Synthesis and biological evaluation of novel pyrazole carboxamide with diarylamine-modified scaffold as potent antifungal agents

Twenty-seven novel pyrazole carboxamides with diarylamine-modified scaffold were designed, synthesized and characterized in detail via ¹H NMR, ¹³C NMR, IR and ESI-HRMS. Preliminary bioassays showed that some of the target compounds exhibited good antifungal activity against Rhizoctonia solani, Rhizoctonia cerealis and Sclerotinia sclerotiorum. Among them, compound 9c-7 exhibited the highest antifungal activities against R. solani, R. cerealis and S. sclerotiorum in vitro with IC₅₀ values of 0.013, 1.608 and 1.874 μg/mL, respectively. Notably, compound 9c-7 still presented the highest fungicidal activities against R. solani in vivo with an IC₅₀ value of 22.21 μg/mL. Molecular docking simulation results reveal that compound 9c-7 binds well to the hydrophobic pockets of the receptor protein succinate dehydrogenase. This study suggests that compound 9c-7 could act as a potential fungicide to be used for further optimization.     

Rapid,High-Yield Purification of Rat Liver Glutathione Peroxidase by High Performance Liquid Chromatography

Abstract

Glutathione peroxidase (GSH:H₂O₂ oxidoreductase, EC 1.11.1.9) was purified 3500-fold from rat liver with a yield of 42% using high performance liquid chromatography. The crucial purification step was size-exclusion chromatography on a Spherogel TSK-3000SW column, and the purified enzyme eluted as a single peak. The enzyme stained as a single band following SDS-gel electrophoresis. The molecular weight of the enzyme was estimated to be 105,000, and the subunit molecular weight determined by SDS-gel electrophoresis was 25,000. Polyacrylamide gel electrophoresis indicated five bands of protein with a broad of enzymatic activity. Isoelectric focusing resulted in a peak of enzymatic activity at pH 6.9 with a shoulder at pH 7.3. The specific activity of the purified enzyme was 1,100 μmol of NADPH oxidized per minute per milligram of protein.     

Composition and antipathogenic activities of the twig essential oil of Chamaecyparis formosensis from Taiwan

In this study, antipathogenic activities of the twig essential oil and its constituents from Chamaecyparis formosensis Matsum were evaluated in vitro against six plant pathogenic fungi. The essential oil from the fresh twigs was isolated using hydrodistillation in a Clevenger-type apparatus, and characterized by GC-FID and GC-MS. Twenty-five compounds were identified, representing 98.9% of the oil. The main components were beta-eudesmol (25.1%), tau-muurolol (21.6%), elemol (15.0%), totarol (14.9%), and alpha-cadinol (12.4%). The twig oil (500 mcirog/mL) showed growth inhibitory activity against the phytopathogenic fungi, Fusarium oxysporum, Pestalotiopsis funereal, and Ganoderma austral, with antifungal indices of 92.7%, 71.1%, and 87.7%, respectively. In addition, the oil suppressed totally the growth of Rhizoctonia solani, Colletotrichum gloeosporioides, and Fusarium solani. In order to ascertain the source compounds of these antipathogenic activities, the main components were individually evaluated. Tau-Muurolol and alpha-cadinol exhibited excellent activity against F. oxysporum, R. solani, C. gloeosporioides, and F. solani, with IC50 < 50 microg/mL. These compounds also efficiently inhibited the mycelial growths of P. funereal and G. austral. Thus, alpha-cadinol and tau-muurolol could be considered as potential natural fungicides for controlling fungal pathogens and worth.     

新型苄基氨基甲酸酯类化合物的合成及其抗菌活性

以邻氯苄胺为原料,经6步反应制得中间体2-氯-5-［1-(氧丙炔基亚氨基)乙基］苄基氨基甲酸甲酯（7）;取代芳基肟经氯化后再分别与7经环合反应合成了一系列新型的苄基氨基甲酸酯类化合物（10a~10l）,其结构经¹H NMR表征。初步的抗菌活性测试表明：在用药量为200 mg·L^-1时,大部分化合物对水稻纹枯病显示出较好的抗菌活性,其中10f的抑菌活性最高,抑制率95%。     

Phytopathogenic fungal inhibitors from celery seeds

Extract of celery (Apium graveolens L.) seeds was investigated against phytopathogenic fungi. The light petroleum extract showed promising inhibition activities in the tests against Rhizoctonia solani and Fusarium oxysporum f. sp. vasinfecum. Chromatographic separation of the extract gave 19 fractions, one of which, QCZ-4, possessed significant inhibitory rates of 64.6%, 88.4% and 54.7% at a concentration of 100 ppm against R. solani, F. oxysporium f. sp. vasinfecum and Alternaria alternata, respectively. Major components in the active fraction were identified by GC-MS as p-(2-aminoethyl)phenol (39.7%), 3-(3,4-dimethybenzoyl) propionic acid (32.6%) and p-heptylphenol (26.9%).     

强阴离子交换色谱结合分子排阻色谱纯化血清中免疫球蛋白和白蛋白

动物血清中免疫球蛋白和白蛋白的等电点分别约为7.8和4.8,根据它们等电点的较大差别,利用Q SepharoseTM-XL强阴离子交换色谱结合分子排阻色谱同时分离纯化这2种蛋白。以0.02 mol/L pH 8.0的Tris-HCl缓冲液平衡离子交换色谱柱并将已稀释10倍的高免疫的兔血清上样,采用pH分段洗脱。在pH 6.0时以0.3 mL/min低流速洗脱得到高纯度的免疫球蛋白,继续在pH 4.0时洗脱,再辅以Sephadex G-75分子排阻色谱可获得纯度大于95%的白蛋白。对纯化后的蛋白进行活性检测,证明所纯化的免疫球蛋白和白蛋白都保持正常的生物活性。蛋白质含量测定说明免疫球蛋白的纯化回收率达到95%以上,而白蛋白的纯化回收率大于90%。该法简便快速,可同时从动物血清中纯化出保持生物活性的免疫球蛋白和白蛋白,纯化效率高。     

Antibacterial and Antifungal Activities of 2‐(substituted ether)‐5‐(1‐phenyl‐5‐(trifluoromethyl)‐1H‐pyrazol‐4‐yl)‐1,3,4‐oxadiazole Derivatives

By replacing the amide bond into 1,3,4‐oxadiazole moiety, a series of 1‐phenyl‐5‐(trifluoromethyl)‐1H‐pyrazole derivatives bearing 1,3,4‐oxadiazole were synthesized and evaluated their antibacterial and antifungal activity. The bioassay results revealed that compounds 7a and 7b showed the strongest antibacterial activity toward pathogen Xanthomonas oryzae pv. oryzae with the EC50 values of 15.0 and 6.4 µg/mL, respectively; compound 6a exhibited comprehensive antifungal activity toward six kinds of fungi; compound 6f could selectively inhibit the growth of Sclertinia sclerotiorum and Rhizoctonia solani with the inhibition rates of 82.5 and 80.3% at the concentrate of 100 µg/mL, respectively; compound 7b exerted good antifungal activity toward Fusarium oxysporum, Cytospora mandshurica, and Rhizoctonia solani with the inhibition rates of 70.8, 69.5, and 71.5%, respectively. The results suggested that this kind of compounds could be further studied as promising antimicrobial agents.     

N-(2-(腈基(4,6-二甲氧基嘧啶-2-基)甲基)苯基)酰胺类衍生物的合成及生物活性

以2-(2-硝基苯基)乙腈及4,6-二甲氧基-2-甲磺酰基嘧啶为起始原料,分别经缩合、还原及酰胺化反应合成了6个未见文献报道的N-(2-(腈基(4,6-二甲氧基嘧啶-2-基)甲基)苯基)酰胺类衍生物,其结构经1H NMR、MS和元素分析进行了确证。 在150 g/hm2的用量条件下,合成化合物未显示出除草活性。 在200 mg/L浓度下,部分化合物对黄瓜灰霉病及水稻纹枯病表现出一定的抑菌作用,其中化合物4d对水稻纹枯病的抑制率为72.33%。     

Rational design of a new one-step purification strategy for Candida antarctica lipase B by ion-exchange chromatography

A fast and efficient one-step method for purification of lipase B from Candida antarctica by ion-exchange chromatography was developed by rational design. The electrostatic properties of the enzyme were calculated and validated by isoelectric focusing and measurement of the titration curve. C. antarctica lipase B shows an unusual pH profile with a broad isoelectric region from pH 4 to 8. At pH 3 C. antarctica lipase B can be bound to a cation-exchange chromatography column and was purified to homogeneity with a purification factor of 2.4. It was stable at pH 3, the residual activity was still 80% after 6 days incubation at 20 degrees C. The broad isoelectric region of C. antarctica lipase B is unique as compared to almost all other alpha/beta-hydrolases which have a well-defined isoelectric point. A search in the lipase engineering database resulted in only one further alpha/beta-hydrolase, the Fusarium solani cutinase, which also has a broad isoelectric region.     

N-苯甲酰基-N’-1,3,4-噻二唑-2-基硫脲的合成及生物活性研究

本文以苯甲酸为原料,经酰氯化、酰化反应得到中间体苯甲酰基异硫氰酸酯,再与2-氨基-1,3,4-噻二唑经加成反应合成并表征了10种N-苯甲酰基-N’-1,3,4-噻二唑-2-基硫脲3a-3j。初步生物活性测试结果表明:当浓度为10 mg/L时,3a～3d对小麦生根和发芽有一定的调节活性。当浓度为100 mg/L时,大部分化合物对黄瓜灰霉病菌、黄瓜炭疽病菌、水稻纹枯病菌、水稻稻瘟病菌有明显的抑制活性,而对棉花枯萎病菌的抑制活性较差。目标化合物的取代基会影响其生长调节活性和抑菌活性。结合量化计算结果讨论了化合物结构对抑菌活性的影响。     

Purification and characterisation of α-amylase produced by mutant strain of Aspergillus oryzae EMS-18

α-Amylase produced by a mutant strain of Aspergillus oryzae EMS-18 has been purified to homogeneity as judged by sodium dodecyle sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme was purified by using 70% ammonium sulphate precipitation followed by anion exchange chromatography on DEAE-Sephadex column and gel filtration on Sephadex G-100. An enzyme purification factor of 9.5-fold was achieved with a final specific activity of 1987.7 U/mg protein and overall yield of 23.8%. The molecular weight of purified α-amylase was estimated to be 48 kDa by SDS-PAGE. The purified enzyme revealed an optimum assay temperature and pH 40°C and 5.0, respectively. Except Ca⁺⁺ all other metal ions such as Mg, Mn, Na, Zn, Ni, Fe, Cu, Co and Ba were found to be inhibitory to enzyme activity.     

High-yield two-step chromatographic procedure for purification of fatty acid-binding protein from human heart.

A high-yield procedure for the purification of cytoplasmic fatty acid-binding protein from human heart (H-FABP) is described. H-FABP was purified by gel permeation chromatography on a Sephacryl S-200 column followed by anion-exchange chromatography on a Sepharose Q fast-flow column at pH 7.0. At this pH H-FABP binds strongly to the column and can be selectively eluted with a salt gradient. The two-step procedure showed a high degree of reproducibility. On average 50 mg of H-FABP was obtained from 150 g of human heart tissue, which corresponds to a recovery of about 50%. Purity was confirmed by gel electrophoresis and isoelectric focusing. Binding of oleic acid to purified H-FABP, using the Lipidex 1000 assay, revealed a maximal binding of 0.75 +/- 0.01 mol fatty acid/mol protein and a dissociation constant of 0.19 +/- 0.01 microM.     

Synthesis of some novel 7-substituted [1,3,4]thiadiazolo[3,2-c][1,3,5]thiadiazine-6,6-dioxides with antifungal evaluation against rice pathogens

Synthesis of a new series of novel [1,3,4]thiadiazolo[3,2-c][1,3,5]thiadiazine-6,6-dioxide by [4 + 2] cycloaddition reaction of 3-benzylidineamino[1,3,4]thiadiazole with sulfene generated in situ from methanesulfonyl chloride in the presence of triethylamine is described. The novel compounds thus synthesized were screened for their antifungal activity against two fungal species, namely Rhizoctonia solani and Dreschslera oryzae. Carbendazim was the standard used for the evaluation of antifungal activity.     

Syntheses and Fungicidal Activities of Thiazole-5-carboxanilides Bearing Thioether Group

In an attempt to find leading thiazole carboxanilides exhibiting fungicidal activities, we designed and synthesized a new series of 2-sulfursubstituted thiazole carboxanilides via the reaction between 2-sulfur substituted thiazole carboxylic acid chlorides and substituted aniline. The fungicidal activities of the title compounds against Rhizoctonia solani were screened and the results were remarkable. The most potent compound 8i, N-[2,6-dichloro-4-(trifluoromethyl)phenyl]-2-methylthio-4-(trifluoromethyl)thiazole-5-carboxamide, was identified. The fungicidal EC₅₀ of compound 8i against Rhizoctonia solani was 1.28 mg/L, being comparable to that of Thifluzamide. The results indicate that compound 8i can be considered as a lead compound for further research.     

吡唑腙及其双杂环化合物的设计、合成及生物活性

利用Vilsmeier-Haak反应得到活性中间体,取代-4-甲酰基吡唑;将其与芳氧乙酰肼反应,合成5个吡唑腙类化合物3,再经关环反应,制得5个吡唑类双杂环化合物4。所有新化合物的结构均经IR,1H NMR,MS和元素分析确证了结构。对新化合物3,4分别进行了棉花枯萎病菌、棉花黄萎病菌、棉花立枯病菌、瓜果腐霉病菌、番茄早疫病菌、向日葵菌核病菌等初步的抑菌活性测试,结果表明,吡唑类双杂环化合物4的抑菌效果明显高于吡唑腙化合物3。在质量浓度为50mg/L时,3d、3e对番茄早疫病菌、向日葵菌核病菌有较好的抑制效果(﹥80%);双杂环化合物4对6种病菌均有明显的抑制效果(﹥70%),其中4d、4e对棉花立枯病菌的抑制率大于95%。     

Novel 1,3,5-thiadiazine-2-thione derivatives containing a hydrazide moiety: Design,synthesis and bioactive evaluation against phytopathogenic fungi in vitro and in vivo

A series of novel 1,3,5-thiadiazine-2-thione derivatives bearing a hydrazide moiety were designed, synthesized and evaluated for their biological activities against phytopathogenic fungi. The antifungal bioassays indicated that the title compound 5b impressively displayed the obvious selectivity and specificity aganist Rhizoctonia solani (Rs) in vitro and in vivo. The above researches provide a significant reference for the further structural optimization of 1,3,5-thiadiazine-2-thione derivatives bearing a hydrazide moiety as potential fungicides.     

Expression,Purification and Characterization of a Bifunctional α‐L‐Arabinofuranosidase/β‐D‐Xylosidase from Trichoderma Koningii G‐39

A gene of α‐L ‐arabinofuranosidase (Abf) from Trichoderma koningii G‐39 was successfully expressed in Pichia pastoris. The recombinant enzyme was purified to > 90% homogeneity by a cation‐exchanged chromatography. The purified enzyme exhibits both α‐L ‐arabinofuranosidase and β‐D ‐xylosidase (Xyl) activities with p‐nitrophenyl‐α‐L ‐arabionfuranoside (pNPAF) and 2,4‐dinitrophenyl‐β‐D ‐xylopyanoside (2,4‐DNPX) as substrate, respectively. The stability and the catalytic feature of the bifunctional enzyme were characterized. The enzyme was stable for at least 2 h at pH values between 2 and 8.3 at room temperature when assayed for Abf and Xyl activities. Enzyme activity decreased dramatically when the pH exceeded 9.5 or dropped below 1.5. The enzyme lost 35% of Abf activity after incubation at 55 °C for 2 h, but retained 95% of Xyl activity, with 2,4‐DNXP as substrate, under the same conditions. Further investigation of the active site topology of both enzymatic functions was performed with the inhibition study of enzyme activities. The results revealed that methyl‐α‐L ‐arabinofuranoside inhibition is noncompetitive towards 2,4‐DNPX as substrate but competitive towards pNPAF. Based on the thermal stability and the inhibition studies, we suggest that the enzymatic reactions of Abf and Xyl are performed at distinct catalytic sites. The recombinant enzyme possesses both the retaining transarabinofuranosyl and transxylopyranosyl activities, indicating both enzymatic reactions proceed through a two‐step, double displacement mechanism.     

Production of Prosaikogenin F,Prosaikogenin G,Saikogenin F and Saikogenin G by the Recombinant Enzymatic Hydrolysis of Saikosaponin and their Anti-Cancer Effect

The saponins of Bupleurum falcatum L., saikosaponins, are the major components responsible for its pharmacological and biological activities. However, the anti-cancer effects of prosaikogenin and saikogenin, which are glycoside hydrolyzed saikosaponins, are still unknown due to its rarity in plants. In this study, we applied two recombinant glycoside hydrolases that exhibit glycoside cleavage activity with saikosaponins. The two enzymes, BglPm and BglLk, were cloned from Paenibacillus mucilaginosus and Lactobacillus koreensis, and exhibited good activity between 30–37 °C and pH 6.5–7.0. Saikosaponin A and D were purified and obtained from the crude B. falcatum L. extract using preparative high performance liquid chromatography technique. Saikosaponin A and D were converted into saikogenin F via prosaikogenin F, and saikogenin G via prosaikogenin G using enzyme transformation with high β-glycosidase activity. The two saikogenin and two prosaikogenin compounds were purified using a silica column to obtain 78.1, 62.4, 8.3, and 7.5 mg of prosaikogenin F, prosaikogenin G, saikogenin F, and saikogenin G, respectively, each with 98% purity. The anti-cancer effect of the six highly purified saikosaponins was investigated in the human colon cancer cell line HCT 116. The results suggested that saikosaponins and prosaikogenins markedly inhibit the growth of the cancer cell line. Thus, this enzymatic technology could significantly improve the production of saponin metabolites of B. falcatum L.     

Catalyst- and solvent-free coupling of 2-methyl quinazolinones and 3-(trifluoroacetyl)coumarins: An environmentally benign access of quinazolinone derivatives

An environmentally benign highly atom-economic protocol for the construction of the CC bond has been developed under catalyst- and solvent-free conditions. This protocol involves the efficient coupling of 2-methyl quinazolinones with 3-(trifluoroacetyl)coumarins for the access of quinazolinone derivatives in excellent yields (up to 90 %). The crystal structure of compound 3di was investigated by X-ray diffraction analysis. The biological activities, such as in vitro antifungal activity of the quinazolinone derivatives against Fusarium graminearum, Fusarium moniliforme, Fusarium oxysporum, Phytophthora parasitica var nicotianae, and Rhizoctonia solani Kuhn, were investigated. The bioassay results indicated that most of the target products exhibited promising fungicidal activities, and compound 3 cl exhibited 95 % fungicidal activity against R. solani, with an EC₅₀ value of 10.6 μg/mL.     

Chemical composition, antifungal and herbicidal effects of essential oil isolated from Chersodoma Argentina (Asteraceae)

Analysis of the hydrodistilled essential oil of the aerial parts of Chersodoma argentina Cabrera by GC-MS and NMR spectroscopy revealed that over 80% consisted of monoterpene hydrocarbons such as alpha-thujene, alpha-pinene and beta-pinene. Contact and headspace volatile exposure assays of the essential oil demonstrated antifungal activity against Sclerotinia sclerotiorum, Sclerotium rolfsii and Rhizoctonia solani with the contact assay showing greater activity than the headspace assay. Herbicidal activity was shown by reduced root growth of Allium porrum, Solanum lycopersicon and Sorghum halepense in both assays.