Article pubs.acs.org/JAFC
Cite This: J. Agric. Food Chem. 2017, 65, 10829−10835
Discovery of a New Fungicide Candidate through Lead Optimization of Pyrimidinamine Derivatives and Its Activity against Cucumber Downy Mildew Aiying Guan,†,‡ Mingan Wang,*,† Jinlong Yang,‡ Lizeng Wang,†,‡ Yong Xie,‡ Jie Lan,‡ and Changling Liu*,‡ †
Department of Applied Chemistry, China Agricultural University, Beijing 100193, People’s Republic of China State Key Laboratory of the Discovery and Development of Novel Pesticide, Shenyang Sinochem Agrochemicals R&D Company Ltd., Shenyang 110021, People’s Republic of China
‡
S Supporting Information *
ABSTRACT: Downy mildew is one of the most highly destructive of the diseases that cause damage to fruits and vegetables. Because of the continual development of resistance, it is important to discover new fungicides with different modes of action from existing fungicides for the control of downy mildew. This study is a continuation of our previous work on the novel pyrimidinamine lead compound, 9, and includes field trials for the identification of the optimal candidate. A new compound, 1c, was obtained, which gave a lower EC50 value (0.10 mg/L) against downy mildew than lead compound 9 (0.19 mg/L) and the commercial fungicides diflumetorim, dimethomorph, and cyazofamid (1.01−23.06 mg/L). Compound 1c displayed similar broad-spectrum fungicidal activity to compound 9 but better field efficacy than compound 9, cyazofamid, and flumorph. The present work indicates that pyrimidinamine compound 1c is a candidate for further development as a commercial fungicide for the control of downy mildew. KEYWORDS: downy mildew, development of resistance, unique mode of action, pyrimidinamine fungicide candidate
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INTRODUCTION Downy mildew is one of the most highly destructive of the diseases that cause serious damage to fruits, vegetables, and other economic crops.1−4 Along with an increase in the standard of living, the demand for a greater quality and quantity of fruits and vegetables increases daily. Consequently, the demand for relevant fungicides against downy mildew is being enhanced. The global market for fungicides against downy mildew was valued at around $1.5 billion.5 Because of the continual development of fungicide resistance, it is important to discover and develop new fungicides with different modes of action from existing fungicides for the control of downy mildew. As the Fungicide Resistance Action Committee (FRAC) reported, the pyrimidinamine fungicides, against which resistance has not been reported, possess a distinct mode of action: inhibiting complex I NADH oxido-reductase, a mode of action unique among commercialized fungicides, except for that of the pyrazole-MET1-like tolfenpyrad.6,7 The only commercial fungicide of this kind, diflumetorim, is not known to possess strong fungicidal activity against downy mildew.8 Thus, an opportunity is presented to discover and develop a pyrimidinamine fungicide for the control of downy mildew. Several research groups are also investigating this type of fungicide for the control of downy mildew.9−11 In our group, new pyrimidinamine derivatives containing an aryloxy pyridine moiety were discovered by employing the intermediate derivatization method (IDM), and an excellent lead compound, 9 (Figure 1), with an EC50 value of 0.19 mg/L against cucumber downy mildew in greenhouse was discovered.12 This study is a continuation of our previous study on novel © 2017 American Chemical Society
pyrimidinamine derivatives. We describe the further design of and SAR studies conducted around the lead compound 9 discovered previously, which include hit-to-lead optimization and field trials used to identify the optimal candidate (Figure 1).
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MATERIALS AND METHODS
All chemicals, such as the starting materials and reagents, were commercially available (Sinopharm Chemical Reagent Company Ltd., Shanghai, China) and used without further purification, except as indicated. Melting points were determined on a M-569 melting point apparatus (Büchi Labortechnik AG, Flawil, Switzerland) and are uncorrected. 1H NMR spectra were recorded with a Mercury 300 MHz spectrometer (Varian, Palo Alto, CA) with deuterochloroform as the solvent and tetramethylsilane (TMS) as the internal standard. Elemental analyses were determined on a MT-3CHN elemental analyzer (Yanaco, Kyoto, Japan). Mass spectra were acquired with an Accurate-Mass-Q-TOF MS 6520 system (Agilent Technologies, Milford, MA) equipped with an electrospray ionization (ESI) source. All plant and bacteria materials were obtained from the Agrochemical Discovery Department of Shenyang Sinochem Agrochemicals R&D Company Ltd. (Shenyang, China). The general synthetic methods for compounds 1a−1q, 2a−2c, 3a, and 3b are shown in Figures 2−4 and their structures are listed in Tables 1−3. The silica-gel chromatography was performed with a column of 254 × 26 mm i.d. using 100−140 mesh silica gel (Sinopharm Chemical Reagent Co. Ltd., Shanghai, China). Received: Revised: Accepted: Published: 10829
August 21, 2017 November 15, 2017 November 18, 2017 November 18, 2017 DOI: 10.1021/acs.jafc.7b03898 J. Agric. Food Chem. 2017, 65, 10829−10835
Article
Journal of Agricultural and Food Chemistry
Figure 1. An overview of the optimization design of new pyrimidinamine derivatives and the discovered potential fungicide candidate.
Figure 2. Synthetic route to compounds 1a−1q.
Figure 3. Synthetic route to compounds 2a−2c.
Figure 4. Synthetic route to compounds 3a and 3b. Synthesis of 5-Chloro-N-(2-(6-(4-chlorophenoxy)pyridin-3yl)ethyl)-6-(difluoromethyl)pyrimidin-4-amine, 1c, and General Procedure for Compounds 1a, 1b, and 1d−q. 4,5-Dichloro-6(difluoromethyl)pyrimidine (0.20 g, 1.0 mmol), prepared according to previous methods, was added to a solution of 2-(6-(4-chlorophenoxy)pyridin-3-yl)ethanamine (SM1) (0.25 g, 1.0 mmol) and potassium carbonate (0.21 g, 1.5 mmol) in 10 mL of DMF.12,13 Then the reaction mixture was heated to 80 °C for 2 h and monitored by TLC until the reaction was complete. The reaction mixture was poured into water and extracted with ethyl acetate. The organic phase was washed successively with water and saturated brine, dried, filtered, and evaporated under reduced pressure. The residue was purified via silicagel chromatography with ethyl acetate/60−90 °C petroleum ether (1:3, v/v) as eluent to obtain compound 1c as a colorless oil (Figure 2): 0.30 g (73%). 1H NMR (300 MHz, CDCl3): δ 2.93 (t, J = 6.9 Hz, 2H, CH2CH2NH), 3.80 (q, 2H, CH2CH2NH), 5.72 (bs, 1H, NH), 6.72 (t, 1JHF = 54 Hz, 1H, CHF2), 6.92 (d, J = 8.4 Hz, 1H, pyridine-3-
H), 7.07 (d, J = 6.9 Hz, 2H, Ph-2,6-2H), 7.35 (d, J = 6.9 Hz, 2H, Ph3,5-2H), 7.58 (dd, J = 8.4, 2.7 Hz, 1H, pyridine-4-H), 8.03 (s, 1H, pyridine-6-H), 8.56 (s, 1H, pyrimidine-2-H). Anal. Calcd (%) for C18H14Cl2F2N4O: C, 52.57; H, 3.43; N, 13.62. Found: C, 52.52; H, 3.47; N, 13.58. HRMS m/z 410.0510 [M + H]+ (calcd [M + H]+ 410.0513). Synthesis of Ethyl (5-Chloro-6-methylpyrimidin-4-yl)(2-(6(4-chlorophenoxy)pyridin-3-yl)ethyl)carbamate, 2c, and General Procedure for Compounds 2a and 2b. Triphosgene (0.45 g, 1.5 mmol) was added to a solution of lead compound 9 (0.37 g, 1.0 mmol), prepared as previously described,12 and triethylamine (0.20 g, 2.0 mmol) in 10 mL of toluene. Then, the reaction mixture was refluxed for 4 h and monitored by TLC until the reaction was complete. The mixture was poured into water and stirred for 0.5 h. The organic phase was successively washed with water and saturated brine, dried, filtered, and evaporated under reduced pressure. The 10830
DOI: 10.1021/acs.jafc.7b03898 J. Agric. Food Chem. 2017, 65, 10829−10835
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Journal of Agricultural and Food Chemistry
follows: Seeds (cucumber: Cucumis sativus L.; wheat: Triticum aestivum L.; and maize: Zea mays L.) were grown to the one-leaf, two-leaf, and two- to three-leaf stages, and then the test solutions were sprayed on the host plants with a homemade sprayer. After 24 h, the leaves of the host plants were inoculated with sporangial suspensions of the fungi CDM (Pseudoperonospora cubensis), WPM (Erysiphe graminis), and SCR (Puccinia polysora Underw), cultured by Shenyang Sinochem Agrochemicals R&D Co. Ltd. (Shenyang, China), each at a concentration of 5 × 105 spores/mL using a PS289 Procon Boy WA double action 0.3 mm airbrush (GSI, Tokyo, Japan). The cucumber plants were stored in a humidity chamber (24 ± 1 °C, RH >95%, dark) and then transferred to a greenhouse (18−30 °C, RH >50− 60%) 24 h after infection. Three replicates were carried out. The activity of each compound was estimated by visual inspection after 7 d, and the screening results were reported in the range of 0% (no control of the fungus) to 100% (complete control of the fungus). The inhibitory activity (%) was estimated as
residue was dissolved in 10 mL of dichloromethane to obtain a solution of SM2 in dichloromethane for use in the next step, directly. The above solution of SM2 (1.0 mmol) was added to a solution of amine or alcohol (R4−H) (1.0 mmol) and triethylamine (0.21 g, 1.5 mmol) in 10 mL of dichloromethane. Then, the reaction mixture was stirred at room temperature for 2 h and monitored by TLC until the reaction was complete. The mixture was poured into water and stirred for 0.5 h. The organic phase was successively washed with water and saturated brine, dried, filtered, and evaporated under reduced pressure. The residue was purified via silica-gel chromatography with ethyl acetate/60−90 °C petroleum ether (1:2, v/v) as eluent to obtain compound 2c as a pale yellow oil (Figure 3): 0.28 g (63%).12,14 1H NMR (300 MHz, CDCl3): δ 1.23 (t, J = 7.5 Hz, 3H, OCH2CH3), 2.64 (s, 1H, CH3), 2.90−2.98 (m, 2H, CH2CH2NH), 4.07 (t, J = 6.9 Hz, 2H, CH2CH2NH), 4.19 (m, 2H, OCH2CH3), 6.82 (d, J = 8.4 Hz, 1H, pyridine-3-H), 7.04 (d, J = 8.4 Hz, 2H, Ph-2,6-2H), 7.33 (d, J = 8.4 Hz, 2H, Ph-3,5-2H), 7.56 (dd, J = 8.4, 2.4 Hz, 1H, pyridine-4-H), 7.98 (s, 1H, pyridine-6-H), 8.79 (s, 1H, pyrimidine-2-H). Anal. Calcd (%) for C21H20Cl2N4O3: C, 56.39; H, 4.51; N, 12.53. Found: C, 56.33; H, 4.56; N, 12.50. HRMS m/z 446.0908 [M + H]+ (calcd [M + H]+ 446.0912). Synthesis of 5-Chloro-N-(4-(4-chlorophenoxy)phenethyl)-6ethylpyrimidin-4-amine, 3b, and General Procedure for Compound 3a. (4-Chlorophenyl)boronic acid (3.74 g, 24.0 mmol), ground 4 Å molecular sieve powder, anhydrous copper acetate (3.82 g, 21.0 mmol), triethylamine (10.1 g, 0.1 mol), and pyridine (7.9 g, 0.1 mol) were added successively to a solution of tertbutyl (4-hydroxyphenethyl)carbamate (SM3) (5.69 g, 24.0 mmol) in 50 mL of dichloromethane. Then, the reaction mixture was stirred overnight at room temperature and monitored by TLC until the reaction was complete. The mixture was filtered; the filtrate was evaporated under reduced pressure. The residue was suspended in ethyl acetate, and the organic phase was washed with saturated brine, dried, filtered, and evaporated under reduced pressure. The residue was purified via silica-gel chromatography with ethyl acetate/60−90 °C petroleum ether (1:2, v/v) as eluent to obtain intermediate SM4 as a white solid (Figure 4): 5.41 g (65%).12,14 Concd HCl (12 mL) was added to a solution of SM4 (3.80 g, 10 mmol) in 50 mL of ethyl acetate. Then, the reaction mixture was stirred for 4 h at room temperature and monitored by TLC until the reaction was complete. The mixture was filtered to obtain intermediate SM5 as a white solid (Figure 4): 2.58 g (91%).12,14 4,5-Dichloro-6-ethylpyrimidine (0.14 g, 1.0 mmol), prepared according to reported methods, was added to a solution of SM5 (0.28 g, 1.0 mmol) and potassium carbonate (0.21 g, 1.5 mmol) in 10 mL of DMF.12,15 Then, the reaction mixture was heated to 80 °C for 2 h and monitored by TLC until the reaction was complete. The reaction mixture was poured into water and extracted with ethyl acetate. The organic phase was washed successively with water and saturated brine, dried, filtered, and evaporated under reduced pressure. The residue was purified via silica-gel chromatography with ethyl acetate/60−90 °C petroleum ether (1:2, v/v) as eluent to obtain compound 3b as a reddish brown solid (Figure 4): 0.32 g (82%), mp 84.7 °C. 1H NMR (300 MHz, CDCl3): δ 1.26 (t, J = 7.5 Hz, 3H, CH2CH3), 2.78 (q, J = 7.5 Hz, 2H, CH2CH3), 2.92 (t, J = 6.9 Hz, 2H, CH2CH2NH), 3.75 (q, 2H, CH2CH2NH), 5.45 (bs, 1H, NH), 6.84− 7.00 (m, 4H, middle-Ph-4H), 7.20 (d, J = 8.4 Hz, 2H, Ph-2,6−2H), 7.29 (d, J = 8.4 Hz, 2H, Ph-3,5-2H), 8.44 (s, 1H, pyrimidine-2-H). Anal. Calcd (%) for C19H17Cl2N3O: C, 60.98; H, 4.58; N, 11.23. Found: C, 61.00; H, 4.51; N, 11.27. HRMS m/z 373.0755 [M + H]+ (calcd [M + H]+ 373.0749). Fungicidal Assay. Fungicidal Assays in a Greenhouse. Each of the test compounds (4 mg) was first dissolved in 5 mL of acetone/ methanol (1:1, v/v), and then 5 mL of water containing 0.1% Tween 80 was added to generate 10 mL stock solutions of concentration 400 mg/L. Serial test solutions were prepared by diluting the above solution (testing range 0.05−400 mg/L). Evaluations of the fungicidal activity of the synthesized compounds against cucumber downy mildew (CDM), wheat powdery mildew (WPM), and southern corn rust (SCR) in vivo were performed as
inhibitory activity (%) = [(viability of the blank control − viability of the treated plant) /viability of the blank control] × 100% The ED50 values were calculated by Duncan’s new multiple-range test (DMRT) using DPS version 14.5. In vitro evaluations of fungicidal activity against rice blast (RB, Pyricularia grisea) and cucumber gray mold (CGM) were conducted as follows:12 A high-throughput screening (HTS) method was used. Under sterile conditions, the tested compounds were diluted to the given concentrations and added into the wells of 96-well culture plates, and then a spore suspension of one of the aforementioned fungi was dropped into the cells. Water was set as the blank control, and three replicates were set for each treatment. The treated culture plates were placed in an incubator (temperature: 24−26 °C). After 24 h, the activities of the tested compounds were evaluated on the basis of germination of the pathogen determined under the microscope. The fungal inhibition rate (%) was estimated as fungal inhibition rate (%) = [(colony diameters of the blank − colony diameters of the treated well) /colony diameters of the blank] × 100%
The fungicidal-test results for compounds 1a−1q, 2a−2c, 3a, and 3b against CDM are listed in Tables 1−3. The test results for compound 1c against WPM, SCR, RB, and CGM are shown in Table 4. Field Trials. Field trials were conducted in Liaoning province, Shenyang (25 m2, at the five-leaf stage).16 The test solution was sprayed on the host plant (Cucumis sativus L.) with a WS-15D knapsack electric sprayer (WishSprayer, Shandong, China). Two spays were carried out with an interval period of 6−7 d. Seven days after the second treatment, the incidence of disease spots in each plot was investigated by randomly selecting 4 samples/plot and 8 plants/ sample. The incidence of the whole plant was recorded by counting the number of diseased leaves and determining the incidence grade. The grade scales were divided into six levels (ratio of leaf-spot area to leaf area): level 0 (no disease), level 1 (51%). The disease index (DI, %) was calculated as DI (%) = ⎡⎣∑ (number of diseased leaves × relative level) /(total number of investigated leaves × the highest level)⎤⎦ × 100%
The inhibitory activity (%) was calculated as 10831
DOI: 10.1021/acs.jafc.7b03898 J. Agric. Food Chem. 2017, 65, 10829−10835
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Journal of Agricultural and Food Chemistry
Table 1. Chemical Structures and Fungicidal Activity of Target Compounds (1a−1q) against Cucumber Downy Mildew
a
Confidence limit. bThe value could not be measured accurately.
modifications on R1, R2, and R3 of the pyrimidine ring (Table 1). For the modification on R1 of the pyrimidine ring when R2 and R3 were fixed as chlorine and hydrogen atoms, respectively, the carbon chain on R1 was extended from methyl to ethyl and propyl, resulting in compounds 1a and 1b. Although these two compounds displayed relatively high activity with EC50 values of 2.65 and 1.99 mg/L, respectively, they were still much less active compared with lead compound 9, which has an EC50 of 0.19 mg/L. However, considering the unique properties of fluorine,17−20 we introduced two fluorine atoms (as CHF2) to obtain compound 1c. This compound exhibited excellent fungicidal activity (EC50 = 0.10 mg/L), which was not only higher than that of compound 9 but also higher than those of the commercial fungicides cyazofamid, dimethomorph, and diflumetorim. Encouraged by this finding, we added one more fluorine atom (compound 1d, R1 = CF3), but this compound was less active (EC50 = 10.75 mg/L). For
inhibitory activity (%) = [(DI of the blank control − DI of the treated plot) /DI of the blank control] × 100%
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RESULTS AND DISCUSSION Synthesis. According to the schemes shown in Figures 2−4, 21 compounds were synthesized with yields of 55−82%. Chemical structures of compounds 1a−1q, 2a−2c, 3a, and 3b are shown in Tables 1−3. The synthesized compounds were characterized by 1H NMR and elemental-analysis HRMS. All data were consistent with the assigned structures. Structure−Activity Relationships (SAR). Modifications on the Pyrimidine Ring. First, to investigate if the substituents on the pyrimidine ring play an important role in the improvement of fungicidal activity, we carried out some 10832
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Journal of Agricultural and Food Chemistry the modification on R2 of the pyrimidine ring when R1 and R3 were fixed as chlorine and hydrogen atoms, respectively, changes around R2 were carried out to explore the electronic properties and spatial characteristics of various substituents. Compound 1e (R2 = H) gave an EC50 of 7.52 mg/L. When the hydrogen atom was replaced with a halogen atom, such as chlorine or bromine, both of which possess lower electronegativity (compounds 1f and 1g, respectively), there was no improvement in activity (EC50 = 9.92 and 10.57 mg/L, respectively). Further replacement of the hydrogen atom with a higher-electron-withdrawing aldehyde group (compound 1h) led to sharply lower activity, with EC50’s ranging from 100 to 400 mg/L. When we replaced the hydrogen atom with typical electron-donating groups, resulting in compounds 1i (CH3), 1j (OCH3), and 1k (NH2), the bioassay results showed that these three compounds were all less efficacious (EC50 = 14.15, 9.09, and 20.50 mg/L, respectively). It appeared that changes to the electronic properties of the R2 substituent did not improve bioactivity. Changes in the spatial characteristics of the R2 substituent (compound 1m with an R2 of 1,3-dioxolane-2-yl and compound 1n with an R2 of 4-Br-phenyl) also did not improve bioactivity. However, when R1 and R2 formed a thienyl ring (compound 1o) or phenyl ring (compound 1p) based on the pyrimidine ring, there were large differences in bioactivity (compound 1o had a high EC50 of 16.24 mg/L, and compound 1p had a low EC50 of 0.31 mg/L), suggesting that R1 and R2 are the key positions in this class of compounds for elaborating fungicidal activity. Finally, for the modification on R3 of the pyrimidine ring when R1 and R2 were fixed as chlorine atoms, compounds 1f (R3 = H, EC50 = 9.92 mg/L) and 1q (R3 = CH3, EC50 = 5.10 mg/L) demonstrated that R3 does not make an obvious contribution to enhancing bioactivity.
Table 3. Chemical Structures and Fungicidal Activity of Target Compounds (3a and 3b) against Cucumber Downy Mildew
a
a
R4
EC50 (mg/L)
95% da
9, lead compound 2a 2b 2c
N(CH3)2 NHC6H5 OC2H5
0.19 10.42 11.13 0.71
0.14−0.26 5.61−19.36 7.17−17.30 0.17−3.05
R1
X
EC50 (mg/L)
95% da
9, lead compound 3a 1a 3b
CH3 CH3 C2H5 C2H5
N CH N CH
0.19 8.62 2.65 6.25−25
0.14−0.26 3.26−22.80 1.61−4.35 b
Confidence limit. bThe value could not be measured accurately.
ring in place of a phenyl ring would produce better activity,21 we synthesized compounds 3a and 3b (X = CH). As predicted, the phenyl-ring analogues were much less efficacious than the corresponding pyridinyl-ring compounds, 9 and 1a (X = N), respectively (Table 3). So far, compound 1c has shown the best fungicidal activity, with an EC50 value of 0.10 mg/L, which is nearly two times better than that of lead compound 9 and significantly better than those of the tested commercial compounds. We next examined the fungicidal spectrum and field-trial potential of our new fungicide candidate, compound 1c. Fungicidal-Spectrum Comparison Test of Compounds 1c and 9. In order to further evaluate the potential of compound 1c, the fungicidal-spectrum test was carried out and compared with the spectrum of the initial lead compound, 9. The results showed that, in addition to fungicidal activity against the cucumber downy mildew control, compounds 1c and 9 also had fungicidal activity against wheat powdery mildew (WPM), southern corn rust (SCR), rice blast (RB), and cucumber gray mold (CGM) (Table 4). Compound 1c displayed good activity against WPM, ranging from 100% at 400 mg/L to 70% at 25 mg/L. Against SCR, compound 1c showed similarly good fungicidal activity, with 100% control at 400 mg/L and 65% at 25 mg/L. Against RB and CGM, compound 1c gave an 80% inhibition rate for both species at 25 mg/L. Although compound 9 displayed slightly better activity than compound 1c against WPM and SCR at 25 mg/L, overall these two compounds exhibited very similar fungicidal spectra. These results suggest that the pyrimidinamine compound 1c possesses a broad spectrum of fungicidal activity, which is worthy of being developed further. Field Trials of Compounds 1c and 9. A field trial is an important assessment indicator in making a strategic decision on whether a bioactive compound meets the requirements for commercial development as a pesticide. The results of field trials in 2017 with compounds 1c and 9 against CDM demonstrated that 10% SC formulations of both compounds displayed excellent efficacies, which were superior to those of the two commercial compounds cyazofamid and flumorph. Compounds 1c and 9 demonstrated 99.5 and 96.0% control efficacy, respectively, each at a concentration of 100 mg/L, whereas cyazofamid at 100 mg/L and flumorph at 200 mg/L demonstrated 81.2 and 67.5%, respectively. Even at 50 mg/L, the field efficacies of compounds 1c and 9 were very good (98.5 and 95.5% control, respectively) (Table 5). Generally speaking, compared with compound 9, compound 1c, which had a much lower EC50 value in the greenhouse tests, was slightly more active against CDM in the field environment.
Table 2. Chemical Structures and Fungicidal Activity of Target Compounds (2a−2c) against Cucumber Downy Mildew
compound
compound
Confidence limit.
Modifications on the Imine−Amide Linkage. Using the original lead compound, 9, as a starting point, we next turned our attention to modifications in the side-chain nitrogen atom by forming a urea or carbamate function (Table 2). Urea compounds 2a and 2b gave a significant decrease in fungicidal activity (EC50 values of 10.42 and 11.13 mg/L, respectively), versus the initial lead compound, 9, (0.19 mg/L); however, carbamate compound 2c approached the activity of lead compound 9 (EC50 = 0.71 mg/L). Although it was more active than the tested controls, it was still much less efficacious than lead compound 9. Replacement of the Pyridinyl Ring with a Phenyl Ring. To confirm our initial design concept that introducing a pyridinyl 10833
DOI: 10.1021/acs.jafc.7b03898 J. Agric. Food Chem. 2017, 65, 10829−10835
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Journal of Agricultural and Food Chemistry Table 4. Fungicidal-Spectrum Test of Compounds 1c and 9 (% Control) WPMc
c
SCRd
RBe
CGMf
compound
400 mg/L
100 mg/L
25 mg/L
400 mg/L
100 mg/L
25 mg/L
25 mg/L
25 mg/L
1c 9
100 100
80 90
75 80
100 100
85 90
65 75
80 80
80 80
WPM: wheat powdery mildew. dSCR: southern corn rust. eRB: rice blast. fCGM: cucumber gray mold. germplasm (Cucumis sativus L.) genetic diversity assessment using ISSR markers. Crop Prot. 2014, 60, 56−61. (2) Romanazzi, G.; Mancini, V.; Feliziani, E.; Servili, A.; Endeshaw, S.; Neri, D. Impact of alternative fungicides on grape downy mildew control and vine growth and development. Plant Dis. 2016, 100, 739− 748. (3) Sireesha, Y.; Velazhahan, R. Biological control of downy mildew of maize caused by Peronosclerospora sorghi under environmentally controlled conditions. J. Appl. Nat. Sci. 2016, 8, 279−283. (4) Cohen, Y.; Ben-Naim, Y. Nocturnal fanning suppresses downy mildew epidemics in sweet basil. PLoS One 2016, 11, e0155330. (5) Mcdougall, P. Oct 2015; crops section − 2014 Market. Phillips McDougall AgriService: Midlothian, UK, p 18. (6) FRAC (Fungicide Resistance Action Committee) Code List ©*2016, fungicides sorted by mode of action (including FRAC code numbering). www.frac.info (accessed Aug 10, 2017). (7) Walter, H. NADH Inhibitors (Complex I). In Modern Crop Protection Compounds; Kraemer, W., Schirmer, U., Jeschke, P., Witschel, M., Eds.; Wiley: Weinheim, Germany, 2012; pp 670−891. (8) Liu, C. L.; Wang, L. Z.; Song, Y. Q.; Sun, X. F.; Lan, J.; Zhang, J. B.; Feng, C.; Chai, B. S.; Sun, Q.; Wang, B.; Liu, S. W. Phenoxyl pyrimidinamine compound and application. CN Patent 103772294, May 7, 2014. (9) Grammenos, W.; Craig, I. R.; Boudet, N.; Mueller, B.; Dietz, J.; Lauterwasser, E. M. W.; Lohmann, J. K.; Montag, J.; Vrettou-Schultes, M. Fungicidal pyrimidine compounds. PCT Int. Appl. WO 2013113778, Aug 8, 2013. (10) Brewster, W.; Klittich, C.; Yao, C. L.; Zhu, Y. M. 5,8-Difluoro-4(2-(4-(heteroaryloxy)-phenyl)ethylamino)quinazolines and their use as agrochemicals. PCT Int. Appl. WO 2010025451, Mar 4, 2010. (11) Brewster, W., Klittich, C., Yao, C. L., Zhu, Y. M. 5,8-Difluoro-4(2-(4-(heteroaryloxy)-phenyl)ethylamino)quinazolines and their use as agrochemicals. PCT Int. Appl. WO 2010025451, June 17, 2010. (12) Guan, A. Y.; Liu, C. L.; Chen, W.; Yang, F.; Xie, Y.; Zhang, J. B.; Li, Z. N.; Wang, M. A. Design, synthesis, and structure−activity relationship of new pyrimidinamine derivatives containing an aryloxy pyridine moiety. J. Agric. Food Chem. 2017, 65, 1272−1280. (13) Dreikorn, B. A.; Suhr, R. G.; Johnson, P. L. Pyridylethoxy-, pyridylethylamino-, and pyridylpropyl-derivatives of quinoline and quinazoline as insecticides and fungicides. PCT Int. Appl. WO 9404527, Mar 3, 1994. (14) Zhang, J. L.; Chen, C. X.; Lei, J. H. A process for preparing fungicide prochloraz. CN Patent 101402608, Nov 2, 2011. (15) Jin, Y.; Wan, Z. H.; Zhang, Q. Compounds. US Patent 8637536, Jan 28, 2014. (16) Si, N. G.; Liu, J. L.; Li, Z. N.; Chen, L.; Zhang, D. M.; Wang, L. Z. Study on bioactivity and application of fungicide enestroburin (I) − cucumber downy mildew. Chin. J. Pestic. 2003, 10, 36−38. (17) Guan, A. Y.; Liu, C. L.; Huang, G.; Li, H. C.; Hao, S. L.; Xu, Y.; Xie, Y.; Li, Z. N. Synthesis and fungicidal activity of fluorine containing chlorothalonil derivatives. J. Fluorine Chem. 2014, 160, 82− 87. (18) Guan, A. Y.; Qin, Y. K.; Wang, J. F.; Li, B. Synthesis and insecticidal activity of novel dihalopropene derivatives containing benzoxazole moiety: a structure - activity relationship study. J. Fluorine Chem. 2013, 156, 120−123. (19) Smart, B. E. Fluorine substituent effects (on bioactivity). J. Fluorine Chem. 2001, 109, 3−11. (20) Maienfisch, P.; Hall, R. G. The importance of fluorine in the life science industry. Chimia 2004, 58, 93−99.
Table 5. Field-Trial Results of Compounds 1c and 9 against Cucumber Downy Mildew (Shenyang, 2017) control (%) compound
mg/L
I
II
III
IV
mean
10% 1c SC
50 100 200 50 100 200 100 200 disease index
98 100 100 95 96 99 82 68 59
99 99 100 96 95 99 80 67 62
99 100 100 97 97 99 82 68 64
98 99 100 94 96 100 81 67 61
98.5 99.5 100.0 95.5 96.0 99.3 81.2 67.5 61.5
10% 9 SC
10% cyazofamid SC 20% flumorph WP CK
In summary, through comprehensive design and an SAR study, a promising pyrimidinamine derivative, compound 1c, was identified. This compound showed high fungicidal activity (EC50 = 0.10 mg/L) in greenhouse tests, a broad fungicide spectrum, and excellent field efficacy, which was slightly better than that of lead compound 9 and much better than those of the tested commercial fungicides. Compound 1c is considered to be a promising candidate for commercial development as a fungicide against downy mildew, and it also should be helpful in managing resistance. Further studies, including continuous structure optimization around 1c, are in progress.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.7b03898. Characterization of compounds 1a, 1b, 1d−1q, 2a, 2b, and 3a (PDF)
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AUTHOR INFORMATION
Corresponding Authors
*Tel.: +86-(0)10-62734093, E-mail:
[email protected] (M.W.). *Tel.: +86-(0)24-85869078, Fax: +86-(0)24-85869137, E-mail:
[email protected] (C.L.). ORCID
Aiying Guan: 0000-0001-9134-9280 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank Dr. Mark Dekeyser (Canada) for assistance with revision of the manuscript. REFERENCES
(1) Innark, P.; Ratanachan, T.; Khanobdee, C.; Samipak, S.; Jantasuriyarat, C. Downy mildew resistant/susceptible cucumber 10834
DOI: 10.1021/acs.jafc.7b03898 J. Agric. Food Chem. 2017, 65, 10829−10835
Article
Journal of Agricultural and Food Chemistry (21) Pennington, L. D.; Moustakas, D. T. The necessary nitrogen atom: a versatile high-impact design element for multiparameter optimization. J. Med. Chem. 2017, 60, 3552−3579.
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DOI: 10.1021/acs.jafc.7b03898 J. Agric. Food Chem. 2017, 65, 10829−10835
