CN113461770B - 具有近红外成像功能的CDDO-Me靶向前药及其制备方法和用途 - Google Patents
具有近红外成像功能的CDDO-Me靶向前药及其制备方法和用途 Download PDFInfo
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Abstract
Description
技术领域
本发明涉及生物医药技术领域,具体为具有近红外成像功能的CDDO-Me靶向前药及其制备方法和用途。
背景技术
癌症严重威胁人类健康和生命,其发病机制目前尚不十分清楚。近年来,越来越多的证据表明,肿瘤的发生、发展及转移与其所处的微环境密切相关[1]。肿瘤微环境是指肿瘤细胞在生长过程中,由肿瘤细胞及细胞外间质相互作用后形成的肿瘤细胞生长的特殊环境。据文献报道,与正常组织相比,肿瘤微环境中活性氧(ROS)的浓度通常是正常细胞的100倍;肿瘤细胞内GSH浓度达到2-10mM,是正常组织细胞外液及血液环境(2-20μM)中的100-1000倍。若能对肿瘤微环境的动态示踪,将有助于发现肿瘤及其转移部位,采取药物治疗和/或加以手术切除。有鉴于此,基于肿瘤微环境开展荧光成像和治疗的药物研究具有重要科学意义。
具有荧光成像功能的抗肿瘤药物的研究和开发现已成为医药领域的研究热点之一,通常策略是将肿瘤选择性成像与治疗合二为一,在做出成像示踪的同时立即给予有效治疗(药物和/或手术),将大大提高肿瘤诊疗效率,减轻患者痛苦,提高其生存率。同时有助于可视化药物在靶部位的分布及释放过程,以研究药物的作用机制,实现肿瘤治疗效果的实时监控及个体化给药等。
五环三萜类化合物具有广泛的药理作用和重要的生物活性,尤其在抗肿瘤、抗炎以及机体免疫调节等方面。其中最典型的一类为齐墩果酸(OA)衍生物2-腈基-3,12-二氧代齐墩果酸-1,9(11)-二烯-28-羧酸甲酯(CDDO-Me),具有强效的抗肿瘤等活性,已进入多个临床研究。然而其安全性已受到严重关注。CDDO-Me的毒副作用可能是由于CDDO-Me的A环上α-氰基-α,β-不饱和酮(CUK)反应性极强,除与靶标作用外,也可能与非靶标蛋白的巯基发生迈克尔加成,从而导致严重副作用。因此有必要研究开发一种针对药效片段CUK的前药保护策略,使CDDO-Me选择性地在靶部位(肿瘤组织)释放,减少其与正常组织产生的不良反应。
发明内容
本发明的目的在于提供具有近红外成像功能的CDDO-Me靶向前药及其制备方法和用途,以解决上述背景技术中提出的问题。
为实现上述目的,本发明提供如下技术方案:具有近红外成像功能的CDDO-Me靶向前药,该靶向前药具有通式I所示结构:
其中,R1选自H、烷基、R2选自烷基、炔基取代的烷基、低聚乙二醇连接的烷基、或者生物素修饰的低聚乙二醇连接的烷基;Y-代表卤负离子、六氟磷酸根负离子、对甲苯磺酸负离子、或者甲磺酸负离子;n选自1-5。
优选的,所述R1优选H、CH3、CH2CH3、R2优选CH3、CH2CH3、炔丙基、炔戊基、其中m=1-10,o=1-6;Y-代表卤负离子、六氟磷酸根负离子、对甲苯磺酸负离子、或者甲磺酸负离子;n选自2-5。
优选的,式I的结构中,R1、R2、Y及n选自如下组合:
R1=H,R2=CH3,Y=Br,n=2;
I1:2-((E)-2-(6-((2-((3-((1-(4-((((((((4aR,6aS,6bR,8aS,12aS,12bR,14bS)-2-氰基-1-羟基-8a-(甲氧羰基)-4,4,6a,6b,11,11,14b-七甲基-13-氧代-1,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-十八烷基氢吡喃-3-基)氧基)甲基)苯氧基)-1-氧丙烷-2-基)硫基)乙基)硫代)丙酰基)氧基)-2,3-二氢-1H-呫吨-4-基)乙烯基)-1,1,3-三甲基-1H-苯并[e]吲哚-3-溴盐;
或者R1=CH3,R2=炔丙基,Y=I,n=2;
I2:2-((E)-2-(6-((2-((3-((1-(4-((((((((4aR,6aS,6bR,8aS,12aS,12bR,14bS)-2-氰基-1-甲氧基-8a-(甲氧羰基)-4,4,6a,6b,11,11,14b-七甲基-13-氧代-1,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-十八烷基氢吡喃-3-基)氧基)甲基)苯氧基)-1-氧丙烷-2-基)硫基)乙基)硫代)丙酰基)氧基)-2,3-二氢-1H-呫吨-4-基)乙烯基)-1,1-二甲基-3-(戊-4-基-1-基)-1H-苯并[e]吲哚-3-碘盐;
或者R1=CH3CH2,R2=CH3CH2,Y=PF6,n=4;
I3:2-((E)-2-(6-((2-((3-((1-(4-((((((((4aR,6aS,6bR,8aS,12aS,12bR,14bS)-2-氰基-1-乙氧基-8a-(甲氧羰基)-4,4,6a,6b,11,11,14b-七甲基-13-氧代-1,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-十八烷基氢吡喃-3-基)氧基)甲基)苯氧基)-1-氧丙烷-2-基)硫基)丁基)硫代)丙酰基)氧基)-2,3-二氢-1H-呫吨-4-基)乙烯基)-3-乙基-1,1-二甲基-1H-苯并[e]吲哚-3-六氟磷酸盐;
I4:2-((E)-2-(6-((2-((4-((1-(4-((((((((4aR,6aS,6bR,8aS,12aS,12bR,14bS)-2-氰基-8a-(甲氧羰基)-4,4,6a,6b,11,11,14b-七甲基-13-氧代-1-(((8-氧代-8-(苯基氨基)辛酰胺基)氧基)-1,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-十八烷基氢吡啶-3-基)氧基)甲基)苯氧基)-1-氧丙烷-2-基)硫)丙基)硫代)丙酰基)氧基)-2,3-二氢-1H-呫吨-4-基)乙烯基)-1,1-二甲基-3-(戊-4-基-1-基)-1H-苯并[e]吲哚-3-碘盐;
I5:2-((E)-2-(6-((2-((3-((1-(4-((((((((4aR,6aS,6bR,8aS,12aS,12bR,14bS)-2-氰基-8a-(甲氧羰基)-4,4,6a,6b,11,11,14b-七甲基-13-氧代-1-(((8-氧代-8-(苯基氨基)辛酰胺基)氧基)-1,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-十八烷基氢吡啶-3-基)氧基)甲基)苯氧基)-1-氧丙烷-2-基)硫代丙基)硫代丙酰基)氧基)-2,3-二氢-1H-呫吨-4-基)乙烯基)-1,1-二甲基-3-(3-(1-(2-(2-(2-(5-((3aR,6S,6aS)-2-氧六氢-1H-噻吩并[3,4-d]咪唑-6-基)戊酰胺基乙氧基)乙氧基)乙基)-1H-1,2,3-三唑-4-基)丙基)-1H-苯并[e]吲哚-3-碘盐。
本发明还提供了具有近红外成像功能的CDDO-Me靶向前药的制备方法,包括如下步骤:
A、首先化合物2和苯并吲哚鎓盐3在催化量的哌啶中加热回流,通过Knoevenagel反应获得化合物4,之后加入BBr3进行脱甲化反应获得化合物5;
B、将等摩尔量的CDDO-Me与R1OH在1.5当量的碳酸钾作用下反应得到中间体6,再与4-(溴甲基)苯酚进行醚化反应得到中间体7;另一方面,硫代乳酸8与二溴烷烃9在冰浴NaOH条件下反应生成硫醚10,然后使用1-乙基-3-(3-二甲基氨基丙基)碳二亚胺(EDC)、4-二甲基氨基吡啶(DMAP)与化合物7通过酯化反应得到中间体11;
C、中间体11和NIR荧光片段5在EDCI和DMAP催化下通过酯化反应获得化合物Ia;此外,化合物Ia与Biotin-N3在N2保护的甲醇和/或DCM条件下,通过TBTA和Cu(CNCH3)4PF6催化,进行“点击”反应获得了Ib;化合物Ia和Ib均属于发明化合物I。
优选的,所述制备方法具体为
合成路线如下所示:
优选的,所述R1优选H、CH3、CH2CH3、R2优选CH3、CH2CH3、炔丙基、炔戊基、其中m=1-10,o=1-6;Y-代表卤负离子、六氟磷酸根负离子、对甲苯磺酸负离子、或者甲磺酸负离子;n选自1-5。
本发明还提供了上述具有近红外成像功能的CDDO-Me靶向前药在制备具有ROS/GSH双重响应的肿瘤选择性近红外荧光成像试剂的用途。
本发明还提供了上述具有近红外成像功能的CDDO-Me靶向前药在制备具有ROS/GSH双重响应的肿瘤选择性药物释放试剂的用途。
优选的,上述CDDO-Me靶向前药在对肺癌、结肠癌、肝癌、乳腺癌、宫颈癌的选择性治疗的应用,且对正常组织损伤较小。
优选的,可实现CDDO-Me原药在体内可视化释放和动态分布的监测作用。
优选的,可实现对体内肿瘤组织治疗效果的实时监控作用。
与现有技术相比,本发明的有益效果是:本发明采用亲核性烷基醇或者羟肟酸类HDAC抑制剂对CDDO-Me的A环上α,β-不饱和酮进行1,4-加成,形成烯醇加成产物,利用具有ROS和GSH双重响应的双巯基异丙酸,一端连接到4-羟基苄基保护的CDDO-Me与烷基醇或者羟肟酸类HDAC抑制剂偶联物,另一端链接到半花菁荧光片段,荧光片段末端引入肿瘤靶向基团生物素,从而设计合成了具有肿瘤微环境ROS/GSH双重响应的诊疗剂I。期望其利用生物素靶向片段递送到肿瘤组织中,同时在肿瘤微环境的氧化、还原应激下,一方面利用高水平的谷胱甘肽(GSH)对CDDO-Me前药和半花菁荧光探针的连接酯键进行GSH裂解,同时释放活性药物中间体和半花菁荧光探针。另一方面,连接酯键linker中的硫醚基团被氧化为砜/亚砜部分,进一步水解释放出活性药物中间体和半花菁荧光探针。活性药物中间体经1,6-消除反应释放出原药CDDO-Me和HDAC抑制剂,从而发挥选择性荧光成像示踪和协同高效肿瘤治疗作用。
附图说明
图1本发明化合物在ROS或GSH响应的荧光光谱数据曲线图;
图2本发明化合物肿瘤细胞选择性成像图;
图3本发明化合物在肿瘤细胞成像示意图;
图4本发明实施例中荷瘤小鼠活体荧光成像图;
图5本发明实施例中荷瘤小鼠的肿瘤组织和正常组织的荧光成像图。
具体实施方式
下面将结合本发明实施例中的附图,对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
实施例1:2-((E)-2-(6-((2-((3-((1-(4-((((((((4aR,6aS,6bR,8aS,12aS,12bR,14bS)-2-氰基-1-羟基-8a-(甲氧羰基)-4,4,6a,6b,11,11,14b-七甲基-13-氧代-1,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-十八烷基氢吡喃-3-基)氧基)甲基)苯氧基)-1-氧丙烷-2-基)硫基)乙基)硫代)丙酰基)氧基)-2,3-二氢-1H-呫吨-4-基)乙烯基)-1,1,3-三甲基-1H-苯并[e]吲哚-3-溴盐(I1)
(E)-2-(2-(2-(6-羟基-2,3-二氢-1H-噻吨-4-基)乙烯基)-1,1,3-三甲基-1H-苯并[e]吲哚-3-溴盐(5a)
将化合物2(2.52mmol)用5mL的甲醇溶解,加入化合物3a(2.52mmol),再加入3滴乙酸和2滴哌啶催化,80℃回流12h。反应完毕后,减压浓缩,柱层析纯化,得到蓝色固体0.81g,将其用DCM(5ml)溶解,N2保护,冰浴,然后在剧烈搅拌下将BBr3(5ml)滴加到该混合液中,反应12h。反应完毕后,将混合液倒入到冰水中,用固体NaHCO3调pH至中性。随后,用CH2Cl2(60mL)萃取水溶液三次,并将有机层经Na2SO4干燥并浓缩,得到蓝色产物5a,产率73%。1HNMR(400MHz,CDCl3)δ8.76(d,J=14.9Hz,1H,ArH),8.26(d,J=8.4Hz,1H,ArH),8.05(d,J=8.9Hz,1H,ArH),8.00(d,J=8.1Hz,1H,ArH),7.83(d,J=8.9Hz,1H,ArH),7.70(m,1H,ArH),7.60(m,1H,CH=C),7.39(d,J=8.3Hz,1H,ArH),7.24(s,1H,CH=C),6.91(dt,J=8.3,2.4Hz,2H,ArH),6.69(d,J=14.9Hz,1H,CH=C),4.80(m,2H,CH2),2.86(m,2H,CH2),2.13(m,2H,CH2),2.12(d,J=4.8Hz,6H,2CH3),1.99(m,2H,CH2),0.9(s,3H,CH3).
2,2'-(乙基双(亚磺酰二基))二丙酸(10a)
将NaOH(84.2mmol,3.48g)用甲醇(40ml)溶解,N2保护,冰浴搅拌10min后,加入硫代乳酸(41.2mmol,4.37g),继续搅拌20min,最后加入1,2-二溴乙烷(20mmol,3.86mg),反应12h。反应完毕后,用2mol/L的盐酸调节pH至4,然后DCM萃取,有机层干燥、减压浓缩得到无色油状物10a,产率97.2%。1H NMR(400MHz,CDCl3)δ10.71(s,2H,2COOH),3.43(q,J=7.1Hz,2H,CH2),2.81(m,4H,2CH2),1.47(d,J=7.2Hz,6H,2CH3).
(4aS,6aR,6bS,8aR,12aS,14aR,14bS)-甲基-11-氰基-12-羟基-10-((4-羟基苄基)氧基)-2,2,6a,6b,9,9,12a-七甲基-14-氧代-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,12,12a,14,14a,14b-十八烷基氢吡啶-4a-羧酸盐(7a)
将CDDO-Me(0.198mmol,100mg)用无水DMF(4ml)溶解,再加入K2CO3(0.396mmol,54.7mg),N2保护,冰浴,反应12h后,将H2O(0.198mmol,11.7mg)用DMF(1ml)溶解,然后在剧烈搅拌下将对羟基溴苄(0.30mmol,55.3mg)滴加到混合液中,反应2h。待反应完毕后,用CH2Cl2(60mL)萃取水溶液三次,并将有机层经Na2SO4干燥并浓缩,柱分离,得到化合物7a,产率60%。
2-(((((1-(4-((((((4aR,6aS,6bR,8aS,12aS,12bR,14bS)-2-氰基-1-羟基-8a-(甲氧羰基)-4,4,6a,6b,11,11,14b-七甲基-13-氧代-1,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-十八碳氢吡啶-3-基)氧基)甲基)苯氧基)-1-氧代丙烷-2-基)硫代)乙基)硫代)丙酸(11a)
将中间体7a(0.26mmol,230mg)用无水DCM(4ml)溶解,再加入EDCI(0.52mmol,183mg)、中间体10a(0.78mmol,181.7mg)、DMAP(0.14mmol,20mg),反应12h。待反应完毕后,DCM萃取,有机层干燥,减压浓缩,柱层析纯化,得到产物11a,产率83%。1HNMR(400MHz,CDCl3)δ6.88(m,2H,2ArH),6.73(m,2H,2ArH),5.85(m,1H,CH),5.35(s,1H,OH),4.97(m,2H,CH2),3.68(s,3H,OCH3),3.38(m,1H,CH),3.30(s,3H,OCH3),2.80(s,1H,CH),2.27(m,2H,2CH),2.13(m,4H,2CH2),1.62(s,6H,2CH3),1.38(m,4H,2CH2),1.32(s,9H,3CH3),1.30(m,4H,2CH2),1.25(s,6H,2CH3),1.13(m,1H,CH).
2-((E)-2-(6-((2-(((((1-(4-((((((((4aR,6aS,6bR,8aS,12aS,12bR,14bS)-2-氰基-1-羟基-8a-(甲氧羰基)-4,4,6a,6b,11,11,14b-七甲基-13-氧代-1,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-十八烷基氢吡喃-3-基)氧基)甲基)苯氧基)-1-氧代丙烷-2-基)硫基)乙基)硫代)丙酰基)氧基)-2,3-二氢-1H-呫吨-4-基)乙烯基)-1,1,3-三甲基-1H-苯并[e]吲哚-3-溴盐(I1)
将中间体11a(0.23mmol,251mg)用无水DCM(4ml)溶解,再加入EDCI(0.46mmol,87.9mg)、中间体5a(0.21mmol,132mg)、DMAP(0.12mmol,18mg),反应12h。待反应完毕后,DCM萃取、干燥、减压浓缩,柱层析纯化,得到最终产物321.8mg,产率81%。1H NMR(400MHz,CDCl3)δ8.76(d,J=14.9Hz,1H,ArH),8.26(d,J=8.4Hz,1H,ArH),8.05(d,J=8.9Hz,1H,ArH),8.00(d,J=8.1Hz,1H,ArH),7.83(d,J=8.9Hz,1H,ArH),7.70(m,1H,ArH),7.60(m,1H,CH=C),7.39(d,J=8.3Hz,1H,ArH),7.24(s,1H,CH=C),7.22(m,2H,2ArH),7.07(m,2H,2ArH),6.91(dt,J=8.3,2.4Hz,2H,ArH),6.69(d,J=14.9Hz,1H,CH=C),5.85(m,1H,CH=C),4.97(m,2H,CH2),4.03(m,2H,CH2),3.54(m,2H,2CH),3.38(m,2H,CH2),3.20(m,1H,CH),2.81(m,6H,3CH2),2.80(s,1H,CH),2.46(m,2H,CH2),2.27(m,2H,2CH),2.20(m,2H,CH2)2.13(m,2H,CH2),2.03(m,2H,CH2),1.62(s,6H,2CH3),1.50(m,2H,CH2),1.38(m,4H,2CH2),1.35(s,6H,2CH3),1.32(s,6H,2CH3),1.30(m,4H,2CH2),1.25(s,6H,2CH3),1.13(m,1H,CH),1.10(m,3H,CH3),0.9(s,6H,2CH3).
实施例2 2-((E)-2-(6-((2-((3-((1-(4-((((((((4aR,6aS,6bR,8aS,12aS,12bR,14bS)-2-氰基-1-甲氧基-8a-(甲氧羰基)-4,4,6a,6b,11,11,14b-七甲基-13-氧代-1,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-十八烷基氢吡喃-3-基)氧基)甲基)苯氧基)-1-氧丙烷-2-基)硫基)甲基)硫代)丙酰基)氧基)-2,3-二氢-1H-呫吨-4-基)乙烯基)-1,1-二甲基-3-(戊-4-基-1-基)-1H-苯并[e]吲哚-3-碘盐(I2)
(E)-2-(2-(6-羟基-6,3-二氢-1H-呫吨-4-基)乙烯基)-1,1-二甲基-3-(戊-4-基-1-基)-1H-苯并[e]吲哚-3-碘盐(5b)
参考实施例1中5a的制备方法,将化合物3b替代方法中的3a,最后得到深蓝色产物5b,产率70%。1H NMR(400MHz,CDCl3)δ8.76(d,J=14.9Hz,1H,ArH),8.26(d,J=8.4Hz,1H,ArH),8.05(d,J=8.9Hz,1H,ArH),8.00(d,J=8.1Hz,1H,ArH),7.83(d,J=8.9Hz,1H,ArH),7.70(m,1H,ArH),7.60(m,1H,CH=C),7.39(d,J=8.3Hz,1H,ArH),7.24(s,1H,CH=C),6.91(dt,J=8.3,2.4Hz,2H,ArH),6.69(d,J=14.9Hz,1H,CH=C),4.80(m,2H,CH2),4.11(m,1H,C≡CH),2.86(m,2H,CH2),2.79(m,2H,CH2),2.57(m,2H,CH2),2.25(m,2H,CH2),2.13(m,2H,CH2),2.12(d,J=4.8Hz,6H,2CH3),1.99(m,2H,CH2).
(4aS,6aR,6bS,8aR,12aS,14aR,14bS)-甲基-11-氰基-12-甲基-10-((4-羟基苄基)氧基)-2,2,6a,6b,9,9,12a-七甲基-14-氧代-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,12,12a,14,14a,14b-十八烷基氢吡啶-4a-羧酸盐(7b)
参考实施例1中7a的制备方法,将反应物甲醇替代方法中的水,最后得到中间体7b,产率70%。
2-(((((1-(4-((((((4aR,6aS,6bR,8aS,12aS,12bR,14bS)-2-氰基-1-甲基-8a-(甲氧羰基)-4,4,6a,6b,11,11,14b-七甲基-13-氧代-1,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-十八碳氢吡啶-3-基)氧基)甲基)苯氧基)-1-氧代丙烷-2-基)硫代)甲基)硫代)丙酸(11b)
参考实施例1中11a的制备方法,将反应物7b替代方法中的7a,最后得到中间体11b,产率88%。1H NMR(400MHz,CDCl3)δ11.0(s,1H,COOH),7.22(m,2H,2ArH),7.07(m,2H,2ArH),4.97(m,2H,CH2),3.68(s,3H,OCH3),3.54(m,2H,2CH),3.30(s,3H,OCH3),3.20(m,1H,CH),2.81(m,8H,4CH2),2.80(s,1H,CH),2.46(m,2H,CH2),2.27(m,2H,2CH),2.13(m,4H,2CH2),1.62(s,6H,2CH3),1.50(m,2H,CH2),1.35(s,6H,2CH3),1.32(s,9H,3CH3),1.30(m,4H,2CH2),0.9(s,6H,2CH3).
2-((E)-2-(6-((2-(((((1-(4-((((((((4aR,6aS,6bR,8aS,12aS,12bR,14bS)-2-氰基-1-羟基-8a-(甲氧羰基)-4,4,6a,6b,11,11,14b-七甲基-13-氧代-1,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-十八烷基氢吡喃-3-基)氧基)甲基)苯氧基)-1-氧代丙烷-2-基)硫基)甲基)硫代)丙酰基)氧基)-2,3-二氢-1H-呫吨-4-基)乙烯基)-1,1-二甲基-3-(戊-4-基-1-基)-1H-苯并[e]吲哚-3-碘盐(I2)
参考实施例1中I1的制备方法,将反应物5b和11b替代方法中的5a和11a,最后得到最终产物I2,产率81%。1H NMR(400MHz,CDCl3)δ8.76(d,J=14.9Hz,1H,ArH),8.26(d,J=8.4Hz,1H,ArH),8.05(d,1H,ArH),8.00(d,1H,ArH),7.83(d,J=8.9Hz,1H,ArH),7.70(m,1H,ArH),7.60(m,1H,CH=C),7.39(d,J=8.3Hz,1H,ArH),7.24(s,1H,CH=C),7.22(m,2H,2ArH),7.07(m,2H,2ArH),6.91(m,2H,ArH),6.69(d,J=14.9Hz,1H,CH=C),5.85(m,1H,CH=C),4.97(m,2H,CH2),4.03(m,2H,CH2),3.68(s,3H,OCH3),3.54(m,2H,2CH),3.30(s,3H,OCH3),3.20(m,1H,CH),2.81(m,8H,4CH2),2.80(s,1H,CH),2.46(m,2H,CH2),2.27(m,2H,2CH),2.13(m,4H,2CH2),1.62(s,6H,2CH3),1.60(m,2H,CH2),1.50(m,2H,CH2),1.38(m,4H,2CH2),1.35(s,6H,2CH3),1.32(s,9H,3CH3),1.30(m,4H,2CH2),1.25(s,6H,2CH3),1.13(m,1H,CH),0.9(s,6H,2CH3).
实施例3 2-((E)-2-(6-((2-((3-((1-(4-((((((((4aR,6aS,6bR,8aS,12aS,12bR,14bS)-2-氰基-1-乙氧基-8a-(甲氧羰基)-4,4,6a,6b,11,11,14b-七甲基-13-氧代-1,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-十八烷基氢吡喃-3-基)氧基)甲基)苯氧基)-1-氧丙烷-2-基)硫基)丁基)硫代)丙酰基)氧基)-2,3-二氢-1H-呫吨-4-基)乙烯基)-3-乙基-1,1-二甲基-1H-苯并[e]吲哚-3-六氟磷酸盐(I3)
(E)-3-乙基-2-(2-(6-羟基-6,3-二氢-1H-呫吨-4-基)乙烯基)-1,1-二甲基-1H-苯并[e]吲哚-3-六氟磷酸盐(5c)
参考实施例1中5a的制备方法,将化合物3c替代方法中的3a,最后得到深蓝色产物5c,产率72%。1H NMR(400MHz,CDCl3)δ8.76(d,J=14.9Hz,1H,ArH),8.26(d,J=8.4Hz,1H,ArH),8.05(d,J=8.9Hz,1H,ArH),8.00(d,J=8.1Hz,1H,ArH),7.83(d,J=8.9Hz,1H,ArH),7.70(m,1H,ArH),7.60(m,1H,CH=C),7.39(d,J=8.3Hz,1H,ArH),7.24(s,1H,CH=C),6.91(dt,J=8.3,2.4Hz,2H,ArH),6.69(d,J=14.9Hz,1H,CH=C),4.80(m,2H,CH2),2.86(m,2H,CH2),2.54(m,2H,CH2),2.13(m,2H,CH2),2.12(d,J=4.8Hz,6H,2CH3),1.99(m,2H,CH2),0.9(s,3H,CH3).
2,2'-(丁烷-1,4-二基双(硫代二基))二丙酸(10b)
参考实施例1中10a的制备方法,将1,4-二溴丁烷替代方法中的1,2-二溴乙烷,最后得到产物10b,产率96%。1H NMR(400MHz,CDCl3)δ10.71(s,2H,2COOH),3.43(q,J=7.1Hz,2H,CH2),2.81(m,4H,2CH2),1.66(m,4H,2CH2),1.47(d,J=7.2Hz,6H,2CH3).
(4aS,6aR,6bS,8aR,12aS,14aR,14bS)-甲基-11-氰基-12-乙基-10-((4-羟基苄基)氧基)-2,2,6a,6b,9,9,12a-七甲基-14-氧代-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,12,12a,14,14a,14b-十八烷基氢吡啶-4a-羧酸盐(7c)
参考实施例1中7a的制备方法,将反应物乙醇替代方法中的水,最后得到中间体7c,产率67%。
2-(((((1-(4-((((((4aR,6aS,6bR,8aS,12aS,12bR,14bS)-2-氰基-1-乙基-8a-(甲氧羰基)-4,4,6a,6b,11,11,14b-七甲基-13-氧代-1,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-十八碳氢吡啶-3-基)氧基)甲基)苯氧基)-1-氧代丙烷-2-基)硫代)丁基)硫代)丙酸(11c)
参考实施例1中11a的制备方法,将反应物7c和10b替代方法中的7a和10a,最后得到中间体11c,产率89%。1H NMR(400MHz,CDCl3)δ11.0(s,1H,COOH),7.22(m,2H,2ArH),7.07(m,2H,2ArH),4.97(m,2H,CH2),3.88(m,2H,CH2),3.68(s,3H,OCH3),3.54(m,2H,2CH),3.20(m,1H,CH),2.81(m,8H,4CH2),2.80(s,1H,CH),2.46(m,2H,CH2),2.27(m,2H,2CH),2.13(m,4H,2CH2),2.3(m,2H,CH2),1.62(s,6H,2CH3),1.50(m,2H,CH2),1.35(s,6H,2CH3),1.32(s,9H,3CH3),1.30(m,4H,2CH2),1.10(s,3H,CH3),0.9(s,6H,2CH3).
2-((E)-2-(6-((2-(((((1-(4-((((((((4aR,6aS,6bR,8aS,12aS,12bR,14bS)-2-氰基-1-羟基-8a-(甲氧羰基)-4,4,6a,6b,11,11,14b-七甲基-13-氧代-1,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-十八烷基氢吡喃-3-基)氧基)甲基)苯氧基)-1-氧代丙烷-2-基)硫基)丁基)硫代)丙酰基)氧基)-2,3-二氢-1H-呫吨-4-基)乙烯基)-1,1-二甲基-3-乙基-1H-苯并[e]吲哚-3-六氟磷酸盐(I3)
参考实施例1中I1的制备方法,将反应物5c和11c替代方法中的5a和11a,最后得到最终产物I3,产率85%。1H NMR(400MHz,CDCl3)δ8.76(d,J=14.9Hz,1H,ArH),8.26(d,J=8.4Hz,1H,ArH),8.05(d,J=8.9Hz,1H,ArH),8.00(d,J=8.1Hz,1H,ArH),7.83(d,J=8.9Hz,1H,ArH),7.70(m,1H,ArH),7.60(m,1H,CH=C),7.39(d,J=8.3Hz,1H,ArH),7.24(s,1H,CH=C),7.22(m,2H,2ArH),7.07(m,2H,2ArH),6.91(dt,J=8.3,2.4Hz,2H,ArH),6.69(d,J=14.9Hz,1H,CH=C),5.85(m,1H,CH=C),4.97(m,2H,CH2),4.03(m,2H,CH2),3.68(s,3H,OCH3),3.54(m,2H,2CH),3.38(m,2H,CH2),3.20(m,1H,CH),2.81(m,8H,4CH2),2.80(s,1H,CH),2.46(m,2H,CH2),2.27(m,2H,2CH),2.13(m,4H,2CH2),2.03(m,2H,CH2),1.62(s,6H,2CH3),1.60(m,2H,CH2),1.50(m,2H,CH2),1.38(m,4H,2CH2),1.35(s,6H,2CH3),1.32(s,9H,3CH3),1.30(m,4H,2CH2),1.25(s,6H,2CH3),1.13(m,1H,CH),1.10(m,3H,CH3),0.9(s,6H,2CH3).
实施例4 2-((E(-2-(6-((2-((3-((1-(4-((((((((4aR,6aS,6bR,8aS,12aS,12bR,14bS)-2-氰基-8a-(甲氧羰基)-4,4,6a,6b,11,11,14b-七甲基-13-氧代-1-(((8-氧代-8-(苯基氨基)辛酰胺基)氧基)-1,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-十八烷基氢吡喃-3-基)氧基)甲基)苯氧基)-1-氧丙烷-2-基)硫代丙基)硫代丙酰基)氧基)-2,3-二氢-1H-呫吨-4-基)乙烯基)-1,1-二甲基-3-(戊-4-1-基)-1H-苯并[e]吲哚-3-碘盐(I4)
(E)-2-(2-(6-羟基-6,3-二氢-1H-呫吨-4-基)乙烯基)-1,1-二甲基-3-(戊-4-基-1-基)-1H-苯并[e]吲哚-3-碘盐(5d)
参考实施例1中5a的制备方法,将化合物3d替代方法中的3a,最后得到深蓝色产物5d,产率75%。
2,2'-(丙烷-1,3-二基双(磺酰二基))二丙酸(10c)
参考实施例1中10a的制备方法,将1,3-二溴丙烷替代方法中的1,2-二溴乙烷,最后得到产物10c,产率92%。1H NMR(400MHz,CDCl3)δ10.71(s,2H,2COOH),3.43(q,J=7.1Hz,2H,CH2),2.90(m,4H,2CH2),2.01(m,2H,CH2),1.47(d,J=7.2Hz,6H,2CH3).
(4aS,6aR,6bS,8aR,12aS,14aR,14bS)-甲基-11-氰基-10-((4-羟基苄氧基)氧基)-2,2,6a,6b,9,9,12a-庚甲基-14-氧代-12-(((8-氧代-8-(苯胺)八甲胺基)氧)-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,12,12a,14,14a,14b-十八碳二氢吡啶-4a-羧酸(7d)
参考实施例1中7a的制备方法,将反应物SAHA替代方法中的水,最后得到中间体7d,产率66%。
2-((3-((1-(4-((((((4aR,6aS,6bR,8aS,12aS,12bR,14bS)-2-氰基-8a-(甲氧羰基)-4,4,6a,6b,11,11,14b-七甲基-13-氧代-1-(((8-氧代-8-(苯氨基)辛酰胺基)氧基)-1,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-十八烷基氢吡喃-3-基)氧基)甲基)苯氧基)-1-氧代丙烷-2-基)硫代)丙基)硫代)丙酸(11d)
参考实施例1中11a的制备方法,将反应物7d和10c替代方法中的7a和10a,最后得到中间体11d,产率89%。
2-((E(-2-(6-((2-((3-((1-(4-((((((((4aR,6aS,6bR,8aS,12aS,12bR,14bS)-2-氰基-8a-(甲氧羰基)-4,4,6a,6b,11,11,14b-七甲基-13-氧代-1-(((8-氧代-8-(苯基氨基)辛酰胺基)氧基)-1,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-十八烷基氢吡喃-3-基)氧基)甲基)苯氧基)-1-氧丙烷-2-基)硫代丙基)硫代丙酰基)氧基)-2,3-二氢-1H-呫吨-4-基)乙烯基)-1,1-二甲基-3-(戊-4-1-基)-1H-苯并[e]吲哚-3-碘盐(I4)
参考实施例1中I1的制备方法,将反应物5d和11d替代方法中的5a和11a,最后得到最终产物I4,产率82%。
实施例5 2-((E)-2-(6-((2-((3-((1-(4-((((((((4aR,6aS,6bR,8aS,12aS,12bR,14bS)-2-氰基-8a-(甲氧羰基)-4,4,6a,6b,11,11,14b-七甲基-13-氧代-1-(((8-氧代-8-(苯基氨基)辛酰胺基)氧基)-1,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-十八烷基氢吡啶-3-基)氧基)甲基)苯氧基)-1-氧丙烷-2-基)硫代丙基)硫代丙酰基)氧基)-2,3-二氢-1H-呫吨-4-基)乙烯基)-1,1-二甲基-3-(3-(1-(2-(2-(2-(5-((3aR,6S,6aS)-2-氧六氢-1H-噻吩并[3,4-d]咪唑-6-基)戊酰胺基乙氧基)乙氧基)乙基)-1H-1,2,3-三唑-4-基)丙基)-1H-苯并[e]吲哚-3-碘盐(I5)
将化合物I4(0.12mmol)用DCM溶解,N2保护,然后加入TBTA(0.54mmol,220.3mg),Cu(CNCH3)4PF6(0.0108mmol,2.7mg)避光搅拌10min后,加入Biotin-N3(0.132mmol,58mg),室温搅拌10h。待反应完毕后,加入DCM萃取、干燥、浓缩,柱层析分离,得到化合物I5,产率85%。1H NMR(400MHz,CDCl3)δ8.75(d,J=15.2Hz,1H,ArH),8.24(d,J=8.5Hz,1H,ArH),8.07(dd,J=23.1,8.5Hz,3H,2ArH,NH),7.85(d,J=9.0Hz,1H,ArH),7.75(m,2H,2ArH),7.61(t,J=7.4Hz,1H,ArH),7.53(d,J=6.3Hz,1H,ArH),7.41(m,2H,ArH),7.15(s,1H,ArH),7.10(m,5H,4ArH,NH),5.84(d,J=6.8Hz,2H,2CH=C),5.12(t,J=7.6Hz,2H,2NH),5.01(s,1H,CH),4.98(m,1H,CH=C),4.17(m,2H,CH2),3.98(d,J=6.0Hz,2H,2CH),3.73(s,2H,CH2),3.69(s,4H,2CH2),3.47(dd,J=14.5,5.2Hz,6H,CH,CH2,CH3),3.28(dd,J=13.7,6.6Hz,2H,2CH),2.94(d,J=3.1Hz,4H,2CH2),2.87(d,J=7.0Hz,3H,CH,CH2),2.82(m,2H,CH2),2.76(s,6H,2CH3),2.50(d,J=14.0Hz,4H,2CH2),2.10(d,J=4.0Hz,8H,CH2),2.04–1.99(m,8H),1.89(m,9H,CH,CH2),1.64(d,J=7.2Hz,6H,2CH3),1.59(s,6H,2CH3),1.52(d,J=6.8Hz,4H,2CH2),1.45(d,J=7.1Hz,5H,CH,2CH2),1.37(s,5H,3CH,CH2),1.21(m,13H,CH,6CH2),1.05(d,J=2.8Hz,4H,2CH2),1.00(s,3H,CH3),0.89(d,J=8.5Hz,9H,3CH3);MS(ESI)m/z[M]+=1978.
实施例6采用MTT法对本发明化合物的肿瘤细胞和正常细胞增殖抑制率测定研究
按常规采用MTT评价了本发明化合物对5种人癌细胞株和人正常细胞株的抗增殖活性。MTT法已广泛用于大规模的抗肿瘤药物筛选、细胞毒性试验等。选择CDDO-Me作为阳性对照药。
人癌细胞株:肝癌细胞HepG2、肺癌细胞A549、结肠癌细胞HCT116、乳腺癌细胞Mcf-7、人宫颈癌细胞Hela。
人正常细胞株:正常肝细胞LO2。
实验方法如下:取处于指数生长期状态良好的细胞一瓶,加入0.25%胰蛋白酶消化,使贴壁细胞脱落,制成每毫升含2×104~4×104个细胞的悬液。取细胞悬液接种于96孔板上,每孔180μL,置恒温CO2培养箱中培养24小时。换液,加入受试化合物I1-I5(化合物用DMSO溶解后用PBS稀释,受试化合物浓度分别为2.5×10-5mol/L),每孔20μL,培养72小时。将MTT加入96孔板中,每孔20μL,培养箱中反应4h。吸去上清液,加入DMSO,每孔150μL,平板摇床上振摇5分钟。用酶联免疫检测仪在波长为570nm处测定每孔的吸收度,计算细胞抑制率。实验结果如表1所示。
细胞抑制率=(阴性对照组OD值-受试物组OD值)/阴性对照组OD值×100%。
本发明所述化合物经过一系列肿瘤细胞和正常细胞抗增殖活性测试,药理实验结果表明(见表1),发现本发明所述化合物I1-I5对大部分肿瘤细胞增殖抑制作用较强,尤其部分化合物比阳性对照药CDDO-Me稍强或相当,表1中大部分化合物在10μM浓度下抑制率大于75%;然而,本发明化合物I1-I5在相同浓度下对人正常肝细胞LO2的细胞毒明显低于多个肿瘤细胞,同时本发明化合物I1-I5在相同浓度下对人正常肝细胞LO2的抑制率明显低于阳性对照药CDDO-Me,由此说明本发明化合物不仅对肿瘤细胞具有显著的抗肿瘤活性,而且对正常细胞毒性较低,具有一定的肿瘤细胞选择性。
表1本发明部分化合物对人肿瘤细胞和正常细胞增殖的抑制率%(10μM)
ND:未检测
实施例7本发明化合物基于肿瘤微环境的ROS和GSH响应性的荧光光谱测试
将本发明荧光化合物溶于含1%DMSO的水溶液中,配制成不同浓度的GSH(0-10mM)和H2O2(0-10mM)的检测液。采用荧光光谱仪测试其荧光发射光谱数据,结果显示本发明荧光化合物最大发射波长在680nm-740nm范围内。
其中化合物I4和I5在715nm左右的荧光峰值随GSH和H2O2浓度的增大而增加,最大荧光峰值与未加GSH和H2O2相差30倍(图1),具有显著的ROS和GSH响应性荧光开关特性,可用于肿瘤微环境的选择性荧光成像。
实施例10本发明化合物的对肿瘤细胞选择性荧光成像研究
通过使用40X物镜的共聚焦激光扫描显微镜(Leica TCS SP8)进行细胞摄取和定位。简而言之,将HepG2、Hela或者A549细胞用1mL培养基以1×105细胞的密度在共聚焦培养皿中于37℃培养24h。然后,将培养基替换为含有10μM的I1、I3的新鲜培养基,并在37℃下孵育1h,然后用PBS洗涤细胞3次。最后,使用共聚焦激光扫描显微镜获得细胞荧光成像的图像。
图2成像结果显示,I1(10μM)在4h后能够清晰地对肝肿瘤细胞HepG2进行荧光成像,而在正常细胞LO2荧光很弱,根据细胞内荧光的量化,HepG2的荧光强度是LO2细胞的6倍,说明本发明化合物能够选择性地对肝肿瘤细胞荧光成像;图3共聚焦荧光图像表明,I3对Hela细胞的荧光成像随着时间推移,成像图像荧光逐步增强。
实施例11本发明化合物的体内荧光成像和抗肿瘤活性研究
为了评估I系列化合物的体内荧光成像和抗肿瘤活性,建立用A549细胞皮下接种的BALB/c裸鼠模型。实体瘤形成后,将裸鼠随机给予PBS、对照药CDDO-Me+SAHA,化合物I5,在21天内每三天给药一次并监测肿瘤大小的变化。
本发明化合物I5的体内抗肿瘤活性研究结果,control组裸鼠存在稳定的肿瘤生长。然而,化合物I5治疗组显着减少了肺肿瘤的体积和重量。用相同摩尔剂量的I5治疗比对照药CDDO-Me+SAHA联合给药组显示出更优的抗肿瘤活性,在I5治疗结束时产生了更显著的肿瘤抑制率。与PBS处理的对照组(0.95±0.10g)相比,经I5治疗的小鼠的肿瘤重量(0.22±0.06g)减少了77%(w/w),而对照药CDDO-Me+SAHA联合给药组处理的对照组肿瘤重量(0.47±0.17g)减少了50%(w/w)。结果表明,本发明化合物I5体内对肺癌肿瘤的生长具有显著的抗肿瘤活性。
将A549荷瘤BALB/c裸鼠用于荧光成像,尾静脉注射I5(40mg/kg,50μL),分别在1h,2h,4h,8h,12h和24h观察本发明化合物在荷瘤小鼠的肿瘤靶向荧光成像情况。首先,将小鼠置于IVIS密闭室内的加热台上,并用2.5%异氟烷维持麻醉。使用配备有激发滤光片:690nm和发射滤光片:720nm的Caliper IVIS Lumina II体内光学成像系统进行所有图像采集。结果表明,本发明化合物能够精准地在荷瘤小鼠的肿瘤部位活体成像,且近红外荧光成像持续时间超过24h(参照图4)。
最后处死小鼠,收集包括心脏,肺,肝,肾,脾,结肠和肿瘤在内的主要器官,并用荧光成像系统成像。实验结果发现本发明化合物能够高选择性地对肿瘤组织成像,肿瘤组织荧光强度显著高于其他正常组织(参照图5)。
综上所述,本发明的具有近红外成像功能的CDDO-Me靶向前药利用具有ROS和GSH双重响应的双巯基异丙酸,获得了具有肿瘤微环境ROS/GSH双重响应的近红外荧光成像特性,在肿瘤组织选择性释放出原药CDDO-Me和HDAC抑制剂SAHA,从而发挥肿瘤选择性荧光成像示踪和协同高效肿瘤治疗作用。
对于本领域技术人员而言,显然本发明不限于上述示范性实施例的细节,而且在不背离本发明的精神或基本特征的情况下,能够以其他的具体形式实现本发明。因此,无论从哪一点来看,均应将实施例看作是示范性的,而且是非限制性的,本发明的范围由所附权利要求而不是上述说明限定,因此旨在将落在权利要求的等同要件的含义和范围内的所有变化囊括在本发明内。不应将权利要求中的任何附图标记视为限制所涉及的权利要求。
Claims (7)
3.根据权利要求1或2所述的具有近红外成像功能的CDDO-Me靶向前药在制备具有ROS/GSH双重响应的肿瘤选择性近红外荧光成像试剂的用途。
4.根据权利要求1或2所述的具有近红外成像功能的CDDO-Me靶向前药在制备具有ROS/GSH双重响应的肿瘤选择性药物释放试剂的用途。
5.根据权利要求3或4所述的用途,其特征在于:该CDDO-Me靶向前药在对肺癌、结肠癌、肝癌、乳腺癌、宫颈癌的选择性荧光成像和治疗的应用。
6.根据权利要求3或4所述的用途,其特征在于:可实现CDDO-Me原药在体内可视化释放和动态分布的监测作用。
7.根据权利要求3或4所述的用途,其特征在于:可实现对体内肿瘤组织治疗效果的实时监控作用的应用。
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