Research Article Received: 27 November 2013
Revised: 24 January 2014
Accepted article published: 18 February 2014
Published online in Wiley Online Library: 1 April 2014
(wileyonlinelibrary.com) DOI 10.1002/ps.3765
Synthesis and antifungal activity of 3-aryl-1,2,4-triazin-6-one derivatives W John Owen,* Michael T Sullenberger, Michael R Loso, Kevin G Meyer and Thomas J Slanec Abstract BACKGROUND: As a result of resistance development in many plant-pathogenic fungi to agricultural fungicides, there is an ongoing need to discover novel antifungal chemistries to help sustain efficient crop production. A fungicide screening program identified 3-phenyl-1-(2,2,2-trifluoroethyl)-1,2,4-triazin-6(1H)-one (5) as a promising new starting point for further activity optimization. A series of analogs were designed, prepared and evaluated in growth inhibition assays using four plant-pathogenic fungi. RESULTS: Thirty nine analogs (compounds 5 to 43) were prepared to explore structure–activity relationships at R1 and R2 , and all targeted structures were characterized by 1 H NMR and MS. All compounds were in vitro tested against three ascomycetes [Leptosphaeria nodorum, Magnaporthe grisea and Zymoseptoria tritici (syn. Mycosphaerella graminicola)] and one basidiomycete (Ustilago maydis) pathogen. When R2 was trifluoroethyl, fungicidal activity was enhanced by a single electron-withdrawing substitution, such as Br, Cl and CF3 in the 3-position at R1 (compounds 9, 10 and 12), of which the 3-bromo compound (10) was the most active (EC50 = 0.08, averaged across four pathogens). More subtle activity improvement was found by addition of a second halogen substituent in the 4-position, with the 3-Br-4-F analog (20) being the most active against the commercially important cereal pathogen Z. tritici. Replacement of the R2 haloalkyl group with benzyl, alkyl (e.g. n-butyl, i-butyl, n-pentyl) and, particularly, CH2 -cycloalkyls (e.g. CH2 -cyclopropyl, CH2 -cyclobutyl) resulted in further activity enhancements against the ascomycete fungi, but was either neutral or detrimental to activity against U. maydis. One of the most active compounds in this series (41) gave control of Z. tritici, with an EC50 of 0.005 ppm, comparable with that of the commercial strobilurin fungicide azoxystrobin (EC50 0.002 ppm). CONCLUSIONS: The present work demonstrated that the 3-phenyl-1,2,4-triazin-6-ones are a novel series of compounds with highly compelling levels of antifungal activity against agriculturally relevant plant-pathogenic fungi. © 2014 Society of Chemical Industry Keywords: fungicide; triazinone; structure–activity relationship; Zymoseptoria tritici; Leptosphaeria nodorum; Magnoporthe grisea; Ustilago maydis
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INTRODUCTION
Pest Manag Sci 2015; 71: 83–90
∗
Correspondence to: W John Owen, Dow AgroSciences, 9330 Zionsville Rd, Indianapolis, IN 46268-1054, USA. E-mail: [email protected] Dow AgroSciences, Indianapolis, IN, USA
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The use of broad-spectrum fungicides to protect crops from epidemics of plant pathogens is an important grower practice towards maintaining high yields and quality of harvested products. However, many plant pathogens have the propensity for developing resistance to fungicides, particularly to those classes of compounds that act at a single defined target site.1 – 5 For delaying onset of resistance development, it is frequently recommended that fungicides be applied in mixtures or in alternation with products of different modes of action (MOA), and that the number and frequency of applications per growing season be kept to a minimum.6 – 8 In spite of such practices, resistant and less sensitive isolates can continue to propagate and accumulate within a crop to a point at which fungicide performance becomes visibly diminished. This has been well exemplified in recent years in the European cereals market, where resistance of Zymoseptoria tritici, the causative agent of wheat leaf blotch, to the strobilurin fungicide class has become widespread.9 Z. tritici isolates carrying a variety of mutations in the CYP51 gene encoding the sterol C14-demethylase target of the azole fungicides have also
emerged and are causing significant sensitivity shifts among European populations.10 – 16 Cases such as these emphasize that continued research to identify new active chemistries with novel mechanisms of action and a broad antifungal spectrum is a critical aspect of resistance management.17 With this in mind, the ongoing fungicide screening program at Dow AgroSciences identified 3-phenyl-1,2,4-triazin-6-ones (Fig. 1) as having antifungal activity against a number of agriculturally relevant plant pathogens in in vitro assays. Subsequently, a synthesis program to optimize the biological activity of this new class of fungicides was initiated. The present paper describes the synthesis of a particular series of 1,2,4-triazin-6-ones, in which the R1 and R2 components were varied, and compares their in vitro antifungal activities against the plant pathogens Leptosphaeria nodorum, Z. tritici, Magnaporthe grisea and Ustilago maydis.
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N N R1
N
R2
Figure 1. Core structure of the 3-phenyl-1,2,4-triazin-6-ones.
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MATERIALS AND METHODS
2.1 General All starting materials and reagents were commercially available and used without further purification. Melting points were determined on a Thomas-Hoover™ melting point apparatus and were uncorrected. NMR spectra were recorded with a Varian 300 MHz spectrometer in CDCl3 with TMS as the internal standard. Electrospray ionization mass spectra (ESIMS) were recorded with a Water’s Micromass ZQ high-performance liquid chromatograph–mass spectrometer (HPLC-MS) or a Water’s Acquity ultraperformance liquid chromatograph–mass spectrometer (UPLC-MS). Infrared spectra (IR) were recorded with a Thermo Scientific Nicolet 6700 FT-IR instrument. 2.2 Synthesis of the title compounds 5 to 43 The general synthetic route for the title compounds is shown in Scheme 1. Representative procedures are given below, and reaction yields were not optimized. New compounds were characterized and verified by 1 H NMR, MS, melting point, and IR for oils and low-melting solids. Many of the structures reported herein were disclosed previously in International Patent Application WO2006/119400A2.18 2.3 Representative procedure for the synthesis of 3-(3-bromo-4-fluorophenyl)-1-(cyclopropylmethyl)-1,2, 4-triazin-6(1H)-one (38) 2.3.1 Synthesis of ethyl 3-bromo-4-fluorobenzimidate hydrochloride (2, R1 = 3-bromo-4-fluoro) To a solution of 3-bromo-4-fluorobenzonitrile (10 g, 50 mmol) in DCM (56 mL) was added ethanol (4.37 mL, 75 mmol), and the mixture was cooled to −5 ∘ C. Dry HCl gas was bubbled in for 1 h, and the resulting mixture was left in the refrigerator for 1 week. The mixture was then added at a rapid dropwise rate to hexanes (300 mL), with stirring, and the resulting solids were collected by filtration and washed with hexanes (100 mL) and ether (100 mL). The solid material was collected and dried under vacuum at room temperature for 3 h to afford 12.6 g (89%) of the title compound as a white solid, mp = 127–128 ∘ C. 1 H NMR (300 MHz, CDCl3 ) 𝛿 12.78 (bs, 1H), 8.67–8.62 (m, 1H), 8.54 (dd, J = 6.3, 2.4 Hz, 1H), 7.34 (t, J = 8.7 Hz, 1H), 4.96 (q, J = 6.9 Hz, 2H), 1.65 (t, J = 7.2 Hz, 3H). ESIMS m/z 248 (M + H)+ .
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2.3.2 Synthesis of benzyl 2-{[(3-bromo-4-fluorophenyl) (ethoxy)methylene]amino}acetate (3, R1 = 3-bromo-4-fluoro) To a suspension of 3-bromo-4-fluorobenzimidate hydrochloride (6.8 g, 24.0 mmol) in diethyl ether (50 mL) was added a saturated aqueous solution of NaHCO3 (50 mL). The mixture was allowed to stir for 5 min and the layers were separated. The ether layer was dried (MgSO4 ), filtered and concentrated in vacuo. The resulting residue was diluted with DCM (80 mL), and solid benzyl glycine p-toluenesulfonic acid salt (8.1 g, 24.0 mmol) was added in one portion. After stirring at room temperature overnight, the white suspension was diluted with ether and water. The layers were
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separated, and the organic layer was dried (MgSO4 ), filtered and concentrated in vacuo to afford 8.9 g (95%) of the title compound as a pink liquid. The residue was carried on without further purification. 1 H NMR (300 MHz, CDCl3 ) 𝛿 7.57 (dd, J = 6.4, 2.1 Hz, 1H), 7.35 (s, 5H), 7.26 (ddd, J = 8.5, 4.6, 2.0 Hz, 1H), 7.11 (t, J = 8.4 Hz, 1H), 5.16 (s, 2H), 4.30 (q, J = 7.1 Hz, 2H), 4.10 (s, 2H), 1.35 (t, J = 7.1 Hz, 3H). ESIMS m/z 394.2 (M + H)+ . 2.3.3 Synthesis of 3-(3-bromo-4-fluorophenyl)-1(cyclopropylmethyl)-4,5-dihydro-1,2,4-triazin-6(1H)-one (4, R1 = 3-bromo-4-fluoro and R2 = cyclopropylmethyl) To a solution of cyclopropylmethylhydrazine hydrochloride19 (1.0 M in EtOH, 3.3 mL, 3.3 mmol) was added triethylamine (1.1 mL, 7.5 mmol). After stirring for 5 min, a solution of 2-{[(3-bromo-4-fluorophenyl)(ethoxy)methylene]amino}acetate (1.18 g, 3.0 mmol) in EtOH (3 mL) was added, and the reaction mixture was heated to 40 ∘ C for 3 h. Upon cooling, the mixture was diluted with EtOAc and washed with water. The organic extract was dried (MgSO4 ), filtered and concentrated in vacuo. The resulting residue was dissolved in DCM (1 mL) followed by hexanes (50 mL) to give a red solid. The solid was collected via filtration to afford 392 mg (40%) of the title compound as a red solid, mp = 109–113 ∘ C. 1 H NMR (300 MHz, CDCl3 ) 𝛿 7.90 (d, J = 6.6 Hz, 1H), 7.61 (bs, 1H), 7.17 (t, J = 8.1 Hz, 1H), 5.06 (bs, 1H), 4.10 (bs, 2H), 3.65 (d, J = 6.9 Hz, 2H), 1.28 (m, 1H), 0.57–0.48 (m, 2H), 0.38 (m, 2H). ESIMS m/z 327.9 (M + H)+ . 2.3.4 Synthesis of 3-(3-bromo-4-fluorophenyl)-1(cyclopropylmethyl)-1,2,4-triazin-6(1H)-one (38) 2,3-Dichloro-5,6-dicyano-p-quinone (DDQ) (115 mg, 0.51 mmol) was added to a solution of 3-(3-bromo-4-fluorophenyl)-1(cyclopropylmethyl)-4,5-dihydro-1,2,4-triazin-6(1H)-one (150 mg, 0.46 mmol) in ethyl acetate (4.6 mL), and the resulting mixture was heated at reflux for 1 h. Upon cooling, the reaction mixture was concentrated in vacuo, and the resulting crude material was diluted with DCM (30 mL) and washed with saturated aqueous Na2 CO3 (2 × 30 mL). The organic extract was dried (MgSO4 ), filtered and concentrated in vacuo. The crude residue was filtered through a small plug of silica gel (DCM as eluent) and concentrated in vacuo to afford 111 mg (74%) of the title compound as a yellow solid, mp = 76–81 ∘ C. 1 H NMR (300 MHz, CDCl3 ) 𝛿 8.45 (s, 1H), 8.37 (dd, J = 6.8, 2.1 Hz, 1H), 8.09 (ddd, J = 8.9, 4.9, 2.3 Hz, 1H), 7.21 (t, J = 8.4 Hz, 1H), 4.02 (d, J = 7.3 Hz, 2H), 1.40 (ddt, J = 10.2, 7.3, 3.8 Hz, 1H), 0.68–0.55 (m, 2H), 0.55–0.43 (m, 2H). ESIMS m/z 325.3 (M + H)+ . The materials listed below (Sections 2.3.5 to 2.3.42) were prepared in a similar fashion. 2.3.5 3-Phenyl-1-(2,2,2-trifluoroethyl)-1,2,4-triazin-6(1H)-one (5) Yielded 400 mg (80%) of a pale-yellow solid, mp = 129–131 ∘ C. 1 H NMR (300 MHz, CDCl3 ) 𝛿 8.57 (s, 1H), 8.15 (dd, J = 6.6, 3.0 Hz, 2H), 7.55–7.38 (m, 3H), 4.77 (q, J = 8.1 Hz, 2H). ESIMS m/z 256.2 (M + H)+ . 2.3.6 3-(2-Fluorophenyl)-1-(2,2,2-trifluoroethyl)-1,2,4-triazin6(1H)-one (6) Yielded 610 mg (82%) of a yellow semi-solid. 1 H NMR (300 MHz, CDCl3 ) 𝛿 8.57 (s, 1H), 7.91 (td, J = 7.8, 1.9 Hz, 1H), 7.52–7.42 (m, 1H), 7.30–7.23 (m, 1H), 7.20 (ddd, J = 11.2, 8.2, 1.2 Hz, 1H). ESIMS m/z 274.2 (M + H)+ . IR (neat) = 3017, 1685, 1574 cm−1 .
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Novel triazinone derivatives as fungicides
www.soci.org HCl NH
N
1.EtOH, HCl, Et2O
R1
O
R1 1
. TsOH
O
R1
2
O
N O
R1
2. BnOC(O)CH2NH2 DCM
O
O
EtOH 3
N
R1
O
3
HN NH2NH-R2
O
N
1. NaHCO3,Et2O
N
DDQ, EtOAc R2
4
O
N N
R1
N
R2
5
Scheme 1. Synthesis of title compounds 5 to 43.
2.3.7 3-(2-Chlorophenyl)-1-(2,2,2-trifluoroethyl)-1,2,4-triazin6(1H)-one (7) Yielded 350 mg (60%) of a yellow semi-solid. 1 H NMR (300 MHz, CDCl3 ) 𝛿 8.57 (s, 1H), 7.60 (m, 1H), 7.49–7.32 (m, 3H), 4.77 (q, J = 7.8 Hz, 2H). ESIMS m/z 290.6 (M + H)+ . IR (neat) = 3305, 1668, 1624, 1434 cm−1 . 2.3.8 3-(3-Fluorophenyl)-1-(2,2,2-trifluoroethyl)-1,2,4-triazin6(1H)-one (8) Yielded 178 mg (65%) of a gray-green solid, mp = 109–111 ∘ C. 1 H NMR (300 MHz, CDCl3 ) 𝛿 8.57 (s, 1H), 7.95 (d, J = 7.6 Hz, 1H), 7.85 (d, J = 9.9 Hz, 1H), 7.46 (dt, J = 13.9, 7.1 Hz, 1H), 7.19 (dd, J = 9.2, 7.2 Hz, 1H), 4.77 (q, J = 8.2 Hz, 2H). ESIMS m/z 274.3 (M + H)+ . 2.3.9 3-(3-Chlorophenyl)-1-(2,2,2-trifluoroethyl)-1,2,4-triazin6(1H)-one (9) Yielded 1.3 g (90%) of a yellow solid, mp = 129–131 ∘ C. 1 H NMR (300 MHz, CDCl3 ) 𝛿 8.56 (s, 1H), 8.14 (t, J = 1.8 Hz, 1H), 8.03 (dt, J = 7.3, 1.6 Hz, 1H), 7.46 (dt, J = 3.7, 1.6 Hz, 1H), 7.42 (dd, J = 11.6, 4.3 Hz, 1H), 4.77 (q, J = 8.2 Hz, 2H). ESIMS m/z 290.6 (M + H)+ . 2.3.10 3-(3-Bromophenyl)-1-(2,2,2-trifluoroethyl)-1,2,4-triazin6(1H)-one (10) Yielded 123 mg (57%) of a light-brown powder, mp = 104–105 ∘ C. 1 H NMR (300 MHz, CDCl3 ) 𝛿 8.56 (s, 1H), 8.30 (s, 1H), 8.09 (d, J = 7.8 Hz, 1H), 7.62 (d, J = 8.1 Hz, 1H), 7.36 (t, J = 8.0 Hz, 1H), 4.77 (q, J = 8.2 Hz, 2H). ESIMS m/z 335.2 (M + H)+ . 2.3.11 3-(m-Tolyl)-1-(2,2,2-trifluoroethyl)-1,2,4-triazin6(1H)-one (11) Yielded 2.4 g (98%) of a gold solid, mp = 97–98 ∘ C. 1 H NMR (300 MHz, CDCl3 ) 𝛿 8.54 (s, 1H), 7.85 (d, J = 7.8 Hz, 1H), 7.43–7.18 (m, 3H), 4.77 (q, J = 8.2 Hz, 2H), 2.39 (s, 3H). ESIMS m/z 270.5 (M + H)+ .
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2.3.14 3-(4-Bromophenyl)-1-(2,2,2-trifluoroethyl)-1,2,4-triazin6(1H)-one (14) Yielded 67 mg (95%) of a yellow solid, mp = 123–125 ∘ C. 1 H NMR (300 MHz, CDCl3 ) 𝛿 8.55 (s, 1H), 8.01 (dt, J = 8.7, 2.1 Hz, 2H), 7.61 (dt, J = 8.7, 2.1 Hz, 2H), 4.76 (q, J = 8.4 Hz, 2H). ESIMS m/z 335.2 (M + H)+ . 2.3.15 3-(p-Tolyl)-1-(2,2,2-trifluoroethyl)-1,2,4-triazin6(1H)-one (15) Yielded 112 mg (41%) of a light-yellow solid, mp = 190–192 ∘ C. 1 H NMR (300 MHz, CDCl3 ) 𝛿 8.54 (s, 1H), 8.03 (d, J = 8.1 Hz, 2H), 7.28 (d, J = 8.1 Hz, 2H), 4.76 (q, J = 8.2 Hz, 2H), 2.42 (s, 3H). ESIMS m/z 270.2 (M + H)+ . 2.3.16 1-(2,2,2-Trifluoroethyl)-3-[4-(trifluoromethyl)phenyl]1,2,4-triazin-6(1H)-one (16) Yielded 27 mg (31%) of a light-brown solid, mp = 91–92 ∘ C. 1 H NMR (300 MHz, CDCl3 ) 𝛿 8.59 (s, 1H), 8.27 (d, J = 8.4 Hz, 2H), 7.74 (d, J = 8.4 Hz, 2H), 4.79 (q, J = 8.2 Hz, 2H). ESIMS m/z 324.2 (M + H)+ . 2.3.17 3-(3,5-Dibromophenyl)-1-(2,2,2-trifluoroethyl)-1,2,4-triazin6(1H)-one (17) Yielded 238 mg (73%) of a light-brown solid, mp = 139–141 ∘ C. 1 H NMR (300 MHz, CDCl3 ) 𝛿 8.56 (s, 1H), 8.23 (s, 2H), 7.78 (s, 1H), 4.77 (q, J = 8.1 Hz, 2H). ESIMS m/z 414.2 (M + H)+ . 2.3.18 3-(3-Bromo-4-methylphenyl)-1-(2,2,2-trifluoroethyl)1,2,4-triazin-6(1H)-one (18) Yielded 111 mg (32%) of a yellow solid, mp = 102–103 ∘ C. 1 H NMR (300 MHz, CDCl3 ) 𝛿 8.55 (s, 1H), 8.31 (s, 1H), 7.97 (d, J = 7.8 Hz, 1H), 7.33 (d, J = 8.1 Hz, 1H), 4.76 (q, J = 8.2 Hz, 2H), 2.46 (s, 3H). ESIMS m/z 349.2 (M + H)+ . 2.3.19 3-(3-Bromo-4-chlorophenyl)-1-(2,2,2-trifluoroethyl)1,2,4-triazin-6(1H)-one (19) Yielded 134 mg (88%) of a yellow solid, mp = 124–125 ∘ C. 1 H NMR (300 MHz, CDCl3 ) 𝛿 8.55 (s, 1H), 8.35 (s, 1H), 8.09 (d, J = 8.5 Hz, 1H),
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2.3.12 1-(2,2,2-Trifluoroethyl)-3-[3-(trifluoromethyl)phenyl]1,2,4-triazin-6(1H)-one (12) Yielded 208 mg (55%) of an off-white solid, mp = 74–75 ∘ C. 1 H NMR (300 MHz, CDCl3 ) 𝛿 8.59 (s, 1H), 8.42 (s, 1H), 8.34 (d, J = 7.8 Hz, 1H), 7.75 (d, J = 7.8 Hz, 1H), 7.62 (t, J = 7.8 Hz, 1H), 4.80 (q, J = 8.2 Hz, 2H). ESIMS m/z 324.5 (M + H)+ .
2.3.13 3-(4-Chlorophenyl)-1-(2,2,2-trifluoroethyl)-1,2,4-triazin6(1H)-one (13) Yielded 460 mg (79%) of a brown solid, mp = 110–115 ∘ C. 1 H NMR (300 MHz, CDCl3 ) 𝛿 8.54 (s, 1H), 8.08 (d, J = 8.6 Hz, 2H), 7.44 (d, J = 8.8 Hz, 2H), 4.76 (q, J = 8.2 Hz, 2H). ESIMS m/z 290.5 (M + H)+ .
www.soci.org 7.52 (t, J = 8.5 Hz, 1H), 4.76 (q, J = 8.1 Hz, 2H). ESIMS m/z 369.7 (M + H)+ . 2.3.20 3-(3-Bromo-4-fluorophenyl)-1-(2,2,2-trifluoroethyl)1,2,4-triazin-6(1H)-one (20) Yielded 447 mg (79%) of a yellow solid, mp = 97–99 ∘ C. 1 H NMR (300 MHz, CDCl3 ) 𝛿 8.55 (s, 1H), 8.37 (dd, J = 6.6, 2.4 Hz, 1H), 8.09 (ddd, J = 8.7, 4.5, 2.1 Hz, 1H), 7.22 (t, J = 8.4 Hz, 1H), 4.76 (q, J = 8.1 Hz, 2H). ESIMS m/z 353.2 (M + H)+ . 2.3.21 3-(3-Bromo-4-fluorophenyl)-1-(1,1,1-trifluoropropan2-yl)-1,2,4-triazin-6(1H)-one (21) Yielded 37 mg (35%) of a brown solid, mp = 129–131 ∘ C. 1 H NMR (300 MHz, CDCl3 ) 𝛿 8.55 (s, 1H), 8.37 (dd, J = 6.6, 2.4 Hz, 1H), 8.09 (ddd, J = 8.7, 4.5, 2.1 Hz, 1H), 7.22 (t, J = 8.4 Hz, 1H), 5.69 (p, J = 7.1 Hz, 1H), 1.87 (d, J = 7.1 Hz, 3H). ESIMS m/z 367.2 (M + H)+ . 2.3.22 3-(3-Bromo-4-fluorophenyl)-1-(3,3,3-trifluoropropyl)1,2,4-triazin-6(1H)-one (22) Yielded 75 mg (18%) of a yellow solid, mp = 80–81 ∘ C. 1 H NMR (300 MHz, CDCl3 ) 𝛿 8.49 (s, 1H), 8.37 (dd, J = 6.9, 2.4 Hz, 1H), 8.09 (ddd, J = 7.2, 4.8, 2.4 Hz, 1H), 7.21 (t, J = 8.7 Hz, 1H), 4.42 (t, J = 7.2 Hz, 2H), 2.77 (m, 2H). ESIMS m/z 367.2 (M + H)+ . 2.3.23 3-(3-Bromo-4-fluorophenyl)-1-[3,3,3-trifluoro-2(trifluoromethyl)propyl]-1,2,4-triazin-6(1H)-one (23) Yielded 35 mg (44%) of a yellow solid, mp = 105–107 ∘ C. 1 H NMR (300 MHz, CDCl3 ) 𝛿 8.52 (s, 1H), 8.37 (dd, J = 6.6, 2.2 Hz, 1H), 8.09 (ddd, J = 8.8, 4.7, 2.2 Hz, 1H), 7.21 (t, J = 8.5 Hz, 1H), 4.62 (d, J = 6.5 Hz, 2H), 3.95 (m, 1H). ESIMS m/z 435.3 (M + H)+ . 2.3.24 3-(3-Bromo-4-fluorophenyl)-1-(2-fluoroethyl)-1,2,4-triazin6(1H)-one (24) Yielded 387 mg (72%) of a yellow solid, mp = 117–119 ∘ C. 1 H NMR (300 MHz, CDCl3 ) 𝛿 8.51 (s, 1H), 8.39 (dd, J = 6.6, 2.2 Hz, 1H), 8.11 (ddd, J = 8.8, 4.7, 2.2 Hz, 1H), 7.21 (t, J = 8.5 Hz, 1H), 4.97 (dd, J = 5.2, 4.4 Hz, 1H), 4.82 (dd, J = 5.2, 4.7 Hz, 1H), 4.50 (dt, J = 25.0, 4.8 Hz, 2H). ESIMS m/z 317.2 (M + H)+ . 2.3.25 3-(3-Bromo-4-fluorophenyl)-1-butyl-1,2,4-triazin-6(1H)one (25) Yielded 220 mg (88%) of a yellow solid, mp = 62–64 ∘ C. 1 H NMR (300 MHz, CDCl3 ) 𝛿 8.44 (s, 1H), 8.37 (dd, J = 6.7, 2.1 Hz, 1H), 8.09 (ddd, J = 8.6, 4.8, 2.2 Hz, 1H), 7.20 (t, J = 8.4 Hz, 1H), 4.16 (t, J = 7.2 Hz, 2H), 1.94–1.78 (m, 2H), 1.53–1.34 (m, 2H), 0.99 (t, J = 7.4 Hz, 3H). ESIMS m/z 327.3 (M + H)+ . 2.3.26 3-(3-Bromo-4-fluorophenyl)-1-pentyl-1,2,4-triazin6(1H)-one (26) Yielded 72 mg (42%) of a yellow semi-solid. 1 H NMR (300 MHz, CDCl3 ) 𝛿 8.45 (s, 1H), 8.37 (dd, J = 6.7, 2.1 Hz, 1H), 8.10 (ddd, J = 8.6, 4.8, 2.2 Hz, 1H), 7.20 (t, J = 8.4 Hz, 1H), 4.15 (t, J = 7.1 Hz, 2H), 1.95–1.81 (m, 2H), 1.51–1.29 (m, 4H), 0.89 (t, J = 7.4 Hz, 3H). ESIMS m/z 341.5 (M + H)+ . IR (neat) = 2953, 1671, 1565 cm−1 .
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2.3.27 3-(3-Bromo-4-fluorophenyl)-1-hexyl-1,2,4-triazin-6(1H)-one (27) Yielded 33 mg (51%) of a yellow wax. 1 H NMR (300 MHz, CDCl3 ) 𝛿 8.44 (s, 1H), 8.37 (dd, J = 6.7, 2.1 Hz, 1H), 8.09 (ddd, J = 8.6, 4.8, 2.2 Hz, 1H), 7.20 (t, J = 8.4 Hz, 1H), 4.16 (t, J = 7.1 Hz, 2H), 1.92–1.79 (m, 2H), 1.48–1.25 (m, 6H), 0.84 (t, J = 7.4 Hz, 3H). ESIMS m/z 355.2 (M + H)+ . IR (neat) = 2952, 1671, 1565 cm−1 .
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2.3.28 3-(3-Bromo-4-fluorophenyl)-1-(2-ethoxyethyl)-1,2,4-triazin6(1H)-one (28) Yielded 216 mg (49%) of a yellow solid, mp = 91–93 ∘ C. 1 H NMR (300 MHz, CDCl3 ) 𝛿 8.47 (s, 1H), 8.39 (dd, J = 6.7, 2.1 Hz, 1H), 8.11 (ddd, J = 8.8, 4.7, 2.2 Hz, 1H), 7.21 (t, J = 8.5 Hz, 1H), 4.38 (t, J = 5.6 Hz, 2H), 3.91 (t, J = 5.6 Hz, 2H), 3.57 (q, J = 7.0 Hz, 2H), 1.19 (t, J = 7.0 Hz, 3H). ESIMS m/z 343.3 (M + H)+ . 2.3.29 3-(3-Bromo-4-fluorophenyl)-1-isopropyl-1,2,4-triazin6(1H)-one (29) Yielded 273 mg (66%) of a yellow solid, mp = 107–108 ∘ C. 1 H NMR (300 MHz, CDCl3 ) 𝛿 8.44 (s, 1H), 8.39 (dd, J = 6.9, 2.2 Hz, 1H), 8.13 (ddd, J = 8.8, 4.7, 2.2 Hz, 1H), 7.21 (t, J = 8.5 Hz, 1H), 5.29–5.17 (m, 1H), 1.48 (d, J = 6.6 Hz, 7H). ESIMS m/z 313.2 (M + H)+ . 2.3.30 3-(3-Bromo-4-fluorophenyl)-1-(sec-butyl)-1,2,4-triazin6(1H)-one (30) Yielded 342 mg (74%) of a yellow solid, mp = 84–86 ∘ C. 1 H NMR (300 MHz, CDCl3 ) 𝛿 8.46 (d, J = 0.8 Hz, 1H), 8.38 (dd, J = 6.6, 2.2 Hz, 1H), 8.12 (ddd, J = 8.8, 4.7, 2.2 Hz, 1H), 7.23 (t, J = 8.4 Hz, 2H), 5.03 (dd, J = 14.6, 6.6 Hz, 1H), 1.88 (dtd, J = 20.1, 13.7, 7.1 Hz, 2H), 1.45 (d, J = 6.6 Hz, 3H), 0.92 (t, J = 7.4 Hz, 3H). ESIMS m/z 327.4 (M + H)+ . 2.3.31 3-(3-Bromo-4-fluorophenyl)-1-cyclopentyl-1,2,4-triazin6(1H)-one (31) Yielded 368 mg (77%) of a yellow solid, mp = 115–117 ∘ C. 1 H NMR (300 MHz, CDCl3 ) 𝛿 8.43 (d, J = 0.8 Hz, 1H), 8.37 (dd, J = 6.9, 2.2 Hz, 1H), 8.10 (ddd, J = 8.5, 4.7, 2.2 Hz, 1H), 7.22 (t, J = 8.5 Hz, 1H), 5.35 (ddd, J = 9.1, 8.0, 4.7 Hz, 1H), 2.23–2.08 (m, 2H), 2.00 (m, 4H), 1.78 (m, 2H). ESIMS m/z 339.2 (M + H)+ . 2.3.32 3-(3-Bromo-4-fluorophenyl)-1-(prop-2-yn-1-yl)1,2,4-triazin-6(1H)-one (32) Yielded 295 mg (95%) of a light-brown solid, mp = 132–137 ∘ C. 1 H NMR (300 MHz, CDCl3 ) 𝛿 8.50 (s, 1H), 8.40 (dd, J = 6.7, 2.1 Hz, 1H), 8.12 (ddd, J = 8.6, 4.8, 2.3 Hz, 1H), 7.21 (t, J = 8.5 Hz, 1H), 4.92 (d, J = 2.8 Hz, 2H), 2.43 (t, J = 2.5 Hz, 1H). ESIMS m/z 309.3 (M + H)+ . 2.3.33 3-(3-Bromo-4-fluorophenyl)-1-(but-2-yn-1-yl)-1,2,4triazin-6(1H)-one (33) Yielded 30 mg (50%) of an off-white solid, mp = 145–147 ∘ C. 1 H NMR (300 MHz, CDCl3 ) 𝛿 8.48 (s, 1H), 8.40 (dd, J = 6.6, 2.3 Hz, 1H), 8.12 (ddd, J = 8.6, 4.8, 2.3 Hz, 1H), 7.21 (t, J = 8.5 Hz, 1H), 4.87 (q, J = 2.5 Hz, 2H), 1.85 (t, J = 2.5 Hz, 3H). ESIMS m/z 323.2 (M + H)+ . 2.3.34 3-(3-Bromo-4-fluorophenyl)-1-(pent-2-yn-1-yl)-1,2,4-triazin6(1H)-one (34) Yielded 369 mg (81%) of a white solid, mp = 97–101 ∘ C. 1 H NMR (300 MHz, CDCl3 ) 𝛿 8.50 (s, 1H), 8.43 (dd, J = 6.9, 2.2 Hz, 1H), 8.14 (ddd, J = 8.5, 4.7, 2.2 Hz, 1H), 7.21 (t, J = 8.5 Hz, 1H), 4.91 (t, J = 2.3 Hz, 2H), 2.25 (qt, J = 7.4, 2.2 Hz, 2H), 1.17 (t, J = 7.6 Hz, 3H). ESIMS m/z 337.3 (M + H)+ . 2.3.35 1-Benzyl-3-(3-bromo-4-fluorophenyl)-1,2,4-triazin-6(1H)-one (35) Yielded 159 mg (80%) of a brown solid, mp = 146–148 ∘ C. 1 H NMR (300 MHz, CDCl3 ) 𝛿 8.45 (s, 1H), 8.36 (dd, J = 6.7, 2.1 Hz, 1H), 8.08 (ddd, J = 8.8, 4.8, 2.3 Hz, 1H), 7.50 (dd, J = 8.0, 1.6 Hz, 2H), 7.40–7.32 (m, 3H), 7.23–7.16 (m, 1H), 5.30 (s, 2H). ESIMS m/z 361.2 (M + H)+ .
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2.3.36 3-(3-Bromo-4-fluorophenyl)-1-(1-phenylethyl)-1,2,4triazin-6(1H)-one (36) Yielded 47 mg (35%) of a yellow oil. 1 H NMR (300 MHz, CDCl3 ) 𝛿 8.42 (s, 1H), 8.35 (dd, J = 6.7, 2.1 Hz, 1H), 8.07 (ddd, J = 8.6, 4.5, 2.0 Hz, 1H), 7.51–7.47 (m, 2H), 7.35 (dt, J = 13.1, 7.1 Hz, 3H), 7.20 (t, J = 8.3 Hz, 1H), 6.23 (q, J = 7.1 Hz, 1H), 1.87 (d, J = 7.1 Hz, 3H). ESIMS m/z 375.2 (M + H)+ . IR (neat) = 2927, 1670, 1567 cm−1 . 2.3.37 3-(3-Bromo-4-fluorophenyl)-1-(2-phenylpropyl)1,2,4-triazin-6(1H)-one (37) Yielded 190 mg (70%) of a yellow semi-solid. 1 H NMR (300 MHz, CDCl3 ) 𝛿 8.38 (s, 1H), 8.16 (dd, J = 6.7, 2.1 Hz, 1H), 7.93 (ddd, J = 8.8, 4.8, 2.3 Hz, 1H), 7.35–7.11 (m, 6H), 4.32 (ddd, J = 21.2, 12.8, 7.8 Hz, 2H), 3.48 (dd, J = 15.2, 7.3 Hz, 1H), 1.38 (d, J = 6.8 Hz, 3H). ESIMS m/z 389.3 (M + H)+ . IR (neat) = 2964, 1670, 1566 cm−1 . 2.3.38 3-(3-Bromo-4-fluorophenyl)-1-[(2-methylcyclopropyl) methyl]-1,2,4-triazin-6(1H)-one (39) Yielded 335 mg (84%) of a white solid, mp = 109–111 ∘ C. 1 H NMR (300 MHz, CDCl3 ) 𝛿 8.45 (s, 1H), 8.38 (dd, J = 6.7, 2.1 Hz, 1H), 8.10 (ddd, J = 8.8, 4.8, 2.3 Hz, 1H), 7.21 (t, J = 8.3 Hz, 1H), 4.02 (ddd, J = 47.2, 13.4, 7.5 Hz, 2H), 1.11 (ddd, J = 11.1, 7.6, 3.3 Hz, 1H), 1.06 (d, J = 5.8 Hz, 3H), 0.97–0.83 (m, 1H), 0.69–0.59 (m, 1H), 0.34 (dt, J = 8.1, 4.9 Hz, 1H). ESIMS m/z 339.4 (M + H)+ . 2.3.39 3-(3-Bromo-4-fluorophenyl)-1-[(2,2-dichlorocyclopropyl) methyl]-1,2,4-triazin-6(1H)-one (40) Yielded 30 mg (41%) of a clear viscous glass. 1 H NMR (300 MHz, CDCl3 ) 𝛿 8.45 (s, 1H), 8.37 (dd, J = 6.8, 2.1 Hz, 1H), 8.09 (ddd, J = 8.9, 4.9, 2.3 Hz, 1H), 7.21 (t, J = 8.5 Hz, 1H), 4.35 (m, 2H), 2.25 (m, 1H), 1.79 (m, 1H), 1.56 (m, 1H). ESIMS m/z 394.2 (M + H)+ . IR (neat) = 2954, 1672, 1566 cm−1 . 2.3.40 3-(3-Bromo-4-fluorophenyl)-1-(cyclobutylmethyl)-1,2,4triazin-6(1H)-one (41) Yielded 282 mg (67%) of a yellow solid, mp = 111–112 ∘ C. 1 H NMR (300 MHz, CDCl3 ) 𝛿 8.46 (d, J = 0.5 Hz, 1H), 8.38 (dd, J = 6.6, 2.2 Hz, 1H), 8.10 (ddd, J = 8.8, 4.7, 2.2 Hz, 1H), 7.22 (t, J = 8.5 Hz, 1H), 4.22 (d, J = 7.4 Hz, 2H), 3.02–2.82 (m, 1H), 2.13 (ddd, J = 10.2, 7.8, 2.6 Hz, 2H), 2.05–1.72 (m, 4H). ESIMS m/z 256.2 (M + H)+ . 2.3.41 3-(3-Bromo-4-fluorophenyl)-1-(cyclopentylmethyl)-1,2,4triazin-6(1H)-one (42) Yielded 36 mg (16%) of an orange wax. 1 H NMR (300 MHz, CDCl3 ) 𝛿 8.46 (d, J = 0.5 Hz, 1H), 8.38 (dd, J = 6.6, 2.2 Hz, 1H), 8.10 (ddd, J = 8.8, 4.7, 2.2 Hz, 1H), 7.21 (t, J = 8.5 Hz, 1H), 4.15 (d, J = 7.4 Hz, 2H), 2.57 (m, 1H), 1.81–1.57 (m, 4H), 1.45–1.21 (m, 4H). ESIMS m/z 353.3 (M + H)+ . IR (neat) = 2952, 1671, 1565 cm−1 . 2.3.42 3-(3-Bromo-4-fluorophenyl)-1-(2-cyclopropylethyl)-1,2,4triazin-6(1H)-one (43) Yielded 35 mg (28%) of an orange solid, mp = 90–91 ∘ C. 1 H NMR (300 MHz, CDCl3 ) 𝛿 8.45 (s, 1H), 8.37 (dd, J = 6.8, 2.1 Hz, 1H), 8.09 (ddd, J = 8.9, 4.9, 2.3 Hz, 1H), 7.21 (t, J = 8.5 Hz, 1H), 4.25 (t, J = 7.3 Hz, 2H), 1.79 (q, J = 7.3 Hz, 2H), 0.79 (m, 1H), 0.49 (m, 2H), 0.09 (m, 2H). ESIMS m/z 339.4 (M + H)+ .
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3
RESULTS AND DISCUSSION
3.1 Synthesis and characterization Scheme 1 describes the synthesis of the target compounds. The standard formation of ethyl imidate salts 2 from substituted benzonitriles (1) was followed by acid-catalyzed transimination with benzyl glycine p-toluenesulfonic acid salt to give glycine imidates 3. Condensation of 3 with substituted hydrazines19 provided the 4,5-dihydro-1,2,4-triazin-6(1H)-one intermediates 4. Subsequent oxidation of the dihydro intermediates with 2,3-dichloro-5,6-dicyano-p-benzoquinone (DDQ) afforded the title compounds (5 to 43). 3.2 In vitro antifungal activity Initial synthesis efforts aimed at optimizing antifungal activity of this chemistry focused on examining the electronic and steric tolerances of the 3-phenyl component by incorporating halo, alkyl and haloalkyl substituents, key examples of which are presented in Table 1. To enable better evaluation of the biological impact of each substituent addition to the 3-phenyl group, the R2 component was held constant as trifluoroethyl. Although the EC50 values indicate some differences in sensitivity among the test fungi, compounds 5 to 16 show a consistent relative efficacy trend across the four-pathogen spectrum that is represented by the values in Table 1 for the four-pathogen mean. To examine the effect of bis-substitution on the phenyl ring while maintaining bromine in the 3-position, a small series of
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2.4 Fungal growth inhibition assays Antifungal assays against L. nodorum, Z. tritici, M. grisea and U. maydis were carried out in 96-well flat-bottomed microtitre
plates (Techno Plastic Products, Trasadingen, Switzerland), with each well containing a total assay volume of 200 𝜇L. The growth medium employed was Difco YNB (6.7 g L−1 , yeast nitrogen base without amino acids; BD Diagnostic Systems, Sparks, MD) supplemented with 2 g of glucose and 3 g each of potassium dihydrogen phosphate and dipotassium hydrogen phosphate L−1 . Initial inoculum density was adjusted to 200 000 spores mL−1 for L. nodorum, 100 000 spores mL−1 for Z. tritici and 40 000 spores mL−1 for M. grisea. For inoculum of U. maydis, a liquid culture inoculated the day before testing was grown to an absorbance reading of 0.2, measured using a Spectronic20D+ spectrophotometer (Milton Roy, Warminster, PA) set at a wavelength of 450 nm, and then diluted 500-fold with the supplemented YNB medium to give a spore concentration of 10 000 spores mL−1 . Serial dilutions of each test compound were prepared using stock solutions in DMSO, and 2 𝜇L aliquots were added to the wells. To enable better comparison of analogs differing significantly in biological activity, each compound was evaluated in three separate tests, each involving an 8 rate dose response, which provided a total of 19 treatment concentrations ranging from 40 to 0.0001 ppm. Immediately after addition of spore suspensions, initial cell density readings were determined using a NepheloStar nephelometer (BMG LABTECH Gmb, Ortenberg, Germany). After incubation in the dark at 90 rpm in a New Brunswick Innova 44 shaking incubator (Eppendorf, Inc., Enfield, CT) for 48 h (L. nodorum, M. grisea and U. maydis) or 72 h (Z. tritici) at 22 ∘ C (L. nodorum, M. grisea and Z. tritici) or 24 ∘ C (U. maydis), conditions under which evaporation from wells was minimal, the plates were read in the NepheloStar a second time to assess growth. Percentage growth inhibition was calculated by reference to control wells containing growth media and inoculum amended with 1% DMSO. Fungicide sensitivities were determined as 50% effective concentration (EC50 ) using a dose–response relationship provided by the logistic regression curve fit tool within Spotfire (TIBCO Software Inc., Palo Alto, CA).
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Table 1. Antifungal activity of 3-substituted phenyl-N-1-trifluoroethyl-1,2,4-triazinones EC50 values (μg mL−1 )
Substituents Compound number 5 6 7 8 9 10 11 12 13 14 15 16 Azoxystrobin Tebuconazole a b
R1
R2
H 2-F 2-Cl 3-F 3-Cl 3-Br 3-CH3 3-CF3 4-Cl 4-Br 4-CF3 4-CH3
L. nodorum
CH2 CF3 CH2 CF3 CH2 CF3 CH2 CF3 CH2 CF3 CH2 CF3 CH2 CF3 CH2 CF3 CH2 CF3 CH2 CF3 CH2 CF3 CH2 CF3
0.91 2.82 10.72 1.35 0.20 0.08 0.25 0.04 0.81 0.08 1.51 2.14 0.011 0.063
M. grisea
Z. tritici
3.47 8.12 16.98 3.89 0.24 0.06 0.25 0.01 0.23 0.17 0.15 0.26 0.009 0.18
0.74 1.78 3.72 0.96 0.06 0.05 0.12 0.06 1.51 2.29 2.24 1.66 0.003 0.03
U. maydis 0.91 2.14 2.09 2.14 0.28 0.13 0.65 0.28 0.28 0.18 0.21 0.69 0.005 0.002
Ascomycete meana
Four-pathogen meanb
1.71 4.24 10.47 2.06 0.16 0.06 0.20 0.04 0.85 0.85 1.30 1.35
1.51 3.72 8.38 2.08 0.19 0.08 0.32 0.10 0.71 0.68 1.03 1.19
Ascomycete meana
Four-pathogen meanb
Mean of the EC50 values for L. nodorum, M. grisea and Z. tritici. Mean of the EC50 values across all four pathogens tested.
Table 2. Antifungal activity of 3-bromophenyl-N-1-trifluoroethyl-1,2,4-triazinones EC50 Values (μg mL−1 )
Substituents Compound number 10 17 18 19 20 Azoxystrobin Tebuconazole a b
R1 3-Br 3,5-Br2 3-Br-4-Me 3-Br-4-Cl 3-Br-4-F
R2 CH2 CF3 CH2 CF3 CH2 CF3 CH2 CF3 CH2 CF3
L. nodorum 0.08 0.17 0.13 0.02 0.06 0.008 0.082
M. grisea
Z. tritici
0.06 0.02 0.01 0.02 0.01 0.011 0.224
0.05 0.15 0.11 0.09 0.04 0.003 0.051
U. maydis 0.13 0.06 0.13 0.07 0.11 0.003 0.002
0.06 0.11 0.08 0.04 0.04
0.08 0.10 0.10 0.05 0.05
Mean of the EC50 values for L. nodorum, M. grisea and Z. tritici. Mean of the EC50 values across all four pathogens tested.
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disubstituted phenyl analogs (17 to 20) of compound 10 were prepared and tested. The addition of a symmetrical bromine atom (17) at the 5-position or a methyl (18), chloro (19) or fluoro (20) group at the 4-position resulted in relatively minor changes to antifungal potency, the magnitude of which varied for the individual pathogens (Table 2). This suggested that substitution at the 3-position of the phenyl was the more critical determinant of antifungal activity. Based on these data, synthetic efforts were then directed at exploring the effects on activity of replacing the trifluoroethyl group at R2 with a variety of alternative chemistries. While all 3-bromophenyl analogs were similarly potent, based on both ascomycete and four-pathogen mean EC50 values (Table 2), the 3-bromo-4-fluorophenyl compound (20) was selected for R2 optimization efforts owing to its stronger performance against Z. tritici, a pathogen of key importance in cereal production. Towards this end, a set of 23 compounds were prepared to explore the impact on antifungal activity of structural variations at R2 by branching, homologation and replacement of the haloalkyl moiety with
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alkyls, branched alkyls, alkynes, benzyls and cycloalkyl groups (Table 3). With the exception of the cyclopentyl derivative (compound 31), all trifluoroethyl replacements resulted in potency reduction relative to compound 20 against U. maydis, with the ethoxyethyl compound (28) reducing activity against U. maydis 30-fold. While a few compounds (e.g. compounds 23, 27 and 31) had U. maydis activity similar to that against the other fungi tested, the majority of the R2 replacements (compounds 21 to 43) resulted in loss of U. maydis potency while maintaining or contributing modest activity enhancements (e.g. compounds 25, 35, 38 and 41) on the ascomycetes. This differential in sensitivity between U. maydis and the other fungi is generally in the 5–10-fold range, but for the most active compounds of the series, the CH2 -cycloalkyls 38 and 41, and the n-pentyl derivative (26), the difference is 40–60 fold. Z. tritici was considerably less sensitive to alkynyls at R2 (compounds 33 and 34 in particular) and L. nodorum to the 2-Me phenethyl analog (compound 37), but, otherwise, the three ascomycetes were similar in sensitivity to compounds 21 to 43.
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Table 3. Antifungal activity of 3-bromo-4-fluorophenyl-1,2,4-triazinones variously substituted at N-1 Substituents (R1 = 3-Br-4-F) Compound number
R2
20 CH2 CF3 21 CH(CH3 )CF3 22 CH2 CH2 CF3 23 CH2 CH(CF3 )2 24 CH2 CH2 F 25 n-Butyl 26 n-Pentyl 27 n-Hexyl 28 CH2 CH2 OCH2 CH3 29 i-Propyl 30 i-Butyl 31 Cyclopentyl 32 2-Propynyl 33 2- Butynyl 34 2-Pentynyl 35 Benzyl 36 𝛼-Me benzyl 37 2-Me phenethyl 38 CH2 -cyclopropyl 39 CH2 -2-Me cyclopropyl 40 CH2 -2,2-di-Cl-cyclopropyl 41 CH2 -cyclobutyl 42 CH2 -cyclopentyl 43 CH2 CH2 -cyclopropyl Azoxystrobin Tebuconazole a b
EC50 values (𝜇g mL−1 ) L. nodorum 0.06 0.04 0.06 0.07 0.36 0.03 0.04 0.62 0.30 0.08 0.04 0.07 0.03 0.03 0.07 0.03 0.08 1.70 0.02 0.05 0.37 0.02 0.05 0.04 0.006 0.048
M. grisea
Z. tritici
0.01 0.09 0.05 0.21 0.20 0.01 0.02 0.25 0.13 0.28 0.10 0.13 0.02 0.02 0.06 0.02 0.27 0.13 0.01 0.02 0.05 0.01 0.03 0.02 0.007 0.209
0.04 0.08 0.05 0.17 0.72 0.01 0.07 0.91 0.56 0.17 0.01 0.03 0.16 0.34 0.87 0.02 0.14 0.85 0.005 0.01 0.98 0.02 0.15 0.02 0.002 0.046
Ascomycete meana
0.11 0.15 0.40 0.17 0.71 0.26 2.51 0.45 3.09 0.83 0.16 0.10 1.00 0.45 0.24 0.19 0.43 0.15 0.51 0.35 1.82 0.44 0.54 0.40 0.002 0.001
0.04 0.07 0.06 0.15 0.43 0.02 0.04 0.59 0.33 0.17 0.05 0.07 0.07 0.13 0.34 0.02 0.17 0.89 0.01 0.03 0.47 0.01 0.08 0.03
Four-pathogen meanb 0.05 0.09 0.14 0.16 0.50 0.08 0.66 0.56 1.02 0.34 0.08 0.08 0.30 0.21 0.31 0.06 0.23 0.71 0.14 0.11 0.80 0.12 0.19 0.12
Ascomycete 4 7 6 10 14 2 4 16 12 11 5 7 7 9 13 2 11 17 1 3 15 1 8 3
U. maydis 2 3 10 5 16 8 20 13 21 17 4 1 18 13 7 6 11 3 14 9 19 12 15 10
Mean of the EC50 values for L. nodorum, M. grisea and Z. tritici. Mean of the EC50 values across all four pathogens tested.
EC50 values generated for each pathogen averaged across compounds 20 to 43 were 0.09, 0.18 and 0.27 for M. grisea, L. nodorum and Z. tritici, respectively, compared with 0.64 for U. maydis. In addition to the greater sensitivity of ascomycetes to the majority of compounds 21 to 43, Table 3 reveals evidence of a divergence of structure–activity relationships between the ascomycete panel and the basidiomycete U. maydis. This is particularly exemplified by compounds 38 and 41, which were the most inhibitory to L. nodorum, M. grisea and Z. tritici, but considerably less active against U. maydis, and by the 2-Me phenethyl derivative (compound 37), which was ranked third most active against U. maydis but was among the weakest against ascomycetes. The reasons for the pathogen sensitivity differences and diverging structure–activity relationships focused on R2 substitution are not known, but may reflect target-site or other biochemical differences among diverse fungi, or differing physical property requirements for target-site access. However, the biochemical target site and mechanism of action of the 3-aryl-1,2,4-triazin-6-ones remain unknown.
4
U. maydis
Fungitoxicity rank
CONCLUSION
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The 3-aryl-1,2,4-triazin-6-ones represent a novel series of chemistry with high levels of in vitro antifungal activity across a spectrum of agriculturally relevant plant-pathogenic fungi.
Structure–activity relationship studies indicated a preference for substitution at the 3-position of the phenyl group attached at the C-3 carbon of the triazinone ring, particularly with electron-withdrawing groups such as Br, Cl and CF3 . Substitutions at other positions on the phenyl ring were less well tolerated. Exploration of the effects of varying the R2 group attached at N-1 of the triazinone ring revealed a diverging structure–activity relationship between the basidiomycete pathogen, U. maydis, and the ascomycetes L. nodorum, M. grisea and Z. tritici. For example, growth inhibition activity on U. maydis was generally reduced by replacement of the trifluoroethyl group at N-1 with various CH2 -cycloalkyls, while the CH2 -cyclopropyl (38) and CH2 -cyclobutyl (41) analogs were the most active against the ascomycete pathogens. With an EC50 of 0.005 ppm on Z. tritici, compound 38 demonstrated antifungal activity on this key cereal pathogen that was comparable with that of the commercial strobilurin fungicide azoxystrobin (EC50 0.002 ppm) and significantly better than that of the triazole tebuconazole (EC50 0.046 ppm). However, 38 was 100 times less active against U. maydis (EC50 0.51 ppm), which was generally the least sensitive pathogen to this triazinone chemistry. The basis of this difference in sensitivity is unknown and warrants further investigation. The in vitro antifungal activity data summarized in this paper represent an initial phase of a broader biological characterization program for
www.soci.org this novel class of fungicides, which included higher-tier in planta greenhouse testing. Results from whole-plant evaluation of the 3-aryl-1,2,4-triazin-6-ones will be presented in a subsequent paper.
ACKNOWLEDGEMENTS The authors would like to acknowledge and thank S Shaber, R Ross, WR Erickson, MJ Kelly, I Morrison, B Rieder, D Weaver and M Yap for their contributions to the development of this chemistry.
REFERENCES 1 Gisi U and Sierotzki H, Fungicide modes of action and resistance in downy mildews. Eur J Plant Pathol 122:157–167 (2008). 2 Gisi U, Hermann D, Ohl L and Steden C, Sensitivity profiles of Mycosphaerella graminicola and Phytophthora infestans populations to different classes of fungicides. Pestic Sci 51:290–298 (1997). 3 Ishii H, Impact of fungicide resistance in plant pathogens on crop disease control and agricultural environment. JARQ 40:205–211 (2006). 4 Fernandez-Ortuno D, Tores JA, Vicente A and Perez-Garcia A, Mechanisms of resistance to QoI fungicides in phytopathogenic fungi. Int Microbiol 11:1–9 (2008). 5 Koller W, Avila-Adame C, Olaya G and Zheng D, Resistance to strobilurin fungicides. ACS Symp Ser 808(Agrochemical Resistance):215–229 (2002). 6 Russell PE, Fungicide resistance – occurrence and management. J Agric Sci 124:317–323 (1995). 7 Brent KJ and Hollomon DW, Fungicide resistance in crop pathogens: how can it be managed? FRAC Monograph No. 1, 2nd edition. FRAC, Brussels, Belgium, 56 pp. (2007). 8 De Waard, MA, Fungicide resistance strategies in winter wheat in the Netherlands. Schriftenreihe der Deutschen Phytomedizinischen Gesellschaft 4 (Proc 10th Int Symp – Systemic Fungicides and Antifungal Compounds, 1992), pp. 183–191 (1993).
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9 Sierotzki H, Parisi S, Steinfeld U, Tenzer I, Poirey S and Gisi U, Mode of resistance to respiration inhibitors at the cytochrome bc(1) enzyme complex of Mycosphaerella fijiensis field isolates. Pest Manag Sci 56(10):833–841 (2000). 10 Mullins JGL, Parker JE, Cools HJ, Togawa RC, Lucas JA, Fraaije BA, et al, Molecular modelling of the emergence of azole resistance in Mycosphaerella graminicola. PloS ONE 6:e20973 (2011). 11 Fraaije BA, Cools HJ, Kim SH, Motteram J, Clark WS and Lucas JA, A novel substitution I381V in the sterol 14 alpha-demethylase (CYP51) of Mycosphaerella graminicola is differentially selected by azole fungicides. Mol Plant Pathol 8(3):245–254 (2007). 12 Cools HJ, Mullins JGL, Fraaije BA, Parker JE, Kelly DE, Lucas JA, et al, Impact of recently emerged sterol 14 alpha-demethylase (CYP51) variants of Mycosphaerella graminicola on azole fungicide sensitivity. Appl Environ Microb 77(11):3830–3837 (2011). 13 Cools HJ, Fraaije BA and Lucas JA, Molecular mechanisms conferring reduced sensitivities to triazoles in UK isolates of Septoria tritici. Phytopathology 94(6):S20–S21 (2004). 14 Cools HJ and Fraaije BA, Update on mechanisms of azole resistance in Mycosphaerella graminicola and implications for future control. Pest Manag Sci 69(2):150-155 (2013). 15 Cools HJ and Fraaije BA, Are azole fungicides losing ground against Septoria wheat disease? Resistance mechanisms in Mycosphaerella graminicola. Pest Manag Sci 64(7):681–684 (2008). 16 Cools HJ, Bayon C, Atkins S, Lucas JA and Fraaije BA, Overexpression of the sterol 14𝛼-demethylase gene (MgCYP51) in Mycosphaerella graminicola isolates confers a novel azole fungicide sensitivity phenotype. Pest Manag Sci 68(7):1034–1040 (2012). 17 Hollomon DW, New modes of action contribute to resistance management, in Fungicide Resistance in Crop Protection: Risk and Management, ed. by Thind TS. CABI, Wallingford, Oxon, UK, pp. 104–115 (2011). 18 Shaber SH, Meyer KG, Niyaz NM, Rieder BJ, Sullenberger MT, Smith FD, et al, Preparation of 4,5-dihydro-1,2,4-triazin-6-ones and 1,2,4-trianzin-6-ones for use as fungicides. WO2006/119400A2 (2006). 19 Meyer KG, Simple preparation of monoalkylhydrazines. Synlett 13:2355–2356 (2004).
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Pest Manag Sci 2015; 71: 83–90
Revised: 24 January 2014
Accepted article published: 18 February 2014
Published online in Wiley Online Library: 1 April 2014
(wileyonlinelibrary.com) DOI 10.1002/ps.3765
Synthesis and antifungal activity of 3-aryl-1,2,4-triazin-6-one derivatives W John Owen,* Michael T Sullenberger, Michael R Loso, Kevin G Meyer and Thomas J Slanec Abstract BACKGROUND: As a result of resistance development in many plant-pathogenic fungi to agricultural fungicides, there is an ongoing need to discover novel antifungal chemistries to help sustain efficient crop production. A fungicide screening program identified 3-phenyl-1-(2,2,2-trifluoroethyl)-1,2,4-triazin-6(1H)-one (5) as a promising new starting point for further activity optimization. A series of analogs were designed, prepared and evaluated in growth inhibition assays using four plant-pathogenic fungi. RESULTS: Thirty nine analogs (compounds 5 to 43) were prepared to explore structure–activity relationships at R1 and R2 , and all targeted structures were characterized by 1 H NMR and MS. All compounds were in vitro tested against three ascomycetes [Leptosphaeria nodorum, Magnaporthe grisea and Zymoseptoria tritici (syn. Mycosphaerella graminicola)] and one basidiomycete (Ustilago maydis) pathogen. When R2 was trifluoroethyl, fungicidal activity was enhanced by a single electron-withdrawing substitution, such as Br, Cl and CF3 in the 3-position at R1 (compounds 9, 10 and 12), of which the 3-bromo compound (10) was the most active (EC50 = 0.08, averaged across four pathogens). More subtle activity improvement was found by addition of a second halogen substituent in the 4-position, with the 3-Br-4-F analog (20) being the most active against the commercially important cereal pathogen Z. tritici. Replacement of the R2 haloalkyl group with benzyl, alkyl (e.g. n-butyl, i-butyl, n-pentyl) and, particularly, CH2 -cycloalkyls (e.g. CH2 -cyclopropyl, CH2 -cyclobutyl) resulted in further activity enhancements against the ascomycete fungi, but was either neutral or detrimental to activity against U. maydis. One of the most active compounds in this series (41) gave control of Z. tritici, with an EC50 of 0.005 ppm, comparable with that of the commercial strobilurin fungicide azoxystrobin (EC50 0.002 ppm). CONCLUSIONS: The present work demonstrated that the 3-phenyl-1,2,4-triazin-6-ones are a novel series of compounds with highly compelling levels of antifungal activity against agriculturally relevant plant-pathogenic fungi. © 2014 Society of Chemical Industry Keywords: fungicide; triazinone; structure–activity relationship; Zymoseptoria tritici; Leptosphaeria nodorum; Magnoporthe grisea; Ustilago maydis
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INTRODUCTION
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∗
Correspondence to: W John Owen, Dow AgroSciences, 9330 Zionsville Rd, Indianapolis, IN 46268-1054, USA. E-mail: [email protected] Dow AgroSciences, Indianapolis, IN, USA
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The use of broad-spectrum fungicides to protect crops from epidemics of plant pathogens is an important grower practice towards maintaining high yields and quality of harvested products. However, many plant pathogens have the propensity for developing resistance to fungicides, particularly to those classes of compounds that act at a single defined target site.1 – 5 For delaying onset of resistance development, it is frequently recommended that fungicides be applied in mixtures or in alternation with products of different modes of action (MOA), and that the number and frequency of applications per growing season be kept to a minimum.6 – 8 In spite of such practices, resistant and less sensitive isolates can continue to propagate and accumulate within a crop to a point at which fungicide performance becomes visibly diminished. This has been well exemplified in recent years in the European cereals market, where resistance of Zymoseptoria tritici, the causative agent of wheat leaf blotch, to the strobilurin fungicide class has become widespread.9 Z. tritici isolates carrying a variety of mutations in the CYP51 gene encoding the sterol C14-demethylase target of the azole fungicides have also
emerged and are causing significant sensitivity shifts among European populations.10 – 16 Cases such as these emphasize that continued research to identify new active chemistries with novel mechanisms of action and a broad antifungal spectrum is a critical aspect of resistance management.17 With this in mind, the ongoing fungicide screening program at Dow AgroSciences identified 3-phenyl-1,2,4-triazin-6-ones (Fig. 1) as having antifungal activity against a number of agriculturally relevant plant pathogens in in vitro assays. Subsequently, a synthesis program to optimize the biological activity of this new class of fungicides was initiated. The present paper describes the synthesis of a particular series of 1,2,4-triazin-6-ones, in which the R1 and R2 components were varied, and compares their in vitro antifungal activities against the plant pathogens Leptosphaeria nodorum, Z. tritici, Magnaporthe grisea and Ustilago maydis.
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N N R1
N
R2
Figure 1. Core structure of the 3-phenyl-1,2,4-triazin-6-ones.
2
MATERIALS AND METHODS
2.1 General All starting materials and reagents were commercially available and used without further purification. Melting points were determined on a Thomas-Hoover™ melting point apparatus and were uncorrected. NMR spectra were recorded with a Varian 300 MHz spectrometer in CDCl3 with TMS as the internal standard. Electrospray ionization mass spectra (ESIMS) were recorded with a Water’s Micromass ZQ high-performance liquid chromatograph–mass spectrometer (HPLC-MS) or a Water’s Acquity ultraperformance liquid chromatograph–mass spectrometer (UPLC-MS). Infrared spectra (IR) were recorded with a Thermo Scientific Nicolet 6700 FT-IR instrument. 2.2 Synthesis of the title compounds 5 to 43 The general synthetic route for the title compounds is shown in Scheme 1. Representative procedures are given below, and reaction yields were not optimized. New compounds were characterized and verified by 1 H NMR, MS, melting point, and IR for oils and low-melting solids. Many of the structures reported herein were disclosed previously in International Patent Application WO2006/119400A2.18 2.3 Representative procedure for the synthesis of 3-(3-bromo-4-fluorophenyl)-1-(cyclopropylmethyl)-1,2, 4-triazin-6(1H)-one (38) 2.3.1 Synthesis of ethyl 3-bromo-4-fluorobenzimidate hydrochloride (2, R1 = 3-bromo-4-fluoro) To a solution of 3-bromo-4-fluorobenzonitrile (10 g, 50 mmol) in DCM (56 mL) was added ethanol (4.37 mL, 75 mmol), and the mixture was cooled to −5 ∘ C. Dry HCl gas was bubbled in for 1 h, and the resulting mixture was left in the refrigerator for 1 week. The mixture was then added at a rapid dropwise rate to hexanes (300 mL), with stirring, and the resulting solids were collected by filtration and washed with hexanes (100 mL) and ether (100 mL). The solid material was collected and dried under vacuum at room temperature for 3 h to afford 12.6 g (89%) of the title compound as a white solid, mp = 127–128 ∘ C. 1 H NMR (300 MHz, CDCl3 ) 𝛿 12.78 (bs, 1H), 8.67–8.62 (m, 1H), 8.54 (dd, J = 6.3, 2.4 Hz, 1H), 7.34 (t, J = 8.7 Hz, 1H), 4.96 (q, J = 6.9 Hz, 2H), 1.65 (t, J = 7.2 Hz, 3H). ESIMS m/z 248 (M + H)+ .
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2.3.2 Synthesis of benzyl 2-{[(3-bromo-4-fluorophenyl) (ethoxy)methylene]amino}acetate (3, R1 = 3-bromo-4-fluoro) To a suspension of 3-bromo-4-fluorobenzimidate hydrochloride (6.8 g, 24.0 mmol) in diethyl ether (50 mL) was added a saturated aqueous solution of NaHCO3 (50 mL). The mixture was allowed to stir for 5 min and the layers were separated. The ether layer was dried (MgSO4 ), filtered and concentrated in vacuo. The resulting residue was diluted with DCM (80 mL), and solid benzyl glycine p-toluenesulfonic acid salt (8.1 g, 24.0 mmol) was added in one portion. After stirring at room temperature overnight, the white suspension was diluted with ether and water. The layers were
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separated, and the organic layer was dried (MgSO4 ), filtered and concentrated in vacuo to afford 8.9 g (95%) of the title compound as a pink liquid. The residue was carried on without further purification. 1 H NMR (300 MHz, CDCl3 ) 𝛿 7.57 (dd, J = 6.4, 2.1 Hz, 1H), 7.35 (s, 5H), 7.26 (ddd, J = 8.5, 4.6, 2.0 Hz, 1H), 7.11 (t, J = 8.4 Hz, 1H), 5.16 (s, 2H), 4.30 (q, J = 7.1 Hz, 2H), 4.10 (s, 2H), 1.35 (t, J = 7.1 Hz, 3H). ESIMS m/z 394.2 (M + H)+ . 2.3.3 Synthesis of 3-(3-bromo-4-fluorophenyl)-1(cyclopropylmethyl)-4,5-dihydro-1,2,4-triazin-6(1H)-one (4, R1 = 3-bromo-4-fluoro and R2 = cyclopropylmethyl) To a solution of cyclopropylmethylhydrazine hydrochloride19 (1.0 M in EtOH, 3.3 mL, 3.3 mmol) was added triethylamine (1.1 mL, 7.5 mmol). After stirring for 5 min, a solution of 2-{[(3-bromo-4-fluorophenyl)(ethoxy)methylene]amino}acetate (1.18 g, 3.0 mmol) in EtOH (3 mL) was added, and the reaction mixture was heated to 40 ∘ C for 3 h. Upon cooling, the mixture was diluted with EtOAc and washed with water. The organic extract was dried (MgSO4 ), filtered and concentrated in vacuo. The resulting residue was dissolved in DCM (1 mL) followed by hexanes (50 mL) to give a red solid. The solid was collected via filtration to afford 392 mg (40%) of the title compound as a red solid, mp = 109–113 ∘ C. 1 H NMR (300 MHz, CDCl3 ) 𝛿 7.90 (d, J = 6.6 Hz, 1H), 7.61 (bs, 1H), 7.17 (t, J = 8.1 Hz, 1H), 5.06 (bs, 1H), 4.10 (bs, 2H), 3.65 (d, J = 6.9 Hz, 2H), 1.28 (m, 1H), 0.57–0.48 (m, 2H), 0.38 (m, 2H). ESIMS m/z 327.9 (M + H)+ . 2.3.4 Synthesis of 3-(3-bromo-4-fluorophenyl)-1(cyclopropylmethyl)-1,2,4-triazin-6(1H)-one (38) 2,3-Dichloro-5,6-dicyano-p-quinone (DDQ) (115 mg, 0.51 mmol) was added to a solution of 3-(3-bromo-4-fluorophenyl)-1(cyclopropylmethyl)-4,5-dihydro-1,2,4-triazin-6(1H)-one (150 mg, 0.46 mmol) in ethyl acetate (4.6 mL), and the resulting mixture was heated at reflux for 1 h. Upon cooling, the reaction mixture was concentrated in vacuo, and the resulting crude material was diluted with DCM (30 mL) and washed with saturated aqueous Na2 CO3 (2 × 30 mL). The organic extract was dried (MgSO4 ), filtered and concentrated in vacuo. The crude residue was filtered through a small plug of silica gel (DCM as eluent) and concentrated in vacuo to afford 111 mg (74%) of the title compound as a yellow solid, mp = 76–81 ∘ C. 1 H NMR (300 MHz, CDCl3 ) 𝛿 8.45 (s, 1H), 8.37 (dd, J = 6.8, 2.1 Hz, 1H), 8.09 (ddd, J = 8.9, 4.9, 2.3 Hz, 1H), 7.21 (t, J = 8.4 Hz, 1H), 4.02 (d, J = 7.3 Hz, 2H), 1.40 (ddt, J = 10.2, 7.3, 3.8 Hz, 1H), 0.68–0.55 (m, 2H), 0.55–0.43 (m, 2H). ESIMS m/z 325.3 (M + H)+ . The materials listed below (Sections 2.3.5 to 2.3.42) were prepared in a similar fashion. 2.3.5 3-Phenyl-1-(2,2,2-trifluoroethyl)-1,2,4-triazin-6(1H)-one (5) Yielded 400 mg (80%) of a pale-yellow solid, mp = 129–131 ∘ C. 1 H NMR (300 MHz, CDCl3 ) 𝛿 8.57 (s, 1H), 8.15 (dd, J = 6.6, 3.0 Hz, 2H), 7.55–7.38 (m, 3H), 4.77 (q, J = 8.1 Hz, 2H). ESIMS m/z 256.2 (M + H)+ . 2.3.6 3-(2-Fluorophenyl)-1-(2,2,2-trifluoroethyl)-1,2,4-triazin6(1H)-one (6) Yielded 610 mg (82%) of a yellow semi-solid. 1 H NMR (300 MHz, CDCl3 ) 𝛿 8.57 (s, 1H), 7.91 (td, J = 7.8, 1.9 Hz, 1H), 7.52–7.42 (m, 1H), 7.30–7.23 (m, 1H), 7.20 (ddd, J = 11.2, 8.2, 1.2 Hz, 1H). ESIMS m/z 274.2 (M + H)+ . IR (neat) = 3017, 1685, 1574 cm−1 .
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Novel triazinone derivatives as fungicides
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N
1.EtOH, HCl, Et2O
R1
O
R1 1
. TsOH
O
R1
2
O
N O
R1
2. BnOC(O)CH2NH2 DCM
O
O
EtOH 3
N
R1
O
3
HN NH2NH-R2
O
N
1. NaHCO3,Et2O
N
DDQ, EtOAc R2
4
O
N N
R1
N
R2
5
Scheme 1. Synthesis of title compounds 5 to 43.
2.3.7 3-(2-Chlorophenyl)-1-(2,2,2-trifluoroethyl)-1,2,4-triazin6(1H)-one (7) Yielded 350 mg (60%) of a yellow semi-solid. 1 H NMR (300 MHz, CDCl3 ) 𝛿 8.57 (s, 1H), 7.60 (m, 1H), 7.49–7.32 (m, 3H), 4.77 (q, J = 7.8 Hz, 2H). ESIMS m/z 290.6 (M + H)+ . IR (neat) = 3305, 1668, 1624, 1434 cm−1 . 2.3.8 3-(3-Fluorophenyl)-1-(2,2,2-trifluoroethyl)-1,2,4-triazin6(1H)-one (8) Yielded 178 mg (65%) of a gray-green solid, mp = 109–111 ∘ C. 1 H NMR (300 MHz, CDCl3 ) 𝛿 8.57 (s, 1H), 7.95 (d, J = 7.6 Hz, 1H), 7.85 (d, J = 9.9 Hz, 1H), 7.46 (dt, J = 13.9, 7.1 Hz, 1H), 7.19 (dd, J = 9.2, 7.2 Hz, 1H), 4.77 (q, J = 8.2 Hz, 2H). ESIMS m/z 274.3 (M + H)+ . 2.3.9 3-(3-Chlorophenyl)-1-(2,2,2-trifluoroethyl)-1,2,4-triazin6(1H)-one (9) Yielded 1.3 g (90%) of a yellow solid, mp = 129–131 ∘ C. 1 H NMR (300 MHz, CDCl3 ) 𝛿 8.56 (s, 1H), 8.14 (t, J = 1.8 Hz, 1H), 8.03 (dt, J = 7.3, 1.6 Hz, 1H), 7.46 (dt, J = 3.7, 1.6 Hz, 1H), 7.42 (dd, J = 11.6, 4.3 Hz, 1H), 4.77 (q, J = 8.2 Hz, 2H). ESIMS m/z 290.6 (M + H)+ . 2.3.10 3-(3-Bromophenyl)-1-(2,2,2-trifluoroethyl)-1,2,4-triazin6(1H)-one (10) Yielded 123 mg (57%) of a light-brown powder, mp = 104–105 ∘ C. 1 H NMR (300 MHz, CDCl3 ) 𝛿 8.56 (s, 1H), 8.30 (s, 1H), 8.09 (d, J = 7.8 Hz, 1H), 7.62 (d, J = 8.1 Hz, 1H), 7.36 (t, J = 8.0 Hz, 1H), 4.77 (q, J = 8.2 Hz, 2H). ESIMS m/z 335.2 (M + H)+ . 2.3.11 3-(m-Tolyl)-1-(2,2,2-trifluoroethyl)-1,2,4-triazin6(1H)-one (11) Yielded 2.4 g (98%) of a gold solid, mp = 97–98 ∘ C. 1 H NMR (300 MHz, CDCl3 ) 𝛿 8.54 (s, 1H), 7.85 (d, J = 7.8 Hz, 1H), 7.43–7.18 (m, 3H), 4.77 (q, J = 8.2 Hz, 2H), 2.39 (s, 3H). ESIMS m/z 270.5 (M + H)+ .
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2.3.14 3-(4-Bromophenyl)-1-(2,2,2-trifluoroethyl)-1,2,4-triazin6(1H)-one (14) Yielded 67 mg (95%) of a yellow solid, mp = 123–125 ∘ C. 1 H NMR (300 MHz, CDCl3 ) 𝛿 8.55 (s, 1H), 8.01 (dt, J = 8.7, 2.1 Hz, 2H), 7.61 (dt, J = 8.7, 2.1 Hz, 2H), 4.76 (q, J = 8.4 Hz, 2H). ESIMS m/z 335.2 (M + H)+ . 2.3.15 3-(p-Tolyl)-1-(2,2,2-trifluoroethyl)-1,2,4-triazin6(1H)-one (15) Yielded 112 mg (41%) of a light-yellow solid, mp = 190–192 ∘ C. 1 H NMR (300 MHz, CDCl3 ) 𝛿 8.54 (s, 1H), 8.03 (d, J = 8.1 Hz, 2H), 7.28 (d, J = 8.1 Hz, 2H), 4.76 (q, J = 8.2 Hz, 2H), 2.42 (s, 3H). ESIMS m/z 270.2 (M + H)+ . 2.3.16 1-(2,2,2-Trifluoroethyl)-3-[4-(trifluoromethyl)phenyl]1,2,4-triazin-6(1H)-one (16) Yielded 27 mg (31%) of a light-brown solid, mp = 91–92 ∘ C. 1 H NMR (300 MHz, CDCl3 ) 𝛿 8.59 (s, 1H), 8.27 (d, J = 8.4 Hz, 2H), 7.74 (d, J = 8.4 Hz, 2H), 4.79 (q, J = 8.2 Hz, 2H). ESIMS m/z 324.2 (M + H)+ . 2.3.17 3-(3,5-Dibromophenyl)-1-(2,2,2-trifluoroethyl)-1,2,4-triazin6(1H)-one (17) Yielded 238 mg (73%) of a light-brown solid, mp = 139–141 ∘ C. 1 H NMR (300 MHz, CDCl3 ) 𝛿 8.56 (s, 1H), 8.23 (s, 2H), 7.78 (s, 1H), 4.77 (q, J = 8.1 Hz, 2H). ESIMS m/z 414.2 (M + H)+ . 2.3.18 3-(3-Bromo-4-methylphenyl)-1-(2,2,2-trifluoroethyl)1,2,4-triazin-6(1H)-one (18) Yielded 111 mg (32%) of a yellow solid, mp = 102–103 ∘ C. 1 H NMR (300 MHz, CDCl3 ) 𝛿 8.55 (s, 1H), 8.31 (s, 1H), 7.97 (d, J = 7.8 Hz, 1H), 7.33 (d, J = 8.1 Hz, 1H), 4.76 (q, J = 8.2 Hz, 2H), 2.46 (s, 3H). ESIMS m/z 349.2 (M + H)+ . 2.3.19 3-(3-Bromo-4-chlorophenyl)-1-(2,2,2-trifluoroethyl)1,2,4-triazin-6(1H)-one (19) Yielded 134 mg (88%) of a yellow solid, mp = 124–125 ∘ C. 1 H NMR (300 MHz, CDCl3 ) 𝛿 8.55 (s, 1H), 8.35 (s, 1H), 8.09 (d, J = 8.5 Hz, 1H),
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2.3.12 1-(2,2,2-Trifluoroethyl)-3-[3-(trifluoromethyl)phenyl]1,2,4-triazin-6(1H)-one (12) Yielded 208 mg (55%) of an off-white solid, mp = 74–75 ∘ C. 1 H NMR (300 MHz, CDCl3 ) 𝛿 8.59 (s, 1H), 8.42 (s, 1H), 8.34 (d, J = 7.8 Hz, 1H), 7.75 (d, J = 7.8 Hz, 1H), 7.62 (t, J = 7.8 Hz, 1H), 4.80 (q, J = 8.2 Hz, 2H). ESIMS m/z 324.5 (M + H)+ .
2.3.13 3-(4-Chlorophenyl)-1-(2,2,2-trifluoroethyl)-1,2,4-triazin6(1H)-one (13) Yielded 460 mg (79%) of a brown solid, mp = 110–115 ∘ C. 1 H NMR (300 MHz, CDCl3 ) 𝛿 8.54 (s, 1H), 8.08 (d, J = 8.6 Hz, 2H), 7.44 (d, J = 8.8 Hz, 2H), 4.76 (q, J = 8.2 Hz, 2H). ESIMS m/z 290.5 (M + H)+ .
www.soci.org 7.52 (t, J = 8.5 Hz, 1H), 4.76 (q, J = 8.1 Hz, 2H). ESIMS m/z 369.7 (M + H)+ . 2.3.20 3-(3-Bromo-4-fluorophenyl)-1-(2,2,2-trifluoroethyl)1,2,4-triazin-6(1H)-one (20) Yielded 447 mg (79%) of a yellow solid, mp = 97–99 ∘ C. 1 H NMR (300 MHz, CDCl3 ) 𝛿 8.55 (s, 1H), 8.37 (dd, J = 6.6, 2.4 Hz, 1H), 8.09 (ddd, J = 8.7, 4.5, 2.1 Hz, 1H), 7.22 (t, J = 8.4 Hz, 1H), 4.76 (q, J = 8.1 Hz, 2H). ESIMS m/z 353.2 (M + H)+ . 2.3.21 3-(3-Bromo-4-fluorophenyl)-1-(1,1,1-trifluoropropan2-yl)-1,2,4-triazin-6(1H)-one (21) Yielded 37 mg (35%) of a brown solid, mp = 129–131 ∘ C. 1 H NMR (300 MHz, CDCl3 ) 𝛿 8.55 (s, 1H), 8.37 (dd, J = 6.6, 2.4 Hz, 1H), 8.09 (ddd, J = 8.7, 4.5, 2.1 Hz, 1H), 7.22 (t, J = 8.4 Hz, 1H), 5.69 (p, J = 7.1 Hz, 1H), 1.87 (d, J = 7.1 Hz, 3H). ESIMS m/z 367.2 (M + H)+ . 2.3.22 3-(3-Bromo-4-fluorophenyl)-1-(3,3,3-trifluoropropyl)1,2,4-triazin-6(1H)-one (22) Yielded 75 mg (18%) of a yellow solid, mp = 80–81 ∘ C. 1 H NMR (300 MHz, CDCl3 ) 𝛿 8.49 (s, 1H), 8.37 (dd, J = 6.9, 2.4 Hz, 1H), 8.09 (ddd, J = 7.2, 4.8, 2.4 Hz, 1H), 7.21 (t, J = 8.7 Hz, 1H), 4.42 (t, J = 7.2 Hz, 2H), 2.77 (m, 2H). ESIMS m/z 367.2 (M + H)+ . 2.3.23 3-(3-Bromo-4-fluorophenyl)-1-[3,3,3-trifluoro-2(trifluoromethyl)propyl]-1,2,4-triazin-6(1H)-one (23) Yielded 35 mg (44%) of a yellow solid, mp = 105–107 ∘ C. 1 H NMR (300 MHz, CDCl3 ) 𝛿 8.52 (s, 1H), 8.37 (dd, J = 6.6, 2.2 Hz, 1H), 8.09 (ddd, J = 8.8, 4.7, 2.2 Hz, 1H), 7.21 (t, J = 8.5 Hz, 1H), 4.62 (d, J = 6.5 Hz, 2H), 3.95 (m, 1H). ESIMS m/z 435.3 (M + H)+ . 2.3.24 3-(3-Bromo-4-fluorophenyl)-1-(2-fluoroethyl)-1,2,4-triazin6(1H)-one (24) Yielded 387 mg (72%) of a yellow solid, mp = 117–119 ∘ C. 1 H NMR (300 MHz, CDCl3 ) 𝛿 8.51 (s, 1H), 8.39 (dd, J = 6.6, 2.2 Hz, 1H), 8.11 (ddd, J = 8.8, 4.7, 2.2 Hz, 1H), 7.21 (t, J = 8.5 Hz, 1H), 4.97 (dd, J = 5.2, 4.4 Hz, 1H), 4.82 (dd, J = 5.2, 4.7 Hz, 1H), 4.50 (dt, J = 25.0, 4.8 Hz, 2H). ESIMS m/z 317.2 (M + H)+ . 2.3.25 3-(3-Bromo-4-fluorophenyl)-1-butyl-1,2,4-triazin-6(1H)one (25) Yielded 220 mg (88%) of a yellow solid, mp = 62–64 ∘ C. 1 H NMR (300 MHz, CDCl3 ) 𝛿 8.44 (s, 1H), 8.37 (dd, J = 6.7, 2.1 Hz, 1H), 8.09 (ddd, J = 8.6, 4.8, 2.2 Hz, 1H), 7.20 (t, J = 8.4 Hz, 1H), 4.16 (t, J = 7.2 Hz, 2H), 1.94–1.78 (m, 2H), 1.53–1.34 (m, 2H), 0.99 (t, J = 7.4 Hz, 3H). ESIMS m/z 327.3 (M + H)+ . 2.3.26 3-(3-Bromo-4-fluorophenyl)-1-pentyl-1,2,4-triazin6(1H)-one (26) Yielded 72 mg (42%) of a yellow semi-solid. 1 H NMR (300 MHz, CDCl3 ) 𝛿 8.45 (s, 1H), 8.37 (dd, J = 6.7, 2.1 Hz, 1H), 8.10 (ddd, J = 8.6, 4.8, 2.2 Hz, 1H), 7.20 (t, J = 8.4 Hz, 1H), 4.15 (t, J = 7.1 Hz, 2H), 1.95–1.81 (m, 2H), 1.51–1.29 (m, 4H), 0.89 (t, J = 7.4 Hz, 3H). ESIMS m/z 341.5 (M + H)+ . IR (neat) = 2953, 1671, 1565 cm−1 .
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2.3.27 3-(3-Bromo-4-fluorophenyl)-1-hexyl-1,2,4-triazin-6(1H)-one (27) Yielded 33 mg (51%) of a yellow wax. 1 H NMR (300 MHz, CDCl3 ) 𝛿 8.44 (s, 1H), 8.37 (dd, J = 6.7, 2.1 Hz, 1H), 8.09 (ddd, J = 8.6, 4.8, 2.2 Hz, 1H), 7.20 (t, J = 8.4 Hz, 1H), 4.16 (t, J = 7.1 Hz, 2H), 1.92–1.79 (m, 2H), 1.48–1.25 (m, 6H), 0.84 (t, J = 7.4 Hz, 3H). ESIMS m/z 355.2 (M + H)+ . IR (neat) = 2952, 1671, 1565 cm−1 .
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2.3.28 3-(3-Bromo-4-fluorophenyl)-1-(2-ethoxyethyl)-1,2,4-triazin6(1H)-one (28) Yielded 216 mg (49%) of a yellow solid, mp = 91–93 ∘ C. 1 H NMR (300 MHz, CDCl3 ) 𝛿 8.47 (s, 1H), 8.39 (dd, J = 6.7, 2.1 Hz, 1H), 8.11 (ddd, J = 8.8, 4.7, 2.2 Hz, 1H), 7.21 (t, J = 8.5 Hz, 1H), 4.38 (t, J = 5.6 Hz, 2H), 3.91 (t, J = 5.6 Hz, 2H), 3.57 (q, J = 7.0 Hz, 2H), 1.19 (t, J = 7.0 Hz, 3H). ESIMS m/z 343.3 (M + H)+ . 2.3.29 3-(3-Bromo-4-fluorophenyl)-1-isopropyl-1,2,4-triazin6(1H)-one (29) Yielded 273 mg (66%) of a yellow solid, mp = 107–108 ∘ C. 1 H NMR (300 MHz, CDCl3 ) 𝛿 8.44 (s, 1H), 8.39 (dd, J = 6.9, 2.2 Hz, 1H), 8.13 (ddd, J = 8.8, 4.7, 2.2 Hz, 1H), 7.21 (t, J = 8.5 Hz, 1H), 5.29–5.17 (m, 1H), 1.48 (d, J = 6.6 Hz, 7H). ESIMS m/z 313.2 (M + H)+ . 2.3.30 3-(3-Bromo-4-fluorophenyl)-1-(sec-butyl)-1,2,4-triazin6(1H)-one (30) Yielded 342 mg (74%) of a yellow solid, mp = 84–86 ∘ C. 1 H NMR (300 MHz, CDCl3 ) 𝛿 8.46 (d, J = 0.8 Hz, 1H), 8.38 (dd, J = 6.6, 2.2 Hz, 1H), 8.12 (ddd, J = 8.8, 4.7, 2.2 Hz, 1H), 7.23 (t, J = 8.4 Hz, 2H), 5.03 (dd, J = 14.6, 6.6 Hz, 1H), 1.88 (dtd, J = 20.1, 13.7, 7.1 Hz, 2H), 1.45 (d, J = 6.6 Hz, 3H), 0.92 (t, J = 7.4 Hz, 3H). ESIMS m/z 327.4 (M + H)+ . 2.3.31 3-(3-Bromo-4-fluorophenyl)-1-cyclopentyl-1,2,4-triazin6(1H)-one (31) Yielded 368 mg (77%) of a yellow solid, mp = 115–117 ∘ C. 1 H NMR (300 MHz, CDCl3 ) 𝛿 8.43 (d, J = 0.8 Hz, 1H), 8.37 (dd, J = 6.9, 2.2 Hz, 1H), 8.10 (ddd, J = 8.5, 4.7, 2.2 Hz, 1H), 7.22 (t, J = 8.5 Hz, 1H), 5.35 (ddd, J = 9.1, 8.0, 4.7 Hz, 1H), 2.23–2.08 (m, 2H), 2.00 (m, 4H), 1.78 (m, 2H). ESIMS m/z 339.2 (M + H)+ . 2.3.32 3-(3-Bromo-4-fluorophenyl)-1-(prop-2-yn-1-yl)1,2,4-triazin-6(1H)-one (32) Yielded 295 mg (95%) of a light-brown solid, mp = 132–137 ∘ C. 1 H NMR (300 MHz, CDCl3 ) 𝛿 8.50 (s, 1H), 8.40 (dd, J = 6.7, 2.1 Hz, 1H), 8.12 (ddd, J = 8.6, 4.8, 2.3 Hz, 1H), 7.21 (t, J = 8.5 Hz, 1H), 4.92 (d, J = 2.8 Hz, 2H), 2.43 (t, J = 2.5 Hz, 1H). ESIMS m/z 309.3 (M + H)+ . 2.3.33 3-(3-Bromo-4-fluorophenyl)-1-(but-2-yn-1-yl)-1,2,4triazin-6(1H)-one (33) Yielded 30 mg (50%) of an off-white solid, mp = 145–147 ∘ C. 1 H NMR (300 MHz, CDCl3 ) 𝛿 8.48 (s, 1H), 8.40 (dd, J = 6.6, 2.3 Hz, 1H), 8.12 (ddd, J = 8.6, 4.8, 2.3 Hz, 1H), 7.21 (t, J = 8.5 Hz, 1H), 4.87 (q, J = 2.5 Hz, 2H), 1.85 (t, J = 2.5 Hz, 3H). ESIMS m/z 323.2 (M + H)+ . 2.3.34 3-(3-Bromo-4-fluorophenyl)-1-(pent-2-yn-1-yl)-1,2,4-triazin6(1H)-one (34) Yielded 369 mg (81%) of a white solid, mp = 97–101 ∘ C. 1 H NMR (300 MHz, CDCl3 ) 𝛿 8.50 (s, 1H), 8.43 (dd, J = 6.9, 2.2 Hz, 1H), 8.14 (ddd, J = 8.5, 4.7, 2.2 Hz, 1H), 7.21 (t, J = 8.5 Hz, 1H), 4.91 (t, J = 2.3 Hz, 2H), 2.25 (qt, J = 7.4, 2.2 Hz, 2H), 1.17 (t, J = 7.6 Hz, 3H). ESIMS m/z 337.3 (M + H)+ . 2.3.35 1-Benzyl-3-(3-bromo-4-fluorophenyl)-1,2,4-triazin-6(1H)-one (35) Yielded 159 mg (80%) of a brown solid, mp = 146–148 ∘ C. 1 H NMR (300 MHz, CDCl3 ) 𝛿 8.45 (s, 1H), 8.36 (dd, J = 6.7, 2.1 Hz, 1H), 8.08 (ddd, J = 8.8, 4.8, 2.3 Hz, 1H), 7.50 (dd, J = 8.0, 1.6 Hz, 2H), 7.40–7.32 (m, 3H), 7.23–7.16 (m, 1H), 5.30 (s, 2H). ESIMS m/z 361.2 (M + H)+ .
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2.3.36 3-(3-Bromo-4-fluorophenyl)-1-(1-phenylethyl)-1,2,4triazin-6(1H)-one (36) Yielded 47 mg (35%) of a yellow oil. 1 H NMR (300 MHz, CDCl3 ) 𝛿 8.42 (s, 1H), 8.35 (dd, J = 6.7, 2.1 Hz, 1H), 8.07 (ddd, J = 8.6, 4.5, 2.0 Hz, 1H), 7.51–7.47 (m, 2H), 7.35 (dt, J = 13.1, 7.1 Hz, 3H), 7.20 (t, J = 8.3 Hz, 1H), 6.23 (q, J = 7.1 Hz, 1H), 1.87 (d, J = 7.1 Hz, 3H). ESIMS m/z 375.2 (M + H)+ . IR (neat) = 2927, 1670, 1567 cm−1 . 2.3.37 3-(3-Bromo-4-fluorophenyl)-1-(2-phenylpropyl)1,2,4-triazin-6(1H)-one (37) Yielded 190 mg (70%) of a yellow semi-solid. 1 H NMR (300 MHz, CDCl3 ) 𝛿 8.38 (s, 1H), 8.16 (dd, J = 6.7, 2.1 Hz, 1H), 7.93 (ddd, J = 8.8, 4.8, 2.3 Hz, 1H), 7.35–7.11 (m, 6H), 4.32 (ddd, J = 21.2, 12.8, 7.8 Hz, 2H), 3.48 (dd, J = 15.2, 7.3 Hz, 1H), 1.38 (d, J = 6.8 Hz, 3H). ESIMS m/z 389.3 (M + H)+ . IR (neat) = 2964, 1670, 1566 cm−1 . 2.3.38 3-(3-Bromo-4-fluorophenyl)-1-[(2-methylcyclopropyl) methyl]-1,2,4-triazin-6(1H)-one (39) Yielded 335 mg (84%) of a white solid, mp = 109–111 ∘ C. 1 H NMR (300 MHz, CDCl3 ) 𝛿 8.45 (s, 1H), 8.38 (dd, J = 6.7, 2.1 Hz, 1H), 8.10 (ddd, J = 8.8, 4.8, 2.3 Hz, 1H), 7.21 (t, J = 8.3 Hz, 1H), 4.02 (ddd, J = 47.2, 13.4, 7.5 Hz, 2H), 1.11 (ddd, J = 11.1, 7.6, 3.3 Hz, 1H), 1.06 (d, J = 5.8 Hz, 3H), 0.97–0.83 (m, 1H), 0.69–0.59 (m, 1H), 0.34 (dt, J = 8.1, 4.9 Hz, 1H). ESIMS m/z 339.4 (M + H)+ . 2.3.39 3-(3-Bromo-4-fluorophenyl)-1-[(2,2-dichlorocyclopropyl) methyl]-1,2,4-triazin-6(1H)-one (40) Yielded 30 mg (41%) of a clear viscous glass. 1 H NMR (300 MHz, CDCl3 ) 𝛿 8.45 (s, 1H), 8.37 (dd, J = 6.8, 2.1 Hz, 1H), 8.09 (ddd, J = 8.9, 4.9, 2.3 Hz, 1H), 7.21 (t, J = 8.5 Hz, 1H), 4.35 (m, 2H), 2.25 (m, 1H), 1.79 (m, 1H), 1.56 (m, 1H). ESIMS m/z 394.2 (M + H)+ . IR (neat) = 2954, 1672, 1566 cm−1 . 2.3.40 3-(3-Bromo-4-fluorophenyl)-1-(cyclobutylmethyl)-1,2,4triazin-6(1H)-one (41) Yielded 282 mg (67%) of a yellow solid, mp = 111–112 ∘ C. 1 H NMR (300 MHz, CDCl3 ) 𝛿 8.46 (d, J = 0.5 Hz, 1H), 8.38 (dd, J = 6.6, 2.2 Hz, 1H), 8.10 (ddd, J = 8.8, 4.7, 2.2 Hz, 1H), 7.22 (t, J = 8.5 Hz, 1H), 4.22 (d, J = 7.4 Hz, 2H), 3.02–2.82 (m, 1H), 2.13 (ddd, J = 10.2, 7.8, 2.6 Hz, 2H), 2.05–1.72 (m, 4H). ESIMS m/z 256.2 (M + H)+ . 2.3.41 3-(3-Bromo-4-fluorophenyl)-1-(cyclopentylmethyl)-1,2,4triazin-6(1H)-one (42) Yielded 36 mg (16%) of an orange wax. 1 H NMR (300 MHz, CDCl3 ) 𝛿 8.46 (d, J = 0.5 Hz, 1H), 8.38 (dd, J = 6.6, 2.2 Hz, 1H), 8.10 (ddd, J = 8.8, 4.7, 2.2 Hz, 1H), 7.21 (t, J = 8.5 Hz, 1H), 4.15 (d, J = 7.4 Hz, 2H), 2.57 (m, 1H), 1.81–1.57 (m, 4H), 1.45–1.21 (m, 4H). ESIMS m/z 353.3 (M + H)+ . IR (neat) = 2952, 1671, 1565 cm−1 . 2.3.42 3-(3-Bromo-4-fluorophenyl)-1-(2-cyclopropylethyl)-1,2,4triazin-6(1H)-one (43) Yielded 35 mg (28%) of an orange solid, mp = 90–91 ∘ C. 1 H NMR (300 MHz, CDCl3 ) 𝛿 8.45 (s, 1H), 8.37 (dd, J = 6.8, 2.1 Hz, 1H), 8.09 (ddd, J = 8.9, 4.9, 2.3 Hz, 1H), 7.21 (t, J = 8.5 Hz, 1H), 4.25 (t, J = 7.3 Hz, 2H), 1.79 (q, J = 7.3 Hz, 2H), 0.79 (m, 1H), 0.49 (m, 2H), 0.09 (m, 2H). ESIMS m/z 339.4 (M + H)+ .
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3
RESULTS AND DISCUSSION
3.1 Synthesis and characterization Scheme 1 describes the synthesis of the target compounds. The standard formation of ethyl imidate salts 2 from substituted benzonitriles (1) was followed by acid-catalyzed transimination with benzyl glycine p-toluenesulfonic acid salt to give glycine imidates 3. Condensation of 3 with substituted hydrazines19 provided the 4,5-dihydro-1,2,4-triazin-6(1H)-one intermediates 4. Subsequent oxidation of the dihydro intermediates with 2,3-dichloro-5,6-dicyano-p-benzoquinone (DDQ) afforded the title compounds (5 to 43). 3.2 In vitro antifungal activity Initial synthesis efforts aimed at optimizing antifungal activity of this chemistry focused on examining the electronic and steric tolerances of the 3-phenyl component by incorporating halo, alkyl and haloalkyl substituents, key examples of which are presented in Table 1. To enable better evaluation of the biological impact of each substituent addition to the 3-phenyl group, the R2 component was held constant as trifluoroethyl. Although the EC50 values indicate some differences in sensitivity among the test fungi, compounds 5 to 16 show a consistent relative efficacy trend across the four-pathogen spectrum that is represented by the values in Table 1 for the four-pathogen mean. To examine the effect of bis-substitution on the phenyl ring while maintaining bromine in the 3-position, a small series of
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2.4 Fungal growth inhibition assays Antifungal assays against L. nodorum, Z. tritici, M. grisea and U. maydis were carried out in 96-well flat-bottomed microtitre
plates (Techno Plastic Products, Trasadingen, Switzerland), with each well containing a total assay volume of 200 𝜇L. The growth medium employed was Difco YNB (6.7 g L−1 , yeast nitrogen base without amino acids; BD Diagnostic Systems, Sparks, MD) supplemented with 2 g of glucose and 3 g each of potassium dihydrogen phosphate and dipotassium hydrogen phosphate L−1 . Initial inoculum density was adjusted to 200 000 spores mL−1 for L. nodorum, 100 000 spores mL−1 for Z. tritici and 40 000 spores mL−1 for M. grisea. For inoculum of U. maydis, a liquid culture inoculated the day before testing was grown to an absorbance reading of 0.2, measured using a Spectronic20D+ spectrophotometer (Milton Roy, Warminster, PA) set at a wavelength of 450 nm, and then diluted 500-fold with the supplemented YNB medium to give a spore concentration of 10 000 spores mL−1 . Serial dilutions of each test compound were prepared using stock solutions in DMSO, and 2 𝜇L aliquots were added to the wells. To enable better comparison of analogs differing significantly in biological activity, each compound was evaluated in three separate tests, each involving an 8 rate dose response, which provided a total of 19 treatment concentrations ranging from 40 to 0.0001 ppm. Immediately after addition of spore suspensions, initial cell density readings were determined using a NepheloStar nephelometer (BMG LABTECH Gmb, Ortenberg, Germany). After incubation in the dark at 90 rpm in a New Brunswick Innova 44 shaking incubator (Eppendorf, Inc., Enfield, CT) for 48 h (L. nodorum, M. grisea and U. maydis) or 72 h (Z. tritici) at 22 ∘ C (L. nodorum, M. grisea and Z. tritici) or 24 ∘ C (U. maydis), conditions under which evaporation from wells was minimal, the plates were read in the NepheloStar a second time to assess growth. Percentage growth inhibition was calculated by reference to control wells containing growth media and inoculum amended with 1% DMSO. Fungicide sensitivities were determined as 50% effective concentration (EC50 ) using a dose–response relationship provided by the logistic regression curve fit tool within Spotfire (TIBCO Software Inc., Palo Alto, CA).
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Table 1. Antifungal activity of 3-substituted phenyl-N-1-trifluoroethyl-1,2,4-triazinones EC50 values (μg mL−1 )
Substituents Compound number 5 6 7 8 9 10 11 12 13 14 15 16 Azoxystrobin Tebuconazole a b
R1
R2
H 2-F 2-Cl 3-F 3-Cl 3-Br 3-CH3 3-CF3 4-Cl 4-Br 4-CF3 4-CH3
L. nodorum
CH2 CF3 CH2 CF3 CH2 CF3 CH2 CF3 CH2 CF3 CH2 CF3 CH2 CF3 CH2 CF3 CH2 CF3 CH2 CF3 CH2 CF3 CH2 CF3
0.91 2.82 10.72 1.35 0.20 0.08 0.25 0.04 0.81 0.08 1.51 2.14 0.011 0.063
M. grisea
Z. tritici
3.47 8.12 16.98 3.89 0.24 0.06 0.25 0.01 0.23 0.17 0.15 0.26 0.009 0.18
0.74 1.78 3.72 0.96 0.06 0.05 0.12 0.06 1.51 2.29 2.24 1.66 0.003 0.03
U. maydis 0.91 2.14 2.09 2.14 0.28 0.13 0.65 0.28 0.28 0.18 0.21 0.69 0.005 0.002
Ascomycete meana
Four-pathogen meanb
1.71 4.24 10.47 2.06 0.16 0.06 0.20 0.04 0.85 0.85 1.30 1.35
1.51 3.72 8.38 2.08 0.19 0.08 0.32 0.10 0.71 0.68 1.03 1.19
Ascomycete meana
Four-pathogen meanb
Mean of the EC50 values for L. nodorum, M. grisea and Z. tritici. Mean of the EC50 values across all four pathogens tested.
Table 2. Antifungal activity of 3-bromophenyl-N-1-trifluoroethyl-1,2,4-triazinones EC50 Values (μg mL−1 )
Substituents Compound number 10 17 18 19 20 Azoxystrobin Tebuconazole a b
R1 3-Br 3,5-Br2 3-Br-4-Me 3-Br-4-Cl 3-Br-4-F
R2 CH2 CF3 CH2 CF3 CH2 CF3 CH2 CF3 CH2 CF3
L. nodorum 0.08 0.17 0.13 0.02 0.06 0.008 0.082
M. grisea
Z. tritici
0.06 0.02 0.01 0.02 0.01 0.011 0.224
0.05 0.15 0.11 0.09 0.04 0.003 0.051
U. maydis 0.13 0.06 0.13 0.07 0.11 0.003 0.002
0.06 0.11 0.08 0.04 0.04
0.08 0.10 0.10 0.05 0.05
Mean of the EC50 values for L. nodorum, M. grisea and Z. tritici. Mean of the EC50 values across all four pathogens tested.
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disubstituted phenyl analogs (17 to 20) of compound 10 were prepared and tested. The addition of a symmetrical bromine atom (17) at the 5-position or a methyl (18), chloro (19) or fluoro (20) group at the 4-position resulted in relatively minor changes to antifungal potency, the magnitude of which varied for the individual pathogens (Table 2). This suggested that substitution at the 3-position of the phenyl was the more critical determinant of antifungal activity. Based on these data, synthetic efforts were then directed at exploring the effects on activity of replacing the trifluoroethyl group at R2 with a variety of alternative chemistries. While all 3-bromophenyl analogs were similarly potent, based on both ascomycete and four-pathogen mean EC50 values (Table 2), the 3-bromo-4-fluorophenyl compound (20) was selected for R2 optimization efforts owing to its stronger performance against Z. tritici, a pathogen of key importance in cereal production. Towards this end, a set of 23 compounds were prepared to explore the impact on antifungal activity of structural variations at R2 by branching, homologation and replacement of the haloalkyl moiety with
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alkyls, branched alkyls, alkynes, benzyls and cycloalkyl groups (Table 3). With the exception of the cyclopentyl derivative (compound 31), all trifluoroethyl replacements resulted in potency reduction relative to compound 20 against U. maydis, with the ethoxyethyl compound (28) reducing activity against U. maydis 30-fold. While a few compounds (e.g. compounds 23, 27 and 31) had U. maydis activity similar to that against the other fungi tested, the majority of the R2 replacements (compounds 21 to 43) resulted in loss of U. maydis potency while maintaining or contributing modest activity enhancements (e.g. compounds 25, 35, 38 and 41) on the ascomycetes. This differential in sensitivity between U. maydis and the other fungi is generally in the 5–10-fold range, but for the most active compounds of the series, the CH2 -cycloalkyls 38 and 41, and the n-pentyl derivative (26), the difference is 40–60 fold. Z. tritici was considerably less sensitive to alkynyls at R2 (compounds 33 and 34 in particular) and L. nodorum to the 2-Me phenethyl analog (compound 37), but, otherwise, the three ascomycetes were similar in sensitivity to compounds 21 to 43.
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Novel triazinone derivatives as fungicides
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Table 3. Antifungal activity of 3-bromo-4-fluorophenyl-1,2,4-triazinones variously substituted at N-1 Substituents (R1 = 3-Br-4-F) Compound number
R2
20 CH2 CF3 21 CH(CH3 )CF3 22 CH2 CH2 CF3 23 CH2 CH(CF3 )2 24 CH2 CH2 F 25 n-Butyl 26 n-Pentyl 27 n-Hexyl 28 CH2 CH2 OCH2 CH3 29 i-Propyl 30 i-Butyl 31 Cyclopentyl 32 2-Propynyl 33 2- Butynyl 34 2-Pentynyl 35 Benzyl 36 𝛼-Me benzyl 37 2-Me phenethyl 38 CH2 -cyclopropyl 39 CH2 -2-Me cyclopropyl 40 CH2 -2,2-di-Cl-cyclopropyl 41 CH2 -cyclobutyl 42 CH2 -cyclopentyl 43 CH2 CH2 -cyclopropyl Azoxystrobin Tebuconazole a b
EC50 values (𝜇g mL−1 ) L. nodorum 0.06 0.04 0.06 0.07 0.36 0.03 0.04 0.62 0.30 0.08 0.04 0.07 0.03 0.03 0.07 0.03 0.08 1.70 0.02 0.05 0.37 0.02 0.05 0.04 0.006 0.048
M. grisea
Z. tritici
0.01 0.09 0.05 0.21 0.20 0.01 0.02 0.25 0.13 0.28 0.10 0.13 0.02 0.02 0.06 0.02 0.27 0.13 0.01 0.02 0.05 0.01 0.03 0.02 0.007 0.209
0.04 0.08 0.05 0.17 0.72 0.01 0.07 0.91 0.56 0.17 0.01 0.03 0.16 0.34 0.87 0.02 0.14 0.85 0.005 0.01 0.98 0.02 0.15 0.02 0.002 0.046
Ascomycete meana
0.11 0.15 0.40 0.17 0.71 0.26 2.51 0.45 3.09 0.83 0.16 0.10 1.00 0.45 0.24 0.19 0.43 0.15 0.51 0.35 1.82 0.44 0.54 0.40 0.002 0.001
0.04 0.07 0.06 0.15 0.43 0.02 0.04 0.59 0.33 0.17 0.05 0.07 0.07 0.13 0.34 0.02 0.17 0.89 0.01 0.03 0.47 0.01 0.08 0.03
Four-pathogen meanb 0.05 0.09 0.14 0.16 0.50 0.08 0.66 0.56 1.02 0.34 0.08 0.08 0.30 0.21 0.31 0.06 0.23 0.71 0.14 0.11 0.80 0.12 0.19 0.12
Ascomycete 4 7 6 10 14 2 4 16 12 11 5 7 7 9 13 2 11 17 1 3 15 1 8 3
U. maydis 2 3 10 5 16 8 20 13 21 17 4 1 18 13 7 6 11 3 14 9 19 12 15 10
Mean of the EC50 values for L. nodorum, M. grisea and Z. tritici. Mean of the EC50 values across all four pathogens tested.
EC50 values generated for each pathogen averaged across compounds 20 to 43 were 0.09, 0.18 and 0.27 for M. grisea, L. nodorum and Z. tritici, respectively, compared with 0.64 for U. maydis. In addition to the greater sensitivity of ascomycetes to the majority of compounds 21 to 43, Table 3 reveals evidence of a divergence of structure–activity relationships between the ascomycete panel and the basidiomycete U. maydis. This is particularly exemplified by compounds 38 and 41, which were the most inhibitory to L. nodorum, M. grisea and Z. tritici, but considerably less active against U. maydis, and by the 2-Me phenethyl derivative (compound 37), which was ranked third most active against U. maydis but was among the weakest against ascomycetes. The reasons for the pathogen sensitivity differences and diverging structure–activity relationships focused on R2 substitution are not known, but may reflect target-site or other biochemical differences among diverse fungi, or differing physical property requirements for target-site access. However, the biochemical target site and mechanism of action of the 3-aryl-1,2,4-triazin-6-ones remain unknown.
4
U. maydis
Fungitoxicity rank
CONCLUSION
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The 3-aryl-1,2,4-triazin-6-ones represent a novel series of chemistry with high levels of in vitro antifungal activity across a spectrum of agriculturally relevant plant-pathogenic fungi.
Structure–activity relationship studies indicated a preference for substitution at the 3-position of the phenyl group attached at the C-3 carbon of the triazinone ring, particularly with electron-withdrawing groups such as Br, Cl and CF3 . Substitutions at other positions on the phenyl ring were less well tolerated. Exploration of the effects of varying the R2 group attached at N-1 of the triazinone ring revealed a diverging structure–activity relationship between the basidiomycete pathogen, U. maydis, and the ascomycetes L. nodorum, M. grisea and Z. tritici. For example, growth inhibition activity on U. maydis was generally reduced by replacement of the trifluoroethyl group at N-1 with various CH2 -cycloalkyls, while the CH2 -cyclopropyl (38) and CH2 -cyclobutyl (41) analogs were the most active against the ascomycete pathogens. With an EC50 of 0.005 ppm on Z. tritici, compound 38 demonstrated antifungal activity on this key cereal pathogen that was comparable with that of the commercial strobilurin fungicide azoxystrobin (EC50 0.002 ppm) and significantly better than that of the triazole tebuconazole (EC50 0.046 ppm). However, 38 was 100 times less active against U. maydis (EC50 0.51 ppm), which was generally the least sensitive pathogen to this triazinone chemistry. The basis of this difference in sensitivity is unknown and warrants further investigation. The in vitro antifungal activity data summarized in this paper represent an initial phase of a broader biological characterization program for
www.soci.org this novel class of fungicides, which included higher-tier in planta greenhouse testing. Results from whole-plant evaluation of the 3-aryl-1,2,4-triazin-6-ones will be presented in a subsequent paper.
ACKNOWLEDGEMENTS The authors would like to acknowledge and thank S Shaber, R Ross, WR Erickson, MJ Kelly, I Morrison, B Rieder, D Weaver and M Yap for their contributions to the development of this chemistry.
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