HI-6人体血清白蛋白纳米粒的制备及其对梭曼中毒小鼠脑AChE重活化作用评价

迅速恢复中毒乙酰胆碱酯酶(acetylcholinesterase,AChE)活性是神经性毒剂中毒救治最关键的环节.血脑屏障的存在限制了具有重活化作用的抗毒药物以有效治疗剂量进入中枢.本研究以已知重活化作用最强的重活化剂酰胺磷定(HI-6)为目标药物,通过去溶剂化法制备了人体血清白蛋白纳米粒,通过静电作用将HI-6分子装载在纳米粒表面,合成装载HI-6的人体血清白蛋白纳米粒;在斑马鱼及神经性毒剂梭曼染毒小鼠上,对药物穿透血脑屏障能力及中枢中毒AChE重活化作用进行了评价.结果表明,装载HI-6人体血清白蛋白纳米粒在物理表征上符合纳米药物基本特征;相对于自由HI-6,HI-6人体血清白蛋白纳米粒能够透过血脑屏障,并将中枢中毒AChE活性提升2倍以上,表明人体血清白蛋白纳米粒成功携带HI-6分子进入中枢.本文建立了一种无毒、高效、小尺寸中枢靶向性纳米粒的制备方法,基于该方法制备的纳米重活化剂,在脑内可有效释放目标药物并迅速发挥解毒作用.     

噁二唑肟醚取代的均三唑水杨醛席夫碱衍生物的合成及抗菌活性

4-氨基-3-苯基-5-巯基-均.三唑(1)在无水乙酸钠作用下与β-氯苯丙酮(2)缩合得氨基三唑硫基苯丙酮(3).化合物3与盐酸羟胺肟化得羰基肟化物(4),采用氯甲基(噁)二唑(5)对化合物4的肟羟基醚化得均三唑席夫碱(噁)二唑肟醚(6a～6e).肟化物6a～6e与水杨醛缩合得到目标物席夫碱7a～7e.初步的体外抗菌实验表明,席夫碱肟醚类化合物7a～7e的活性明显高于氨基肟醚类化合物6a～6e,且对革兰氏阳性(Sa)的活性高于对革兰氏阴性菌(Ec)的.     

4-偕胺肟双吡啶季铵碘盐的合成

双吡啶醛肟季铵盐类对某些有机磷酸酯类中毒具有明显的对抗作用,因此合成了一类4-偕胺肟双吡啶季铵碘盐化合物(1a～1n),以期找出更有效的抗毒剂. CONR_2取代在3位:NR_2=NH_2(a),N(CH_3)_2(b),N(C_2H_5)2(c),NHC_3H_(7-n)(d),NHC_3H_(7-i)(e),NHC_4H_9n(f),NHC_4H_(9-i)(g), CONR_2取代在4位:NR_2=NH_2(1),NHC_3H_(7-n)(m),这类新化合物的合成是以4-吡啶甲酸(2)制得4-氰基吡啶(3),再与盐酸羟胺作用制得4-吡啶偕胺肟(4),然后与α,α’-二氯甲醚作用制得N-氯甲氧甲基-4-吡啶偕胺肟氯盐(简称单氯盐,5),最后与相应的N-取代吡啶甲酰胺(6)作用而得.     

重化活化剂作用机理——季铵和非季铵型重活化剂的构效关系研究

本文根据新的重活化理论模型讨论了季铵和非季铵两类重活化剂的构效关系及其作用机理。在构效关系研究基础上,对重活化剂的分子结构进行了基本设计:(1)最好是叔胺型,不仅有利于通过血脑屏障,进入中枢,更有利于药物与受体之间的轨道作用,季铵的电荷作用太强,不利于轨道作用的充分发挥。(2)应该存在足够大的π-π共轭体系,具有较高的HOMO能级或较低的LUMO能级。(3)活性基团之间的距离,肟基氧到季铵或叔胺氮的距离D1应接近3—6;π-π共轭体系到肟羟基氧的距离D2约为7—10。(4)活性基团最好是柔性结构(有利于构象调节),构象改变又不要明显地影响前线轨道能级的变化(以保持轨道作用的最佳状态)。     

有机磷化合物的研究(LVII)——二苯(氧)基氯膦对肟基的自由基加成——合成1-氨基取代三烷基氧化膦及烷基膦酸酯的新途径

二苯基氯膦或二苯氧基氯膦对醛(酮)肟的反应可作为合成1-氨基烷基二苯基氧化膦或1-氨基烷基膦酸二苯酯的新方法,具有条件温和、操作方便及得率高的优点。EPR研究结果揭示了这类反应属自由基机理。     

脂肪酰胺水解酶催化三联体膦酰化失活的机理：一个模型体系的理论研究

甲基花生四烯基氟代膦酸酯(MAFP)是脂肪酰胺水解酶(FAAH)的一个抑制剂. FAAH的丝氨酸241(Ser241)-丝氨酸217(Ser217)-赖氨酸142(Lys142)催化三联体被MAFP膦酰化后将导致FAAH失活. 本文采用B3LYP/6-311G(d,p)和MP2/6-311G(d,p)方法及一个简化的计算模型体系对这个膦酰化抑制反应进行理论研究. 考虑了两种反应途径. Path A涉及FAAH的催化三联体的所有残基, 是一个分步的加成-消除过程, 形成两性离子的三角双锥中间体, 其中第一步反应是决速步骤. 在这个反应途径中, Ser217和Lys142对亲核试剂Ser241起到碱催化活化的作用, 而Ser217充作Lys142和Ser241之间的桥梁. 此外, 溶剂中的一个水分子作为Lys142和MAFP间的“氢桥”具有关键的作用, 通过给出和接收质子促进了长距离的质子转移. Path B是催化三联体中的残基Lys142被突变为丙氨酸以后的膦酰化反应, 也是一个分步过程. 水的本体溶剂效应通过极化连续介质模型(PCM)估算. 计算结果显示膦酰化反应的Path A是优势途径, 在水溶液中其决速步骤的活化能垒为64.9 kJ·mol-1. FAAH催化三联体中残基Lys142的变异会降低膦酰化反应的速率, 这与实验结果相一致.     

E-、Z-2’-(1-咪唑基)-O-(α-甲基-二氯苄基)-2,4-二氯苯乙酮肟硝酸盐的合成与抑菌活性

以间二氯苯、氯乙酰氯和咪唑为原料 ,利用成肟和引入咪唑基顺序不同 ,首先合成了 E-,Z-2 -(1 -咪唑基 ) -2 ,4 -二氯苯乙酮肟 ( E、IZ) ,从二氯苯合成了 α-氯 -二氯取代乙苯 ( A、 B和 C) ,咪唑基 -二氯苯乙酮肟顺反异构体 ( Z、 E)分别与 α-氯 -二氯乙苯反应 ,生成 E-、Z-2 -(1 -咪唑基 ) -O-(α-甲基 -二氯苄基 ) -2 ,4 -二氯苯乙酮肟硝酸盐共 6种新化合物 ,产率为 5 7.5 %～ 64 .5 %,新化合物结构经元素分析、IR和 1H NMR表征 .初步实验表明 ,各化合物对水稻苗腐根有不同程度抑制活性 .     

过渡金属催化肟类化合物的反应研究进展

按照反应类型的不同,对近年来过渡金属催化肟类化合物反应的研究进展进行了综述,主要包括酮肟的还原酰化反应,肟类化合物的催化偶联反应,胺化反应以及肟类化合物与炔烃的环化反应.     

苯基氧肟酸、正辛基氧肟酸与希土配合物的制备和性质

在乙醇一水混合溶液中,采用沉淀法制得了三十个苯基氧肟酸、正辛基氧肟酸与除钜以外的镧系元素及钇的配合物.通过元素分析、红外光谱、紫外光谱、质谱和差热热重分析研究了它们的组成和性质.它们的组成可分别确定为:M(C_6H_5C(O)NHO)_3和M(n-C_7H_(15)C(O)NHO)_3;光谱研究说明配体失去羟基质子后与希土离子形成了O、O五元螯合物,质谱和差热热重分析进一步说明螯合环在质子轰击与加热至220℃时均不易断裂,非常稳定。     

不对称环磷酸肟酯的合成和生物活性研究

通过反式-2-氯-2-氧-4-苯基-5,5-二甲基-1,3,2-二氧磷杂环己烷(Ⅱ)和α-氰基-芳基甲醛肟(Ⅰa-Ⅰf)在相转移催化条件下反应,制得新型的1,3,2-二氧磷杂环磷酸肟酯(Ⅲa_Ⅲf),产物中的非对映异构体经¹HNMR、³¹PNMR和单晶X射线衍射确证.但化合物Ⅱ与芳基甲醛肟反应,得到芳基甲腈和环磷酸,这可能是生成不稳定的环磷酸醛肟酯(Ⅳ)经Beckmann裂解所致.生物活性测试表明,化合物cis-Ⅲd具有很好的抗烟草花叶病毒活性.     

Improvement of acetylcholinesterase-based assay for organophosphates in way of identification by reactivators

Organophosphates present serious fulmination in several aspects of human life. Detection of organophosphates is frequently based on following acetylcholinesterase (AChE) inhibition. Although limit of detection and sensitivity for AChE-based assays seem to be intriguing, the identification of organophosphates is not currently efficient in this way. We introduce an improvement of AChE-based assay by reactivators using a selective come-back of AChE activity after previous inhibition. We have chosen four organophosphates: paraoxon-ethyl, paraoxon-methyl, trichlorfon, methamidophos as representative pesticides and the three most available reactivators: HI-6, obidoxime, pralidoxime. Reactivation was realized in the 96-wells photometric microplates and activity of human recombinant AChE was followed by reaction of Ellman's reagent with one of enzyme digestion product: thiocholine. Distinguishing of reactivation efficacy was judged by the independent two population t-test. The most significant identification was based on methamidophos inhibited AChE reactivation by HI-6 or pralidoxime and paraoxon-ethyl inhibited AChE by obidoxime; moreover, identification of trichlorfon and paraoxon-methyl was possible, too. Practical impact of described method is discussed.     

Reversal of Tabun Toxicity Enabled by a Triazole-Annulated Oxime Library—Reactivators of Acetylcholinesterase

Acetylcholinesterase (AChE), an enzyme that degrades the neurotransmitter acetylcholine, when covalently inhibited by organophosphorus compounds (OPs), such as nerve agents and pesticides, can be reactivated by oximes. However, tabun remains among the most dangerous nerve agents due to the low reactivation efficacy of standard pyridinium aldoxime antidotes. Therefore, finding an optimal reactivator for prophylaxis against tabun toxicity and for post-exposure treatment is a continued challenge. In this study, we analyzed the reactivation potency of 111 novel nucleophilic oximes mostly synthesized using the CuAAC triazole ligation between alkyne and azide building blocks. We identified several oximes with significantly improved in vitro reactivating potential for tabun-inhibited human AChE, and in vivo antidotal efficacies in tabun-exposed mice. Our findings offer a significantly improved platform for further development of antidotes and scavengers directed against tabun and related phosphoramidate exposures, such as the Novichok compounds.     

First efficient uncharged reactivators for the dephosphylation of poisoned human acetylcholinesterase

Nerve agents are highly toxic organophosphorus compounds with strong inhibition potency against acetylcholinesterase (AChE). Herein, we describe two first extremely promising uncharged reactivators for poisoned human AChE with a superior or similar in vitro ability to reactivate the enzyme as compared to that of HI-6, obidoxime, TMB-4 and HL?-7.     

Chemoselective Hydrogenation of 6-Alkynyl-3-fluoro-2-pyridinaldoximes: Access to First-in-Class 6-Alkyl-3-Fluoro-2-pyridinaldoxime Scaffolds as New Reactivators of Sarin-Inhibited Human Acetylcholinesterase with Increased Blood–Brain Barrier Permeability

Novel 6-alkyl- and 6-alkenyl-3-fluoro-2-pyridinaldoximes have been synthesised by using a mild and efficient chemoselective hydrogenation of 6-alkynyl-3-fluoro-2-pyridinaldoxime scaffolds, without altering the reducible, unprotected, sensitive oxime functionality and the C−F bond. These novel 6-alkyl-3-fluoro-2-pyridinaldoximes may find medicinal application as antidotes to organophosphate poisoning. Indeed, one low-molecular-weight compound exhibited increased affinity for sarin-inhibited acetylcholinesterase (hAChE) and greater reactivation efficiency or resurrection for sarin-inhibited hAChE, compared with those of 2-pyridinaldoxime (2-PAM) and 1-({[4-(aminocarbonyl)pyridinio]methoxy}methyl)-2-[(hydroxyimino)methyl]pyridinium chloride (HI-6), two pyridinium salts currently used as antidote by several countries. In addition, the uncharged 3-fluorinated bifunctional hybrid showed increased in vitro blood–brain barrier permeability compared with those of 2-PAM, HI-6 and obidoxime. These promising features of novel low-molecular-weight alkylfluoropyridinaldoxime open up a new era for the design, synthesis and discovery of central non-quaternary broad spectrum reactivators for organophosphate-inhibited cholinesterases.     

Study of Inhibition,Reactivation and Aging Processes of Pesticides Using Graphene Nanosheets/Gold Nanoparticles‐Based Acetylcholinesterase Biosensor

Organophosphate (OP) and carbamate pesticides exert their toxicity via attacking the hydroxyl moiety of serine in the ‘active site’ of acetylcholinesterase (AChE). In this paper we developed a stable AChE biosensor based on self‐assembling AChE to graphene nanosheet (GN)‐gold nanoparticles (AuNPs) nanocomposite electrode for investigation of inhibition, reactivation and aging processes of different pesticides. It is confirmed that pesticides can inhibit AChE in a short time. OPs poisoning is treatable with oximes while carbarmates exposure is insensitive to oximes. The proposed electrochemical approach thus provides a new simple tool for comparison of pesticide sensitivity and guide of therapeutic intervention.     

Aging of cholinesterases phosphylated by tabun proceeds through O-dealkylation

Human butyrylcholinesterase (hBChE) hydrolyzes or scavenges a wide range of toxic esters, including heroin, cocaine, carbamate pesticides, organophosphorus pesticides, and nerve agents. Organophosphates (OPs) exert their acute toxicity through inhibition of acetylcholinesterase (AChE) by phosphorylation of the catalytic serine. Phosphylated cholinesterase (ChE) can undergo a spontaneous, time-dependent process called "aging", during which the OP-ChE conjugate is dealkylated. This leads to irreversible inhibition of the enzyme. The inhibition of ChEs by tabun and the subsequent aging reaction are of particular interest, because tabun-ChE conjugates display an extraordinary resistance toward most current oxime reactivators. We investigated the structural basis of oxime resistance for phosphoramidated ChE conjugates by determining the crystal structures of the non-aged and aged forms of hBChE inhibited by tabun, and by updating the refinement of non-aged and aged tabun-inhibited mouse AChE (mAChE). Structures for non-aged and aged tabun-hBChE were refined to 2.3 and 2.1 A, respectively. The refined structures of aged ChE conjugates clearly show that the aging reaction proceeds through O-dealkylation of the P(R) enantiomer of tabun. After dealkylation, the negatively charged oxygen forms a strong salt bridge with protonated His438N epsilon2 that prevents reactivation. Mass spectrometric analysis of the aged tabun-inhibited hBChE showed that both the dimethylamine and ethoxy side chains were missing from the phosphorus. Loss of the ethoxy is consistent with the crystallography results. Loss of the dimethylamine is consistent with acid-catalyzed deamidation during the preparation of the aged adduct for mass spectrometry. The reported 3D data will help in the design of new oximes capable of reactivating tabun-ChE conjugates.     

New methods in synthesis of acetylcholinesterase reactivators and evaluation of their potency to reactivate cyclosarin-inhibited AChE

Nine potential AChE reactivators were synthesized using modification of currently known synthetic pathways. Their potency to reactivate AChE inhibited by cyclosarin nerve agent was tested in vitro. According to the previous results, 1,4-bis(2-hydroxyiminomethylpyridinium)butane dibromide seems to be the most potent AChE reactivator. The reactivation potency of these compounds depends on structural factors such as presence of quaternary nitrogens, length of the linking chain between both pyridinium rings, and position of the oxime moiety at the pyridinium ring.     

In Vitro Screening of Blood-Brain Barrier Penetration of Monoquaternary Acetylcholinesterase Reactivators

The HPLC method for prediction of drug penetration into the CNS was examined in our study. In this in vitro study, we determined the ability of monoquaternary AChE reactivators to penetrate through blood-brain barrier (BBB). Three reactivators varying in the position of oxime group on the pyridinium ring (2-PAM, 3-PAM and 4-PAM) were evaluated. As a result, the position of the oxime group on the pyridine ring plays a role for penetration of oximes into brain. The best position of oxime group seems to be position four. The location on the position two and three is not favorable for penetrating through BBB.     

Targeted synthesis of 1-(4-hydroxyiminomethylpyridinium)-3-pyridiniumpropane dibromide--a new nerve agent reactivator

Preparation of 1-(4-hydroxy-iminomethylpyridinium)-3-pyridiniumpropane dibromide is described. This compound represents a new acetylcholinesterase (AChE) reactivator, which has no substituents on the second pyridinium ring as found in other commonly used AChE reactivators. The reactivation ability of this reactivator was tested on tabun- and cyclosarin-inhibited AChE. According to the results obtained, the new compound (without substitution and with decreased molecule size) showed increased reactivation potency in case of cyclosarin inhibited AChE. A potent oxime for treatment of tabun and cyclosarin-caused intoxications was thus obtained via slight modification of the reactivator structure (compared to trimedoxime and K027).