CN115010643A - 苯磺酰胺修饰的七甲川吲哚花菁小分子和制备方法及用途 - Google Patents
苯磺酰胺修饰的七甲川吲哚花菁小分子和制备方法及用途 Download PDFInfo
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- CN115010643A CN115010643A CN202210631570.5A CN202210631570A CN115010643A CN 115010643 A CN115010643 A CN 115010643A CN 202210631570 A CN202210631570 A CN 202210631570A CN 115010643 A CN115010643 A CN 115010643A
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- benzenesulfonamide
- heptamethine
- indole
- tumor
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Abstract
本发明涉及一种苯磺酰胺修饰的七甲川吲哚花菁小分子和制备方法及用途，所述七甲川吲哚花菁小分子由苯磺酰胺衍生物侧链与吲哚七甲川链共价连接得到。本发明设计将具有内质网靶向的苯磺酰胺基团和具有近红外成像、肿瘤靶向的多功能吲哚七甲川花菁染料分子共价连接，通过苯磺酰胺环上具有推电子和拉电子的不同电子效应基团调节分子的电子云密度分布和前线轨道分子能级，实现808nm近红外光照射下激发出染料分子最大的光热(PTT)和光动力(PDT)效能，同时光热能量释放伴随产生光声信号，进而实现近红外和光声双模态成像。
Description
技术领域
本发明属于生物医学工程技术领域，特别涉及一种苯磺酰胺修饰的七甲川吲哚花菁小分子和制备方法及用途。
背景技术
当今社会，癌症已经成为危害人类生命健康的重大疾病，在全世界范围内每年造成了成千上万的人死亡，对人类的生命健康安全造成了重大的威胁。目前临床上主要使用化疗或放疗手段来治疗恶性肿瘤，但大多数化疗药物不具有肿瘤靶向性，对正常组织细胞也具有毒害作用，而高能射线放疗也存在对非肿瘤的正常组织或器官造成不可逆的辐射损伤，危害人体健康。此外，化疗或放疗方式缺乏对肿瘤组织的显影功能，不利于对肿瘤侵袭的病灶组织的精准医疗，同时对转移瘤也缺乏有效的治疗手段。因此，对肿瘤的实时诊断显影和精准治疗对当今医学仍然是巨大的挑战。
近年来，光疗(phototherapy)作为一种局部的、非侵袭性的肿瘤治疗手段已受到极大的关注。由于其局部光照治疗的特点，可极大减少全身治疗对人体的伤害，降低毒副作用。肿瘤光疗是依靠光敏剂在激光照射下产生光热(PTT)和光动力(PDT)两种效应的治疗方式。在激光照射下，光敏剂吸收光能然后转化为热能或通过系间跨越产生大量的活性氧，进而导致肿瘤细胞死亡。此外，一个理想的光敏剂还应具有低毒副作用，成像功能好和肿瘤优先蓄积能力的特性。然而，单纯的光疗对肿瘤的杀伤效果还略显不足，尤其是对转移瘤的抑制更加不尽人意。
内质网(endoplasmic reticulum stress,ER)作为细胞内蛋白质合成和折叠的场所，对维持细胞的新陈代谢和稳态具有重要的作用。内质网功能的紊乱能引起内质网应激反应发生，进而引发细胞免疫原性死亡。近年来，有文献(W.Li,J.Yang,L.Luo,M.Jiang,B.Qin,H.Yin,C.Zhu,X.Yuan,J.Zhang,Z.Luo,Du Y,Q.Li,Y.Lou,Y.Qiu,J.You,Nat Commun2019,10,3349.)报道了纳米材料包裹光敏剂，靶向肿瘤细胞内质网，在激光照射下能产生光热和光动力效果，诱导肿瘤细胞内质网应激，引起肿瘤细胞免疫免疫原性死亡发生。然而，纳米材料存在重复性差和难以大规模制备的缺点，使其在走向临床应用还有很长一段距离。
发明内容
本发明的目的是提供了一种苯磺酰胺衍修饰的七甲川吲哚花菁小分子和制备方法及用途。所述七甲川吲哚花菁小分子由苯磺酰胺衍生物侧链与吲哚七甲川链共价连接得到。本发明将具有内质网靶向的苯磺酰胺基团和具有近红外成像、肿瘤靶向的多功能吲哚七甲川花菁染料分子共价连接，通过苯磺酰胺环上具有推电子和拉电子的不同电子效应基团调节分子的电子云密度分布和前线轨道分子能级，实现808nm近红外光照射下激发出染料分子最大的光热(PTT)和光动力(PDT)效能，同时光热能量释放伴随产生光声信号，进而实现近红外和光声双模态成像。所述苯磺酰胺衍生修饰的七甲川吲哚花菁小分子是以肿瘤内质网为靶向，在近红外荧光成像引导下，同步实现精确高效的光热、光动力和免疫多模态协同抗肿瘤作用的多功能有机小分子。本发明所述七甲川吲哚花菁小分子对原位瘤和远端瘤的生长均表现很好的抑制作用，本发明所述七甲川吲哚花菁小分子合成方法简单，反应条件温和，易于操作，具有良好的转化和应用前景。
本发明的技术方案如下：
苯磺酰胺修饰的七甲川吲哚花菁小分子，由肿瘤靶向的七甲川吲哚花菁共轭链和苯磺酰胺修饰的N-烷基侧链构成，其具有以下结构通式：
其中，n＝1-10；R1，R2、R3、R4、R5和R6为氢、卤素、烷基、烷氧基、乙烯基、羧基、磺酸基、羟基、氨基、硝基、氰基、醛基、甲酰基、芳基中的任意一种；X－为卤素离子、烷基磺酸根、四氟硼酸根、高氯酸根中任意的一种。
所述卤素离子为碘离子、氯离子、溴离子。
所述卤素为氟、氯、溴和碘；所述烷基为甲基、乙基、丙基、丁基，异丙基、叔丁基，环戊基；所述烷氧基为甲氧基、乙氧基、丙氧基；所述芳基为苯基和萘基。
上述的苯磺酰胺物修饰的七甲川吲哚花菁小分子的制备方法，有以下步骤：
1)N,N-二甲基甲酰胺(DMF)为反应溶剂，冰浴下依次缓慢滴加三氯氧磷(POCl3)和环己酮，再缓慢升温至45℃反应6h。反应结束后，反应液经过冰水沉淀，抽滤，滤饼经乙酸乙酯重结晶后得缩合剂A；
2)苯磺酰氯衍生物和伯胺衍生物溶于二氯甲烷(DCM)，冰浴下缓慢滴加三乙胺(TEA)，滴加完毕后室温反应过夜。反应结束后，反应液旋转蒸发除去溶剂，柱层析分离得到苯磺酰胺衍生物B；
或者将苯磺酰胺衍生物和末端卤代烷烃衍生物溶于乙腈(MeCN)，在碳酸钾(K2CO3)作用下60℃加热反应，反应结束后柱层析分离制得苯磺酰胺衍生物B；
3)乙腈(MeCN)为反应溶剂，2,3,3-三甲基-3H吲哚衍生物和苯磺酰胺衍生物B在氮气保护下110℃加热回流反应16h，反应结束后，旋转蒸发浓缩得到残留物，经乙酸乙酯洗涤后，得到吲哚季铵盐C；
4)无水乙醇为反应溶剂，乙酸钠为碱性添加剂，将步骤1)所得缩合剂A与步骤3)所得吲哚季铵盐C在氮气保护下于70℃反应3h，反应液减压浓缩得到残留物，经柱层析分离得到苯磺酰胺衍生物修饰的七甲川吲哚花菁染料D。
步骤1)中所述环己酮：POCl3：DMF的摩尔比为3:1:2。
步骤2)中所述苯磺酰氯衍生物：伯胺衍生物：三乙胺的摩尔比为1.2:1:2-1.5:1:2.5；
步骤2)中所述苯磺酰胺衍生物：卤代烷烃衍生物：碳酸钾的摩尔比为1:3:1-1:5:1。
步骤4)中所述缩合剂A与吲哚季铵盐C的摩尔比为2:1-3:1。
步骤2)所述柱层析的洗脱剂为正己烷和乙酸乙酯混合溶剂；所述正己烷：乙酸乙酯的体积比为＝5:1-3:1。
步骤4)所述柱层析的洗脱剂为二氯甲烷和甲醇混合溶剂；所述二氯甲烷：甲醇的体积比为＝20:1-10:1。
上述苯磺酰胺修饰的七甲川吲哚花菁小分子在制备诊断肿瘤显影剂中的应用；所述显影剂用于在肿瘤内质网蓄积、荧光和光声双模态成像。
上述苯磺酰胺修饰的七甲川吲哚花菁小分子在制备治疗肿瘤光疗和免疫药物中的应用；所述药物在808nm激光照射下产生光热和光动力，引发肿瘤细胞免疫原性死亡。
本发明所述苯磺酰胺修饰的七甲川吲哚花菁小分子毒副作用小，具有近红外荧光和光声双模态成像功能，能靶向蓄积肿瘤细胞内质网。
本发明所述肿瘤光疗和免疫治疗是指苯磺酰胺修饰的七甲川吲哚花菁小分子在808nm激光照射下能产生光热和光动力作用，引起内质网应激反应，进而引发肿瘤细胞的免疫原性死亡，能显著抑制原位和远端肿瘤的生长。
本发明所述苯磺酰胺修饰的七甲川吲哚花菁小分子制备方法简单，反应条件温和，原料价廉易得，易于实施。
申请人前期的研究发现七甲川吲哚花菁骨架可选择性地高效蓄积在肿瘤细胞内，将经典的内质网靶向基团(苯磺酰胺)引入到七甲川吲哚花菁骨架侧链，同时基于计算机辅助和密度泛函计算(DFT)，通过在苯磺酰胺环上引入不同电子特性的基团，合理地设计合成了一种具有低细胞毒性，同时还具有肿瘤内质网蓄积、荧光和光声双模态成像、显著的光热和光动力效能以及对原位和远端肿瘤光免疫治疗作用的苯磺酰胺修饰的七甲川吲哚花菁小分子。
研究表明，所述小分子能蓄积于肿瘤细胞内质网，在808nm激光照射下，能产生光热和光动力效应，引发内质网应激，诱导肿瘤细胞免疫原性死亡(ICD)，进而对原位和远端肿瘤生长表现显著的抑制作用。
本发明所述小分子经尾静脉注射，靶向蓄积肿瘤部位，在近红外光照射下能发射出荧光，具有显影作用，同时对原位瘤用808nm激光照射，原位瘤和远端肿瘤的生长显著抑制。此外，光热过程伴随产生光声信号，有助于该类小分子用于深部肿瘤病灶组织成像。因此，所述小分子有望用于肿瘤实时诊断显影以及光疗和免疫协同治疗。
申请人实验表明，所述苯磺酰胺修饰的七甲川吲哚花菁小分子具有荧光和光声双模态成像以及肿瘤细胞内质网蓄积特性，808nm激光照射原位瘤，能同时显著抑制原位瘤和远端肿瘤的生长，可以作为精准治疗肿瘤的小分子药物。
下面对本发明的较佳实施例进行详细阐述，以使本发明的优点和特征能更易被本领域人员理解。任何本领域人员根据本发明的技术方案及其发明构思进行等同替换或改变均属于本发明保护范畴。
附图说明
图1为实施例6中化合物D1-D9的荧光光谱；
图2为实施例7中化合物D1-D5的体外光热效应曲线；
图3为实施例8中化合物D1-D5的体外光动力SOSG荧光强度；
图4为实施例9中化合物D1-D5的内质网蓄积图；
图5为实施例10中化合物D5的肿瘤靶向近红外成像图；
图6为实施例11中各处理组中免疫原性死亡(ICD)标志物相关因子表达；
图7为实施例12中各处理组中DC细胞成熟度流式图；
图8为实施例13中化合物D5对原位和远端肿瘤生长抑制曲线；
图9为实施例14中治疗21天后化合物D5与对照组的原位和远端肿瘤体积大小图。
具体实施方式
实施例中所采用的试剂与仪器：
除了溶剂乙腈、无水乙醇和N,N-二甲基甲酰胺(DMF)为除水处理后的超干溶剂，其他所使用的化学试剂和溶剂均购买于sigma-aldrich、Adamas或Greagent试剂公司并直接使用。
所有反应在氮气及避光条件下进行，用硅胶薄层层析版监测反应进程。硅胶薄层层析板使用乳山市太阳干燥剂有限公司生产的高效薄层层析硅胶板(型号GF-254)，采用254nm荧光或日光下目视直接进行检测。
采用烟台市芝罘黄务硅胶开发试验厂所生产的柱层析硅胶(200-300目)分离纯化染料分子。层析所用有机溶剂均为精馏级分析纯试剂。
所有化合物核磁氢谱(1H NMR)由美国Agilent公司生产的600MHz核磁共振谱仪测定，TMS作内标，用CDCl3和CD3OD作溶剂，δ值单位为ppm。
高分辨质谱(HRMS)由德国Bruker Ultraflextreme MALDI-TOF质谱仪测定；紫外吸收光谱由日本SHIMADZU公司紫外可见荧光分光光度计UV-3600采集；荧光光谱由美国Thermo Fisher公司的Lumina Fluorescence Spectrometer测定。
下述实施例中所使用的实验方法如无特殊说明，均为常规方法。下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
实施例1化合物A的合成
于250mL圆底烧瓶中加入50mL N,N-二甲基甲酰胺(DMF),冰浴下依次缓慢滴加34mL三氯氧磷(POCl3)和环己酮10g,0.1mol，再缓慢升温至45℃反应6h。反应结束后，反应液经过冰水沉淀，抽滤，滤饼经乙酸乙酯重结晶后得缩合剂A，直接用于下一步反应。
实施例2化合物B1-B7的合成
于100mL圆底烧瓶中加入苯磺酰氯衍生物(见上述化学反应结构式)
15mmol和3-溴丙胺氢溴酸盐10mmol,2.19g，20mL二氯甲烷(DCM)溶解，冰浴下缓慢滴加三乙胺25mmol,3.5mL，滴加完毕后室温反应过夜。反应结束后，反应液旋转蒸发除去溶剂，柱层析分离得到苯磺酰胺衍生物B1-B7。
其中化合物B1-B5,B7的核磁氢谱(1H NMR)结果如下所示：
B1:1H NMR(600MHz,CDCl3)δ7.88(d,J＝7.2Hz,2H),7.59(t,J＝7.2Hz,1H),7.52(t,J＝7.8Hz,2H),5.13(t,J＝6.0Hz,1H),3.40(t,J＝6.0Hz,2H),3.11(q,J＝6.6Hz,2H),2.04–1.99(m,2H)ppm.
B2:1H NMR(600MHz,CDCl3)δ7.75(d,J＝7.8Hz,2H),7.32(d,J＝8.4Hz,2H),4.65(t,J＝6.0Hz,1H),3.42(t,J＝6.0Hz,2H),3.11(q,J＝6.6Hz,2H),2.43(s,3H),2.05–2.01(m,2H)ppm.
B3:1H NMR(600MHz,CDCl3)δ8.38(d,J＝9.0Hz,2H),8.07(d,J＝9.0Hz,2H),4.95(t,J＝6.0Hz,1H),3.43(t,J＝6.0Hz,2H),3.21(q,J＝6.6Hz,2H),2.09–2.05(m,2H)ppm.
B4:1H NMR(600MHz,CDCl3)δ8.16–8.15(m,1H),7.89–7.87(m,1H),7.78–7.74(m,2H),5.43(t,J＝6.0Hz,1H),3.46(t,J＝6.0Hz,2H),3.28(q,J＝6.6Hz,2H),2.13–2.09(m,2H)ppm.
B5:1H NMR(600MHz,CDCl3)δ9.12(s,1H),8.60(s,1H),8.29(d,J＝9.6Hz,1H),7.01(d,J＝9.6Hz,1H),3.66(q,J＝6.6,2H),3.54(t,J＝5.4Hz,2H),2.34–2.30(m,2H)ppm.
B7:1H NMR(600MHz,CDCl3)δ8.38(d,J＝9.0Hz,2H),8.06(d,J＝9.0Hz,2H),4.75(t,J＝6.0Hz,1H),3.38(t,J＝6.6Hz,2H),3.07(q,J＝6.6Hz,2H),1.88(dt,J＝14.4,6.6Hz,2H),1.70–1.65(m,2H)ppm.
实施例3化合物B8的合成
于100mL圆底烧瓶中加入1,10-二溴癸烷(12mmol)和4-硝基苯磺酰胺4mmol,0.81g，16mL乙腈(MeCN)溶解，升温至60℃,薄层色谱监测反应进程。反应结束后，反应液旋转蒸发除去溶剂，柱层析分离得到苯磺酰胺衍生物B8，直接用于下一步反应。
实施例4化合物C1-C9的合成
于100mL圆底烧瓶中加入2,3,3-三甲基-3H吲哚5.12mmol,822μL和苯磺酰胺衍生物B1-B8 3.41mmol，20mL乙腈(MeCN)为反应溶剂，在氮气保护下110℃加热回流反应16h。反应结束后，旋转蒸发浓缩得到残留物，经乙酸乙酯洗涤后，得到吲哚季铵盐C1-C9，直接用于下一步反应。
实施例5化合物D1-D9的合成
于50mL圆底烧瓶中加入吲哚季铵盐C1-C9 0.535mmol和缩合剂A 36.9mg,0.214mmol，10mL无水乙醇为反应溶剂，乙酸钠48.3mg,0.589mmol为碱性添加剂，氮气保护下于70℃反应3h，反应液减压浓缩得到残留物，经柱层析分离得到苯磺酰胺衍生物修饰的七甲川吲哚花菁诊疗剂D1-D9。
其中化合物D1-D5的核磁氢谱(1H NMR)结果如下所示：
D1:1H NMR(600MHz,CDCl3)δ8.29(d,J＝14.3Hz,2H),7.98(d,J＝7.2Hz,4H),7.51–7.42(m,8H),7.33(dd,J＝15.6,7.8Hz,4H),7.20(dd,J＝17.4,7.8Hz,4H),6.30(d,J＝13.8Hz,2H),4.32(t,J＝7.8Hz,4H),3.13(q,J＝6.0Hz,4H),2.73(t,J＝6.0Hz,4H),2.01–2.07(m,4H),1.93–1.89(m,2H),1.67(s,12H)ppm.
D2:1H NMR(600MHz,CDCl3)δ8.29(d,J＝14.4Hz,2H),7.84(d,J＝8.4Hz,4H),7.35–7.31(m,6H),7.26–7.18(m,8H),6.30(d,J＝13.8Hz,2H),4.32(t,J＝7.8Hz,4H),3.12(q,J＝6.0Hz,4H),2.74(t,J＝6.0Hz,4H),2.34(s,6H),2.10–2.06(m,4H),1.94–1.90(m,2H),1.66(s,12H)ppm.
D3:1H NMR(600MHz,CDCl3)δ8.31(d,J＝14.4Hz,2H),8.26–8.22(m,10H),7.36–7.33(m,4H),7.22(t,J＝7.2Hz,2H),7.17(d,J＝7.8Hz,2H),6.31(d,J＝14.4Hz,2H),4.31(t,J＝8.4Hz,4H),3.14(q,J＝6.0Hz,4H),2.74(t,J＝6.0Hz,4H),2.14–2.09(m,4H),1.91(t,J＝6.0Hz,2H),1.68(s,12H)ppm.
D4:1H NMR(600MHz,CDCl3)δ8.31–8.28(m,4H),7.74–7.68(m,6H),7.45(t,J＝6.0Hz,2H),7.36–7.33(m,4H),7.24–7.19(m,4H),6.25(d,J＝14.4Hz,2H),4.31(t,J＝7.8Hz,2H),3.33(q,J＝6.0Hz,4H),2.67(t,J＝6.0Hz,4H),2.16–2.11(m,4H),1.87–1.83(m,2H),1.68(s,12H)ppm.
D5:1H NMR(600MHz,CD3OD)δ8.74(d,J＝1.8Hz,2H),8.58(dd,J＝8.4,2.4Hz,2H),8.44(d,J＝13.8Hz,2H),8.30(d,J＝9.0Hz,2H),7.53(d,J＝7.2Hz,2H),7.42(t,J＝7.8Hz,2H),7.34–7.28(m,4H),6.33(d,J＝14.4Hz,2H),4.25(t,J＝7.8Hz,4H),3.25(t,J＝6.6Hz,4H),2.74(t,J＝6.0Hz,4H),2.10–2.05(m,4H),1.99–1.92(m,2H),1.74(s,12H)ppm.
化合物D1-D9的产率和高分辨质谱(HRMS)结果如下表所示:
实施例6化合物D1-D9的荧光特性
用万分之一分析天平精确称量苯磺酰胺衍生物修饰的七甲川吲哚花菁小分子D1-D9，配成10mM的二甲基亚砜(DMSO)溶液，备用。测试时，用DMSO稀释成4μM的染料溶液，用Lumina Fluorescence Spectrometer荧光光谱仪测定化合物D1-D9的荧光发射光谱。如图1所示，测试结果表明所有化合物的最大荧光发射波长均位于近红外区(700-900nm)，表现出近红外荧光发射特性。
实施例7化合物D1-D5的体外光热效应
用DMSO将吲哚菁绿(ICG)和化合物D1-D5配置成10mM的储备液，-20℃保存。从以上配置的D1-D5化合物的储存液中各吸取0.6μL溶于1.5mL磷酸盐缓冲液(PBS，10mM,PH＝7.4)中，配制成4μM的检测液，分别加入1.5mL离心管中。将装有检测液的离心管置于808nm、1.0W/cm2的近红外激光(Laser)下照射5分钟，每隔30秒记录温度的变化，结果见图2：化合物D1-D5中，D5的升温效果最为优异(ΔT＝21℃)，并且化合物D2-D5升温效果显著优于临床批准使用的非肿瘤靶向性的吲哚菁绿(ICG)，表明本发明制备的所述化合物具有较优异的光热特性，为实现肿瘤光热治疗提供可行性。
实施例8化合物D1-D5的体外光动力效应
于2mL的浓度为4μM的各种花菁染料水溶液中分别加入SOSG探针(10％甲醇水溶液配制)，使SOSG的浓度为5μM，混匀，再分别于1W/cm2的808nm激光下照射5分钟，随后以495nm波长激发，立即测定在500-600nm的荧光发射波谱，以525nm处的最大发射峰荧光强度来量化单态氧的生成。如图3所示，实验结果显示与空白溶液为对照，化合物D1-D5在接受近红外808nm激光照射后均能够产生大量的单线态氧，其中以化合物D5单线态氧生成能力最强，为实现肿瘤光动力治疗提供可行性。
实施例9化合物D1-D5的内质网蓄积特性
将乳腺癌(4T1)细胞消化计数，于激光共聚焦小皿中每孔接种1×105个细胞，放置于37℃恒温细胞培养箱过夜。向细胞中加入浓度为1μM的化合物D1-D5，孵育4h后用内质网探针ER-Tracker Green处理细胞，再孵育30分钟用PBS洗涤3次，用4％多聚甲醛固定10min，再用PBS洗涤3次后用激光共聚焦显微镜观察化合物D1-D5的荧光信号(红色，激发波长504nm，发射波长511nm)、ER-Tracker Green(绿色，激发波长633nm，发射波长780nm)。如图4所示，化合物D1-D5在体外培养条件可被4T1细胞高效摄取，均能蓄积到肿瘤细胞内质网，显示出所设计合成的苯磺酰胺衍生物修饰的七甲川吲哚花菁小分子具有优异的内质网蓄积能力。
实施例10化合物D5的肿瘤靶向能力
利用4T1细胞建立了Balb/c小鼠(雌性)大腿外侧皮下荷瘤模型。经尾静脉给予0.25mg/kg的化合物D5，利用小动物活体成像系统(
Trilogy,Gene Co.,Ltd)进行近红外荧光实时成像，观察不同时间点化合物D5在小鼠体内的代谢分布。如图5结果所示，化合物D5展现出较好的肿瘤选择性蓄积特性，并在48小时于肿瘤部位蓄积达到峰值，对肿瘤的诊断和精确光学治疗具有重要的意义。
实施例11化合物D5在细胞水平诱导免疫反应
同时设置对照组PBS组、Laser组和PBS+Laser组，4T1细胞与4μM D5在共聚焦小皿中37℃共孵育24h后，用PBS洗涤，然后用近红外激光808nm照射5分钟(1W/cm2)，再孵育24h后，细胞用4％多聚甲醛在室温下固定15分钟，与增强免疫染色渗透缓冲液孵育5分钟。然后细胞与阻断缓冲液孵育60分钟以去除多余的蛋白结合位点，并与抗钙网蛋白、HMGB1和CHOP的一抗(Abcam,Cambridge,MA,USA)在4℃条件下孵育过夜。随后，将细胞与兔抗IgG Fab2Fluor(R)488或抗小鼠IgG Fab2 Fluor(R)555二抗体(Cell Signaling Technology,Danvers,MA,USA)在室温黑暗条件下孵育60分钟。用DAPI(1:1000)复染细胞核10min，然后在CLLSM下成像。如图6所示，免疫原性死亡(ICD)标志物相关因子CHOP和CRT表达明显增多，HMGB1表达显著减少，充分证明化合物D5在近红外808nm激光照射下能诱导肿瘤细胞发生免疫反应。
实施例12化合物D5光疗后促进DC细胞成熟
从4周大的雌性Balb/c小鼠中分离提取小鼠骨髓源树突状细胞(BMDCs)。然后用含有粒细胞-巨噬细胞集落刺激因子(GM-CSF)(25ng mL-1)和IL-4(10ng mL-1)的PRMI-1640培养基(R&D Systems,Minneapolis,MN,USA)培养BMDCs。第6天获得未成熟的DCs细胞，与各处理组(PBS组、光照Laser组、给药D5组和给药D5加光照Laser组)的4T-1细胞上清共培养过夜。接下来，采集DCs细胞并与抗小鼠FITC-CD11c、抗小鼠PE-CD86、抗小鼠APC-CD86共染色(BioLegend,San Diego,CA,USA)。最后采用流式细胞仪检测成熟DCs细胞(CD11c+CD80+CD86+)的百分率。如图7所示，给药D5加光照Laser组(D5+Laser组)DC细胞的成熟度显著高于其他三个对照组，表明在激光照射下化合物D5能促进DC细胞成熟，进而诱导肿瘤细胞的免疫反应。
实施例13化合物D5的肿瘤光疗和免疫治疗应用
利用4T1细胞建立了Balb/c小鼠(雌性)左侧(远端肿瘤)和右侧(原位肿瘤)大腿外侧皮下荷瘤模型，分为(1)PBS组，(2)单独给药化合物D5组，(3)PBS加光照Laser组和(4)给药化合物D5加光照Laser组，每组10只。经尾静脉给予组别(2)和(4)小鼠以2.5mg/kg的化合物D5，48小时后再分别只给予组别(3)和(4)小鼠右侧大腿部位的肿瘤(原位肿瘤)1W/cm2的808nm激光照射5分钟，连续观察测量上述4组小鼠体重和两侧肿瘤的体积大小变化。如图8和图9结果所示，只有组别4中小鼠原位和远端肿瘤生长都明显受到抑制，甚至发生肿瘤消融，而其余三组小鼠两侧肿瘤生长没有明显观察到抑制现象，显示出在808nm激光照射下，化合物D5表现出对肿瘤良好的光疗和免疫治疗效果，为开发一种有效的肿瘤光免疫诊疗剂提供了可能性。
Claims (11)
1.一种苯磺酰胺修饰的七甲川吲哚花菁小分子，该小分子由肿瘤靶向的七甲川吲哚花菁共轭链和苯磺酰胺修饰的N-烷基侧链构成，其具有以下结构通式：
其中，n＝1-10；R1，R2、R3、R4、R5和R6为氢、卤素、烷基、烷氧基、乙烯基、羧基、磺酸基、羟基、氨基、硝基、氰基、醛基、甲酰基、芳基中的任意一种；X－为碘离子、氯离子、溴离子、烷基磺酸根、四氟硼酸根、高氯酸根中任意的一种；
优选地，所述卤素离子为碘离子、氯离子、溴离子。
2.根据权利要求1所述的小分子，其特征在于：所述卤素为氟、氯、溴和碘；所述烷基为甲基、乙基、丙基、丁基，异丙基、叔丁基，环戊基；所述烷氧基为甲氧基、乙氧基、丙氧基；所述芳基为苯基和萘基。
3.权利要求1或2所述的苯磺酰胺修饰的七甲川吲哚花菁小分子的制备方法，其特征在于，有以下步骤：
1)N,N-二甲基甲酰胺(DMF)为反应溶剂，冰浴下依次缓慢滴加三氯氧磷(POCl3)和环己酮，再缓慢升温至45℃反应6h，反应液经过冰水沉淀，抽滤，滤饼经乙酸乙酯重结晶后得缩合剂A；
2)苯磺酰氯衍生物和伯胺衍生物溶于二氯甲烷(DCM)，冰浴下缓慢滴加三乙胺(TEA)，滴加完毕后室温反应过夜，反应液旋转蒸发除去溶剂，柱层析分离得到苯磺酰胺衍生物B；
3)乙腈(MeCN)为反应溶剂，2,3,3-三甲基-3H吲哚衍生物和苯磺酰胺衍生物B在氮气保护下110℃加热回流反应16h，旋转蒸发浓缩得到残留物，经乙酸乙酯洗涤后，得到吲哚季铵盐C；
4)无水乙醇为反应溶剂，乙酸钠为碱性添加剂，将步骤1)所得缩合剂A与步骤3)所得吲哚季铵盐C在氮气保护下于70℃反应3h，反应液减压浓缩得到残留物，经柱层析分离得到苯磺酰胺衍生物修饰的七甲川吲哚花菁染料D。
4.根据权利要求3所述的方法，其特征在于：步骤2)还可以将苯磺酰胺衍生物和末端卤代烷烃衍生物溶于乙腈(MeCN)，在碳酸钾(K2CO3)作用下60℃加热反应。反应结束后柱层析分离制得苯磺酰胺衍生物B。
5.根据权利要求3所述的方法，其特征在于：步骤1)中所述环己酮：POCl3：DMF的摩尔比为3:1:2。
6.根据权利要求3所述的方法，其特征在于：步骤2)中所述苯磺酰氯衍生物：伯胺衍生物：三乙胺的摩尔比为1.2:1:2-1.5:1:2.5；
所述苯磺酰胺衍生物：卤代烷烃衍生物：碳酸钾的摩尔比为1:3:1-1:5:1。
7.根据权利要求3所述的方法，其特征在于：步骤4)中所述吲哚季铵盐B与缩合剂C的摩尔比为2:1-3:1。
8.根据权利要求3所述的方法，其特征在于：步骤2)所述柱层析的洗脱剂为正己烷和乙酸乙酯混合溶剂；
优选地，所述正己烷：乙酸乙酯的体积比为＝5:1-3:1。
9.根据权利要求3所述的方法，其特征在于：步骤4)所述柱层析的洗脱剂为二氯甲烷和甲醇混合溶剂；
优选地，所述二氯甲烷：甲醇的体积比为＝20:1-10:1。
10.权利要求1或2所述苯磺酰胺修饰的七甲川吲哚花菁小分子在制备诊断肿瘤显影剂中的应用；
优选地，所述显影剂用于在肿瘤内质网蓄积、荧光和光声双模态成像。
11.权利要求1或2所述苯磺酰胺修饰的七甲川吲哚花菁小分子在制备治疗肿瘤光疗和免疫药物中的应用；
优选地，所述药物在808nm激光照射下产生光热和光动力，引发肿瘤细胞免疫原性死亡。
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