發(fā)布時(shí)間：2018-08-09 18:06

【摘要】：先導(dǎo)化合物的發(fā)現(xiàn)和優(yōu)化是新藥研發(fā)的重要環(huán)節(jié)。本論文運(yùn)用了多種策略來發(fā)現(xiàn)和優(yōu)化抗真菌和抗腫瘤先導(dǎo)結(jié)構(gòu)，主要包括：(1)通過基于結(jié)構(gòu)的藥物設(shè)計(jì)技術(shù)優(yōu)化本實(shí)驗(yàn)室發(fā)現(xiàn)的高活性唑類抗真菌先導(dǎo)結(jié)構(gòu)；(2)通過基于細(xì)胞的表型篩選技術(shù)發(fā)現(xiàn)咔啉類抗真菌先導(dǎo)結(jié)構(gòu)，對(duì)其結(jié)構(gòu)優(yōu)化發(fā)現(xiàn)全新作用機(jī)制的抗真菌新化學(xué)實(shí)體；(3)通過藥物結(jié)構(gòu)優(yōu)化技術(shù)，系統(tǒng)完成了傳統(tǒng)中藥有效成分吳茱萸堿的構(gòu)效關(guān)系和藥理活性研究，發(fā)現(xiàn)高活性抗腫瘤候選分子；(4)通過有機(jī)小分子催化的不對(duì)稱串聯(lián)反應(yīng)技術(shù)，，發(fā)展了三項(xiàng)含硫骨架合成新方法，構(gòu)建得到類藥性分子庫，并篩選發(fā)現(xiàn)具有廣譜抗腫瘤活性的全新先導(dǎo)結(jié)構(gòu)。 一、新型抗真菌先導(dǎo)結(jié)構(gòu)的設(shè)計(jì)、合成與活性研究 (一)抗真菌靶標(biāo)CYP51的同源模建和唑類先導(dǎo)結(jié)構(gòu)的優(yōu)化設(shè)計(jì) 真菌羊毛甾醇14α-去甲基化酶(CYP51)是抗真菌藥物的重要靶點(diǎn)，其抑制劑唑類抗真菌藥物已經(jīng)廣泛應(yīng)用于臨床。但由于真菌CYP51是跨膜蛋白，提取純化比較困難，目前還沒有晶體結(jié)構(gòu)報(bào)道。本研究首次以人CYP51為模板，同源模建了白念珠菌CYP51(CA-CYP51)的三維模型，并進(jìn)行了分子動(dòng)力學(xué)優(yōu)化。對(duì)CA-CYP51模建結(jié)構(gòu)的準(zhǔn)確度進(jìn)行了系統(tǒng)的計(jì)算評(píng)價(jià)，包括蛋白的Pro-check、Profiles-3D、分子對(duì)接驗(yàn)證和富集性試驗(yàn)測試。結(jié)果表明，所建立的模型具有較高的精確性，可以用于指導(dǎo)新型唑類抗真菌藥物的合理設(shè)計(jì)。 本課題組前期發(fā)現(xiàn)含有N-甲基側(cè)鏈的唑類化合物表現(xiàn)出優(yōu)秀的體外抗真菌活性。在此基礎(chǔ)上，通過基于結(jié)構(gòu)的藥物設(shè)計(jì)技術(shù)對(duì)其進(jìn)行了結(jié)構(gòu)優(yōu)化和構(gòu)效關(guān)系研究，主要考察了含氮側(cè)鏈上不同的取代基團(tuán)對(duì)抗真菌活性的影響，設(shè)計(jì)合成了25個(gè)新化合物。構(gòu)效關(guān)系表明，N上的取代基團(tuán)以氫原子和甲基為最優(yōu)，并且取代基團(tuán)會(huì)影響側(cè)鏈在CYP51活性位點(diǎn)中的伸展，進(jìn)而影響抗真菌活性。其中化合物A1和A14較對(duì)照藥氟康唑相比，表現(xiàn)出相當(dāng)或更優(yōu)的體外抗真菌活性。通過分子對(duì)接闡明了目標(biāo)化合物與CA-CYP51的作用模式，并合理解釋了構(gòu)效關(guān)系，為進(jìn)一步合理設(shè)計(jì)新型唑類抗真菌藥物提供了有價(jià)值的信息。 (二)咔啉類抗真菌先導(dǎo)結(jié)構(gòu)的發(fā)現(xiàn)、優(yōu)化和生物活性研究 基于細(xì)胞表型或者功能的篩選是發(fā)現(xiàn)先導(dǎo)結(jié)構(gòu)的重要途徑。與針對(duì)具體靶點(diǎn)的分子水平篩選不同的是，細(xì)胞水平篩選不僅能夠直接發(fā)現(xiàn)具有藥理活性的分子，而且有可能發(fā)現(xiàn)全新結(jié)構(gòu)類型和全新作用機(jī)制的先導(dǎo)結(jié)構(gòu)，對(duì)新靶點(diǎn)和新藥的發(fā)現(xiàn)具有重要作用。本研究中對(duì)課題組內(nèi)部化合物庫進(jìn)行了體外抗真菌細(xì)胞水平的篩選，發(fā)現(xiàn)具有β-咔啉骨架結(jié)構(gòu)的化合物表現(xiàn)出廣譜的抗真菌活性。進(jìn)一步對(duì)其進(jìn)行結(jié)構(gòu)優(yōu)化，設(shè)計(jì)合成了27個(gè)新化合物。體外抗真菌測試表明，部分化合物的抗真菌活性優(yōu)于先導(dǎo)結(jié)構(gòu)。其中化合物C27活性最優(yōu)，與對(duì)照藥氟康唑相當(dāng)。對(duì)其進(jìn)行了深入的藥理學(xué)評(píng)價(jià)，發(fā)現(xiàn)化合物C27對(duì)氟康唑敏感菌和耐藥菌都具有殺真菌活性，能夠有效抑制真菌生物被膜和真菌菌絲的形成，而氟康唑無此效應(yīng)。協(xié)同抗真菌實(shí)驗(yàn)表明，化合物C27與氟康唑具有很好的協(xié)同抗真菌效果。采用透射電鏡和GC-MS方法對(duì)C27的抗真菌作用機(jī)制進(jìn)行了初步研究，發(fā)現(xiàn)該類化合物具有與氟康唑不同的作用機(jī)制，可能干擾了真菌細(xì)胞壁的生物合成途徑。上述結(jié)果表明，對(duì)咔啉類抗真菌化合物進(jìn)行深入研究，對(duì)于解決真菌的耐藥性問題具有重要意義。 二、新型吳茱萸堿衍生物的設(shè)計(jì)、合成和抗腫瘤活性研究 天然產(chǎn)物一直是抗腫瘤新藥研發(fā)的重要來源。在前期工作中，本課題組通過基于結(jié)構(gòu)的虛擬篩選，首次報(bào)道了天然產(chǎn)物吳茱萸堿是拓?fù)洚悩?gòu)酶的抑制劑。但它的體外抗腫瘤活性還比較低，分子作用靶點(diǎn)還不明確，有待深入的研究。 本研究對(duì)吳茱萸堿進(jìn)行了系統(tǒng)的結(jié)構(gòu)修飾，考察了引入取代基和改變分子骨架對(duì)抗腫瘤活性的影響，總共設(shè)計(jì)合成了139個(gè)新型吳茱萸堿衍生物，并在分子、細(xì)胞和動(dòng)物水平進(jìn)行了藥理活性測試。結(jié)果顯示，部分化合物對(duì)多種腫瘤株的GI50小于3nM，表現(xiàn)出廣譜、高效的體外抗腫瘤活性。裸鼠體內(nèi)腸癌和肺癌模型顯示，部分高活性衍生物表現(xiàn)出很好的體內(nèi)抗腫瘤效果。例如在裸鼠腸癌模型中，化合物E135在2mg/kg條件下抑瘤率達(dá)到50.39%，并表現(xiàn)低毒和高耐受性特點(diǎn)。細(xì)胞凋亡實(shí)驗(yàn)顯示，高活性化合物(E38, E112和E133)能夠誘導(dǎo)A549腫瘤細(xì)胞凋亡，使細(xì)胞周期阻滯于G2/M期。在分子作用靶點(diǎn)上，發(fā)現(xiàn)吳茱萸堿衍生物是首次報(bào)道的Top1/Top2/微管蛋白的三靶點(diǎn)抑制劑。其中化合物E112和E135對(duì)微管蛋白的抑制活性(IC50分別為5.3μM和4.5μM)優(yōu)于對(duì)照藥秋水仙堿(IC50為10.8μM)。吳茱萸堿衍生物多靶點(diǎn)抗腫瘤作用特點(diǎn)對(duì)于提高腫瘤化療效果和克服腫瘤耐藥性問題具有重要的意義。 三、基于有機(jī)合成方法學(xué)構(gòu)建類藥性骨架和抗腫瘤先導(dǎo)結(jié)構(gòu)的發(fā)現(xiàn) 近年來，有機(jī)小分子催化的不對(duì)稱串聯(lián)反應(yīng)發(fā)展迅速。這類反應(yīng)僅需通過一步反應(yīng)即能夠以較好的反應(yīng)收率和高立體選擇性構(gòu)建手性骨架，具有環(huán)境友好和原子經(jīng)濟(jì)性等特點(diǎn)，成為有機(jī)合成方法學(xué)的研究熱點(diǎn)。 3,4-二氫-2H-硫代吡喃骨架是藥物活性分子中的優(yōu)勢骨架。本研究首次采用有機(jī)小分子催化的硫-[3+3]環(huán)合反應(yīng)，一步構(gòu)建了含有兩個(gè)手性中心的二氫硫代吡喃骨架。反應(yīng)具有很好的反應(yīng)收率(51%-84%)和較好的立體選擇性(最高值20:1dr,99%ee)，并通過Nazarov反應(yīng)構(gòu)建得到含有四個(gè)手性中心的全新類藥性骨架。 在此基礎(chǔ)上，首次采用有機(jī)小分子催化的Michael-Michael串聯(lián)反應(yīng)，以較好的反應(yīng)收率(58%-78%)、中等的非對(duì)映選擇性(最高值為5.2:1dr)和高對(duì)映選擇性(最高值99%ee)，一步構(gòu)建了含有四個(gè)手性中心的四氫硫代吡喃骨架。通過簡單的化學(xué)轉(zhuǎn)化，能夠得到結(jié)構(gòu)更加復(fù)雜、骨架更加新穎的類藥性分子。 螺吲哚酮骨架是天然產(chǎn)物和藥物活性分子中的優(yōu)勢骨架，成為近年來藥物研究的熱點(diǎn)結(jié)構(gòu)。采用有機(jī)小分子催化的方法將吲哚酮和四氫硫代吡喃骨架相結(jié)合，通過Michael-Michael串聯(lián)反應(yīng)一步構(gòu)建了含有四個(gè)手性中心的吲哚酮螺四氫硫代吡喃骨架。反應(yīng)具有很好的反應(yīng)收率(55%-74%)和高立體選擇性(dr30:1,ee≥99%)。通過簡單的化學(xué)轉(zhuǎn)化，能夠得到骨架更加新穎、結(jié)構(gòu)更加復(fù)雜的結(jié)構(gòu)。 基于上述三類骨架建立了一個(gè)小型化合物庫，并進(jìn)行了體外抗腫瘤活性測試。結(jié)果表明，吲哚酮螺四氫硫代吡喃類衍生物表現(xiàn)出廣譜的抗腫瘤活性。其中化合物3b的體外抗腫瘤活性總體要優(yōu)于對(duì)照藥nutlin-3，具有深入研究的價(jià)值。 在本部分研究中，通過發(fā)展有機(jī)合成方法學(xué)構(gòu)建了三種含硫類藥性骨架，并通過體外抗腫瘤活性篩選首次發(fā)現(xiàn)吲哚酮螺四氫硫代吡喃衍生物具有廣譜抗腫瘤的特點(diǎn)。這項(xiàng)工作體現(xiàn)了有機(jī)合成方法學(xué)與藥物化學(xué)的相結(jié)合，為新藥發(fā)現(xiàn)提供了一種新思路，并為抗腫瘤化學(xué)生物研究提供了分子探針。 四、總結(jié) 綜上所述，本研究將多種先導(dǎo)物發(fā)現(xiàn)和優(yōu)化策略應(yīng)用于抗真菌和抗腫瘤新藥發(fā)現(xiàn)中，總共設(shè)計(jì)合成了191個(gè)新化合物，并發(fā)現(xiàn)四種結(jié)構(gòu)類型的抗真菌或抗腫瘤活性化合物。本論文的創(chuàng)新性主要體現(xiàn)在如下三個(gè)方面：(1)首次發(fā)現(xiàn)咔啉類化合物是全新作用機(jī)制的抗真菌先導(dǎo)結(jié)構(gòu)，發(fā)現(xiàn)化合物C27在克服真菌耐藥性方面具有潛在的應(yīng)用價(jià)值；(2)首次發(fā)現(xiàn)并證實(shí)吳茱萸堿衍生物是Top1/Top2/微管蛋白的三靶點(diǎn)抑制劑，并獲得了高效、低毒和廣譜的抗腫瘤新化學(xué)實(shí)體，為開發(fā)具有自主知識(shí)產(chǎn)權(quán)的抗腫瘤原創(chuàng)藥物奠定了基礎(chǔ)；(3)將有機(jī)合成方法學(xué)和藥物化學(xué)緊密結(jié)合，提出了先導(dǎo)化合物發(fā)現(xiàn)新策略。通過有機(jī)小分子催化的不對(duì)稱串聯(lián)反應(yīng)，快速構(gòu)建了三類含硫優(yōu)勢分子骨架，并發(fā)現(xiàn)吲哚酮螺四氫硫代吡喃類衍生物具有廣譜的抗腫瘤活性。本論文研究工作為開發(fā)具有自主知識(shí)產(chǎn)權(quán)的抗真菌和抗腫瘤創(chuàng)新藥物奠定了基礎(chǔ)。
[Abstract]:The discovery and optimization of pilot compounds is an important link in the development of new drugs. This paper uses a variety of strategies to discover and optimize antifungal and antitumor precursor structures, including: (1) optimize the antifungal precursor structure of the highly active azoles found in our laboratory through structural based drug design technology; (2) through cell based phenotype Screening techniques found the anti fungal precursor structure of carbazoline, optimized its structure and found a new antifungal entity with new mechanism. (3) through the optimization of drug structure, the structure-activity relationship and pharmacological activity of evodiazeroid from traditional Chinese medicine were systematically studied, and highly active antitumor candidate molecules were found; (4) through organic matter. Three new methods for the synthesis of sulphur containing skeletons have been developed by the small molecule catalyzed asymmetric series reaction technology, and the molecular library of drug like molecules has been constructed, and a new pilot structure with broad-spectrum antitumor activity has been screened.
1. Design, Synthesis and Activity of New Antifungal Pilot Structures
(1) Homology Modeling of Antifungal Target CYP51 and Optimization of Azole Pilot Structure
Fungi wool sterol 14 alpha demethylation enzyme (CYP51) is an important target for antifungal drugs, and its inhibitors, azole antifungal agents, have been widely used in clinical practice. However, because fungal CYP51 is a transmembrane protein, it is difficult to extract and purify the crystal structure. At present, there is no crystal structure report. This study was based on human CYP51 as a template for the first time, and a homologous model of Candida albicans C was built. The three-dimensional model of YP51 (CA-CYP51) was optimized by molecular dynamics. The accuracy of the CA-CYP51 model construction was evaluated systematically, including the protein Pro-check, Profiles-3D, molecular docking verification and enrichment test. The results show that the model has high accuracy and can be used to guide the new type of azole resistance. The rational design of fungal drugs.
The research group found that the azoles containing N- methyl side chain showed excellent antifungal activity in vitro. On this basis, structural optimization and structure-activity relationship were studied by structural based drug design technology, and the effect of different substituent groups on the nitrogen side chain was mainly investigated and the design and synthesis were designed. 25 new compounds. The structure-activity relationship shows that the substituent groups on N are optimal with hydrogen atoms and methyl groups, and the substituent groups affect the extension of the side chain in the CYP51 active site, and then affect the antifungal activity. The compounds A1 and A14 show the antifungal activity in phase or better in vitro compared with the control drug fluconazole. The mode of action of target compounds and CA-CYP51 is clarified, and the structure-activity relationship is explained reasonably, which provides valuable information for the further rational design of new azole antifungal drugs.
(2) Discovery, Optimization and Biological Activity of Caroline Antifungal Pilot Structures
Screening for phenotype or function based on cell phenotype is an important approach to the discovery of pilot structures. Unlike molecular level screening aimed at specific targets, cell level screening is not only able to detect molecules with pharmacological activity directly, but also may discover new structure types and new mechanisms, and new targets and new drugs. In this study, the level of antifungal cells in the compound library was screened in this study. It was found that the compounds with beta carbazoline skeleton structure showed broad-spectrum antifungal activity. Further, the structure was optimized and 27 new compounds were designed and synthesized. In vitro antifungal test showed that the compound was partial. The antifungal activity of the compound was superior to the pilot structure, in which the activity of compound C27 was the best, which was equivalent to the control drug fluconazole. The pharmacological evaluation of the compound was carried out. It was found that compound C27 had fungicidal activity to fluconazole sensitive and drug resistant bacteria, and could effectively inhibit the formation of fungal biofilm and fungal mycelium, and fluconazole had no such effect. Synergistic antifungal experiment showed that compound C27 had a good synergistic antifungal effect with fluconazole. The mechanism of anti fungal action of C27 was preliminarily studied by transmission electron microscope and GC-MS method. It was found that the compounds have different mechanisms of action with fluconazole, which may interfere with the biosynthesis pathway of the cell wall of fungi. The results showed that further study on carbaline antifungal compounds was of great significance to solve the problem of fungal resistance.
2. Design, Synthesis and Antitumor Activity of Novel Evodiamine Derivatives
Natural products have always been an important source of research and development of new antitumor drugs. In the early work, we first reported the natural product evodialkali as a topoisomerase inhibitor based on the structure based virtual screening. But its antitumor activity in vitro is still relatively low and the molecular target target is not clear.
In this study, the structure of evodiodipine was systematically modified, and the effects of introducing substituents and changing the molecular framework against the tumor activity were investigated. A total of 139 new evodiwood derivatives were designed and synthesized, and the pharmacological activities were tested at the molecular, cell and animal levels. The results showed that some of the compounds were GI50 in a variety of tumor strains. Less than 3nM, showing broad-spectrum and efficient antitumor activity in vitro. The model of colorectal cancer and lung cancer in nude mice showed that some highly active derivatives showed good antitumor effect in vivo. For example, in the nude mouse model, compound E135 was 50.39% under the condition of 2mg/kg, and showed low toxicity and high tolerance. The results showed that the highly active compounds (E38, E112 and E133) could induce apoptosis of A549 tumor cells and block the cell cycle in G2/M phase. At the molecular target, it was found that evodipine derivatives were the three target inhibitors of Top1/Top2/ microtubules for the first time. The inhibitory activity of compounds E112 and E135 to microtubule protein (IC50 was 5., respectively). 3 mu M and 4.5 mu M) are superior to the control drug colchicine (IC50 is 10.8 M). The antitumor characteristics of evodiine derivatives at multiple targets are of great significance in improving the effect of tumor chemotherapy and overcoming the problem of tumor resistance.
3. Discovery of Drug-like Skeleton and Antitumor Pilot Structures Based on Organic Synthesis Methodology
In recent years, the asymmetric series reaction catalyzed by small organic molecules has developed rapidly. This kind of reaction only needs to build chiral skeletons with good reaction yield and high stereoselectivity by one step reaction, which has the characteristics of environment-friendly and atomic economy. It has become a hot topic in the research of organic synthesis methodology.
3,4- two hydrogen -2H- thiothiolan skeleton is the dominant skeleton in drug active molecules. In this study, a sulfur -[3+3] cyclization reaction catalyzed by small organic molecules was used for the first time to construct a two hydrogen thiothiolan skeleton containing two chiral centers. The reaction has a good reaction yield (51%-84%) and a better stereoselectivity (maximum 20:1dr, 99%ee). A novel drug-like skeleton containing four chiral centers was constructed by Nazarov reaction.
On this basis, the Michael-Michael series reaction catalyzed by organic small molecules has been used for the first time, with a good reaction yield (58%-78%), medium non enantioselectivity (the maximum value of 5.2:1dr) and high enantioselectivity (the highest value 99%ee). One step is to construct a four thiothiolan skeleton containing four chiral centers. Drug-like molecules with more complex structure and novel skeleton can be obtained.
The cytoskeleton of spiro indolone is the dominant skeleton in natural products and drug active molecules. It has been a hot structure in drug research in recent years. Using organic small molecular catalysis, indolone and four thiothiolan skeleton were combined and four chiral centers containing four chiral centers were constructed by one step Michael-Michael series reaction. The cytoskeleton has a good reaction yield (55%-74%) and high stereoselectivity (dr30:1, EE > 99%). Through simple chemical conversion, a more novel structure and more complex structure of the skeleton can be obtained.
Based on the above three kinds of skeletons, a small compound library was established and the antitumor activity in vitro was tested. The results showed that the indolonone four thiothioparan derivatives showed broad-spectrum antitumor activity. The overall anti tumor activity of compound 3B was better than that of the control drug nutlin-3 in vitro, which was of great value for further study.
In this part, three kinds of sulfur containing insecticide skeleton were constructed by the development of organic synthesis method, and the characteristics of broad spectrum antitumor characteristics were found for the first time by the screening of anti tumor activity in vitro. This work embodies the combination of organic synthesis methodology and drug chemistry and the discovery of new drugs. It provides a new idea and a molecular probe for the study of anti-tumor chemistry and biology.
Four, summary
To sum up, a variety of pilot discovery and optimization strategies are applied to the discovery of antifungal and antitumor drugs. A total of 191 new compounds have been designed and four structural types of antifungal or antitumor compounds have been found. The innovation of this paper is mainly reflected in the following three aspects: (1) the first discovery of carbazolin The compound is an antifungal precursor structure of the new mechanism of action. It has been found that compound C27 has potential application value in overcoming fungal resistance. (2) it was first discovered and confirmed that evodipine derivatives are the three target inhibitors of microtubulin, and have obtained high efficient, low toxic and broad-spectrum anti-tumor chemical entities. The independent intellectual property rights have laid the foundation for antitumor original drugs; (3) a new strategy for the discovery of pilot compounds is proposed by combining organic synthesis methodology with drug chemistry. A rapid construction of three kinds of sulfur containing dominant molecular skeletons through asymmetric tandem reactions catalyzed by organic small molecules and the discovery of indolonone four hydrogen thiothioles derivatives are found. Biology has broad-spectrum antitumor activity. The research work of this paper lays a foundation for the development of innovative antifungal and antitumor drugs with independent intellectual property rights.
【學(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R914

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