Discovery of the imidazo[1,5-a][1,2,4]-triazolo[1,5-d][1,4]benzodiazepine scaffold as a novel,potent and selective GABAA α5 inverse agonist series

Through iterative design cycles we have discovered a number of novel new classes where the imidazo[1,5-a][1,2,4]-triazolo[1,5-d][1,4]benzodiazepine was deemed the most promising GABA_A α5 inverse agonist class with potential for cognitive enhancement. This class combines a modest subtype binding selectivity with inverse agonism and has the most favourable molecular properties for further lead optimisation towards a central nervous system (CNS) acting medicine.     

Pyrazolo[1,5-a]pyrimidines, triazolo[1,5-a]pyrimidines and their tricyclic derivatives as corticotropin-releasing factor 1 (CRF₁) receptor antagonists

To identify structurally novel CRF1 receptor antagonists, a series of bicyclic core antagonists, pyrazolo[1,5-a]pyrimidines, triazolo[1,5-a]pyrimidines, imidazo[1,2-a]pyrimidines and pyrazolo[1,5-a][1,3,5]triazines were designed, synthesized and evaluated as CRF1 receptor antagonists. Compounds 2-27 showed binding affinity (IC(50)=4.2-418 nM) and antagonist activity (EC(50)=4.0-889 nM). Compound 5 was found to show oral efficacy in an Elevated Plus Maze test in rats. Further chemical modification of them led us to discovery of the tricyclic core antagonists pyrazolo[1,5-a]pyrrolo[3,2-e]pyrimidines. The discovery process of these compounds is presented, as is the study of the structure-activity relationship.     

Antagonists of 5-HT₆ receptors. Substituted 3-(phenylsulfonyl)pyrazolo[1,5-a]pyrido[3,4-e]pyrimidines and 3-(phenylsulfonyl)pyrazolo[1,5-a]pyrido[4,3-d]pyrimidines-Synthesis and 'structure-activity' relationship

Synthesis and biological evaluation of a new series of structurally unrestricted and intramolecular hydrogen bond restricted derivatives of 3-(phenylsulfonyl)pyrazolo[1,5-a]pyrido[3,4-e]pyrimidines (angular tricyclics) and 3-(phenylsulfonyl)pyrazolo[1,5-a]pyrido[4,3-d]pyrimidines (linear tricyclics) are described. Structurally restricted derivatives are highly potent and selective blockers of 5-HT(6) receptors with little difference between angular or linear shape of the tricyclic core, the angular species being only slightly more potent. The angular representative of 3-(phenylsulfonyl)pyrazolo[1,5-a]pyrido[3,4-e]pyrimidines, 5, can be considered as more favorable candidate for further development as it shows only weak 5-HT(2B) blocking activity (IC(50)=6.16 μM as compared with IC(50)=1.8 nM for 5-HT(6) receptors) and very low hERG potassium channel blocking potency (IC(50)=54.2 μM). The linear analog, 11, is less favorable as while showing no binding to the 5-HT(2B) receptor at concentrations of up to 10 μM, it exhibits quite a high potency to block the hERG channel (IC(50)=0.5 μM).     

New inhibitors of fungal 17β-hydroxysteroid dehydrogenase based on the [1,5]-benzodiazepine scaffold

The synthesis and activity of a new series of non-steroidal inhibitors of 17β-hydroxysteroid dehydrogenase that are based on a 1,5-benzodiazepine scaffold are presented. Their inhibitory potential was screened against 17β-hydroxysteroid dehydrogenase from the fungus Cochliobolus lunatus (17β-HSDcl), a model enzyme of the short-chain dehydrogenase/reductase superfamily. Some of these compounds are potent inhibitors of 17β-HSDcl activity, with IC₅₀ values in the low micromolar range and represent promising lead compounds that should be further developed and investigated as inhibitors of human 17β-HSD isoforms, which are the enzymes associated with the development of many hormone-dependent and neuronal diseases.     

Tetrahydropyrazolo[1,5-a]Pyrimidine-3-Carboxamide and N-Benzyl-6′,7′-Dihydrospiro[Piperidine-4,4′-Thieno[3,2-c]Pyran] Analogues with Bactericidal Efficacy against Mycobacterium tuberculosis Targeting MmpL3

Mycobacterium tuberculosis is a major human pathogen and the causative agent for the pulmonary disease, tuberculosis (TB). Current treatment programs to combat TB are under threat due to the emergence of multi-drug and extensively-drug resistant TB. As part of our efforts towards the discovery of new anti-tubercular leads, a number of potent tetrahydropyrazolo[1,5-a]pyrimidine-3-carboxamide (THPP) and N-benzyl-6′,7′-dihydrospiro[piperidine-4,4′-thieno[3,2-c]pyran] (Spiro) analogues were recently identified against Mycobacterium tuberculosis and Mycobacterium bovis BCG through a high-throughput whole-cell screening campaign. Herein, we describe the attractive in vitro and in vivo anti-tubercular profiles of both lead series. The generation of M. tuberculosis spontaneous mutants and subsequent whole genome sequencing of several resistant mutants identified single mutations in the essential mmpL3 gene. This ‘genetic phenotype’ was further confirmed by a ‘chemical phenotype’, whereby M. bovis BCG treated with both the THPP and Spiro series resulted in the accumulation of trehalose monomycolate. In vivo efficacy evaluation of two optimized THPP and Spiro leads showed how the compounds were able to reduce >2 logs bacterial cfu counts in the lungs of infected mice.     

Discovery of pyrazolo[1,5-a]pyridines as p110α-selective PI3 kinase inhibitors

We have made a novel series of pyrazolo[1,5-a]pyridines as PI3 kinase inhibitors, and demonstrated their selectivity for the p110α isoform over the other Class Ia PI3 kinases. We investigated the SAR around the pyrazolo[1,5-a]pyridine ring system, and found compound 5x to be a particularly potent example (p110α IC(50) 0.9nM). This compound inhibits cell proliferation and phosphorylation of Akt/PKB, a downstream marker of PI3 kinase activity, and showed in vivo activity in an HCT-116 human xenograft model.     

Synthesis and evaluation of 2′H-spiro[cyclohexane-1,3′-imidazo[1,5-a]pyridine]-1′,5′-dione derivatives as Mnk inhibitors

Post-translational modulation of eIF4E through phosphorylation by Mnks is highly integral to the pathogenesis of different cancers. Therefore, inhibition of Mnks offers a strategy for cancer treatment. Herein, a series of 2′H-spiro[cyclohexane-1,3′-imidazo[1,5-a]pyridine]-1′,5′-dione derivatives is presented as Mnk inhibitors. Some of them showed sub-micromolar to low nanomolar inhibitory activities against Mnk1/2 with a high level of selectivity for both kinases over CDKs. Biochemical assays revealed that compounds 4c and 4t are non-ATP-competitive inhibitors of Mnks. Lead compound 4t demonstrated a high selectivity for Mnk1/2 over a selection of 51 kinases, and displayed anti-proliferative activities against a panel of cancer cell lines. However, this compound in combination with our in-house CDK4/6 inhibitor 83 did not show a synergistic effect in A2780 ovarian cancer cells, suggesting that caution be exercised in the selection of an agent to be combined with an Mnk inhibitor.     

生物工程技术讲座[八]

应用微细胞检测基因产物的方法由于质粒和噬菌体等的DAN分子能在细胞内进行自我复制,以及DNA重组技术的普及,质粒DNA做为异源性DNA分子的载体,在基因工程中目前已被广泛应用。在克隆或分析真核生物或原核生物的多种不同基因时,常用检测基因表达来确定其存在。把含有某一目的基因的DNA片段和载体质粒的DNA进行重组,再通过重组DNA在受体细胞中的转录、翻译、     

光合作用中的[H]与呼吸作用中的[H]相同吗？

答：这里的[H]并不是指氢离子（H＋）,而是指辅酶中的氢（还原性氢）,[H]的产生与利用即NADH、FADH。和NADPH的产生与利用。光合作用与呼吸作用中产生和利用的[H]不同,光合作用产生及利用NADPH．而呼吸作用产生与利用的[H]主要是NADH与FADH2。     

Optimization of a 1,5-dihydrobenzo[b][1,4]diazepine-2,4-dione series of HIV capsid assembly inhibitors 2: Structure–activity relationships (SAR) of the C3-phenyl moiety

Detailed structure–activity relationships of the C3-phenyl moiety that allow for the optimization of antiviral potency of a series of 1,5-dihydrobenzo[b][1,4]diazepine-2,4-dione inhibitors of HIV capsid (CA) assembly are described. Combination of favorable substitutions gave additive SAR and allowed for the identification of the most potent compound in the series, analog 27. Productive SAR also transferred to the benzotriazepine and spirobenzodiazepine scaffolds, providing a solution to the labile stereocenter at the C3 position. The molecular basis of how compound 27 inhibits mature CA assembly is rationalized using high-resolution structural information. Our understanding of how compound 27 may inhibit immature Gag assembly is also discussed.     

Novel pyrazolo[1,5-a]pyridines with improved aqueous solubility as p110α-selective PI3 kinase inhibitors

As part of our investigation into pyrazolo[1,5-a]pyridines as novel p110α selective PI3 kinase inhibitors, we report a range of analogues with improved aqueous solubility by the addition of a basic amine. The compounds demonstrated comparable p110α potency and selectivity to earlier compounds but with up to 1000× greater aqueous solubility, as the hydrochloride salts. The compounds also displayed good activity in a cellular assay of PI3 kinase activity.     

云南高黎贡山[虫脩]目昆虫五新种及污色无翅刺[虫脩]雄性的发现（[虫脩]：异[虫脩]科，[虫脩]科）

记述云南高黎贡山[虫脩]目异[虫脩]科和[虫脩]科5个新种,即刺胸阿异[虫脩]Aruanoidea notata,sp．nov．,高山短肛[虫脩]Baculum altissimum,sp．nov．,光亮拟短肛[虫脩]Parabaculum laevigatum,sp．nov．,双角刺[虫脩]Cnipsomorpha biangulatus,sp．nov．,黑缘介[虫脩]Interphasma marginatum,sp．nov．,并且首次记述了污色无翅刺蟾Cnipsomorpha colorantis（Chen et He）的雄性。模式标本分别存放于北京林业大学与中国林业科学研究院昆虫标本馆。     

绿藻[FeFe]氢化酶

该文介绍了绿藻[FeFe]氢化酶的研究现状,包括酶的结构、催化中心、金属簇的性质,以及对氧的敏感性和可能的解决办法。并且对已报道的绿藻[FeFe]氢化酶基因及其调控等问题作了介绍。     

P[4]、P[6]和P[8]型轮状病毒VP8*核心区蛋白的表达和纯化

为进一步深入研究我国人群中轮状病毒主要流行的P[4]、P[6]和P[8]型毒株的受体结合特点及结构基础,本研究将P[4]、P[6]和P[8]型毒株VP8*核心区(VP8*core)基因分别构建到原核表达载体pGEX4T-1和pET30a中,利用大肠杆菌表达获得轮状病毒P[4]、P[6]和P[8]型毒株VP8*核心区蛋白,并通过亲和层析分别纯化得到VP8*core-GST融合蛋白和VP8*-his标签蛋白,SDS-PAGE显示蛋白大小分别为46kDa和20kDa。综上,本研究成功构建了pGEX4T-1-VP8*core和pET30a-VP8*core重组质粒,利用原核表达系统得到VP8*coreGST融合蛋白和VP8*core-his蛋白,为VP8*蛋白的功能和结构研究提供基础。     

Novel pyrazolo[1,5-a]pyridines as p110α-selective PI3 kinase inhibitors: Exploring the benzenesulfonohydrazide SAR

Structure-activity relationship studies of the pyrazolo[1,5-a]pyridine class of PI3 kinase inhibitors show that substitution off the hydrazone nitrogen and replacement of the sulfonyl both gave a loss of p110α selectivity, with the exception of an N-hydroxyethyl analogue. Limited substitutions were tolerated around the phenyl ring; in particular the 2,5-substitution pattern was important for PI3 kinase activity. The N-hydroxyethyl compound also showed good inhibition of cell proliferation and inhibition of phosphorylation of Akt/PKB, a downstream marker of PI3 kinase activity. It had suitable pharmacokinetics for evaluation in vivo, and showed tumour growth inhibition in two human tumour cell lines in xenograft studies. This work has provided suggestions for the design of more soluble analogues.     

Structure–activity relationship study of pyrimido[1,2-c][1,3]benzothiazin-6-imine derivatives for potent anti-HIV agents

3,4-Dihydro-2H,6H-pyrimido[1,2-c][1,3]benzothiazin-6-imine (PD 404182) is an antiretroviral agent with submicromolar inhibitory activity against human immunodeficiency virus-1 (HIV-1) and HIV-2 infection. In the current study, the structure–activity relationships of accessory groups at the 3- and 9-positions of pyrimido[1,2-c][1,3]benzothiazin-6-imine were investigated for the development of more potent anti-HIV agents. Several different derivatives containing a 9-aryl group were designed and synthesized using Suzuki–Miyaura cross-coupling and Ullmann coupling reactions. Modification of the m-methoxyphenyl or benzo[d][1,3]dioxol-5-yl group resulted in improved anti-HIV activity. In addition, the 2,4-diazaspiro[5.5]undec-2-ene-fused benzo[e][1,3]thiazine derivatives were designed and tested for their anti-HIV activities. The most potent 9-(benzo[d][1,3]dioxol-5-yl) derivative was two–threefold more effective against several strains of HIV-1 and HIV-2 than the parent compound, PD 404182.     

Combined use of [TBA][L-ASP] and hydroxypropyl-β-cyclodextrin as selectors for separation of Cinchona alkaloids by capillary electrophoresis

In this paper, a new capillary electrophoresis (CE) separation and detection method was developed for the chiral separation of the four major Cinchona alkaloids (quinine/quinidine and cinchonine/cinchonidine) using hydroxypropyl-β-cyclodextrin (HP-β-CD) and chiral ionic liquid ([TBA][L-ASP]) as selectors. Separation parameters such as buffer concentrations, pH, HP-β-CD and chiral ionic liquid concentrations, capillary temperature, and separation voltage were investigated. After optimization of separation conditions, baseline separation of the three analytes (cinchonidine, quinine, cinchonine) was achieved in fewer than 7 min in ammonium acetate background electrolyte (pH 5.0) with the addition of HP-β-CD in a concentration of 40 mM and [TBA][L-ASP] of 14 mM, while the baseline separation of cinchonine and quinidine was not obtained. Therefore, the first-order derivative electropherogram was applied for resolving overlapping peaks. Regression equations revealed a good linear relationship between peak areas in first-order derivative electropherograms and concentrations of the two diastereomer pairs. The results not only indicated that the first-order derivative electropherogram was effective in determination of a low content component and of those not fully separated from adjacent ones, but also showed that the ionic liquid appeared to be a very promising chiral selector in CE.     

Development of 13H-benzo[f]chromeno[4,3-b][1,7]naphthyridines and their salts as potent cytotoxic agents and topoisomerase I/IIα inhibitors

A novel series of 35 angularly fused pentacyclic 13H-benzo[f]chromeno[4,3-b][1,7]naphthyridines and 13H-benzo[f]chromeno[4,3-b][1,7]naphthyridin-5-ium chlorides were designed and synthesized. Their cytotoxic activities were investigated against six human cancer cell lines (NCIH23, HCT15, NUGC-3, ACHN, PC-3, and MDA-MB-231). Among all screened compounds; 28, 30, 34, 35, 46, 48, 52, and 53 compounds exhibited potent cytotoxic activities against all tested human cancer cell lines. Further, these potent lead cytotoxic agents were evaluated against human Topoisomerase I and IIα inhibition. Among them, the compound 48 exhibited dual Topoisomerase I and IIα inhibition especially at 20?μM concentrations the compound 48 exhibited 1.25?times more potent Topoisomerase IIα inhibitory activity (38.3%) than the reference drug etoposide (30.6%). The compound 52 also exhibited excellent (88.4%) topoisomerase I inhibition than the reference drug camptothecin (66.7%) at 100?μM concentrations. Molecular docking studies of the compounds 48 and 52 with topo I discovered that they both intercalated into the DNA single-strand cleavage site where the compound 48 have van der Waals interactions with residues Arg364, Pro431, and Asn722 whilst the compound 52 have with Arg364, Thr718, and Asn722 residues. Both the compounds 48 and 52 have π–π stacking interactions with the stacked DNA bases. The docking studies of the compound 48 with topo IIα explored that it was bound to the topo IIα DNA cleavage site where etoposide was situated. The benzo[f]chromeno[4,3-b][1,7]naphthyridine ring of the compound 48 was stacked between the DNA bases of the cleavage site with π–π stacking interactions and there were no hydrogen bond interactions with topo IIα.     

Structure–activity relationship and interaction studies of new SIRT1 inhibitors with the scaffold of 3-(furan-2-yl)-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazole

SIRT1 is a NAD⁺-dependent deacetylase. It deacetylates a broad range of substrates and is involved in multiple diseases such as type 2 diabetes and cancer. Here we discovered a new class of SIRT1 inhibitors with the scaffold of 3-(furan-2-yl)-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazole. The inhibitors up-regulate acetyl p53 level in human breast cells MCF-7. The docking simulations indicated that the scaffold and the R-substituents of the inhibitors bind in the C and D pocket of SIRT1, respectively, which was supported by the structure–activity relationship and SIRT1 mutagenesis studies. We propose that binding of the inhibitors repels the entering of the nicotinamide moiety of NAD⁺ to the C pocket, prevents its transformation to the productive conformation and therefore inhibits the deacetylation catalyzed by SIRT1.     

光合作用与呼吸作用中的[H]

光合作用与呼吸作用所涉及的[H]指NADH、FADH2与NADPH中的还原性氢。光合作用与呼吸作用中产生和利用的[H]不同,光合作用产生及利用NADPH,而呼吸作用产生与利用的[H]主要是NADH与FADH2。