Fungicide

In the previous report, Metyltetraprole inhibited electron transfer from succinate to cytochrome c and also from nicotinamide adenine dinucleotide (NADH) to ubiquinone.23 Thus, the target site of metyltetraprole was estimated to be the cytochrome bc1 complex.23 However, there are two pockets for small molecule inhibitors in Complex III (the Qo and Qi sites).6 Therefore, it is still unclear whether Metyltetraprole inhibit Qo or Qi sites, although it is more likely to be a Qo site inhibitor because of its structural similarity with QoIs. Therefore, we tried to confirm this using radiolabeled metyltetraprole and an existing QoI, pyraclostrobin. We found that pyraclostrobin (1 ppm) decreased the levels of radioactivity in the crude submitochondrial fraction corresponding to the amount of Metyltetraprole, if the fraction was prepared from the cultures of wild-type Z. tritici (Fig. 6). Although more than 50% radioactivity remained in the filter, possibly because of the non-specific adhesion of radioactive metyltetraprole to the filter or pellets, the decrease in radioactivity indicated that the binding of metyltetraprole was blocked by the presence of the QoI pyraclostrobin. This decrease induced by pyraclostrobin was smaller when the submitochondrial fraction was prepared from the culture of the G143A mutant, probably because of the low affinity of pyraclostrobin to the G143A mutant cytochrome b.6 Therefore, the result was in line with our estimation that the binding site of metyltetraprole in Z. tritici mitochondria is likely to be the Qo site of the cytochrome bc1 complex, as well as pyraclostrobin, a known QoI.[1]

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Background: Metyltetraprole is a new fungicide with a unique tetrazolinone-moiety and a similar side chain to a known quinone outside inhibitor (QoI), pyraclostrobin. In this study we describe a unique bioactivity of metyltetraprole on QoI-resistant strains of Zymoseptoria tritici and Pyrenophora teres. Results: Metyltetraprole exhibited potent antifungal activity against Ascomycetes; it was especially effective against Z. tritici and P. teres in seedling pot tests. Metyltetraprole was also effective in field tests with QoI-resistant mutants. Antifungal activity tests using field strains of Z. tritici and P. teres showed that the performance of metyltetraprole was unaltered by QoI, succinate dehydrogenase inhibitor (SDHI), and sterol 14α-demethylation inhibitor (DMI) resistance. However, the mitochondrial activity test indicated that the compound inhibits the respiratory chain via complex III. Conclusion: Metyltetraprole is a novel fungicide that is highly effective against a wide range of fungal diseases, including important cereal diseases. Although Metyltetraprole most likely inhibits the respiratory chain via complex III, it remains effective against QoI resistant strains. Therefore, metyltetraprole is considered as a novel fungicidal agent for controlling diseases affecting cereal crops and overcoming pathogen resistance to existing fungicides. [2]

Field efficacy [2]
The above results demonstrate that Metyltetraprole can inhibit the growth of Z. tritici and P. teres on potted plants in a greenhouse. We also conducted field trials in Europe to evaluate the potential of metyltetraprole as an agricultural fungicide under field conditions, with three commercial agricultural fungicides (pyraclostrobin, prothioconazole, and fluxapyroxad) as references.

In comparison with pyraclostrobin, the efficacy of Metyltetraprole against septoria leaf blotch was high and stable (Fig. 4(A)). Based on the general information, we presumed the existence of resistant isolates to QoI fungicides and therefore conducted a sensitivity analysis of field isolates in 2015. We isolated five strains from each field and tested the antifungal activity of azoxystrobin with the microtiter plate method. Only 2/30 strains had an EC50 < 0.1 mg L−1, indicating that the resistant strains accounted for >90% of the population in the tested fields (Supporting Information Table S3). Metyltetraprole showed higher efficacy than pyraclostrobin against Z. tritici.

Metyltetraprole also showed a high, stable degree of efficacy against net blotch, in contrast to the variable efficacy of pyraclostrobin (Fig. 4(B)). Sensitivity tests on YBA plates amended with 0.5 mg L−1 of azoxystrobin were conducted in 2015; strains showing less than 20% growth compared to the untreated plate were categorized as sensitive strains. We conducted four trials in 2015 and found that pyraclostrobin showed lower efficacy in two fields (3.4% in trial 3 and 51.3% in trial 1 vs. 84.4% in trial 2 and 97.3% in trial 4). The percentage of resistant strains isolated from trials 2 and 4 was < 20%; however, 56% of isolates in trial 3 were resistant (Supporting Information Table S4). Since isolates from trial 1 were contaminated, the sensitivity data obtained from this trial were excluded. Nonetheless, our results indicate that the efficacy of pyraclostrobin was declined in fields with a higher proportion of strains of resistant biotypes whereas that of metyltetraprole was stable. Thus, metyltetraprole can control both Z. tritici and P. teres even in the presence of high proportions of QoI‐resistant strains in trial fields.

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Seedling pot test using QoI‐resistant strain [2]
We evaluated the efficacy of Metyltetraprole against the G143A mutant of Z. tritici under greenhouse conditions. Pyraclostrobin showed complete control of wild‐type strain at 83 g ha−1; however, the efficacy against the G143A mutant was significantly reduced (Fig. 5). In contrast, metyltetraprole controlled the G143A mutant to a degree comparable to the sensitive strain. These results indicate that the antifungal efficacy of metyltetraprole is unaffected by the presence of pyraclostrobin‐resistant strains.

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Antifungal activity test against fungicide‐resistant strains [2]
The antifungal activity of Metyltetraprole was further assessed on field isolates of Z. tritici and P. teres, which showed low sensitivity to representative fungicides including pyraclostrobin (QoI), fluxapyroxad (SDHI), and prothioconazole (DMI) (Tables 2 and 3). Resistant field isolates were collected from various locations in Europe (Supporting Information Table S2).

EC50 values of Metyltetraprole against Z. tritici field strains ranged from 0.0025 to 0.0088 mg L−1 and were similar to that against a wild type strain Set1, which is sensitive to QoI, SDHI, and DMI fungicides. The RF (i.e. ratio of the EC50 value of a field isolates to that of Set 1) of metyltetraprole was <3. For example, Set 15‐3 was a triple‐resistant strain with RF values of 188 to pyraclostrobin, 20 to fluxapyroxad, and 66 to prothioconazole, although the EC50 of metyltetraprole against this strain was 0.0047 mg L−1. The RF value of 1.3 indicated that both Set 15‐3 and Set 1 were sensitive to metyltetraprole (Table 2).

P. teres strains resistant to representative fungicides were also sensitive to Metyltetraprole (Table 3). Pt 15‐1 was resistant to pyraclostrobin and prothioconazole, with RF values of 14.5 and 6.3, respectively. Meanwhile, Pt 15‐2 was resistant to fluxapyroxad and prothioconazole, with RF values of 83.1 and 9.6, respectively. On the other hand, metyltetraprole showed similar antifungal activity levels against field strains and the sensitive strain Pt 6. The EC50 values of the other field strains were also similar to that of the wild‐type, that is, 0.007–0.015 mg L−1 and RF < 3. These data indicate that the activity of metyltetraprole is almost unaffected by the strains that show resistance to pyraclostrobin, fluxapyroxad, and prothioconazole.

[14C]-Metyltetraprole displacement assay [1]
To study possible Metyltetraprole displacement with pyraclostrtobin at the Qo site, a crude submitochondrial fraction of Z. tritici was prepared as previously described.23 The protein amount in the submitochondrial fraction was estimated with DC Protein Assay Kit. The crude submitochondrial fraction containing 5 × 102 μg total protein was mixed in 1.0 mL reaction buffer (0.1 M K-phosphate buffer of pH 7.4, 0.3 mM EDTA of pH 8.0, 20 mM succinate in water of pH 7.4 adjusted with NaOH, 1 mM KCN, and 1% DMSO) in tubes with a filter unit (UltrafreeMC-PLHCC 250/pk for Metabolome Analysis). Aliquots of [14C]Metyltetraprole were added to aliquots of the crude submitochondrial fraction in the reaction buffer to give 1 ppm of final concentrations (minimum concentration for the stable radioactivity detection); after incubation at 18–23 °C for 20 min to reach equilibrium, aliquots of pyraclostrobin were added to give final concentrations of 0.1 ppm or 1 ppm, respectively. After incubation at 18–23 °C for 20 min to reach equilibrium, the tubes were centrifuged at 12,000g for 120 min and the supernatant was removed. The filter unit trapping the pellet of submitochondrial fraction was picked up from the tube and subjected to liquid scintillation counting.

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In vitro assay for mitochondrial electron transport activity [2]
The succinate‐cytochrome c reductase (SCR) assay was carried out as previously described. Metyltetraprole and the other QoI fungicides were added to the SCR reaction mixtures as DMSO solutions. The final concentration of DMSO was 0.1%. The inhibitory activity of each fungicide was determined as the fungicide concentration required for 50% inhibition (IC50).

Mycelial growth assay [2]
The antifungal activity of Metyltetraprole against Z. tritici, Ramularia collo‐cygni, P. teres, Pyrenophora tritici‐repentis, Parastagonospora nodorum, Botrytis cinerea, Colletotrichum graminicola, Microdochium nivale, Rhizoctonia solani, Ustilago maydis, Aphanomyces cochlioides, Pythium irregulare, and Phytophthora capsici was evaluated by two different methods under the incubation conditions detailed in Supporting Information Table S1.

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96‐well microtiter plate method [2]
Growth of Z. tritici, R. collo‐cygni, P. nodorum, and U. maydis was evaluated on 96‐well microtiter plates. Conidia of Z. tritici, crushed mycelia of R. collo‐cygni, conidia of P. nodorum, or yeast‐like cell of U. maydis were harvested in distilled water and the density was adjusted with the appropriate medium (Supporting Information Table S1) to 1 × 104 mL−1 conidia, crushed mycelia, or yeast‐like cell, respectively. A 100‐fold dilution series of Metyltetraprole was prepared as a stock solution in DMSO and a 1‐µL aliquot was added to each well for a total of 11 test concentrations. A 99‐µL volume of prepared inoculum or medium without conidia/mycelia (blank) was added to each well. The final concentrations of Metyltetraprole were 3, 1, 0.3, 0.1, 0.03, 0.01, 0.003, 0.001, 0.0003, 0.0001, and 0 mg L−1. The incubation conditions are shown in Supporting Information Table S1. Growth was measured by optical density at a wavelength of 600 nm with a microplate reader SH‐9000 Lab. Optical density values were corrected by the value for the blank well. The 50% effective concentration (EC50) was determined by probit analysis. One unit of experiment has four replicates of each concentration of Metyltetraprole.

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Agar plate method [2]
P. teres, P. tritici‐repentis, B. cinerea, C. graminicola, M. nivale, R. solani, A. cochlioides, P. irregulare, and P. capsici were cultured on agar media (see Supporting Information Table S1) amended with a series of concentrations of Metyltetraprole (3, 1, 0.3, 0.1, 0.03, 0.01, 0.003, 0.001, 0.0003, 0.0001, and 0 mg L−1). Mycelium radial length was measured at designated days after inoculation and EC50 values were calculated. One unit of experiment has four replicates of each concentration of metyltetraprole.

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Cross‐resistance test [2]
Z. tritici and P. teres strains used for the cross‐resistance test were isolated from infected leaves collected from the fields. Detached leaves were kept in humid conditions and spore formation was induced. A single spore was collected under the microscope and grown on potato dextrose agar (PDA) medium (39 g PDA in 1 L water). Sampling locations of isolates are listed in Supporting Information Table S2. Growth on fungicide‐containing medium was evaluated with the microtiter plate method (Z. tritici) or plated‐medium method (P. teres). Criteria for resistance were as follows: Z. tritici strains have EC50 > 1 mg L−1 against azoxystrobin (QoI),27 ≥0.5 mg L−1 against fluxapyroxad (SDHI),16 and >1 mg L−1 against bromuconazole (DMI).28 P. teres strains showed >20% growth in comparison to the untreated control on Yeast Bacto Acetate (YBA) medium plates containing 0.5 mg L−1 azoxystrobin, 5 mg L−1 boscalid (SDHI), and 1 mg L−1 bromuconazole. The tested concentrations of Metyltetraprole, pyraclostrobin, fluxapyroxad, and prothioconazole‐desthio were 3, 1, 0.3, 0.1, 0.03, 0.01, 0.003, 0.001, 0.0003, 0.0001, and 0 mg L−1. The EC50 value was calculated based on the average inhibition rate of four replicates. Resistance factor (RF) was calculated using the formula RF = (EC50 of field isolate)/(EC50 of reference isolate).

Field trial [2]
The field data presented in this report are based on 38 Z. tritici trials (13 in France, 9 in Germany, 8 in UK, 4 in Ireland, 3 in Italy, and 1 in Belgium) and 27 P. teres trials (13 in France, 2 in UK, 4 in Italy, 2 in Poland, and 1 each in Ireland, Austria, Hungary, Czech, Romania, and Bulgaria) conducted from 2015 to 2017. The trials were carried out by contractor companies according to the guidelines of the European and Mediterranean Plant Protection Organization (http://pp1.eppo.int/) of the year of study. The field efficacy of Metyltetraprole at 120 g active ingredient ha−1 was tested, with pyraclostrobin (Comet; BASF) at 220 g active ingredient ha−1, prothioconazole, and fluxapyroxad (IMTREX 62.5 g L−1 EC; BASF) serving as reference treatments. The water volume was 200–250 L ha−1. All chemicals were applied with a hand‐held boom sprayer with conventional nozzles at T1 and T2 fungicide application timings. Disease severity was assessed and the percentage of disease control was calculated relative to the infection level of corresponding untreated leaves. Mean percentage of disease control was determined from the data of penultimate leaf at individual trials.

[1]. Discovery of metyltetraprole: Identification of tetrazolinone pharmacophore to overcome QoI resistance. Bioorg Med Chem. 2020 Jan 1;28(1):115211.

[2]. Metyltetraprole, a novel putative complex III inhibitor, targets known QoI-resistant strains of Zymoseptoria tritici and Pyrenophora teres. Pest Manag Sci. 2019 Apr;75(4):1181-1189.

Metyltetraprole is a member of the class of tetrazoles that is 1-methyl-4-phenyltetrazole in which the phenyl group has been substituted at positions 2 and 3 by [1-(p-chlorophenyl)-1H-pyrazol-3-yl]oxy}methyl and methyl groups, respectively. A quinone outside inhibitor, it is a fungicide that can be used to control a broad range of diseases, including Septoria leaf blotch in wheat. It has a role as an antifungal agrochemical and a quinone outside inhibitor. It is a member of tetrazoles, a pyrazole pesticide and a member of monochlorobenzenes.

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Quinone outside inhibitors (QoIs) are one of the major agricultural fungicide groups used worldwide. However, the development of resistance by different pathogenic species associated with specific mutation at the target gene site is becoming a critical issue for the sustainable use of QoIs. The authors aimed to design a novel QoI molecule to overcome the aforementioned issue. A rational approach to avoid steric hindrance between the QoI molecule and the mutated target site was successfully employed. The resulting compound, Metyltetraprole, is characterized by 3-substituted central ring with a tetrazolinone moiety, the key structure to retain potent activity against QoI-resistant mutants. Metyltetraprole is a promising new fungicide under commercial development, and its development in this study has paved the way to overcoming resistance to QoI fungicides.[1]

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This study aimed to overcome the qualitative resistance associated with QoI through the use of an innovative technology. Metyltetraprole is the first member of a new generation of QoIs, tetrazolinone fungicides. The use of a diverse sample collection, highly efficient microtiter test system, and careful investigation of the structure–activity relationship were key factors for success. As reported earlier, metyltetraprole shows stable efficacy in greenhouses and fields, even in the presence of G143A QoI-resistant mutants. Fungicide resistance is a growing threat to sustainable agriculture Despite the declining use of QoI, we believe that it is important to maintain the diversity of fungicides in spraying programs so that distinct fungicide classes can be mixed or alternated, to avoid the rapid development of resistance to a single class. Metyltetraprole is the first of a new innovative generation of QoIs and represents a new option for fungicide spraying programs. We hope that this report serves as a useful reference to researchers combating fungicide resistance.[1]

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In this study, we reported the unique profile of Metyltetraprole, showing stable antifungal activity and efficacy in both the greenhouse and field against QoI‐resistant disease pathogens while targeting the same Qo site. We believe metyltetraprole is the first molecule which overcomes the cross‐resistance among QoI fungicides in the disease management of cereal production, therefore it can be used as a new tool for the sustainable management of crop pathogens. We also expect that further novel compounds with a similar tetrazolinone structure can be discovered as new types of highly effective agricultural fungicides. Our findings contribute to ongoing efforts to minimize losses of economically important crops through improved management of relevant diseases. [2]

C19H17CLN6O2

396.83

396.11

1472649-01-6

89881183

Typically exists as solid at room temperature

4.6

0

5

5

28

585

0

CC1=C(C(=CC=C1)N2C(=O)N(N=N2)C)COC3=NN(C=C3)C4=CC=C(C=C4)Cl

XUQQRGKFXLAPNV-UHFFFAOYSA-N

InChI=1S/C19H17ClN6O2/c1-13-4-3-5-17(26-19(27)24(2)22-23-26)16(13)12-28-18-10-11-25(21-18)15-8-6-14(20)7-9-15/h3-11H,12H2,1-2H3

1-[2-[[1-(4-chlorophenyl)pyrazol-3-yl]oxymethyl]-3-methylphenyl]-4-methyltetrazol-5-one

metyltetraprole; Metyltetraprole [ISO]; Pavecto; 44WE6KNK7M; CHEBI:141152; 1-(2-((1-(4-Chlorophenyl)pyrazol-3-yl)oxymethyl)-3-methylphenyl)-4-methyltetrazol-5-one; 1-(2-(((1-(4-Chlorophenyl)-1H-pyrazol-3-yl)oxy)methyl)-3-methylphenyl)-1,4-dihydro-4-methyl-5H-tetrazol-5-one; ...; 1472649-01-6;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)