CN104803993B - 一种具有Aβ亲和力的荧光化合物及其用途 - Google Patents
一种具有Aβ亲和力的荧光化合物及其用途 Download PDFInfo
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Abstract
一种具有Aβ亲和力的荧光化合物及其用途,本发明涉及通式(I)表示的具有Aβ斑块亲和力的荧光化合物(n取1、2或3)以及含有该衍生物的组合物。本发明还涉及其制法和其在显像Aβ斑块的方法中的应用。
Description
技术领域
本发明涉及特异性分子识别诊断试剂领域,更具体地说,涉及一种具有淀粉样斑块亲和力的荧光化合物,以及该类化合物在淀粉样斑块的显像方法中的应用。
背景技术
阿尔茨海默病(Alzheimer's Disease,AD)是一种进行性发展的致死性神经退行性疾病。目前我国已提前进入老龄化社会,大约有600万人患AD,数量居世界各国之首,且患病人数以每年30万以上的速度递增。我国65岁以上人群患病率达7.2%,且AD患者正在向年轻化趋势发展。AD已成为仅次于心脏病、癌症、中风的导致老人死亡的第四大杀手。AD不但严重影响中老年人的生活质量,而且给患者家庭和社会带来沉重的经济负担。由于AD病因复杂,确切的发病机理目前仍不清楚,临床上主要通过评价患者的认知功能损伤来诊断,确诊患者多已进入病程的中晚期而延误了治疗。缺乏有效的检测手段,已成为AD早期诊断与治疗的重大障碍。
在AD患者脑内,以β-淀粉样蛋白(Aβ)为主要成分的β-淀粉样斑块(Aβ斑块)是AD最为显著的病理性标志之一。Aβ斑块在AD发病前的数十年已开始出现,是AD最早的神经组织退化标志和重要病理学特征,淀粉样级联假说提出神经细胞外纤维状Aβ斑块的形成是AD病理发生发展的关键事件,并最终导致认知功能障碍(Hardy J, et al. Science. 2002,297(5580), 353–356)。近年来,Aβ斑块形成的预防和逆转成为治疗AD的目标之一,抑制Aβ斑块在脑内的产生和蓄积的药物和疗法得到了广泛研究。
除了AD外,Aβ斑块也存在于其他疾病,如脑淀粉样血管病、淀粉样心肌病、淀粉样多发性神经病、系统性老年淀粉样变和伴有淀粉样变的遗传性脑出血等。因此,研发具有特异性结合Aβ斑块的配体(Aβ分子探针)引人关注。利用Aβ分子探针和分子成像技术进行Aβ斑块的显像,可实现无创、实时的Aβ斑块的体内示踪与检测,进而为AD等疾病的早期诊断、疗效监测以及治疗药物的研究等提供极大便利。
过去的数年间,已有较多放射性Aβ分子探针进入临床试验阶段,如[11C]PIB(Klunk WE,et al. Ann. Neurol. 2004, 55(3), 306–319)、[18F]GE-067(Koole M, etal. J. Nucl. Med. 2009,50(5), 818-822)、[18F]BAY-94-9172(Rowe CC, et al.Lancet.Neurol. 2008, 7(2), 129-135)、[18F]AV-45(Wong DF, et al. J. Nucl. Med.2010, 51(6), 913-920)等。但是放射性显像剂的应用也受一些因素限制,比如放射性显像剂所发出的射线对人体具有一定的辐射损伤、需要医疗机构配套有生产放射性核素的回旋加速器、放射性显像剂须专业技术人员标记配制等。
相较之下,光学成像具有安全无放射性、数据采集时间短以及成本低廉等诸多优势,近年在医学诊断等中的应用受到广泛重视;尤其是新兴的近红外荧光成像技术,在其光谱范围内,生物组织的自发荧光干扰较小,组织背景荧光信号很低,且穿透组织距离可高达数厘米。因此,研发具有Aβ斑块亲和力的荧光显像剂(分子探针),将具有非常重要的科学意义和实际价值。然而目前Aβ荧光分子探针的研究总体上进展不大。基于上述技术背景,有必要提出一种新的化合物,以做为Aβ斑块的荧光显像剂应用于AD等疾病的早期诊断、疗效监测以及治疗药物的研究。
发明内容
本发明要解决的技术问题在于,现有荧光显像(造影)剂的荧光团常具有较大的分子结构且带有电荷,因而不适合应用于脑内Aβ斑块的显像剂。本发明提供一种小分子荧光化合物,其结构中的推电子基团和拉电子基团,形成推-拉电子作用的共轭结构,并通过碳碳双键增加π-π*跃迁的共轭体系,使化合物分子产生的荧光向近红外光区移动,同时其结构整体又满足了与Aβ斑块的亲和力。
本发明的目的在于提供具有Aβ斑块亲和力的荧光化合物及其应用。
为了实现本发明目的,本发明的具有Aβ斑块亲和力的荧光化合物,统称为“PTAD类化合物”,其结构如式(I)所示:
其中,
n取1、2或3;
本发明还提供所述式(I)的化合物的制备方法,包括以下步骤:
取1.0 mmol和1.0 mmol,溶于10-50mL乙腈,再加入200 μL哌啶,搅拌下回流3-24小时;蒸去乙腈,残余物用水洗涤,再用乙酸乙酯萃取,无水硫酸镁干燥过夜;乙酸乙酯层旋蒸得粗产物。粗产物经柱层析(洗脱剂为乙酸乙酯:正己烷)分离,得所述的具有Aβ斑块亲和力的荧光化合物;其中m取0、1或2。
所述的制备过程如下所示:
其中,
m取0、1或2;
n取1、2或3。
所述制备过程使用的化合物为市售化合物,为现有化合物,可通过下式所示的过程制得:
其中,
p取0或1;
m取1或2;
本发明也提供一种诊断组合物,所述组合物包括式(I)所示的化合物与药学上可接受的载体制成的制剂。“药学上可接受的载体”包括各种赋形剂和稀释剂。本发明所述的在组合物中药学上可接受的载体可包括液体,如水、生理盐水、甘油和乙醇。
本发明提供Aβ斑块的染色方法。在本染色方法的第一步中,将可检测量的式(I)所示的化合物引入组织或患者中。化合物通常是诊断组合物的部分并且通过本领域技术人员公知的方法给药至组织或患者。在本发明的优选实施方案中,以可检测量的式(I)所示的化合物引入患者并且在足以使化合物与Aβ斑块结合的时间之后,非创伤性地检测化合物。在本发明的另一实施方案中,将式(I)所示的化合物引入患者,经足量的时间以使化合物与Aβ斑块结合,自患者取组织样品,并脱离患者检测组织中的化合物。在本发明的第三个实施方案中,自患者取组织样品并且将式(I)所示的化合物引入该组织样品。在足以使该化合物结合至Aβ斑块的时间之后,检测化合物。
本发明进一步提供显像Aβ斑块的方法。在本显像方法的第一步中,将可检测量的式(I)所示的化合物引入组织或患者中。化合物通常是诊断组合物的部分并且通过本领域技术人员公知的方法给药至组织或患者。在本发明的优选实施方案中,以可检测量的式(I)所示的化合物引入患者并且在足以使化合物与Aβ斑块结合的时间之后,非创伤性地检测化合物。在本发明的另一实施方案中,将式(I)所示的化合物引入患者,经足量的时间以使化合物与Aβ斑块结合,自患者取组织样品,并脱离患者检测组织中的化合物。在本发明的第三个实施方案中,自患者取组织样品并且将式(I)所示的化合物引入该组织样品。在足以使该化合物结合至Aβ斑块的时间之后,检测化合物。
可以通过整体的或局部的给药途径将式(I)所示的化合物给药至患者。例如,可以将化合物给药至患者以使其递送至全身。可选地,可以将化合物给药至关注的特定的器官或者组织。例如,为了诊断或追踪患者的AD的进程,需要定位和定量脑内的淀粉样斑块。
术语“组织”是指患者身体的一部分。组织的实例包括脑、心、肺、肝、肾、脾、胰腺、胃、肠、血管、动脉。可检测量是通过选用的检测方法所需的化合物的量。本领域的技术人员可以容易地确定为了提供检测需要引入患者的化合物的量。例如,可以给患者增加的化合物的量直至通过选用的方法检测到化合物。
术语“患者”是指人类和其他动物。本领域技术人员也熟知如何确定足以使化合物与Aβ斑块结合的时间。通过将可检测量的式(I)所示的化合物引入患者,然后在给药后的各时间处检测化合物,可以容易地测定所需的时间。
术语“结合”是指化合物和Aβ斑块之间的化学相互作用。结合的实例包括共价键、离子键、亲水-亲水相互作用、疏水-疏水相互作用和络合物。
本发明所述的显像手段为光学显像。
本发明提供式(I)所示的化合物在淀粉样沉积相关疾病的诊断和治疗药物研究中的应用。所述的淀粉样沉积相关疾病是阿尔茨海默病或脑淀粉样血管病。
脑活体内显像剂的关键前提之一是具有穿过完整的血脑屏障的能力。正常小鼠活体成像试验表明,本发明中的荧光化合物能够有效地穿过血脑屏障,并具有良好的脑摄取量和脑内清除速率等优点。
此外,本发明中的化合物具有良好的荧光性质,部分化合物的荧光发射波长达到近红外光区;体外荧光染色实验表明该类化合物能够选择性结合并荧光标记AD转基因小鼠脑内Aβ斑块,有望成为一种用于检测体内Aβ斑块的新型荧光显像剂。
附图说明
图1是本发明的具有Aβ斑块亲和力的荧光化合物的化学结构式。
图2是实施例一的化合物(PTAD-1)与Aβ1-42聚集体混合前后的荧光发射光谱。
图3-A是实施例一的化合物(PTAD-1)对AD转基因小鼠脑切片的荧光染色标记。
图3-B是实施例一的化合物(PTAD-2)对AD转基因小鼠脑切片的荧光染色标记。
图4是实施例一的化合物(PTAD-1)尾静脉注入正常小鼠后各时间点脑的荧光信号。
图5是实施例一的化合物(PTAD-1)尾静脉注入AD转基因小鼠和正常小鼠对照后60min时间点脑部活体成像。
图6是实施例一的化合物(PTAD-1)尾静脉注入AD转基因小鼠和正常小鼠对照后60min时间点脑部离体成像。
具体实施方式
以下通过实施例更详细地说明本发明,但本发明不限于所述实施例。常见的且对本领域技术人员而言显而易见的各种条件和参数的其他适当的修饰和改变处于本发明的精神和范围之内。若未特别指明,实施例中所用的技术手段为本领域技术人员所熟识的常规手段,所用原料均为市售商品。
实施例
实施例一:PTAD类化合物的合成
本发明的PTAD类化合物通过下述的合成路径合成。
合成PTAD-1
称取2,6-二甲基-4-吡喃亚基丙二腈(172.0 mg, 1.00 mmol)和5-(二甲氨基)-2-噻吩甲醛 (155.0 mg, 1.00 mmol) ,溶于10 mL乙腈,再加入200 μL哌啶,搅拌下回流16小时;蒸去乙腈,残余物用水洗涤,再用乙酸乙酯萃取,无水硫酸镁干燥过夜;乙酸乙酯层旋蒸得粗产物。粗产物经柱层析(洗脱剂为乙酸乙酯:正己烷= 5 : 1)分离,得64.9 mg PAD-1,结构如下,产率为21.0%。MS: m/z 310 (M++H)。1H NMR (400 MHz, CDCl3):δ2.34 (s, 3H),3.07 (s, 6H), 5.85 (s, 1H), 5.98 (d, 1H, J = 9.6 Hz), 6.46 (d, 2H, J = 10.4Hz), 7.03 (s, 1H), 7.42 (d, 1H, J = 10.6 Hz)。
合成PTAD-2
称取2,6-二甲基-4-吡喃亚基丙二腈(172.0 mg, 0.50 mmol)和 (181 mg, 1.00 mmol) ,溶于10 mL乙腈,再加入200 μL哌啶,搅拌下回流10小时;蒸去乙腈,残余物用水洗涤,再用乙酸乙酯萃取,无水硫酸镁干燥过夜;乙酸乙酯层旋蒸得粗产物。粗产物经柱层析(洗脱剂为乙酸乙酯:正己烷= 3 : 1)分离,得53.2mg PAD-2, 结构如下,产率为15.9%。MS: m/z 336 (M++H)。1H NMR (400 MHz, CDCl3):δ2.37 (s, 3H), 3.07 (s, 6H), 5.81 (s, 1H), 5.91 (d, 1H, J = 9.6 Hz), 6.47 (d,2H, J = 9.6 Hz), 6.70 (d, 2H, J = 8.8 Hz), 7.10 (s, 1H), 7.43 (d, 1H, J = 9.8Hz)。
实施例二:荧光激发波长与荧光发射波长的测定
本发明中的化合物具有良好的荧光性质。为了考察本发明中的化合物的荧光性质,具体实施步骤为:精密称取本发明中的化合物适量,溶解于甲醇,并用甲醇稀释至1 μmol·L-1。应用荧光分光光度计进行荧光检测。固定激发/发射波长并400-750 nm连续扫描发射/激发波长,绘制波行图像。本发明实施例中的部分化合物的最大激发波长与最大发射波长见表1。
表1 实施例中的部分化合物的最大荧光激发波长与最大发射波长。
PTAD-1 | |
λex(nm) | 530 |
λem (nm) | 620 |
实施例三:Aβ1-42聚集体混合后的化合物荧光光谱
本发明中的化合物与Aβ聚集体结合后具有荧光增强的性质。为了考察本发明中的化合物与Aβ混合后的荧光光谱行为的变化,具体实施步骤为:精密称取本发明中的化合物适量,溶解于乙醇,并用PBS稀释至1 μmol·L-1。应用荧光分光光度计进行荧光检测。固定激发/发射波长并400-750 nm连续扫描发射/激发波长,绘制波行图像。选用Aβ1-42蛋白在37℃水浴中培育Aβ聚集体,用于模拟人脑内的Aβ聚集体。将化合物(1 μmol·L-1)与Aβ1-42聚集体(2.75 μmol·L-1)混合,应用荧光分光光度计进行荧光检测。固定激发/发射波长并400-750nm连续扫描发射/激发波长,绘制波行图像。本发明中的化合物与Aβ聚集体结合后具有荧光增强的性质。其中,本发明实施例中的化合物DPAD-1与Aβ1-42聚集体混合前后的荧光光谱行为见图2。PTAD-1与Aβ1-42聚集体混合后的荧光强度大幅增强,为化合物自身荧光强度的29倍。
实施例四:体外荧光染色实验
本发明中的化合物能够选择性结合Aβ斑块,并有效地荧光标记Aβ斑块。为了考察对Aβ斑块的标记能力,具体实施步骤为:配制浓度为1 μmol·L-1的本发明中的化合物,分别滴加覆盖AD转基因小鼠脑切片(10 μm),室温培育10分钟;切片按40%乙醇(2分钟)、40%乙醇(2分钟)、纯水(30 秒)的顺序进行漂洗,风干后应用荧光显微镜观察。本发明中的化合物能够选择性结合Aβ斑块,并有效地荧光标记Aβ斑块。其中,本发明实施例中的化合物PTAD-1和PTAD-2的染色结果如图3。
实施例五:正常小鼠脑离体成像实验
本发明中的化合物能够有效地穿过血脑屏障,并具有良好的脑初始摄取量和清除速率。为了考察血脑屏障通过能力,并同时考察血脑屏障通过能力以及脑内消除速度,具体实施步骤为:取本发明中的化合物(2.0 mg/kg,含20%二甲亚砜和80%丙二醇),经尾静脉注射入正常小鼠(n = 5)体内,分别于注射后5、30、60以及120 min时断头取脑。取未注射药液的小鼠脑作为空白对照。利用光学成像系统进行小鼠脑的离体成像。确定整脑为感兴趣区域(ROI)并计算荧光信号。调节空白对照脑信号为0 (sum = 0)。以最大荧光信号值为1,计算各时间点的相对信号值,并绘制各时间点脑的荧光信号曲线。本发明中的化合物能够有效地穿过血脑屏障进入脑内,且具有良好的脑初始摄取量和清除速率。其中,本发明实施例中的化合物PTAD-1的结果如图4所示。化合物注入正常小鼠后脑内迅速显示荧光信号;随着时间延长,荧光强度逐渐减弱,60 min时荧光信号已基本消除出脑外。
实施例六:转基因小鼠活体成像实验
本发明中的化合物能够有效地穿过血脑屏障、选择性结合AD转基因小鼠脑内Aβ斑块、并有效地荧光标记Aβ斑块。为了考察对活体脑内Aβ斑块的标记能力,具体实施步骤为:取本发明中的化合物(2.0 mg/kg,含20%二甲亚砜和80%丙二醇),经尾静脉注射入AD转基因小鼠(APP/PS1)及野生型对照小鼠体内,利用光学成像系统进行小鼠活体成像。确定整脑为感兴趣区域(ROI)并计算荧光信号。本发明中的化合物能够选择性结合并有效地荧光标记转基因小鼠脑内Aβ斑块。其中,本发明实施例中的化合物PTAD-1的结果如图5所示。化合物注入小鼠后60 min时APP/PS1转基因小鼠脑内荧光信号明显高于正常小鼠对照。
实施例七:转基因小鼠离体成像实验
为了进一步确认APP/PS1转基因小鼠活体成像中的高荧光信号来自于荧光标记Aβ斑块,具体实施步骤为:取本发明中的化合物(2.0 mg/kg,含20%二甲亚砜和80%丙二醇),经尾静脉注射入APP/PS1转基因小鼠及野生型对照小鼠体内,于注射后60 min时断头取脑并制作脑切片。风干后应用荧光显微镜观察。本发明中的化合物能够选择性荧光标记APP/PS1转基因小鼠脑内Aβ斑块,正常对照组未见明显荧光标记。其中,本发明实施例中的化合物PTAD-1的结果如图6。
虽然,上文中已经用一般性说明及具体实施方案对本发明做了详尽的描述,但在本发明基础上,可以对之做一些修改或改进,这对本领域技术人员而言是显而易见的。因此,在不偏离本发明精神的基础上所做的这些修改或改进,均属于本发明要求保护的范围。
Claims (4)
1.一种具有脑内β-淀粉样斑块亲和力的荧光化合物,其特征在于:其结构如式(I)所示:
其中,n取2或3。
2.权利要求1所述的化合物的制备方法,包括以下步骤:取1.0mmol和1.0mmol溶于10-50mL乙腈,再加入200μL哌啶,搅拌下回流3-24小时;蒸去乙腈,残余物用水洗涤,再用乙酸乙酯萃取,无水硫酸镁干燥过夜;乙酸乙酯层旋蒸得粗产物;粗产物经柱层析分离,洗脱剂为乙酸乙酯:正己烷,得所述的与Aβ斑块具有亲和力的化合物;其中m取1或2。
3.一种用于显像β-淀粉样斑块的诊断组合物,其包含权利要求1所述的化合物与药学上可接受的载体制成的制剂。
4.权利要求1所述的化合物在制备淀粉样沉积相关疾病的诊断和治疗药物中的应用,其特征在于:所述的淀粉样沉积相关疾病是阿尔茨海默病或脑淀粉样血管病。
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