CN116589404A - 一种靶向β淀粉样蛋白的光敏剂及其制备方法与应用 - Google Patents
一种靶向β淀粉样蛋白的光敏剂及其制备方法与应用 Download PDFInfo
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Abstract
本发明公开了一种靶向β淀粉样蛋白的光敏剂及其制备方法与应用。该光敏剂为A‑D‑A构型光敏剂,其结构式如式I所示。本发明中合成的光敏剂的发射波长较长,在与β淀粉样蛋白结合后荧光信号明显增强,并且能有效产生单线态氧,即其可以靶向标记Aβ蛋白,用于Aβ蛋白的荧光成像;且本发明中合成的光敏剂在与阿尔兹海默症(AD)模型小鼠脑部的β淀粉样蛋白结合后,能有效改善AD小鼠的认知能力,降低Aβ介导的神经毒性,促进Aβ斑块的清除,即可用于阿尔兹海默症的早期诊断和治疗。
Description
技术领域
本发明属于生物医药领域,特别涉及一种靶向β淀粉样蛋白的光敏剂及其制备方法与应用。
背景技术
阿尔茨海默病(AD)作为一种不可逆的神经退行性疾病,主要表现为认知功能发生障碍和渐进性记忆缺失。AD的研究已经受到学者的广泛关注,但其具体致病机制尚未明确。目前被国内外公认的学说主要包括:β淀粉样蛋白异常沉积学说、Tau蛋白过度磷酸化学说、免疫炎性反应学说、线粒体功能紊乱学说、氧化应激学说等。淀粉样蛋白(amyloidβ-protein,Aβ)学说在目前AD的早期诊断和治疗中起到非常关键的作用,靶向β淀粉样蛋白的单克隆抗体药物Leqembi在2023年已经获得FDA加速批准上市。该学说认为淀粉样前体蛋白(APP)在β位点(Asp1)被β-分泌酶切割产生具有99个氨基酸的C-末端片段(CTF),再通过γ-分泌酶进一步形成长短不同的Aβ片段,在正常生理条件下Aβ的形成和降解处于一种平衡的状态,而AD患者脑中Aβ蛋白的生成和清除具有不协调的特点,在其大脑皮层或海马区过量的Aβ错误折叠形成具有神经毒性的低聚物和不溶性纤维,从而导致认知能力的下降。
近年来,光氧化Aβ蛋白,成为一项新兴的AD药物干预策略。目前较多学者认为,Aβ蛋白侧链的光氧化会导致其自组装成富含β-折叠的聚集体的倾向降低,其中Met残基最容易受到氧化应激的影响,Met35中硫的电子对被氧原子占据产生亚砜或砜,导致β链的疏水性降低,Aβ蛋白C末端结构域的灵活性增加,从而抑制Aβ蛋白的聚集。Aβ蛋白侧链上的His13和His14也可以通过光氧化转化为脱氢-2-咪唑啉,从而阻碍肽的构象变化和Aβ单体之间的分子间组装。在这种情况下,Met和His的光敏化修饰可能导致金属离子催化的ROS(Aβ诱导细胞毒性的关键参与者)生成减少,显著降低其细胞毒性。另外,也有报道光氧化后的β淀粉样蛋白更容易被小胶质细胞所清除。
目前已经报道的光氧化Aβ蛋白的光敏剂诊疗分子主要包括有机小分子光敏剂(硫黄素、姜黄素类、玫瑰红、亚甲基蓝等)、金属配合物以及配体光敏剂。自从20世纪30年代以来,喹啉类化合物主要作为抗疟疾的药物。随着光敏剂的发展,喹啉类化合物因为具有很好的成药性,并且喹啉类光敏剂具有发射波长在近红外,有效产生活性氧等特点,在光敏剂领域具有很大的潜在价值。
发明内容
本发明的首要目的在于克服现有技术的缺点与不足,提供一种靶向β淀粉样蛋白的光敏剂。
本发明的另一目的在于提供所述靶向β淀粉样蛋白的光敏剂的制备方法。
本发明的再一目的在于提供所述靶向β淀粉样蛋白的光敏剂的应用。
本发明的目的通过下述技术方案实现:
一种靶向β淀粉样蛋白的光敏剂,为A-D-A构型光敏剂,包括吸电子基团A,π共轭部分B,给电子基团C,π共轭部分D,以及吸电子基团E;其中,A部分主要为1-甲基-6-二甲氨基喹啉,B部分为乙烯连接桥,C部分为三苯胺和四苯乙烯等衍生基团,D部分为乙烯连接桥,E部分主要为1-甲基-6-二甲氨基喹啉;优选为具有如下所示的结构:
式中,R选自下述任一基团:
所述的靶向β淀粉样蛋白的光敏剂的制备方法,包括如下步骤:
(1)将N,N-二甲基-1,4-对苯二胺(对二甲氨基苯胺)、巴豆醛和甲苯加入到盐酸溶液中,在保护性气体氛围、115±5℃条件下进行回流反应,待反应结束后经分离纯化得到化合物1,其结构式如式II所示:
(2)将化合物1加入到有机溶剂中,然后加入碘甲烷,在保护性气体氛围、80±5℃条件下进行回流反应,待反应结束后经分离纯化得到化合物2,其结构式如式III所示:
(3)将化合物2和醛类配体化合物加入到有机溶剂中,加入乙酸,于80±5℃条件下进行回流反应(缩合反应),待反应结束后经纯化得到所述靶向β淀粉样蛋白的光敏剂。
步骤(1)和(2)中所述的保护性气体为氮气和氩气中的至少一种。
步骤(1)中所述的N,N-二甲基-1,4-对苯二胺和巴豆醛的摩尔比为1:1~2;优选为1:1.5~2。
步骤(1)中所述的甲苯的用量为按每克N,N-二甲基-1,4-对苯二胺配比8~10ml甲苯计算;优选为按每克N,N-二甲基-1,4-对苯二胺配比8ml甲苯计算。
步骤(1)中所述的盐酸溶液的浓度为6mol/L。
步骤(1)中所述的反应在搅拌条件下进行;所述反应的时间为4~6小时。
步骤(1)中所述的分离纯化优选为通过如下步骤实现:将反应结束后的反应液萃取除去甲苯,然后在冰浴条件下调节pH至中性,最后经二氯甲烷萃取后采用硅胶柱层析纯化,得到化合物1。
所述的硅胶柱层析的条件为:石油醚与乙酸乙酯的体积比为20:1,硅胶200~300目。
步骤(2)和(3)中所述的有机溶剂优选为乙醇。
步骤(2)中所述的化合物1与碘甲烷的摩尔比为1:1~5;优选为1:2~3。
步骤(2)中所述的反应在搅拌条件下进行;所述反应的时间为8~12小时(优选为10小时)。
步骤(2)中所述的分离纯化优选为通过如下步骤实现:将反应结束后的反应液旋转蒸发去除溶剂,然后通过硅胶柱层析纯化,得到化合物1。
所述的硅胶柱层析的条件为:二氯甲烷与甲醇的体积比为40:1,硅胶200~300目。
步骤(3)中所述的醛类配体化合物为如下式中的任意一种:
步骤(3)中所述的化合物2与醛类配体化合物的摩尔比为4~5:1;优选为4:1。
步骤(3)中所述的反应在搅拌条件下进行;所述反应的时间为3~5小时(优选为5小时)。
步骤(3)中所述的乙酸的用量为按其在体系的终浓度为体积百分比0.75%(终浓度为7.5μL/mL)添加计算。
步骤(3)中所述的纯化为采用薄层色谱进行纯化;所述的薄层色谱的条件如下:层析硅胶的目数为200~300目,展开剂为二氯甲烷与甲醇按体积比为100:1配比得到的展开剂。
所述的靶向β淀粉样蛋白的光敏剂在制备Aβ荧光成像剂中的应用。
所述的靶向β淀粉样蛋白的光敏剂在制备诊断和/或治疗阿尔兹海默症的产品中的应用。
所述的产品包括光敏剂、检测或诊断试剂、检测或诊断试剂盒、治疗药物等。
所述的治疗药物包括Aβ光氧化治疗药物。
所述的光氧化治疗优选为利用520nm、1000mW的激光照射10±2min。
所述的β淀粉样蛋白优选为Aβ1-42聚集体。
本发明相对于现有技术具有如下的优点及效果:
1、本发明合成了一种新型A-π-D-π-A喹啉鎓类化合物,以6-二甲氨基-1-甲基喹啉作为化合物母体,在已有的标记Aβ蛋白的光敏剂中尚未发现报道,其可应用于标记Aβ蛋白的荧光成像和光氧化治疗,用于早期阿尔兹海默症的诊断和治疗。
2、本发明中合成的β淀粉样蛋白的靶向光敏剂的发射波长较长,能特异性检测β淀粉样蛋白,在与β淀粉样蛋白结合后荧光信号明显增强,并且能有效产生单线态氧,对Aβ蛋白侧链进行光氧化,导致其自组装成富含β-折叠的聚集体的倾向降低,而光氧化后的Aβ蛋白更容易被小胶质细胞所清除,从而显著降低其神经毒性。
3、本发明中合成A-π-D-π-A构型的2-取代芳乙烯基喹啉类衍生物,以喹啉乙烯衍生物作为荧光团,可以靶向标记Aβ蛋白,且在与阿尔兹海默症(AD)模型小鼠脑部的β淀粉样蛋白结合后,能有效改善AD小鼠的认知能力,对于阿尔兹海默症诊疗试剂的发展具有很重要的指导意义。
附图说明
图1为本发明中的光敏剂的合成路线图(图中,a:巴豆醛(Crotonaldehyde),HCl:H2O=1:1,115℃;b:CH3I,EtOH,80℃;c:不同醛类配体,CH3COOK,80℃)。
图2为本发明中合成的光敏剂的结构式。
图3为本发明中合成的类光敏剂与β淀粉样蛋白的荧光光谱图。
图4为本发明中合成的光敏剂产生单线态氧的能力图。
图5为本发明中合成的光敏剂(QM-TA-2)对AD模型小鼠的水迷宫实验;其中,(A)为潜伏期;(B)为穿越虚拟平台次数;(C)为目标平台停留时间;(D)为目标平台停留距离;(E)为不同组小鼠的代表性游泳路径。
图6为本发明中合成的光敏剂(QM-TA-2)对AD模型小鼠的HE染色图;其中,A为小鼠脑部HE染色结果;B为小鼠脑部HE染色变性细胞的统计结果。
图7为本发明中合成的光敏剂(QM-TA-2)对AD模型小鼠的尼氏染色图;其中,A为不同组别的小鼠脑部尼氏染色图像;B为不同组别的小鼠脑部存活细胞定量统计结果。
图8为本发明者合成的光敏剂(QM-TA-2)对AD模型小鼠的免疫组化图;其中,A为小鼠脑部Aβ斑块的免疫组化图像;B为小鼠脑部Aβ斑块的定量统计结果。
具体实施方式
下面结合实施例对本发明作进一步详细的描述,但本发明的实施方式不限于此。除非特别说明,本发明采用的试剂、方法和设备为本技术领域常规试剂、方法和设备。下列实施例中未注明具体实验条件的试验方法,通常按照常规实验条件或按照制造厂所建议的实验条件。除非特别说明,本发明所用试剂和原材料均可通过市售获得。
实施例1
1.实施化合物的制备(合成路线图见图1):
(1)化合物1的合成步骤:
称取5.8g N,N-二甲基-1,4-对苯二胺(42.60mmol),充分搅拌溶解在浓度为6M的盐酸溶液(250mL)中,加入巴豆醛6.5ml,在氩气保护下加入40ml的纯甲苯溶液,在115℃的温度下进行回流反应,每隔1h监测反应的进程,直至反应结束。直接将反应液倒入分液漏斗中进行萃取,除去上层甲苯,将水相在冰浴条件下调节pH至中性,最后经二氯甲烷萃取,采用硅胶柱层析(石油醚:乙酸乙酯=20:1,v/v;硅胶200~300目)纯化得到黄色固体化合物1。
核磁氢谱表征:
N,N,2-trimethylquinolin-6-amine:Yellow solid;Yield:32.21%;1H NMR(400MHz,Chloroform-d):δ=7.91(dd,J=14.3,8.9Hz,2H),7.36(dd,J=9.3,2.8Hz,1H),7.18(d,J=8.4Hz,1H),6.81(d,J=2.9Hz,1H),3.07(s,6H),2.70(s,3H).
(2)化合物2的合成步骤:
称取2.6g(13.96mmol)化合物1溶解于40mL乙醇中,在氩气保护下滴加碘甲烷4.86g(34.24mmol),在80℃条件下回流反应10h。反应结束后使用旋转蒸发仪器除去溶剂,进一步通过硅胶柱层析纯化(二氯甲烷:甲醇=40:1,v/v;硅胶200~300目),得到橙黄色固体化合物2,产率为25.06%。
6-(dimethylamino)-1,2-dimethylquinolin-1-ium:Yellow solid;Yield:25.06%;1H NMR(400MHz,DMSO-d6):δ=8.73(d,J=8.6Hz,1H),8.35(d,J=9.7Hz,1H),7.86(d,J=8.6Hz,1H),7.77(dd,J=9.9,3.0Hz,1H),7.27(d,J=3.0Hz,1H),4.36(s,3H),3.13(d,J=1.2Hz,6H),2.95(s,3H).
(3)A-D-A型化合物的合成步骤:
称取化合物2和不同的醛类配体化合物(摩尔比为4:1)溶解在乙醇中,加入适量乙酸(150μL,终浓度为7.5μL/mL),在80℃的温度下回流5h,通过制备的薄层色谱纯化,以涂布于支持板上的1mm层析硅胶(200~300目)作为固定相,以及二氯甲烷:甲醇(100:1,v/v)混合溶液作为展开剂,在室温下得到深蓝色的固体。
2,2'-((1E,1'E)-((phenylazanediyl)bis(4,1-phenylene))bis(ethene-2,1-diyl))bis(6-(dimethylamino)-1-methylquinolin-1-ium):Blue solid;Yield:54.21%;1H NMR(400MHz,DMSO-d6)δ8.70(d,J=9.1Hz,2H),8.35(dd,J=9.5,5.5Hz,4H),8.02(d,J=15.8Hz,2H),7.89(d,J=8.5Hz,4H),7.80–7.63(m,4H),7.47(t,J=7.7Hz,2H),7.37–7.02(m,8H),4.48(s,6H),3.35(s,12H).13C NMR(101MHz,DMSO-d6)δ150.73,149.69,149.10,143.65,141.51,132.09,130.88,130.61,130.34,126.64,123.39,122.98,121.14,120.40,117.88,106.23,56.51,19.01.
2,2'-((1E,1'E)-((2,2-diphenylethene-1,1-diyl)bis(4,1-phenylene))bis(ethene-2,1-diyl))bis(6-(dimethylamino)-1-methylquinolin-1-ium):Blue solid;Yield:46.32%;1H NMR(400MHz,DMSO-d6)δ8.72(d,J=9.1Hz,2H),8.34(dd,J=14.3,9.5Hz,4H),7.92(d,J=15.9Hz,2H),7.83–7.64(m,8H),7.37–6.89(m,16H),4.48(s,6H),3.15(s,12H).
2,2'-((1E,1'E)-(((4-methoxyphenyl)azanediyl)bis(4,1-phenylene))bis(ethene-2,1-diyl))bis(6-(dimethylamino)-1-methylquinolin-1-ium):Blue solid;Yield:52.45%;1H NMR(400MHz,DMSO-d6)δ8.69(d,J=9.1Hz,2H),8.48–8.28(m,4H),8.07(d,J=15.6Hz,2H),7.89(d,J=8.5Hz,4H),7.72(dd,J=9.7,2.9Hz,2H),7.63(d,J=15.8Hz,2H),7.29(d,J=2.9Hz,2H),7.19(d,J=8.9Hz,2H),7.14(m,4H),7.06(d,J=9.0Hz,2H),5.08(s,3H),3.82(s,3H),3.15(s,12H).
上述合成得到的化合物QM-TA-2、QM-TB-2和QM-OTA-2的激发波长和发射波长如表1所示:
表1光敏剂的激发波长和发射波长
名称 | λex(nm) | λem(nm) |
QM-TA-2 | 542 | 720 |
QM-TB-2 | 494 | 678 |
QM-OTA-2 | 545 | 674 |
λex:探针的激发波长;
λem:探针的最大发射波长。
2.光敏剂对Aβ聚集体的荧光响应:
分别配制光敏剂(QM-TA-2、QM-TB-2和QM-OTA-2)和Aβ1-42聚集体(β-Amyloid(1-42),上海强耀生物科技有限公司)的最终浓度分别为1μM和10μM,然后混合均匀后在室温下孵化10分钟,通过荧光分光光度计检测荧光光谱,以PBS缓冲液为对照,3次重复。结果如图3所示。
3.测定光敏剂产生的单线态氧:
以9,10-蒽二基-双(亚甲基)二甲酸(ABDA)为指示剂,检测单线态氧的产生,并通过商业光敏剂玫瑰红(RB)进行对照。首先制备ABDA(终浓度100μM)和不同光敏剂(终浓度10μM)的混合溶液。再通过白光(10mW/cm2)照射不同时间(0~500s),记录ABDA的特征峰378nm处的吸光度。结果如图4所示。
4.抗AD药效评价;
(1)将小鼠分为以下几组(n=6/组):(1)Wt:静脉注射0.9%盐水的Wt小鼠(C57BL6,11月龄,雄性);(2)AD:静脉注射0.9%盐水的Tg小鼠(C57BL6,APPsw/PSEN1,11月龄,雄性,Huafukang Biotechnology Co.,Ltd.);(3)AD+Light:静脉注射0.9%盐水,注射后1h时用激光(520nm,1000mW)照射Tg鼠10min;(4)AD+QM-TA-2:静脉注射QM-TA-2;(5)AD+QM-TA-2+Light:静脉注射QM-TA-2,注射后1h用激光(520nm,1000mW)照射10min;其中,QM-TA-2连续15天定期溶解在0.9%盐水中,然后静脉注射5mg/kg/day。照射前后,使用热红外成像相机监测头部的局部温度。
(2)通过水迷宫(MWM)测试对不同组别小鼠(n=6/组)进行行为学研究。实验设备为圆形水箱(直径120厘米,高45厘米,水温20±1℃,深度32厘米,通添加无毒的白色TiO2将水变成不透明溶液)。将一个平台(直径9厘米,高30厘米)隐藏在水面下2.0厘米处。对不同组别的小鼠进行连续5天的训练,小鼠有90秒找到隐藏的平台,如果没有,老鼠被引导进入平台,逃生潜伏期被记录为90秒。每次试用计算机化数字化视频跟踪系统记录逃离水迷宫的潜伏期。在第5天训练试验的24小时后,进行了实验测试,平台从池中移出。通过视频跟踪软件记录平台穿越的时间(原站台位置)、平均游泳速度、所用时间和目标象限内的游泳路径长度以及虚拟平台,分析老鼠的记忆巩固程度。
(3)HE染色:每组6只老鼠被错位处死。将大脑切除,并在4%的甲醛中粘合24小时,然后嵌入石蜡中,石蜡切片按常规脱蜡补水,苏木精染色5分钟,伊红(武汉塞维尔生物科技有限公司)染色1分钟,然后以分级系列酒精脱水(70%、95%和100%),用二甲苯制成透明,密封。采用计算机图像分析方法对海马组织病理异常进行了研究。利用ImageJ软件在三个具有代表性的小鼠图像中计算变性细胞指数。
(4)尼氏(Nissl)染色:在10mM PBS缓冲液中用0.5%(v/v)的甲苯紫罗兰溶液(含有少量冰醋酸)冲洗4μm的薄片15分钟两次,10~15分钟,用蒸馏水冲洗,在分级乙醇(70%、95%和100%,体积百分数)中脱水,用二甲苯和沉淀液浸泡。利用计算机图像分析对海马Cortex和CA1、CA3、DG区的细胞进行计数。使用ImageJ软件在每只小鼠的三个代表性图像中计算出存活神经元的百分比。
(5)免疫组化:在微波炉(中热8分钟,停止加热8分钟,中低热7分钟)(P70D20TL-P4,格兰仕微波炉电器有限公司)进行抗原修复,在室温下冷却后,用PBS(pH 7.4)洗涤两次。然后在室温下以3%(v/v)的氢气溶液在黑暗条件下进行25分钟的淬火,用PBS(PH 7.4)中洗涤两次,每次5分钟,在阻断溶液牛血清白蛋白(BSA)中进一步孵化30分钟。阻断后,以1:100(v/v)稀释β-淀粉样蛋白1-16(6E10)单克隆抗体(武汉塞维尔生物科技有限公司)并隔夜孵育,再以1:500(v/v)稀释二抗(武汉塞维尔生物科技有限公司)并孵育50min。PBS洗涤两次,在3,3′-二氨基联苯胺四盐酸盐(DAB)中孵化,用蒸馏水洗涤,在分级乙醇(70%、80%、90%、95%和100%,体积百分数)中脱水,置于二甲苯中,然后进行覆盖。利用ImageJ软件,计算了大脑中Aβ斑块沉积在每只小鼠三张代表性图像中Aβ沉积面积相对于整个面积的百分比。
结果如图5~8所示:
Morris水迷宫实验的结果(图5)显示,给药加光照后的AD小鼠与野生型小鼠类似,均采用趋向式搜索策略(图5(E)中的I和V),而其他组采用边缘式搜索策略,表明光氧化治疗后的小鼠和野生型小鼠具有更好的空间学习记忆。通过比较不同组小鼠的潜伏期,穿过虚拟平台次数,停留目标象限和虚拟平台的时间距离(图5(E)中的II、III、IV),进一步证明光氧化治疗可以明显改善AD小鼠的认知障碍,有效提高其空间记忆能力。
在行为学测试后,我们收集每组六只小鼠的脑切片进行HE染色,尼氏染色,免疫组化染色研究光氧化治疗后对神经元细胞以及Aβ斑块的影响。我首先采用HE染色法来评价各组小鼠大脑皮质层和海马神经元的退化程度。HE染色结果如图6所示,药物处理加光照组与AD组小鼠相比,皮质层和海马CA1、CA3、DG区细胞分裂、核固缩、形态不规则、HE染色偏深等的凋亡坏死细胞量明显减少,从统计分析结果来看,药物加光照处理后,变性细胞指数与野生组相当,远低于AD组小鼠,以及单用520nm激光处理或单用QM-TA-2处理的AD小鼠。通过尼氏染色进一步评估QM-TA-2介导的光氧化治疗对神经元损伤的保护作用(图7),尼氏染色图以及统计分析结果表明,药物加光照组含有较多的尼氏小体神经元,与野生小鼠相似,而AD组,以及单独给药或者单独光照组均表现出明显的尼氏小体减少。HE染色以及尼氏染色结果均表明,QM-TA-2介导的光氧化Aβ治疗可以降低Aβ介导的神经毒性,保护神经元细胞。我们进一步通过免疫组化染色来考察QM-TA-2介导的光氧化对Aβ含量的影响(图8),给药加光照处理后AD小鼠中Aβ斑块的数量显著减少,明显低于AD组,以及单独给药或者单独光照组,表明QM-TA-2介导的光氧化可以促进Aβ斑块的清除。
上述实施例为本发明较佳的实施方式,但本发明的实施方式并不受上述实施例的限制,其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化,均应为等效的置换方式,都包含在本发明的保护范围之内。
Claims (10)
1.一种靶向β淀粉样蛋白的光敏剂,其特征在于:所述的光敏剂为A-D-A构型光敏剂,包括吸电子基团A,π共轭部分B,给电子基团C,π共轭部分D,以及吸电子基团E;其中,A部分为1-甲基-6-二甲氨基喹啉,B部分为乙烯连接桥,C部分为三苯胺和四苯乙烯等衍生基团,D部分为乙烯连接桥,E部分为1-甲基-6-二甲氨基喹啉。
2.根据权利要求1所述的靶向β淀粉样蛋白的光敏剂,其特征在于:所述的光敏剂具有如下所示的结构:
式中,R选自下述任一基团:
3.根据权利要求1或2所述的靶向β淀粉样蛋白的光敏剂的制备方法,其特征在于,包括如下步骤:
(1)将N,N-二甲基-1,4-对苯二胺、巴豆醛和甲苯加入到盐酸溶液中,在保护性气体氛围、115±5℃条件下进行回流反应,待反应结束后经分离纯化得到化合物1,其结构式如式II所示:
(2)将化合物1加入到有机溶剂中,然后加入碘甲烷,在保护性气体氛围、80±5℃条件下进行回流反应,待反应结束后经分离纯化得到化合物2,其结构式如式III所示:
(3)将化合物2和醛类配体化合物加入到有机溶剂中,加入乙酸,于80±5℃条件下进行回流反应,待反应结束后经纯化得到所述靶向β淀粉样蛋白的光敏剂。
4.根据权利要求3所述的靶向β淀粉样蛋白的光敏剂的制备方法,其特征在于:
步骤(3)中所述的醛类配体化合物为如下式中的任意一种:
5.根据权利要求3所述的靶向β淀粉样蛋白的光敏剂的制备方法,其特征在于:
步骤(1)中所述的N,N-二甲基-1,4-对苯二胺和巴豆醛的摩尔比为1:1~2;
步骤(2)中所述的化合物1与碘甲烷的摩尔比为1:1~5;
步骤(3)中所述的化合物2与醛类配体化合物的摩尔比为4~5:1。
6.根据权利要求3所述的靶向β淀粉样蛋白的光敏剂的制备方法,其特征在于:
步骤(1)中所述的分离纯化通过如下步骤实现:将反应结束后的反应液萃取除去甲苯,然后在冰浴条件下调节pH至中性,最后经二氯甲烷萃取后采用硅胶柱层析纯化,得到化合物1;其中所述的硅胶柱层析的条件为:石油醚与乙酸乙酯的体积比为20:1,硅胶200~300目;
步骤(2)中所述的分离纯化通过如下步骤实现:将反应结束后的反应液旋转蒸发去除溶剂,然后通过硅胶柱层析纯化,得到化合物1;其中所述的硅胶柱层析的条件为:二氯甲烷与甲醇的体积比为40:1,硅胶200~300目;
步骤(3)中所述的纯化为采用薄层色谱进行纯化;所述的薄层色谱的条件如下:层析硅胶的目数为200~300目,展开剂为二氯甲烷与甲醇按体积比为100:1配比得到的展开剂。
7.根据权利要求3所述的靶向β淀粉样蛋白的光敏剂的制备方法,其特征在于:
步骤(1)和(2)中所述的保护性气体为氮气和氩气中的至少一种;
步骤(1)中所述的甲苯的用量为按每克N,N-二甲基-1,4-对苯二胺配比8~10ml甲苯计算;
步骤(1)中所述的盐酸溶液的浓度为6mol/L;
步骤(1)中所述的反应在搅拌条件下进行;所述反应的时间为4~6小时;
步骤(2)和(3)中所述的有机溶剂为乙醇;
步骤(2)中所述的反应在搅拌条件下进行;所述反应的时间为8~12小时;
步骤(3)中所述的反应搅拌条件下进行;所述反应的时间为3~5小时;
步骤(3)中所述的乙酸的用量为按其在体系的终浓度为体积百分比0.75%添加计算。
8.权利要求1或2所述的靶向β淀粉样蛋白的光敏剂在制备Aβ荧光成像剂中的应用。
9.权利要求1或2所述的靶向β淀粉样蛋白的光敏剂在制备诊断和/或治疗阿尔兹海默症的产品中的应用。
10.根据权利要求9所述的应用,其特征在于:所述的β淀粉样蛋白为Aβ1-42聚集体。
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