CN110193086A - 一种la-gflg-dox偶联物及其制备方法与用途 - Google Patents
一种la-gflg-dox偶联物及其制备方法与用途 Download PDFInfo
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Abstract
本发明涉及医药领域中抗肿瘤药物及其制备方法与用途,具体公开了一种LA‑GFLG‑DOX偶联物及其制备方法与用途,所述LA‑GFLG‑DOX偶联物为硫辛酸‑甘氨酸‑苯丙氨酸‑亮氨酸‑甘氨酸‑阿霉素分子通过酰胺键顺次连接形成的化合物。本发明在保留DOX对肿瘤细胞毒性的同时,利用LA的抗氧化能力,可以抵抗DOX产生的活性氧带来的对正常细胞的损伤。通过抗肿瘤活性检测及对正常细胞的毒副作用的实验可以看出,所述LA‑GFLG‑DOX偶联物对肿瘤细胞有明显的抑制作用,且可以减少对正常细胞的损伤,对降低DOX的毒副作用是有益的。
Description
技术领域
本发明涉及医药领域中抗肿瘤药物及其制备方法与用途,尤其涉及一种LA-GFLG-DOX偶联物及其制备方法与用途。
背景技术
硫辛酸(lipoic acid,LA)是一种天然存在的抗氧化剂,属于B族维生素。它具有良好的脂溶性和水溶性,在水溶性和非水溶性条件下都可以发挥其抗氧化作用。硫辛酸可以分布在生物体内各个组织,也是人体中不可缺少的抗氧化物质,被称为“万能抗氧剂”。硫辛酸的抗氧化能力是维生素C和维生素E的400倍,在美、日等国已经被批准可以作为食品和保健品的功能成分。在细胞内,硫辛酸主要存在于细胞的线粒体中,清除体内的自由基等活性氧,抑制氧化应激,对氧化损伤有比较强的修复作用。因此,在预防及治疗与自由基有关的疾病如肿瘤、糖尿病、动脉粥样硬化、退化性神经系统疾病等发挥着重要的作用。
阿霉素(doxorubicin,DOX)作为一种蒽环类抗生素,是高效广谱的抗肿瘤药物。阿霉素可以嵌插在肿瘤细胞DNA的碱基对之间,阻碍DNA的复制而达到抗肿瘤作用。但在其治疗过程中,DOX本身缺乏肿瘤靶向性,在杀死肿瘤细胞的同时,也会对正常的组织、细胞带来伤害,如心脏毒性、骨髓抑制、听力损伤等毒副作用。这严重限制和影响了DOX的肿瘤治疗效果。DOX的毒性主要源于其在体内产生的活性氧带来的氧化应激和氧化损伤。
GFLG是由甘氨酸,苯丙氨酸,亮氨酸,甘氨酸通过酰胺键连接形成的小分子四肽化合物。研究发现,GFLG可被组织蛋白酶B特异性地识别并水解,而组织蛋白酶B在多种肿瘤细胞的溶酶体中高表达,在正常细胞中低表达,所以GFLG是一个具有良好应用前景的酶响应的小分子化合物。由于GFLG的酶响应特点,通常将其用作载体和药物之间的连接臂,使其在正常细胞中药物释放量很少,而在肿瘤细胞中释放药物,从而减少了对正常细胞的损伤作用。正是由于GFLG的这一特点,目前有一些GFLG和DOX的偶联物,在GFLG一端又修饰了其他多肽衍生物以提高其抗肿瘤特性,如中国专利文献CN102127154A和CN103768613。但是,在GFLG一端修饰LA以降低DOX毒副作用的偶联物目前尚无报道。
发明内容
针对DOX的毒副作用,结合LA的抗氧化特性,以及GFLG可被肿瘤细胞内高表达的组织蛋白酶B特异性水解切断的特点,本发明提供了一种新的化合物LA-GFLG-DOX偶联物及其制备方法与用途,在保留DOX对肿瘤细胞毒性的同时,利用LA的抗氧化能力,抵抗DOX产生的活性氧带来的对正常细胞的损伤。
本发明一方面提供了一种LA-GFLG-DOX偶联物,所述LA-GFLG-DOX偶联物为硫辛酸-甘氨酸-苯丙氨酸-亮氨酸-甘氨酸-阿霉素分子通过酰胺键顺次连接,其结构式如式(Ⅰ)所示。
本发明的另一方面提供了所述LA-GFLG-DOX偶联物的制备方法,包括以下步骤:
(1)通过缩合反应合成全保护的四肽化合物R1-GFLG-R2;
(2)对步骤(1)中所得化合物的氨基端脱除保护基连接硫辛酸,得到化合物LA-GFLG-R2;
(3)对步骤(2)中所得化合物的羧基端脱除保护基并活化连接上阿霉素得到化合物LA-GFLG-DOX。
其中,R1为与四肽化合物GFLG氨基端连接的保护基,选自叔丁氧羰基、苄氧羰基、对甲苯磺酰基、三苯甲基、甲酰基中任一种;R2为与四肽化合物GFLG羧基端连接的保护基,选自甲氧基、苄酯、对硝基苄酯,或形成酰胺、酰肼。
优选地,R1为叔丁氧羰基,R2为甲氧基。
进一步地,硫辛酸在氮气保护下对其羧基端活化,与GFLG的氨基端连接,得到LA-GFLG,接下来再次对GFLG中甘氨酸的羧基端活化,与阿霉素连接,得到LA-GFLG-DOX。
进一步地,Boc-GFLG-OMe在制备过程中每个氨基酸的氨基端均以叔丁氧羰基保护,并在酸性条件下脱除;羧基端均以甲氧基保护,并在碱性条件下脱除,通过酰胺键将四种氨基酸连接得到Boc-GFLG-OMe。
在本发明的一实施例中,R1为叔丁氧羰基,R2为甲氧基,LA-GFLG-DOX偶联物的制备方法包括以下步骤:
(1)合成全保护的四肽化合物R1-GFLG-R2;
(2)LA-GFLG-OMe的合成:将Boc-Gly-Phe-Leu-Gly-OMe溶解后加入等体积的三氟乙酸,室温搅拌,反应液旋干;将硫辛酸和N,N’-羰基二咪唑溶解后在氮气保护下搅拌得到羧基端活化的硫辛酸;氮气保护下,将活化的硫辛酸溶液与Gly-Phe-Leu-Gly-OMe混合,以N,N-二异丙基乙胺调节pH值在8-9,室温搅拌过夜;反应结束后,萃取得到目标产物;
(3)LA-GFLG-OH的合成:LA-Gly-Phe-Leu-Gly-OMe溶解于甲醇,加入碱性溶液;室温搅拌,调节反应溶液pH为7,以旋转蒸发仪旋干甲醇,旋干后的反应物溶解于超纯水;调节该水溶液pH为3,乙酸乙酯萃取,收集乙酸乙酯层并旋干;
(4)LA-GFLG-DOX的合成:LA-Gly-Phe-Leu-Gly-OH溶解于四氢呋喃,继续加入N-羟基丁二酰亚胺,1-乙基-(3-二甲基氨基丙基)碳酰二亚胺盐酸盐,冰浴条件下加入至茄瓶中,保持4℃反应过夜;反应液以旋转蒸发仪旋干除去溶剂,剩余物重新加入二氯甲烷溶解,转移至分液漏斗,二氯甲烷层水洗三次,旋干得到黄色油状物;阿霉素盐酸盐溶解于二甲基甲酰胺,加入黄色油状物,室温避光搅拌;反应结束后,反应液以0.1%三氟乙酸水溶液稀释,二氯甲烷萃取,得到的二氯甲烷层旋干。
进一步地,步骤(2)中所述硫辛酸和N,N’-羰基二咪唑的摩尔比为1:1-1:1.5;所述在氮气保护下搅拌时间为1.5h-4h。
进一步地,步骤(3)中所述碱性溶液选自碱金属的氢氧化物或碳酸盐进一步地,步骤(4)中LA-Gly-Phe-Leu-Gly-OH与N-羟基丁二酰亚胺、1-乙基-(3-二甲基氨基丙基)碳酰二亚胺盐酸盐的摩尔比为;1:1-1:1.5和1:1-1.5;LA-Gly-Phe-Leu-Gly-OH与阿霉素盐酸盐的摩尔比为1:0.3-1.05。
本发明还提供了所述LA-GFLG-DOX偶联物的抗肿瘤用途。
本发明所述LA-GFLG-DOX偶联物具有以下有益效果:
(1)通过细胞毒性实验表明所述LA-GFLG-DOX偶联物对肿瘤细胞有明显的抑制作用,且随着给药时间延长,对细胞的抑制作用增强,说明所述LA-GFLG-DOX偶联物具有抗肿瘤活性;
(2)从细胞水平分析,所述LA-GFLG-DOX偶联物对正常人支气管内皮细胞16HBE的毒性较小,说明所述LA-GFLG-DOX偶联物对于肿瘤细胞与正常细胞有一定的选择性,可以减少对正常细胞的损伤,对降低DOX的毒副作用是有益的。
附图说明
图1本发明一实施例中LA-GFLG-DOX偶联物的合成路线图;
图2本发明一实施例中LAX-GFLG-DOX的1H NMR谱;
图3本发明一实施例中LAX-GFLG-DOX偶联物的质谱(ES+);
图4本发明一实施例中LAX-GFLG-DOX偶联物的质谱(ES-);
图5本发明一实施例中给药LAX-GFLG-DOX偶联物分别孵育24(A),48(B),72(C)小时对于肿瘤细胞MCF-7细胞生长抑制的影响;
图6本发明一实施例中给药相同浓度阿霉素和LAX-GFLG-DOX偶联物分别孵育24(A),48(B),72(C)小时对于正常细胞16HBE细胞生长抑制的影响。
具体实施方式
下面结合附图对本发明内容作进一步详细描述:
实施例1:
一、LA-GFLG-DOX偶联物的制备
合成路线如图1所示:
1.Boc-GF-OMe的合成
室温下,Boc-Gly-OH(10.0mmol)溶解于四氢呋喃,冰浴下,加入1-羟基苯并三唑(10.0mmol)和1-乙基-(3-二甲基氨基丙基)碳酰二亚胺盐酸盐(10.0mmol),反应30分钟。继续加入HCl·Phe-OMe(10.0mmol),以N,N-二异丙基乙胺调节pH值在8-9,室温搅拌过夜。反应结束后,旋转蒸发仪旋干溶剂,得到的油状物以30mL乙酸乙酯溶解并转移至100mL分液漏斗。乙酸乙酯层以50mL 5%NaHCO3萃取洗涤三次,50mL 5%NaHSO4溶液萃取洗涤三次,50mL饱和NaCl溶液萃取洗涤三次,50mL 5%NaHCO3萃取洗涤三次,50mL饱和NaCl溶液萃取洗涤三次,得到的乙酸乙酯层旋干。
核磁共振氢谱及质谱解析结果:1HNMR(300MHz,DMSO-d6)δ:1.38(s,9H,-C(CH3)3),2.94(dd,1H,CH2-Phe),3.04(dd,1H,-CH2-Phe),3.57(m,2H,-CH2-N),3.60(s,3H,-OCH3),4.51(dd,1H,-CH-N),6.92(d,1H,NH),7.20-7.31(m,5H,Phe),8.22(d,1H,NH-Boc).m/z=337.27[M+H]+,m/z=359.25[M+Na]+,m/z=375.23[M+K]+.
2.Boc-GF-OH的合成
室温下,Boc-Gly-Phe-OMe(10.0mmol)溶解于甲醇,加入0.35g NaOH。室温搅拌,以1M HCl调节反应溶液pH为7,以旋转蒸发仪旋干甲醇,旋干后的反应物溶解于20mL超纯水。该水溶液以1M HCl调节反应溶液pH为3,乙酸乙酯萃取,收集乙酸乙酯层并旋干。
核磁共振氢谱及质谱解析结果:1HNMR(300MHz,DMSO-d6)δ:1.34(s,9H,-C(CH3)3),3.02(m,1H,-CH2-Phe),3.07(m,1H,-CH2-Phe),3.59(m,2H,-CH2-N),4.44(m,1H,-CH-N),6.91(s,1H,NH),7.23-7.28(m,5H,Phe),8.00(d,1H,NH-Boc),12.77(s,1H,-COOH).m/z=323.02[M+H]+,m/z=344.98[M+Na]+.
3.Boc-LG-OMe的合成
室温下,Boc-Leu-OH(10.0mmol)溶解于四氢呋喃,冰浴下,加入1-羟基苯并三唑(10.0mmol)和1-乙基-(3-二甲基氨基丙基)碳酰二亚胺盐酸盐(10.0mmol),反应30分钟。继续加入HCl·Gly-OMe(10.0mmol),以N,N-二异丙基乙胺调节pH值在8-9,搅拌过夜。反应结束后,旋转蒸发仪旋干溶剂,得到的油状物以30mL乙酸乙酯溶解并转移至100mL分液漏斗。乙酸乙酯层以50mL5%NaHCO3萃取洗涤三次,50mL 5%NaHSO4溶液萃取洗涤三次,50mL饱和NaCl溶液萃取洗涤三次,50mL 5%NaHCO3萃取洗涤三次,50mL饱和NaCl溶液萃取洗涤三次,得到的乙酸乙酯层旋干。
核磁共振氢谱及质谱解析结果:1HNMR(300MHz,DMSO-d6)δ:0.89(d,6H,-C(CH3)2),1.42(s,9H,-C(CH3)3),1.45(m,2H,-CH2-),1.65(m,1H,-CH-),3.62(s,3H,-OCH3),3.86(m,2H,-CH2-N),4.02(m,1H,-CH-N),6.89(d,1H,NH),8.21(t,1H,NH-Boc).m/z=325.08[M+Na]+.
4.LG-OMe的合成
Boc-Leu-Gly-OMe(5.80mmol)溶解于二氯甲烷,加入等体积的三氟乙酸,室温搅拌,反应液旋干。
核磁共振氢谱及质谱解析结果:1HNMR(300MHz,DMSO-d6)δ:0.91(d,6H,-C(CH3)2),1.53(m,2H,-CH2-),1.75(m,1H,-CH-),3.65(s,3H,-OCH3),3.96(m,1H,-CH-N),7.83(s,2H,NH2),8.24(s,1H,NH).m/z=203.05[M+H]+.
5.Boc-GFLG-OMe的合成
室温下,Boc-Gly-Phe-OH(5.80mmol)溶解于四氢呋喃,冰浴下,加入1-羟基苯并三唑(5.80mmol)和1-乙基-(3-二甲基氨基丙基)碳酰二亚胺盐酸盐(5.80mmol),反应30分钟。继续加入Leu-Gly-OMe(5.60mmol),以N,N-二异丙基乙胺调节pH值在8-9,室温搅拌过夜。反应结束后,旋转蒸发仪旋干溶剂,得到的油状物以30mL乙酸乙酯溶解并转移至100mL分液漏斗。乙酸乙酯层以50mL 5%NaHCO3萃取洗涤三次,50mL 5%NaHSO4溶液萃取洗涤三次,50mL饱和NaCl溶液萃取洗涤三次,50mL 5%NaHCO3萃取洗涤三次,50mL饱和NaCl溶液萃取洗涤三次,得到的乙酸乙酯层旋干。
核磁共振氢谱及质谱解析结果:1HNMR(300MHz,DMSO-d6)δ:0.84(m,6H,-C(CH3)2),1.28(m,9H,-C(CH3)3),1.50(m,2H,-CH2-),1.59(m,1H,-CH-),2.79,2.99(m,2H,-CH2-Phe),3.46(m,2H,-CH2-N),3.84(s 3H,-OCH3),3.86(m,2H,-CH2-N),4.35(m,1H,-CH-),4.55(m,1H,-CH-),6.92(s,1H,NH),7.22(s,5H,Phe),7.84(d,1H,NH),8.12(d,1H,NH),8.23(d,1H,NH).m/z=529.34[M+Na]+.
6.LA-GFLG-OMe的合成
Boc-Gly-Phe-Leu-Gly-OMe(2.70mmol)溶解于二氯甲烷,加入等体积的三氟乙酸,室温搅拌,反应液旋干。硫辛酸(2.70mmol)和N,N’-羰基二咪唑(2.90mmol)于100mL三颈瓶,加入20mL已干燥的三氯甲烷至完全溶解,氮气保护下搅拌两个小时得到羧基端活化的硫辛酸。氮气保护下,活化的硫辛酸溶液与Gly-Phe-Leu-Gly-OMe混合,以N,N-二异丙基乙胺调节pH值在8-9,室温搅拌过夜。反应结束后,向三颈瓶中加入100mL超纯水,反应液分层,收集三氯甲烷层溶液,水层继续以三氯甲烷萃取,得到的三氯甲烷层旋干。
核磁共振氢谱及质谱解析结果:1HNMR(300MHz,DMSO-d6)δ:0.84(m,6H,-C(CH3)2),1.5-2.1(m,11H,-CH2-),2.44-3.36(m,5H),3.50-3.52(m,2H,-CH2-),3.66(s,3H,-CH3),3.60-3.69(m,2H,-CH2-),3.83(m,2H,-CH2-),4.32(m,1H,-CH-),4.44(m,1H,-CH-),7.23(s,5H,Phe),8.02-8.22(m,4H,NH).m/z=595.11[M+H]+,m/z=617.05[M+Na]+.
7.LA-GFLG-OH的合成
LA-Gly-Phe-Leu-Gly-OMe(1.50mmol)溶解于甲醇,加入0.20g NaOH。室温搅拌,以1M HCl调节反应溶液pH为7,以旋转蒸发仪旋干甲醇,旋干后的反应物溶解于20mL超纯水。该水溶液以1M HCl调节反应溶液pH为3,乙酸乙酯萃取,收集乙酸乙酯层并旋干。
核磁共振氢谱及质谱解析结果:1H NMR(300MHz,DMSO-d6)δ:0.88(m,6H,-C(CH3)2),1.5-2.1(m,11H,-CH2-),2.39-3.23(m,5H,-CH-,-CH2-),3.51-3.58(m,2H,-CH2-),3.60(m,2H,-CH2-),3.67(m,2H,-CH2-),4.53(m,1H,-CH-),4.32(m,1H,-CH-),7.23(s,5H,Phe),8.01-8.15(m,4H,NH),12.54(s,1H,-COOH).m/z=603.09[M+Na]+.
8.LA-GFLG-DOX的合成
LA-Gly-Phe-Leu-Gly-OH(281μmol)溶解于四氢呋喃,继续加入N-羟基丁二酰亚胺(361μmol),1-乙基-(3-二甲基氨基丙基)碳酰二亚胺盐酸盐(342μmol),冰浴条件下加入至茄瓶中,保持4℃反应过夜。反应液以旋转蒸发仪旋干除去溶剂,剩余物重新加入二氯甲烷溶解,转移至100mL分液漏斗,二氯甲烷层水洗三次,旋干得到黄色油状物。阿霉素盐酸盐(111μmol)溶解于N,N-二甲基甲酰胺,加入黄色油状物,室温避光搅拌。反应结束后,反应液以0.1%三氟乙酸水溶液稀释,二氯甲烷萃取,得到的二氯甲烷层旋干。
核磁共振氢谱(图2)及质谱(图3和图4)解析结果:1H NMR(300MHz,DMSO-d6)δ:0.81(m,6H,-C(CH3)2),1.13(d,3H,CH3-C-DOX),1.2-1.8(15H,6CH2-LA,-CH2-CH–Leu),2.0-2.5(4H,2–CH2–DOX),2.6-3.0(10H,–CH2–DOX,2–CH–DOX,3–CH2–(Gly and Phe)),3.98(s,3H,H3CO–DOX),3.9-5.4(8H,–HC–DOX,–CH–Leu,–CH–Phe),5.0-5.5(2H,HO–DOX),7.20(s,5H,–HC–benzene ring in Phe),7.47(d,1H,–HC–benzene ring in DOX),7.64(t,1H,–HC–benzene ring in DOX),7.90(d,1H,–HC–benzene ring in DOX),7.9-8.3(4H,–HC–amidebond),13.26(s,1H,HO–DOX),14.02(s,1H,HO–DOX).m/z=1128.02[M+Na]+,m/z=1144.95[M+K]+。
二、LA-GFLG-DOX偶联物抗肿瘤活性检测及对正常细胞的毒副作用
检测本发明的化合物采用肿瘤细胞增殖实验,采用常用MTT法,具体操作如下:
1.样品配制:将LA-GFLG-DOX偶联物配制成1%浓度的DMSO溶液,以培养基稀释。
2.细胞株:肿瘤细胞MCF-7细胞、正常人支气管内皮细胞16HBE细胞,均由本实验室复苏,冻存及传代。
3.收集对数生长期的细胞,进行细胞消化,离心后去上清,培养基重悬,计数,种于96孔板,每孔加入100μl 5×104个/mL细胞,放入37℃、5%CO2孵箱静置培养。待细胞贴壁后,吸弃培养液,进行给药,在37℃、5%CO2孵箱培养24h,48h,72h后,吸弃给药培养液,PBS洗两次,加入100μl新鲜培养液,加入20μl MTT(5mg/mL)溶液,放入37℃、5%CO2孵箱静置培养4h。吸弃96孔板内液体,每孔加入100μl DMSO,在振荡器摇晃15min。用酶标仪检测570nm处OD值(光密度),记录结果。各组细胞抑制率和生长活力如图5和图6所示。IC50值用origin软件拟合得到,列于表1。
表1.LA-GFLG-DOX偶联物的抗肿瘤活性数据
从表1中可以看出,LA-GFLG-DOX对肿瘤细胞MCF-7在孵育24小时、48小时和72小时后的半数有效抑制率分别为:7.12±1.00,2.77±0.42和0.32±0.02μg/mL。说明LA-GFLG-DOX有抗肿瘤活性,并随孵育时间延长其抗肿瘤效果越好。同时,由图6可看出,LA-GFLG-DOX偶联物对正常人支气管内皮细胞16HBE细胞毒性较小,细胞活力基本在80%以上,无法检测和计算其IC50值,说明LA-GFLG-DOX对正常细胞有较好的生物相容性,毒性较小。这也说明LA-GFLG-DOX有益于降低DOX对正常细胞的毒性。
Claims (10)
1.一种LA-GFLG-DOX偶联物,其特征在于,该偶联物为硫辛酸-甘氨酸-苯丙氨酸-亮氨酸-甘氨酸-阿霉素分子通过酰胺键顺次连接,其结构式如式(Ⅰ)所示:
2.一种LA-GFLG-DOX偶联物的制备方法,其特征在于,包括以下步骤:
(1)通过缩合反应合成全保护的四肽化合物R1-GFLG-R2;
(2)对步骤(1)中所得化合物的氨基端脱除保护基连接硫辛酸,得到化合物LA-GFLG-R2;
(3)对步骤(2)中所得化合物的羧基端脱除保护基并活化连接上阿霉素得到化合物LA-GFLG-DOX;其中,
R1为与四肽化合物GFLG氨基端连接的保护基,R2为与四肽化合物GFLG羧基端连接的保护基。
3.如权利要求2所述的一种LA-GFLG-DOX偶联物的制备方法,其特征在于,R1选自叔丁氧羰基、苄氧羰基、对甲苯磺酰基、三苯甲基、甲酰基中任一种;R2选自甲氧基、苄酯、对硝基苄酯,或形成酰胺、酰肼。
4.如权利要求2所述的一种LA-GFLG-DOX偶联物的制备方法,其特征在于,R1为叔丁氧羰基,R2为甲氧基。
5.如权利要求2所述的一种LA-GFLG-DOX偶联物的制备方法,其特征在于,LA在氮气保护下对其羧基端活化,与GFLG的氨基端连接,得到LA-GFLG,接下来再次对GFLG中甘氨酸的羧基端活化,与DOX连接,得到LA-GFLG-DOX。
6.如权利要求4所述的一种LA-GFLG-DOX偶联物的制备方法,其特征在于,Boc-GFLG-OMe在制备过程中每个氨基酸的氨基端均以叔丁氧羰基保护,并在酸性条件下脱除;羧基端均以甲氧基保护,并在碱性条件下脱除,通过酰胺键将四种氨基酸连接得到Boc-GFLG-OMe。
7.如权利要求4或6所述的一种LA-GFLG-DOX偶联物的制备方法,其特征在于,包括以下步骤:
(1)合成全保护的四肽化合物R1-GFLG-R2;
(2)LA-GFLG-OMe的合成:将Boc-Gly-Phe-Leu-Gly-OMe溶解后加入等体积的三氟乙酸,室温搅拌,反应液旋干;将硫辛酸和N,N,-羰基二咪唑溶解后在氮气保护下搅拌得到羧基端活化的硫辛酸;氮气保护下,将活化的硫辛酸溶液与Gly-Phe-Leu-Gly-OMe混合;反应结束后,萃取得到目标产物;
(3)LA-GFLG-OH的合成:LA-Gly-Phe-Leu-Gly-OMe溶解于甲醇,加入碱性溶液;室温搅拌,调节反应溶液pH至中性,以旋转蒸发仪旋干甲醇,旋干后的反应物溶解于超纯水;乙酸乙酯萃取,收集乙酸乙酯层并旋干;
(4)LA-GFLG-DOX的合成:LA-Gly-Phe-Leu-Gly-OH溶解于四氢呋喃,继续加入N-羟基丁二酰亚胺,1-乙基-(3-二甲基氨基丙基)碳酰二亚胺盐酸盐,冰浴条件下加入至茄瓶中,反应过夜;反应液以旋转蒸发仪旋干除去溶剂,剩余物重新加入二氯甲烷溶解,转移至分液漏斗,二氯甲烷层水洗三次,旋干得到黄色油状物;阿霉素盐酸盐溶解于N,N-二甲基甲酰胺,加入黄色油状物,室温避光搅拌;反应结束后,反应液以三氟乙酸水溶液稀释,二氯甲烷萃取,得到的二氯甲烷层旋干。
8.如权利要求7所述制备方法,其特征在于,步骤(2)中所述硫辛酸和N,N,-羰基二咪唑的摩尔比为1:1-1:1.5;所述在氮气保护下搅拌时间为1.5h-4h;步骤(2)中还包括将活化的硫辛酸溶液与Gly-Phe-Leu-Gly-OMe混合后,以N,N-二异丙基乙胺调节pH值在8-9,室温搅拌过夜;
步骤(4)中LA-Gly-Phe-Leu-Gly-OH与N-羟基丁二酰亚胺、1-乙基-(3-二甲基氨基丙基)碳酰二亚胺盐酸盐的摩尔比为;1:1-1:1.5和1:1-1:1.5;
LA-Gly-Phe-Leu-Gly-OH与阿霉素盐酸盐的摩尔比为1:0.3-1:0.5。
9.如权利要求7或8所述制备方法,其特征在于,步骤(3)中所述碱性溶液选自碱金属的氢氧化物或碳酸盐。
10.如权利要求1所述的一种LA-GFLG-DOX偶联物的抗肿瘤用途。
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