Synthesis of Novel 4-Acyloxy-2'-Bromo-6'-Chloropodophyllotoxin Derivatives Displaying Significant Insecticidal Activity Against Mythimna Separata.

In order to explore novel natural product-based insecticidal agent, two important intermediates (2 and 3) and 4-acyloxy-2'-bromo-6'-chloropodophyllotoxin derivatives (4 a-f and 5 a-f) were designed and prepared, and their structures were confirmed by 1H-NMR, 13C NMR, HRMS, ESI-MS, optical rotation and melting point (mp). The stereochemical configuration of compound 4 b was unambiguously confirmed by single-crystal X-ray diffraction. Moreover, we evaluated the insecticidal activity of target compounds 4 a-f and 5 a-f against a serious agricultural pest of Mythimna separata by using the leaf-dipping method. Among all tested compounds, compounds 4 d, 5 d and 5 f exhibited stronger insecticidal activity with a final mortality rate exceeding 60 %. Especially compound 5 d exhibited the best insecticidal activity, with a final mortality rate of 74.1 %. It has been proven that introducing bromine or chlorine atoms at the C-2', C-2' and C-6' positions of the E ring of podophyllotoxin can produce more potent compounds. In addition, the configuration of the C-4 position is important for insecticidal activity, and 4ß-configuration is optimal. This will pave the way for further design, structural modification, and development of derivatives of podophyllotoxin as insecticidal agents.

Design, synthesis, and anti-oomycete activity of 3-acyloxymaltol/ethyl maltol derivatives.

Twenty 3-acyloxymaltol/ethyl maltol derivatives (7a-j and 8a-j) were synthesized and evaluated in vitro for their anti-oomycete activity against Phytophthora capsici, respectively. Among all of twenty derivatives, more than half of the compounds 7f, 7h, 8a-h and 8j had anti-oomycete activity higher than the positive control zoxamide (EC50 = 22.23 mg/L), and the EC50 values of 18.66, 20.32, 12.80, 16.18, 10.59, 14.98, 16.80, 10.36, 15.32, 12.64, and 13.59 mg/L, respectively. Especially, compounds 8c and 8f exhibited the best anti-oomycete activity against P. capsici with EC50 values of 10.59 and 10.36 mg/L, respectively. Overall, hydroxyl group of maltol/ethyl maltol is important active modification site.

Assessment of reproductive toxicity in adult zebrafish (Danio rerio) following sublethal exposure to penthiopyrad.

Penthiopyrad (PO), a succinate dehydrogenase inhibitor (SDHI) fungicide, poses a potential risk to fish. Here, we investigated the adverse effects of PO on endocrine regulation and reproductive capacity in zebrafish during a 21-d sublethal exposure to PO concentrations ranging from 0.02 to 2.00 mg/L. Following exposure to PO (0.20 and 2.00 mg/L), female-specific effects including follicle necrosis, structural disturbance of the yolk follicle, fusion of cortical follicles appeared in ovarian tissue of adult females, which led to a significant reduction in fertility. Correspondingly, 0.20 and 2.00 mg/L PO led to a marked reduction in the GSI values of females, and 2.00 mg/L PO caused a 31% decline in the proportion of perinucleolar oocytes (PCO) in oocytes. In addition, testosterone (T) level was obviously suppressed and 17ß-estradiol (E2) level was increased in females after exposure to 2.00 mg/L PO. Male zebrafish treated with 0.20 and 2.00 mg/L of PO exhibited significant interstitial enlargement, edema in the testes, and reduced diameter of seminiferous tubules, along with a thinner basement membrane. The effects of PO on males were associated with significant increase in E2 level, suggesting that PO has an estrogenic effect on male fish. Greater E2 levels in serum were further supported by increased transcription levels of genes linked to the hypothalamic-pituitary-gonad-liver (HPGL) axis. Notably, transcription levels of cyp19a, er2b, era, and cyp19b was remarkably increased, exhibiting a clear link with variations in E2 levels. Overall, the present study demonstrates that PO induces reproductive impairment in zebrafish by promoting steroidogenesis.

Resistance risk assessment of Fusarium pseudograminearum from wheat to prothioconazole.

Fusarium crown rot (FCR), primarily caused by Fusarium pseudograminearum, poses significant threats to cereal crops worldwide. Prothioconazole is a demethylation inhibitor (DMI) fungicide used to control FCR. However, the risk of resistance in F. pseudograminearum to prothioconazole has not yet been evaluated. In this study, the sensitivity of a total of 255 F. pseudograminearum strains obtained from Henan Province, China to prothioconazole were determined by the mycelial growth inhibition. The results showed that the effective concentration to 50% growth inhibition (EC50) of these strains ranged from 0.4228 µg/mL to 2.5284 µg/mL, with a mean EC50 value of 1.0692 ± 0.4527 µg/mL (mean ± SD). Thirty prothioconazole-resistant mutants were obtained out of six selected sensitive parental strains by means of fungicide taming. The resistant mutants exhibited defects in vegetative growth, conidia production, and pathogenicity on wheat seedlings compared to their parental strains. Under ion, cell wall, and temperature stress conditions but not osmotic stress, all the mutants exhibited decreased growth rates compared with their parental strains, which was consistent with the control treatment. Cross-resistance test showed that there was a cross-resistance relationship between prothioconazole and four DMI fungicides, including prochloraz, metconazole, tebuconazole and hexaconazole, but no cross-resistance was observed between prothioconazole and carbendazim, phenamacril, fludioxonil, or azoxystrobin. Although no site mutation occurred on Cyp51a and Cyp51b genes, the constitutive expression level of the Cyp51a gene was significantly increased in all mutants. After being treated with prothioconazole, the Cyp51a and Cyp51b genes were significantly increased in both the resistant mutants and their parents. These results suggested that the resistance to prothioconazole of the mutants may be attributed to the changes of the relative expression level of Cyp51a and Cyp51b genes. Taken together, these results could provide a theoretical basis for the scientific use of prothioconazole in the field and fungicide resistance management strategies.

Synthesis, Anti-Oomycete and Anti-fungal Activities of Novel Cinchona Alkaloid Derivatives Containing Sulfonate Moiety.

Using cinchona alkaloid as the lead compound, twenty-four cinchona alkaloid sulfonate derivatives (1 a-l, 2 a-c, 3 a-c, 4 a-c, and 5 a-c) were designed and prepared by modifying their C9 position, and structurally confirmed by 1 H-NMR, 13 C-NMR, HR-MS and melting points. Moreover, the stereochemical configurations of compounds 1 f and 1 l were unambiguously confirmed by single-crystal X-ray diffraction. Furthermore, we determined the anti-oomycete and anti-fungal activities of these target compounds against Phytophthora capsici and Fusarium graminearum in vitro. The results showed that two compounds 4 b and 4 c exhibited prominent anti-oomycete activity, and the median effective concentration (EC50 ) values of 4 b and 4 c against P. capsici were 22.55 and 16.32 mg/L, respectively. This study suggested that when the C9 position of cinchona alkaloid sulfonate derivatives is in the S configuration and the 6'-position methoxy group is not present, the anti-oomycete activity is superior. In addition, five compounds 1 e, 1 f, 1 k, 3 c and 4 c displayed significant anti-fungal activity, with EC50 values of 43.64, 45.07, 80.18, 48.58 and 41.88 mg/L against F. graminearum, respectively. This result indicates that only when a specific substituent is introduced into the structural framework of the target compound, the corresponding compound exhibits significant inhibitory activity against fungi.

Synthesis of Hinokitiol Sulfonate Derivatives and Their Anti-Oomycete and Nematicidal Activities.

In order to explore novel natural product-based anti-oomycete and nematicidal agents, sixteen unreported 2-sulfonyloxyhinokitiol derivatives were prepared using the principle of active splicing, and structurally confirmed by proton nuclear magnetic resonance (1 H-NMR), carbon-13 nuclear magnetic resonance (13 C-NMR), high-resolution mass spectrometry (HRMS), and melting point. Moreover, we evaluated the title compounds as anti-oomycete and nematicidal agents against two serious agricultural pests of Phytophthora capsici and Meloidogyne incongnita. Among the sixteen hinokitiol esters tested: (1) Compounds 3a and 3m exhibited the most potent anti-oomycete activity compared to zoxamide against P. capsici, and the median effective concentration (EC50 ) values of 3a, 3m, and zoxamide were 18.64, 21.11, and 23.15 mg/L, respectively; Further studies showed that the existence of seven membered ring and carbonyl group was the necessary condition for the high anti-oomycete activity of hinokitiol. (2) Compounds 3n and 3p exhibited more promising nematicidal activity than hinokitiol, and the median lethal concentration (LC50 ) values of 3n, 3p and 1 against M. incongnita were 0.2111, 0.2079, and 0.3933 mg/L, respectively. This result will pave the way for further modification of hinokitiol to develop potential new fungicides and nematicides.

Synthesis and Anti-Oomycete Preliminary Mechanism of Sulfonate Derivatives of Ethyl Maltol.

To discover novel molecules with unique mechanism against plant pathogenic oomycetes, sixteen new sulfonate derivatives of ethyl maltol (3a-p) were synthesized by structural modification of 2-ethyl-3-hydroxy-4H-pyran-4-one, and their anti-oomycete activity against a serious agricultural disease, Phytophthora capsici Leonian was determined in this study. Among all tested compounds, derivatives 3e, 3m and 3p exhibited the most potent anti-oomycete activity against P. capsici with EC50 values of 19.40, 21.04 and 31.10 mg/L, respectively; especially 3e and 3m showed the best promising and pronounced anti-oomycete activity than zoxamide (EC50 =26.87 mg/L). The results further proved that 4-tert-butylphenylsulfonyl group, 3-nitro-4-chlorophenylsulfonyl group and 8-quinolinesulfonyl group introduced at the hydroxy position of ethyl maltol or maltol were necessary for obtaining the most potent compounds. Further mechanism studies of P. capsici treated with 3e demonstrated that this compound can affect the growth of mycelia by disrupting the integrity of the membrane, and the higher the concentration of the compound is, the greater the degree of membrane integrity damage. These important results will pave the way for further modification of ethyl maltol to develop potential new fungicides.

Synthesis and Anti-Oomycete Activity of Sulfonate Derivatives of Fenjuntong.

In order to discover highly active fungicides, sixteen novel sulfonate derivatives of Fenjuntong were synthesized by structural modification of 2'-hydroxybutyrophenone, and their anti-oomycete activity against Phytophthora capsici Leonian was determined in this study. Among all tested compounds, compound 3b displayed more significant anti-oomycete activity than the precursor Fenjuntong against P. capsici, and the EC50 values of 3b and Fenjuntong were 84.50 and 517.25 mg/L, respectively. By comparing the anti-oomycete activity of compounds 3a-p, I-a-p, and II-a-p, the following conclusions were drawn: (1) Hydroxy group is well tolerated, and sulfonylation of hydroxy group enhances its anti-oomycete activity. (2) The proper length of the ketone carbonyl chain is very important for their anti-oomycete activity. (3) The presence of a site methoxy group in the structural skeleton is closely related to the anti-oomycete activity. These important results will pave the way for further modification of Fenjuntong to develop potential new fungicides.

Sensitivity and Resistance Risk Assessment of Fusarium graminearum from Wheat to Prothioconazole.

Fusarium head blight (FHB), caused mainly by Fusarium graminearum, is one of the most devastating diseases of wheat. Prothioconazole is a broad-spectrum demethylation inhibitor fungicide with excellent efficacy against FHB. In this study, 235 strains of F. graminearum collected from different regions of Henan Province of China in 2016, 2017, and 2018 were randomly selected. The sensitivity of F. graminearum to prothioconazole was determined by the mycelial growth inhibition method. The results showed that the half maximal effective concentration (EC50) values of F. graminearum to prothioconazole ranged from 0.4742 to 3.4403 µg/ml, and the average EC50 value was 1.7758 ± 0.6667 µg/ml. The sensitivity frequency distribution presented a consequent unimodal curve, and thus the average EC50 value can be established as the baseline sensitivity of F. graminearum to prothioconazole. Ten strains of prothioconazole-resistant mutants were obtained by fungicide taming, and the resistance factor of the mutants ranged from 5.71 to 12.32. The genetic stability assay showed that resistance can be inherited stably for 10 generations. All mutants displayed different degrees of defects in vegetative growth, conidia formation, and pathogenicity compared with the parental strain. These results indicated that F. graminearum has a low risk of resistance to prothioconazole. Cross-resistance assay showed that no cross-resistance was found between prothioconazole and carbendazim, tebuconazole, phenamacril, and pydiflumetofen. Among all mutants, sequence analysis showed that no mutation site was found in cyp51A and cyp51B. Real-time PCR assays showed that the expression levels of cyp51A and cyp51B of the mutants were significantly increased after prothioconazole treatment for 24 h. In summary, our study provided a theoretical basis for the resistance risk assessment of F. graminearum to prothioconazole and scientific application of prothioconazole in controlling FHB.

Synthesis of novel 3/5(3,5)-(di)nitropaeonol hydrazone derivatives as nematicidal agents.

Eighteen novel 3/5(3,5)-(di)nitropaeonol hydrazone derivatives were prepared, and their structures well characterized by 1H NMR, HRMS, and mp. Due to the steric hindrance, the substituents on the C = N double bond of all hydrazine compounds (except E/Z = 4/1 for IV-1g, IV-1l, IV-2b, and E/Z = 3/2 for IV-1n, IV-3a) adopted E configuration. Among all compounds, four compounds 2, 4, IV-1j, and IV-1n exhibited potent nematicidal activity than their precursor paeonol, especially 5-nitropaeonol (2) and 3,5-dinitropaeonol (4) displayed the most potent nematicidal activity Heterodera glycines in vivo with LC50 values of 32.3307 and 36.7074 mg/L, respectively.

Synthesis and Anti-Oomycete Activity of 1-Sulfonyloxy/Acyloxydihydroeugenol Derivatives.

Endeavor to discover biorational natural products-based fungicides, two series (26) of novel 1-sulfonyloxy/acyloxydihydroeugenol derivatives (3a-p and 5a-j) were prepared and assessed for their fungicidal activity against P. capsici Leonian, in vitro. Results of fungicidal activity revealed that, among all compounds, especially compounds 3a, 5c, and 5e displayed the most potent anti-oomycete activity against P. capsici with EC50 values of 69.33, 68.81, and 67.77 mg/L, respectively. Overall, the anti-oomycete activities of 1-acyloxydihydroeugenol derivatives (5a-j) were higher than that of 1-sulfonyloxydihydroeugenol derivatives (3a-p). It is proved that the introduction of the acyl group at hydroxy position of dihydroeugenol is more beneficial to improve its anti-oomycete activity than that of the sulfonyl group. These preliminary results will pave the way for further modification of dihydroeugenol in the development of potential new fungicides.

Resistance to Boscalid in Botrytis cinerea From Greenhouse-Grown Tomato.

Gray mold, caused by the fungus Botrytis cinerea Pers ex Fr., is one of the most destructive spoilage diseases, severely affecting tomato production in Henan Province, China. Spraying fungicides from the flowering to the harvest stage is a necessary measure to reduce losses associated with B. cinerea infection. However, B. cinerea has developed resistance to fungicides in many countries. Boscalid is a succinate dehydrogenase inhibitor (SDHI) fungicide and was registered for the control of gray mold. In this study, a total of 269 B. cinerea isolates were collected from tomato in commercial greenhouses in different locations of Henan Province in 2014 and 2015. The sensitivity and resistance of B. cinerea field isolates were determined based on mycelial growth. The effective concentration 50 ranged from 0.11 to 15.92 µg/ml and 0.16 to 8.54 µg/ml, in 2014 and 2015, respectively. The frequency of low resistance to boscalid was 12.6 and 7.6%, and moderate resistance was 2.7 and 1.3% in 2014 and 2015, respectively. No highly resistant isolates were found in Henan Province, China. Mycelial growth, mycelial dry weight, spore production, and pathogenicity were not significantly different between resistant and sensitive phenotypes of the B. cinerea isolates. The results of cross-resistance testing showed no correlation between boscalid and carbendazim, procymidone, pyrimethanil, fluazinam, or fluopyram. In this study, the succinate dehydrogenase genes B (sdhB), C (sdhC), and D (sdhD) were analyzed and compared in sensitive and low-resistance and moderately resistant B. cinerea isolates to boscalid. Results showed that point mutations occurred simultaneously at sdhC amino acid positions 85 (G85A), 93 (I93V), 158 (M158V), and 168 (V168I) in 4 out of 10 sensitive isolates and 23 of 26 low-resistance and 5 of 5 moderately resistant B. cinerea isolates to boscalid. No point mutations were found in the sdhB and sdhD genes of all isolates. Furthermore, no point mutations were found in sdhB, sdhC, and sdhD genes in 3 of 26 low-resistance B. cinerea isolates to boscalid. Therefore, we speculate that the simultaneous point mutations in the sdhC gene may not be related to the resistance of B. cinerea to boscalid. These results suggested that there might be a substitution mechanism for the resistance of B. cinerea to the SDHI fungicide boscalid.

Synthesis of sulfonate derivatives of carvacrol and thymol as anti-oomycetes agents.

Two series of sulfonate derivatives of carvacrol and thymol were synthesized and screened in vitro for their anti-oomycete activity against Phytophthora capsici, respectively. Among all of 32 derivatives, five compounds 3a, 4a, 4k, 3n, and 4n exhibited more potent anti-oomycete activity against P. capsici with EC50 values of 66.66, 62.94, 68.65, 61.24, and 52.91 mg/L, respectively. This suggested that introduction of different substitutions at the hydroxyl position of 1/2 could have remarkable effect on anti-oomycete activity. Overall, when R1 = isopropyl and R2 = methyl, the anti-oomycete activities of the compounds were higher than that of the corresponding compounds of R1 = methyl and R2 = isopropyl.[Formula: see text].

Synthesis of novel 9R/S-acyloxy derivatives of cinchonidine and cinchonine as insecticidal agents.

Endeavor to discover biorational natural products-based insecticides, two series (27) of novel 9R/S-acyloxy derivatives of cinchonidine and cinchonine were prepared and assessed for their insecticidal activity against Mythimna separata in vivo by the leaf-dipping method at 1 mg/mL. Among all the compounds, especially derivatives 6l and 6o exhibited the best insecticidal activity with final mortality rates of 75.0% and 71.4%, respectively. Overall, a free 9-hydroxyl group is not a prerequisite for insecticidal activity and C9-substitution is well tolerated; the configuration of C8/9 position is important for insecticidal activity, and 9S-configuration is optimal; 6'-OCH3 moiety is not necessary, removal of it is also acceptable. [Formula: see text].

Synthesis, anti-oomycete activity, and SAR studies of paeonol derivatives.

Three series of sulfonate derivatives of paeonol were synthesized and screened in vitro for their anti-oomycete activity against P. capsici, respectively. Among all the compounds, 4m displayed the best promising and pronounced anti-oomycete activity against P. capsici than zoxamide, with the EC50 values of 24.51 and 26.87 mg/L, respectively. The results show that acetyl and 4-OCH3 are two necessary groups. The existence of these two sites is closely related to the anti-oomycete activity. Relatively speaking, hydroxyl group is well tolerated, and the results showed that after modification of hydroxyl group with sulfonyl, the anti-oomycete activity was significantly increased. [Formula: see text].

Synthesis of Novel (9S)-Acyloxy Derivatives of Quinidine and Dihydroquinidine as Insecticidal Agents.

Endeavor to discover biorational natural products-based insecticides, two series (30) of novel (9S)-acyloxy derivatives of quinidine and dihydroquinidine were prepared and assessed for their insecticidal activity against Mythimna separata in vivo by the leaf-dipping method at 1 mg/mL. Among all the compounds, especially four derivatives exhibited the best insecticidal activity with final mortality rates of 71.4 %, 75.0 %, 71.4 %, and 75.0 %, respectively. Relatively speaking, 9-hydroxy group is well tolerated, and the results showed that after modification of the hydroxy group with an acyloxy group, the insecticidal activity was significantly increased; the configuration at C8/9 position is important for insecticidal activity, and the (9S)-configuration is optimal; modification of the out-ring double bond is acceptable, and hydrogenation of the double bond enhances insecticidal activity. These preliminary results will pave the way for further modification of quinidine in the development of potential new insecticides.

Synthesis of sulfonate derivatives of maltol and their biological activity against Phytophthora capsici and Bursaphelenchus xylophilus in vitro.

Sixteen sulfonate derivatives of maltol were synthesized and screened in vitro for their anti-oomycete and nematicidal activity against Phytophthora capsici and Bursaphelenchus xylophilus, respectively. Among all the compounds, 3e, 3m, and 3p exhibited the most promising and pronounced anti-oomycete activity against P. capsici than zoxamide, and the EC50 values of 25.42, 18.44, 23.69, and 27.99 mg/L, respectively; compounds 3e, 3m, 3n, and 3p exhibited potent nematicidal activity with LC50 values ranging from 1 to 2 mg/L, especially 3m and 3n showed the best promising and pronounced nematicidal activity, with LC50 values of 1.1762 and 1.2384 mg/L, respectively. [Formula: see text].

Synthesis and insecticidal activity of sulfonate derivatives of sesamol against Mythimna separata in vivo.

A series of sulfonate derivatives of sesamol were synthesized and evaluated for their insecticidal activity against a crop-threatening agricultural pest, the pre-third-instar larvae of Mythimna separata in vivo. Among all the target compounds, compounds 3b, 3g, 3h, and 3p exhibited more promising insecticidal activity than sesamol and toosendanin, and the final mortality rates (FMRs) of 3b, 3g, 3h, 3p, 1, and toosendanin were 60.7%/60.7%/67.9%/53.6%/32.1%/50.0%, respectively. Especially compound 3h exhibited the most potent insecticidal activity with FMRs of 67.9%. This suggested that a 4-fluorophenylsulfonyl group introduced at the hydroxyl position of sesamol was necessary for obtaining the most potent compound.[Formula: see text].

Controlled synthesis of N, N-dimethylarylsulfonamide derivatives as nematicidal agents.

Gramine can be intelligently and efficiently supplied with N, N-dimethylamino group and then reacted with the corresponding sulfonyl chlorides to synthesize N, N-dimethylarylsulfonamides. We herein designed and controlled synthesis of N, N-dimethylarylsulfonamide derivatives, and first reported the results of the nematicidal activity of 15 title compounds 3a-o against Meloidogyne incongnita in vitro, respectively. Among all of the title derivatives, compounds 3a, 3c, 3k, and 3o exhibited potent nematicidal activity with median lethal concentration (LC50) values ranging from 0.22 to 0.26 mg/L. Most noteworthy, N, N-dimethyl-4-methoxyphenylsulfonamide (3c) and N, N-dimethyl-8-quinolinesulfonamide (3o) showed the best promising and pronounced nematicidal activity, with LC50 values of 0.2381 and 0.2259 mg/L, respectively.

Carbendazim Resistance of Fusarium graminearum From Henan Wheat.

Fusarium head blight, also called scab, is caused by Fusarium graminearum and is one of the most important destructive diseases of wheat. The frequency of carbendazim resistance in 1,132 isolates of F. graminearum recovered from fields in different regions of Henan Province in 2016, 2017, and 2018 was determined. A total of 31 F. graminearum isolates resistant to carbendazim were detected, including 30 moderately resistant isolates and one highly resistant isolate. The frequency of resistance of F. graminearum isolates to carbendazim was 2.7%. The range of effective concentration (EC50) values of 1,101 sensitive isolates and 30 moderately resistant isolates was 0.08 to 0.98 µg ml-1 and 2.73 to 13.28 µg ml-1, respectively. The mean ± SD EC50 value was 0.55 ± 0.13 µg ml-1 and 5.61 ± 2.58 µg ml-1, respectively. The EC50 value of the highly resistant isolate was 21.12 µg ml-1. Point mutation types of the carbendazim-resistant isolates were characterized by cloning the ß2-tubulin gene of 31 resistant isolates. Three point mutation types at amino acids F167Y, E198Q, and E198L in the ß2-tubulin gene of resistant isolates were identified. Among 31 resistant isolates, the frequency of point mutation types in F167Y, E198Q, and E198L of the ß2-tubulin gene was 71.0, 25.8, and 3.2%, respectively. The data indicate that F. graminearum has developed resistance to carbendazim in Henan Province, and single point mutations at amino acid F167Y were the predominant type of mutation detected.