Chemicals and instruments
All chemicals and solvents were obtained from commercial suppliers and were used without further purification. The melting points were determined on a WRR melting point apparatus (Shanghai Jingke Industrial Co. Ltd., PR China) and were uncorrected. Thin-layer chromatography (TLC) was performed on silica gel 60 F254 (Qingdao Marine Chemical Ltd., P. R. China). Column chromatography (CC) purification was performed over silica gel (200–300 mesh, Qingdao Marine Chemical Ltd.). 1H NMR spectrum were recorded in CDCl3 solution on a Bruker 600 MHz spectrometer (Bruker Co., Switzerland), using tetramethylsilane (TMS) as an internal standard, and chemical shift values (δ) were given in parts per million (ppm). The following abbreviations were used to designate chemical shift multiplicities: s = singlet, d = doublet, t = triplet, q = quartet, m = multiple. MS data were obtained using a APEX IV Fourier-transform mass spectrometry (Bruker).
Synthesis of hydroxybenzoic acid esters
The compound 2-hydroxybenzoic acid (15 mmol) and its corresponding alcohol (30 mL) were added into a 50 mL round-bottom flask, and cooled at 0 °C. An aliquot of 2 mL of 98% H2SO4 was slowly added. The reaction was stirred at reflux temperature for 12 h and monitored by thin-layer chromatography (TLC) until the 2-hydroxybenzoic acid was completely consumed. The mixture was evaporated under vacuum, neutralized with water and 5% NaHCO3 aqueous solution, extracted by ether 3 times, dried over Na2SO4, concentrated in vacuum, and used in next step without purification. The compounds 3-hydroxybenzoic acid esters and p-hydroxybenzoic acid esters were also synthesized by this method.
Synthesis of phenazine-1-carbonyl chloride
Phenazine-1-carboxylic acid (10 mmol) and N,N-dimethylformamide (0.1 mmol) were added in 30 mL of dry CH2Cl2, and cooled at 0 °C. A solution of 15 mmol of oxalyl chloride in 20 mL of dry CH2Cl2 was then slowly added. The reaction was stirred at reflux temperature for 12 h, then cooled to room temperature and evaporated under vacuum. The residue was dissolved in 10 mL of dry CH2Cl2 and used in next step without purification.
General procedure for hydroxybenzoic acid ester conjugates of phenazine-1-carboxylic acid 5a–5p
Phenazine-1-carbonyl chloride (10 mmol) dissolved in 10 mL of dry CH2Cl2 was added dropwise to a solution of compound 2-hydroxybenzoic acid methyl ester (10 mmol), and triethylamine (12 mmol) as the attaching acid agent in CH2Cl2, The mixture was stirred at room temperature for 4 h until the reaction was complete (indicated by TLC), then quenched with water and 5% Na2CO3 aqueous solution, dried over Na2SO4, filtered and concentrated in vacuum. The obtained crude extract was purified by recrystallizing from the solution of EtOAc-DCM (1:1) to give pure conjugate 5a. Conjugates 5b–5p were also synthesized by this method.
2-(Methoxycarbonyl)phenyl phenazine-1-carboxylate (5a)
Yellow solid; yield: 89.5%; m.p. 141–142 °C; 1H-NMR (600 MHz, CDCl3) δ: 8.69 (d, J = 7.2 Hz, 1H), 8.49 (d, J = 8.8 Hz, 1H), 8.36 (dd, J = 6.0, 3.6 Hz, 1H), 8.28 (dd, J = 6.6, 3.6 Hz, 1H), 8.14 (dd, J = 7.8, 1.2 Hz, 1H), 7.98 (dd, J = 8.4, 7.2 Hz, 1H), 7.94–7.87 (m, 2H), 7.74–7.68 (m, 1H), 7.51 (d, J = 7.8 Hz, 1H), 7.43 (t, J = 7.8 Hz, 1H), 3.86 (s, 3H). HRMS calcd for C21H14N2O4 [M+H]+: 359.1026, found 359.1027.
2-(Ethoxycarbonyl)phenyl phenazine-1-carboxylate (5b)
Yellow solid; yield: 92.3%; m.p. 143–144 °C; 1H-NMR (600 MHz, CDCl3) δ: 8.74–8.69 (m, 1H), 8.48 (dd, J = 8.4, 1.2 Hz, 1H), 8.36 (dd, J = 6.6, 3.6 Hz, 1H), 8.28 (dd, J = 6.6, 3.6 Hz, 1H), 8.14 (dd, J = 7.8, 1.2 Hz, 1H), 7.98 (dd, J = 8.4, 7.2 Hz, 1H), 7.93–7.86 (m, 2H), 7.74–7.66 (m, 1H), 7.50 (d, J = 7.8 Hz, 1H), 7.42 (t, J = 7.8 Hz, 1H), 4.33 (q, J = 7.2 Hz, 2H), 1.27 (t, J = 7.2 Hz, 3H). HRMS calcd for C22H16N2O4 [M+H]+: 373.1183, found 373.1182.
2-(Propoxycarbonyl)phenyl phenazine-1-carboxylate (5c)
Yellow solid; yield: 97.5%; m.p. 102–103 °C; 1H-NMR (600 MHz, CDCl3) δ 8.72 (dd, J = 6.6, 1.2 Hz, 1H), 8.48 (dd, J = 8.4, 1.2 Hz, 1H), 8.40–8.31 (m, 1H), 8.32–8.21 (m, 1H), 8.14 (dd, J = 7.8, 1.8 Hz, 1H), 7.98 (dd, J = 8.4, 7.2 Hz, 1H), 7.94–7.79 (m, 2H), 7.73–7.59 (m, 1H), 7.51 (d, J = 7.2 Hz, 1H), 7.43 (dd, J = 11.4, 4.2 Hz, 1H), 4.23 (t, J = 6.6 Hz, 2H), 1.74–1.41 (m, 2H), 0.92 (t, J = 7.2 Hz, 3H). HRMS calcd for C23H18N2O4 [M+H]+: 387.1339, found 387.1338.
2-(Isopropoxycarbonyl)phenyl phenazine-1-carboxylate (5d)
Yellow solid; yield: 90.5%; m.p. 125–126 °C; 1H-NMR (600 MHz, CDCl3) δ 8.73 (d, J = 6.6 Hz, 1H), 8.48 (d, J = 8.4 Hz, 1H), 8.36 (dd, J = 6.6, 3.6 Hz, 1H), 8.27 (dd, J = 6.6, 3.6 Hz, 1H), 8.12 (dd, J = 7.8, 1.2 Hz, 1H), 7.98 (dd, J = 8.4, 7.2 Hz, 1H), 7.93–7.86 (m, 2H), 7.71–7.66 (m, 1H), 7.50 (d, J = 7.8 Hz, 1H), 7.41 (t, J = 7.8 Hz, 1H), 5.30–5.30 (m, 1H), 1.27 (d, J = 6.6 Hz, 6H). HRMS calcd for C23H18N2O4 [M+H]+: 387.1339, found 387.1340.
2-(Butoxycarbonyl)phenyl phenazine-1-carboxylate (5e)
Yellow solid; yield: 94.1%; m.p. 89–90 °C; 1H-NMR (600 MHz, CDCl3) δ 8.72 (dd, J = 6.9, 1.4 Hz, 1H), 8.48 (dd, J = 8.7, 1.4 Hz, 1H), 8.39–8.34 (m, 1H), 8.30–8.25 (m, 1H), 8.13 (dd, J = 7.9, 1.7 Hz, 1H), 7.98 (dd, J = 8.4, 6.6 Hz, 1H), 7.93–7.86 (m, 2H), 7.72–7.67 (m, 1H), 7.51 (dd, J = 7.8, 1.2 Hz, 1H), 7.47–7.37 (m, 1H), 4.27 (t, J = 6.7 Hz, 2H), 1.72–7.57 (m, 2H), 1.42–1.31 (m, 2H), 0.84 (t, J = 7.2 Hz, 3H). HRMS calcd for C24H20N2O4 [M+H]+: 401.1496, found 401.1497.
3-(Methoxycarbonyl)phenyl phenazine-1-carboxylate (5f)
Yellow solid; yield: 95.0%; m.p. 120–121 °C; 1H-NMR (600 MHz, CDCl3) δ 8.48 (t, J = 7.2 Hz, 2H), 8.39–8.34 (m, 1H), 8.30–8.25 (m, 1H), 8.14 (s, 1H), 8.03 (d, J = 7.8 Hz, 1H), 7.98–7.89 (m, 3H), 7.70–7.66 (m, 1H), 7.59 (t, J = 7.8 Hz, 1H), 3.98 (s, 3H). HRMS calcd for C21H14N2O4 [M+H]+: 359.1026, found 359.1027.
3-(Ethoxycarbonyl)phenyl phenazine-1-carboxylate (5g)
Yellow solid; yield: 96.5%; m.p. 109–110 °C; 1H-NMR (600 MHz, CDCl3) δ 8.55–8.41 (m, 2H), 8.39–8.30 (m, 1H), 8.31–8.24 (m, 1H), 8.14 (s, 1H), 8.04 (d, J = 7.8 Hz, 1H), 7.98–7.85 (m, 3H), 7.67 (d, J = 7.8 Hz, 1H), 7.59 (t, J = 7.8 Hz, 1H), 4.44 (q, J = 7.2 Hz, 2H), 1.44 (t, J = 7.2 Hz, 3H). HRMS calcd for C22H16N2O4 [M+H]+: 373.1183, found 373.1182.
3-(Propoxycarbonyl)phenyl phenazine-1-carboxylate (5h)
Yellow solid; yield: 95.2%; m.p. 87–88 °C; 1H-NMR (600 MHz, CDCl3) δ 8.51–8.43 (m, 2H), 8.38–8.32 (m, 1H), 8.27 (dd, J = 6.0, 4.2 Hz, 1H), 8.13 (s, 1H), 8.04 (d, J = 7.8 Hz, 1H), 7.97–7.86 (m, 3H), 7.67 (dd, J = 7.8, 1.2 Hz, 1H), 7.59 (t, J = 7.8 Hz, 1H), 4.34 (t, J = 6.6 Hz, 2H), 1.88–1.82 (m, 1H), 1.06 (t, J = 7.8 Hz, 3H). HRMS calcd for C23H18N2O4 [M+H]+: 387.1339, found 387.1340.
3-(Butoxycarbonyl)phenyl phenazine-1-carboxylate (5i)
Yellow solid; yield: 95.5%; m.p. 97–98 °C; 1H-NMR (600 MHz, CDCl3) δ 8.47 (d, J = 7.8 Hz, 2H), 8.39–8.31 (m, 1H), 8.29–8.23 (m, 1H), 8.15–8.09 (m, 1H), 8.03 (d, J = 7.8 Hz, 1H), 7.97–7.85 (m, 3H), 7.66 (dd, J = 7.8, 2.0 Hz, 1H), 7.58 (t, J = 7.8 Hz, 1H), 5.37–5.25 (m, 1H), 1.41 (d, J = 6.6 Hz, 6H). HRMS calcd for C23H18N2O4 [M+H]+: 387.1339, found 387.1340.
3-(Butoxycarbonyl)phenyl phenazine-1-carboxylate (5j)
Yellow solid; yield: 95.2%; m.p. 87–88 °C; 1H-NMR (600 MHz, CDCl3) δ 8.48 (dd, J = 7.8, 3.6 Hz, 2H), 8.39–8.33 (m, 1H), 8.31–8.25 (m, 1H), 8.12 (s, 1H), 8.04 (d, J = 7.8 Hz, 1H), 7.98–7.90 (m, 3H), 7.67 (dd, J = 7.8, 2.4 Hz, 1H), 7.59 (t, J = 7.8 Hz, 1H), 4.39 (t, J = 6.6 Hz, 2H), 1.83–1.77 (m, 2H), 1.56–1.48 (m, 2H), 1.01 (t, J = 7.2 Hz, 3H). HRMS calcd for C24H20N2O4 [M+H]+: 401.1496, found 401.1495.
4-(Methoxycarbonyl)phenyl phenazine-1-carboxylate (5k)
Yellow solid; yield: 95.0%; m.p. 164–165 °C; 1H-NMR (600 MHz, CDCl3) δ 8.54–8.43 (m, 2H), 8.37–8.33 (m, 1H), 8.31–8.26 (m, 1H), 8.24–8.18 (m, 2H), 7.97–7.90 (m, 3H), 7.57–7.51 (m, 2H), 3.97 (s, 3H). HRMS calcd for C21H14N2O4 [M+H]+: 359.1026, found 359.1025.
4-(Ethoxycarbonyl)phenyl phenazine-1-carboxylate (5l)
Yellow solid; yield: 98.1%; m.p. 123–125 °C; 1H-NMR (600 MHz, CDCl3) δ 8.51–8.46 (m, 2H), 8.38–8.32 (m, 1H), 8.31–8.26 (m, 1H), 8.21 (t, J = 5.4 Hz, 2H), 7.98–7.89 (m, 3H), 7.53 (t, J = 5.4 Hz, 2H), 4.43 (q, J = 7.2 Hz, 2H), 1.44 (t, J = 7.2 Hz, 3H). HRMS calcd for C22H16N2O4 [M+H]+: 373.1183, found 373.1182.
4-(Propoxycarbonyl)phenyl phenazine-1-carboxylate (5m)
Yellow solid; yield: 98.1%; m.p. 95 °C; 1H-NMR (600 MHz, CDCl3) δ 8.61–8.40 (m, 2H), 8.41–8.30 (m, 1H), 8.31–8.27 (m, 1H), 8.27–8.15 (m, 2H), 8.02–7.82 (m, 3H), 7.64–7.46 (m, 2H), 4.33 (t, J = 6.6 Hz, 2H), 1.89–1.79 (m, 2H), 1.07 (t, J = 7.2 Hz, 3H). HRMS calcd for C23H18N2O4 [M+H]+: 387.1339, found 387.1340.
4-(Butoxycarbonyl)phenyl phenazine-1-carboxylate (5n)
Yellow solid; yield: 97.5%; m.p. 119–120 °C; 1H-NMR (600 MHz, CDCl3) δ 8.52–8.44 (m, 2H), 8.36–8.31 (m, 1H), 8.30–8.25 (m, 1H), 8.23–8.18 (m, 2H), 7.96–7.89 (m, 3H), 7.55–7.51 (m, 2H), 5.33–5.28 (m, 1H), 1.41 (d, J = 6.6 Hz, 6H). HRMS calcd for C23H18N2O4 [M+H]+: 387.1339, found 387.1340.
4-(Butoxycarbonyl)phenyl phenazine-1-carboxylate (5o)
Yellow solid; yield: 99.0%; m.p. 89–90 °C; 1H-NMR (600 MHz, CDCl3) δ 8.53–8.39 (m, 2H), 8.36–8.31 (m, 1H), 8.29–8.25 (m, 1H), 8.24–8.19 (m, 2H), 7.96–7.87 (m, 3H), 7.56–7.51 (m, 2H), 4.38 (t, J = 6.6 Hz, 2H), 1.88–1.76 (m, 2H), 1.57–1.48 (m, 2H), 1.02 (t, J = 7.2 Hz, 3H). HRMS calcd for C24H20N2O4 [M+H]+: 401.1496, found 401.1497.
4-(Octyloxycarbonyl)phenyl phenazine-1-carboxylate (5p)
Yellow solid; yield: 97.1%; m.p. 57–59 °C; 1H-NMR (600 MHz, CDCl3) δ 8.60–8.40 (m, 2H), 8.43–8.31 (m, 1H), 8.31–8.24 (m, 1H), 8.25–8.18 (m, 2H), 8.02–7.85 (m, 3H), 7.63–7.46 (m, 2H), 4.36 (t, J = 6.6 Hz, 2H), 1.88–1.76 (m, 2H), 1.53–1.43 (m, 2H), 1.42–1.26 (m, 8H), 0.91 (t, J = 6.6 Hz, 3H). HRMS calcd for C28H28N2O4 [M+H]+: 457.2122, found 457.2123.
Biological assays
Compounds were screened for their in vitro fungicidal activity against Rhizoctonia solani, Fusaium graminearum, Altemaria solani, Fusarium oxysporum, Sclerotinia sclerotiorum and Pyricularia oryzae with the mycelium growth rate test.
The method for testing the primary biological activity was performed aseptically with pure cultures. Synthesized compounds were dissolved in 100% acetone, and the solutions were diluted with aqueous 1% Tween 80 and were then added to sterile potato dextrose agar (PDA). The target final concentration of each compound was 50 μg/mL. The control blank assay was performed with 1 mL of sterile water. Mycelial plugs 6 mm in diameter were obtained with a cork borer and placed on the amended PDA. The culture plates were incubated at 28 °C. The diameter of the mycelia was measured after 72 h. Acetone in sterile aqueous 1% Tween 80 served as the negative control, whereas phenazine-1-carboxylic acid served as positive controls. Each sample was screened with three replicates, and each colony diameter of the three replicates was measured four times. All statistical analysis was performed using EXCEL 2010 software. The log dose–response curves allowed determination of the EC50 for the bioassay using probit analysis. The 95% confidence limits for the range of EC50 values were determined by the least-square regression analysis of the relative growth rate (% control) against the logarithm of the compound concentration. The relative inhibition rate of the circle mycelium compared to blank assay was calculated via the following equation:
$${\text{Relative}}\;{\text{inhibition}}\;{\text{rate}}\;\left( \% \right) = \left[ {{{\left( {{\text{CK}} - {\text{PT}}} \right)} \mathord{\left/ {\vphantom {{\left( {{\text{CK}} - {\text{PT}}} \right)} {\left( {{\text{CK}} - 6\;{\text{mm}}} \right)}}} \right. \kern-0pt} {\left( {{\text{CK}} - 6\;{\text{mm}}} \right)}}} \right] \times 100\%$$
where CK is the extended diameter of the circle mycelium during the blank assay; and PT is the extended diameter of the circle mycelium during testing.
Plant materials and fungal growth condition
Seeds of rice (Feng liang you xiang No. 1), with high rates of germination, were grown in plastic pots of 20 cm diameter and kept in a greenhouse under a temperature of 26–28 °C, with 10 plant per pot. After 4 weeks the four-leaf stage plants were used in the experiments. Rhizoctonia solani was cultured for 4 days at 28 °C on potato dextrose agar (PDA), under aseptic conditions. Spore concentration was adjusted with sterile distilled water to 105 spores/mL.
Chemical treatment of plants
Chemical treatments of plants were carried out as described by Makandar and others [34, 35]. Briefly, a stock solution of 10 mmol/L for testing conjugate 5c (highest fungicidal activity against Rhizoctonia solani) was prepared in water and diluted to a final concentration of 200 μmol/L. Rice plants at the four-leaf of the similar size were sprayed with a concentration of 200 μmol/L of test conjugate 5c, PCA and of salicylic acid (SA). A blank water control was also applied under the same conditions. There were four treatments as follows: (1) PCA, (2) SA, (3) conjugate 5c, and water-treated control. Each treatment consisted of three pots each containing 10 rice seedlings, and were arranged in a completely randomized design and replicated four times. In all treatments, spraying was done 24 h prior to inoculation.
Fungal inoculation and disease rating
Plants were treated with chemicals and 24 h later, point inoculations of rice leaf sheaths were done with needle injection of 10 μL of the 105 spores/mL suspension at the four-leaf stage of seedlings of rice. For each replication of each treatment, 30 leaf sheaths were inoculated. The inoculated plants were covered with black plastic bags and kept in a growth room maintained at 90% relative humidity near 90% at 26–28 °C for 24 h. Plants were evaluated for rice sheath blight disease as percent leaf sheath infected with Rhizoctonia solani at 14 days after inoculation. All statistical analyses were performed using EXCEL 2010 software. The disease reduction was calculated as follows:
$${\text{Disease}}\;{\text{reduction}}\;\left( \% \right) = \left[ {{{\left( {{\text{CK}} - {\text{PT}}} \right)} \mathord{\left/ {\vphantom {{\left( {{\text{CK}} - {\text{PT}}} \right)} {\text{CK}}}} \right. \kern-0pt} {\text{CK}}}} \right] \times 100\%$$
where CK is the percent disease in inoculated plants treated with water while PT is the disease rating for inducer treatments.