CN111978372B - Rgd序列肽修饰的六环哌嗪二酮,其制备,抗肿瘤活性及应用 - Google Patents
Rgd序列肽修饰的六环哌嗪二酮,其制备,抗肿瘤活性及应用 Download PDFInfo
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- CN111978372B CN111978372B CN201910430304.4A CN201910430304A CN111978372B CN 111978372 B CN111978372 B CN 111978372B CN 201910430304 A CN201910430304 A CN 201910430304A CN 111978372 B CN111978372 B CN 111978372B
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- tetrahydro
- carboline
- beta
- obzl
- piperazine
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Abstract
Description
技术领域
本发明涉及下式的9-(CH2CO-Arg-Gly-Asp-AA)-四氢-β-咔啉[3:4]并哌嗪-2,5-二酮并哌啶 [4:5]并咪唑。涉及它们的制备方法和在制备抗肿瘤药物中的应用。本发明属于生物医药领域。
背景技术
肿瘤已经成为严重威胁人类健康的常见病。例如2015年新增的肿瘤患者大约有392.9万, 其中有233.8万肿瘤患者死亡。平均每天有超过1万人被确诊为肿瘤。目前,临床应用治疗癌症的方法主要有放射疗法,化学疗法,抗体治疗和免疫治疗等。然而由于严重的副作用,药物治疗后产生多药耐药性以及昂贵的治疗价格,使得癌症的治疗再度陷入困境。发明新型的抗肿瘤药物是药物研究的前沿之一。
四氢-β-咔啉-3-羧酸是具有多种生物活性的药效团,环组氨酸也是具有多种生物活性的药效团。在一项关联发明中,发明人发现四氢-β-咔啉-3-羧酸与环组氨酸两个药效团融合形成的下式左的四氢-β-咔啉[3:4]并哌嗪-2,5-二酮并哌啶[4:5]并咪唑是P-选择素抑制剂,具有抗肿瘤生长的作用。在进一步的研究中,发明人认识到在四氢-β-咔啉[3:4]并哌嗪-2,5-二酮并哌啶[4:5] 并咪唑的9位引入CH2CO-Arg-Gly-Asp-AA(式中AA为Ser残基,Phe残基及Val残基)生成的下式右的9-(CH2CO-Arg-Gly-Asp-AA)-四氢-β-咔啉[3:4]并哌嗪-2,5-二酮并哌啶[4:5]并咪唑可具有更强的抗肿瘤作用。根据这种认识,发明人提出了本发明。
发明内容
本发明的第一个内容是提供下式的9-(CH2CO-Arg-Gly-Asp-AA)-四氢-β-咔啉[3:4]并哌嗪 -2,5-二酮并哌啶[4:5]并咪唑(式中AA为Ser残基,Phe残基及Val残基)。
本发明的第二个内容是提供制备9-(CH2CO-Arg-Gly-Asp-AA)-四氢-β-咔啉[3:4]并哌嗪-2,5-二酮并哌啶[4:5]并咪唑的方法,该方法包括:
(1)L-Trp在硫酸催化条件下与甲醛进行Pictet-Spengler反应,制备3S-1,2,3,4-四氢-β-咔啉-3-羧酸(1);
(2)在N,N-二甲基甲酰胺中1与(Boc)2O反应,制备3S-2-叔丁氧羰基-1,2,3,4-四氢-β-咔啉-3-羧酸(2);
(3)L-His在硫酸催化条件下与甲醛进行Pictet-Spengler反应,制备6S-4,5,6,7-四氢-咪唑[4:5]并哌啶-6-羧酸(3);
(4)在甲醇和氯化亚砜中由3制备6S-4,5,6,7-四氢-咪唑[4:5]并哌啶-6-羧酸甲酯(4);
(5)以3-二乙氧基磷酰基-1,2,3-苯唑4(H)-酮作缩合剂,在无水四氢呋喃中2与4偶联制备2-叔丁氧羰基-四氢-β-咔啉-3-甲酰-哌啶[4:5]并咪唑-6-羧酸甲酯(5);
(6)脱除5的叔丁氧羰基保护基,溶于甲醇之后加N-甲基吗啉调pH为9制备四氢-β-咔啉[3:4]并哌嗪-2,5-二酮并哌啶-[4:5]并咪唑(6);
(7)在二氯甲烷中6与三苯基氯甲烷反应,制备1-三苯甲基-四氢-β-咔啉[3:4]并哌嗪-2,5-二酮并哌啶[4:5]并咪唑(7);
(8)在N,N-二甲基甲酰胺中7与溴乙酸苄酯反应,制备1-三苯甲基-9-乙酸苄酯-四氢-β-咔啉- [3:4]并哌嗪-2,5-二酮并哌啶[4:5]并咪唑(8);
(9)8脱苄基制备1-三苯甲基-9-乙酸-四氢-β-咔啉[3:4]并哌嗪-2,5-二酮并哌啶[4:5]并咪唑(9);
(10)在N,N-二甲基甲酰胺中9与Arg(NO2)-Gly-Asp(OBzl)-AA-OBzl偶联,制备1-三苯甲基-9- [CH2CO-Arg(NO2)-Gly-Asp(OBzl)-AA-OBzl]-四氢-β-咔啉[3:4]并哌嗪-2,5-二酮并哌啶[4:5]并咪唑(10a-c,式中AA为Ser残基,Phe残基及Val残基);
(11)10a-c脱保护制备9-(CH2CO-Arg-Gly-Asp-AA)-四氢-β-咔啉[3:4]并哌嗪-2,5-二酮并哌啶 [4:5]并咪唑(11a-c,式中AA为Ser残基,Phe残基及Val残基)。
本发明的第三个内容是评价9-(CH2CO-Arg-Gly-Asp-AA)-四氢-β-咔啉[3:4]并哌嗪-2,5-二酮并哌啶[4:5]并咪唑(式中AA为Ser残基,Phe残基及Val残基)对S180小鼠肿瘤生长的抑制作用。
附图说明
图1 9-(CH2CO-Arg-Gly-Asp-AA)-四氢-β-咔啉[3:4]并哌嗪-2,5-二酮并哌啶[4:5]并咪唑(式中AA为Ser残基,Phe残基及Val残基)的合成路线。i)甲醛(37%),浓硫酸(98%),水,氨水溶液 (25%);ii)N,N-二甲基甲酰胺,(Boc)2O,三乙胺;iii)甲醇,氯化亚砜;iv)四氢呋喃,3-二乙氧基磷酰基-1,2,3-苯唑4(3H)-酮,三乙胺;v)氯化氢的乙酸乙酯溶液(4M),甲醇,N-甲基吗啉;vi) 二氯甲烷,三苯基氯甲烷;vii)N,N-二甲基甲酰胺,氢化钠(60%),溴乙酸苄酯;viii)甲醇,钯碳,氢气;ix)N,N-二甲基甲酰胺,四氢呋喃,1-羟基苯并三唑,二环己基碳二亚胺,N-甲基吗啉; x)甲醇,氢氧化钠水溶液(2M);xi)氯化氢的乙酸乙酯溶液(4M);xii)三氟醋酸,三氟甲磺酸。
具体实施方式
为了进一步阐述本发明,下面给出一系列实施例。这些实施例完全是例证性的,它们仅用来对本发明进行具体描述,不应当理解为对本发明的限制。
实施例1制备Boc-Arg(NO2)-Gly-OBzl
0℃及搅拌下将1.595g(5.0mmol)Boc-L-Arg(NO2)溶于20mL四氢呋喃,然后加0.675g (5.0mmol)1-羟基苯并三唑和1.236g(6.0mmol)二环己基碳二亚胺,反应0.5h。之后加1.854 g(5.5mmol)Tos·Gly-OBzl,最后反应液用N-甲基吗啉调pH为8。得到的溶液室温搅拌6h至 TLC显示Boc-L-Arg(NO2)完全消失。反应混合物过滤,滤液减压浓缩,得到的淡黄色油状物用60mL乙酸乙酯溶解,得到的溶液依次用饱和碳酸氢钠水溶液洗(20mL×3),饱和氯化钠水溶液洗(20mL×3),5%硫酸氢钾水溶液洗(20mL×3),饱和氯化钠水溶液洗(20mL×3),饱和碳酸氢钠水溶液洗(20mL×3),饱和氯化钠水溶液洗(20mL×3),乙酸乙酯层用无水硫酸钠干燥 12h,过滤,滤液减压浓缩,得到2.120g(90%)标题化合物,为无色粉末,ESI-MS(m/e):467 [M+H]+。
实施例2制备Boc-Arg(NO2)-Gly
0℃及搅拌下将2.120g(5.0mmol)Boc-Arg(NO2)-Gly-OBzl用15mL甲醇溶解,加氢氧化钠水溶液(2M)调pH为11,搅拌至TLC显示Boc-Arg(NO2)-Gly-OBzl完全消失。反应溶液先用饱和硫酸氢钾水溶液调pH为中性,减压浓缩,再用饱和硫酸氢钾水溶液调pH为2,用乙酸乙酯萃取(50mL×3),合并乙酸乙酯层并用饱和氯化钠水溶液洗(30mL×3)。乙酸乙酯层用无水硫酸钠干燥12h,过滤,滤液减压浓缩得到1.542g(82%)标题化合物,为无色油状产物。 ESI-MS(m/e):375[M-H]-。
实施例3制备Boc-Asp(OBzl)-Ser-OBzl
用实施例1的方法从1.615g(5.0mmol)Boc-L-Asp(OBzl)和1.271g(5.5mmol)L-Ser-OBzl 得到2.520g(92%)标题化合物,为无色油状产物。ESI-MS(m/e):501[M+H]+。
实施例4制备Asp(OBzl)-Ser-OBzl
0℃及搅拌下将2.520g(5.0mmol)Boc-Asp(OBzl)-Ser-OBzl用30mL氯化氢的乙酸乙酯溶液(4M)溶解,搅拌4h,TLC显示Boc-Asp(OBzl)-Ser-OBzl完全消失。反应混合物减压浓缩, 残留物用20mL无水乙酸乙酯溶液溶解。得到的溶液再减压浓缩。该操作重复3次。得到的固体用无水乙醚洗(10mL×3),得到1.930g(88%)标题化合物,为淡黄色油状物。ESI-MS(m/e): 401[M+H]+。
实施例5制备Boc-Arg(NO2)-Gly-Asp(OBzl)-Ser-OBzl
用实施例1的方法从1.542g(5.2mmol)Boc-Arg(NO2)-Gly和1.930g(5.0mmol) Asp(OBzl)-Ser-OBzl得到2.450g(69%)标题化合物,为无色粉末。ESI-MS(m/e):757[M-H]-。
实施例6制备Arg(NO2)-Gly-Asp(OBzl)-Ser-OBzl
用实施例4的方法从2.450g(3.2mmol)Boc-Arg(NO2)-Gly-Asp(OBzl)-Ser-OBzl得到1.820 g(85%)标题化合物,为无色粉末。ESI-MS(m/e):659[M+H]+。
实施例7制备Boc-Asp(OBzl)-Phe-OBzl
用实施例1的方法从1.615g(5.0mmol)Boc-L-Asp(OBzl)和1.604g(5.5mmol)L-Phe-OBzl 得到1.993g(69%)标题化合物,为无色油状产物。ESI-MS(m/e):561[M+H]+。
实施例8制备Asp(OBzl)-Phe-OBzl
用实施例4的方法从2.850g(5.0mmol)Boc-Asp(OBzl)-Phe-OBzl得到2.230g(88%)标题化合物,为无色粉末。ESI-MS(m/e):461[M+H]+。
实施例9制备Boc-Arg(NO2)-Gly-Asp(OBzl)-Phe-OBzl
用实施例1的方法从1.542g(5.2mmol)Boc-Arg(NO2)-Gly和2.340g(5.0mmol) Asp(OBzl)-Phe-OBzl得到2.924g(74%)标题化合物,为无色粉末。ESI-MS(m/e):819[M+H]+。
实施例10制备Arg(NO2)-Gly-Asp(OBzl)-Phe-OBzl
用实施例4的方法从2.924g(3.6mmol)Boc-Arg(NO2)-Gly-Asp(OBzl)-Phe-OBzl得到2.362 g(92%)标题化合物,为无色粉末。ESI-MS(m/e):719[M+H]+。
实施例11制备Boc-Asp(OBzl)-Val-OBzl
用实施例1的方法从1.615g(5.0mmol)Boc-L-Asp(OBzl)和1.342g(5.5mmol)L-Val-OBzl 得到1.669g(65%)标题化合物,为无色油状产物。ESI-MS(m/e):513[M+H]+。
实施例12制备Asp(OBzl)-Val-OBzl
用实施例4的方法从2.560g(5.0mmol)Boc-Asp(OBzl)-Val-OBzl得到1.978g(96%)标题化合物,为无色粉末。ESI-MS(m/e):413[M+H]+。
实施例13制备Boc-Arg(NO2)-Gly-Asp(OBzl)-Val-OBzl
用实施例1的方法从1.542g(5.2mmol)Boc-Arg(NO2)-Gly和2.060g(5.0mmol) Asp(OBzl)-Val-OBzl得到1.940g(62%)标题化合物,为无色粉末。ESI-MS(m/e):771[M+H]+。
实施例14制备Arg(NO2)-Gly-Asp(OBzl)-Val-OBzl
用实施例4的方法从1.940g(2.5mmol)Boc-Arg(NO2)-Gly-Asp(OBzl)-Val-OBzl得到1.589 g(95%)标题化合物,为无色粉末。ESI-MS(m/e):671[M+H]+。
实施例15制备3S-1,2,3,4-四氢-β-咔啉-3-羧酸(1)
0℃及搅拌下向200mL蒸馏水中加入0.1mL浓硫酸(98%),之后加入2.04g(10.0mmol) L-Trp并搅拌至固体溶解,最后加入5mL甲醛水溶液(37%)并室温搅拌6h。TLC显示L-Trp 完全消失。0℃及搅拌下向反应混合物中加入氨水溶液(25%)调pH为7。反应混合物室温静置30min,过滤收集生成的沉淀,得到2.03g(93%)标题化合物,为淡黄色固体。ESI-MS(m/e): 217[M+H]+;1H NMR(300MHz,DMSO-d6):δ/ppm=12.141(s,1H),10.944(s,1H),7.436(s,1 H),7.321(s,1H),7.032(d,2H),4.207(m,3H),2.822(m,2H)。
实施例16制备3S-2-叔丁氧羰基-1,2,3,4-四氢-β-咔啉-3-羧酸(2)
向0.972g(4.5mmol)3S-1,2,3,4-四氢-β-咔啉-3-羧酸(1)中加入10mL N,N-二甲基甲酰胺, 搅拌使固体悬浮。0℃及搅拌下先往该悬浮液中加入1.275g(1.3mmol)(Boc)2O,再加入三乙胺调pH为10。得到的溶液室温搅拌至TLC显示化合物1完全消失。反应混合物减压浓缩,得到的淡黄色油状物用40mL乙酸乙酯溶解,得到的乙酸乙酯溶液先用5%硫酸氢钾水溶液洗 (50mL×3)再用饱和氯化钠水溶液洗(50mL×3),乙酸乙酯层用无水硫酸钠干燥12h。过滤,滤液减压浓缩,得到的淡黄色固体在15mL二氯甲烷中超声,使固体均匀分散。过滤,得到1.106 g(77%)标题化合物,为无色固体。ESI-MS(m/e):315[M-H]-;1H NMR(300MHz,DMSO-d6): δ/ppm=12.769(s,1H),10.868(d,1H),7.416(d,J=7.5Hz,1H),7.283(m,1H),7.048(t,J= 7.2Hz,1H),6.966(t,J=7.2Hz,1H),5.101(m,1H),4.716(t,J=12.9Hz,1H),4.394(m,1H), 3.297(d,J=16.2Hz,1H),2.959(m,1H),1.461(d,J=9.9Hz,9H)。
实施例17制备6S-4,5,6,7-四氢-咪唑[4:5]并哌啶-6-羧酸(3)
0℃及搅拌下向2.50g(16.1mmol)L-His与10mL蒸馏水的溶液中加入0.4mL浓硫酸(98%)使L-His逐渐溶解。向得到的溶液中加3mL甲醛水溶液(37%)并于60℃及加热6h,TLC显示L-His完全消失。反应混合物于0℃搅拌,之后加25%氨水溶液调pH为7。室温静置30min,过滤。收集的固体用水及丙酮各洗三次,得到2.649g(98%)标题化合物,为无色固体。ESI-MS(m/e):168[M+H]+;1H NMR(300MHz,DMSO-d6):δ/ppm=12.016(s,1H),7.548(s,1 H),5.040(d,J=13.8Hz,1H),4.789(d,J=14.1Hz,1H),4.277(m,1H),3.761(m,2H)。
实施例18制备6S-4,5,6,7-四氢-咪唑[4:5]并哌啶-6-羧酸甲酯(4)
0℃及搅拌下向120mL甲醇中滴加8mL氯化亚砜并搅拌40min。之后,加入5.01g(30mmol)6S-4,5,6,7-四氢-咪唑[4:5]并哌啶-6-羧酸(3)。反应混合物室温搅拌至TLC显示化合物3 完全消失。反应混合物减压浓缩,残留物用50mL甲醇溶解。溶液减压浓缩,残留物再用50mL 甲醇溶解。该操作重复3次。固体用无水乙醚洗(30mL×3),得到7.129g(93%)标题化合物, 为无色粉末。ESI-MS(m/e):182[M+H]+;1H NMR(300MHz,DMSO-d6):δ/ppm=12.667(s,1 H),9.051(s,1H),4.710(m,1H),4.333(m,2H),3.819(s,3H),3.302(m,1H),3.154(m,1H)。
实施例19制备2-叔丁氧羰基-四氢-β-咔啉-3-甲酰哌啶[4:5]并咪唑-6-羧酸甲酯(5)
将2.212g(7.0mmol)3S-2-叔丁氧羰基-1,2,3,4-四氢-β-咔啉-3-羧酸(2),2.134g(8.4mmol) 6S-4,5,6,7-四氢-咪唑[4:5]并哌啶-6-羧酸甲酯(4)以及2.512g(8.4mmol)3-二乙氧基磷酰基 -1,2,3-苯唑4(3H)-酮用30mL四氢呋喃溶解。0℃与搅拌下向溶液中加3mL(21.0mmol)三乙胺,室温搅拌至TLC显示化合物4完全消失。滤去不溶物,滤液减压浓缩。得到的棕黄色糖浆状物用80mL二氯甲烷溶解,溶液用10%的碳酸钠水溶液洗(50mL×3)及饱和氯化钠水溶液洗(50mL×3)。二氯甲烷层用无水硫酸钠干燥12h,过滤。滤液减压浓缩,得到3.126g棕黄色固体。该固体经柱层析纯化,得到1.633g(52%)标题化合物,为无色粉末。ESI-MS(m/e): 502[M+Na];1H NMR(300MHz,DMSO-d6):δ/ppm=11.917(s,1H),10.823(s,1H),7.570(s,1 H),7.436(m,1H),7.277(m,1H),7.068-6.911(m,2H),5.682-5.397(m,2H),4.896-4.435(m,4 H),3.966(m,1H),3.633(d,J=5.7Hz,1H),3.477(s,1H),3.396(s,1H),3.333(s,4H), 3.186-2.813(m,5H),1.453-1.290(d,9H);13C NMR(125MHz,DMSO-d6):δ/ppm=171.27, 171.08,154.94,136.39,135.69,135.40,131.09,126.91,121.18,118.85,117.91,111.37,104.55, 103.23,80.36,55.38,52.80,51.08,50.42,50.24,28.50,28.44,28.35,28.19,21.80。
实施例20制备四氢-β-咔啉[3:4]并哌嗪-2,5-二酮并哌啶[4:5]并咪唑(6)
0℃及搅拌下将1.633g(3.4mmol)2-叔丁氧羰基-四氢-β-咔啉-3-甲酰哌啶[4:5]并咪唑-6- 羧酸甲酯(5)用25mL氯化氢的乙酸乙酯溶液(4M)溶解。得到的溶液在0℃搅拌4h,TLC显示化合物5完全消失。将反应混合物减压浓缩,残留物用20mL干燥的乙酸乙酯溶液溶解。得到的溶液再减压浓缩。该操作重复3次。得到的固体用无水乙醚洗(30mL×3),得到1.244g (95%)四氢-β-咔啉-3-甲酰哌啶[4:5]并咪唑-6-羧酸甲酯,为棕黄色粉末。ESI-MS(m/e):380 [M+H]+。
0℃及搅拌下将棕黄色四氢-β-咔啉-3-甲酰哌啶[4:5]并咪唑-6-羧酸甲酯粉末先用20mL 甲醇溶解,再加N-甲基吗啉调pH为9,室温搅拌至TLC显示棕黄色粉末完全消失。反应混合物减压浓缩,得到的深棕色糖浆状物通过硅胶柱层析纯化,得到0.854g(72%)标题化合物, 为淡黄色粉末。FT-ESI-MS(m/e):348.1447[M+H]+;Mp:210-211℃;1H NMR(300MHz,DMSO-d6):δ/ppm=12.003(s,1H),11.017(s,1H),7.565(s,1H),7.414(d,J=7.8Hz,1H),7.345(d,J=8.1Hz,1H),7.072(t,J=7.5Hz,1H),6.978(t,J=7.2Hz,1H),5.427(d,J=16.5 Hz,1H),5.200(d,J=15.6Hz,1H),4.493-4.455(m,2H),4.268(d,J=16.5Hz,1H),4.063(m, 1H),3.251(m,1H),3.075(m,1H),2.816(t,J=12.6Hz,2H);13C NMR(125MHz,DMSO-d6): δ/ppm=164.88,164.52,136.42,135.25,130.18,126.76,121.61,119.24,118.15,111.61,106.10, 56.41,56.20,28.98,28.01。
实施例21制备1-三苯甲基-四氢-β-咔啉[3:4]并哌嗪-2,5-二酮并哌啶[4:5]并咪唑(7)
0℃及搅拌下将0.500g(1.4mmol)四氢-β-咔啉[3:4]并哌嗪-2,5-二酮并哌啶[4:5]并咪唑(6) 用15mL二氯甲烷溶解。之后,先加300μL(2.1mmol)三乙胺,再加0.400g(1.4mmol)三苯基氯甲烷。得到的溶液室温搅拌15h,TLC显示化合物6完全消失。加50mL冰水猝灭反应,水层用二氯甲烷萃取(20mL×3)。合并的二氯甲烷层用饱和氯化钠水溶液洗(20mL×3),用无水硫酸钠干燥12h,过滤,滤液减压浓缩,得到的棕黄色糖浆状物质经硅胶柱层析纯化,得到 0.297g(35%)标题化合物,为无色粉末。ESI-MS(m/e):612[M+Na];1HNMR(300MHz, DMSO-d6):δ/ppm=10.968(s,1H),7.442(s,10H),7.329(d,J=7.9Hz,1H),7.247(s,1H), 7.133-6.952(m,8H),5.373(d,J=16.5Hz,1H),4.394(d,J=16.0Hz,1H),4.309-4.158(m,3H), 3.193(d,J=14.8Hz,1H),3.013(d,J=14.3Hz,1H),2.755(m,3H);13CNMR(125MHz, DMSO-d6):δ/ppm=164.85,164.08,141.47,138.10,136.35,135.01,130.15,129.90,128.91, 128.80,128.59,126.68,124.60,121.67,119.32,118.08,111.65,105.56,74.82,66.51,56.16,55.49, 46.52,41.11,30.43,27.98。
实施例22制备1-三苯甲基-9-乙酸苄酯-四氢-β-咔啉[3:4]并哌嗪-2,5-二酮并哌啶[4:5]并咪唑 (8)
0℃及搅拌下将0.747g(1.3mmol)1-三苯甲基-四氢-β-咔啉[3:4]并哌嗪-2,5-二酮并哌啶 [4:5]并咪唑(7)用5mL N,N-二甲基甲酰胺溶解。之后,加入0.101g(2.6mmol)氢化钠(60%)并搅拌5min。之后,加300μL(2.0mmol)溴乙酸苄酯。反应混合物室温搅拌5h,TLC显示化合物7完全消失。加50mL冰水猝灭反应,水层用乙酸乙酯萃取(30mL×3)。合并的乙酸乙酯层用饱和氯化钠水溶液洗(20mL×3),用无水硫酸钠干燥12h。过滤,滤液减压浓缩,得到的淡黄色油状物经硅胶柱层析纯化,得到0.386g(41%)标题化合物。为无色粉末。ESI-MS(m/e):760 [M+Na];1H NMR(300MHz,DMSO-d6):δ/ppm=7.498-7.365(m,16H),7.253(s,1H),7.139-7.027(m,8H),5.443(d,J=16.7Hz,1H),5.188(s,4H),4.384(d,J=15.8Hz,1H),4.232 (m,2H),4.129(d,J=16.4Hz,1H),2.232(d,J=14.9Hz,1H),3.028(d,J=14.3Hz,1H), 2.893-2.694(m,4H);13C NMR(125MHz,DMSO-d6):δ/ppm=170.81,169.37,164.34,164.32, 141.35,137.87,137.28,136.10,135.40,131.40,130.01,128.92,128.90,128.75,128.62,128.38, 126.58,123.93,121.94,119.97,118.50,109.94,106.93,75.02,66.89,60.23,56.28,56.07,44.93, 39.17,31.23,27.80,21.24,14.56。
实施例23制备1-三苯甲基-9-乙酸-四氢-β-咔啉[3:4]并哌嗪-2,5-二酮并哌啶[4:5]并咪唑(9)
将0.491g(0.6mmol)1-三苯甲基-9-乙酸苄酯-四氢-β-咔啉[3:4]并哌嗪-2,5-二酮并哌啶[4:5] 并咪唑(8)用20mL甲醇溶解。之后,加入5mg Pd/C,用水泵抽气3min除瓶内空气,通氢气。该操作重复三次。反应混合物室温通氢气6h至TLC显示化合物8完全消失。反应混合物滤除Pd/C,滤液减压浓缩,得到0.406g(94%)标题化合物,为无色粉末。ESI-MS(m/e):646 [M-H]-;1H NMR(300MHz,DMSO-d6):δ/ppm=12.901(s,1H),7.459-7.012(m,20H),5.402 (m,2H),4.982(s,2H),4.386(d,J=16.2Hz,1H),4.224(m,2H),4.138(d,J=17.1Hz,1H), 3.225(d,J=16.2Hz,1H),3.028(d,J=13.5Hz,1H),2.767(m,2H);13C NMR(125MHz,DMSO-d6):δ/ppm=170.83,164.78,164.32,144.25,141.31,137.84,137.25,135.29,131.38, 130.01,129.51,128.93,128.81,128.76,128.74,128.35,126.71,126.47,123.97,121.83,119.78, 118.41,109.82,106.55,75.07,56.37,56.29,56.24,56.04,55.91,44.94,39.14,31.15,27.80。
实施例24制备1-三苯甲基-9-[CH2CO-Arg(NO2)-Gly-Asp(OBzl)-Ser-OBzl]-四氢-β-咔啉[3:4] 并哌嗪-2,5-二酮并哌啶[4:5]并咪唑(10a)
用实施例1的方法从0.587g(0.8mmol)1-三苯甲基-9-乙酸-四氢-β-咔啉[3:4]并哌嗪-2,5- 二酮并哌啶[4:5]并咪唑(9)及0.629g(0.9mmol)Arg(NO2)-Gly-Asp(OBzl)-Ser-OBzl得到0.528g (45%)标题化合物,为淡黄色粉末。ESI-MS(m/e):1310[M+Na]+;1HNMR(300MHz,DMSO-d6): δ/ppm=8.573(d,J=7.5Hz,2H),8.350(s,1H),8.272(d,J=7.5Hz,2H),7.900(s,2H), 7.444-7.341(m,24H),7.252(s,1H),7.136-6.988(m,9H),5.499(d,J=15.0Hz,1H),5.094(m, 5H),4.816(m,3H),4.381(m,3H),4.174(m,3H),3.724(m,4H),3.122(m,5H),2.755(m,5 H),2.573(d,J=8.7Hz,1H),1.724(s,1H),1.526(m,4H);13CNMR(125MHz,DMSO-d6): δ/ppm=175.87,174.43,172.02,171.94,171.01,170.62,170.30,169.68,169.09,168.06,167.97, 167.38,164.37,164.30,142.98,141.33,137.87,137.25,136.48,136.36,136.05,135.38,131.70, 130.00,128.92,128.86,128.83,128.76,128.50,128.43,128.41,128.35,128.09,128.01,127.08, 126.88,126.44,123.95,121.75,119.70,118.36,109.84,106.28,106.25,75.02,66.78,66.42,66.16,63.35,61.54,58.20,56.31,56.06,55.49,55.01,52.70,50.88,49.51,48.58,46.14,42.29,42.14, 36.85,35.06,32.76,31.21,29.94,27.86,25.11。
实施例25制备1-三苯甲基-9-[CH2CO-Arg(NO2)-Gly-Asp(OBzl)-Phe-OBzl]-四氢-β-咔啉[3:4] 并哌嗪-2,5-二酮并哌啶[4:5]并咪唑(10b)
用实施例1的方法从0.587g(0.8mmol)1-三苯甲基-9-乙酸-四氢-β-咔啉[3:4]并哌嗪-2,5- 二酮并哌啶[4:5]并咪唑(9)及0.693g(0.9mmol)Arg(NO2)-Gly-Asp(OBzl)-Phe-OBzl得到0.493 g(40%)标题化合物,为淡黄色粉末。ESI-MS(m/e):1370[M+Na];1HNMR(300MHz, DMSO-d6):δ/ppm=8.506(d,J=7.5Hz,1H),8.413(d,J=7.2Hz,1H),8.250(m,2H),7.856(s, 2H),7.500(d,J=7.5Hz,1H),7.423-6.975(m,42H),5.337(d,J=16.8Hz,1H),5.167(d,J= 15.6Hz,1H),5.055(m,5H),4.791-4.711(m,3H),4.485(m,1H),4.314(m,3H),4.056(d,J= 13.8Hz,2H),3.711(m,2H),3.218(d,J=14.1Hz,1H),3.112(m,2H),2.993(m,2H),2.683(m, 1H),2.384(m,2H),2.201(d,J=13.8Hz,1H),1.705(s,1H),1.705(s,1H),1.427(m,4H);13C NMR(125MHz,DMSO-d6):δ/ppm=171.88,171.47,170.86,170.21,168.96,167.83,164.69, 163.93,141.47,138.16,137.32,137.20,136.46,136.10,134.91,131.78,129.89,129.57,128.86, 128.82,128.77,128.62,128.50,128.46,128.37,128.33,127.08,126.34,124.64,121.84,119.76, 118.25,109.89,105.78,74.83,66.54,66.19,56.19,55.67,55.55,54.37,52.59,49.43,46.09,42.19,41.15,40.64,40.57,40.41,40.24,39.26,36.95,36.78,30.51,30.05,27.96.
实施例26制备1-三苯甲基-9-[CH2CO-Arg(NO2)-Gly-Asp(OBzl)-Val-OBzl]-四氢-β-咔啉[3:4] 并哌嗪-2,5-二酮并哌啶[4:5]并咪唑(10c)
用实施例1的方法从0.587g(0.8mmol)1-三苯甲基-9-乙酸-四氢-β-咔啉[3:4]并哌嗪-2,5- 二酮并哌啶[4:5]并咪唑(9)及0.650g(0.9mmol)Arg(NO2)-Gly-Asp(OBzl)-Val-OBzl得到0.564g (47%)标题化合物,为淡黄色粉末。ESI-MS(m/e):1322[M+Na];1HNMR(300MHz,DMSO-d6): δ/ppm=8.526(d,J=7.5Hz,1H),8.285(m,2H),8.179(d,J=7.8Hz,1H),7.871(s,1H),7.496 (d,J=7.5Hz,1H),7.369(m,20H),7.240(s,1H),7.094(m,8H),5.335(d,J=16.5Hz,1H), 5.111(m,5H),4.763(m,3H),4,315(m,3H),4.180(m,1H),4.061(m,2H),3.720(m,2H), 3.385(d,J=6.9Hz,1H),3.180(m,3H),2.703(m,1H),2.378(t,J=13.5Hz,1H),2.195(d,J=15.0Hz,1H),2.064(m,1H),1.704(m,1H),1.426(m,4H),0.842(d,J=6.3Hz,6H);13C NMR (125MHz,DMSO-d6):δ/ppm=171.93,171.88,171.53,171.09,170.24,169.18,169.16,167.97, 167.88,164.71,164.38,164.29,163.93,141.45,141.32,138.16,137.88,137.25,137.20,136.46, 136.26,135.35,134.88,131.76,131.70,129.99,129.89,128.92,128.87,128.83,128.77,128.63, 128.57,128.51,128.45,128.37,128.23,127.99,126.45,126.33,124.64,123.96,121.84,121.75,119.70,118.35,109.83,106.25,105.77,75.03,74.84,66.42,66.18,60.24,58.12,56.31,56.19, 56.06,55.54,52.71,49.54,46.12,42.20,40.64,36.65,31.20,30.24,29.95,27.86,19.36,18.57.
实施例27制备9-(CH2CO-Arg-Gly-Asp-Ser)-四氢-β-咔啉[3:4]并哌嗪-2,5-二酮并哌啶[4:5]并咪唑(11a)
0℃及搅拌下将0.100g(0.07mmol)1-三苯甲基-9-[CH2CO-Arg(NO2)-Gly-Asp(OBzl)-Ser- OBzl]-四氢-β-咔啉[3:4]并哌嗪-2,5-二酮并哌啶[4:5]并咪唑(10a)与1mL三氟乙酸和0.3mL三氟甲磺酸混合,搅拌0.5h至TLC显示10a完全消失。反应混合物在0℃及搅拌下用水泵抽气5min,加25mL冰冷的乙醚,溶液3000rpm离心5min,弃上清,再加25mL冰冷的乙醚, 溶液3000rpm离心5min,弃上清,得到的墨绿色固体经C18纯化,得到15mg(22%)标题化合物,为黄色粉末。FT-ESI-MS(m/e):819.3267[M-H]-;Mp:172-173℃;1H NMR(300MHz,DMSO-d6):δ/ppm=12.008(s,1H),10.006(s,1H),8.622(m,3H),7.564(s,1H),7.391(m,3H), 7.129(m,1H),7.031(m,2H),5.568(d,J=16.2Hz,1H),5.230(d,J=15.3Hz,1H),4.941(m,2 H),4.439(m,3H),4.263(m,2H),4.014(m,2H),3.877(m,1H),3.608(m,3H),3.454-3.056(m, 7H),2.812(m,2H),1.976(s,1H),1.624(m,3H);13C NMR(125MHz,DMSO-d6):δ/ppm= 174.72,173.29,172.68,171.00,169.06,167.97,167.25,164.73,164.45,157.72,141.33,140.36, 137.24,135.93,135.26,131.86,126.45,121.76,119.67,118.32,109.83,109.19,106.22,62.53, 56.43,56.20,55.71,52.90,50.19,46.11,42.99,40.87,37.53,30.44,27.95,25.03;IR(cm-1): 3247.09,3059.03,1647.83,1515.80,1463.31,1384.59,1326.16,1245.76,740.73,673.29。
实施例28制备9-(CH2CO-Arg-Gly-Asp-Phe)-四氢-β-咔啉[3:4]并哌嗪-2,5-二酮并哌啶[4:5]并咪唑(11b)
用实施例27的方法从0.100g(0.07mmol)1-三苯甲基-9-[CH2CO-Arg(NO2)- Gly-Asp(OBzl)-Phe-OBzl]-四氢-β-咔啉[3:4]并哌嗪-2,5-二酮并哌啶[4:5]并咪唑(10b)得到14mg (21%)标题化合物,为黄色粉末。FT-ESI-MS(m/e):879.3617[M-H]-;Mp:146-148℃;1HNMR (300MHz,DMSO-d6):δ/ppm=12.053(s,1H),10.162(s,1H),8.822(d,J=7.2Hz,1H),8.744 (d,J=6.6Hz,1H),8.466(d,J=12.9Hz,1H),7.566(s,1H),7.402(m,3H),7.165(m,6H), 7.009(m,3H),5.561(d,J=16.8Hz,1H),5.225(d,J=15.0Hz,1H),5.035(m,1H),4.891(m,2 H),4.413(m,4H),4.222(m,2H),4.018(d,J=15.6Hz,1H),3.908(m,1H),3.605(m,2H), 2.918(m,9H),1.905(s,1H),1.558(m,3H);13C NMR(125MHz,DMSO-d6):δ/ppm=173.73, 170.55,169.07,167.90,167.17,164.73,164.56,164.45,157.77,141.33,140.37,138.72,137.24, 135.94,135.28,131.88,129.86,128.69,128.32,126.37,121.75,119.68,109.19,106.22,56.43, 56.14,55.05,52.74,50.72,46.12,42.88,40.82,40.65,40.58,39.32,39.14,37.89,37.21,30.38, 27.94,24.63;IR(cm-1):3294.86,3053.21,1644.55,1514.94,1455.36,1381.50,1321.48,1241.62, 1186.08,740.73,700.67。
实施例29制备9-(CH2CO-Arg-Gly-Asp-Val)-四氢-β-咔啉[3:4]并哌嗪-2,5-二酮并哌啶[4:5]并咪唑(11c)
用实施例27的方法从0.100g(0.07mmol)1-三苯甲基-9-[CH2CO-Arg(NO2)- Gly-Asp(OBzl)-Val-OBzl]-四氢-β-咔啉[3:4]并哌嗪-2,5-二酮并哌啶[4:5]并咪唑(10c)得到16mg (25%)标题化合物,为黄色粉末。FT-ESI-MS(m/e):831.3595[M-H]-;Mp:176-177℃;1HNMR (300MHz,DMSO-d6):δ/ppm=12.094(s,1H),10.325(s,1H),9.021(d,J=6.3Hz,1H),8.738 (s,1H),8.638(m,1H),7.570(s,1H),7.427(m,1H),7.050(m,4H),5.552(d,J=14.7Hz,1H), 5.229(d,J=14.7Hz,1H),4.971(m,2H),4.432(m,4H),4.241(m,1H),4.022(m,3H),3.532 (d,J=19.2Hz,1H),2.856(m,6H),2.308(d,J=16.8Hz,1H),2.033(m,2H),1.621(m,3H), 0.842(d,J=6.6Hz,6H);13C NMR(125MHz,DMSO-d6):δ/ppm=174.02,172.80,170.54, 168.86,167.99,167.35,164.72,164.57,164.46,164.42,157.86,141.28,140.41,137.27,135.91, 135.26,131.84,126.40,125.74,121.73,119.63,118.26,117.17,115.53,110.63,109.94,109.17, 106.18,56.45,56.14,52.64,50.68,46.47,46.05,42.82,37.92,31.43,30.65,27.94,27.55,24.99, 19.76,19.72,18.53,18.43;IR(cm-1):3249.90,2963.30,1651.33,1515.31,1463.21,1385.72, 1326.66,1245.34,1187.49,740.73,667.67。
实施例30评价化合物11a-c的抗肿瘤生长活性
1)体重20±2g的清洁级雄性ICR小鼠,购自北京维通利华动物实验技术有限公司。
2)阳性对照为阿霉素及化合物6,各12只小鼠;空白对照为生理盐水,12只小鼠;化合物 11a-c,12只小鼠。
3)阿霉素腹腔注射,剂量为2μmol/kg/天,连续给药10天;化合物6口服,剂量为1μmol/kg/ 天,连续给药10天;生理盐水口服,剂量为0.1mL/10g,连续给药10天;化合物11a-c口服, 剂量为0.1μmol/kg/天,连续给药10天。
4)采用移植性S180小鼠肉瘤模型进行评价,瘤源为小鼠S180肉瘤细胞,传自北京大学医学部动物实验中心。无菌条件下取出生长旺盛的小鼠S180腹水瘤液,1000r/min离心3min,弃上清,残余细胞用生理盐水洗,再离心,弃上清。残余细胞用生理盐水吹打使悬浮均匀,取出少量细胞悬液用台盼蓝染色(0.2%),可染色的死细胞计数,确保活细胞数>90%。用生理盐水稀释细胞悬液,制备密度为1~2×107个/mL的细胞悬液接种于小鼠右侧腋下,每只小鼠接种0.2 mL。(4大方格内活细胞数/4)×104×稀释倍数,细胞存活率=[活细胞数/(活细胞数+死细胞数)]×100%。
每日对密度为1~2×107个/mL的细胞悬液接种小鼠观察,7天后小鼠右侧腋下长出绿豆大小的实体瘤,按照肿瘤体积将小鼠随机分组,使各组小鼠的肿瘤体积均匀分布。之后,各组小鼠连续给药10天,第十一天记录各组小鼠的体重。之后麻醉,采血供测定P-选择素用。小鼠颈椎脱臼处死,钝性分离小鼠腋下肿瘤,统计各组小鼠的肿瘤重量,经t检验,p<0.05即有统计学差异。结果列入表1。可以看到,在0.1μmol/kg/天剂量下化合物11a-c治疗小鼠的瘤重显著小于生理盐水治疗小鼠的瘤重。此外,在0.1μmol/kg/天剂量下化合物11a-c治疗小鼠的瘤重与1μmol/kg/天剂量下化合物6治疗小鼠的瘤重没有显著性差异。在口服剂量只有阿霉素腹腔注射剂量1/20时,化合物11b的抗肿瘤活性仍然与阿霉素同样优秀,说明本发明具有突出的技术效果。
表1化合物11a-c对S180荷瘤小鼠肿瘤生长的影响
a)与生理盐水比p<0.01;b)与生理盐水比p<0.01,与化合物6比p>0.05;c)与生理盐水比 p<0.01,与化合物6及阿霉素比p>0.05;n=12.
实施例31评价化合物11a-c对S180荷瘤小鼠血清中P-选择素含量水平的影响
实施例30采集的S180小鼠的全血经1000rpm离心10min,收集血清,按照试剂盒描述的操作用小鼠P-选择素酶联免疫实验测定血清中的P-选择素含量。酶标包被板设P-选择素标准品孔,生理盐水治疗的小鼠血清孔,阿霉素治疗的小鼠血清孔和化合物6及11a-c治疗的小鼠血清孔。标准品孔中加50μL不同浓度的P-选择素标准品(用来自试剂盒的标准品稀释制备)。在生理盐水治疗的小鼠血清孔,阿霉素治疗的小鼠血清孔和化合物6及11a-c治疗的小鼠血清孔中先加40μL样品稀释液,再加10μL血清(最终稀释度为5倍)。各孔再加100μL 酶标试剂,用封板膜封板,置37℃温育60min。试剂盒提供的洗涤液用蒸馏水稀释20倍,备用。小心揭掉封板膜,弃液体,甩干,各孔加满洗涤液,静置30s后弃去洗涤液。该操作重复 5次,拍干。各孔先加试剂盒提供的50μL显色剂A,再加试剂盒提供的50μL显色剂B,轻轻震荡,使混匀,37℃避光显色15min。之后,各孔加50μL试剂盒提供的终止液,终止显色反应(此时蓝色立转黄色),以空白孔为参照调零,在450nm波长下测各孔的吸光度。测定时设空白对照孔。空白对照孔除不加样品及酶标试剂外,其余各步操作不变。以P-选择素标准品的浓度为横坐标,吸光度值为纵坐标绘制P-选择素标准曲线,模拟直线回归方程。将生理盐水治疗的小鼠血清样品的吸光度,阿霉素治疗的小鼠血清样品的吸光度和化合物6及11a-c治疗的小鼠血清样品的吸光度代入方程式,计算血清样品中P-选择素浓度。数据以均值±SD pg/mL表示,经t检验,p<0.05即有统计学差异。结果列入表2。可以看出,化合物6及11a-c 有效地降低S180小鼠血清中P-选择素含量,而阿霉素没有这种活性。可见,P-选择素是化合物6及11a-c显示抗肿瘤活性的靶点。这同样是本发明的突出的技术效果。
表2化合物11a-c对S180荷瘤小鼠血清P-选择素含量的影响
a)与生理盐水及阿霉素比p<0.05;b)与生理盐水及阿霉素比p<0.01;n=12。
Claims (3)
2.权利要求1所述的9-(CH2CO-Arg-Gly-Asp-AA)-四氢-β-咔啉[3:4]并哌嗪-2,5-二酮并哌啶[4:5]并咪唑的制备方法, 该方法包括:
(1) L-Trp在硫酸催化条件下与甲醛进行Pictet-Spengler反应, 制备3S-1,2,3,4-四氢-β-咔啉-3-羧酸(1);
(2)在N,N-二甲基甲酰胺中1与(Boc)2O反应, 制备3S-2-叔丁氧羰基-1,2,3,4-四氢-β-咔啉-3-羧酸(2);
(3) L-His在硫酸催化条件下与甲醛进行Pictet-Spengler反应, 制备6S-4,5,6,7-四氢-咪唑[4:5]并哌啶-6-羧酸(3);
(4) 在甲醇和氯化亚砜中由3制备6S-4,5,6,7-四氢-咪唑[4:5]并哌啶-6-羧酸甲酯(4);
(5) 以3-二乙氧基磷酰基-1,2,3-苯唑4(H)-酮作缩合剂,在无水四氢呋喃中2与4偶联制备2-叔丁氧羰基-四氢-β-咔啉-3-甲酰-哌啶[4:5]并咪唑-6-羧酸甲酯(5);
(6) 脱除5的叔丁氧羰基保护基, 溶于甲醇之后加N-甲基吗啉调pH为9制备四氢-β-咔啉[3:4]并哌嗪-2,5-二酮并哌啶- [4:5]并咪唑(6);
(7) 在二氯甲烷中6与三苯基氯甲烷反应, 制备1-三苯甲基-四氢-β-咔啉[3:4]并哌嗪-2,5-二酮并哌啶[4:5]并咪唑(7);
(8) 在N, N-二甲基甲酰胺中7与溴乙酸苄酯反应, 制备1-三苯甲基-9-乙酸苄酯-四氢-β-咔啉- [3:4]并哌嗪-2,5-二酮并哌啶[4:5]并咪唑(8);
(9) 8脱苄基制备1-三苯甲基-9-乙酸-四氢-β-咔啉[3:4]并哌嗪-2,5-二酮并哌啶[4:5]并咪唑(9);
(10) 在N, N-二甲基甲酰胺中9分别与Arg(NO2)-Gly-Asp(OBzl)-AA-OBzl偶联, 制备1-三苯甲基-9-[CH2CO-Arg(NO2)-Gly-Asp(OBzl)-AA-OBzl]-四氢-β-咔啉[3:4]并哌嗪-2,5-二酮并哌啶[4:5]并咪唑(10a-c);
(11) 10a-c脱保护制备9-(CH2CO-Arg-Gly-Asp-AA)-四氢-β-咔啉[3:4]并哌嗪-2,5-二酮并哌啶[4:5]并咪唑(11a-c)。
3.权利要求1所述的9-(CH2CO-Arg-Gly-Asp-AA)-四氢-β-咔啉[3:4]并哌嗪-2,5-二酮并哌啶[4:5]并咪唑在制备抗肿瘤药物中的应用。
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