CN109517032B - 一种四肽化合物及制备方法和用途 - Google Patents

一种四肽化合物及制备方法和用途 Download PDF

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CN109517032B
CN109517032B CN201811616048.XA CN201811616048A CN109517032B CN 109517032 B CN109517032 B CN 109517032B CN 201811616048 A CN201811616048 A CN 201811616048A CN 109517032 B CN109517032 B CN 109517032B
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达朝山
杜智宏
秦文娟
陶宝秀
白彦兵
林行
张连春
殷杭华
余建新
刘学愚
姜伟林
金文究
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Hangzhou Xinfu Science & Technology Co ltd
Lanzhou University
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Abstract

本发明公开一种四肽化合物及其制备方法及用途。本发明的四肽化合物如

Description

一种四肽化合物及制备方法和用途
技术领域
本发明涉及一种四肽化合物及制备方法和用途。确切讲本发明是一种四肽化合物及制备方法与用途,特别是一种可应用于催化脂肪醛与马来酰亚胺的不对称共轭加成反应的化合物及制备方法与用途。
背景技术
光学活性化合物是自然界的普遍现象,在细胞和生物体的生命活动过程中发挥着无可替代的作用。许多药物都有光学活性,手征性是药物的一个重要要素。用手性催化剂催化不对称反应是合成手性化合物最为经济、绿色的方法。不对成催化包含了三个领域,即手性配体-金属复合物催化剂、有机小分子催化剂和生物酶催化反应。由于绿色和良好的经济性能,生物酶催化一直是不对称催化的热点研究领域,但是由于酶蛋白结构的复杂性、易失活、专一性等因素限制了其快速应用。多肽是按照酶催化机理设计并合成的一类新型有机催化剂,具有结构简单、易合成、底物范围广泛、适应反应类型多、反应条件温和、结构稳定等诸多优势,是天然酶的优良模拟物,是化学生物学研究的重点方向。
已有报道证明,人工合成的短肽可以较好地替代天然蛋白酶,并模拟酶催化一系列化学反应,被广泛地应用于催化如Aldol、Michael、Stetter、叠氮化、Strecker、Baylis-Hillman、烯丙基取代、氰氢化、环氧化、氢化和酰基化等诸多不对称反应中,合成多种光学活性化合物,有效地克服了蛋白酶催化反应底物专一性和反应类型单一的缺陷,拓展了反应的类型和底物,适合于多种反应条件,并基本保持了生物催化反应绿色、经济的优点[(1)刘爱香,傅尧,刘磊,郭庆祥,有机化学,2007,27,1195-1219.(2)Davie,E.A.C.;Mennen,S.M.;Xu,Y.;Miller,S.J.Chem.Rev.2007,107,5759-5812.(3)Wennemers,H.Chem.Commun.,2011,47,12036–12041.(4)Lewandowski,B.;Wennemers,H.Curr.Opin.Chem.Biol.2014,22,40–46.(5)Kelly,D.R.;Roberts,S.M.Biopolymers2006,84,74–89.(6)Akagawa,K.;Kudo,K.Acc.Chem.Res.2017,50,2429-2439.(7)Ball,Z.T.Acc.Chem.Res.2012,46,560-570.]。因而,发现新型高效的短肽催化剂模拟蛋白酶催化有机化学反应,一直是手性化合物合成领域的热点。
在不对称催化条件下,亲核试剂与N-取代马来酰亚胺的不对称共轭加成反应合成的手性3-取代琥珀酰亚胺,经过还原可以方便地转变成一系列手性吡咯化合物。手性吡咯化合物是许多生物活性化合物的核心骨架,不对称合成手性吡咯化合物一直是有机化学与药物化学研究的一个核心课题。此外,手性3-取代的琥珀酰亚胺经过水解反应也可以方便地转换为手性琥珀酸,是很重要的生理活性化合物以及手性合成砌块。醛作为亲核试剂与马来酰亚胺的不对称共轭加成产物,额外具有一个很容易衍生为其它官能团的有效基团,使得该反应具有重要的应用价值。因而持续开发高效的手性催化剂催化脂肪醛与马来酰亚胺的不对称共轭加成反应,是许多研究机构追求的一个目标。总结文献报道,成功应用于这一反应的催化剂基本上都是手性有机催化剂,绝大部分都是手性环己二胺为原料衍生的伯胺类硫脲或方酰胺催化剂,取得了理想的产率和对映选择性[(8)Xue,F.;Liu,L.;Zhang,S.;Duan,W.;Wang,W.Chem.Eur.J.2010,16,7979-7982;(9)Ma,Z.-W.;Liu,Y.-X.;Zhang,W.-J.;Tao,Y.;Zhu,Y.;Tao,J.-C.;Tang,M.-S.Eur.J.Org.Chem.2011,6747-6754.(10)Orlandi,S.;Pozzi,G.;Ghisetti,M.;Benaglia,M.NewJ.Chem.2013,37,4140-4147.(11)Yu,F.;Jin,Z.;Huang,H.;Ye,T.;Liang,X.;Ye,J.Org.Biomol.Chem.2010,8,4767-4774.(12)Bai,J.-F.;Peng,L.;Wang,L.-L.;Wang,L.-X.;Xu,X.-Y.Tetrahedron 2010,66,8928-8932.(13)Ma,Z.-W.;Liu,Y.-X.;Li,P.-L.;Ren,H.;Zhu,Y.;Tao,J.-C.;Tetrahedron:Asymmetry2011,22,1740-1748.(14)Ma,Z.-W.;Liu,X.-F.;Liu,J.-T.;Liu,Z.-J.;Tao,J.-C.Tetrahedron Letters 2017,58,4487-4490.]。金鸡纳碱硫脲和2-氯苯基甘氨酸作为一个高效的双催化剂体系,同样取得了理想的产率和对映选择性[(15)Muramulla,S.;Ma,J.-A.;Zhao,J.C.-G.Adv.Synth.Catal.2013,355,1260-1264]。金鸡纳碱衍生的手性伯胺在添加三苯基膦的条件下,其催化产率与对映选择性均非常理想[(16)Yang,W.;Jiang,K.-Z.;Lu,X.;Yang,H.-M.;Li,L.;Lu,Y.;Xu,L.-W.Chem.AsianJ.2013,8,1182-1190]。在添加碱金属条件下发现,β-苯丙氨酸、天冬氨酸-α-叔丁酯以及侧链叔丁基保护的苏氨酸、异亮氨酸催化这一反应都取得了很好的不对称催化效果[(17)Kokotos,C.G.Org.Lett.2013,15,2406-2409.(18)Nugent,T.C.;Sadiq,A.;Bibi,A.;Heine,T.;Liu L.;Vankova,N.;Bassil,B.S.Chem.Eur.J.2012,18,4088-4098]。也有一例二肽Ala-Ala催化该丙醛与马来酰亚胺反应的报道,其产物的ee值只有38%[(19)Zhao,G.-L.;Xu,Y.;Sundén,H.;Eriksson,L.;Sayah,M.;Córdova,A.Chem.Commun.2007,734–735.]。因而开发应用于这一反应的高效多肽催化剂,是一个具有挑战性的工作。
发明内容
本发明提供一种可克服现有技术不足,可以高效率、不对称地催化脂肪醛与马来酰亚胺的共轭加成反应的化合物。
本发明所述的四肽化合物如式1所示TP,
其中:R1和R2分别是C1~C6的任意直链烷基、支链烷基、环烷基、羟基取代烷基、巯基取代烷基、甲硫基取代烷基、氨基取代烷基、胍基取代烷基、芳基、芳甲基或杂原子芳基中的任一种。
优选地,本发明的四肽化合物,其R1和R2分别是C1~C4的任意直链烷基、支链烷基、环己基、苯基、苯甲基中的任一种。
更为具体地,本发明所述的四肽化合物,其R1=i-Pr,R2=i-Bu,即:其氨基酸序列为H2N-D-Val-Pro-Gly-D-Leu-OH,命名为TP-1。
或者,本发明所述的四肽化合物,其R1=t-Bu,R2=i-Bu,即:其氨基酸序列为H2N-D-Tle-Pro-Gly-D-Leu-OH,命名为TP-2。
或者,本发明所述的四肽化合物,其R1=Ph,R2=i-Bu,即:其氨基酸序列为H2N-D-Phg-Pro-Gly-D-Leu-OH,命名为TP-3。
或者,本发明所述的四肽化合物,其R1=c-hex,R2=i-Bu,即:其氨基酸序列为H2N-D-Chg-Pro-Gly-D-Leu-OH,命名为TP-4。
或者,本发明所述的四肽化合物,其R1=Bn,R2=i-Bu,即:其氨基酸序列为H2N-D-Phe-Pro-Gly-D-Leu-OH,命名为TP-5。
或者,本发明所述的四肽化合物,其R1=Bn,R2=Bn,即:其氨基酸序列为H2N-D-Phe-Pro-Gly-D-Phe-OH,命名为TP-6。
本发明的四肽化合物制备方法,其反应历程如式2、式3、式4和式5所示,即:
将甘氨酸1、氨基酸2、缩合试剂C-1和添加剂A-1加入溶剂S-1搅拌反应,得到产物3,再将产物3和脱保护剂D-1加入溶剂S-2搅拌反应,得到产物4;
将产物4、脯氨酸5、缩合试剂C-2和添加剂A-2加入溶剂S-3中搅拌反应,得到产物6,再将产物6和脱保护剂D-2加入溶剂S-4中搅拌反应,得到产物7;
将产物7、氨基酸8、缩合试剂C-3和添加剂A-3加入溶剂S-5中搅拌反应,得到产物9,再将产物9和脱保护剂D-3加入溶剂S-6中搅拌反应,得到产物10;
将产物10和催化剂Pd/C加入溶剂S-7中,通入压力为1~30Mpa氢气进行反应,得到终产物四肽TP,其中:
R3是芳基甲基;
R4、R5、R6是Boc(叔丁氧甲酰基)、Cbz(苄氧甲酰基)或Fmoc(芴甲氧甲酰基)中的任一种;
缩合试剂C-1、C-2和C-3是碳二亚胺、烷氧甲酰氯或R7OCOCl中的任一种,R7是C1~C4的任意直链烷基或支链烷基;
添加剂A-1、A-2和A-3是指R8R9R10N或NMM(N-甲基吗啉)或TMEDA(4-二甲氨基吡啶)或HOBt(1-羟基苯并三氮唑),R8、R9、R10是C1~C4的任意直链烷基或支链烷基;
溶剂S-1、S-2、S-3、S-4、S-5、S-6是甲苯、苯、二氯甲烷、二氯乙烷、乙醚、四氢呋喃、乙二醇二甲醚、甲基叔丁基醚、环氧六烷、乙酸乙酯、乙酸甲酯、乙腈或丙腈中的任一种或任数种的组合,溶剂S-7是甲醇、乙醇、丙醇或丁醇中的任一种或任数种的组合;
脱保护剂D-1、D-2和D-3是甲基磺酸、三氟甲磺酸、苯磺酸、对甲苯磺酸、三氟乙酸、三氯乙酸、盐酸、硫酸、哌啶、吗啉、四氢吡咯、二氢吡咯、吡咯、二乙胺、二丙胺、二丁胺、二异丙胺或二异丁胺中的任一种。
优选地,本发明所述的化合物制备方法是其中:
R3是苄基;
R4、R5、R6是指Boc或Cbz;
缩合试剂C-1、C-2和C-3是DCC(N,N'-二环己基碳二亚胺)或DIC(N,N'-二异丙基碳二亚胺)或烷氧甲酰氯R7OCOCl,R7是Et或i-Pr或i-Bu;
添加剂A-1、A-2和A-3是TEA(三乙胺)、DIPEA(二异丙基乙胺)、NMM或HOBt中的任一种;
溶剂S-1、S-2、S-3、S-4、S-5和S-6是二氯甲烷或四氢呋喃,溶剂S-7是甲醇或乙醇;
脱保护剂D-1、D-2和D-3是甲基磺酸或三氟甲磺酸或三氟乙酸。
优选地,本发明所述的化合物制备方法是其中:
R3是苄基;
R4、R5和R6是Boc;
缩合试剂C-1、C-2和C-3是DCC或DIC或烷氧甲酰氯R7OCOCl,R7是Et或i-Pr或i-Bu;
添加剂A-1、A-2和A-3是指TEA、DIPEA、NMM或HOBt中的任一种;
溶剂S-1、S-2、S-3、S-4、S-5、S-6是指二氯甲烷或四氢呋喃,溶剂S-7是指甲醇;
脱保护剂D-1、D-2和D-3是指三氟乙酸;
氢气压力是8Mpa。
本发明所述的化合物可用于酮与芳香醛的Aldol反应,或者用于如式6所示催化脂肪醛与马来酰亚胺的不对称共轭加成反应。即:将式6所示的脂肪醛11、
马来酰亚胺12和四肽TP加入含溶剂S-8的反应容器中搅拌,得到共轭加成反应的产物3-取代琥珀酰亚胺(R)-P,其中:
R11是氢或C1~C6的任意直链烷基或支链烷基或苯基或苯甲基,R12是C1~C6的任意直链烷基或支链烷基或苯基或苯甲基,或-R11-R12-是环己基或环戊基,R13是氢或苯基或取代苯基或直链烷基或环烷基或芳甲基;
四肽TP是如式1所示含两个末端D-型-α-伯氨基酸的四肽;
在上述反应中,溶剂S-8是指二氯甲烷、氯仿、二氯乙烷、乙酸乙酯、乙酸甲酯、甲苯、苯、二甲苯、乙醚、四氢呋喃、乙二醇二甲醚、甲基叔丁基醚、乙腈、丙腈、乙醇、甲醇、丙醇、丁醇中的任一种或多种。
优选地:
R11是氢或甲基或乙基,R12是甲基或乙基或异丙基或正丁基,或-R11-R12-是环己基或环戊基,R13是氢或甲基或环己基或苯甲基或苯基或取代苯基,取代苯基是指对甲基苯基或对氯苯基或对溴苯基或对氟苯基或对硝基苯基或对甲氧基苯基;
四肽TP是前述TP-1~TP-6中的任一种;
溶剂S-8是二氯甲烷、乙腈、乙醇、四氢呋喃、二甲基亚砜、甲苯中的任一种。
更优选地四肽TP是TP-3;溶剂S-8是乙腈。
本发明用于制备式1示化合物TP的中间化合物为:如式3示6或者如式3示7或者如式4示9或者如式4示10。
本发明的化合物在不对称催化的脂肪醛与马来酰亚胺共轭反应过程中可以高产率、高对映选择性地催化脂肪醛与马来酰亚胺的共轭加成反应。根据相关实验,本发明的四肽化合物可以高效率、不对称地催化脂肪醛与马来酰亚胺的共轭加成反应,反应的产率达到98%,对映选择性最高达到了99%,应用前景良好。
具体实施方式
本发明以下结合实施例进行解说。
一、四肽化合物TP的制备
本发明式1示四肽化合物TP的制备的反应历程参见前述的式2至式5,其制备方法参见发明内容部分,更详细地制备过程如下:
(一)合成中间化合物3
1.1Boc-Gly-D-Leu-OBn(3a)
参见上式,把Boc-Gly-OH 1(8.75g,50mmol)加入到100mL的无水THF中,氩气保护下在冰盐浴中搅拌5min,使反应液的温度下降到–15℃,加入NMM(6.2mL,55mmol),接着把氯甲酸异丁酯(7mL,55mmol)缓慢滴加到反应液中。搅拌5min后,加入事先用无水DMF溶解的D-Leu-OBn 2a对甲苯磺酸盐(19.7g,50mmol)和NMM(6.2mL)的溶液,在–15℃下继续搅拌反应0.5h,撤走冰盐浴,自然恢复反应液至室温,反应过夜。待反应结束后,抽滤除去不溶物,滤液减压浓缩,再用乙酸乙酯溶解,乙酸乙酯相依次用1M NaOH溶液、水、1M盐酸溶液和少量饱和食盐水洗涤,无水硫酸钠干燥后,减压浓缩得到Boc-Gly-D-Leu-OBn(3a)19g,为淡黄色油状液体,产率100%.[α]D 20=+5.0(c 1.0,CHCl3);1H NMR(200MHz,CDCl3)δ7.34–7.28(m,5H),6.94–6.90(d,J=6.8Hz,1H),5.51(m,1H),5.15(s,2H),4.69–4.64(m,1H),3.83–3.81(m,2H),1.62–1.51(m,3H),1.44(s,9H),0.91–0.89(m,6H);13C NMR(100MHz,CDC13)δ172.6,169.4,156.0,135.3,128.5,128.4,128.2,128.0,80.0,66.9,50.7,44.1,41.1,28.1,24.7,22.7,21.7;ESI-MS calcd for[C20H30N2O5+H+]379.2,found:379.3.。
或者如上式,把Boc-Gly-OH 1(8.75g,50mmol)加入到100mL的无水THF中,氩气保护下在冰盐浴中搅拌5min,使反应液的温度下降到–15℃,加入NMM(6.2mL,55mmol),接着把氯甲酸乙酯(5.3mL,55mmol)缓慢滴加到反应液中。搅拌5min后,加入事先用无水DMF溶解的D-Leu-OBn 2a对甲苯磺酸盐(19.7g,50mmol)和NMM(6.2mL)的溶液,在–15℃下继续搅拌反应半小时,撤走冰盐浴,自然恢复反应液至室温,反应过夜。待反应结束后,抽滤除去不溶物,滤液减压浓缩,再用乙酸乙酯溶解,乙酸乙酯相依次用1MNaOH溶液、水、1M盐酸溶液和少量饱和食盐水洗涤,无水硫酸钠干燥后,减压浓缩得到Boc-Gly-D-Leu-OBn(3a)17.30g,为淡黄色油状液体,产率91%。
或者参见上式,把Boc-Gly-OH 1(8.75g,50mmol)加入到100mL的无水THF中,氩气保护下在冰盐浴中搅拌5min,使反应液的温度下降到–15℃,加入NMM(6.2mL,55mmol),接着把氯甲酸异丙酯(6.3mL,55mmol)缓慢滴加到反应液中。搅拌5min后,加入事先用无水DMF溶解的D-Leu-OBn 2a对甲苯磺酸盐(19.7g,50mmol)和NMM(6.2mL)的溶液,在–15℃下继续搅拌反应0.5h,撤走冰盐浴,自然恢复反应液至室温,反应过夜。待反应结束后,抽滤除去不溶物,滤液减压浓缩,再用乙酸乙酯溶解,乙酸乙酯相依次用1MNaOH溶液、水、1M盐酸溶液和少量饱和食盐水洗涤,无水硫酸钠干燥后,减压浓缩得到Boc-Gly-D-Leu-OBn(3a)17.67g,为淡黄色油状液体,产率93%。
或者如上式,把Boc-Gly-OH(8.75g,50mmol)加入到100mL的无水THF中,氩气保护下在冰盐浴中搅拌5min,使反应液的温度下降到–15℃,加入TEA(三乙胺,7.6mL,55mmol),接着把氯甲酸异丁酯(7mL,55mmol)缓慢滴加到反应液中。搅拌5min后,加入事先用无水DMF溶解的D-Leu-OBn对甲苯磺酸盐(19.7g,50mmol)和TEA(7.6mL)的溶液,在–15℃下继续搅拌反应0.5h,撤走冰盐浴,自然恢复反应液至室温,反应过夜。待反应结束后,抽滤除去不溶物,滤液减压浓缩,再用乙酸乙酯溶解,乙酸乙酯相依次用1MNaOH溶液、水、1M盐酸溶液和少量饱和食盐水洗涤,无水硫酸钠干燥后,减压浓缩得到Boc-Gly-D-Leu-OBn(3a)16.48g,为淡黄色油状液体,产率87%。
或者如上式,把Boc-Gly-OH 1(8.75g,50mmol)加入到100mL的无水THF中,氩气保护下在冰盐浴中搅拌5min,使反应液的温度下降到–15℃,加入DIPEA(二异丙基乙胺,9.6mL,55mmol),接着把氯甲酸异丁酯(7mL,55mmol)缓慢滴加到反应液中。搅拌5min后,加入事先用无水DMF溶解的D-Leu-OBn 2a对甲苯磺酸盐(19.7g,50mmol)和DIPEA(9.6mL)的溶液,在–15℃下继续搅拌反应0.5h,撤走冰盐浴,自然恢复反应液至室温,反应过夜。待反应结束后,抽滤除去不溶物,滤液减压浓缩,再用乙酸乙酯溶解,乙酸乙酯相依次用1M NaOH溶液、水、1M盐酸溶液和少量饱和食盐水洗涤,无水硫酸钠干燥后,减压浓缩得到Boc-Gly-D-Leu-OBn(3a)15.47g,为淡黄色油状液体,产率81%。
1.2合成Boc-Gly-D-Phe-OBn(3b)
把Boc-Gly-OH 1(8.75g,50mmol)加入到100mL的无水THF中,氩气保护下在冰盐浴中搅拌5min,使反应液的温度下降到-15℃,加入NMM(6.2mL,55mmol),接着把氯甲酸异丁酯(7mL,55mmol)缓慢滴加到反应液中。搅拌5min后,加入事先用无水DMF溶解的D-Phe-OBn 2b的对甲苯磺酸盐(21.35g,50mmol)和NMM(6.2mL)的溶液,在-15℃下继续搅拌反应0.5h,撤走冰盐浴,自然恢复反应液至室温反应过夜。待反应结束后,抽滤除去不溶物,滤液减压浓缩,再用乙酸乙酯溶解,乙酸乙酯相依次用1M NaOH溶液、水、1M盐酸溶液和少量饱和食盐水洗涤,无水硫酸钠干燥后,减压浓缩得到淡黄色油状物的Boc-Gly-D-Phe-OBn(3b),石油醚和乙酸乙酯柱层析纯化得到白色固体19.16g,产率93%。White solid,Mp 73.5-75℃;[α]D 25=-11.6(c 0.5,CHCl3);1H NMR(600MHz,CDCl3)δ7.35(m,3H),7.30–7.27(m,1H),7.23–7.18(m,2H),7.13–6.86(m,2H),6.63(d,J=5.7Hz,1H),5.16-5.08(m,2H),4.92(dd,J=6,12Hz,1H),3.86–3.71(m,2H),3.15–3.05(m,2H),1.44(d,J=8.5Hz,9H).13C NMR(150MHz,CDCl3)δ171.1,169.1,135.5,135.0,129.3,128.6,128.6,128.5,128.5,128.4,128.2,127.1,77.2,77.0,76.8,67.3,53.1,41.4,37.9,28.3(s),24.8,22.8,21.8;ESI-MScalcd for[C23H28N2O5+H+]413.2,found 413.2.。
(二)合成中间化合物4
2.1合成NH2-Gly-D-Leu-OBn(4a)
把上面反应得到的二肽Boc-Gly-D-Leu-OBn 3a(19g)溶于50mL二氯甲烷中,反应液冷却至0℃,向其中缓慢滴加TFA(三氟乙酸,50mL),自然恢复反应溶液至室温下搅拌5h后,把反应液减压浓缩除去TFA,残留物用二氯甲烷溶解,用1.0M NaOH水溶液在0℃条件下调pH=9.0左右,分离有机相,水相用二氯甲烷(20mL×3)萃取。合并有机相,依次用少量饱和食盐水洗涤有机相、无水硫酸钠干燥后,减压浓缩,得到产物NH2-Gly-D-Leu-OBn(4a)为无色油状液体,产率13.25g(95%)。无需纯化,直接用于下一步反应。
或者如上式,把上面反应得到的二肽Boc-Gly-D-Leu-OBn 3a(19g)溶于50mL二氯甲烷中,反应液冷却至0℃,向其中缓慢滴加甲基磺酸(50mL),自然恢复反应溶液至室温下搅拌5h后,用1.0M NaOH水溶液在0℃条件下调pH=9.0左右,分离有机相,水相用二氯甲烷(20mL×3)萃取。合并有机相,依次用少量饱和食盐水洗涤有机相、无水硫酸钠干燥后,减压浓缩,得到产物NH2-Gly-D-Leu-OBn(4a)为无色油状液体,产率12.83g(92%)。无需纯化,直接用于下一步反应。
或者如上式,把上面反应得到的二肽Boc-Gly-D-Leu-OBn 3a(19g)溶于50mL二氯甲烷中,反应液冷却至0℃,向其中缓慢滴加三氟甲磺酸(50mL),自然恢复反应溶液至室温下搅拌5h后,用1.0M NaOH水溶液在0℃条件下调pH=9.0左右,分离有机相,水相用二氯甲烷(20mL×3)萃取。合并有机相,依次用少量饱和食盐水洗涤有机相、无水硫酸钠干燥后,减压浓缩,得到产物NH2-Gly-D-Leu-OBn(4a)为无色油状液体,产率12.7g(91%)。无需纯化,直接用于下一步反应。
2.2合成H2N-Gly-D-Phe-OBn(4b)
把上面反应得到的二肽Boc-Gly-D-Phe-OBn 3b(18.2g,44mmol)溶于44mL二氯甲烷中,反应液冷却至0℃,向其中缓慢滴加TFA(44mL),自然恢复反应溶液至室温下搅拌5h后,把反应液减压浓缩除去TFA,残留物用二氯甲烷溶解,用1M NaOH水溶液在0℃条件下调pH=9.0左右,分离有机相,水相用二氯甲烷(20mL×3)萃取。合并有机相,依次用少量饱和食盐水洗涤有机相、无水硫酸钠干燥后,减压浓缩,得到产物NH2-Gly-D-Leu-OBn(4b)为无色油状液体,产率12.5g(91%)。无需纯化,直接用于下一步反应。
(三)中间化合物6的合成
3.1合成Boc-Pro-Gly-D-Leu-OBn(6a)
将Boc-Pro-OH 5(9.68g,45mmol)加入到100mL的无水THF中,氩气保护下在冰盐浴中搅拌5min使反应液的温度下降到–15℃,加入NMM(5.6mL,50mmol),把氯甲酸异丁酯(6.4mL,50mmol)缓慢滴加到反应液中,搅拌5min后,加入预先用无水THF溶解的二肽NH2-Gly-D-Leu-OBn 4a(12.56g,45mmol)和NMM(5.6mL)的溶液。冰盐浴下继续搅拌30min,撤走冰盐浴,使反应自然恢复至室温,反应搅拌过夜至反应结束,抽滤出去不溶物,滤液减压浓缩,残余油状物用乙酸乙酯溶解,然后依次用1.0M NaOH溶液、水、1.0M盐酸溶液和饱和食盐水洗涤,无水硫酸钠干燥,减压浓缩,得到淡黄色油状液体的Boc-Pro-Gly-D-Leu-OBn(6a),用石油醚和乙酸乙酯柱层析纯化得到20.33g产物,产率95%。White solid,Mp:104–105℃;[α]D 20=-27(c 1.0,CHCl3);1HNMR(400MHz,CDCl3)δ7.38–7.32(m,5H),7.22(br,1H),7.03(br,1H),5.08–5.07(dd,J=12.8Hz,J=21.6Hz,2H),4.57–4.55(m,1H),4.25–4.11(m,2H),3.86–3.81(dd,J=4.8Hz,J=17.2Hz,1H),3.50–3.40(m,2H),2.15–2.11(m,2H),1.92–1.89(m,2H),1.60–1.73(m,3H),1.44(s,9H),0.92–0.89(m,6H);13C NMR(100MHz,CDCl3)δ172.5,169.3,155.8,128.5,128.1,128.0,109.7,80.8,66.7,60.9,51.0,47.4,42.8,40.1,29.5,28.3,24.7,24.6,22.8,21.5;ESI-MS calcd for[C25H37N3O6+H+]476.3,found:476.3.。
3.2合成Boc-Pro-Gly-D-Phe-OBn(6b)
将Boc-Pro-OH 5(7.53g,35mmol)加入到100mL的无水THF中,氩气保护下在冰盐浴中搅拌5min使反应液的温度下降到-15℃,加入NMM(4.3mL,38.5mmol),把氯甲酸异丁酯(4.9mL,38.5mmol)缓慢滴加到反应液中,搅拌5min后,加入预先用无水THF溶解的二肽NH2-Gly-D-Phe-OBn 4b(10.92g,35mmol)和NMM(4.3mL)的溶液。冰盐浴下继续搅拌30min,撤走冰盐浴,使反应自然恢复至室温,反应搅拌过夜至反应结束,抽滤出去不溶物,滤液减压浓缩,残余油状物用乙酸乙酯溶解,然后依次用1M NaOH溶液、水、1M盐酸溶液和饱和食盐水洗涤,无水硫酸钠干燥,减压浓缩用石油醚和乙酸乙酯柱层析纯化得到Boc-Pro-Gly-D-Phe-OBn(6b)产物,17.1g,产率96%。White solid,Mp 100-102℃;[α]D 25=-66.0(c 0.5,CHCl3);1H NMR(600MHz,CDCl3)δ7.34(d,J=6.5Hz,2H),7.27(s,1H),7.21(t,J=7.3Hz,2H),7.13–6.89(m,3H),6.54(m,1H),5.12(t,J=14.8Hz,2H),4.84(s,1H),4.22(s,1H),3.92(d,J=33.6Hz,2H),3.51–3.33(m,2H),3.17-3.05(m,2H),2.23–2.00(m,2H),1.86(s,2H),1.45(s,9H).13C NMR(150MHz,CDCl3)δ172.5,171.1,168.8,136.1,135.3,129.3,129.2,128.6,128.5,128.4,127.0,80.7,77.3,77.0,76.8,67.1,60.6,53.6,47.32,43.0,37.6,28.4,24.6;ESI-MS calcd for[C28H35N3O6+H+]510.2,found 510.2.。
(四)中间化合物7的合成
4.1合成NH2-Pro-Gly-D-Leu-OBn(7a)
将合成的三肽Boc-Pro-Gly-D-Leu-OBn 6a(19g,40mmol)完全溶于CH2Cl2(40mL)中,在0℃下缓慢滴加TFA(40mL),恢复反应液至室温下,搅拌5h。将反应液减压浓缩尽量除去TFA,残留物用CH2Cl2溶解,再用1MNaOH溶液在0℃下中和至约pH=9.0左右。分离出有机相,水相用CH2Cl2(20mL×3)萃取。合并有机相,饱和食盐水洗一次,无水硫酸钠干燥,减压浓缩,得到13.54g无色油状液体的NH2-Pro-Gly-D-Leu-OBn(7a),产率为90%,无需纯化,直接用于下一步反应。
4.2合成Pro-Gly-D-Phe-OBn(7b)
将合成的三肽Boc-Pro-Gly-D-Phe-OBn 6b(16.5g,32mmol)完全溶于CH2Cl2(32mL)中,在0℃下缓慢滴加TFA(32mL),恢复反应液至室温下,搅拌5h。将反应液减压浓缩尽量除去TFA,残留物用CH2Cl2溶解,再用1MNaOH溶液在0℃下中和至约pH=9.0左右。分离出有机相,水相用CH2Cl2(20mL×3)萃取。合并有机相,饱和食盐水洗一次,无水硫酸钠干燥,减压浓缩,得到11.8g无色油状液体的Pro-Gly-D-Phe-OBn(7b),产率为90%。无需纯化,直接用于下一步反应。
(五)中间化合物9的合成
5.1合成Boc-D-Val-Pro-Gly-D-Leu-OBn(9a)
把HOBt(4.45g,33mmol),Boc-D-Val-OH 8a(6.45g,30mmol)加入到150mL的圆底烧瓶中,氩气保护,加入60mL干燥的THF,0℃搅拌待瓶内温度降至0℃时,缓慢滴加DIC(N,N'-二异丙基碳二亚胺,5.1mL,33mmol),0.5h之后加入事先用THF溶解好的三肽Pro-Gly-D-Leu-OBn 7a(11.28g,30mmol)。室温搅拌48h后滤去不溶物,滤液减压浓缩干。残留物用乙酸乙酯溶解,然后依次用1M NaOH溶液、水、1M盐酸溶液和饱和食盐水洗涤,无水硫酸钠干燥,减压浓缩后用石油醚和乙酸乙酯柱层析纯化,得到14.84g白色固体四肽Boc-D-Val-Pro-Gly-D-Leu-OBn(9a),产率86%。White solid,Mp:123.5–125.5℃;[α]D 20=+15(c 0.7,CHCl3);1H NMR(400MHz,CDCl3)δ7.36–7.30(m,5H),7.24–7.23(m,1H),5.34–5.32(d,J=6.8Hz,1H),5.20–5.13(dd,J=12.4Hz,J=14.8Hz,2H),4.64–4.59(m,1H),4.50–4.50(t,J=6.0Hz,l H),4.20–4.14(dd,J=7.2Hz,J=16.8Hz,1H),4.07(m,1H),3.98–3.94(m,1H),3.65–3.56(m,2H),2.21–2.16(m,2H),2.09–1.99(m,2H),1.95–1.90(m,1H),1.76–1.61(m,3H),1.40(s,9H),1.01–1.00(d,J=6.8Hz,3H),0.97–0.95(d,J=6.8Hz,3H),0.92–0.88(m,6H);13C NMR(100MHz,CDCl3)δ172.6,171.7,169.0,156.9,135.7,128.4,128.0,127.8,80.3,66.4,61.0,58.2,50.7,47.6,43.1,40.7,30.1,29.2,28.1,24.6,23.4,22.8,21.7,19.1,18.8;ESI-MS calcd for[C30H46N4O7+H+]575.3,found575.3.。
或者如上式示,把HOBt(4.45g,33mmol),Boc-D-Val-OH 8a(6.51g,30mmol)加入到150mL的圆底烧瓶中,氩气保护,加入60mL干燥的THF,0℃搅拌待瓶内温度降至0℃时,缓慢滴加用DMF溶解的DCC(N,N'-二环己基碳二亚胺,6.8g,33mmol),0.5h之后加入事先用THF溶解好的三肽Pro-Gly-D-Leu-OBn 7a(11.28g,30mmol)。室温搅拌48h后滤去不溶物,滤液减压浓缩干。残留物用乙酸乙酯溶解,然后依次用1M NaOH溶液、水、1M盐酸溶液和饱和食盐水洗涤,无水硫酸钠干燥,减压浓缩后用石油醚和乙酸乙酯柱层析纯化,得到12.42g白色固体四肽Boc-D-Val-Pro-Gly-D-Leu-OBn(9a),产率72%。
5.2合成Boc-D-Tle-Pro-Gly-D-Leu-OBn(9b)
把HOBt(4.45g,33mmol),Boc-D-Tle-OH 8b(6.93g,30mmol)加入到150mL的圆底烧瓶中,氩气保护,加入60mL干燥的THF,0℃搅拌待瓶内温度降至0℃时,缓慢滴加DIC(5.1mL,33mmol),0.5h之后加入事先用THF溶解好的三肽Pro-Gly-D-Leu-OBn 7a(11.28g,30mmol)。室温搅拌48h后滤去不溶物,滤液减压浓缩干。残留物用乙酸乙酯溶解,然后依次用1MNaOH溶液、水、1M盐酸溶液和饱和食盐水洗涤,无水硫酸钠干燥,减压浓缩后用石油醚和乙酸乙酯柱层析纯化,得到15.02g白色固体的四肽Boc-D-Tle-Pro-Gly-D-Leu-OBn(9b),产率85%。White solid,Mp 168–169.4℃;[α]D 25=+9.0(c 0.5,CHCl3);1H NMR(600MHz,CDCl3)δ7.47(d,J=7.7Hz,1H),7.35(d,J=4.4Hz,3H),7.33–7.28(m,1H),7.08–7.03(m,1H),5.48(d,J=7.1Hz,1H),5.19–5.12(m,2H),4.62-4.55(m,1H),4.46(dd,J=7.5,4.9Hz,1H),4.22(dd,J=17.2,7.9Hz,1H),4.15–4.10(m,1H),4.07(d,J=7.1Hz,1H),3.65–3.59(m,1H),3.56(dd,J=17.1,5.1Hz,1H),2.20–2.15(m,2H),2.09–2.03(m,1H),2.00(dd,J=11.3,5.3Hz,1H),1.77–1.56(m,4H),1.40(s,9H),1.04(s,9H),0.88(dd,J=12.8,6.4Hz,6H).13CNMR(150MHz,CDCl3)δ172.7,172.2,171.7,169.1,135.91,128.5,128.1,128.0,77.24,77.0,76.8,66.4,61.3,59.7,50.9,48.3,43.2,40.7,33.9,29.4,28.2,26.5,24.8,24.7,22.8,21.8;ESI-MS calcd for[C31H48N4O7+H+]588.7,found588.7.。
5.3合成Boc-D-Phg-Pro-Gly-D-Leu-OBn(9c)
把HOBt(4.45g,33mmol),Boc-D-Phg-OH 8c(7.53g,30mmol)加入到150mL的圆底烧瓶中,氩气保护,加入60mL干燥的THF,0℃搅拌待瓶内温度降至0℃时,缓慢滴加DIC(5.1mL,33mmol),0.5h之后加入事先用THF溶解好的三肽Pro-Gly-D-Leu-OBn 7a(11.28g,30mmol)。室温搅拌48h后滤去不溶物,滤液减压浓缩干。残留物用乙酸乙酯溶解,然后依次用1M NaOH溶液、水、1M盐酸溶液和饱和食盐水洗涤,无水硫酸钠干燥,减压浓缩,得到15.52g四肽Boc-D-Phg-Pro-Gly-D-Leu-OBn(9c),产率85%。White solid,Mp142–144℃;[α]D 25=-2.0(c0.5,CHCl3);1H NMR(600MHz,CDCl3)δ7.45–7.39(m,2H),7.38–7.32(m,6H),7.27(s,1H),7.22(d,J=7.0Hz,1H),5.72(d,J=5.2Hz,2H),5.38-5.30(m,1H),5.17(d,J=3.3Hz,2H),4.66–4.61(m,1H),4.47–4.39(m,1H),4.04(dd,J=16.8,6.6Hz,1H),3.98–3.94(m,1H),3.83(dd,J=12.7,6.3Hz,1H),3.38–3.29(m,1H),2.21–2.16(m,1H),2.09–1.99(m,2H),1.75–1.70(m,2H),1.39(d,J=4.9Hz,9H),1.13(d,J=6.5Hz,4H),0.96–0.90(m,6H).13CNMR(150MHz,CDCl3)δ172.5,171.5,169.0,135.7,129.2,129.1,128.5,128.4,128.1(dd,J=12.3,9.2Hz),80.5,77.2,77.0,76.8,66.8,61.2,51.0,47.3,43.4,42.1,41.1,28.2,24.6,23.4,22.8,21.8;ESI-MS calcd for[C33H44N4O7+H+]609.3,found609.3。
5.4合成四肽Boc-D-Chg-Phe-Pro-Gly-D-Leu-OBn(9d)
把HOBt(4.45g,33mmol),Boc-D-Chg-OH 8d(7.71g,30mmol)加入到150mL的圆底烧瓶中,氩气保护,加入60mL干燥的THF,0℃搅拌待瓶内温度降至0℃时,缓慢滴加DIC(5.1mL,33mmol),0.5h之后加入事先用THF溶解好的三肽Pro-Gly-D-Leu-OBn 7a(11.28g,30mmol)。室温搅拌48h后滤去不溶物,滤液减压浓缩干。残留物用乙酸乙酯溶解,然后依次用1M NaOH溶液、水、1M盐酸溶液和饱和食盐水洗涤,无水硫酸钠干燥,减压浓缩,石油醚和乙酸乙酯柱层析纯化得到四肽Boc-D-Chg-Phe-Pro-Gly-D-Leu-OBn(9d)15.5g,产率84%。Whitesolid,Mp:160–161℃;[α]D 20=-10(c 0.62,CHCl3);1H NMR(400MHz,CDCl3)δ7.37–7.30(m,5H),5.28–5.26(d,J=6.4Hz,1H),5.21–5.13(m,2H),4.66–4.60(m,1H),4.50–4.47(m,1H),4.20–4.07(m,2H),4.00–3.96(m,1H),3.64–3.56(m,2H),2.21–2.16(m,2H),2.10–1.98(m,2H),1.87–1.84(m,1H),1.79–1.56(m,8H),1.39(s,9H),1.28–1.17(m,4H),1.31–1.11(d,J=6.8Hz,2H),1.06–0.99(m,2H),0.92–0.89(m,6H);13C NMR(100MHz,CDCl3)δ172.5,171.7,169.0,157.0,135.7,128.4,128.0,127.8,80.3,66.3,60.9,57.6,50.7,47.6,43.1,41.8,40.8,39.5,29.4,29.2,28.1,26.0,25.7,25.6,24.6,23.4,22.8,21.7;ESI-MS calcd for[C33H50N4O7+H+]614.4,found:614.4.。
5.5合成Boc-D-Phe-Pro-Gly-D-Leu-OBn(9e)
把HOBt(4.45g,33mmol),Boc-D-Phe-OH 8e(7.95g,30mmol)加入到150mL的圆底烧瓶中,氩气保护,加入60mL干燥的THF,0℃搅拌待瓶内温度降至0℃时,缓慢滴加DIC(5.1mL,33mmol),0.5h之后加入事先用THF溶解好的三肽Pro-Gly-D-Leu-OBn 7a(11.28g,30mmol)。室温搅拌48h后滤去不溶物,滤液减压浓缩干。残留物用乙酸乙酯溶解,然后依次用1M NaOH溶液、水、1M盐酸溶液和饱和食盐水洗涤,无水硫酸钠干燥,减压浓缩,石油醚和乙酸乙酯柱层析纯化得到四肽Boc-D-Phe-Pro-Gly-D-Leu-OBn(9e),16.07g,产率86%。White solid,Mp:160-161℃;[α]D 20=-10(c 0.62,CHCl3);1H NMR(400MHz,CDCl3)δ7.40-7.19(m,10H),5.50(br,1H),5.18(s,1H),4.65-4.64(m,1H),4.41-4.38(m,1H),4.32-4.30(m,1H),4.16-4.10(m,1H),3.69-3.61(m,2H),2.68-2.65(m,1H),2.05-2.02(m,1H),1.91-1.56(m,8H),1.39(s,9H),1.14-1.12(m,2H),0.96-0.91(m,6H);13C NMR(100MHz,CDCl3)δ172.6,172.0,171.5,169.0156.3,135.9,135.7,129.2,128.6,128.5,128.1,127.9,127.2,80.5,66.5,60.9,54.4,50.7,47.1,43.1,42.0,40.9,38.1,28.8,28.1,24.6,24.4,23.4,22.9,21.7;ESI-MS calcd for[C34H46N4O7+H+]623.3,found 623.3.。
5.6合成Boc-D-Phe-Pro-Gly-D-Phe-OBn(9f)
把HOBt(3.71g,27.5mmol),Boc-D-Phe-OH 8e(6.63,25mmol)加入到150mL的圆底烧瓶中,氩气保护,加入50mL干燥的THF,0℃搅拌待瓶内温度降至0℃时,缓慢滴加DIC(4.25mL,27.5mmol),0.5h之后加入事先用THF溶解好的三肽Pro-Gly-D-Phe-OBn 7b(10.23g,25mmol)。室温搅拌48h后滤去不溶物,滤液减压浓缩干。残留物用乙酸乙酯溶解,然后依次用1MNaOH溶液、水、1M盐酸溶液和饱和食盐水洗涤,无水硫酸钠干燥,减压浓缩后用石油醚和乙酸乙酯柱层析纯化得到四肽Boc-D-Phe-Pro-Gly-D-Phe-OBn(9f),13.45g,产率82%。White solid,Mp 138-139.6℃;[α]D 25=-49.0(c 0.5,CHCl3);1H NMR(600MHz,CDCl3)δ7.33–7.28(m,4H),7.25–7.18(m,8H),7.16–7.13(m,3H),5.45(d,J=6.5Hz,1H),5.13–5.08(m,2H),4.88(dd,J=14.4,6.6Hz,1H),4.46–4.41(m,1H),4.33(dd,J=7.8,3.7Hz,1H),3.98(dd,J=17.0,6.9Hz,1H),3.73(dd,J=17.0,5.7Hz,1H),3.66–3.585(b,1H),3.15(dd,J=6.7,2.6Hz,2H),2.95(t,J=7.7Hz,2H),2.67(dd,J=16.8,7.6Hz,1H),2.08–2.02(m,1H),1.84–1.75(b,3H),1.57–1.51(b,1H),1.39(s,9H).13C NMR(150MHz,CDCl3)δ171.9,171.5,171.4,168.8,136.2,135.4,129.4,129.3,128.6,128.5,128.4,128.3,128.2,127.2,126.8,80.5,77.2,77.0,76.8,66.9,60.8,53.6,47.1,43.2,38.2,28.7,28.3,24.4;ESI-MS calcd for[C37H44N4O7+H+]657.3,found 657.3.。
(六)中间化合物10的合成
6.1合成H2N-D-Val-Pro-Gly-D-Leu-OBn(10a)
把前面合成的四肽Boc-D-Val-Pro-Gly-D-Leu-OBn 9a(12.65g,22mmol)溶于CH2Cl2(20mL)中,然后在0℃下缓慢滴加TFA(20mL),室温下搅拌5h。将反应液减压浓缩尽量除去TFA,残留物用CH2Cl2溶解,再用1M NaOH溶液在0℃下中和至约pH=9.0左右。分离有机相,水相用CH2Cl2(20mL×3)萃取。合并有机相,饱和食盐水洗一次,无水硫酸钠干燥,减压浓缩,得到10.03g无色油状液体的H2N-D-Val-Pro-Gly-D-Leu-OBn(10a),产率为96%。无需纯化,直接用于下一步反应。
6.2合成H2N-D-Tle-Pro-Gly-D-Leu-OBn(10b)
把前面合成的四肽Boc-D-Tle-Pro-Gly-D-Leu-OBn 9b(12.96g,22mmol)溶于CH2Cl2(25mL)中,然后在0℃下缓慢滴加TFA(25mL),室温下搅拌5h。将反应液减压浓缩尽量除去TFA,残留物用CH2Cl2溶解,再用1MNaOH溶液在0℃下中和至约pH=9.0左右。分离有机相,水相用CH2Cl2(20mL×3)萃取。合并有机相,饱和食盐水洗一次,无水硫酸钠干燥,减压浓缩,得到10.22g无色油状液体的H2N-D-Tle-Pro-Gly-D-Leu-OBn(10b),产率为95%。无需纯化,直接用于下一步反应。
6.3合成H2N-D-Phg-Pro-Gly-D-Leu-OBn(10c)
把前面合成的四肽Boc-D-Phg-Pro-Gly-D-Leu-OBn 9c(13.4g,22mmol)溶于CH2Cl2(25mL)中,然后在0℃下缓慢滴加TFA(25mL),室温下搅拌5h。将反应液减压浓缩尽量除去TFA,残留物用CH2Cl2溶解,再用1M NaOH溶液在0℃下中和至约pH=9.0左右。分离有机相,水相用CH2Cl2(20mL×3)萃取。合并有机相,饱和食盐水洗一次,无水硫酸钠干燥,减压浓缩,得到10.3g无色油状液体的H2N-D-Phg-Pro-Gly-D-Leu-OBn(10c),产率为92%。无需纯化,直接用于下一步反应。
6.4合成四肽H2N-D-Chg-Pro-Gly-D-Leu-OBn(10d)
把前面合成的四肽Boc-D-Chg-Pro-Gly-D-Leu-OBn 9d(13.53g,22mmol)溶于CH2Cl2(25mL)中,然后在0℃下缓慢滴加TFA(25mL),室温下搅拌5h。将反应液减压浓缩尽量除去TFA,残留物用CH2Cl2溶解,再用1M NaOH溶液在0℃下中和至约pH=11.0左右。分离有机相,水相用CH2Cl2(20mL×3)萃取。合并有机相,依次用饱和食盐水洗涤、无水硫酸钠干燥,减压浓缩,得到无色油状液体的H2N-D-Chg-Pro-Gly-D-Leu-OBn(10d)10.6g,产率为94%。无需纯化,直接用于下一步反应。
6.5合成H2N-D-Phe-Pro-Gly-D-Leu-OBn(10e)
把前面合成的四肽Boc-D-Phe-Pro-Gly-D-Leu-OBn 9e(13.7g,22mmol)溶于CH2Cl2(25mL)中,然后在0℃下缓慢滴加TFA(25mL),室温下搅拌5h。将反应液减压浓缩尽量除去TFA,残留物用CH2Cl2溶解,再用1M NaOH溶液在0℃下中和至约pH=9.0左右。分离有机相,水相用CH2Cl2(20mL×3)萃取。合并有机相,依次用饱和食盐水洗涤、无水硫酸钠干燥,减压浓缩,得到无色油状液体的H2N-D-Phe-Pro-Gly-D-Leu-OBn(10e),10.5g,产率为91%。无需纯化,直接用于下一步反应。
6.6合成H2N-D-Phe-Pro-Gly-D-Phe-OBn(10f)
把前面合成的四肽Boc-D-Phe-Pro-Gly-D-Phe-OBn 9f(13.12g,20mmol)溶于CH2Cl2(25mL)中,然后在0℃下缓慢滴加TFA(25mL),室温下搅拌5h。将反应液减压浓缩尽量除去TFA,残留物用CH2Cl2溶解,再用1M NaOH溶液在0℃下中和至约pH=11.0左右。分离有机相,水相用CH2Cl2(20mL×3)萃取。合并有机相,饱和食盐水洗一次,无水硫酸钠干燥,减压浓缩,得到10.62g无色油状液体的H2N-D-Phe-Pro-Gly-D-Phe-OBn(10f),产率为94%。无需纯化,直接用于下一步反应。
以下是本发明的TP-1至TP-6具体化合物的制备方法。
实施例1
合成四肽TP-1:NH2-D-Val-Pro-Gly-D-Leu-OH
把前面合成的NH2-D-Val-Pro-Gly-D-Leu-OBn 10a(10.0g,21mmol)溶于甲醇(20mL),转移到100mL的高压反应釜中,然后加入500mg Pd/C(5%)。室温下通入氢气至8Mpa,搅拌6小时。过滤除去Pd/C,滤液减压浓缩,得到产物四肽NH2-D-Val-Pro-Gly-D-Leu-OH(TP-1),8.0g,产率100%。White solid,Mp:152–154℃;[α]D 20=–58.0(c 0.85,H2O);1HNMR(600MHz,D2O)δ4.55–4.50(m,1H),4.31–4.25(m,1H),4.04(d,J=17.1Hz,1H),3.90(d,J=17.1Hz,1H),3.87-3.83(m,1H),3.77-3.73(m,1H),2.41–2.33(m,2H),2.11-2.04(m,3H),1.74–1.60(m,4H),1.13(d,J=7.2Hz,3H),1.05(d,J=12Hz,3H),0.95(d,J=6Hz 3H),0.91(d,J=6Hz,3H).13C NMR(150MHz,D2O)δ179.7,174.3,170.4,168.9,61.1,57.1,53.5,48.1,42.4,40.5,29.2,28.7,24.5,24.3,22.4,20.5,17.9,15.9;HRMS calcd for[C18H32N4O5+H+]385.2445,found 385.2438.
或者把前面合成的NH2-D-Val-Pro-Gly-D-Leu-OBn(10.0g)溶于乙醇(20mL),转移到100mL的高压反应釜中,然后加入500mg Pd/C(5%)。室温下通入氢气至8Mpa,搅拌6小时。过滤除去Pd/C,滤液减压浓缩,得到产物四肽NH2-D-Val-Pro-Gly-D-Leu-OH(TP-1),为白色固体,7.45g,产率93%。
实施例2
合成TP-2:H2N-D-Tle-Pro-Gly-D-Leu-OH
把前面合成的NH2-D-Tle-Pro-Gly-D-Leu-OBn 10b(10.0g,20mmol)溶于甲醇(20mL),转移到100mL的高压反应釜中,然后加入500mg Pd/C(5%)。室温下通入氢气至8Mpa,搅拌6小时。过滤除去Pd/C,滤液减压浓缩,得到产物四肽NH2-D-Tle-Pro-Gly-D-Leu-OH(TP-2),7.8g,产率98%。White solid,Mp:204–205℃,[α]D 20=–28.0(c 1.0,DMF);1HNMR(600MHz,D2O)4.38–4.34(m,1H),4.12–4.02(m,2H),3.81(q,J=17.1Hz,2H),3.72–3.63(m,2H),3.21(d,J=0.4Hz,1H),2.24–2.18(m,1H),1.91(s,3H),1.57–1.52(m,1H),1.50–1.42(m,2H),0.97(s,9H),0.76(dd,J=22.8,5.8Hz,6H);13C NMR(150MHz,D2O)δ179.8,174.3,170.4,168.2,61.0,59.1,53.7,49.0,42.4,40.6,33.9,29.3,25.3,24.5,24.26,22.4,20.5;HRMS calcd for[C19H30N4O5+H+]399.2602,found 399.2599。
实施例3.
合成TP-3:H2N-D-Phg-Pro-Gly-D-Leu-OH
把前面合成的NH2-D-Phg-Pro-Gly-D-Leu-OBn 10c(10.0g,19.5mmol)溶于甲醇(20mL),转移到100mL的高压反应釜中,然后加入500mg Pd/C(5%)。室温下通入氢气至8Mpa,搅拌6h。过滤除去Pd/C,滤液减压浓缩,得到产物四肽NH2-D-Phg-Pro-Gly-D-Leu-OH(TP-3),7.8g,产率96%。White solid,Mp:150–153℃,[α]D 20=–94.0(c 1.0,H2O);1H NMR(600MHz,D2O)δ7.62–7.42(m,5H),5.47(s,1H),4.50(dd,J=9.0,3.8Hz,1H),4.31(dd,J=10.6,3.2Hz,1H),4.09(d,J=17.2Hz,1H),3.89(d,J=17.2Hz,1H),3.80–3.71(m,1H),3.07–3.00(m,1H),2.25–2.15(m,1H),2.05–1.94(m,2H),1.86–1.72(m,2H),1.69–1.53(m,2H),1.01–0.86(m,6H);13C NMR(150MHz,D2O)δ179.9,174.3,170.3,167.4,130.6,129.8,129.6,128.9,128.5,61.5,56.4,53.6,47.5,42.3,40.6,28.9,24.6,24.2,22.4,20.6;HRMScalcd for[C21H30N4O5+H+]419.2289,found:419.2285.。
实施例4
合成TP-4:H2N-D-Chg-Pro-Gly-D-Leu-OH
把前面合成的NH2-D-Chg-Pro-Gly-D-Leu-OBn 10d(10.0g,19.4mmol)溶于甲醇(20mL),转移到100mL的高压反应釜中,然后加入500mg Pd/C(5%)。室温下通入氢气至8Mpa,搅拌6小时。过滤除去Pd/C,滤液减压浓缩,得到产物四肽NH2-D-Chg-Pro-Gly-D-Leu-OH(TP-4),7.94g,产率97%。White solid,Mp:167–169℃,[α]D 20=–96.0(c 1.0,H2O);1HNMR(600MHz,D2O)δ4.55–4.46(m,1H),4.33–4.20(m,2H),4.03(d,J=17.1Hz,1H),3.93–3.70(m,3H),2.40–2.32.(m,1H),2.13–1.94(m,3H),1.86–1.57(m,7H),1.37–1.07(m,5H),0.98–0.86(m,6H);13C NMR(150MHz,D2O)δ179.6,174.3,170.4,168.8,61.1,56.6,53.9,53.4,48.1,42.4,40.5,38.4,29.3,28.5,27.1,25.1,24.5,24.2,22.4,20.5;HRMS calcdfor[C21H36N4O5+H+]425.2758,found 425.2748.。
实施例5
合成TP-5:NH2-D-Phe-Pro-Gly-D-Leu-OH
把前面合成的NH2-D-Phe-Pro-Gly-D-Leu-OBn 9e(10.0g,19.1mmol)溶于甲醇(20mL),转移到100mL的高压反应釜中,然后加入500mg Pd/C(5%)。室温下通入氢气至8Mpa,搅拌6小时。过滤除去Pd/C,滤液减压浓缩,得到产物四肽NH2-D-Phe-Pro-Gly-D-Leu-OH(TP-5),为白色固体,7.7g,产率93%。White solid,Mp:198-200℃;[α]D 20=–98.0(c1.0,H2O);1H NMR(600MHz,D2O)7.45-7.39(m,3H),7.35(d,J=6.9Hz,2H),4.62-4.57(m,1H),4.37(dd,J=8.8,4.6Hz,1H),4.26(dd,J=10.5,3.5Hz,1H),4.01(d,J=17.1Hz,1H),3.88(d,J=17.2Hz,1H),3.64–3.57(m,1H),3.30-3.18(m,2H),2.86-2.80(m,1H),2.16–2.08(m,1H),1.97-1.85(m,2H),1.73–1.56(m,4H),0.92(dd,J=28.0,6.0Hz,6H);13C NMR(150MHz,D2O)δ179.8,174.1,170.3,168.5,133.3,129.4,129.1,128.1,61.0,53.6,53.0,47.7,42.4,40.5,36.3,29.1,24.5,24.0,22.4,20.5;HRMS calcd for[C22H32N4O5+H+]433.2445,found419.2439.。
实施例6.
合成TP-6:NH2-D-Phe-Pro-Gly-D-Phe-OH
把前面合成的NH2-D-Phe-Pro-Gly-D-Phe-OBn 10f(10.2g,18mmol)溶于甲醇(20mL),转移到100mL的高压反应釜中,然后加入500mg Pd/C(5%)。室温下通入氢气至8Mpa,搅拌6小时。过滤除去Pd/C,滤液减压浓缩,得到产物四肽NH2-D-Phe-Pro-Gly-D-Phe-OH(TP-6),为白色固体,7.8g,产率92%。Mp 182.5–184.4℃,[α]D 20=–50.0(c 0.5,DMF);1HNMR(600MHz,DMSO)δ8.50–8.16(m,2H),7.41–7.03(m,10H),4.38(m,3H),3.76–3.64(m,1H),3.50–3.32(m,2H),3.20–2.79(m,4H),1.87–1.60(m,3H),1.40–1.26(m,1H),0.98(d,J=6.5Hz,1H);13C NMR(150MHz,DMSO)δ175.0,171.4,170.2,168.3,139.3,136.0,129.9,129.7,128.9,128.3,127.5,126.2,61.2,52.9,47.1,42.1,40.4,40.2,40.1,39.9,39.7,39.6,38.8,29.3,24.4,23.7;HRMS calcd for[C25H30N4O5+H+]467.2288,found:467.2289.。
二、化合物TP的应用例
实施例7
在洁净的5mL圆底烧瓶中加入准确称量的本发明的四肽催化剂TP(0.025mmol)、N-苯基马来酰亚胺(87mg,0.5mmol)和1.0mL干燥重蒸过的甲苯,在冰水浴和磁力搅拌条件下加入重蒸的异丁醛(92μL,1.0mmol),恢复反应液至室温下继续搅拌反应,通过TLC检测至反应结束后,加3~4滴饱和氯化铵溶液,用乙酸乙酯萃取(10mL×3),少量饱和食盐水洗涤,无水硫酸钠干燥,减压浓缩,残余油状物利用石油醚和乙酸乙酯柱层析纯化,获得纯产物。
本发明的TP-1~TP-6六种四肽催化剂催化异丁醛与N-苯基马来酰亚胺的不对称共轭加成反应的评价,结果如下表1所示。由结果可知,TP-3和TP-5的不对称催化效率最高。
表1.四肽催化剂催化异丁醛与N-苯基马来酰亚胺的不对称共轭加成反应.a
a分离产率,ee由手性HPLC确定。
实施例8.
溶剂对四肽TP-5催化异丁醛与N-苯基马来酰亚胺不对称共轭加成反应的影响。
在洁净的5mL圆底烧瓶中加入准确称量的四肽催化剂TP-5(0.025mmol,10.8mg)、N-苯基马来酰亚胺(87mg,0.5mmol)和1.0mL干燥重蒸过的反应溶剂,在冰水浴下和磁力搅拌条件下加入重蒸的异丁醛(92μL,1.0mmol),恢复反应液至室温下继续搅拌反应,通过TLC检测至反应结束后,加3~4滴饱和氯化铵溶液,用乙酸乙酯萃取(10mL×3),少量饱和食盐水洗涤,无水硫酸钠干燥,减压浓缩,残余油状物利用石油醚和乙酸乙酯柱层析纯化,获得纯产物。
各种溶剂对反应的产率和ee值影响,结果见下表2所示。结果显示:与甲苯相比较,乙腈为溶剂时产率和ee值都显著提高。
表2.溶剂对四肽TP-5催化异丁醛与N-苯基马来酰亚胺的不对称共轭加成反应的影响a
entry solvent hours yield% ee
1 CH2Cl2 40h 80 99
2 MeCN 40h 95 98
3 EtOH 40h 91 88
4 THF 40h 90 97
5 DMSOb -- -- ---
6 toluene 72h 55 91
a分离产率,ee由手性HPLC确定。b没有检测到反应。
实施例9.
观察催化剂用量对不对称共轭加成反应的催化效率的影响。
在洁净的5mL圆底烧瓶中加入准确称量的四肽催化剂TP-5或TP-3(0.025mmol)、N-苯基马来酰亚胺(87mg,0.5mmol)和1.0mL干燥重蒸过的反应溶剂,在冰水浴下和磁力搅拌条件下加入重蒸的异丁醛(92μL,1.0mmol),恢复反应液至室温下继续搅拌反应,通过TLC检测至反应结束后,加3~4滴饱和氯化铵溶液,用乙酸乙酯萃取(10mL×3),少量饱和食盐水洗涤,无水硫酸钠干燥,减压浓缩,残余油状物利用石油醚和乙酸乙酯柱层析纯化,获得纯产物。
反应产率和ee值如下表3所示。结果显示,使用2.5mol%用量的TP-3四肽为催化剂,以乙腈为溶剂,可以获得最优的不对称催化效果。
表3.四肽使用量对不对称共轭加成反应的影响a.
Entry TP(mmol%) solvent hours yield% ee
1 TP-5(5%) MeCN 40h 95 98
2 TP-5(2.5%) MeCN 40h 95 98
3 TP-5(1%) MeCN 40h 56 98
4 TP-3(2.5%) MeCN 40h 98 99
a分离产率,ee由手性HPLC确定。
实施例10
观察四肽TP-3催化脂肪醛与马来酰亚胺不对称共轭加成反应的底物适应范围。
在洁净的5mL圆底烧瓶中加入准确称量的四肽催化剂2.5%TP-3(0.0125mmol,5.3mg)、马来酰亚胺(0.5mmol)和1.0mL干燥重蒸过的乙腈,在冰水浴下和磁力搅拌条件下加入重蒸的脂肪醛(1.0mmol),恢复反应液至室温下继续搅拌反应,通过TLC检测至反应结束后,加3~4滴饱和氯化铵溶液,用乙酸乙酯萃取(10mL×3),少量饱和食盐水洗涤,无水硫酸钠干燥,减压浓缩,残余油状物利用石油醚和乙酸乙酯柱层析纯化,获得纯产物。
结果如表4所示。
表4.四肽TP-3催化的脂肪醛与马来酰亚胺的不对称共轭加成反应.a
a分离产率,ee由手性HPLC确定。
产物P-1~P-17表征如下。
P-1,[α]D 25=-6.0(c=1.0,CHCl3);1H NMR(600 MHz,CDCl3)δ9.51(s,1H),
7.48-7.44(m,2H),7.40–7.36(m,1H),7.29-7.25(m,2H),3.14(dd,J=9.6,5.6Hz,1H),2.96(dd,J=18.6,9.6 Hz,1H),2.61(dd,J=18.4,5.6 Hz,1H),1.32(s,3H),1.28(s,3H).。
P-2,[α]D 25=+2.0(c=1.0,CHCl3);1H NMR(600 MHz,CDCl3)δ9.49(s,1H),7.28
–7.24(m,2H),7.17-7.12(m,2H),3.11(dd,J=9.6,5.6 Hz,1H),2.97(dd,J=18.3,9.6 Hz,1H),2.61(dd,J=18.4,5.6 Hz,1H),1.35(s,3H),1.28(s,3H).。
P-3,[α]D 25=+3.0(c=1.0,CHCl3);1H NMR(600 MHz,CDCl3)δ9.49(s,1H),7.49
–7.40(m,2H),7.28–7.22(m,2H),3.11(dd,J=9.6,5.6 Hz,1H),2.97(dd,J=18.3,9.6 Hz,1H),2.62(dd,J=18.3,5.6 Hz,1H),1.36(s,3H),1.29(s,3H).。
P-4,[α]D 25=+3.0(c=1.0,CHCl3);1H NMR(600 MHz,CDCl3)δ9.49(s,1H),7.62
–7.57(m,1H),7.23–7.15(m,2H),3.11(dd,J=9.6,5.4 Hz,1H),2.97(dd,J=18.4,9.6 Hz,1H),2.62(dd,J=18.4,5.6 Hz,1H),1.36(s,3H),1.29(s,3H).。
P-5,[α]D 25=+6.0(c=1.0,CHCl3);1H NMR(600 MHz,CDCl3)δ9.46(s,1H),8.35
–8.29(m,2H),7.61–7.54(m,2H),3.10(dd,J=9.6,5.4 Hz,1H),3.00(dd,J=18.6,9.6 Hz,1H),2.65(dd,J=18.6,5.4 Hz,1H),1.41(s,3H),1.30(s,3H).。
P-6,[α]D 25=+5.0(c=1.0,CHCl3);1H NMR(600 MHz,CDCl3)δ9.52(s,1H),7.21
–7.15(m,2H),6.99–6.95(m,2H),3.82(s,3H),3.13(dd,J=9.6,5.4 Hz,1H),2.96(dd,J=18.4,9.6 Hz,1H),2.60(dd,J=18.6,5.4 Hz,1H),1.32(s,3H),1.28(s,3H).。
P-7,[α]D 25=+6.0(c=1.0,CHCl3);1H NMR(600 MHz,CDCl3)δ9.52(s,1H),7.27
(d,J=7.2 Hz,2H),7.14(d,J=8.4 Hz,2H),3.14(dd,J=9.6,5.4 Hz,1H),2.97(dd,J=18.4,9.6 Hz,1H),2.61(dd,J=18.4,5.5 Hz,1H),2.37(s,3H),1.32(s,3H),1.28(s,3H).。
P-8,[α]D 25=+20.0(c=1.0,CHCl3);1H NMR(600 MHz,CDCl3)δ9.63(s,1H),
7.48(t,J=7.8 Hz,2H),7.40(t,J=7.5 Hz,1H),7.34–7.28(m,2H),3.31-3.26(m,1H),3.12–3.06(m,1H),2.93(dd,J=18.0,9.7 Hz,1H),2.60(dd,J=18.0,5.7 Hz,1H),1.40(d,J=7.8 Hz,3H).。
P-9,[α]D 25=+16.0(c=1.0,CHCl3);1H NMR(600 MHz,CDCl3)δ9.78(s,1H),
7.50–7.45(m,2H),7.41–7.36(m,1H),7.31-7.28(m,2H),3.17(dd,J=7.2,3.7Hz,1H),3.11–2.99(m,1H),2.87(dd,J=18.0,9.6 Hz,1H),2.73(dd,J=18.0,6.0 Hz,1H),2.36–2.28(m,1H),1.26(d,J=6.6 Hz,3H),1.10(d,J=6.6 Hz,3H).。
P-10,[α]D 25=+41.0(c=1.0,CHCl3);1H NMR(600 MHz,CDCl3)δ9.75(s,1H),
7.47(t,J=6Hz,2H),7.41-7.37(m,1H),7.32-7.26(m,2H),3.36-3.32(m,1H),3.03–2.96(m,2H),2.57(dd,J=18.0,5.4 Hz,1H),1.94-1.88(m,1H),1.70-1.62(m,1H),1.51–1.45(m,2H),1.42-1.36(m,2H),0.93(t,J=6 Hz,3H).。
P-11,[α]D 25=-40.0(c=1.0,CHCl3);1H NMR(600 MHz,CDCl3)δ9.39(s,1H),
7.49-7.46(m,2H),7.40-7.37(m,1H),7.34–7.29(m,2H),3.05-2.94(m,2H),2.58(dd,J=18,5.4 Hz,1H),2.37–2.27(m,1H),2.14–2.03(m,2H),1.88–1.69(m,5H).。
P-12,[α]D 25=-5.0(c=1.0,CHCl3);1H NMR(600 MHz,CDCl3)δ9.55(s,1H),
7.47(t,J=7.7 Hz,2H),7.40-7.38(m,1H),7.31–7.27(m,2H),3.22(dd,J=9.0,6.0Hz,1H),2.87(dd,J=18.0,9.6 Hz,1H),2.68(dd,J=18.2,5.9 Hz,1H),1.99–1.85(m,3H),1.67–1.49(m,6H).。
P-13,[α]D 25=+10.0(c=1.0,CHCl3);1H NMR(600 MHz,CDCl3)δ9.63(s,1H),
7.47(t,J=7.8 Hz,2H),7.39(t,J=7.5 Hz,1H),7.29–7.25(m,2H),3.25(dd,J=9.6,6.0 Hz,1H),2.97(dd,J=18.4,9.6 Hz,1H),2.69(dd,J=18.4,6.0 Hz,1H),2.02–1.83(m,3H),1.74(dq,J=15.0,7.5 Hz,1H),1.03–0.97(m,6H).。
P-14,[α]D 25=-9.0(c=1.0,CHCl3);1H NMR(600 MHz,CDCl3)δ9.51(s,1H),
3.04(dd,J=9.6,5.4 Hz,1H),2.99(s,3H),2.82(dd,J=18.3,9.4 Hz,1H),2.45(dd,J=18.3,5.4 Hz,1H),1.22(d,J=6.4 Hz,6H).。
P-15,[α]D 25=-11.0(c=1.0,CHCl3);1H NMR(600 MHz,cdcl3)δ9.51(s,1H),
3.98–3.91(m,1H),2.97(dd,J=9.5,5.2 Hz,1H),2.74(dd,J=18.3,9.5 Hz,1H),2.37(dd,J=18.3,5.2 Hz,1H),2.16-2.08 m,2H),1.80(d,J=13.4 Hz,2H),1.66-1.53(m,3H),1.32-1.23(m,2H),1.23–1.12(m,7H).。
P-16,[α]D 25=-14.0(c=1.0,CHCl3);1H NMR(600 MHz,CDCl3)δ9.48(s,1H),
7.37–7.34(m,2H),7.31-7.28(m,2H),4.65(q,J=14.4 Hz,2H),3.03(dd,J=9.4,5.4 Hz,1H),2.81(dd,J=18.3,9.4 Hz,1H),2.45(dd,J=18.3,5.4 Hz,1H),1.16(d,J=2.3 Hz,6H).。
P-17,[α]D 25=+5.0(c=1.0,CHCl3);1H NMR(600MHz,CDCl3)δ9.46(s,1H),8.81(s,1H),3.07(dd,J=9.6,5.4Hz,1H),2.83(dd,J=18.4,9.6Hz,1H),2.48(dd,J=18.4,5.4Hz,1H),1.21(d,J=6.9Hz,6H).。

Claims (3)

1.一种如式1示四肽化合物,
其特征在于R1=i-Pr,R2=i-Bu,其氨基酸序列为H2N-D-Val-Pro-Gly-D-Leu-OH,命名为TP-1。
2.权利要求1中所述化合物TP-1的制备方法,其特征在于:
1)如式2或式3或式4或式5或式6中任一反应制备出化合物3a,
2)再以式7或式8或式9中任一方法制备出化合物4a,
3)再以式10和式11示反应历程制备得到化合物7a,
4)以式12或式13所示反应的任一种制备得到化合物9a,
5)以式14和式15示反应历程制备得到目标化合物TP-1,
3.权利要求1所述的四肽化合物用于催化脂肪醛与马来酰亚胺的不对称共轭加成反应。
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