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Synthesis of N-substituted phthalimides and their antifungal activity against Alternaria solani and Botrytis cinerea
Microbial Pathogenesis 95 (2016) 186e192
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Microbial Pathogenesis journal homepage: www.elsevier.com/locate/micpath
Synthesis of N-substituted phthalimides and their antifungal activity against Alternaria solani and Botrytis cinerea Le Pan a, b, Xiuzhuang Li b, Chengwen Gong c, Hui Jin b, Bo Qin b, * a
University of Chinese Academy of Sciences, People's Republic of China Key Laboratory of Chemistry of Northwestern Plant Resources of CAS and Key Laboratory for Natural Medicine of Gansu Province, Lanzhou Institute of Chemical Physics, Chinese Academy of Sciences, Lanzhou 730000, People's Republic of China c Institute of Chinese Medicinal Herbs, Gansu Academy of Agricultural Sciences, Lanzhou 730070, People's Republic of China b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 2 July 2015 Received in revised form 15 September 2015 Accepted 10 April 2016 Available online 12 April 2016
As organosulfur and organophosphorus agents, phaltane and phosmet are facing great challenges for the environmental contamination, mammalian toxicity and increasing resistance with long term use. It is efficient and meaningful to develop phthalimide-based alternatives with non-sulfur and nonphosphorus groups. A series of N-substituted phthalimides were synthesized and their antifungal activity against two disastrous phytopathogenic fungi, Alternaria solani and Botrytis cinerea was evaluated in vitro. Most of them showed significant antifungal activity against both of fungi, or either of them selectively. N-vinylphthalimide (4) and 8-[4-(phthalimide-2-yl) butyloxy] quinoline (13) were identified as the most promising candidates against B. cinerea and A. solani with the IC50 values of 7.92 mg/mL and 10.85 mg/mL respectively. The brief structure-activity relationships have revealed that vinyl, quinolyl, bromide alkyl and benzyl substitutions were appropriate substituents and coupling functional moieties indirectly with optimum alkyl chain was efficient to prepare phthalimides related fungicides. © 2016 Elsevier Ltd. All rights reserved.
Keywords: N-substituted phthalimides Synthesis IC50 values Antifungal activity
1. Introduction Plant pathogenic fungi have been listed as one of the disastrous plant pathogens worldwide due to resulting in great agricultural losses [1e3]. Up to now, scientific farming practices and biological methods are advocated on the disease control, but the available methods are restricted to some specific cultivars and the narrow usage range has constrained their application [4,5]. Therefore, chemical pesticides have still been the common options on disease control and great effort has been desired to develop novel compounds with highly inhibitory effects on phytopathogenic fungi. Phthalimide subunit existed widely in both natural and synthetic compounds with broad activities against AIDS, cancer and inflammation [6,7]. They were also reported to be the crucial structure of numerous biological inhibitors, such as human protein kinase CK2 inhibitor, COX inhibitors, histone deacetylase inhibitors and etc [8e10]. In agriculture application, phthalimide has represented a famous scaffold for the development of efficient pesticides. Phaltane and phosmet are effective chemical agents in plant
* Corresponding author. E-mail address: [email protected] (B. Qin). http://dx.doi.org/10.1016/j.micpath.2016.04.012 0882-4010/© 2016 Elsevier Ltd. All rights reserved.
disease control [11] (Fig. 1), but as organosulfur and organophosphorus compounds, their applications are facing great challenges for the environmental contamination, mammalian toxicity and increasing resistance with long term use [12e14]. Thus, the development non-sulfur and non-phosphorus phthalimide derivatives has been a meaningful and promising way to discover novel alternatives with new or synergic action mode. Moreover, many phthalimide derivatives has been reported to show antimicrobial activity recently, however as far as we knew, few of them was applied in controlling phytopathogenic fungi [15,16]. Based on this, we focused on coupling phthalimide with various functional moieties to prepare a series of N-substituted phthalimides and evaluating their fungicidal activity with two disastrous phytopathogenic fungi Alternaria solani (A. solani, a universal phytopathogenic fungus, can cause destructive diseases called “early blight” in tomato and potato.) and Botrytis cinerea (B. cinerea, a major necrotrophic fungus, severely limits the storage of fruits.) to explore for the effective chemical candidates for the plant fungal disease control (see Fig. 2).
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dibromopropane or 1, 4-dibromobutane (6.0 mmol) was added respectively. The mixture was stirred in reflux and the reaction was monitored by TLC. After cooling down, the mixture was separated and filtered out. The solvent was concentrated in vacuum to afford crude products, which were recrystallized from ethyl acetate to obtain compounds 1~3. Fig. 1. The structure of phaltane and phosmet.
2. Material and methods Chemicals. All the reactants were of AR grades and used without purification. Reactions were monitored by silica-coated TLC plates (Silica Gel 60 F254). Column chromatography (Silica gel: 200e300 mesh) were used for purification. Melting points were detected with a X-4 melting point apparatus (Beijing Tech Instrument Co. Ltd., China). Nuclear magnetic resonance (1H and 13 C NMR) spectra were performed on Bruker AM-400BB (400 MHz) spectrometer (Bruker, Karlsruhe, Germany) with TMS as internal standard. EIMS spectra were measured on an HP 5988A spectrometer. ESI-HRMS was recorded using a Bruker micrOTOF-Q II. The purity of compound was monitored by silica-coated TLC plates. 2.1. Synthesis of target compounds 1e25 2.1.1. General procedure for the preparation of compounds 1~3 To a solution of phthalimide (5.0 mmol) and potassium carbonate (10.0 mmol) in acetone (50.0 mL), 1, 2-dibromoethane, 1, 3-
2.1.1.1. N-(2-bromoethyl) phthalimide (1). White solid; Yield: 86%; m.p. 80e81 C; 1H NMR (CHCl3-d, 400 MHz) d: 3.76 (t, 2H, J ¼ 6.8 Hz, H-11), 4.07 (t, 2H, J ¼ 6.8 Hz, H-10), 7.69 (dd, 2H, J ¼ 5.6 Hz, 3.2 Hz, H-5, H-8), 7.82 (dd, 2H, J ¼ 5.6 Hz, 3.2 Hz, H-6, H7); 13C NMR (CHCl3-d, 100 MHz) d: 27.85, 37.78, 123.32, 131.96, 133.95, 168.24. EIMS, m/z 253 [M]þ. 2.1.1.2. N-(3-bromanylpropyl) phthalimide (2). White solid; Yield: 84%; m.p. 75e76 C; 1H NMR (CHCl3-d, 400 MHz) d: 2.31e2.40 (m, 2H, H-11), 3.51 (t, 2H, J ¼ 6.0 Hz, H-12), 4.07 (t, 2H, J ¼ 6.0 Hz, H-10), 7.70 (dd, 2H, J ¼ 5.6 Hz, 3.2 Hz, H-5, H-8), 7.84 (dd, 2H, J ¼ 5.6 Hz, 3.2 Hz, H-6, H-7); 13C NMR (CHCl3-d, 100 MHz) d: 28.66, 35.94, 36.93, 123.74, 132.16, 133.48, 168.36. EIMS, m/z 267 [M]þ. 2.1.1.3. N-(4-bromobutyl) phthalimide(3). White solid; Yield: 82%; m.p. 73e74 C; 1H NMR (CHCl3-d, 400 MHz) d: 2.02e2.12 (m, 4H, H11, H-12), 3.65 (t, 2H, J ¼ 6.4 Hz, H-13), 3.93 (t, 2H, J ¼ 6.4 Hz, H-10), 7.92 (dd, 2H, J ¼ 5.6 Hz, 3.2 Hz, H-5, H-8), 8.05 (dd, 2H, J ¼ 5.6 Hz, 3.2 Hz, H-6, H-7); 13C NMR (CHCl3-d, 100 MHz) d: 27.55, 28.93, 33.83, 37.88, 123.36, 132.26, 133.71, 168.26. EIMS, m/z 281 [M]þ.
Fig. 2. Representative pictures of A. solani and B. cinerea treated with compound 13 and 4 at the concentration of 100 mg/mL for 96 h.
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2.1.2. General procedure for the preparation of compounds 4 and 5 Potassium hydroxide (0.5 mmol) and quinoline (0.1 mmol) was added to a solution of compound 1 or 2 (0.5 mmol) in ethanol (15.0 mL) respectively. The mixture was refluxed to the end of the reaction. After that, the mixture was cooled down and separated. The solvent was evaporated to prepare crude products, which were purified by chromatography (CHCl3/MeOH 50/1) to give compounds 4 and 5. 2.1.2.1. N-vinylphthalimide (4). White solid; Yield: 31%; m.p. 85e86 C; 1H NMR (CHCl3-d, 400 MHz) d: 5.07 (d, 1H, J ¼ 10.0 Hz, H11), 6.11 (d, 1H, J ¼ 16.4 Hz, H-11), 6.90 (dd, 1H, J ¼ 16.4 Hz, 10.0 Hz, H-10), 7.77 (dd, 2H, J ¼ 5.6 Hz, 3.2 Hz, H-5, H-8), 7.79 (dd, 2H, J ¼ 5.6 Hz, 3.2 Hz, H-6, H-7); 13C NMR (CHCl3-d, 100 MHz) d: 104.56, 123.67, 123.84, 131.67, 134.49, 166.50. EIMS, m/z 173 [M]þ. 2.1.2.2. N-propenylphthalimide (5). White solid; Yield: 33%; m.p. 69e70 C; 1H NMR (CHCl3-d, 400 MHz) d: 5.19 (d, 1H, J ¼ 10.0 Hz, H12), 5.49 (d, 1H, J ¼ 13.6 Hz, H-12), 5.56 (s, 2H, H-10), 5.90e6.05 (m, 1H, H-11), 7.74 (dd, 2H, J ¼ 5.6 Hz, 3.2 Hz, H-5, H-8), 7.87 (dd, 2H, J ¼ 5.6 Hz, 3.2 Hz, H-6, H-7); 13C NMR (CHCl3-d, 100 MHz) d: 39.54, 116.23, 123.23, 130.51, 132.16, 133.93, 165.74. EIMS, m/z 187 [M]þ. 2.1.3. General procedure for the preparation of compounds 6~8 To a solution of phthalimide (1.0 mmol) and potassium carbonate (2.0 mmol) in acetonitrile (15.0 mL), compounds 1, 2 or 3 (1.0 mmol) was added respectively. The mixture was refluxed with stirring for 6 h. Then it was cooled down and filtered out. The solvent was evaporated to get crude products, which were recrystallized from ethyl acetate to obtain compounds 6~8 with high yield. 2.1.3.1. Bis-N-(phthalimido)-1, 2-ethane (6). White solid; Yield: 80%; m.p. 233e234 C; 1H NMR (CHCl3-d, 400 MHz) d: 4.04 (s, 4H, H-10, H-11), 7.71 (dd, 4H, J ¼ 5.6 Hz, 3.2 Hz, H-5, H-50 , H-8, H-80 ), 7.81 (dd, 4H, J ¼ 5.6 Hz, 3.2 Hz, H-6, H-60 , H-7, H-70 ); 13C NMR (CHCl3-d, 100 MHz) d: 35.85, 123.36, 131.99, 133.99, 168.22. EIMS, m/ z 320 [M]þ. 2.1.3.2. Bis-N-(phthalimido)-1, 3-propylene (7). White solid; Yield: 72%; m.p. 189e190 C; 1H NMR (CHCl3-d, 400 MHz) d: 2.09e2.17 (m, 2H, H-11), 3.79 (t, 4H, J ¼ 7.2 Hz, H-10, H-12), 7.73 (dd, 4H, J ¼ 5.6 Hz, 3.2 Hz, H-5, H-50 , H-8, H-80 ), 7.86 (dd, 4H, J ¼ 5.6 Hz, 3.2 Hz, H-6, H-60 , H-7, H-70 ); 13C NMR (CHCl3-d, 100 MHz) d: 27.69, 35.77, 123.29, 132.11, 133.97, 168.22. EIMS, m/z 334 [M]þ. 2.1.3.3. Bis-N-(phthalimido)-1, 4-butane (8). White solid; Yield: 75%; m.p. 220e221 C; 1H NMR (CHCl3-d, 400 MHz) d: 1.74e1.78 (m, 4H, H-11, H-12), 3.75 (t, 4H, J ¼ 6.8 Hz, H-10, H-13), 7.73 (dd, 4H, J ¼ 5.6 Hz, 3.2 Hz, H-5, H-50 , H-8, H-80 ), 7.85 (dd, 4H, J ¼ 5.6 Hz, 3.2 Hz, H-6, H-60 , H-7, H-70 ); 13C NMR (CHCl3-d, 100 MHz) d: 25.06, 37.42, 123.25, 132.13, 133.93, 168.36. EIMS, m/z 348 [M]þ. 2.1.4. General procedure for the preparation of compounds 9~11 To a solution of 6-methoxy-4-methylquinolin-2(1H)-one (0.5 mmol) in methylbenzene (15.0 mL), compounds 1, 2 or 3 (0.5 mmol) was added respectively. The reaction was conducted at 90 C in the presence of KOH (1.0 mmol), KI (0.05 mmol) and TBAB (0.05 mmol) and monitored with TLC. After the reaction, the mixture was cooled down and separated. The solvent was evaporated to prepare crude products, which were purified by chromatography (CHCl3/MeOH 100/1) to give compounds 9~11. 2.1.4.1. N-[2-(6-methoxy-4-methylquinolin-2-on-1-yl)ethyl]phthalimide (9). White solid; Yield: 48%; m.p. 215e216 C; 1H NMR
(CHCl3-d, 400 MHz) d: 2.52 (s, 3H, CH3), 3.94 (s, 3H, OCH3), 4.12 (t, 2H, d ¼ 5.6 Hz, H-10), 4.81 (t, 2H, d ¼ 5.6 Hz, H-11), 6.69 (s, 1H, H30 ), 7.06 (d, 1H, J ¼ 2.8 Hz, H-50 ), 7.36 (m, 1H, H-70 ), 7.62 (dd, 2H, J ¼ 5.6 Hz, 3.2 Hz, H-5, H-8), 7.72 (dd, 2H, J ¼ 5.6 Hz, 3.2 Hz, H-6, H7), 7.77 (d, 1H, J ¼ 8.4 Hz, H-80 ); 13C NMR (CHCl3-d, 100 MHz) d: 19.13, 23.47, 37.09, 40.19, 54.83, 101.42, 111.94, 123.67, 127.41, 132.07, 132.21, 136.44, 136.53, 147.79, 156.55, 161.25, 167.69. MS (ESI) m/z 363.1 ([MþH]þ). 2.1.4.2. N-[3-(6-methoxy-4-methylquinolin-2-on-1-yl) propyl] phthalimide (10). White solid; Yield: 34%; m.p. 189e190 C; 1H NMR (CHCl3-d, 400 MHz) d: 1.88e1.92 (m, 2H, H-11), 2.45 (s, 3H, CH3), 3.88 (s, 3H, OCH3), 4.35 (t, 2H, J ¼ 5.6 Hz, H-12), 4.49 (t, 2H, J ¼ 5.6 Hz, H-10), 6.58 (s, 1H, H-30 ), 6.79 (s, 1H, H-50 ), 7.21 (d, 1H, J ¼ 2.8 Hz, H-70 ), 7.41 (dd, 2H, J ¼ 5.2 Hz, 3.2 Hz, H-5, H-8), 7.63 (dd, 2H, J ¼ 5.2 Hz, 3.2 Hz, H-6, H-7), 7.89 (d, 1H, H-80 ); 13C NMR (CHCl3d, 100 MHz) d: 19.34, 23.47, 37.88, 40.18, 55.58, 102.96, 107.40, 112.54, 116.69, 119.82, 127.30, 128.18, 128.37, 129.38, 146.36, 156.26, 160.74, 168.89. MS (ESI) m/z 377.1 ([MþH]þ). 2.1.4.3. N-[4-(6-methoxy-4-methylquinolin-2-on-1-yl) butyl] phthalimide (11). White solid; Yield: 45%; m.p. 185e186 C; 1H NMR (CHCl3-d, 400 MHz) d: 1.57 (m, 2H, H-11), 1.93 (m, 2H, H-12), 2.63 (s, 3H, CH3), 3.81 (t, 2H, J ¼ 6.8 Hz, H-10), 3.94 (s, 3H, OCH3), 4.61 (t, 2H, J ¼ 6.8 Hz, H-13), 6.80 (s, 1H, H-30 ), 7.18 (d, 1H, J ¼ 2.8 Hz, H-50 ), 7.33 (d, 1H, J ¼ 9.2 Hz, H-70 ), 7.73 (dd, 2H, J ¼ 5.2 Hz, 3.2 Hz, H-5, H-8), 7.86 (dd, 2H, J ¼ 5.2 Hz, 3.2 Hz, H-6, H-7), 8.02 (d, 1H, J ¼ 7.2 Hz, H80 ); 13C NMR (CHCl3-d, 100 MHz) d: 18.87, 25.47, 25.57, 37.60, 37.88, 55.58, 103.47, 113.24, 123.19, 123.24, 125.81, 132.14, 132.19, 133.86, 133.91, 139.94, 156.06, 160.53, 168.44. MS (ESI) m/z 391.2 ([MþH]þ). 2.1.5. General procedure for the preparation of compounds 12 and 13 To a solution of N-(4-bromobutyl) phthalimide (3) (1 mmol) in methylbenzene (15.0 mL), 7-hydroxy-4-methylcoumarin (1 mmol) or 8-hydroxyquinoline (1 mmol) was added. The mixture was heated to 90 C with stirring in the presence of KOH (2.0 mmol), KI (0.06 mmol) and TBAB (0.06 mmol). After the reaction, the mixture was cooled down and separated. The solvent was evaporated to prepare crude products, which were purified by chromatography (CHCl3/MeOH 50/1) to give compounds 12 and 13. 2.1.5.1. 4-Methyl-7-[4-(isoindole-1, 3-dione-2-yl) butyloxy] benzopyran-2-one (12). White solid; Yield: 51%; m.p. 181e182 C; 1H NMR (CHCl3-d, 400 MHz) d:1.90e1.92 (m, 4H, H-11, H-12), 2.41 (s, 2H, CH3), 3.80 (t, 2H, J ¼ 6.4 Hz, H-10), 4.08 (t, 2H, J ¼ 6.4 Hz, H-13), 6.15 (s, 1H, H-30 ), 6.80 (d, 1H, J ¼ 2.4 Hz, H-80 ), 6.86 (dd, 1H, J ¼ 8.4 Hz, 2.4 Hz, H-60 ), 7.48e7.50 (d, 1H, J ¼ 8.4 Hz, H-50 ), 7.73e7.75 (m, 2H, H-5, H-8), 7.86e7.88 (m, 2H, H-6, H-7); 13C NMR (CHCl3-d, 100 MHz) d: 18.66, 25.27, 26.39, 37.56, 67.77, 101.47, 111.97, 112.59, 113.58, 123.26, 125.49, 132.11, 133.98, 152.48, 155.29, 161.29, 161.98, 168.43. MS (ESI) m/z 378.1 ([MþH]þ). 2.1.5.2. 8-[4-(phthalimide-2-yl) butyloxy] quinoline (13). White solid; Yield: 60%; m.p. 127e128 C; 1H NMR (CHCl3-d, 400 MHz) d:1.99e2.05 (m, 2H, H-11), 2.09e2.14 (m, 2H, H-12), 3.84 (t, 2H, J ¼ 5.8 Hz, H-10), 4.33 (t, 2H, J ¼ 5.8 Hz, H-13), 7.13 (d, 1H, J ¼ 8.0 Hz, H-70 ), 7.42 (d, 1H, J ¼ 8.0 Hz, H-30 ), 7.48e7.52 (m, 2H, H-50 , H-60 ), 7.73 (dd, 2H, J ¼ 5.6 Hz, 3.2 Hz, H-5, H-8), 7.86 (dd, 2H, J ¼ 5.6 Hz, 3.2 Hz, H-6, H-7), 8.21 (d, 1H, J ¼ 8.0 Hz, H-40 ), 9.01 (d, 1H, J ¼ 2.4 Hz, H-80 ); 13C NMR (CHCl3-d, 100 MHz) d: 25.23, 25.27, 37.59, 68.39, 109.42, 119.55, 121.56, 123.21, 127.21, 129.57, 132.16, 133.91, 148.63, 148.66, 150.24, 168.45. MS (ESI) m/z 347.1 ([MþH]þ).
L. Pan et al. / Microbial Pathogenesis 95 (2016) 186e192
2.1.6. General procedure for the preparation of compounds 14~25 Compounds 14~25 were prepared by the condensation reaction between phthalic anhydride and related primary amine. To a solution of phthalic anhydride (0.5 mmol) in acetic acid (10 mL), related primary amine (0.5 mmol) was added respectively. The mixture was refluxed at 120 C for 3e4 h. Then 20 mL distilled water was added into the mixture after being cooled down and the solid precipitate was filtered out to get crude compounds, which were recrystallized in ethanol to prepare compounds 14~25. 2.1.6.1. N-phenylphthalimide (14). White solid; Yield: 88%; m.p. 209e210 C; 1H NMR (CHCl3-d, 400 MHz) d: 7.42e7.48 (m, 3H, H-30 , H-40 , H-50 ), 7.52e7.56 (m, 2H, H-20 , H-60 ), 7.82 (dd, 2H, J ¼ 7.2 Hz, 3.2 Hz, H-5, H-8), 7.99 (dd, 2H, J ¼ 7.2 Hz, 3.2 Hz, H-6, H-7); 13C NMR (CHCl3-d, 100 MHz) d: 123.77, 126.60, 128.11, 129.13, 131.83, 134.39, 167.29. EIMS, m/z 223 [M]þ. 2.1.6.2. N-(phenylmethyl) phthalimide (15). White solid; Yield: 90%; m.p. 118e119 C; 1H NMR (CHCl3-d, 400 MHz) d: 4.88 (s, 2H, H-10), 7.30e7.36 (m, 3H, H-30 , H-40 , H-50 ), 7.46 (d, 2H, J ¼ 7.2 Hz, H-20 , H60 ), 7.73 (dd, 2H, J ¼ 5.2 Hz, 3.2 Hz, H-5, H-8), 7.87 (dd, 2H, J ¼ 5.2 Hz, 3.2 Hz, H-6, H-7); 13C NMR (CHCl3-d, 100 MHz) d: 41.64, 123.36, 127.83, 128.62, 128.69, 132.18, 133.98, 168.05. EIMS, m/z 237 [M]þ. 2.1.6.3. N-(4-methoxyphenyl) phthalimide (16). Purple solid; Yield: 82%; m.p. 158e159 C; 1H NMR (CHCl3-d, 400 MHz) d: 3.88 (s, 3H, OCH3), 7.05 (dd, 2H, J ¼ 7.2 Hz, 2.0 Hz, H-30 , H-50 ), 7.36 (d, 2H, J ¼ 8.8 Hz, H-20 , H-60 ), 7.81 (dd, 2H, J ¼ 5.6 Hz, 3.2 Hz, H-5, H-8), 7.98 (dd, 2H, J ¼ 5.6 Hz, 3.2 Hz, H-6, H-7); 13C NMR (CHCl3-d, 100 MHz) d: 55.54, 114.52, 123.68, 127.95, 131.89, 134.30, 159.29, 167.58. EIMS, m/ z 253 [M]þ. 2.1.6.4. N-(benzamide-4-yl) phthalimide (17). White solid; Yield: 78%; m.p. 264e265 C; 1H NMR (CHCl3-d, 400 MHz) d: 7.64 (d, 2H, J ¼ 8.2 Hz, H-30 , H-50 ), 7.85 (dd, 2H, J ¼ 7.2 Hz, 3.2 Hz, H-5, H-8), 7.98 (d, 2H, J ¼ 8.2 Hz, H-20 , H-60 ); 8.01 (dd, 2H, J ¼ 7.2 Hz, 3.2 Hz, H-6, H7); 13C NMR (CHCl3-d, 100 MHz) d: 123.85, 126.25, 128.20, 129.27, 131.82, 132.26, 139.13, 167.53, 168.78. EIMS, m/z 266 [M]þ. 2.1.6.5. N-(quinolin-5-yl) phthalimide (18). White solid; Yield: 86%; m.p. 217e218 C; 1H NMR (CHCl3-d, 400 MHz) d: 7.48 (dd, 1H, J ¼ 8.8 Hz, 4.0 Hz, H-80 ), 7.59 (d, 1H, J ¼ 7.2 Hz, H-30 ), 7.88e7.92 (m, 3H, H-5, H-8, H-70 ), 8.03e8.07 (m, 3H, H-6, H-7, H-40 ), 8.34 (d, 1H, J ¼ 8.4 Hz, H-20 ), 9.01e9.02 (m, 1H, H-60 ); 13C NMR (CHCl3-d, 100 MHz) d: 121.79, 121.80, 124.15, 125.80, 125.81, 127.75, 127.76, 128.20, 129.41, 131.84, 134.79, 150.23, 167.49. EIMS, m/z 274 [M]þ. 2.1.6.6. N-(isoquinolin-5-yl) phthalimide (19). White solid; Yield: 83%; m.p. 238e239 C; 1H NMR (CHCl3-d, 400 MHz) d: 7.56 (d, 1H, J ¼ 6.0 Hz, H-60 ), 7.79e7.87 (m, 2H, H-70, H-80 ), 7.90 (dd, 2H, J ¼ 5.6 Hz, 3.2 Hz, H-5, H-8), 8.06 (dd, 2H, J ¼ 5.6 Hz, 3.2 Hz, H-6, H7), 8.23 (d, 1H, J ¼ 8.0 Hz, H-40 ), 8.58 (d, 1H, J ¼ 6.0 Hz, H-30 ), 9.46 (s, 1H, H-10 ); 13C NMR (CHCl3-d, 100 MHz) d: 116.62, 124.21, 127.69, 127.77, 129.29, 129.79, 131.80, 132.22, 133.65, 134.86, 141.89, 151.86, 167.27. EIMS, m/z 274 [M]þ. 2.1.6.7. N-(quinolin-8-yl) phthalimide (20). White solid; Yield: 83%; m.p. 227e228 C; 1H NMR (CHCl3-d, 400 MHz) d: 7.48 (dd, 1H, J ¼ 8.4 Hz, 4.4 Hz, H-60 ), 7.72 (d, 1H, J ¼ 8.0 Hz, H-50 ), 7.84 (dd, 2H, J ¼ 5.2 Hz, 3.2 Hz, H-5, H-8), 7.90 (dd, 1H, J ¼ 7.2 Hz, 3.2 Hz, H-30 ), 7.99e8.04 (m, 3H, H-6, H-7, H-40 ), 8.28 (dd, 1H, J ¼ 8.4 Hz, 1.2 Hz, H70 ), 8.89e8.90 (m, 1H, H-20 ); 13C NMR (CHCl3-d, 100 MHz) d: 121.92, 123.91, 126.32, 129.36, 129.61, 129.73, 130.46, 132.53, 134.19, 136.55, 136.58, 150.73, 167.91. EIMS, m/z 274 [M]þ.
189
2.1.6.8. N-(5-carboxylhloropyridine-2-yl) phthalimide (21). White solid; Yield: 80%; m.p. 164e165 C;1H NMR (DMSO-d6, 400 MHz) d: 7.70 (m, 1H, H-50 ), 7.79e8.02 (m, 4H, H-5, H-6, H-7, H8), 8.49 (d, 1H, J ¼ 8.0 Hz, H-40 ), 9.11 (d, 1H, J ¼ 1.2 Hz, H-20 ), 13.58 (s, 1H, COOH); 13C NMR (DMSO-d6, 100 MHz) d: 112.26, 119.86, 124.58, 132.35, 134.02, 140.76, 150.61, 152.35, 165.88, 167.72. EIMS, m/z 268 [M]þ. 2.1.6.9. N-(2-chloropyridine-4-yl) phthalimide (22). White solid; Yield: 81%; m.p. 156e157 C; 1H NMR (CHCl3-d, 400 MHz) d: 7.65 (dd, 1H, J ¼ 5.6 Hz, 1.6 Hz, H-30 ), 7.76 (d, 1H, J ¼ 1.6 Hz, H-50 ), 7.88 (dd, 2H, J ¼ 7.2 Hz, 3.2 Hz, H-5, H-8), 8.03 (dd, 2H, J ¼ 7.2 Hz, 3.2 Hz, H-6, H-7), 8.54 (d, 1H, J ¼ 5.6 Hz, H-60 ); 13C NMR (CHCl3-d, 100 MHz) d: 117.71, 119.49, 124.32, 131.22, 135.18, 141.84, 150.38, 152.29, 165.93. EIMS, m/z 258 [M]þ. 2.1.6.10. 2-(phthalimide-2-yl)-3-(1H-indol-3-yl) propanoic acid (23). White solid; Yield: 85%; m.p. 195e196 C; 1H NMR (DMSO-d6, 400 MHz) d: 3.73e3.87 (m, 2H, H-11), 5.36 (dd, 1H, J ¼ 10.8 Hz, 5.2 Hz, H-10), 7.05 (s, 1H, H-20 ), 7.10 (t, 1H, J ¼ 8.0 Hz, H-50 ), 7.16 (t, 1H, J ¼ 7.2 Hz, H-60 ), 7.62 (d, 1H, J ¼ 8.0 Hz, H-70 ), 7.69 (dd, 2H, J ¼ 5.2 Hz, 3.2 Hz, H-5, H-8), 7.78 (dd, 2H, J ¼ 5.2 Hz, 3.2 Hz, H-6, H7), 7.94 (d, 1H, H-40 ); 13C NMR (DMSO-d6, 100 MHz) d: 24.71, 52.32, 110.99, 111.08, 118.51, 119.66, 122.22, 122.55, 123.48, 131.69, 134.07, 136.10, 167.47, 172.53. EIMS, m/z 334 [M]þ. 2.1.6.11. N-(aminoantipyrine-4-yl) phthalimide (24). White solid; Yield: 87%; m.p. 214e215 C; 1H NMR (CHCl3-d, 400 MHz) d: 2.25 (s, 3H, CH3), 3.27 (s, 3H, NCH3), 7.33e7.37 (m, 1H, H-40 ), 7.47e7.50 (m, 4H, H-20 , H-30 , H-50 , H-60 ), 7.79 (dd, 2H, J ¼ 5.2 Hz, 3.2 Hz, H-5, H-8), 7.96 (dd, 2H, J ¼ 5.2 Hz, 3.2 Hz, H-6, H-7); 13C NMR (CHCl3-d, 100 MHz) d: 11.15, 35.54, 102.23, 123.83, 124.86, 127.33, 129.29, 132.25, 134.29, 134.39, 152.78, 160.77, 166.91. EIMS, m/z 333 [M]þ. 2.1.6.12. 2-(4-methyl-2-oxidanylidene-chromen-7-yl) phthalimide (25). White solid; Yield: 80%; m.p. 255e256 C; 1H NMR (CHCl3-d, 400 MHz) d: 2.50 (s, 3H, CH3), 6.37 (s, 1H, H-30 ), 7.52 (dd, 1H, J ¼ 9.6 Hz, 2.0 Hz, H-60 ), 7.58 (d, 1H, J ¼ 2.0 Hz, H-80 ), 7.76 (d, 1H, J ¼ 8.4 Hz, H-50 ), 7.86 (dd, 2H, J ¼ 5.6 Hz, 3.2 Hz, H-5, H-8), 8.02 (dd, 2H, J ¼ 5.6 Hz, 3.2 Hz, H-6, H-7); 13C NMR (CHCl3-d, 100 MHz) d: 18.69, 114.74, 115.52, 121.80, 124.09, 125.05, 131.51, 134.77, 134.82, 151.73, 153.68, 160.37, 166.63. EIMS, m/z 305 [M]þ. 2.2. Antifungal bioassay Two destructive phytopathogenic fungi (A. solani and B. cinerea) were applied to test the antifungal activity of the prepared compounds in vitro by measuring mycelial inhibition of radial growth. All compounds were dissolved in DMSO as stock solutions and diluted to a testing concentration of 100 mg/mL with PDA medium under 50 C. The final solutions were poured into sterilized Petri dishes (6 cm). After solidification, a mycelia disk (diameter: 0.6 cm) of active fungi was inoculated in the center of the PDA Petri dishes. The dishes were parafilmed and incubated at 25 C for 5 days. DMSO mixed with PDA to the equivalent concentrations was used as negative control, and carbendazim was served as positive control. Growth inhibition was calculated by the formula: Inhibition rate (%) ¼ (C-T) 100/C (C: the mean colony diameter in control; T: the colony diameter in treatments). The measurement was carried out with three replicates, and the results were shown as mean values (±SD). The effective compounds were further tested under a series of lower concentrations to calculate the IC50 values [17,18].
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L. Pan et al. / Microbial Pathogenesis 95 (2016) 186e192
Scheme 1. Synthesis of phthalimide derivatives (1e13).
Scheme 2. Synthesis of phthalimide derivatives (14e25).
3. Results and discussion A series of phthalimide coupled derivatives were efficiently synthesized according to the proposed methods, and the general synthesis was depicted in Schemes 1 and 2. Compounds 1~3 were synthesized by the N-alkylation of phthalimide with appropriate dibromoalkane [19,20]. Under the similar condition, compounds 1~3 condensed with phthalimide to give compounds 6~8. But the coupling of compounds 1~3 with 6-methoxy-4-methylquinolin2(1H)-one could not be realized in the presence of potassium carbonate, a complex alkaline system of potassium hydroxide, potassium iodide and tetrabutyl ammonium bromide (TBAB) was applied to prepare 9~11. Compounds 12 and 13 were prepared under the similar condition by the reaction between compound 3 and 7-hydroxy-4-methylcoumarin or 8-hydroxyquinoline. With the presence of potassium hydroxide and quinoline in ethanol, compounds 1 and 2 eliminated the hydrogen bromide to prepare compounds 4 and 5 respectively (Scheme 1).
Phthalic anhydride and related primary amine were employed to prepare compounds 14~25 effectively [21,22] (Scheme 2). The antifungal activity of prepared compounds against A. solani and B. cinerea was initially assayed at 100 mg/mL (Table 1), at which level, most of the prepared compounds showed significant fungitoxicity against either or both of pathogenic fungi. Among these tested derivatives, compounds 1, 2, 3, 4, 13 and 15 exhibited the strong activity against both A. solani and B. cinerea with over 50% inhibition of mycelial growth. Compounds 6, 8, 11 and 18 selectively inhibited 50.00e57.14% of mycelia growth against A. solani, whereas compound 16 showed stronger inhibitory effects against B. cinerea (53.19% of inhibition) than A. solani (with 37.62% inhibition). The effective compounds with over 50% inhibition of mycelial growth were further tested at a series of lower concentrations and their IC50 values were calculated (Table 2). The results indicated that compound 4 has the lowest IC50 value against B. cinerea (7.92 mg/mL), and compounds 13 showed the strongest antifungal activity against A. solani with IC50 values of 10.85 mg/mL.
L. Pan et al. / Microbial Pathogenesis 95 (2016) 186e192 Table 1 Antifungal activity of the prepared compounds at 100 mg/mL (96 h of incubation). The inhibition of radial growth (%, mean ± SD, N ¼ 3)
Compd.
A. solani 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 DMSO(1%) Carbendazim
64.29 59.52 69.05 78.57 48.14 50.00 42.86 54.76 40.48 47.62 52.38 45.71 71.43 41.77 54.76 37.62 27.60 57.14 49.38 38.10 40.48 19.05 15.30 40.48 33.33 0 93.88
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
B. cinerea 78.72 ± 10.35 51.94 ± 6.83 74.47 ± 7.69 93.62 ± 6.92 45.07 ± 15.08 34.04 ± 10.76 31.91 ± 13.09 48.94 ± 3.26 31.91 ± 12.85 36.17 ± 8.60 38.30 ± 2.30 42.55 ± 12.26 91.49 ± 22.30 39.18 ± 11.33 65.96 ± 6.20 53.19 ± 15.38 33.15 ± 9.79 25.53 ± 3.84 27.66 ± 7.69 6.38 ± 4.26 2.13 ± 0.96 14.89 ± 7.92 0 38.30 ± 9.14 27.66 ± 13.27 1.03 ± 1.16 95.16 ± 9.47
14.26 3.85 10.57 14.35 10.90 12.24 5.23 21.57 7.23 2.81 7.14 11.42 8.53 7.45 2.14 18.44 12.45 22.16 21.42 2.17 12.43 9.85 10.89 12.85 15.71
± 17.29
Table 2 IC50 values of effective compounds against two harmful phytopathogenic fungi (96 h of incubation). Compd.
1 2 3 4 7 8 11 13 15 16 18 Carbendazim a
IC50 (95% cL)a (mg/mL) A. solani
B. cinerea
45.83 50.17 48.12 16.95 164.06 112.38 171.48 10.85 65.19 e 85.71 1.74
33.33 129.58 64.69 7.92 e 178.40 e 35.65 91.55 98.43 e 1.90
(29.35e62.97) (32.08e76.56) (21.35e69.78) (4.84e29.85) (107.65e221.25) (87.54e156.16) (143.35e236.59) (3.62e20.84) (38.42e86.93) (69.23e107.82) (0.23e3.03)
(19.35e57.61) (105.31e159.93) (43.81e79.78) (3.08e16.40)
191
two moieties were coupled directly. On phenyl substitution, N(phenylmethyl) phthalimide (15) was more effective than N-phenylphthalimides (14) and the introduction of methoxyl group at para-position (16) could increase the activity, whereas benzamide substitution (17) contributed less. It was found that direct coupling with other functional moieties of pyridine (21, 22), tryptophane (23), antipyrine (24) and coumarin (25) made little contribution to the activity. The results indicated that the appropriate functional groups with indirect coupling by optimum carbon chain might be promising to prepare effective fungicide. 4. Conclusion In summary, N-substituted pthalimide derivatives were efficiently synthesized by modified condensation reactions. With screening for their antifungal activity against A. solani and B. cinerea, N-vinyl-phthalimide (4) and 8-[4-(phthalimide-2-yl) butyloxy]quinoline (13) were found to be the most effective among the prepared compounds and proposed to be promising candidates for the further development. With studying the activity contribution of functional moieties, it clearly suggested that the optimum substitution at N atom of pthalimide could bring about significant increase of activity, which would be helpful for the discovery of efficacious fungicides. Funding This work was supported by the National Natural Science Foundation of China (No. 31570354, 31070386, 21302195 and 31300290), Agricultural Biotechnology Research and Development Program of Gansu Province (GNSW-2015-25), Cooperation Program to Gansu Province of Lanzhou Branch of the Chinese Academy of Sciences, 135 key cultivation program of the Chinese Academy of Sciences, and the Province-academy Cooperation Program of Henan Province of China (NO. 102106000021). Notes The authors declare no competing financial interest.
(130.90e229.47)
Appendix A. Supplementary data (21.92e49.25) (49.76e124.85) (65.31e140.40)
Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.micpath.2016.04.012.
(0.39e2.60)
95% confidence limit.
Concerning the phytotoxicity, we took a preliminary test on the growth inhibition of tomato seedlings with effective phthalimides derivatives. The results shows that the compounds exhibited insignificant inhibitory effects on seedling growth in comparing with negative control (data not shown here). For their possible use, more comprehensive work should be conducted to evaluate their efficacy and safety. The types of functional groups are intensive responsible for the activity. N-vinyl substituted phthalimide (4) exhibited strong antifungal activity against both A. solani and B. cinerea, and Nbromoalkylation of phthalimides (1~3) significantly increased the inhibitory effects, regardless of the side chain length. However, the chain length of quinoline coupled phthalimides was found to influence the activity deeply, such as compound 13, with butoxy chain between quinoline and phthalimides, exhibited much stronger toxicity against both fungi than compound 20, of which
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Microbial Pathogenesis journal homepage: www.elsevier.com/locate/micpath
Synthesis of N-substituted phthalimides and their antifungal activity against Alternaria solani and Botrytis cinerea Le Pan a, b, Xiuzhuang Li b, Chengwen Gong c, Hui Jin b, Bo Qin b, * a
University of Chinese Academy of Sciences, People's Republic of China Key Laboratory of Chemistry of Northwestern Plant Resources of CAS and Key Laboratory for Natural Medicine of Gansu Province, Lanzhou Institute of Chemical Physics, Chinese Academy of Sciences, Lanzhou 730000, People's Republic of China c Institute of Chinese Medicinal Herbs, Gansu Academy of Agricultural Sciences, Lanzhou 730070, People's Republic of China b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 2 July 2015 Received in revised form 15 September 2015 Accepted 10 April 2016 Available online 12 April 2016
As organosulfur and organophosphorus agents, phaltane and phosmet are facing great challenges for the environmental contamination, mammalian toxicity and increasing resistance with long term use. It is efficient and meaningful to develop phthalimide-based alternatives with non-sulfur and nonphosphorus groups. A series of N-substituted phthalimides were synthesized and their antifungal activity against two disastrous phytopathogenic fungi, Alternaria solani and Botrytis cinerea was evaluated in vitro. Most of them showed significant antifungal activity against both of fungi, or either of them selectively. N-vinylphthalimide (4) and 8-[4-(phthalimide-2-yl) butyloxy] quinoline (13) were identified as the most promising candidates against B. cinerea and A. solani with the IC50 values of 7.92 mg/mL and 10.85 mg/mL respectively. The brief structure-activity relationships have revealed that vinyl, quinolyl, bromide alkyl and benzyl substitutions were appropriate substituents and coupling functional moieties indirectly with optimum alkyl chain was efficient to prepare phthalimides related fungicides. © 2016 Elsevier Ltd. All rights reserved.
Keywords: N-substituted phthalimides Synthesis IC50 values Antifungal activity
1. Introduction Plant pathogenic fungi have been listed as one of the disastrous plant pathogens worldwide due to resulting in great agricultural losses [1e3]. Up to now, scientific farming practices and biological methods are advocated on the disease control, but the available methods are restricted to some specific cultivars and the narrow usage range has constrained their application [4,5]. Therefore, chemical pesticides have still been the common options on disease control and great effort has been desired to develop novel compounds with highly inhibitory effects on phytopathogenic fungi. Phthalimide subunit existed widely in both natural and synthetic compounds with broad activities against AIDS, cancer and inflammation [6,7]. They were also reported to be the crucial structure of numerous biological inhibitors, such as human protein kinase CK2 inhibitor, COX inhibitors, histone deacetylase inhibitors and etc [8e10]. In agriculture application, phthalimide has represented a famous scaffold for the development of efficient pesticides. Phaltane and phosmet are effective chemical agents in plant
* Corresponding author. E-mail address: [email protected] (B. Qin). http://dx.doi.org/10.1016/j.micpath.2016.04.012 0882-4010/© 2016 Elsevier Ltd. All rights reserved.
disease control [11] (Fig. 1), but as organosulfur and organophosphorus compounds, their applications are facing great challenges for the environmental contamination, mammalian toxicity and increasing resistance with long term use [12e14]. Thus, the development non-sulfur and non-phosphorus phthalimide derivatives has been a meaningful and promising way to discover novel alternatives with new or synergic action mode. Moreover, many phthalimide derivatives has been reported to show antimicrobial activity recently, however as far as we knew, few of them was applied in controlling phytopathogenic fungi [15,16]. Based on this, we focused on coupling phthalimide with various functional moieties to prepare a series of N-substituted phthalimides and evaluating their fungicidal activity with two disastrous phytopathogenic fungi Alternaria solani (A. solani, a universal phytopathogenic fungus, can cause destructive diseases called “early blight” in tomato and potato.) and Botrytis cinerea (B. cinerea, a major necrotrophic fungus, severely limits the storage of fruits.) to explore for the effective chemical candidates for the plant fungal disease control (see Fig. 2).
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dibromopropane or 1, 4-dibromobutane (6.0 mmol) was added respectively. The mixture was stirred in reflux and the reaction was monitored by TLC. After cooling down, the mixture was separated and filtered out. The solvent was concentrated in vacuum to afford crude products, which were recrystallized from ethyl acetate to obtain compounds 1~3. Fig. 1. The structure of phaltane and phosmet.
2. Material and methods Chemicals. All the reactants were of AR grades and used without purification. Reactions were monitored by silica-coated TLC plates (Silica Gel 60 F254). Column chromatography (Silica gel: 200e300 mesh) were used for purification. Melting points were detected with a X-4 melting point apparatus (Beijing Tech Instrument Co. Ltd., China). Nuclear magnetic resonance (1H and 13 C NMR) spectra were performed on Bruker AM-400BB (400 MHz) spectrometer (Bruker, Karlsruhe, Germany) with TMS as internal standard. EIMS spectra were measured on an HP 5988A spectrometer. ESI-HRMS was recorded using a Bruker micrOTOF-Q II. The purity of compound was monitored by silica-coated TLC plates. 2.1. Synthesis of target compounds 1e25 2.1.1. General procedure for the preparation of compounds 1~3 To a solution of phthalimide (5.0 mmol) and potassium carbonate (10.0 mmol) in acetone (50.0 mL), 1, 2-dibromoethane, 1, 3-
2.1.1.1. N-(2-bromoethyl) phthalimide (1). White solid; Yield: 86%; m.p. 80e81 C; 1H NMR (CHCl3-d, 400 MHz) d: 3.76 (t, 2H, J ¼ 6.8 Hz, H-11), 4.07 (t, 2H, J ¼ 6.8 Hz, H-10), 7.69 (dd, 2H, J ¼ 5.6 Hz, 3.2 Hz, H-5, H-8), 7.82 (dd, 2H, J ¼ 5.6 Hz, 3.2 Hz, H-6, H7); 13C NMR (CHCl3-d, 100 MHz) d: 27.85, 37.78, 123.32, 131.96, 133.95, 168.24. EIMS, m/z 253 [M]þ. 2.1.1.2. N-(3-bromanylpropyl) phthalimide (2). White solid; Yield: 84%; m.p. 75e76 C; 1H NMR (CHCl3-d, 400 MHz) d: 2.31e2.40 (m, 2H, H-11), 3.51 (t, 2H, J ¼ 6.0 Hz, H-12), 4.07 (t, 2H, J ¼ 6.0 Hz, H-10), 7.70 (dd, 2H, J ¼ 5.6 Hz, 3.2 Hz, H-5, H-8), 7.84 (dd, 2H, J ¼ 5.6 Hz, 3.2 Hz, H-6, H-7); 13C NMR (CHCl3-d, 100 MHz) d: 28.66, 35.94, 36.93, 123.74, 132.16, 133.48, 168.36. EIMS, m/z 267 [M]þ. 2.1.1.3. N-(4-bromobutyl) phthalimide(3). White solid; Yield: 82%; m.p. 73e74 C; 1H NMR (CHCl3-d, 400 MHz) d: 2.02e2.12 (m, 4H, H11, H-12), 3.65 (t, 2H, J ¼ 6.4 Hz, H-13), 3.93 (t, 2H, J ¼ 6.4 Hz, H-10), 7.92 (dd, 2H, J ¼ 5.6 Hz, 3.2 Hz, H-5, H-8), 8.05 (dd, 2H, J ¼ 5.6 Hz, 3.2 Hz, H-6, H-7); 13C NMR (CHCl3-d, 100 MHz) d: 27.55, 28.93, 33.83, 37.88, 123.36, 132.26, 133.71, 168.26. EIMS, m/z 281 [M]þ.
Fig. 2. Representative pictures of A. solani and B. cinerea treated with compound 13 and 4 at the concentration of 100 mg/mL for 96 h.
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2.1.2. General procedure for the preparation of compounds 4 and 5 Potassium hydroxide (0.5 mmol) and quinoline (0.1 mmol) was added to a solution of compound 1 or 2 (0.5 mmol) in ethanol (15.0 mL) respectively. The mixture was refluxed to the end of the reaction. After that, the mixture was cooled down and separated. The solvent was evaporated to prepare crude products, which were purified by chromatography (CHCl3/MeOH 50/1) to give compounds 4 and 5. 2.1.2.1. N-vinylphthalimide (4). White solid; Yield: 31%; m.p. 85e86 C; 1H NMR (CHCl3-d, 400 MHz) d: 5.07 (d, 1H, J ¼ 10.0 Hz, H11), 6.11 (d, 1H, J ¼ 16.4 Hz, H-11), 6.90 (dd, 1H, J ¼ 16.4 Hz, 10.0 Hz, H-10), 7.77 (dd, 2H, J ¼ 5.6 Hz, 3.2 Hz, H-5, H-8), 7.79 (dd, 2H, J ¼ 5.6 Hz, 3.2 Hz, H-6, H-7); 13C NMR (CHCl3-d, 100 MHz) d: 104.56, 123.67, 123.84, 131.67, 134.49, 166.50. EIMS, m/z 173 [M]þ. 2.1.2.2. N-propenylphthalimide (5). White solid; Yield: 33%; m.p. 69e70 C; 1H NMR (CHCl3-d, 400 MHz) d: 5.19 (d, 1H, J ¼ 10.0 Hz, H12), 5.49 (d, 1H, J ¼ 13.6 Hz, H-12), 5.56 (s, 2H, H-10), 5.90e6.05 (m, 1H, H-11), 7.74 (dd, 2H, J ¼ 5.6 Hz, 3.2 Hz, H-5, H-8), 7.87 (dd, 2H, J ¼ 5.6 Hz, 3.2 Hz, H-6, H-7); 13C NMR (CHCl3-d, 100 MHz) d: 39.54, 116.23, 123.23, 130.51, 132.16, 133.93, 165.74. EIMS, m/z 187 [M]þ. 2.1.3. General procedure for the preparation of compounds 6~8 To a solution of phthalimide (1.0 mmol) and potassium carbonate (2.0 mmol) in acetonitrile (15.0 mL), compounds 1, 2 or 3 (1.0 mmol) was added respectively. The mixture was refluxed with stirring for 6 h. Then it was cooled down and filtered out. The solvent was evaporated to get crude products, which were recrystallized from ethyl acetate to obtain compounds 6~8 with high yield. 2.1.3.1. Bis-N-(phthalimido)-1, 2-ethane (6). White solid; Yield: 80%; m.p. 233e234 C; 1H NMR (CHCl3-d, 400 MHz) d: 4.04 (s, 4H, H-10, H-11), 7.71 (dd, 4H, J ¼ 5.6 Hz, 3.2 Hz, H-5, H-50 , H-8, H-80 ), 7.81 (dd, 4H, J ¼ 5.6 Hz, 3.2 Hz, H-6, H-60 , H-7, H-70 ); 13C NMR (CHCl3-d, 100 MHz) d: 35.85, 123.36, 131.99, 133.99, 168.22. EIMS, m/ z 320 [M]þ. 2.1.3.2. Bis-N-(phthalimido)-1, 3-propylene (7). White solid; Yield: 72%; m.p. 189e190 C; 1H NMR (CHCl3-d, 400 MHz) d: 2.09e2.17 (m, 2H, H-11), 3.79 (t, 4H, J ¼ 7.2 Hz, H-10, H-12), 7.73 (dd, 4H, J ¼ 5.6 Hz, 3.2 Hz, H-5, H-50 , H-8, H-80 ), 7.86 (dd, 4H, J ¼ 5.6 Hz, 3.2 Hz, H-6, H-60 , H-7, H-70 ); 13C NMR (CHCl3-d, 100 MHz) d: 27.69, 35.77, 123.29, 132.11, 133.97, 168.22. EIMS, m/z 334 [M]þ. 2.1.3.3. Bis-N-(phthalimido)-1, 4-butane (8). White solid; Yield: 75%; m.p. 220e221 C; 1H NMR (CHCl3-d, 400 MHz) d: 1.74e1.78 (m, 4H, H-11, H-12), 3.75 (t, 4H, J ¼ 6.8 Hz, H-10, H-13), 7.73 (dd, 4H, J ¼ 5.6 Hz, 3.2 Hz, H-5, H-50 , H-8, H-80 ), 7.85 (dd, 4H, J ¼ 5.6 Hz, 3.2 Hz, H-6, H-60 , H-7, H-70 ); 13C NMR (CHCl3-d, 100 MHz) d: 25.06, 37.42, 123.25, 132.13, 133.93, 168.36. EIMS, m/z 348 [M]þ. 2.1.4. General procedure for the preparation of compounds 9~11 To a solution of 6-methoxy-4-methylquinolin-2(1H)-one (0.5 mmol) in methylbenzene (15.0 mL), compounds 1, 2 or 3 (0.5 mmol) was added respectively. The reaction was conducted at 90 C in the presence of KOH (1.0 mmol), KI (0.05 mmol) and TBAB (0.05 mmol) and monitored with TLC. After the reaction, the mixture was cooled down and separated. The solvent was evaporated to prepare crude products, which were purified by chromatography (CHCl3/MeOH 100/1) to give compounds 9~11. 2.1.4.1. N-[2-(6-methoxy-4-methylquinolin-2-on-1-yl)ethyl]phthalimide (9). White solid; Yield: 48%; m.p. 215e216 C; 1H NMR
(CHCl3-d, 400 MHz) d: 2.52 (s, 3H, CH3), 3.94 (s, 3H, OCH3), 4.12 (t, 2H, d ¼ 5.6 Hz, H-10), 4.81 (t, 2H, d ¼ 5.6 Hz, H-11), 6.69 (s, 1H, H30 ), 7.06 (d, 1H, J ¼ 2.8 Hz, H-50 ), 7.36 (m, 1H, H-70 ), 7.62 (dd, 2H, J ¼ 5.6 Hz, 3.2 Hz, H-5, H-8), 7.72 (dd, 2H, J ¼ 5.6 Hz, 3.2 Hz, H-6, H7), 7.77 (d, 1H, J ¼ 8.4 Hz, H-80 ); 13C NMR (CHCl3-d, 100 MHz) d: 19.13, 23.47, 37.09, 40.19, 54.83, 101.42, 111.94, 123.67, 127.41, 132.07, 132.21, 136.44, 136.53, 147.79, 156.55, 161.25, 167.69. MS (ESI) m/z 363.1 ([MþH]þ). 2.1.4.2. N-[3-(6-methoxy-4-methylquinolin-2-on-1-yl) propyl] phthalimide (10). White solid; Yield: 34%; m.p. 189e190 C; 1H NMR (CHCl3-d, 400 MHz) d: 1.88e1.92 (m, 2H, H-11), 2.45 (s, 3H, CH3), 3.88 (s, 3H, OCH3), 4.35 (t, 2H, J ¼ 5.6 Hz, H-12), 4.49 (t, 2H, J ¼ 5.6 Hz, H-10), 6.58 (s, 1H, H-30 ), 6.79 (s, 1H, H-50 ), 7.21 (d, 1H, J ¼ 2.8 Hz, H-70 ), 7.41 (dd, 2H, J ¼ 5.2 Hz, 3.2 Hz, H-5, H-8), 7.63 (dd, 2H, J ¼ 5.2 Hz, 3.2 Hz, H-6, H-7), 7.89 (d, 1H, H-80 ); 13C NMR (CHCl3d, 100 MHz) d: 19.34, 23.47, 37.88, 40.18, 55.58, 102.96, 107.40, 112.54, 116.69, 119.82, 127.30, 128.18, 128.37, 129.38, 146.36, 156.26, 160.74, 168.89. MS (ESI) m/z 377.1 ([MþH]þ). 2.1.4.3. N-[4-(6-methoxy-4-methylquinolin-2-on-1-yl) butyl] phthalimide (11). White solid; Yield: 45%; m.p. 185e186 C; 1H NMR (CHCl3-d, 400 MHz) d: 1.57 (m, 2H, H-11), 1.93 (m, 2H, H-12), 2.63 (s, 3H, CH3), 3.81 (t, 2H, J ¼ 6.8 Hz, H-10), 3.94 (s, 3H, OCH3), 4.61 (t, 2H, J ¼ 6.8 Hz, H-13), 6.80 (s, 1H, H-30 ), 7.18 (d, 1H, J ¼ 2.8 Hz, H-50 ), 7.33 (d, 1H, J ¼ 9.2 Hz, H-70 ), 7.73 (dd, 2H, J ¼ 5.2 Hz, 3.2 Hz, H-5, H-8), 7.86 (dd, 2H, J ¼ 5.2 Hz, 3.2 Hz, H-6, H-7), 8.02 (d, 1H, J ¼ 7.2 Hz, H80 ); 13C NMR (CHCl3-d, 100 MHz) d: 18.87, 25.47, 25.57, 37.60, 37.88, 55.58, 103.47, 113.24, 123.19, 123.24, 125.81, 132.14, 132.19, 133.86, 133.91, 139.94, 156.06, 160.53, 168.44. MS (ESI) m/z 391.2 ([MþH]þ). 2.1.5. General procedure for the preparation of compounds 12 and 13 To a solution of N-(4-bromobutyl) phthalimide (3) (1 mmol) in methylbenzene (15.0 mL), 7-hydroxy-4-methylcoumarin (1 mmol) or 8-hydroxyquinoline (1 mmol) was added. The mixture was heated to 90 C with stirring in the presence of KOH (2.0 mmol), KI (0.06 mmol) and TBAB (0.06 mmol). After the reaction, the mixture was cooled down and separated. The solvent was evaporated to prepare crude products, which were purified by chromatography (CHCl3/MeOH 50/1) to give compounds 12 and 13. 2.1.5.1. 4-Methyl-7-[4-(isoindole-1, 3-dione-2-yl) butyloxy] benzopyran-2-one (12). White solid; Yield: 51%; m.p. 181e182 C; 1H NMR (CHCl3-d, 400 MHz) d:1.90e1.92 (m, 4H, H-11, H-12), 2.41 (s, 2H, CH3), 3.80 (t, 2H, J ¼ 6.4 Hz, H-10), 4.08 (t, 2H, J ¼ 6.4 Hz, H-13), 6.15 (s, 1H, H-30 ), 6.80 (d, 1H, J ¼ 2.4 Hz, H-80 ), 6.86 (dd, 1H, J ¼ 8.4 Hz, 2.4 Hz, H-60 ), 7.48e7.50 (d, 1H, J ¼ 8.4 Hz, H-50 ), 7.73e7.75 (m, 2H, H-5, H-8), 7.86e7.88 (m, 2H, H-6, H-7); 13C NMR (CHCl3-d, 100 MHz) d: 18.66, 25.27, 26.39, 37.56, 67.77, 101.47, 111.97, 112.59, 113.58, 123.26, 125.49, 132.11, 133.98, 152.48, 155.29, 161.29, 161.98, 168.43. MS (ESI) m/z 378.1 ([MþH]þ). 2.1.5.2. 8-[4-(phthalimide-2-yl) butyloxy] quinoline (13). White solid; Yield: 60%; m.p. 127e128 C; 1H NMR (CHCl3-d, 400 MHz) d:1.99e2.05 (m, 2H, H-11), 2.09e2.14 (m, 2H, H-12), 3.84 (t, 2H, J ¼ 5.8 Hz, H-10), 4.33 (t, 2H, J ¼ 5.8 Hz, H-13), 7.13 (d, 1H, J ¼ 8.0 Hz, H-70 ), 7.42 (d, 1H, J ¼ 8.0 Hz, H-30 ), 7.48e7.52 (m, 2H, H-50 , H-60 ), 7.73 (dd, 2H, J ¼ 5.6 Hz, 3.2 Hz, H-5, H-8), 7.86 (dd, 2H, J ¼ 5.6 Hz, 3.2 Hz, H-6, H-7), 8.21 (d, 1H, J ¼ 8.0 Hz, H-40 ), 9.01 (d, 1H, J ¼ 2.4 Hz, H-80 ); 13C NMR (CHCl3-d, 100 MHz) d: 25.23, 25.27, 37.59, 68.39, 109.42, 119.55, 121.56, 123.21, 127.21, 129.57, 132.16, 133.91, 148.63, 148.66, 150.24, 168.45. MS (ESI) m/z 347.1 ([MþH]þ).
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2.1.6. General procedure for the preparation of compounds 14~25 Compounds 14~25 were prepared by the condensation reaction between phthalic anhydride and related primary amine. To a solution of phthalic anhydride (0.5 mmol) in acetic acid (10 mL), related primary amine (0.5 mmol) was added respectively. The mixture was refluxed at 120 C for 3e4 h. Then 20 mL distilled water was added into the mixture after being cooled down and the solid precipitate was filtered out to get crude compounds, which were recrystallized in ethanol to prepare compounds 14~25. 2.1.6.1. N-phenylphthalimide (14). White solid; Yield: 88%; m.p. 209e210 C; 1H NMR (CHCl3-d, 400 MHz) d: 7.42e7.48 (m, 3H, H-30 , H-40 , H-50 ), 7.52e7.56 (m, 2H, H-20 , H-60 ), 7.82 (dd, 2H, J ¼ 7.2 Hz, 3.2 Hz, H-5, H-8), 7.99 (dd, 2H, J ¼ 7.2 Hz, 3.2 Hz, H-6, H-7); 13C NMR (CHCl3-d, 100 MHz) d: 123.77, 126.60, 128.11, 129.13, 131.83, 134.39, 167.29. EIMS, m/z 223 [M]þ. 2.1.6.2. N-(phenylmethyl) phthalimide (15). White solid; Yield: 90%; m.p. 118e119 C; 1H NMR (CHCl3-d, 400 MHz) d: 4.88 (s, 2H, H-10), 7.30e7.36 (m, 3H, H-30 , H-40 , H-50 ), 7.46 (d, 2H, J ¼ 7.2 Hz, H-20 , H60 ), 7.73 (dd, 2H, J ¼ 5.2 Hz, 3.2 Hz, H-5, H-8), 7.87 (dd, 2H, J ¼ 5.2 Hz, 3.2 Hz, H-6, H-7); 13C NMR (CHCl3-d, 100 MHz) d: 41.64, 123.36, 127.83, 128.62, 128.69, 132.18, 133.98, 168.05. EIMS, m/z 237 [M]þ. 2.1.6.3. N-(4-methoxyphenyl) phthalimide (16). Purple solid; Yield: 82%; m.p. 158e159 C; 1H NMR (CHCl3-d, 400 MHz) d: 3.88 (s, 3H, OCH3), 7.05 (dd, 2H, J ¼ 7.2 Hz, 2.0 Hz, H-30 , H-50 ), 7.36 (d, 2H, J ¼ 8.8 Hz, H-20 , H-60 ), 7.81 (dd, 2H, J ¼ 5.6 Hz, 3.2 Hz, H-5, H-8), 7.98 (dd, 2H, J ¼ 5.6 Hz, 3.2 Hz, H-6, H-7); 13C NMR (CHCl3-d, 100 MHz) d: 55.54, 114.52, 123.68, 127.95, 131.89, 134.30, 159.29, 167.58. EIMS, m/ z 253 [M]þ. 2.1.6.4. N-(benzamide-4-yl) phthalimide (17). White solid; Yield: 78%; m.p. 264e265 C; 1H NMR (CHCl3-d, 400 MHz) d: 7.64 (d, 2H, J ¼ 8.2 Hz, H-30 , H-50 ), 7.85 (dd, 2H, J ¼ 7.2 Hz, 3.2 Hz, H-5, H-8), 7.98 (d, 2H, J ¼ 8.2 Hz, H-20 , H-60 ); 8.01 (dd, 2H, J ¼ 7.2 Hz, 3.2 Hz, H-6, H7); 13C NMR (CHCl3-d, 100 MHz) d: 123.85, 126.25, 128.20, 129.27, 131.82, 132.26, 139.13, 167.53, 168.78. EIMS, m/z 266 [M]þ. 2.1.6.5. N-(quinolin-5-yl) phthalimide (18). White solid; Yield: 86%; m.p. 217e218 C; 1H NMR (CHCl3-d, 400 MHz) d: 7.48 (dd, 1H, J ¼ 8.8 Hz, 4.0 Hz, H-80 ), 7.59 (d, 1H, J ¼ 7.2 Hz, H-30 ), 7.88e7.92 (m, 3H, H-5, H-8, H-70 ), 8.03e8.07 (m, 3H, H-6, H-7, H-40 ), 8.34 (d, 1H, J ¼ 8.4 Hz, H-20 ), 9.01e9.02 (m, 1H, H-60 ); 13C NMR (CHCl3-d, 100 MHz) d: 121.79, 121.80, 124.15, 125.80, 125.81, 127.75, 127.76, 128.20, 129.41, 131.84, 134.79, 150.23, 167.49. EIMS, m/z 274 [M]þ. 2.1.6.6. N-(isoquinolin-5-yl) phthalimide (19). White solid; Yield: 83%; m.p. 238e239 C; 1H NMR (CHCl3-d, 400 MHz) d: 7.56 (d, 1H, J ¼ 6.0 Hz, H-60 ), 7.79e7.87 (m, 2H, H-70, H-80 ), 7.90 (dd, 2H, J ¼ 5.6 Hz, 3.2 Hz, H-5, H-8), 8.06 (dd, 2H, J ¼ 5.6 Hz, 3.2 Hz, H-6, H7), 8.23 (d, 1H, J ¼ 8.0 Hz, H-40 ), 8.58 (d, 1H, J ¼ 6.0 Hz, H-30 ), 9.46 (s, 1H, H-10 ); 13C NMR (CHCl3-d, 100 MHz) d: 116.62, 124.21, 127.69, 127.77, 129.29, 129.79, 131.80, 132.22, 133.65, 134.86, 141.89, 151.86, 167.27. EIMS, m/z 274 [M]þ. 2.1.6.7. N-(quinolin-8-yl) phthalimide (20). White solid; Yield: 83%; m.p. 227e228 C; 1H NMR (CHCl3-d, 400 MHz) d: 7.48 (dd, 1H, J ¼ 8.4 Hz, 4.4 Hz, H-60 ), 7.72 (d, 1H, J ¼ 8.0 Hz, H-50 ), 7.84 (dd, 2H, J ¼ 5.2 Hz, 3.2 Hz, H-5, H-8), 7.90 (dd, 1H, J ¼ 7.2 Hz, 3.2 Hz, H-30 ), 7.99e8.04 (m, 3H, H-6, H-7, H-40 ), 8.28 (dd, 1H, J ¼ 8.4 Hz, 1.2 Hz, H70 ), 8.89e8.90 (m, 1H, H-20 ); 13C NMR (CHCl3-d, 100 MHz) d: 121.92, 123.91, 126.32, 129.36, 129.61, 129.73, 130.46, 132.53, 134.19, 136.55, 136.58, 150.73, 167.91. EIMS, m/z 274 [M]þ.
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2.1.6.8. N-(5-carboxylhloropyridine-2-yl) phthalimide (21). White solid; Yield: 80%; m.p. 164e165 C;1H NMR (DMSO-d6, 400 MHz) d: 7.70 (m, 1H, H-50 ), 7.79e8.02 (m, 4H, H-5, H-6, H-7, H8), 8.49 (d, 1H, J ¼ 8.0 Hz, H-40 ), 9.11 (d, 1H, J ¼ 1.2 Hz, H-20 ), 13.58 (s, 1H, COOH); 13C NMR (DMSO-d6, 100 MHz) d: 112.26, 119.86, 124.58, 132.35, 134.02, 140.76, 150.61, 152.35, 165.88, 167.72. EIMS, m/z 268 [M]þ. 2.1.6.9. N-(2-chloropyridine-4-yl) phthalimide (22). White solid; Yield: 81%; m.p. 156e157 C; 1H NMR (CHCl3-d, 400 MHz) d: 7.65 (dd, 1H, J ¼ 5.6 Hz, 1.6 Hz, H-30 ), 7.76 (d, 1H, J ¼ 1.6 Hz, H-50 ), 7.88 (dd, 2H, J ¼ 7.2 Hz, 3.2 Hz, H-5, H-8), 8.03 (dd, 2H, J ¼ 7.2 Hz, 3.2 Hz, H-6, H-7), 8.54 (d, 1H, J ¼ 5.6 Hz, H-60 ); 13C NMR (CHCl3-d, 100 MHz) d: 117.71, 119.49, 124.32, 131.22, 135.18, 141.84, 150.38, 152.29, 165.93. EIMS, m/z 258 [M]þ. 2.1.6.10. 2-(phthalimide-2-yl)-3-(1H-indol-3-yl) propanoic acid (23). White solid; Yield: 85%; m.p. 195e196 C; 1H NMR (DMSO-d6, 400 MHz) d: 3.73e3.87 (m, 2H, H-11), 5.36 (dd, 1H, J ¼ 10.8 Hz, 5.2 Hz, H-10), 7.05 (s, 1H, H-20 ), 7.10 (t, 1H, J ¼ 8.0 Hz, H-50 ), 7.16 (t, 1H, J ¼ 7.2 Hz, H-60 ), 7.62 (d, 1H, J ¼ 8.0 Hz, H-70 ), 7.69 (dd, 2H, J ¼ 5.2 Hz, 3.2 Hz, H-5, H-8), 7.78 (dd, 2H, J ¼ 5.2 Hz, 3.2 Hz, H-6, H7), 7.94 (d, 1H, H-40 ); 13C NMR (DMSO-d6, 100 MHz) d: 24.71, 52.32, 110.99, 111.08, 118.51, 119.66, 122.22, 122.55, 123.48, 131.69, 134.07, 136.10, 167.47, 172.53. EIMS, m/z 334 [M]þ. 2.1.6.11. N-(aminoantipyrine-4-yl) phthalimide (24). White solid; Yield: 87%; m.p. 214e215 C; 1H NMR (CHCl3-d, 400 MHz) d: 2.25 (s, 3H, CH3), 3.27 (s, 3H, NCH3), 7.33e7.37 (m, 1H, H-40 ), 7.47e7.50 (m, 4H, H-20 , H-30 , H-50 , H-60 ), 7.79 (dd, 2H, J ¼ 5.2 Hz, 3.2 Hz, H-5, H-8), 7.96 (dd, 2H, J ¼ 5.2 Hz, 3.2 Hz, H-6, H-7); 13C NMR (CHCl3-d, 100 MHz) d: 11.15, 35.54, 102.23, 123.83, 124.86, 127.33, 129.29, 132.25, 134.29, 134.39, 152.78, 160.77, 166.91. EIMS, m/z 333 [M]þ. 2.1.6.12. 2-(4-methyl-2-oxidanylidene-chromen-7-yl) phthalimide (25). White solid; Yield: 80%; m.p. 255e256 C; 1H NMR (CHCl3-d, 400 MHz) d: 2.50 (s, 3H, CH3), 6.37 (s, 1H, H-30 ), 7.52 (dd, 1H, J ¼ 9.6 Hz, 2.0 Hz, H-60 ), 7.58 (d, 1H, J ¼ 2.0 Hz, H-80 ), 7.76 (d, 1H, J ¼ 8.4 Hz, H-50 ), 7.86 (dd, 2H, J ¼ 5.6 Hz, 3.2 Hz, H-5, H-8), 8.02 (dd, 2H, J ¼ 5.6 Hz, 3.2 Hz, H-6, H-7); 13C NMR (CHCl3-d, 100 MHz) d: 18.69, 114.74, 115.52, 121.80, 124.09, 125.05, 131.51, 134.77, 134.82, 151.73, 153.68, 160.37, 166.63. EIMS, m/z 305 [M]þ. 2.2. Antifungal bioassay Two destructive phytopathogenic fungi (A. solani and B. cinerea) were applied to test the antifungal activity of the prepared compounds in vitro by measuring mycelial inhibition of radial growth. All compounds were dissolved in DMSO as stock solutions and diluted to a testing concentration of 100 mg/mL with PDA medium under 50 C. The final solutions were poured into sterilized Petri dishes (6 cm). After solidification, a mycelia disk (diameter: 0.6 cm) of active fungi was inoculated in the center of the PDA Petri dishes. The dishes were parafilmed and incubated at 25 C for 5 days. DMSO mixed with PDA to the equivalent concentrations was used as negative control, and carbendazim was served as positive control. Growth inhibition was calculated by the formula: Inhibition rate (%) ¼ (C-T) 100/C (C: the mean colony diameter in control; T: the colony diameter in treatments). The measurement was carried out with three replicates, and the results were shown as mean values (±SD). The effective compounds were further tested under a series of lower concentrations to calculate the IC50 values [17,18].
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L. Pan et al. / Microbial Pathogenesis 95 (2016) 186e192
Scheme 1. Synthesis of phthalimide derivatives (1e13).
Scheme 2. Synthesis of phthalimide derivatives (14e25).
3. Results and discussion A series of phthalimide coupled derivatives were efficiently synthesized according to the proposed methods, and the general synthesis was depicted in Schemes 1 and 2. Compounds 1~3 were synthesized by the N-alkylation of phthalimide with appropriate dibromoalkane [19,20]. Under the similar condition, compounds 1~3 condensed with phthalimide to give compounds 6~8. But the coupling of compounds 1~3 with 6-methoxy-4-methylquinolin2(1H)-one could not be realized in the presence of potassium carbonate, a complex alkaline system of potassium hydroxide, potassium iodide and tetrabutyl ammonium bromide (TBAB) was applied to prepare 9~11. Compounds 12 and 13 were prepared under the similar condition by the reaction between compound 3 and 7-hydroxy-4-methylcoumarin or 8-hydroxyquinoline. With the presence of potassium hydroxide and quinoline in ethanol, compounds 1 and 2 eliminated the hydrogen bromide to prepare compounds 4 and 5 respectively (Scheme 1).
Phthalic anhydride and related primary amine were employed to prepare compounds 14~25 effectively [21,22] (Scheme 2). The antifungal activity of prepared compounds against A. solani and B. cinerea was initially assayed at 100 mg/mL (Table 1), at which level, most of the prepared compounds showed significant fungitoxicity against either or both of pathogenic fungi. Among these tested derivatives, compounds 1, 2, 3, 4, 13 and 15 exhibited the strong activity against both A. solani and B. cinerea with over 50% inhibition of mycelial growth. Compounds 6, 8, 11 and 18 selectively inhibited 50.00e57.14% of mycelia growth against A. solani, whereas compound 16 showed stronger inhibitory effects against B. cinerea (53.19% of inhibition) than A. solani (with 37.62% inhibition). The effective compounds with over 50% inhibition of mycelial growth were further tested at a series of lower concentrations and their IC50 values were calculated (Table 2). The results indicated that compound 4 has the lowest IC50 value against B. cinerea (7.92 mg/mL), and compounds 13 showed the strongest antifungal activity against A. solani with IC50 values of 10.85 mg/mL.
L. Pan et al. / Microbial Pathogenesis 95 (2016) 186e192 Table 1 Antifungal activity of the prepared compounds at 100 mg/mL (96 h of incubation). The inhibition of radial growth (%, mean ± SD, N ¼ 3)
Compd.
A. solani 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 DMSO(1%) Carbendazim
64.29 59.52 69.05 78.57 48.14 50.00 42.86 54.76 40.48 47.62 52.38 45.71 71.43 41.77 54.76 37.62 27.60 57.14 49.38 38.10 40.48 19.05 15.30 40.48 33.33 0 93.88
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
B. cinerea 78.72 ± 10.35 51.94 ± 6.83 74.47 ± 7.69 93.62 ± 6.92 45.07 ± 15.08 34.04 ± 10.76 31.91 ± 13.09 48.94 ± 3.26 31.91 ± 12.85 36.17 ± 8.60 38.30 ± 2.30 42.55 ± 12.26 91.49 ± 22.30 39.18 ± 11.33 65.96 ± 6.20 53.19 ± 15.38 33.15 ± 9.79 25.53 ± 3.84 27.66 ± 7.69 6.38 ± 4.26 2.13 ± 0.96 14.89 ± 7.92 0 38.30 ± 9.14 27.66 ± 13.27 1.03 ± 1.16 95.16 ± 9.47
14.26 3.85 10.57 14.35 10.90 12.24 5.23 21.57 7.23 2.81 7.14 11.42 8.53 7.45 2.14 18.44 12.45 22.16 21.42 2.17 12.43 9.85 10.89 12.85 15.71
± 17.29
Table 2 IC50 values of effective compounds against two harmful phytopathogenic fungi (96 h of incubation). Compd.
1 2 3 4 7 8 11 13 15 16 18 Carbendazim a
IC50 (95% cL)a (mg/mL) A. solani
B. cinerea
45.83 50.17 48.12 16.95 164.06 112.38 171.48 10.85 65.19 e 85.71 1.74
33.33 129.58 64.69 7.92 e 178.40 e 35.65 91.55 98.43 e 1.90
(29.35e62.97) (32.08e76.56) (21.35e69.78) (4.84e29.85) (107.65e221.25) (87.54e156.16) (143.35e236.59) (3.62e20.84) (38.42e86.93) (69.23e107.82) (0.23e3.03)
(19.35e57.61) (105.31e159.93) (43.81e79.78) (3.08e16.40)
191
two moieties were coupled directly. On phenyl substitution, N(phenylmethyl) phthalimide (15) was more effective than N-phenylphthalimides (14) and the introduction of methoxyl group at para-position (16) could increase the activity, whereas benzamide substitution (17) contributed less. It was found that direct coupling with other functional moieties of pyridine (21, 22), tryptophane (23), antipyrine (24) and coumarin (25) made little contribution to the activity. The results indicated that the appropriate functional groups with indirect coupling by optimum carbon chain might be promising to prepare effective fungicide. 4. Conclusion In summary, N-substituted pthalimide derivatives were efficiently synthesized by modified condensation reactions. With screening for their antifungal activity against A. solani and B. cinerea, N-vinyl-phthalimide (4) and 8-[4-(phthalimide-2-yl) butyloxy]quinoline (13) were found to be the most effective among the prepared compounds and proposed to be promising candidates for the further development. With studying the activity contribution of functional moieties, it clearly suggested that the optimum substitution at N atom of pthalimide could bring about significant increase of activity, which would be helpful for the discovery of efficacious fungicides. Funding This work was supported by the National Natural Science Foundation of China (No. 31570354, 31070386, 21302195 and 31300290), Agricultural Biotechnology Research and Development Program of Gansu Province (GNSW-2015-25), Cooperation Program to Gansu Province of Lanzhou Branch of the Chinese Academy of Sciences, 135 key cultivation program of the Chinese Academy of Sciences, and the Province-academy Cooperation Program of Henan Province of China (NO. 102106000021). Notes The authors declare no competing financial interest.
(130.90e229.47)
Appendix A. Supplementary data (21.92e49.25) (49.76e124.85) (65.31e140.40)
Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.micpath.2016.04.012.
(0.39e2.60)
95% confidence limit.
Concerning the phytotoxicity, we took a preliminary test on the growth inhibition of tomato seedlings with effective phthalimides derivatives. The results shows that the compounds exhibited insignificant inhibitory effects on seedling growth in comparing with negative control (data not shown here). For their possible use, more comprehensive work should be conducted to evaluate their efficacy and safety. The types of functional groups are intensive responsible for the activity. N-vinyl substituted phthalimide (4) exhibited strong antifungal activity against both A. solani and B. cinerea, and Nbromoalkylation of phthalimides (1~3) significantly increased the inhibitory effects, regardless of the side chain length. However, the chain length of quinoline coupled phthalimides was found to influence the activity deeply, such as compound 13, with butoxy chain between quinoline and phthalimides, exhibited much stronger toxicity against both fungi than compound 20, of which
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