7.4Hz,2H,H-15),7.72(t,J=7.8Hz,2H,H-14),7.49(d,J=8.5Hz,2H,H-10),7.41(t,J=7.8Hz,2H,H-13),7.37-7.26(m,3H,H-18,H-12),4.56(m,1H,H-9),4.30(t,J=8.3Hz,1H,H-7),4.23(dd,J=17.6,7.4Hz,2H,H-6),3.82-3.75(m,1H,H-8),3.14-2.99(m,3H,H-4,H-5),2.94(dd,J=13.3,9.4Hz,1H,H-4),1.87(m,1H,H-3),0.96(t,J=7.2Hz,3H,H-2),0.74(dd,J=19.1,6.8Hz,6H,H-1);HR MS formula:C31H34N4O6,calcd:558.2478,found:558.2372.
TM2-B3:m.p.205.9~208.8℃;1H NMR(600MHz,DMSO-d6)δ:8.10(d,J=8.8Hz,2H,H-11),7.98-7.79(m,4H,H-15,H-17,H-2),7.72(t,J=7.7Hz,2H,H-14),7.55(d,J=8.4Hz,2H,H-10),7.42(t,J=6.8Hz,2H,H-13),7.33-7.31(m,3H,H-12,H-16),4.63-4.54(m,1H,H-9),4.33-4.17(m,3H,H-7,H-6),3.86-3.72(m,1H,H-8),3.12-2.90(m,4H,H-4,H-5),1.86(dt,J=15.9,7.9Hz,1H,H-3),1.39-1.30(m,2H,H-18),0.82-0.65(m,9H,H-1,H-19);HR MS formula:C32H36N4O6,calcd:572.2635,found:572.2531.
TM2-B4:m.p.203.1~206.3℃;1H NMR(600MHz,DMSO-d6)δ:8.40(d,J=8.6Hz,1H,H-15),8.09(d,J=8.6Hz,2H,H-10),7.89(d,J=6.8Hz,2H,H-14),7.76-7.67(m,3H,H-9,H-17),7.54-7.46(m,3H,H-13,H-15),7.41(d,J=7.4Hz,2H,H-11),7.34-7.30(m,2H,H-12),4.61-4.45(m,1H,H-5),4.38-4.14(m,3H,H-7,H-8),3.88-3.68(m,2H,H-6,H-3),3.20(dd,J=13.8,4.5Hz,1H,H-4),2.86(dd,J=13.4,11.0Hz,1H,H-4),1.71-1.74(m,1H,H-2),1.07(d,J=6.6Hz,3H,H-18),0.96(d,J=6.6Hz,3H,H-18),0.46-0.66(m,6H,H-1);13C NMR(151MHz,DMSO-d6)δ:171.79,169.71,156.83,147.00,146.66,144.20,141.19,131.06,128.10,127.51,125.73,123.50,120.55,66.30,61.28,54.07,47.13,41.06,37.80,29.99,22.65,19.22,18.82;HR MS formula:C32H36N4O6,calcd:572.2635,found:572.2529.
TM2-B5:m.p.186.6~189.5℃;1H NMR(600MHz,DMSO-d6)δ:8.16-8.05(m,3H,H-10,H-16),8.01(d,J=8.0Hz,1H,H-15),7.95-7.85(m,3H,H-13,H-18),7.72(t,J=7.7Hz,2H,H-12),7.49(d,J=8.4Hz,2H,H-9),7.42(t,J=7.1Hz,2H,H-12),7.36-7.30(m,2H,H-11),4.64-4.49(m,1H,H-5),4.36-4.05(m,3H,H-7,H-8),3.91-3.73(m,1H,H-3),3.07-2.93(m,4H,H-6,H-4),1.90-1.83(m,1H,H-2),1.32-1.25(m,2H,H-17),1.19-1.13(m,2H,H-18),0.83-0.70(m,9H,H-1,H-19);HR MS formula:C33H38N4O6,calcd:586.2791,found:586.2689.
TM2-B6:m.p.219.2~221.3℃;1H NMR(600MHz,DMSO-d6)δ:8.20-8.06(m,4H,H-9,H-14,H-19),7.89(d,J=7.3Hz,2H,H-13),7.72(t,J=7.9Hz,2H,H-12),7.50(d,J=8.4Hz,2H,H-8),7.41(t,J=7.9Hz,2H,H-11),7.31(m,3H,H-10,H-15),4.61(m,1H,H-4),4.34-4.16(m,3H,H-6,H-7),3.94(t,J=5.4Hz,2H,H-17),3.86-3.74(m,1H,H-3),3.25(dd,J=14.0,5.7Hz,2H,H-16),3.08(dd,J=13.5,5.4Hz,1H,H-5),2.94(dd,J=13.5,9.2Hz,1H,H-5),1.97(s,3H,H-18),1.87(m,1H,H-2),0.75(dd,J=15.8,6.7Hz,6H,H-1);13C NMR(151MHz,DMSO-d6)δ:171.44,171.00,170.70,156.47,146.56,144.40,141.19,131.04,128.09,127.51,125.76,125.71,123.49,120.55,66.11,62.91,60.78,53.64,47.19,38.11,30.68,21.06,19.52,18.70.
TM2-B7:m.p.127.2~130.0℃;1H NMR(600MHz,DMSO-d6)δ:8.29(d,J=8.3Hz,1H,H-16),8.08(d,J=8.6Hz,2H,H-10),7.89(d,J=7.5Hz,2H,H-14),7.73(t,J=7.2Hz,2H,H-13),7.51(d,J=8.6Hz,2H,H-9),7.41(t,J=7.5Hz,2H,H-12),7.34-7.30(m,3H,H-11,H-15),4.99(dd,J=14.4,8.3Hz,1H,H-5),4.33-4.17(m,3H,H-7,H-8),3.83(dd,J=9.0,7.7Hz,1H,H-3),3.09(dd,J=13.5,5.8Hz,1H,H-4),2.97-2.88(m,4H,H-6,H-4),2.79(s,3H,H-6),1.85(dt,J=13.7,6.8Hz,1H,H-2),0.76(dd,J=18.9,6.7Hz,6H,H-1);13C NMR(151MHz,DMSO-d6)δ:171.20,170.37,156.37,146.70,146.37,144.43,141.20,131.20,128.09,127.51,125.69,123.47,120.55,66.12,60.65,49.77,47.21,37.69,37.05,35.69,30.78,19.56,18.68;HR MS formula:C31H34N4O6,calcd:558.2478,found:558.2380.
TM2-B8:m.p.131~133.3℃;1H NMR(600MHz,DMSO-d6)δ:8.34(d,J=8.5Hz,1H,H-17),8.09(d,J=8.4Hz,2H,H-11),7.89(d,J=7.5Hz,2H,H-15),7.72(t,J=6.6Hz,2H,H-14),7.52(d,J=8.4Hz,2H,H-10),7.41(t,J=7.4Hz,2H,H-13),7.36-7.23(m,3H,H-12,H-16),4.92(dd,J=15.0,7.9Hz,1H,H-6),4.34-4.14(m,3H,H-8,H-9),3.90-3.80(m,1H,H-3),3.36(dt,J=13.2,6.7Hz,1H,H-5),3.27-3.16(m,3H,H-5),3.08(dd,J=13.3,6.4Hz,1H,H-7),2.96(dd,J=13.2,8.2Hz,1H,H-7),1.86(m,1H,H-2),0.97(m,6H,H-4),0.79-0.71(m,6H,H-1);13C NMR(151MHz,DMSO-d6)δ:171.20,169.73,156.36,146.74,146.27,144.41,141.20,131.24,128.09,127.50,125.71,123.55,120.55,66.11,60.47,49.62,47.20,41.69,38.20,30.93,19.46,18.68,14.81,13.15;HR MS formula:C33H38N4O6,calcd:586.2791,found:586.2686.
TM2-B9:m.p.185.3~187.1℃;1H NMR(600MHz,DMSO-d6)δ:8.35(d,J=8.3Hz,1H,H-16),8.08(d,J=8.4Hz,2H,H-10),7.89(d,J=7.6Hz,2H,H-14),7.72(t,J=6.8Hz,2H,H-13),7.52(d,J=8.5Hz,2H,H-10),7.41(t,J=7.4Hz,2H,H-12),7.29-7.33(m,3H,H-11,H-15),5.01(dd,J=14.5,8.0Hz,1H,H-5),4.18-4.26(m,3H,H-7,H-8),3.83(dd,J=15.5,7.5Hz,1H,H-3),3.46-3.52(m,4H,H-17),3.32-3.41(m,4H,H-6),3.10(dd,J=13.5,6.0Hz,1H,H-4),2.96(dd,J=13.4,8.3Hz,1H,H-4),1.86(dq,J=13.4,6.7Hz,1H,H-2),0.76(dd,J=18.0,6.7Hz,6H,H-1);13C NMR(151MHz,DMSO-d6)δ:171.23,169.19,156.39,146.72,146.25,144.42,141.20,131.29,128.09,127.51,125.70,123.48,120.55,66.50,66.15,60.64,49.33,47.20,46.08,42.48,37.69,30.77,19.59,18.65;HR MS formula:C33H36N4O7,calcd:600.2584,found:600.2661.
TM2-B10:m.p.199.5~201.9℃;1H NMR(600MHz,DMSO-d6)δ:8.28(d,J=8.1Hz,1H,H-17),8.09(d,J=8.5Hz,2H,H-15),7.89(d,J=7.5Hz,2H,H-13),7.72(t,J=7.0Hz,2H,H-12),7.52(d,J=8.5Hz,2H,H-14),7.41(t,J=7.4Hz,2H,H-11),7.35-7.26(m,3H,H-10,H-16),4.77(dd,J=14.5,8.1Hz,1H,H-7),4.80(m,2H,H-8,H-9),3.89-3.80(m,1H,H-3),3.53(dt,J=13.3,6.7Hz,1H,H-5),3.24(m 3H,H-4,H-5),3.10(dd,J=13.4,6.0Hz,1H,H-6),2.95(dd,J=13.3,8.5Hz,1H,H-6),1.86(m,1H,H-2),1.80-1.61(m,4H,,H-18,H-4),0.75(dd,J=19.3,6.7Hz,6H,H-1);13C NMR(151MHz,DMSO-d6)δ:171.78,171.39,156.40,146.78,145.97,144.39,141.18,131.01,128.10,127.49,125.76,123.60,120.54,66.16,64.83,60.48,53.21,47.16,36.82,30.75,30.51,19.50,18.90,18.72,13.89;HR MS formula:C33H36N4O6,calcd:584.2635,found:584.2529.
TM2-B11:m.p.111.2~114.5℃;1H NMR(600MHz,DMSO-d6)δ:8.29(d,J=8.5Hz,1H,H-18),8.08(d,J=8.6Hz,2H,H-16),7.89(d,J=7.5Hz,2H,H-14),7.73(t,J=6.6Hz,2H,H-13),7.51(d,J=8.6Hz,2H,H-15),7.41(t,J=7.5Hz,2H,H-12),7.36-7.25(m,3H,H-11,H-17),5.02(dd,J=14.6,8.1Hz,1H,H-8),4.33-4.13(m,3H,H-9,H-10),3.84(dd,J=9.1,7.4Hz,1H,H-3),3.53-3.38(m,2H,H-5),3.09(dd,J=13.4,6.1Hz,1H,H-7),2.93(dd,J=13.4,8.2Hz,1H,H-7),1.86(m,1H,H-2),1.52-1.29(m,6H,H-5,H-6),0.84(m,2H,H-4),0.76(dd,J=16.6,6.9Hz,6H,H-1);13C NMR(151MHz,DMSO-d6)δ:171.17,168.56,156.40,146.65,146.36,144.39,141.18,131.25,128.10,127.51,125.67,123.46,120.53,66.17,60.65,49.43,47.18,46.40,43.00,37.98,30.80,26.44,25.70,24.34,19.58,18.59;HR MS formula:C34H38N4O6,calcd:598.2791,found:598.2692.
TM2-B12:m.p.211.6~212.5℃;1H NMR(600MHz,DMSO-d6)δ:8.03-8.10(m,4H,H-10,H-14),7.89(d,J=6.3Hz,2H,H-11),7.72(d,J=8.1Hz,2H,H-9),7.48(d,J=8.5Hz,1H,H-16),7.41-7.42(m,3H,H-13,H-15),7.37-7.29(m,3H,H-12,H-18),4.52(dd,J=14.3,8.6Hz,1H,H-5),4.21-4.32(m,3H,H-7,H-8),3.79(dd,J=15.9,7.5Hz,1H,H-3),3.06(dd,J=13.5,5.2Hz,1H,H-4),2.93(dd,J=13.4,9.3Hz,1H,H-4),2.62-2.54(m,1H,H-6),1.86(dt,J=14.2,7.0Hz,1H,H-2),0.74(dd,J=20.1,6.8Hz,6H,H-1),0.60(d,J=7.1Hz,2H,H-17),0.32(dd,J=18.2,3.3Hz,2H,H-17).
TM2-B13:m.p.187.5~191.5℃;1H NMR(600MHz,DMSO-d6)δ:8.07-8.10(m,3H,H-10,H-16),7.90-7.86(m,3H,H-14,H-18),7.72(t,J=7.8Hz,2H,H-13),7.49(d,J=8.5Hz,2H,H-9),7.41(t,J=7.3,2H,H-12),7.38-7.29(m,3H,H-11,H-15),4.58(dd,J=14.4,8.6Hz,1H,H-5),4.35-4.16(m,3H,H-7,H-8),3.95(dd,J=13.3,6.6Hz,1H,H-3),3.82-.75(m,1H,H-6),3.06(dd,J=13.4,5.3Hz,1H,H-4),2.94(dd,J=13.4,9.2Hz,1H,H-4),1.87(dd,J=13.7,6.8Hz,1H,H-2),1.73(ddd,J=19.4,12.8,6.1Hz,2H,H-17),1.62-1.41(m,4H,H-18),1.34(dd,J=13.2,7.0Hz,1H,H-19),1.23(dd,J=12.9,6.9Hz,1H,H-19),0.75(dd,J=18.7,6.7Hz,6H,H-1).
TM2-B14:m.p.105.26~107.8℃;1H NMR(600MHz,DMSO-d6)δ:8.35(d,J=8.6Hz,1H,H-16),8.10(d,J=8.6Hz,2H,H-10),7.89(d,J=7.5Hz,2H,H-14),7.72(t,J=6.6Hz,2H,H-13),7.52(d,J=8.6Hz,2H,H-9),7.41(t,J=7.5Hz,2H,H-12),7.40-7.22(m,4H,H-11,H-17,H-16),4.92(dd,J=15.0,8.1Hz,1H,H-5),4.31-4.19(m,3H,H-7,H-8),3.91-3.79(m,1H,H-3),3.41-3.36(m,2H,H-18),3.25-3.17(m,3H,H-18,H-6),3.08(dd,J=13.3,6.4Hz,1H,H-4),2.96(dd,J=13.2,8.2Hz,1H,H-4),1.85(dt,J=13.5,6.8Hz,1H,H-2),1.01-0.91(m,6H,H-20,H-19),0.76(dd,J=20.6,6.7Hz,6H,H-1);13C NMR(151MHz,DMSO-d6)δ:171.23,169.73,156.37,146.72,146.24,144.15,141.18,131.24,128.10,127.51,125.70,123.55,120.55,66.11,60.48,49.62,47.19,41.70,38.18,33.80,30.91,19.44,18.67,14.79,13.13.
TM2-B15:m.p.224.0~228.5℃;1H NMR(600MHz,DMSO-d6)δ:11.28(s,1H,H-17),8.29(d,J=8.3Hz,1H,H-16),8.11(d,J=8.4Hz,2H,H-10),7.89(d,J=7.2Hz,2H,H-14),7.72(t,J=7.8Hz,2H,H-13),7.54-7.36(m,4H,H-12,H-9),7.37-7.23(m,3H,H-11,H-15),4.41(dd,J=14.8,7.9 Hz,1H,H-5),4.33-4.14(m,3H,H-7,H-8),3.87-3.80(m,1H,H-3),3.48(s,3H,H-6),2.95-3.06(m,2H,H-4),1.87(dt,J=13.9,6.9Hz,1H,H-2),0.77(dd,J=13.9,6.7Hz,6H,H-1);13C NMR(151MHz,DMSO)δ:171.52,167.24,156.43,146.76,144.39,141.18,131.65,131.07,128.10,127.52,125.70,123.59,120.54,66.12,63.57,60.59,51.42,47.18,37.85,30.80,19.49,18.70.
TM2-B16:m.p.169.4~172.1℃;1H NMR(600MHz,DMSO-d6)δ:8.12(d,J=8.4Hz,1H,H-17),8.09(d,8.5Hz,2H,H-16),7.89(d,J=7.4Hz,3H,H-14,H-18),7.72(t,J=7.5Hz,2H,H-13),7.50(d,J=8.5Hz,2H,H-15),7.41(dd,J=7.2,4.5Hz,2H,H-12),7.36-7.29(m,3H,H-19,H-11),4.60(dd,J=14.1,8.7Hz,1H,H-10),4.29(m,1H,H-7),4.22(d,J=6.4Hz,2H,H-8),3.80(t,J=8.4Hz,1H,H-9),3.20-3.06(m,3H,H-6,H-4),2.93(dd,J=13.5,9.3Hz,1H,H-6),2.21(d,J=3.5Hz,2H,H-3),2.11(s,6H,H-5),1.87(m,1H,H-2),0.75(dd,J=16.5,6.7Hz,6H,H-1);13CNMR(151MHz,DMSO-d6)δ:171.42,170.53,156.46,141.20,139.91,137.92,131.08,129.38,127.74,127.51,125.70,123.46,121.82,120.46,66.15,60.86,58.40,53.67,45.55,37.25,30.66,19.78,19.52,18.70,17.14;HR MS formula:C33H39N5O6,calcd:601.6927,found:601.2927.
TM2-B17:m.p.156.4~158.6℃;1H NMR(600MHz,DMSO-d6)δ:8.16-8.07(m,3H,H-10,H-16),7.97(t,J=5.3Hz,1H,H-17),7.90(d,J=7.5Hz,2H,H-14),7.72(t,J=7.5Hz,2H,H-13),7.52-7.48(m,2H,H-9),7.42(t,J=7.4Hz,2H,H-12),7.34(ddd,J=11.6,7.3,2.3Hz,3H,H-11,H-15),4.57(dd,J=14.4,8.5Hz,1H,H-5),4.34-4.17(m,3H,H-7,H-8),3.81(dd,J=8.7,7.5Hz,1H,H-3),3.11-3.01(m,3H,H-4,H-6),2.98-2.90(m,1H,H-4),2.16-2.08(m,8H,H-20,H-19),1.88(dd,J=13.5,6.7Hz,1H,H-2),1.46(dt,J=14.2,7.0Hz,2H,H-18),0.75(dd,J=17.5,6.8Hz,6H,H-1);13C NMR(151MHz,DMSO-d6)δ:170.65,146.72,146.60,143.06,139.89,137.90,131.05,129.40,127.76,123.59,121.82,120.46,110.10,60.02,56.89,53.54,45.35,38.40,37.33,31.40,27.17,19.71,19.52,17.16;HR MS formula:C34H41N5O6,calcd:615.3057,found:615.3132.
TM2-B18:m.p.112.3~115.6℃;1H NMR(600MHz,DMSO-d6)δ:8.32(d,J=8.3Hz,1H),8.09(d,J=8.5Hz,2H),7.89(d,J=7.5Hz,2H),7.77–7.69(m,2H),7.51(t,J=8.9Hz,2H),7.41(t,J=7.4Hz,2H),7.31(dt,J=15.6,7.8Hz,3H),5.02(dd,J=14.8,7.8Hz,1H),4.40-4.11(m,3H),3.91–3.74(m,1H),3.50–3.32(m,4H),3.08(dd,J=13.3,6.3Hz,1H),2.95(dd,J=13.2,8.2Hz,1H),2.21–2.01(m,6H),1.86(dt,J=13.7,6.7Hz,1H),0.76(dd,J=17.2,6.7Hz,6H,H-);13C NMR(151 MHz,DMSO-d6)δ:171.18,169.12,146.23,143.07,141.19,139.90,137.91,131.32,129.39,127.75,123.60,121.83,120.47,60.04,54.64,49.01,47.19,45.96,45.36,41.94,37.86,31.56,30.81,19.85,18.66,17.19;HR MS formula:C34H39N5O6,calcd:613.2900,found:613.2927.
TM2-B19:m.p.113.2~116.1℃;1H NMR(600MHz,DMSO-d6)δ:8.30(d,J=8.2Hz,1H,H-16),8.08(d,J=8.4Hz,2H,H-10),7.89(d,J=7.4Hz,2H,H-14),7.72(d,J=7.1Hz,2H,H-11),7.51(d,J=8.2Hz,2H,H-9),7.41(t,J=7.2Hz,2H,H-13),7.35-7.27(m,5H,H-19),7.25-7.22(m,3H,H-15,H-12),5.01(dd,J=14.5,7.6Hz,1H,H-5),4.3-4.17(m,3H,H-7,H-8),3.87-3.78(m,1H,H-3),3.48(s,2H,H-6),3.42-3.34(m,4H,H-18),3.08(dd,J=13.3,6.0Hz,1H,H-4),2.95(dd,J=13.1,8.2Hz,1H,H-4),2.22-2.17(m,4H,H-17),1.85(dd,J=13.4,6.7Hz,1H,H-2),0.75(dd,J=23.6,6.7Hz,6H,H-1);HR MS formula:C40H43N5O6,calcd:689.3213,found:689.3287.
二、L-對(duì)硝基苯丙氨酰胺二肽衍生物的PPRE激動(dòng)活性檢測(cè)
將HepG2肝癌細(xì)胞按1.5×104/孔接種于96孔板,用含100U/mL鏈霉素和青霉素的低糖DMEM培養(yǎng)基在37℃、5%CO2條件下培養(yǎng)過(guò)夜,參照轉(zhuǎn)染試劑說(shuō)明書進(jìn)行質(zhì)粒轉(zhuǎn)染。轉(zhuǎn)染的質(zhì)粒包括帶有PPRE和螢火蟲熒光素酶(Luc)報(bào)告基因的質(zhì)粒pPPRE-Luc,以及用作轉(zhuǎn)染內(nèi)參照的帶有海腎熒光素酶的質(zhì)粒phRL-TK。轉(zhuǎn)染24h后換用含待測(cè)樣品的培養(yǎng)基,同時(shí)設(shè)立空白對(duì)照(未轉(zhuǎn)染的細(xì)胞)、陰性對(duì)照(轉(zhuǎn)染的細(xì)胞不加樣品)和陽(yáng)性對(duì)照(轉(zhuǎn)染的細(xì)胞加入馬來(lái)酸羅格列酮),繼續(xù)培養(yǎng)24h后,用雙熒光素酶報(bào)告基因檢測(cè)試劑盒檢測(cè)熒光素酶活性。根據(jù)檢測(cè)到的化學(xué)發(fā)光強(qiáng)度L值計(jì)算激動(dòng)率。激動(dòng)率=[(L1樣品-L1空白)/(L1陰性-L1空白)]/[(L2樣品-L2空白)/(L2陰性-L2空白)]×100%,其中L1為螢火蟲熒光素酶的化學(xué)發(fā)光強(qiáng)度,L2為內(nèi)參照海腎熒光素酶的化學(xué)發(fā)光強(qiáng)度。相對(duì)激動(dòng)率=樣品的絕對(duì)激動(dòng)率/陽(yáng)性對(duì)照的絕對(duì)激動(dòng)率×100%。樣品檢測(cè)濃度為10μg/mL,陽(yáng)性對(duì)照馬來(lái)酸羅格列酮的檢測(cè)濃度為0.70μg/mL。初篩時(shí)每個(gè)樣品設(shè)雙復(fù)孔,重復(fù)檢測(cè)兩次。實(shí)驗(yàn)結(jié)果見(jiàn)表4。
表4 TM2-B的PPRE激動(dòng)活性初篩結(jié)果
相對(duì)激動(dòng)率大于100%的樣品進(jìn)一步測(cè)定半數(shù)效應(yīng)濃度(EC50),測(cè)定時(shí)每個(gè)樣品梯度稀釋六個(gè)濃度,每個(gè)濃度設(shè)雙復(fù)孔。根據(jù)激動(dòng)率,應(yīng)用Xlfit軟件中的4 Parameter Logistic Model計(jì)算EC50。實(shí)驗(yàn)結(jié)果見(jiàn)表5。
表5 EC50測(cè)定結(jié)果
上述實(shí)驗(yàn)結(jié)果顯示,本發(fā)明合成的L-對(duì)硝基苯丙氨酰胺二肽衍生物大部分都具有PPAR激動(dòng)活性,其中化合物TM2-B8、TM2-B15和TM2-B18相對(duì)于羅格列酮的PPRE激動(dòng)率分別達(dá)到270.96%、108.53%和132.76%,EC50分別為0.4380μg/mL、1.9534μg/mL和6.8027μg/mL,這些化合物都可以用于制備抗糖尿病的藥物,在糖尿病治療領(lǐng)域具有潛在的應(yīng)用前景。
最后說(shuō)明的是,以上優(yōu)選實(shí)施例僅用以說(shuō)明本發(fā)明的技術(shù)方案而非限制,盡管通過(guò)上述優(yōu)選實(shí)施例已經(jīng)對(duì)本發(fā)明進(jìn)行了詳細(xì)的描述,但本領(lǐng)域技術(shù)人員應(yīng)當(dāng)理解,可以在形式上和細(xì)節(jié)上對(duì)其作出各種各樣的改變,而不偏離本發(fā)明權(quán)利要求書所限定的范圍。