Synthesis and fungicidal activities of novel benzothiophene-substituted oxime ether strobilurins

Twenty-one novel benzothiophene-substituted oxime ether strobilurins, which employed a benzothiophene group to stabilise the E-styryl group in Enoxastrobin (an unsaturated oxime strobilurin fungicide developed by Shenyang Research Institute of Chemical Industry, China) were designed and synthesised. The biological assay indicated that most compounds exhibited good or excellent fungicidal activities, especially against Colletotrichum lagenarium and Puccinia sorghi Schw. In addition, methyl 3-methoxypropenoate oxime ethers and N-methoxy-carbamic acid methyl esters exhibited good in vivo fungicidal activities against Erysiphe graminis, Colletotrichum lagenarium and Puccinia sorghi Schw. under the tested concentrations. Notably, (E,E)-methyl 3-methoxy-2-(2-((((6-chloro-1-(1H-benzo[b]thien-2-yl)ethylidene)amino)oxy)methyl)phenyl)propenoate (5E) exhibited more potent in vivo fungicidal activities against nearly all of the tested fungi at a concentration of 0.39 mg/L compared to Enoxastrobin.     

Antifungal and fungicidal activities of tea extract and catechin against Trichophyton

We examined tea extract, (-) epigallocatechin gallate (EGCg) and theaflavin digallate (TF3) for their antifungal and fungicidal activities against Trichophyton mentagrophytes, T. rubrum, Candida albicans and Cryptococcus neoformans. Tea extract (2.5%) inhibited completely the growth of both T. mentagrophytes and T. rubrum. EGCg at 2.5 mg/ml failed to inhibit their growth, whereas TF3 at 0.5 mg/ml inhibited the growth. EGCg (1mg/ml) showed no fungicidal activity against Trichophyton. TF3 (1mg/ml) killed Trichophyton by a long time contact (72-96 hrs). Tea extract showed a fungicidal activity against Trichophyton in a dose- and contact time-dependent manner. It did not inhibit the growth of C. albicans, but at a high concentration, inhibited slightly the growth of C. neoformans. It had no fungicidal activity against C. albicans or C. neoformans.     

Synthesis and fungicidal activity of 1-(alpha-tert-butylcinnamoyl)imidazoles

Several 1-(alpha-tert-butylcinnamoyl)imidazoles were prepared to examine their fungicidal activity. The (Z)-4-chlorocinnamoyl derivative was prepared from (anti)-2-tert-butyl-3-(4-chlorophenyl)-3-hydroxypropanoic acid by treating with 1,1'-carbonyldiimidazole and a subsequent beta-elimination reaction at an elevated temperature. The (Z)-isomer of the 4-chlorocinnamoyl derivative showed good fungicidal activity against Erysiphe graminis and Botrytis cinerea in pot tests, whereas the corresponding (E)-isomer derived from the (Z)-isomer through photoisomerization was much less active.     

Structural Optimization,Fungicidal Activities Evaluation,DFT Study and Structure-Activity Relationship of Dopamine Derivatives with Benzothiazole Fragment from Polyrhachis dives

In our previous work, two dopamine derivatives with benzothiazole fragment were isolated and identified from Polyrhachis dives (P. dives). Based on their characteristic structure, we used them as lead compound to carry out structural optimization and subsequent fungicidal evaluation. Here 20 dopamine derivatives with benzothiazole fragment were designed and synthesized by a facile method, and their structures were characterized by ¹H-NMR, ¹³CNMR and HMRS. In bioassays, most of the title compounds possess potential fungicidal activities against Altenaia alternala (A. alternala) and Botrytis cinerea (B. cinerea). Especially, (E)-N-(2-(benzo[d]thiazol-6-yl)ethyl)-3-(p-tolyl)acrylamide and (E)-N-(2-(benzo[d]thiazol-6-yl)ethyl)-3-(4-(trifluoromethyl)phenyl)acrylamide displayed 29.3 mg/L and 10.7 mg/L EC₅₀ value against A. alternala, respectively, which possessed equivalent fungicidal activities level to hymexazol.     

Synthesis and biological activities of 2-oxocycloalkylsulfonamides

A series of novel 2-oxocycloalkylsulfonamides (4) were synthesized and their structures confirmed by IR, (1)H NMR, and elemental analysis. The bioassay showed that they have fair to excellent fungicidal activities against Botrytis cinerea Pers and Sclerotinia sclerotiorum. Among them, compounds 4A(10), 4A(11), 4A(12), 4B(2), and 4B(3), the EC(50) values of which were 2.12, 3.66, 3.96, 2.38, and 2.43 microg/mL, respectively, displayed excellent fungicidal activity against B. cinerea Pers, and are comparable with commercial fungicide procymidone (the EC(50) value is 2.45 microg/mL). 3D QSAR against B. cinerea Pers was studied, a statistically significant and chemically meaningful CoMFA model was developed and some compounds which have a high predicted activity were forecasted. In addition, the bioassay also showed that the compounds have good inhibitory activities against human tumor cells HL-60, BGC-823, Bel-7402 and KB. It is interesting to point out that the antitumor activities of compounds 4 are in accordance with their fungicidal activity to a great extent: compounds having relatively best antitumor activities (4A(10), 4A(11), 4A(12), and 4B(3)) also displayed excellent fungicidal activity.     

Sulfation of tibolone metabolites by human postmenopausal liver and small intestinal sulfotransferases (SULTs)

Sulfation is a major pathway in humans for the biotransformation of steroid hormones and structurally related therapeutic agents. Tibolone is a synthetic steroid used for the treatment for climacteric symptoms and postmenopausal osteoporosis. Sulfation inactivates the hydroxylated metabolites, 3alpha-hydroxytibolone (3alpha-OH-tibolone) and 3beta-hydroxytibolone (3beta-OH-tibolone), and contributes to the regulation of tissue responses to tibolone. We detected SULT1A1, SULT1A3, SULT1E1 and SULT2A1 mRNA expression by RT-PCR in postmenopausal liver and small intestine. Liver pool (n=5) SULT activities measured with tibolone substrates reflected COS-1 expressed SULT2A1 and SULT1E1 activities. Liver SULT2A1 activity (1.8 +/- 0.3 units/mg protein, n = 8, mean +/- SEM), and activities with 3alpha-OH-tibolone (0.6 +/- 0.1, n = 8) and 3beta-OH-tibolone (0.9 +/- 0.2, n = 8) were higher than SULT1E1 activities (<0.05, n = 10). SULT1E1 activities were low or not detected in many samples. Mean small intestinal activities were 0.03 +/- 0.01 with 3alpha-OH-tibolone and 0.04 +/- 0.01 with 3beta-OH-tibolone (n = 3). In conclusion, SULT2A1 is the major endogenous enzyme responsible for sulfation of the tibolone metabolites in human postmenopausal tissues. The results support the occurrence of pre-receptor enzymatic regulation of hydroxytibolone metabolites and prompt further investigation of the tissue-selective regulation of tibolone effects.     

Stereoselective synthesis and fungicidal activities of (E)-alpha-(methoxyimino)-benzeneacetate derivatives containing 1,3,4-oxadiazole ring

Fifteen novel (E)-alpha-(methoxyimino)-benzeneacetate derivatives, the analogues of strobilurins, which contain two pharmacophoric substructures of (E)-methyl methoxyiminoacetate moiety and 1,3,4-oxadiazole ring were stereoselectively synthesized. It was first found that the coupling reaction could give stereoselectively the key intermediate (E) and (Z)-methyl 2-(hydroxyimino)-2-o-tolylacetate 2 with a ratio of 14:1. The preliminary bioassays indicated that all the compounds 1 showed potent fungicidal activity against Rhizoctonia solani, Botrytis cinereapers, Gibberella zeae, Physalospora piricola and Bipolaris mayclis, and all of the tested compounds 1a-1o had more potent fungicidal activities against R. solani than Kresoxim-methyl.     

Design, synthesis, and biological activity of novel 4-(3,4-dimethoxyphenyl)-2-methylthiazole-5-carboxylic acid derivatives

Novel 4-(3,4-dimethoxyphenyl)-2-methylthiazole-5-carboxylic acid derivatives (11-23) were synthesized from 3,4-dimethoxyacetophenone (5) in six-step procedure. Their biological activities were evaluated in the greenhouse. Some of the compounds had shown fungicidal and insecticidal activities at 375 g ai/ha and 600 g ai/ha, respectively.     

Synthesis and fungicidal activity of novel pyrazole derivatives containing 5-Phenyl-2-Furan

Pyrazole constitutes an important heterocyclic family covering a broad range of synthetic as well as natural products that exhibit numerous chemical, biological, agrochemical and pharmacological properties. In order to explore compounds with good fungicidal activity, a series of new pyrazole derivatives containing 5-phenyl-2-furan were designed and synthesized. In vitro and in vivo fungicidal activities were evaluated and the compound ethyl-1-(5-phenylfuran-2-carbonyl)-5-propyl-1H-pyrazole-3-carboxylate (I8) displayed significant fungicidal activity against various fungi, especially against P. infestans. The structures of the novel pyrazole derivatives were confirmed by ¹H NMR, ¹³C NMR, MS, elemental analysis and X-ray single crystal diffraction. Further study showed that compound I8 might act on the synthesis of cell walls from morphological and ultrastructural studies by SEM and TEM. The results also revealed that compound I8 could block the nutritional transportation leading to cells senescence and death. These results suggested that the novel pyrazole derivatives proved to be promising lead compounds.     

Elucidation of antifungal metabolites produced by Pseudomonas aurantiaca IB5-10 with broad-spectrum antifungal activity

Antifungal metabolites were isolated from a culture of Pseudomonas aurantiaca IB5-10. Chemical structures of the metabolites were elucidated as phenazine-1-carboxylic acid (PCA; 1), 2-hydroxyphenazine (2-OH-PHZ; 2), and cyclo-(L-Pro-L-Val; 3), respectively, based on spectroscopic methods. Among them, 3 was isolated for the first time from this strain. The antifungal activities of 1-3 were evaluated against a variety of plant pathogens. To the best of our knowledge, the antifungal activities of 3 against plant fungal pathogens have been evaluated for the first time in this work. PCA (1) showed the most potent antifungal activities against Phytophthora capsici, Rhizoctonia solani AG-1(IA), and Pythium ultimum with MICs (microgram/ml) of less than 1.0, 1.3, and 2.0, respectively. On the other hand, 2-OH-PHZ (2) showed potent antifungal activity against R. solani AG-1(IA) with the MIC (microgram/ml) of 2.0, whereas it showed moderate antifungal activity against P. ultimum with the MIC (microgram/ml) of 50.0. In addition, 3 showed antifungal activity against only R. solani AG- 1(IA).     

Fungicidal effect and the mode of action of piscidin 2 derived from hybrid striped bass

Piscidin 2 (P2), a 22-residue cationic peptide isolated from the mast cells of hybrid striped bass, has potent antibacterial activities. However, its antifungal properties are not completely understood. In the current study, we investigated the antifungal effects and mode of action of P2. P2 exhibited potent antifungal activity against human pathogenic fungi. To understand the fungicidal properties of P2, we focused on a membrane-active mechanism of the peptide by in vivo and in vitro testing. Flow cytometric analysis using bis-(1,3-dibutylbarbituric acid) trimethine oxonol [DiBAC₄(3)] and protoplast regeneration experiments showed that P2 caused fungal membrane damage. Furthermore, fluorescence analysis using 1,6-diphenyl-1,3,5-hexatriene (DPH) revealed that P2 created pores in fungal membranes. These results were confirmed with dye leakage tests by using liposomes composed of phosphatidylcholine/phosphatidylserine (3:1, w/w), which mimicked fungal membranes. The present study indicated that P2 exerts its fungicidal effects by perturbing membrane activities.     

Identification and characterization of endoglucanases for fungicidal activity in <Emphasis Type="Italic">Anabaena laxa</Emphasis> (Cyanobacteria)

A cyanobacterial strain (Anabaena laxa RPAN8) exhibiting fungicidal activity and β-1,3 and 1,4 endoglucanase activities was selected for identifying the gene(s) involved. Functional analyses of the genomic library revealed that four clones (8, 64, 116, and 248) of RPAN8 exhibited fungicidal activity and induced structural deformities in the cell wall of the growing mycelia of Pythium aphanidermatum. Higher expression of fungicidal and β-1,4 endoglucanase activities, along with low expression of β-1,3 endoglucanase activity, was recorded in two E. coli clones (8 and 64). Clones 8 and 64 exhibited identical sequences while clones 116 and 248 were also similar. Bioinformatic analyses were undertaken only for the two non-identical clones 8 and 116 which showed open reading frames (ORFs) of 348 (end 1) and 656 amino acid residues (end 2), respectively. The amino acid sequence analyses revealed that the end 1 encoding endoglucanases belonged to peptidase M20 family while end 2 showed significant similarities with several known genes. The putative promoters and ribosomal binding sites were identified and amino acid exchanges were observed in both end 1 and 2. The presence of signal peptides of 24 and 20 amino acid residues respectively revealed the secretory nature of these proteins.     

Effects ofN,N-bis(3-aminopropyl)dodecylamine on antioxidant enzyme activities, mitochondrial morphology and metabolism inAspergillus niger

The activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSP) as well as of succinate dehydrogenase (SDG), NADH dehydrogenase (NDG) and fumarate hydratase (FHT) were examined in relation to mitochondrial ultrastructure changes in Aspergillus niger exposed to N,N-bis(3-aminopropyl)dodecylamine (Apd) that was shown to exhibit fungicidal activity. There was a progressive increase in SOD, CAT and GSP activities 1 and 4 h after 0.05 and 0.1 % Apd application. However, this was followed by a pronounced activity decrease when 0.05 % Apd treatment was prolonged by 1 d. The destructive effect on fungal morphology was observed when this fungicidal agent was applied at the concentration of 0.1 % for 1 d. In the treated hyphae mitochondria degenerated after all organelles. The morphological malformations of mitochondria had an impact on their metabolic state; however, the activities of SDG, NDG and FHT were affected to a different extent. In A. niger the fungicidal effect of Apd could be mediated by oxidative stress impairing the vital mitochondria-related cellular functions.     

Structural identification of methyl protodioscin metabolites in rats' urine and their antiproliferative activities against human tumor cell lines

Methyl protodioscin (MPD), a furostanol saponin, is a preclinical drug shown potent antiproliferative activities against most cell lines from leukemia and solid tumors. The metabolites of MPD in rats' urine after single oral doses of 80 mg/kg were investigated in this research. Ten metabolites were isolated and purified by liquid-liquid extraction, open-column chromatography, medium-pressure liquid chromatography, and preparative high-performance liquid chromatography. The structural identification of the metabolites was carried out by high resolution mass spectra, NMR spectroscopic methods including (1)H NMR, (13)C NMR and 2D NMR, as well as chemical ways. The 10 metabolites were elucidated to be dioscin (M-1), pregna-5,16-dien-3beta-ol-20-one-O-alpha-l-rhamnopyranosyl-(1-->2)-[alpha-l-rhamnopyranosyl-(1-->4)]-beta-d-glucopyranoside (M-2), diosgenin (M-3), protobioside (M-4), methyl protobioside (M-5), 26-O-beta-d-glucopyrannosyl(25R)-furan-5-ene-3beta, 22alpha, 26-trihydroxy-3-O-alpha-l-rhamnopyranosyl-(1-->4)-beta-d-glucopyranoside(M-6),26-O-beta-d-glucopyranosyl(25R)-furan-5-ene-3beta,26-dihydroxy-22-methoxy-3-O-alpha-l-rhamnopyranosyl-(1-->4)-beta-d-glucopyranoside (M-7), prosapogenin A of dioscin (M-8), prosapogenin B of dioscin (M-9), and diosgenin-3-O-beta-d-glucopyranoside (M-10), respectively. M-1 was the main urinary metabolite of MPD in rats. Some metabolites showed potent antiproliferative activities against HepG2, NCI-H460, MCF-7 and HeLa cell lines in vitro.     

Tibolone and its delta-4, 7α-methyl norethisterone metabolite are reversible inhibitors of human aromatase

Tibolone is used for the treatment of climacteric symptoms and osteoporosis in menopausal women. After ingestion, it is rapidly converted to a number of metabolites including 3α- and 3β-hydroxy derivatives and the delta-4, 7α-methylnorethisterone (7α-MeNET) metabolite, which is rapidly cleared from circulation. Tibolone and some of its metabolites act in a tissue-selective manner to inhibit steroid sulphatase (STS) and 17β-hydroxysteroid dehydrogenase Type 1 (17β-HSD1) activities but also stimulate steroid sulphotransferase and 17β-HSD2 activities. In the present study we have examined whether the ability of tibolone and its 7α-MeNET metabolites to regulate the activities of enzymes involved in oestrogen formation or inactivation extends to another key enzyme involved in oestrogen synthesis, the aromatase, which converts androstenedione to oestrone. Using JEG-3 choriocarcinoma cells, which have a high level of aromatase activity, tibolone and 7α-MeNET, but not the 3α- or 3β-hydroxy metabolites, were found to inhibit aromatase activity in intact cells and also lysates prepared from these cells (up to 61% inhibition at 10 μM). An investigation into the nature of aromatase inhibition by these compounds revealed that they inhibit aromatase activity by a reversible mechanism. Tibolone and 7α-MeNET also inhibited aromatase activity in MCF-7 breast cancer cells, which have a much lower level of aromatase activity than JEG-3 cells. It is concluded that, in addition to inhibiting STS and 17β-HSD1, tibolone and 7α-MeNET may exert some of their tissue-selective effects in regulating oestrogen synthesis by also inhibiting aromatase activity.     

HP (2-20) derived from the amino terminal region of helicobacterpylori ribosomal protein L1 exerts its antifungal effects by damaging the plasma membranes of Candida albicans.

The fungicidal effects of the peptide HP (2-20). derived from the N-terminal sequence of Helicobacter pylori ribosomal protein L1 (RPL1). have been investigated. HP (2-20) displays a strong fungicidal activity against various fungi, without haemolytic activity against human erythrocyte cells, and the fungicidal activity is inhibited by Ca2+ and Mg2+ ions. In order to investigate the fungicidal mechanism(s) of HP (2-20). the amount of intracellular trehalose was measured in C. albicans. It was found that the amounts of intracellular trehalose were decreased when HP (2-20) was used. The action of the peptide against fungal cell membranes was further examined by the potassium-release test; HP (2-20) was found to increase the amount of K+ released from the cells. Furthermore, HP (2-20) caused significant morphological changes, as shown by scanning electron microscopy, and by testing the membrane disrupting activity using liposomes (phosphatidyl choline/cholesterol; 10: 1, w/w). Our results suggest that HP (2-20) may exert its antifungal activity by disrupting the structure of cell membranes, via pore formation or direct interaction with the lipid bilayers.     

Synthesis of novel 2-phenylsulfonylhydrazono-3-(2',3',4',6'-tetra-O-acetyl-beta-d-glucopyranosyl)thiazolidine-4-ones from thiosemicarbazide precursors

To develop novel biologically active organic compounds possessing a sugar moiety, a series of 2-phenylsulfonylhydrazono-3-(2',3',4',6'-tetra-O-acetyl-beta-d-glucopyranosyl)thiazolidine-4-one were synthesized via reaction of the thiosemicarbazide with ethyl bromoacetate. Their chemical structures were characterized by (1)H and (13)C NMR spectroscopy, elemental analysis and MS. The bioassay results indicated that some of these compound exhibit moderate fungicidal and herbicidal activities. Furthermore, the effect of various solvents at reflux temperature on the reactions of ethyl bromoacetate with the related thiosemicarbazides was investigated.     

In vitro antioxidative activity of (-)-epicatechin glucuronide metabolites present in human and rat plasma

Recently we identified four conjugated glucuronide metabolites of epicatechin, (-)-epicatechin-3'-O-glucuronide (E3'G), 4'-O-methyl-(-)-epicatechin-3'-O-glucuronide (4'ME3'G), (-)-epicatechin-7-O-glucuronide (E7G) and 3'-O-methyl-(-)-epicatechin-7-O-glucuronide (3'ME7G) from plasma and urine. E3'G and 4'ME3'G were isolated from human urine, while E7G and 3'ME7G were isolated from rats that had received oral administration of (-)-epicatechin (Natsume et al. (2003), Free Radic. Biol. Med. 34, 840-849). It has been suggested that these metabolites possess considerable in vivo activity, and therefore we carried out a study to compare the antioxidant activities of the metabolites with that of the parent compound. This was achieved by measuring superoxide scavenging activity, reduction of plasma TBARS production and reduced susceptibility of low-density-lipoprotein (LDL) to oxidation. (-)-Epicatechin was found to have more potent antioxidant activity than the conjugated glucuronide metabolites. Both (-)-epicatechin and E7G had marked antioxidative properties with respect to superoxide radical scavenging activity, plasma oxidation induced by 2,2'-azobis-(2-aminopropane) dihydrochloride (AAPH) and LDL oxidation induced by copper ions or 2,2'-azobis(4-methoxy-2,4-dimethylvaleronitrile) (MeO-AMVN). In contrast, the other metabolites had light antioxidative activities over the range of physiological concentrations found in plasma.     

亚致死剂量乙草胺对蚯蚓的毒性效应及敏感生物标记物

本研究以赤子爱胜蚓为受试生物,采用外源添加污染物的方法,将受试生物暴露于含亚致死剂量乙草胺(添加浓度分别为1、2、4、8 mg·kg^-1)的土壤中7 d,研究蚯蚓生长抑制率、细胞色素P450同工酶(CYP1A2、2C9和3A4)活力及代谢组学对乙草胺的响应,从个体、酶、小分子标记物3个层次探讨亚致死剂量乙草胺对蚯蚓的毒性效应,初步推断其毒性作用阈值,筛选敏感生物标记物,探讨其致毒机理。结果表明: 乙草胺暴露下,与对照相比,蚯蚓体重抑制率无明显差异,但CYP1A2、2C9和3A4活力受到明显抑制,10组小分子代谢物(1, 6-二磷酸果糖、胞苷酸、尿苷酸、腺苷酸、腺苷、黄嘌呤、延胡索酸、二羟基戊二酸、鸟氨酸与16-羟二十烷四烯酸)水平显著降低;另有6组小分子代谢物(腺苷琥珀酸、琥珀酸、精氨酸、色氨酸、天冬酰胺与苯丙氨酸)水平在2～8 mg·kg^-1乙草胺暴露下显著升高。乙草胺暴露导致蚯蚓受到氧化损伤,糖酵解功能减弱,三羧酸循环失衡,嘌呤及嘧啶代谢紊乱,氨基酸代谢受损。与个体水平的受试终点相比,CYP同工酶活力与上述16个小分子代谢物对乙草胺暴露的响应更为敏感。建议将CYP同工酶(1A2、2C9及3A4)活力与上述小分子代谢物为一组生物标记物,可以多指标、多层次联合诊断土壤乙草胺污染的生态毒性效应。其诊断结果将更为精准。