三肽四氮唑类20S蛋白酶体抑制剂的设计、合成与活性研究

泛素-蛋白酶体途径是细胞内降解蛋白质的一种主要方式,由20S蛋白酶体来完成蛋白质的降解。本文在已经报道的肽类抑制剂的基础上,设计合成了一类三肽四氮唑化合物,通过1H NMR、MS以及元素分析对化合物结构进行了表征。活性评价结果表明,有3个目标化合物(6b、6d和6h)具有较好的抑制20S蛋白酶体类胰凝乳蛋白酶的活性。分子对接研究显示,这类新型C端基肽类化合物能通过与活性位点非共价相互作用而与蛋白酶体结合。     

新型β-肽环氧酮类蛋白酶体抑制剂的合成与活性评价

目的 设计合成新型的β-肽类蛋白酶体抑制剂,并对其活性进行评价。方法 根据先导化合物Carfilzomib与蛋白酶体的作用方式,保留其与蛋白酶体结合的关键环氧酮片段,并结合β-氨基酸的特点,采用氨基酸替换、生物电子等排等药物设计的方法,设计一类结构新颖的蛋白酶体抑制剂;采用缩合、氧化、还原等反应,合成系列目标化合物;通过体外酶抑制活性实验检验化合物活性。结果 合成了8个结构新颖的β-肽环氧酮类衍生物,化合物结构经1H-NMR、ESI-MS确证,部分化合物体现了一定的蛋白酶体抑制活性。结论 β-氨基酸作为一种重要的α-氨基酸替换结构,有望能够丰富短肽类蛋白酶体抑制剂的结构类型。     

新型非共价结合拟肽类蛋白酶体抑制剂的合成及活性评价

目的 设计、合成系列非共价结合拟肽类蛋白酶体抑制剂,并对其进行活性评价。方法 根据非共价结合蛋白酶体抑制剂与蛋白酶体的结合特点,采用氨基酸替换、生物电子等排等经典的药物设计方法,选取邻氯苄胺作为化合物的羧基末端基团,同时在肽骨架结构中引入六元环以增强肽类化合物的稳定性,设计并合成了一系列短肽非共价结合类蛋白酶体抑制剂,并通过体外蛋白酶体活性抑制实验评价该类化合物的活性。结果 共合成了8个具有全新结构的二肽和三肽化合物,其结构经¹H-NMR、ESI-MS确证,该类化合物对蛋白酶体具有中等的抑制活性。结论 肽链的长短及氨基末端不同的取代基对化合物的蛋白酶体抑制活性都有影响,8个化合物在体外对蛋白酶体都具有不同程度的抑制活性。本研究丰富了蛋白酶体抑制剂的结构类型,为该类化合物的深入研究奠定了基础。     

四氢异喹啉-噁二唑类非共价型蛋白酶体抑制剂研究初探

目的探索四氢异喹啉-噁二唑类非共价型蛋白酶体抑制剂的可行性。方法基于拼合原理,将新型非共价型蛋白酶体抑制剂PI-1833(5)的1,2,4-噁二唑靶头与本研究组发现的四氢异喹啉脲拟肽骨架相拼合,设计了四氢异喹啉-噁二唑类非共价型蛋白酶体抑制剂,合成了N-((3-苯基-1,2,4-噁二唑-5-基)甲基)-3,4-二氢异喹啉-2(1H)-甲酰异丙胺(8),并采用MTT法评价其对人胃癌细胞MGC-803的生长抑制活性;利用分子对接的方法分析化合物与20S蛋白酶体的结合状态。结果与结论化合物8在10μmol·L~(-1)浓度下没有显示出抗肿瘤活性;通过比较化合物5和8与20S蛋白酶体的对接结果,发现化合物8不产生活性的主要原因可能是噁二唑和四氢异喹啉之间的连接臂柔性不够,提示进一步研究的重点应该是对连接臂的优化。     

真菌来源的蛋白酶体抑制剂F04W2166A的研究

目的 通过蛋白酶体抑制剂筛选模型从微生物代谢产物中筛选出具有蛋白酶体抑制活性的化合物.方法 对1株筛选得到的阳性真菌进行放大发酵,通过硅胶和Sephadex LH-20柱色谱等分离手段进行单体化合物分离.结合理化性质和质谱及核磁共振等数据的分析,确定其化学结构.测定化合物的蛋白酶体抑制活性以及抗肿瘤细胞增殖活性.结果 从1株具有活性的轮枝孢霉菌F04W2166中得到活性化合物F04W2166A,并确定其结构与环缩酚酞类己知化合物Pullularin C相同.体外酶抑制活性说明该化合物对蛋白酶体有很强的抑制活性,其IC_50为3.1μg/mL.进一步的抗肿瘤细胞增殖活性结果证明该化合物对人结肠癌细胞株HT-29和人乳腺癌细胞株MDA-MB-231的增殖均有很高的抑制活性,其IC_50分别为28.3和271.6ng/mL.结论 F04W2166A是一个环缩酚酞类的蛋白酶体抑制剂,具有强的体外抗肿瘤细胞增殖活性.     

二肽基肽酶-4抑制剂研究进展

二肽基肽酶-4抑制剂为一类常用的2型糖尿病治疗药物,可通过抑制二肽基肽酶-4活性延长胰高血糖素样肽-1的作用时间和促进胰岛素分泌,从而降低血糖水平。根据化学结构类型的不同,将二肽基肽酶-4抑制剂分为拟肽类和非拟肽类抑制剂,并分别介绍其药理活性、构效关系及药动学特性等方面的研究进展。     

7-(咪唑-4-烷基酰胺基)-1,3-二氢-1-羧基烷基-5-苯基-2H-1,4-苯并二氮杂-2-酮一类新的法呢基蛋白转移酶抑制剂

目的设计并合成新结构类型的法呢基蛋白转移酶抑制剂。方法本文结合法呢基蛋白转移酶(FTase)的作用机理和已有FTase抑制剂结构特征,设计了一类以苯并二氮杂为分子骨架,一端连接有可与锌离子配位结合的咪唑基,另一端连接不同长度的末端含羧基的侧链的化合物。此类化合物模拟了FTase配体之一CAAX四肽片段,共合成10个此类新化合物(6～12,16～18),并对其进行体外生物活性测定。结果 所有新目的化合物均经1HNMR和HRMS方法确证结构。结论对FTase抑制活性测定结果表明其中5个化合物(9,10,16～18)有较强的抑制活性。     

选择性组蛋白去乙酰化酶 SIRT2抑制剂的合成及活性研究

目的设计合成新型选择性人沉默调节蛋白2(SIRT2)抑制剂并评价其抗肿瘤活性。方法首先以非典型组蛋白去乙酰化酶SIRT2作为靶标,在前期研究基础上,设计合成了6个N-(3-或4-取代苯基)-2-(芳基杂基)乙酰胺衍生物,并测试其体外对SIRT2的抑制活性;对活性最佳的化合物测试其对SIRT1和SIRT3的抑制活性从而评价其选择性;采用MTT法评价优选化合物1c对10种肿瘤细胞的抑制活性。结果与结论 6个目标化合物对SIRT2均有抑制作用,其中化合物1c活性最好,在50、5μmol·L~(-1)的抑制率分别为95%、71%,且该化合物对SIRT2具有较好的选择性;细胞水平抗肿瘤活性测试表明,化合物1c对肝癌细胞HUH7有较好的抑制作用,在50、25μmol·L~(-1)时的抑制率分别为92%和78%。该研究为特异性SIRT2小分子抑制剂的开发及靶向SIRT2的抗肿瘤药物研究奠定了一定基础。     

新型肽脱甲酰基酶(PDF)抑制剂的研究(Ⅰ) --含异噁唑的丁二酰异羟肟酸衍生物的设计与合成

目的：发现新的肽脱甲酰基酶（PDF）抑制剂先导化合物.方法：在异噁唑环杂原子作为电子对供体替代羰基氧原子与受体形成氢键的新思路指导下,设计并合成了一系列新型含异噁唑的β-戊基-丁二酰异羟肟酸衍生物,其结构经核磁共振（NMR）、质谱（MS）等证明,并对部分该类化合物进行了体外抑菌实验.结果：体外抑菌实验表明,该类化合物具有一定的抗菌活性.结论：以异噁唑环杂原子作为电子对供体替代肽分子中的羰基氧原子与受体形成氢键的设想是成立的,为进一步优化本类化合物结构,乃至对类肽新药的设计具有重要意义.     

新型神经氨酸酶抑制剂的设计合成和药理活性测试

目的 为防控流感,设计合成新型神经氨酸酶抑制剂,并进行药理活性测试。方法 基于苯甲酸类神经氨酸酶抑制剂的结构,拼合具有抗病毒活性的酰基硫脲片段,利用前药原理设计并合成了I和Ⅱ系列共28个新型神经氨酸酶抑制剂并进行药理活性筛选。药理活性测试选用流感病毒菌株A/FM/1/47（H1N1）,分别进行神经氨酸酶单一浓度（10μmol·L^-1）抑制率和病毒抑制活性测试。结果 药理结果显示化合物I-2,I-5,I-11和I-12具有较好的病毒抑制活性,其中I-11的活性最佳,病毒抑制率达到70.50%,优于oseltamivir。结论 通过对28个化合物进行初步构效关系研究,为后续此类新型神经氨酸酶抑制剂的研究提供基础。     

Design,Synthesis and Biological Evaluation of Peptidyl Epoxyketone Proteasome Inhibitors Composed of β‐amino Acids

A series of novel di‐ and tripeptidyl epoxyketone derivatives composed of β‐amino acids were designed, synthesized and evaluated for their proteasome inhibitory activities and anti‐proliferation activities against two multiple myeloma cell lines RPMI 8226 and NCI‐H929 and normal cells (peripheral blood mononucleated cells). Among these tested compounds, tripeptidyl analogues showed much more potent activities than dipeptides, and four tripeptidyl compounds exhibited proteasome inhibitory activities with IC₅₀ values ranging from 0.97 ± 0.05 to 1.85 ± 0.11 μm . In addition, all the four compounds showed anti‐proliferation activities with IC₅₀ values at low micromolar levels against two multiple myeloma cell lines and weak activities against normal cells. Furthermore, Western blot analysis was performed to verify the proteasome inhibition induced by compounds 21d and 21e . All the experimental results validated that the β‐amino acid building block has the potential for the development of proteasome inhibitors.     

Study on the dipeptide vinyl sulfonamide derivatives as proteasome inhibitor

On the basis of the Michael-addition mechanism of classical proteasome inhibitors, six dipeptide vinyl sulfonamide and dipeptide vinyl sulfonate derivatives were designed and synthesized. Moreover, an efficient method for the synthesis of g-amino vinyl sulfonamides, key intermediates to the target molecules, was developed via the Wittig-Horner reaction of peptide aldehyde with Wittig reagents derived from methanesulfonamides.     

Trypanocidal activity of β-lactone-γ-lactam proteasome inhibitors

Four β-lactone- γ-lactam proteasome inhibitors of natural origin were tested for their trypanocidal activities in vitro using culture-adapted bloodstream forms of Trypanosoma brucei. All four compounds displayed activities in the nanomolar range. The most trypanocidal compounds with 50% growth inhibition (GI(50)) values of around 3?nM were the bromine and iodine analogues of salinosporamide A, a potent proteasome inhibitor produced by the marine actinomycete Salinispora tropica. In general, trypanosomes were more susceptible to the compounds than were human HL-60 cells. The data support the potential of β-lactone- γ-lactam proteasome inhibitors for rational anti-trypanosomal drug development.     

Novel tetra-acridine derivatives as dual inhibitors of topoisomerase II and the human proteasome

Acridine derivatives, such as amsacrine, represent a well known class of multi-targeted anti-cancer agents that generally interfere with DNA synthesis and inhibit topoisomerase II. But in addition, these tricyclic molecules often display secondary effects on other biochemical pathways including protein metabolism. In order to identify novel anti-cancer drugs, we evaluated the mechanism of action of a novel series of bis- and tetra-acridines. As expected, these molecules were found to interact with DNA and inhibit the topoisomerase II-mediated DNA decatenation. Interestingly when tested on human tumour cells either sensitive (HL-60) or resistant (HL-60/MX2) to topoisomerase II inhibitors, these molecules proved equicytotoxic against the two cell lines, suggesting that they do not only rely on topoisomerase II inhibition to exert their cytotoxic effects. In order to identify alternative targets, we tested the capacity of acridines 1-9 to inhibit the proteasome machinery. Four tetra-acridines inhibited the proteasome in vitro, with IC(50) values up to 40 times lower than that of the reference proteasome inhibitor lactacystin. Moreover, unlike peptide aldehydes used as reference inhibitors for the proteasome, these new acridine compounds demonstrated a good selectivity towards the proteasome, when tested against four unrelated proteases. A cellular assay based on the degradation of a proteasome protein substrate indicated that at least two of the tetra-acridines maintained this proteasome inhibition activity in a cellular context. This is the first report of tetra-acridines that demonstrate dual topoisomerase II and proteasome inhibition properties. This new dual activity could represent a novel anti-cancer approach to circumvent certain forms of tumour resistance.     

磺胺类黄酮衍生物20S蛋白酶体抑制剂的设计、合成与活性评价

本文通过计算机辅助设计,设计并合成了一系列磺胺类黄酮衍生物作为非共价20S蛋白酶体抑制剂,并对其生物活性进行了测试。与先导化合物相比（β5亚基的IC50值为14.0μM）,化合物仅表现出局部的改善,但仍可作为一类潜在的20S蛋白酶体抑制剂。     

连续荧光监测法测定蛋白酶体活性及其在筛选蛋白酶体抑制剂中的应用

目的:建立用连续荧光监测法测定蛋白酶体活性的方法,研究其在筛选蛋白酶体抑制剂中的应用价值。方法:用特异性荧光多肽底物Suc-Leu-Leu-Val-Tyr-AMC(Suc-LLVT-AMC)、Z-Val-Val-Arg-AMC(ZVVA-AMC)、Z-Leu-Leu-Glu-βNA(ZLLG-βNA),分别测定蛋白酶体的糜凝乳蛋白酶样(chy-motrypsin-like,CT-L)、胰蛋白酶样(trypsin-like,T-L)和肽基谷氨酰肽水解酶样(PGPH-like,PGPH-L)活性,对测定条件优化,并进行方法学评价。用该法测定了硼替佐米(PS-341)和四嗪二甲酰胺(ZG-DHu-1)对纯20S蛋白酶体和B16、Namalwa细胞蛋白酶体提取物抑制作用。结果:连续荧光监测法测定蛋白酶体活性的最适pH为8.2;0.3g/LSDS仅对CT-L活性有激活作用;CT-L、T-L、PGPH-L的Km分别为3.55、4.12、4.88μmol/L;酶反应进程曲线的线性期达20min;线性范围为测定纯20S蛋白酶体时,其蛋白浓度分别达100、120、100μg/L,测定B16细胞蛋白酶体提取物时,其蛋白浓度均达2...     

Trypanocidal effect of alpha',beta'-epoxyketones indicates that trypanosomes are particularly sensitive to inhibitors of proteasome trypsin-like activity

Previous studies have shown that the proteasome of Trypanosoma brucei is a candidate for novel chemotherapy of African sleeping sickness. In this study, two potent and highly selective alpha',beta'-epoxyketones peptide proteasome inhibitors, epoxomicin and YU101, have been tested for their trypanocidal activities in vitro using culture-adapted bloodstream forms of T. brucei. Both inhibitors displayed promising anti-trypanosomal activities with ED(50) and ED(90) values in the low to mid nanomolar range. Based on MIC values, epoxomicin exhibited a selectivity index approaching those of commercially available drugs. Enzymatic analyses of proteasomal peptidase activities revealed that, compared with mammalian cells, trypanosomes are particular sensitive to inhibition of the trypsin-like activity of the proteasome. In conclusion, the data suggests that proteasome inhibitors targeting the trypsin-like activity are the rational choice for future anti-trypanosomal drug development.     

Recent advances in antiviral activity of benzo/heterothiadiazine dioxide derivatives

Benzo/heterothiadiazine dioxides have been identified as important fused heterocyclic systems possessing a broad spectrum of biological activities and potential pharmacological applications. Recently, a large number of structurally novel compounds derived from these heterocycle scaffolds were identified as antiviral agents. Especially, substituted benzo/heterothiadiazine dioxide derivatives have been shown to inhibit the replication of HCMV, VZV, HCV and HIV. Of particular interest, some potent HCV polymerase inhibitors possess a benzothiadiazine dioxide scaffold, which is critical for the anti-HCV potency through strong hydrogen bond formation of the SO(2)NH group with the active site of the enzyme, as shown by X-ray crystallography. Also, some compounds belonging to the benzothiadiazine dioxide class have been found to be potent antiviral agents against HCMV and VZV. Moreover, some novel heterothiadiazine dioxide derivatives have been synthesized and evaluated as potential HIV inhibitors with lower toxicity and/or increased activity against drug-resistant virus strains. No systematic review is available in the literature on these thiadiazine derivatives in the design of potent antiviral inhibitors. In this article, we review the recent advances in the antiviral profile of this kind of compounds, as well as the impact of structural modifications and the structure-activity relationship (SAR).     

Preliminary studies of new proteasome inhibitors in the tumor targeting approach: synthesis and in vitro cytotoxicity

The proteasome is a multicatalytic protease that plays a critical role in the cell. The control of proteasomes could, thus, provide a weapon for the treatment of cancer. Therefore, we have synthesized six new peptide aldehyde inhibitors of the proteasome linked to the N-(2-diethylaminoethyl)benzamide (BZA-CO) structure, in order to target the cytotoxic activity to malignant melanoma cells. Biological studies demonstrated the influence of length and composition of the amino acid chain on the cytotoxicity of our compounds. Among them, compound 19 presents the highest cytotoxicity (IC50 = 0.64 +/- 0.07 micromol): this cytotoxicity was maintained in the presence of BZA-CO but decreased 8-fold compared to the control MG132. Fluorescence activated cell sorter (FACS) and cytotoxic activity analysis demonstrated the selectivity of compound 19 for melanoma cells. Finally, western blottings of ubiquitinated proteins in IPC227F cells as well as proteasome assays confirmed that the cytotoxicity was linked to an inhibition of the proteasome activity.