Fluorine analogs of dicamba and tricamba herbicides; synthesis and their pesticidal activity

4.1 General

2,5-Difluorophenol, 2,4,5-trifluorophenol, dimethyl sulfate, boron tribromide, as well as n-butyllithium and LDA solutions were commercially available and used as received.

Chloromethylmethyl ether (MOMCl) was synthesized according to ref. [20]. Gaseous HCl was passed through a mixture of methanol (44 g, 1.375 mol, 55.6 mL) and 37.5% aqueous solution of formaldehyde (90 g, 1.125 mol) for 6 h in accord with the literature procedure. MOMCl (49.13 g, 54.25%, b.p. 55–60 °C/760 mmHg) (lit. 55–60 °C/760 mmHg [20]) was obtained.

TLC was carried out on silica gel Merck Alurolle 5562 or Alufolien 5554; typical mobile phases: hexane-ethyl acetate (9:1); benzene-ethyl acetate (9:1); benzene-methanol (9:1). TLC visualization was achieved using UV 254 nm light and/or I₂ vapor. Column chromatography was performed on silica gel 0.040–0.063 mm, 230–400 mesh: Merck 1.09385.1000 or Zeochem 60 hyd. mobile phases: benzene, benzene-ethyl acetate (95:5, 9:1, 4:1). EI MS data (70 eV) were recorded on an AMD 604 and Agilent Technologies 5975 B mass spectrometers. EI HR MS data were recorded by using an AMD 604 mass spectrometer. IR spectra were recorded on an FT/IR Jasco 420 spectrophotometer. ¹H, ¹³C and ¹⁹F NMR data were collected using a Varian UNITYplus 300 or 500 spectrophotometers (at 200 or 500 MHz for ¹H, respectively).

4.2 1,4-Difluoro-2-methoxybenzene (2,5-difluoroanisole) (2)

A mixture of 2,5-difluorophenol (1, 11.96 g, 0.092 mol), triturated anhydrous potassium carbonate (15.25 g, 0.11 mol), dimethyl sulfate (13.92 g, 10.45 mL, 0.11 mol) and acetone (240 mL) was stirred at reflux for 2 h. After cooling, the precipitate was filtered off and washed with acetone (10 mL). The filtrate was evaporated. Water (150 mL) was added to the residue (16.5 g) and the mixture was extracted with diethyl ether. The ether layer was washed with water, ammonium hydroxide (15%, 150 mL, stirring for 1.5 h), and with water to pH 7–8 again. The ether layer was dried over anhydrous magnesium sulfate. Filtration of the drying agent and evaporation of the solvent gave the crude product (14.4 g) which was distilled under reduced pressure to give 2.5-difluoroanisole (2, 10.7 g, 81.4%, colorless oil, b.p. 59–60 °C/15 mm Hg (lit. 72–73 °C/25 mm Hg [21]). – MS (EI, 70 eV): m/z (%) = 144 (96) [M]⁺, 129 (47), 115 (12), 114 (15), 113 (11), 101 (100), 96 (4), 95 (6), 81 (10), 75 (15), 63 (19), 57 (11), 51 (10), 50 (6). – IR (cm⁻¹, film): ν = 3100, 3010, 2980, 2946, 2920, 2890, 2850, 1626, 1514, 1453, 1420, 1325, 1290, 1250, 1210, 1192, 1150, 1101, 1101, 1031, 950, 836, 786, 715, 608.

4.3 3,6-Difluoro-2-methoxybenzoic acid (3)

1,4-Difluoro-2-methoxybenzene (2,5-difluoroanisole, 2, 5.56 g, 0.039 mol) in anhydrous THF (15 mL) was cooled to −60 °C. A solution of LDA 1.5 m (30.5 mL, 0.045 mol) was added with a syringe via septum in portions (9, 4, 4, 6, 7.5 mL). Because the temperature of the reaction mixture rose rapidly, adding of LDA was carried out at such a rate that the temperature of the reaction mixture did not increase above −50 °C. After adding LDA, the reaction mixture was stirred for 30 min at −70 °C. Gaseous carbon dioxide was bubbled through the reaction mixture for 1 h. The temperature was maintained at −78 to −50 °C. The reaction mixture was allowed to slowly warm to 0 °C and then was carefully acidified with 30% H₂SO₄ to pH 3–4. The aqueous layer was extracted with ether (2 × 50 mL). The organic layer was concentrated. The residue was dissolved in 2 m NaOH (50 mL) and washed with hexane to remove unreacted 2,5-difluoroanisole (2). The aqueous layer was acidified with 30% H₂SO₄ to pH 3–4 and left for 12 h in a refrigerator. The precipitate (7.14 g) was dissolved in ethyl acetate. The solution was dried over anhydrous magnesium sulfate, and after removing the drying agent, concentrated. Crude 3,6-difluoro-2-methoxybenzoic acid (3, 5.43 g) was purified by a double column chromatography (eluent: hexane-ethyl acetate 4:1). 3,6-Difluoro-2-methoxybenzoic acid (3) was obtained as a colorless solid, 3.87 g, 53.3%, m.p. 81–82 °C (lit. 82–83 °C [10]). – MS (EI, 70 eV): m/z (%) = 188 (56) [M]⁺, 171 (22), 170 (18), 169 (8), 159 (32), 156 (34), 141 (100), 128 (29), 115 (13), 114 (7), 113 (15), 112 (14), 101 (21), 100 (20), 99 (12). – ¹H NMR (300 MHz, CDCl₃): δ = 12.27 (1 H, s, COOH), 7.20 (1H, td, J = 10.0, 5.1 Hz, Ph), 6.84 (1H, td, J = 8.85, 3.6 Hz, Ph), 4.06 (3H, s, OMe). – ¹³C NMR (75.5 MHz, CDCl₃): δ = 62.30 (d, J = 6.3 Hz, OCH3), 110.83 (dd, J = 24.1, 7.4 Hz), 116.02 (d, J = 17.5 Hz), 119.71 (dd, J = 21.8, 10.3 Hz), 146.38 (dd, J = 13.7, 5.6 Hz), 151.55 (dd, J = 244.8, 3.5 Hz), 155.81 (dd, J = 251.8, 2.4 Hz), 168.28 (s, COOH). –¹⁹F NMR (282 MHz, CDCl₃): δ = −116.96 (m), −133.94 (tm, J = 13.8 Hz). – IR (cm⁻¹, KBr): ν = 430, 2962, 2680, 2580, 1705, 1622, 1489, 1429, 1320, 1291, 1239, 1059, 969, 913, 819, 764.

4.4 3,6-Difluoro-2-hydroxybenzoic acid (3,6-difluoro-salicylic acid) (4)

To a solution of 3,6-difluoro-2-methoxybenzoic acid (3, 0.5 g, 0.0027 mol) in anhydrous dichloromethane (20 mL), 1.0 m solution of boron tribromide (5.4 mL, 2 eq.) in dichloromethane was added with magnetic stirring, using a syringe, at 0 °C, under argon atmosphere. The reaction mixture was stirred for 2 h at 0 °C, then was allowed to slowly warm to room temperature and the stirring was continued for 12 h. In order to quench the reaction, methanol (approx. 5 mL) was added dropwise at 0 °C. During the methanol addition, the temperature rose to 17 °C. Water was added to the reaction mixture. The layers were separated. The organic layer was washed with brine and dried over anhydrous sodium sulfate. Filtration of the drying agent and concentration gave the crude product (0.5 g), which was purified by column chromatography with hexane-ethyl acetate 20:1, 9:1, 4:1 as eluents. The fractions containing the product were combined and concentrated. The residue was washed with hexane to give 3,6-difluoro-2-hydroxybenzoic acid (4, 0.2 g, 42.6%, HPLC: 99.95%, m.p.: 154–155 °C (lit. 154–155 °C [11])). – MS (EI, 70 eV): m/z (%) = 174 (38) [M]⁺, 156 (100), 128 (54), 100 (44), 81 (12). – ¹H NMR (300 MHz, acetone-d₆): δ = 11.58 (bs), 7.41 (1H, ddd, J = 10.35, 9.2, 4.8 Hz), 6.70 (1H, ddd, J = 10.35, 9.2, 3.6 Hz). – ¹³C NMR (75.5 MHz, acetone-d₆): δ = 170.99 (t, J = 3.28 Hz, COOH), 158.90 (dd, J = 256.2, 2.76 Hz), 152.15 (dd, 1 J = 4.65, 4.38 Hz), 148.63 (dd, J = 240.28, 3.66 Hz), 122.04 (dd, J = 19.78, 11.55 Hz), 106.45 (dd, J = 25.82, 6.72 Hz), 105.23 (dd, J = 15.44, 3.05 Hz). – ¹⁹F NMR (282.5 MHz, acetone-d₆): δ = −111 (ddd, J = 17.37, 10.45, 4.7 Hz), −142.12 (ddd, J = 17.44 Hz, 10.38, 3.53 Hz). – IR (cm⁻¹, KBr): ν = 3427, 3100, 1715, 1662, 1640, 1495, 1445, 1249, 1181, 1035, 810, 760.

4.5 1,2,4-Trifluoro-5-methoxybenzene (2.4.5-trifluoroanisole) (6)

A mixture of 2,4,5-trifluorophenol (5, 5.03 g, 0.034 mol), triturated anhydrous potassium carbonate (8.28 g, 0.06 mol), dimethyl sulfate (5.68 mL, 0.06 mol, 7.56 g), and acetone (90 mL) was stirred at reflux for 2 h. After cooling, the precipitate was filtered and washed with acetone (10 mL). The filtrate was evaporated. Water (55 mL) was added to the residue (7.6 g) and the mixture was extracted with diethyl ether. The ether layer was washed with water, ammonium hydroxide (15%, 55 mL, stirring for 1.5 h) and with water to pH 7–8 again. The ether layer was dried over anhydrous magnesium sulfate. Filtration of the drying agent and evaporation of the solvent afforded the crude 1,2,4-trifluoro-5-methoxybenzene (6, 3.84 g, 69.7%, GC: 97.8%, colorless solid, m.p. 37–38 °C (lit. m.p. 35–37 °C [22], 36–37 °C [23])) which was used without purification for the next step. – GC/MS: GC: t.r. = 6.164 min. – MS (EI, 70 eV): m/z (%) = 163(8), 162 (94) [M]⁺,148 (5), 147 (71), 131 (6), 120 (6), 119 (100), 81 (16), 69 (9). – IR (cm⁻¹, KBr): ν = , 2962, 1640, 1529, 1261, 1096, 1020, 802.

4.6 2,3,6-Trifluoro-5-methoxybenzoic acid (7)

4.7 2,3,6-Trifluoro-5-hydroxybenzoic acid (8)

To a solution of 2,3,6-trifluoro-5-methoxybenzoic acid (7, 1.0 g, 0.0049 mol) in anhydrous dichloromethane (20 mL), 1.0 m solution of boron tribromide (9.7 mL, 2 eq.) in dichloromethane was added with magnetic stirring using a syringe, at 0 °C under argon atmosphere. The reaction mixture was stirred for 2 h at 0 °C, then was allowed to slowly warm to room temperature and the stirring was continued for 12 h. In order to quench the reaction, methanol (approx. 5 mL) was added dropwise at 0 °C. During the methanol addition, the temperature rose to 17 °C. Water was added to the reaction mixture. The layers were separated. The organic layer was washed with brine and dried over anhydrous sodium sulfate. Filtration of the drying agent and concentration gave the crude product (0.36 g, m.p. 97–98 °C). The aqueous layer was extracted with ethyl acetate. After evaporation of the ethyl acetate, an additional 0.82 g of the product was obtained (0.42 g after washing with hexane and drying). The combined crude products were purified by column chromatography with hexane-ethyl acetate 95:5, 9:1, 4:1. The fractions containing the product were combined and concentrated. The residue was washed with hexane to give 2,3,6-trifluoro-5-hydroxybenzoic acid (0.26 g, m.p. 134–137 °C and 0.13 g, m.p. 138–142 °C (lit. 136–137 °C [11]). Yield of 8 in total 0.39 g, 41.4%. – MS (EI, 70 eV): m/z (%) = 193 (8), 192 (100) [M]⁺, 175 (39), 149 (8), 148 (20), 147 (25), 128 (8), 119 (23), 100 (42), 99 (30), 75 (9), 69 (12). – ¹H NMR (300 MHz, CDCl₃): δ = 10.02 (2H, bs, COOH, OH), 7.00 (1H, dt, J = 11.7, 7.95 Hz, Har). – ¹³C NMR (75.5 MHz, acetone-d₆): δ = 161.73 (d, J = 13.25 Hz, COOH), 147.25 (ddd, J = 243.19, 13.55, 3.78 Hz, C–F), 145.39 (ddd, J = 246.92, 4.57, 3.25 Hz, C–F), 142.68 (ddd, J = 14.80, 9.74, 3.47 Hz), 141.43 (ddd, J = 246.53, 15.50, 5.51 Hz, C–F), 114.21 (t, J = 17.86 Hz), 108.76 (dd, J = 21.71, 3.59 Hz). – ¹⁹F NMR (282.5 MHz, acetone-d₆): δ = −141.08 (ddd, J = 13.56, 7.91, 5.79 Hz), −142.58 (ddd, J = 22.11, 13.28, 11.51 Hz), −150.45 (ddd, J = 22.18, 7.77, 5.93 Hz). – IR (cm⁻¹, KBr): ν = 3228, 1700, 1650, 1620, 1499, 1416, 1271, 1170, 946, 707.

4.8 1,2,4-Trifluoro-5-(methoxymethoxy)benzene (9)

2,4,5-Trifluorophenol (5 g, 0.031 mol), and N-ethyl-diisopropylamine (5.1 mL, 0.034 mol) in methylene chloride (23 mL) were placed in a reaction flask. The reaction mixture was cooled to 0 °C and MOMCl (2.6 mL, 0.034 mol) was added dropwise at 0 °C. The reaction mixture was stirred for 2 h at room temperature. The reaction mixture was poured into 3 m sodium hydroxide (60 mL) and extracted with diethyl ether. The organic layer was dried over anhydrous magnesium sulfate, and concentrated. The oily residue was obtained (5.68 g) and distilled under reduced pressure. 1,2,4-Trifluoro-5-(methoxymethoxy) benzene was obtained (3.67 g, 56.6%, GC/MS 97.5%, b.p. 70–72 °C/10 mm Hg (lit. 61–62 °C/8 mmHg, [11])). – nD20 = 1.4475 (lit. nD20 = 1.4407 [11]). – MS (EI, 70 eV): m/z (%) = 192 (17) [M]⁺, 162 (4), 161 (12), 148 (5), 147 (8),133 (4), 131 (11), 119 (20), 99 (4), 81 (13), 45 (100). – IR (cm⁻¹, film): ν = 3800, 2963, 2840, 1523, 1431, 1218, 1157, 1076, 981, 853, 688.

4.9 2,3,5-trifluoro-6-hydroxybenzoic acid (3,5,6-trifluorosalicylic acid) (10)

1,2,4-Trifluoro-5-(methoxymethoxy)benzene (9, 1.8 g, 0.0094 mol) was placed in anhydrous diethyl ether (30 mL). under argon. The solution was cooled to −64 °C. 2.5 m n-butyllithium solution (0.0104 mol, 4.5 mL) was added in portions with a syringe through septum. The temperature of the reaction mixture rose to −61 °C. n-Butyllithium solution were added dropwise in five portions (4 × 1, 1 × 0.5 mL). After completion of the addition, the reaction mixture was stirred for 4 h at −65 °C. Gaseous carbon dioxide was passed through the reaction mixture for 1 h. The reaction mixture initially rose to −45 °C, then it maintained at approx. −65 °C. A copious amount of white precipitate was observed. The reaction mixture was allowed to warm to 0 °C, Water (10 mL) was added carefully. Aqueous layer was extracted with ether (after evaporation of the ether layer, residue was approx. 0.135 g). The aqueous layer was acidified with concentrated HCl to pH 1, stirred for 5 min. and extracted with ether. The combined ether layers were dried over anhydrous sodium sulfate. After evaporation of the solvent, a solid residue (1.49 g) was obtained. In order to remove the OMOM protection, the residue was dissolved in 6 mL of diethyl ether. A 2 n HCl (6 mL) was added and stirred at room temperature for 7 h (TLC control: benzene-acetone, 1:1). Diethyl ether layer was evaporated. Concentrated HCl (0.2 mL) was added to the residue and the mixture was stirred at room temperature for 12 h. The mixture was extracted with methylene chloride. Drying of the organic layer over anhydrous sodium sulfate and evaporation gave 2,3,5-trifluoro-6-hydroxybenzoic acid (10, 1.03 g, 57.2%, colorless solid, m.p. 170–178 °C, which was subjected without further purification to the methylation to 11. For analytical purposes, a sample of about 0.25 g was subjected to purification by column chromatography. The crude acid 10 was dissolved in ethyl acetate, silica gel (1 g) was added and evaporated to dryness. The sample applied on the gel was placed on the top of a chromatography column. Elution with hexane, hexane-ethyl acetate 9:1, 4:1, 1:1, ethyl acetate, methanol gave the product 10 (0.17 g) which was stirred with hexane at room temperature. The solid was filtered off. The filtrate was concentrated. 2,3,5-Trifluoro-6-hydroxybenzoic acid was obtained (10, 0.13 g, colorless solid, m.p. 173–174 °C. An analytical sample purified by column chromatography had an m.p. of 178–180 °C (lit. 173 °C [24]; 166–168 °C [11]; 165–167 °C [19]). – MS (EI, 70 eV): m/z (%) = 192 (39) [M]⁺, 175 (20), 174 (100), 147 (8), 146 (50), 119 (21), 118 (65), 99 (27), 75 (8), 69 (8), 68 (8), 45 (8). – ¹H NMR (300 MHz, acetone-d₆): δ = 11.39 (1H, bs, COOH), 7.61 (1H, td, J = 10.5, 7.2 Hz, Har). – ¹³C NMR (75.5 MHz, acetone-d₆): δ = 170.03 (dd, J = 6.64, 3.32 Hz, COOH), 147.81 (dt, J = 14.345, 2.53 Hz), 147.26 (ddd, J = 243.808, 9.7, 3.7 Hz), 146.91 (ddd, J = 256.9, 14.25, 4.1 Hz), 143.10 (ddd, J = 239.66, 14.9, 10.7 Hz), 111.69 (td, J = 23.2, 1.58 Hz), 106.13 (dd, J = 11.627, 3.775 Hz). – ¹⁹F NMR (282.5 MHz, acetone-d₆): δ = −138.50 (ddd, J = 22.18, 15.11, 7.2 Hz), −139.49 (ddd, J = 15.1, 10.6, 2.0 Hz), −148.89 (ddd, J = 22.0, 10.5, 2.26 Hz). – IR (cm⁻¹, KBr): ν = 3440, 3100, 1682, 1644, 1496, 1445, 1269, 1178, 956, 714.

4.10 2,3,5-Trifluoro-6-methoxybenzoic acid (11)

To 2,3,5-trifluoro-6-hydroxybenzoic acid (10, 0.7 g, 0.0036 mol), acetone (12 mL), triturated anhydrous potassium carbonate (1.24 g, 0.009 mol), dimethyl sulfate (0.85 mL, 1.134 g, 0.009 mol) was added and refluxed for 7.5 and stirred 12 h at room temperature. The precipitate was then filtered off, the filtrate was concentrated and the crude methyl 2,3,5-trifluoro-2-methoxybenzoate (0.9 g) was obtained and subjected to basic hydrolysis with 40% potassium hydroxide (0.51 mL) in methanol (1 mL). The reaction mixture was stirred under reflux for 3 h. TLC analysis showed only partial hydrolysis (pH approx.7). Additional amount of 40% potassium hydroxide (0.3 mL) and methanol (1 mL) were added and the hydrolysis was continued for 1 h. TLC analysis showed complete hydrolysis of the ester. Methanol was evaporated, the residue was acidified with concentrated HCl. The product was extracted with ethyl acetate (3 × 15 mL) and methylene chloride (3 × 15 mL). Drying over anhydrous magnesium sulfate and concentration afforded 2,3,5-trifluoro-6-methoxybenzoic acid (11, 0.7 g, colorless solid, 93.2%, m.p. 86–89 °C). For analytical purposes a sample of approx. 0.25 g was subjected to purify by column chromatography. The crude 2,3,5-trifluoro-6-methoxybenzoic acid (11) was dissolved in warm benzene and applied to the top of the chromatography column. Elution with benzene, benzene-ethyl acetate 4:1, hexane-ethyl acetate 1:1 gave 2,3,5-trifluoro-6-methoxybenzoic acid (11, colorless solid, 0.22 g, m.p. 87–89 °C). – MS (EI, 70 eV): m/z (%) = 207 (6), 206 (62) [M]⁺, 189 (16), 188 (12), 177 (28), 175 (12), 174 (37), 160 (28), 159 (100), 149 (8), 147 (18), 146 (33), 143 (14), 135 (10), 133 (16), 131 (18), 130 (17), 119 (54), 118 (33), 113 (13), 99 (34), 81 (26), 80 (12), 75 (15), 69 (11), 68 (13), 45 (9). – HRMS (EI, 70 eV): m/z = 206.0193 (calcd 206.0191 for C₈H₅F₃O₃, [M]⁺). – ¹H NMR (300 MHz, CDCl₃): δ = 11.84 (1H, s, COOH), 7.14 (1H, td, J = 10.2, 7.5 Hz, Har), 4.02 (3H, d, J = 1.5 Hz, OCH₃). – ¹³C NMR (75.5 MHz, CDCl₃): δ = 166.91 (t, J = 3.29 Hz, COOH), 150.71 (ddd, J = 248.72, 9.11, 3.6 Hz), 145.70 (ddd, J = 248.94, 14.13, 10.5 Hz), 144.41 (ddd, J = 254.93, 14.8, 4.0 Hz), 142.15 (dt, J = 13.44, 3.7 Hz), 117.22 (dd, J = 13.70, 2.68 Hz), 108.71 (ddd, J = 24.59, 21.86, 1.27 Hz), 62.78 (dd, J = 5.51, 0.53 Hz, OCH₃). – ¹⁹F NMR (282.5 MHz, CDCl₃): δ = −130.1 (t, J = 12 Hz), −138.42 (dd, J = 22.18, 9.8 Hz), −140.54 (ddd, J = 21.89, 14.13, 7.49 Hz). IR (cm⁻¹, KBr): ν = 3430, 3073, 1713, 1498, 1410, 1304, 1006, 868, 725.

4.11 2,3,5-Trichloro-6-methoxybenzoic acid (tricamba)

To 2,3,5-trichloro-6-hydroxybenzoic acid (4.8 g, 0.02 mol), acetone (67 mL), triturated anhydrous potassium carbonate (6.9 g, 0.05 mol), dimethyl sulfate (4.7 mL, 6.3 g, 0.05 mol) was added and refluxed for 7.5 h. The precipitate was filtered off, the filtrate was concentrated and the crude methyl 2,3,5-trichloro-2 methoxybenzoate (6.03 g) was obtained and subjected to basic hydrolysis. with 40% sodium hydroxide (3.1 mL) in methanol (5.6 mL). The reaction mixture was stirred under reflux. After about 1 h the reaction mixture solidified. Water (about 5 mL) was added and heating was continued for 5 h under reflux. Methanol was evaporated, the residue was acidified with concentrated HCl. The product was extracted with ethyl acetate. After drying over anhydrous magnesium sulfate and concentration, the residue (8.13 g) was purified by column chromatography. Elution with hexane, hexane-ethyl acetate 4:1, 1:1, acetone gave 4.1 g of a colorless solid, which was washed with hexane and dried. 2,3,5-Trichloro-6-methoxybenzoic acid was obtained (3.0 g, 58.8%, GC 99.5%, HPLC 98.3%, m.p. 135–138 °C (lit. 137–139 °C [25])) – GC-MS (EI, 70 eV): m/z (%) = 258 (29), 256 (92), 254 (95) [M]⁺, 241 (19), 239 (43), 23 (15), 237 (39), 236 (12), 227 (29), 226 (18), 225 (37), 224 (39), 222 (33), 213 (18), 212 (15), 211 (51), 210 (40), 209 (100), 208 (410, 207 (92), 196 (25), 194 (26), 185 (22), 183 (39), 181 (29), 176 (19), 175 (31), 169 (30), 167 (30), 149 (17), 147 (31), 146 (17), 145 (25), 143 (17), 133 (35), 131 (47), 111 (150, 109 (27), 108 (27), 107 (17), 97 (24), 96 (37), 74 (180), 73 (22), 61 (34). – ¹H NMR (300 MHz, CDCl₃): δ = 10.19 (1H, bs, COOH), 7.60 (1H, s, Ph), 3.98 (s, 3H, OCH₃). – ¹³C NMR (75.5 MHz, CDCl₃): δ = 168.94 (s, COOH), 152.34 (s), 132.33 (s), 130.54 (s), 129.22 (s), 128.07 (s), 127.40 (s), 62.62 (s, OCH3). – IR (cm⁻¹, KBr): ν = 2949, 1713, 1649, 1461, 1421, 1243, 1136, 1014, 875, 685.

4.12 Herbicidal bioassay, pre- and post-emergence experiments

Herbicidal activity was evaluated using different weed species in pot experiments under controlled conditions. Polyethylene pots, 3.5 L capacity, were filled with 0.75 kg of soil (physicochemical characteristic: sandy clay, pH/KCl/6.7; organic matter 2.8%) and were wetted with water. Seeds of the weed species were planted in soil (0.5 cm depth). Plants were grown to the two-leaf stage under normal glasshouse propagation conditions (temperature, 20 ± 5 °C; lighting, 14 h photoperiod of daylight supplemented by lamps, 400 W). The pots were watered overhead. The test compounds were dissolved in an appropriate volume of acetone-water solution (1:3) with the addition of Tween 20 (0.05% v/v) to give the required dose of the tested substance. All treatments were applied as a pre-emergence or post-emergence spray at a volume rate of 300 L ha⁻¹ using track laboratory sprayer (nozzle TeeJet60, pressure 0.2 MPa). There were three replicate pots per treatment arranged in a randomized block design. Pots after spraying were transferred to the growth chamber (temperature, day/night 20/15 °C; lighting, 16 h photoperiod, white fluorescent tubes giving 200 μmol m⁻² s⁻² PAR [photosynthetic active radiation]). A visual assessment of phytotoxicity separately for each species was made (18 or 25 days after treatment for pre- and post-emergence experiments) as a percentage compared to the untreated plants. Results (degree [%] of a visual observation of a phytotoxicity weed damage for a weed species used against control in doses: 2000, 300, 150 g ha⁻¹) are expressed as a fraction: a numerator means a result (in %) of a pre-emergence (via soil) treatment, a denominator means a result (in %) of a post-emergence (on leaves) treatment.

4.13 Fungistatic bioassay in vitro

Fungitoxicity of the tested compounds against phytopathogenic fungi was assessed in vitro using agar growth medium poison technique. PDA media in 100 mm Petri plates containing the acetone solutions of the tested compounds in the defined concentrations were infected with agar disks with thin mycelium of fungi cultures, and allowed the solvent to evaporate. Linear growth of each colony was determined after 3–5 days. The effect of each compound on mycelial growth was assessed by calculating the percentage of growth reduction of a fungus colony; percentage of linear growth reduction = ([colony diameter of a control plate − colony diameter of a tested plat]/[colony diameter of a control plate]) × 100.