1、Ne 神经化学DOI - Neurochemistry DOI.uroDOI: 10.1002/anie.201209885Development of a Platinum Complex as an anti-Amyloid Agent for the Therapy of Alzheimer_s Disease铂复杂的发展作为一个种抗体代理Alzheimer_s疾病的治疗Vijaya B. Kenche, Lin W. Hung, Keyla Perez, Irene Volitakes, Guiseppe Ciccotosto,Jeffrey Kwok, Nicole Critch,
2、Nikki Sherratt, Mikhalina Cortes, Varsha Lal, Colin L. Masters,Kazuma Murakami, Roberto Cappai, Paul A. Adlard, and Kevin J. Barnham*林Vijaya b . Kenche w .挂Keyla佩雷斯,Irene Volitakes Guiseppe Ciccotosto,妮可不久后,尼基中,杰弗里郭Mikhalina议会,Varsha Lal,科林l大师Kazuma村上,Roberto Cappai Paul a .埃德拉德合作,凯文jBarnham *Alzhei
3、mer_s disease (AD) is an age-related neurodegenerative disease. Its pathological indicators include extracellular amyloid plaques, the main constituent of which is the amyloid b-peptide (Ab), and neurofibrillary tangles composed of hyperphosphorylated tau protein.1 Current evidence suggests that the
4、 aggregation of Abs drives the disease process, as various forms of aggregated Ab have been shown to be toxic,2 resulting in the development of a variety of therapeutic strategies that target Ab.36 To date, most Ab aggregation inhibitors have been designed to target the hydrophobic central and C-ter
5、minal regions of Ab, which are in general conjugated polyaromatic molecules that are very hydrophobic.7 Herein, we report a different approach to the design of aggregation inhibitors of Ab and demonstrate that this approach can modify Ab in vivo. Ab contains a metalbinding motif with three histidine
6、 residues (6, 13, and 14) near the N terminus, and the interaction of this site with zinc and copper modulates the aggregation and toxicity of Ab.8,9 We have previously taken advantage of the metal-binding ability of Ab to show that commercially available PtII complexes of 1,10-phenanthroline ligand
7、s target this site, thus inhibiting Ab aggregation in vitro.10 For a variety of reasons, including lack of novelty, cumbersome multi-step synthetic procedures,11, 12 and poor bioavailability, these complexes are unsuitable for in vivo studies. Therefore we sought to identify new Pt complexes that ar
8、e suitable for in vivo studies.Alzheimer_s病(AD)是一种与年龄相关的神经退行性疾病。其病理指标包括细胞外淀粉样斑块,主要成分是淀粉样b-peptide(Ab),和神经原纤维缠结由hyperphosphorylated tau蛋白质。1目前证据表明,Abs驱动疾病的聚合过程中,由于各种形式的聚合Ab是有毒的,2的发展导致各种各样的治疗策略,目标Ab。(3 - 6)到目前为止,大多数Ab聚合抑制剂设计针对Ab的疏水核心和c端区域,这是一般共轭聚芳分子非常疏水。7在此,我们报告一个不同的方法聚合抑制剂的设计Ab和证明这种方法可以修改Ab体内。Ab包含met
9、albinding主题有三个组氨酸残基(6、13和14)在N终点站附近,和这个网站的交互调节Ab的聚合和毒性的锌和铜。(8、9)我们曾利用Ab的metal-binding能力表明商用PtII复合物的10-phenanthroline配体目标这个网站,从而抑制Ab聚合体外。10出于各种原因,包括缺乏新颖性,繁琐的多步合成程序,(11、12)和生物利用度差,不适合这些复合物在体内研究。因此我们试图确定新工党复合物适合体内研究。The 8-(1H-benzoimidazol-2-yl)-quinoline (8-BQ)13 scaffold (Scheme 1) was identified as
10、suitable for generating the desired platinum complexes. The ligand requires few steps to synthesize and provides the large aromatic surface area required for a Pt complex to target Ab. Surprisingly, although the initial synthesis of 8-BQ was first reported14 over 100 years ago, the coordination chem
11、istry of this ligand has not been widely explored and Pt complexes of 8-BQ are novel. Additionally, the presence of an NH functionality on the imidazole moiety of 8-BQ allows easy modification of the ligand through a conventional one-step substitution reaction.Attachment of different groups to this
12、position can be used to modulate the solubility and other pharmacokinetic properties of the complexes. We chose the N,N-dimethylaminoethyl group because it improves drug solubility and stability in aqueous media.15 Herein, we report the synthesis of the 8-BQ ligand (Scheme 1) and its coordination to
13、 PtII and PtIV complexes (Schemes 2 and 3, respectively).(8)- 1 h-benzoimidazol-2-yl -quinoline(8-BQ)13脚手架(方案1)被确认为适合生成所需的铂配合物。合成的配体需要几个步骤,并提供所需的大型芳香表面积Pt复杂目标Ab。令人惊讶的是,尽管最初的合成8-BQ首次报道14在100多年前,这个配体的配位化学还没有广泛地探讨和Pt复合物8-BQ的小说。此外,NH的存在功能的咪唑啉8-BQ允许容易修改的配体通过传统的单步取代反应。附件不同群体的位置可以用来调节复合物的溶解度和其他药代动力学属性。我们选择
14、了N N-dimethylaminoethyl组,因为它可以提高药物溶解度和稳定水媒体。15,我们报告的合成8-BQ配体(方案1)和其协调PtII PtIV复合物(方案2和3,分别)。The desired 8-BQ ligand 3 was obtained in two synthetic steps, a condensation between 8-quinolinecarboxaldehyde (1)16 and 1,2-phenylenediamine followed by a conventional base-catalyzed N alkylation of benzimi
15、dazole 2. A 1:3 mixture of 1 and 1,2-diphenylamine was dissolved in an equimolar mixture of THF and water (saturated with O2 gas) and heated to reflux for 24 hours to give 2 in 60% yield. Here, molecular oxygen was used to form the required benzimidazole moiety by oxidative cyclo-dehydrogenation of
16、an intermediate Schiff_s base.17 The N alkylation was performed by refluxing the mixture of 2, 2-chloro-N,N-dimethylethylamine hydrochloride, and Cs2CO3 in dimethylformamide for 16 hours to give the desired ligand 3 in 85% yield. 所需的8-BQ配体3合成步骤,得到了在两个之间的缩合8-quinolinecarboxaldehyde(1)16,2-phenylenedi
17、amine紧随其后的是一个传统的苯并咪唑2 base-catalyzed N烷基化。1:3的混合1和1,2-diphenylamine溶解在一个克分子数相等的四氢呋喃和水的混合物(与O2气体饱和)和加热回流24小时给2 60%的收益率。在这里,使用分子氧氧化cyclo-dehydrogenation形成所需的苯并咪唑啉的一个中间Schiff_s基地。17N烷基化是由回流冷却的混合2,2-chloro-N,N-dimethylethylamine盐酸盐,在二甲基甲酰胺Cs2CO3 16小时给所需的配体3 85%的收益率。The PtII complex of 3 was prepared by
18、adopting the reported method for synthesis of Pt(1,10-phenanthrolines) complexes18 (Scheme 2). Equimolar amounts of 3 and PtCl2(DMSO)219 were stirred in methanol for 48 hours atroom temperature to give 4 as a yellow solid in approximately 60% yield. The complex was characterized by 1H NMR and 195Pt
19、NMR spectroscopy and ESI/MS (see the Supporting Information). 3制备的PtII复杂采用合成的报道方法Pt(10-phenanthrolines)复合物18(方案2)。克分子数相等的数量的3和PtCl2(DMSO)219在甲醇搅拌48小时atroom温度给4黄色固体在大约60%的收益率。复杂的特点是1 h NMR和195 pt核磁共振光谱和ESI / MS(见支持信息)。The toxicity of Ab is linked to peptide aggregation. Timedependent aggregation of A
20、b into fibrillar structures can be monitored by fluorescence of thioflavin T (ThT), which gives a characteristic signal when bound to amyloid.10 Complex 4 inhibited ThT fluorescence in a dose-dependent manner (Figure 1a), while the uncomplexed ligand 3 had no effect on ThT fluorescence (data not sho
21、wn). Negative-staining electron microscopy confirmed that 4 inhibited the formation of fibrils (see Figure S1 in the Supporting Information).Ab与肽聚集的毒性。改变聚合的Ab成纤维结构可以通过荧光监控thioflavin T(钛合金),使特征信号时绑定到淀粉样蛋白。10复杂4剂量依赖性地抑制阻氢荧光(图1),而uncomplexed配体3没有影响荧光(数据未显示)。Negative-staining电镜证实4抑制纤维的形成(见图S1在支持信息)。Fibr
22、illar amyloid is the end result of the Ab aggregation process, but it is now thought that Ab toxicity is due to smaller soluble oligomeric forms of Ab. We used two methods, SDSPAGE and surface-enhanced laser desorption/ionization time-of-flight (SELDI-TOF) mass spectrometry to determine whether 4 al
23、tered the oligomeric profile of Ab. In the first instance, Ab was aggregated in PBS buffer with and without 4 at 37 8C over 20 hours with samples taken periodically and analyzed by SDS-PAGE Western blotting using the 4G8 antibody (epitope residues 1724 of Ab; see Figure S1 in the Supporting Informat
24、ion). The blot from Ab that was aged for 20 hours in the absence of 4 showed extensive “smearing” of immunoreactivity, consistent with peptide aggregation. This“smearing” and therefore the degree of aggregation was significantly reduced by co-incubation of Ab with 4. We have previously shown that sm
25、all oligomeric forms of Ab can be observed using SELDI-TOF MS and that these oligomeric forms of Ab correlate with the toxicity of peptides.20 When Ab was incubated both with and without 4, two new signals appeared in the spectrum after 4 hours in the presence of the drug. The signals were shifted f
26、rom the mass of Ab (4515_1 Da) by 547 Da and 512 Da, and these differences in molecular weight are consistent with 4 loosing either one or both chloride atoms as the Pt complex binds to Ab.Significantly, while a signal corresponding to the dimer of Ab was detectable in the mass spectrum of untreated
27、 Ab, this signal was absent from the spectrum of Ab incubated with 4(Figure 1b,c).纤维状的淀粉样蛋白是Ab聚合过程的最终结果,但现在认为Ab毒性是由于较小的可溶性低聚物的形式的Ab。我们使用了两个方法,SDSPAGE和表面增强激光解吸/电离飞行时间质谱(SELDI-TOF)来确定是否4 Ab的低聚物的改变。首先,Ab在PBS缓冲与聚合 ,没有4 37岁8 c 与样本超过 20小时定期和sds - page蛋白免疫印迹分析了使用4 g8抗体(抗原决定部位残留17-24 Ab;参见图S1在支持信息)。从 Ab的污点,
28、20个小时没有4岁显示广泛的免疫反应性的“诽谤”,符合肽聚合。这种“模糊”,因此聚合程度显著降低co-incubation Ab的4。我们先前已经表明,小型低聚物的形式的Ab可以观察到使用SELDI-TOF女士和这些低聚物的形式的Ab与多肽的毒性。20和没有4 Ab是孵化时,出现了两个新的信号频谱在4小时后药物的存在。被转移的信号质量的Ab(4515 _1 Da)到547年达512,而这些不同的分子量也符合4失去一个或两个氯原子Ab.Significantly Pt 复杂的绑定,而信号对应于Ab 的二聚物的质谱检测未经处理的Ab,这个信号频谱的缺席Ab孵化4(图 1 b,c)。After th
29、e demonstration that 4 inhibited Ab aggregation, its ability to inhibit Ab toxicity in primary mouse cortical neuronal cell cultures was assessed. Treatment of neurons with Ab42 (10 mm) for 4 days reduced cell viability to 77%, as measured by the MTS assay (Figure 2a). Co-incubation of Ab42 (10 mm)
30、with 4 (10 mm) significantly increased cell viability to 94% (Figure 2a). Complex 4 was not toxic at the concentrations tested. Long-term potentiation (LTP) in rodent hippocampal slices is a measure of synaptic plasticity that focuses on activity-dependent persistent increases in synaptic strength,
31、and is considered to be the biochemical basis of learning and memory.21, 22 High-frequency stimulation of a mouse hippocampal slice gives an LTP of (128_6)% (Figure 2b). Incubation of the hippocampal slice with Ab42 (2 mm) for 30 minutes before stimulus significantly reduced LTP to (91_4)% (Figure 2
32、 c). Addition of 4 (2 mm) to thesolution of Ab42 inhibited its effects on LTP (133_11)%; Figure 2c).证明4 Ab抑制聚合后,它能够抑制Ab毒性的主要鼠皮层神经细胞培养是评估。治疗神经元与Ab42(10毫米)4天细胞生存能力减少到77%,以MTS试验(图2)。Co-incubation Ab42(10毫米)和4(10毫米)显著增加细胞生存能力提高到94%(图2)。复杂4不是有毒浓度测试。长期势差(LTP)在啮齿动物海马切片是一个衡量突触可塑性,着重于依赖性活动持久的突触强度的增加,和被认为是学习和
33、记忆的生化基础。(21、22)高频刺激小鼠海马切片了LTP的(128 _6)%(图2 b)。孵化的海马切片Ab42(2毫米)30分钟前刺激明显减少了LTP(91 _4)%(图2摄氏度)。4(2毫米)解决Ab42抑制其对LTP的影响(图2(133 _11)%;c)。The ultimate objective of this work was to generate an orally administered anti-amyloid compound for in vivo testing. To prepare an orally bioavailable Pt complex capab
34、le of inhibiting Ab aggregation, a prodrug strategy was adopted, as previously used in the development of JM216 (Satraplatin), an orally bioavailable PtIV anticancer complex.23 Substitution reactions for PtIV complexes are kinetically very slow,24, 25 in a biological context this means the complexes
35、 are very stable and able to survive the acidic environment of the stomach and to cross gut membranes intact. Having crossed these mem-branes, the prodrug is then reduced by natural reductants, such as glutathione, to the active PtII complex.23 这项工作的最终目标是生成一个口头管理种抗体体内测试的化合物。准备一个口服生物利用率Pt复杂能够抑制Ab聚合,前
36、体药物的策略是采用,正如前面的开发中使用JM216(Satraplatin),一个口服生物利用率PtIV抗癌复杂。23替代kinetically PtIV复合物的反应很慢,(24、25)在生物环境中这意味着复合物是非常稳定的,能够在酸性环境交叉的胃和肠道膜完好无损。这些mem-branes交叉,然后减少天然药物前体的还原剂,如谷胱甘肽、活动PtII复杂。23Generally, PtIV complexes are prepared by oxidation of PtII complexes utilizing agents such as Cl2 26 and H2O2,27 but in
37、 this instance, attempts to oxidize 4 failed as the 8-BQ ligand was prone to oxidative degradation. Therefore, a direct reaction of equimolar amounts of 3 with H2PtCl6 in DMSO at room temperature for 4 days was adopted, giving the desired product 5 in 60% yield (Scheme 3). Attempts to use higher tem
38、peratures to shorten the reaction time led to an intractable mixture of compounds. Complex 5 was characterized by 1H NMR and 195Pt NMR spectroscopy and ESI/MS (see the Supporting Information).一般来说,PtIV复合物准备PtII配合物的氧化利用代理如Cl226和过氧化氢,27但在这种情况下,试图氧化4失败8-BQ配位体是容易氧化降解。因此,直接反应的克分子数相等的金额3 H2PtCl6在DMSO在室温下采
39、用4天,提供所需的产品5 60%的收益率(方案3)。尝试使用高温缩短反应时间导致了棘手的化合物的混合物。复杂的以1 h NMR和195 pt核磁共振光谱和ESI / MS(见支持信息)。To ensure that the PtIV prodrug strategy results in higher levels of bioavailable drug, we treated wild-type mice with either 4 or 5 at 15 mgkg_1 (note that on a molar basis, the animals treated with 5 recei
40、ved slightly less drug). Blood and brain tissues were collected at 5, 30, and 60 minutes post dosing, plasma was separated from the blood, and the brain tissue homogenized. As a surrogate measure of drug levels, plasma and brain homogenates were analyzed for levels of Pt by inductively coupled plasm
41、a mass spectrometry (ICP-MS). As predicted, the levels of Pt in the plasma were significantly higher in the animals treated with 5 than 4 (1.7_0.8) and (0.3_0.1) mmol of Pt, respectively) after 60 minutes. The higher level of Pt in the blood was also reflected by higher levels of Pt in the brain aft
42、er 60 minutes (0.006_0.002) and (0.003_0.001) mg of Pt per g of body weight). These data areconsistent with the PtIV prodrug being more bioavailable.确保PtIV前体药物策略导致更高水平的生物药物,我们对待野生型小鼠4或5 15 mgkg_1(注意在分子基础上,动物治疗5得到的药物略低于)。血液和大脑组织收集在5日,30日,60分钟后加药,血液中的血浆分离,脑组织均质。作为替代措施的药物的水平,分析了等离子体和脑homogenates水平Pt的电感
43、耦合等离子体质谱法(icp - ms)。正如预测的那样,Pt在等离子体的水平明显高于动物的处理5比4(1.7 _0.8)和Pt(0.3 _0.1)中毒,分别)后60分钟。血液中更高层次的Pt也反映在更高水平的Pt 60分钟后的大脑(_0.002(0.006)和(0.003 _0.001)毫克每克体重Pt)。这些数据是符合PtIV前体药物被更多的生物利用率。To ensure that 5 can be reduced to the active PtII complex in vivo, 5 was mixed with glutathione, and the reduction of Pt
44、IV to PtII was monitored with 195Pt NMR spectroscopy. The 195Pt chemical shift of 5 is _316 ppm, typical of a PtIV complex. When mixed with glutathione for 20 minutes, this resonance disappeared and a new resonance was visible at _2365 ppm, consistent with a PtII species (see the Supporting Informat
45、ion). 确保5可以减少活动PtII复杂的体内,与谷胱甘肽5不一,减少PtIV PtII监控了195 pt核磁共振光谱学。195年工党5是_316 ppm的化学位移,典型的PtIV复杂。当与谷胱甘肽20分钟混合时,这个共振消失了,一个新的共振在_2365 ppm是可见的,符合PtII物种(见支持信息)。We initially tested 5 at a dose of 15 mgkg_1d_1 by oral gavage for 18 weeks in the Tg2576 mouse model of AD.28 We found that treatment with 5 reduc
46、ed the number of Ab plaques by 29%, although this result did not reach statistical significance (see Figure S2a in the Supporting Information).While there was no change in Ab levels detected by Western blot, when the same tissue was analyzed by SELDI-TOF MS using the 4G8 antibody (epitope residues 1
47、721 of Ab), the changes in the spectra of treated and untreated animals were quite pronounced (see Figure S2b and c in the Supporting Information). Perhaps most significantly, signals at 9260 and 12200 Da, which were visible in the vehicle-treated group, were significantly reduced by treatment with
48、5 (see Figure S2d and e in the Supporting Information). Species with these approximate molecular weights have previously been implicated as “naturally derived” toxic forms of Ab.29我们最初的剂量测试5 15 mgkg_1d_1 Tg2576口服填喂法18周的小鼠模型的广告。28我们发现治疗5 Ab斑块的数量减少了29%,尽管这个结果没有达到统计学意义(参见图S2a支持信息)。虽然没有改变Ab水平检测到免疫印迹,当同一
49、组织分析了SELDI TOF女士使用4 g8抗体(Ab)17 - 21型表位残基区间,处理和未经处理的动物的光谱变化非常明显(见图开通和c在支持信息)。或许最重要的是,在9260年和12200年,可见vehicle-treated组,显著降低了治疗5(参见图S2d和e在支持信息)。物种与这些近似分子量以前与自然“派生”有毒的Ab形式。29Next we tested 5 in the APP/PS1 mouse model of AD.30, 31 Analysis of brain tissue using SELDI-TOF MS showed that treatment led to a statistically significant 40% reduction in Ab42 levels (p=0.025; Figure 3a,b). This result was accompanied by a statistically significant 26% decrease in plaque number in the treated ani
